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EFFECTS OF LOW LEVEL LASER (DIODE) THERAPY ON HUMAN BONE REGENERATION:

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EFFECTS OF LOW LEVEL LASER (DIODE) THERAPY ON HUMAN BONE REGENERATION: Powered By Docstoc
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Gono-Bishwabidyalay (Gono-University),Mirzanagar, Savar, Dhak1344, Bangladesh. April, 2010.
1
 Dept.of Medical Radiation Physics, Kreiskrankenhaus Gummersbach, Teaching Hospital of the University of
Cologne, 51643 Gummersbach, Germany.
2
  Dept. of Medical Physics and Biomedical Engineering, Gono-Bishwabidyalay (Gono University), Nayarhat,
Savar, Dhaka- 1344, Bangladesh.
3
  Department of Orthopedic and Traumatology, Shaheed Suhrawardy Medical Colleg e Hospital, Dhaka-
1207, Bangladesh.
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2|Page
Declaration


I declare that I am the sole author of this thesis and that the work
presented here has not previously been submitted as an exercise for a degree
or other qualification at any university.
It consists entirely of my own work, except where references indicate
otherwise.




Dr. Md. Nazrul Islam. 10th April/2010.




                                                                3|Page
o    Acknowledgments




I wish to express my deepest gratitude and thanks to my guide and thesis supervisor Professor Golam Abu Zakaria, for his
kind directions, inspiring guidance, and invaluable discussion throughout the course. Without his patience and
encouragement, this work would never be fulfilled. I also express my heartfelt respect to my co-guides Prof. F. H. Sirazee ,
ex. head of the department, and Associate professor, Dr. P C Debenarh, Department of Orthopedic & Traumatology,
Shaheed Suhrawardy Medical College Hospital. Dhaka-1207 and Prof. Nurul Islam dept. of Medical Physics and
Biomedical Engineering Gono Bishwabidyalay (Gono University) Nayarhat, Savar, Dhaka for heartiest co -
operation and advice throughout the whole research period.
I would also like to express my profoundest gratitude to my thesis advisory board / working team members, Associate
Prof. Dr Sheikh Abbasuddin Ahmed, assistant Prof Dr. Kazi Shamimuzzaman, Dr. Zia Uddin,consultant, Dr. Subir Hossain
Shuvro, assistant registrar, Dr. Abdul Hannan of Orthopedic & Tramatology Department, assistant Prof Dr. Quamrul Akter
Sanju of Surgry department, Shaheed Suhrawardy Medical College Hospital and Dr. Sayed Shaheedul Islam Assistant
professor, NITOR, Dhaka.
Special thanks are due to Prof. Khadiza Begum, ex-director, Prof. A. K.M Mujibur Raman, director, Dr. Mir Mahamuda
Khanam, assistant director of SsMCH, Prof. S. M. Idris Ali, ex. head of the department and vice-principal,SSMC and Prof.
Abdul kader Khan, ex-principal and head of the department, surgery, SSMC/ SsMCH, associate professor, Dr. Mostaq
Hossain Tuhin of Surgery department, assistant. Prof. Asraf Uddin, head o f the department ,Radiology & Imaging
department of SsMCH, and to all my friends and well-wishers, specially to Mr. Sinha Abu Khalid CEO, LabNucleon, Md.
Masud Rana, medical Physicist, National Cancer Institute & Hospital, Md. Anwarul Islam, medical physicist, Squrae
Hospital & Mr. Kumaresh Chandra Pal, medical physicist of Gono Biswabidyalay, Dhaka-1344, and Md Shakilur Rahman,
senior scientific officer of Bangladesh Atomic Energy Commission for their kind and nice co-operation throughout the
course of this work.
My deepest admiration and sincerest love to and my laser machine technician Mr. Mamun, laser operator Ms. Jannant &
Ms. Chewty of LabNucleon, and Md. Abdul Aziz, Mr. Polash, Mr. Abul- kasem, Ms. Fatema, Ms Farida, Mr. Malek of
Orthpedic and Traumatology Department, and Kazi Murad Hossain of Shaheed Suhrawardy Medical Hospital, Dhaka-
1207, Bangladesh for their continuous efforts to successfully complete this project.
Finally, I owe much and pay my heartiest thanks, especially to those who rendered their hind assistance during my study
period. Words cannot be expressed my feeling of love, I am deeply indebted to my wife Habiba Islam, my loving sons-
Sayem Islam labib & Talaat Islam Syiam for all their support and understanding. In the whole course of this work, they
gave me a sweet working atmosphere, which I can‘t find words to express.




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Dedication

To my parents, Mr. Alauddin Sikder & Ms. Munira Begum- for fostering
and encouraging my interest in science
                                 &
Mother in-law, late Ms. Anowara Begum and my wife Ms. Habiba Islam
Happy for their tremendous and unbelievable mental support during my
post-graduate period.




Sponsored by-

This research project is jointly sponsored by-
Lab-Nucleon & Acme Laboratories, Dhaka,
Bangladesh.




                                                           5|Page
Abstract



Thesis Title:

EFFECTS OF LOW LEVEL LASER (DIODE) THERAPY ON
HUMAN BONE REGENERATION:
BY-
DR. MD NAZRUL ISLAM




Objective:
Laser (Semiconductor diode, Ga-Al-As, 830nm) is effective in human bone regeneration,
i.e. it enhances bone fracture healing.


Background Data:
Tissue healing is a complex process that involves both local and systemic responses, and the healing process of bone is
much slower than that of soft tissues which is a great challenge of medical science. The use of Laser Therapy (LLLT) for
wound healing has been shown to be effective in modulating both local and systemic response by enhancing- cellular &
mitochondrial ion exchange, bone mineralization, nitric oxide formation, lymphatic circulation, osteoblast proliferation,
effects on osteoblast gene expression, osteoclast inhibition (prevents bone mineral resorption) and by bone engraftment
on synthetic materials.
Methods:
40 (Twenty in laser & Twenty in control group) otherwise healthy men and women with, closed appendicular bone fracture
(Radius/ ulna, or Femur / Tibial shaft /Clavicle / Meta carpal /Meta-tarsal) was enrolled for fracture management by laser
therapy adjunctive to regular management, and was assed by clinical and radiological findings (X-ray)/at 2nd , 3rd, 4th and
6th week post fracture: assessment included fracture line/margins, fracture gap, external callus appearance, callus-to-
cortex ratio, bridging, and radiologic union as well as clinical assessment of the fracture- compliance of patient, and
onwards follow-up of patients, in comparison to controlled group.
Results:
Early significant bone regeneration /callus formation achieved by early application of Low Level laser therapy (Ga-Al-As,
830 nm) on human fractured long (appendicular) bone.
Conclusions:
Treatment with 830 nm diode laser has substantially reduced the fracture healing time as well as improved the
quality/quantity of callus formation of the patient, thus enhancing fracture healing. Laser biostimulative effects on bone
could be a new dimension for bone regeneration which significantly reduce healing period, lessen cost of treatment, and
enhance patient compliance in medical science.




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Contents:

Chapter-1                                                                           Page- 8- 13
Introduction, Background and literature review
1.1 Introduction
1.2 Background
1.3 Literature review

Chapter-2                                                                           Page- 14-30
Cell & Bone
2.1 Cell
2.2 Bone
2.3 Bone of Fracture
2.4 Healing of fracture

Chapter-3                                                                           Page- 31-41
Laser & Laser System
3.1 Laser
3.2 Laser principle
3.3 Components of a laser system
3.4 Laser Machine
3.5 Measurement of Laser Energy

Chapter-4                                                                           Page- 42-68
Biophysical Aspects, Laser- Tissue Interaction, Mechanisms and Bone Regeneration.
4.1 Biophysical Aspects & light transport theory
4.2 Laser - Tissue Interaction
4.3 The Mechanisms of Low Level Laser Therapy
4.4: Effects of Laser on Biological Cell/Tissue healing
     Laser on hard tissue & Bone stimulation/ Regeneration
4.5 Medical application of Low Level Laser

Chapter-5                                                                            Page- 69-74
Materials & Methods
5.1 Materials
5.2 Methods

Chapter-6                                                                             Page-75-78
Observation & Result
Chapter-7                                                                            Page- 79-80
Discussion
Chapter-8                                                                            Page- 81-81
Conclusion
Chapter-9                                                                            Page- 82-86
References

Chapter-10                                                                           Page-87-97
Appendices-
10.1 Figure & Table list
10.2 Laser Books & Articles
10.3 Datasheet.
10.4 Publications & Presentation:
10.5 Biography & pictures




                                                                                       7|Page
Chapter-1
Introduction, Background and literature review

1.1 Introduction
1.2 Background
1.3 Literature review

1.1 Introduction

Optimizing the results of fracture treatment requires a holistic view of both patients and treatment. The nature of the
patient determines the priority targets for outcome, which differ widely between the elderly and the young, and between
the victims of high and low energy trauma. The efficacy of treatment depends on the overall process of care and
rehabilitation as well as the strategy adopted to achieve bone healing.
The rational basis for fracture treatment is the interaction between three elements, (I) the cell biology of bone
regeneration, (ii) the revascularization of devitalized bone and soft tissue adjacent to the fracture; and (iii) the mechanical
environment of the fracture. The development of systems for early fracture stabilization has been an advance. However,
narrow thinking centered only on the restoration of mechanical integrity leads to poor strategy - the aim is to optimize the
environment for bone healing Future advances may come from the adjuvant use of molecular stimuli to bone regeneration.
Restoring function to a patient who has had a fracture requires the physician/ surgeon to handle a heady mix of
mechanical and biological issues. In real life, it also requires considerable input of time into practice organization, given
the large numbers of patients and the almost universal inadequacy of resource, if each individual patient is to receive
timely and appropriate intervention.
There is a perception, not least among fracture surgeons themselves, that the mechanical issues have been over-
emphasized in the past. The bonesetter's art consisted basically of- providing- anatomical realignment and external
support for as long as nature then took to restore internal structural competence by bone healing. This was slow and
unkind to soft tissues, particularly neighboring joints, so the development of materials, bio-mechanical understanding and
surgical technique launched a swing towards invasive interventions aimed at immediate restoration of internal structural
integrity.
The principles of AO treatment, drummed into a generation of orthopedic trainees, were anatomical open reduction, rigid
internal fixation and early rehabilitation of soft tissues without- external splint. But the scale of invasion required to achieve
these aims brought a steady trickle of serious problems - most notably infected non-unions, sometimes in cases which
surgeons knew they could safely have treated by simpler methods.
Furthermore, there was increasing realization that the abolition of inter-fragmentary motion implied a commitment to
primary cortical union as the only route for healing and a closure of the natural routes of callus formation. From various
directions, less invasive alternatives were developed: functional bracing, external fixation (including the remarkable Ilizarov
circular fixator developed in the USSR, which evades the bone only with fine wires) and closed intra-medullary nailing.
Now the science is taking another step, further in the direction from mechanics to biology. If the mechanical environment
influences bone regeneration and hence fracture healing, how, at a cellular- level, does it do so? - What molecular signals-
produce the response? If we know the signals, can we deliver them in the form of recombinant growth factors and hurry
the- cellular response down the right path? The evolution has been first to use nature, then to ignore her,
then to remember her, and now to outdo her.Optimal fracture treatment requires the following: (I) a definition of what
optimal treatment means and a way of measuring the extent to which it is achieved; (ii) a review of what we know about-
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the natural healing process that we want to harness or improve upon; and (iii) analysis of how to apply the above to clinical
practice.
During the last decade, it was discovered that low-power laser irradiation has stimulatory effects on bone tissue, in the
microscopic (cell proliferation [1-5] and gene expression [6]) and macroscopic [1, 2, 4, 12, 13-20] biological systems.
In order to understand the effects of laser therapy, its mechanism of action in the cell needs to be established. Many
explanations have been proposed         {7-11].   Studies have shown that porphyrins and cytochromes, natural photoacceptors
located in the cell, are the main contributors to laser-tissue interaction [7-11]. Porphyrins and cytochromes absorb the light
into the cell, resulting in the production of singlet 1O2. The singlet oxygen then stimulates the redox activity in the
mitochondria, enhances chemiosmosis, DNA production and calcium-ion influx into the cytoplasm, thereby causing mitosis
and cell proliferation.
The purpose of this review is to analyze the effects of low power laser irradiation on bone cells and bone fracture repair,
examine what has been done so far, and propose areas warranting further exploration.




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1.2 Background



Bone and fracture healing is an important homeostatic process that depends on specialized cell activation and bone

immobility during injury repair       [1, 2].   Fracture reduction and fixation are a prerequisite to healing but a variety of additional

factors such as age, nutrition, and medical co-morbidities can mediate the healing process                                [3, 4]..Different   methods have

been investigated in attempts to accelerate the bone-healing process. Most studies have concentrated on drugs, fixation

methods or surgical techniques; however, there is a potential role for adjunctive modalities that affect the bone-healing

process.



Laser is an acronym for ―Light Amplification by stimulated Emission of Radiation ‖ [5]. The first laser was demonstrated in

1960 and since then it has been used for surgery, diagnostics, and therapeutic medical applications [6]. The physiological

effects of low level lasers occur at the cellular level                   [7, 8],   and can stimulate or inhibit biochemical and physiological

proliferation activities by altering intercellular communication                     [9].   Early work on physical agents as mediators of bone

healing was performed by Yasuda, Noguchi and Sata who studied the electrical stimulation effects on bone healing in the

mid 1950s     [1, 10].   In subsequent years, others repeated this work in humans                     [1, 11]   and a variety of physical agents have

been investigated as potential mediators of bone healing [12, 23, 14, 15, and 16]. With increasing availability of lasers in the early

1970s, the potential to investigate its use as a modality to affect the healing of different connective tissues became

possible   [17, 18, and 19].   In 1971, a short report by Chekurov stated that laser is an effective modality in bone healing

acceleration     [19].    Subsequently, other researchers studied bone healing after laser irradiation using histological,

histochemical, and radiographic measures                [18, 19, 20, 21, 22, 23, and 24].   These studies have demonstrated mixed results where

some observed an acceleration of fracture healing [19, 21, 22, 23, 24], while others reported delayed fracture healing after low-

level laser irradiation [20, 25].




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1.3. Review of literature


      RELATED PREVIOUS WORKS

As far as is known, the first attempt at treating bone fracture with infrared light was reported by Shugaharov and Voronkov.
In 1974 they used low level laser radiation (infrared wavelengths) on fracture sites observing intramedullary
osteosynthesis.1
Gatev studied the effect of stimulating repair of fractures with He-Ne laser. The majority of patients had fractures of the
distal radius treated with a plaster cast. On the 5th to 8th day after injury a hole was cut out of the cast over the fracture
site and laser radiation applied at 632 nm, 2 mW/ cm2. Evaluations were made based on radiographic evidence and
clinical assessment. Results showed statistically significant differences [p<0.001] from the control group in favor of light
treated fractures.2
A 1990 case study looked at a non-union long bone fracture refractive to treatment over a period of 8 months. A 24 year-
old patient was treated conservatively for displaced fracture of the diaphysis of both bones of the right forearm. When
secondary displacement occurred the fracture was operated on with use of a compression plate for the radius and a single
Rush Rod for the ulna. Eight months after the injury the radiological and clinical examination showed signs of delayed
union of both fractures. A diode laser emitting 890 nm wavelengths near infrared light with average output of 3 mW and an
energy deposition of 1.8 Joules/cm2 was applied 3 times per week. After 4 weeks of treatment the signs of callus
formation appeared.
After another 5 weeks the radiogram showed complete remodeling of the ulnar bone and union in the radius. No side
effects were observed.




                      Figure1.3.1: Before & after laser therapy.

A 15 year old male athlete presented with an avulsion fracture with involvement of the inferior aspect of Anterior Superior
Iliac Spine. ASIS injury was non-weight bearing. Patient was taking 3200 mg ibuprofen daily.
Normal prognosis is 4-6 weeks non weight bearing followed by 6 weeks of rehab and additional 10 weeks before return to
sport (running). Protocol followed for this case: initiated daily infrared light treatments, 890 nm, 20 Joules/cm2, 20 min
treatments. Rehab begun on third visit. Discontinued ibuprofen after third treatment. Discharged from treatments after 24
visits and orthopedist released patient at 100% to return to running. Total time reduced from 22 weeks (normal prognosis)
to 5 weeks.4
An 18 year old high school athlete presented with a non union tibial fracture. The patient had previously fractured the
same site, taking 15 months to heal. Re-fracture was fixed with a compression plate. After 2 years the patient still showed
edema and pain with radiographic evidence on non union.
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Once daily treatments (5 days per week) were initiated with infrared light, 890 nm, 20 Joules/cm2, 20 min per treatment.
After 44 days radiographic analysis showed pannus formation over the set screws. After 86 days radiograph showed
complete fracture healing.24 Note pannus formation over screws.5
A patient presented with a non union 5th metatarsal fracture of the left foot. The patient was treated conservatively with
immobilization and non weight bearing. After 3 months no progress was evident from radiographic and clinical
assessments.
Daily treatments (5 days per week) were initiated with infrared light, 890 nm, 20 Joules/cm2, 20 min per treatment. After
three weeks radiographic and clinical assessment showed complete healing. 6
Study conducted by Maawan Khadra7 et all, 2004, The aim of this in vitro study was to investigate the effect of low-level
laser therapy (LLLT) on the attachment,       proliferation, differentiation and production of transforming growth factor-X1
(TGF-b1) by human osteoblast-like cells (HOB). Cells derived from human mandibular bone were exposed to Ga-Al-As
diode- laser at dosages of 1.5 or 3 J/cm2 and then seeded onto titanium discs. Non-irradiated cultures served as controls.
After 1, 3 and 24 h, cells were stained and the attached cells were counted under a light microscope. In order to
investigate the effect of LLLT on cell proliferation after 48, 72 and 96 h, cells were cultured on titanium specimens for 24 h
and then exposed to laser irradiation for three consecutive days.
Specific alkaline phosphates activity and the ability of the cells to synthesize osteocalcin after 10 days were investigated
using p-nitrophenylphosphate as a substrate and the ELSA-OST-NAT immunoradiometric kit, respectively. Cellular
production of TGF-b1 was measured by an enzyme-linked immunosorbent assay (ELISA), using commercially available
kits. LLLT significantly enhanced cellular attachment.Greater cell proliferation in the irradiated groups was- observed first
after 96 h. Osteocalcin synthesis and TGF-b1 production were significantly greater (Po0:05) on the samples exposed to 3
J/cm2. However, alkaline phosphatase activity did not differ significantly among the three groups. These results showed
that in response to- LLLT, HOB cultured on titanium implant material had a tendency towards increased cellular
attachment, proliferation, differentiation and production of TGF-b1, indicating that in vitro LLLT can modulate the activity of
cells and tissues surrounding implant material.
Study conducted by Chauhan and Sarin 8 in 2006, Low level laser therapy of stress fracture of tibia in a
prospective randomized trial and found complete resolution of pain and tenderness, and return to painless
ambulation was taken as end point of therapy. Standard treatment of Stress fracture includes rest, compression,
elevation and passive stretching.
Low level laser therapy (LLLT) has been described in treatment of joint conditions, tendophaties, musculofascial pains and
dermatological conditions. 68 cases were enrolled, 34 each in control and test group. Control cases were treated with
placebo and test group with laser-therapy. Complete resolution of pain and tenderness, and return to painless ambulation
was taken as end point of therapy in both groups. The test group showed earlier resolution of symptoms and- painless
ambulation with less recurrence. LLLT appears beneficial in treatment of stress fracture in this study.
Study conducted by S.Teixeira et al,9 2006, they concluded surface characterized by a homogeneous reproducible
microtopography, microtexure and microchemistry.
Surface topographic changes induced by the laser treatment Hydroxyapatite have been of different types where compared
at different scales, as both they have produced a major enhancement on the actual surface area. The result showed that
the surface topography of the substrate is attractive to the- cells, since they adhere to the cells very strongly to the
surface, being their philopodia attached between the valleys of the laser induced columnar texture.
Another study was conduct by Dimitrov et al10, 2009 , Department of Operative dentistry and Endodontics, Faculty of
Dental Medicine- Sofia, Medical University, Sofia and Department of Biochemistry, Faculty of Medicine,
Medical University, Sofia, effect of laser irradiation with different wavelength on the proliferations activity of human pulp
fibroblast cells, depending of irradiation- parameters and hard tissue thickness.
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There was determined marked stimulatory effect on- proliferation activity of human pulp fibroblast cells upon direct
irradiation with infrared laser and lower upon irradiation through different sections of dental hard tissue.
Upon irradiation with Helium-neon laser was determined inhibitory effect on the proliferation activity. It‘s possible that a
part of the mesenchymal pulp cells were differentiated into another cells. That will be explained with Western blot analysis
in the second part of our investigation. Forthcoming investigations will explain the vitality of isolated mesenchymal pulp
cells, their- identification and differentiation possibility, the permeability of laser beam through different sections of dentin
and enamel, power density of passed light, time and number of exposures in order to achieve the optimal effect on
proliferation.




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Chapter-2


Cell & Bone

2.1   Cell
2.2   Bone
2.3   Bone Fracture
2.4   Healing of fracture

2.1 Cell

The cell is the structural and functional unit of all known living organism. It is the smallest unit of an organism that is
classified as living, and is often called the building block of life. There are two types of cells: eukaryotic and prokaryotic.
Prokaryotic cells are usually independent, while eukaryotic cells are often found in multicellular organisms.
Cell Structure and functions
Each cell is a self-contained and self-maintaining entity: it can take in nutrients, convert these nutrients into
energy, carry out specialized functions, and reproduce as necessary. Each cell stores its own set of
instructions for carrying out each of these activities.
All cells share several common abilities:
      Reproduction by cell division.
      Metabolism, including taking in raw materials, building cell components, creating energy molecules
      and releasing byproducts.
      Synthesis of proteins, the functional workhorses of cells, such as enzymes. A typical mammalian cell
      contains up to 10,000 different proteins.
      Response to external and internal stimuli such as changes in temperature, pH or nutrient levels.
      Traffic of vesicles.

Cellular components




                      Figure: 2. 1.1- Eukaryotic cell components.

All cells whether prokaryotic or eukaryotic have a membrane, which envelopes the cell, separates its interior from the
surroundings, strictly controls what moves in and out and maintains the electric potential of the cell. Inside the membrane
is a salty cytoplasm (the substance which makes up most of the cell volume). All cells possess DNA, the hereditary
material of genes and RNA, which contain the- information necessary to express various proteins such as enzyme, the
cell's primary machinery. Within the cell at any given time are various additional bimolecular organelles.


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The vital components of a cell are-
O Cell membrane
O Mitochondria –


Cell membrane
The outer lining of a eukaryotic cell is called the plasma membrane. This membrane serves to separate and protect a cell
from its surrounding environment and is made mostly from a double layer of lipids (fat-like molecules) and proteins.
Embedded within this membrane are a variety of other molecules that act as channels and pumps, moving different
molecules into and out of the cell.
Cell membrane: Structure and Function
Structure




                       Figure: 2 1.2-Cell membrane



Components of the cell membrane
•It consists of two layers of phospholipids molecules.
•The head composed of protein and lipid.
•The heads are soluble in water (hydrophilic)
•The tails are insoluble in water (hydrophobic)
•They meet in the interior of the membrane.


Cytoskeleton - a cell's scaffold the cytoskeleton is an important, complex, and dynamic cell component. There are a

great number of proteins associated with the cytoskeleton, each controlling a cell‘s structure by directing, bundling, and
aligning filaments. It acts to organize and maintains the cell's shape; anchors organelles in place; helps during
endocytosis, the uptake of external materials by a cell; and moves parts of the cell in processes of growth and motility.

Cytoplasm - a cell's inner space- Inside the cell there is a large fluid-filled space called the cytoplasm. This refers both

to the mixture of ions and fluids in solution within the cell, and the organelles contained in it which are separated from this
intercellular "soup" by their own membranes. The cytosol refers only to the fluid, and not to the organelles.
It normally contains a large number of organelles, and is the home of the cytoskeleton. The cytoplasm also contains many
salts and is an excellent conductor of electricity, creating the- perfect environment for the mechanics of the cell. The
function of the cytoplasm, and the organelles which reside in it, are critical for a cell's survival.


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Genetic material-

Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most organisms use
DNA for their long term information storage, but a few viruses have RNA as their genetic material. The biological
information contained in an organism is encoded in its DNA or RNA sequence.
Function
1. Separates between cytoplasm & ECF.
2. Maintain cell internal environment...
3. Transport of molecules in & out the cell.
4. Controls ions distribution between cytoplasm and ECF.
5. It contains protein receptors for hormones &chemical transmitter.
6. Generates membrane potentials.

Mitochondria– the power generators

Mitochondria are self-replicating organelles that occur in various numbers, shapes, and sizes in the cytoplasm of all
eukaryotic cells. Mitochondria play a critical role in generating energy in the eukaryotic cell. Mitochondria generate the
cell's energy by the process of oxidative phosphorylation, utilizing oxygen to release energy stored in cellular nutrients
(typically pertaining to glucose) to generate ATP. Mitochondria multiply by splitting in two.




                    Figure: 2.1.3- Simplified structure of mitochondrion

Mitochondria-Structure and Function:
A mitochondrion contains outer and inner membranes composed of phospholipids bilayers and proteins.[6] The two
membranes, however, have different properties. Because of this double-membraned organization, there are five distinct
compartments within the mitochondrion. There is the outer mitochondrial membrane, the intermembrane space (the-
space between the outer and inner membranes), the inner mitochondrial membrane, the cristae space (formed by
enfolding of the inner membrane), and the matrix (space within the inner membrane)..The vital functional unit of
mitochondrion, respiratory chain is embedded in-between the Outer & inner membrane.
The processes that happen in the mitochondrion are
    Energy conversion
    pyruvate oxidation,
    the Krebs cycle,
    the metabolism of amino acids, fatty acids, and steroids,
    NADH and FADH2: the electron transport chain.
    Heat production
    Storage of calcium ions
     Generation of adenosine triphosphate (ATP).
    The membrane also maintains the cell potential.
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Mitochondria-Metabolism




                    Figure: 2 1.4-Main pathways of cellular and mitochondrial energy metabolism.


The two main metabolic pathways, i.e. glycolysis and oxidative phosphorylation are linked by the enzyme complex
pyruvate dehydrogenase. Briefly, glucose is transported inside the cell and oxidized to pyruvate. Under aerobic conditions,
the complete oxidation- of pyruvate occurs- through the TCA cycle to produce NADH, H+ and/or FADH2. On the figure we
also figured in pink the glutamine oxidation pathway, as observed in cancer cells.


Mitochondrial respiratory chain.




                    Figure: 2.1.5: Schematic diagram of mitochondrial respiratory chain.

The mitochondrial respiratory chain consists of four enzyme complexes (complexes I- IV) and two intermediary substrates
(coenzyme Q and cytochrome c). The NADH+H+ and FADH2 produced by the intermediate metabolism are oxidized
further by the mitochondrial respiratory chain to establish an electrochemical gradient of protons, which is finally used by
the F1F0-ATP synthase (complex V) to produce ATP, the only form of energy used by the cell.




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2.2- Bone




General aspects of bone

At the molecular level, bone is one of the few materials in the body that contains a mineral-like component in addition to
the organic components, others include dentin and enamel.
Bone is a composite material, which consists of organic matrix (mainly collagen) and inorganic hydroxyapatite (HA). Water
accounts for about 20% of the wet weight of cortical bone, HA makes up approximately 45%, and organic substances
account for the remaining 35%1. Cortical bone, which surrounds all bones, primarily serves the supportive and- protective
function of bone, whereas trabecular (cancellous) bone is mostly responsible for the metabolic function.


Functions of Bones
     o    Mechanical Protection — Bones can serve to protect internal organs, such as the skull protecting the brain or
          the ribs protecting the heart and lungs.
     o    Shape — Bones provide a frame to keep the body supported.
     o    Movement — Bones, skeletal muscles, tendons, ligaments and joints function together to generate and transfer
          forces so that individual body parts or the whole- body can be manipulated in three-dimensional space.
     o    Sound transduction — Bones are important in the mechanical aspect of overshadowed hearing.
     o    Synthetic
     o    Blood production — The marrow, located within the medullary cavity of long bones and interstices of cancellous
          bone, produces blood cells in a process called haematopoiesis.
     o    Metabolic
     o    Mineral storage — Bones act as reserves of minerals important for the body, most notably calcium and
          phosphorus.
     o    Growth factor storage — Mineralized bone matrix stores important growth factors such as insulin-like growth
          factors, transforming growth factor, bone morphogenetic proteins and others.
     o    Fat Storage — the yellow bone marrow acts as a storage reserve of fatty acids
     o    Acid-base balance — Bone buffers the blood against excessive pH changes by absorbing or releasing alkaline
          salts.
     o    Detoxification — Bone tissues can also store heavy metals and other foreign elements, removing them from the
          blood and reducing their effects on other tissues. These can later be gradually released for excretion. [Citation
          needed]
     o    Endocrine organ - Bone controls phosphate metabolism by releasing fibroblast growth factor - 23 (FGF-23),
          which acts on kidney to reduce phosphate reabsorption.




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Types of Bone (General)




           Table: 2.2.6-Schematic diagram of bone structure


 Types of Bone(Macroscopic)




          Figure: 2.2 .7-A femur head with a cortex of compact bone and medulla of trabecular bone.

Bone in human bodies is generally classified /categorized morphologically into two types, 1) Cortical bone, also known as
compact bone and 2) Trabecular bone, also known as cancellous- or spongy bone. These two types (Compact &
Trabecular) are classified as on the basis of porosity and the unit microstructure. .Trabecular bone accounts for 20% of
total bone mass but has nearly ten times the surface area of compact bone. Compact bone is, as the name suggests, a
compacted and stiff material with- a relatively low porosity. Trabecular bone is a more porous structure comprised of small
struts and plates called-
Trabecular. Cortical bone is much denser with a porosity ranging between 5% and 10%% while the porosity of trabecular
bone ranges from 50% to 90%. Cortical bone is found primary is found in the shaft of long bones and- forms the outer
shell around cancellous bone at the end of joints and the vertebrae. Trabecular bone is found in the medullary cavity of flat
and short bones, and in the epiphysis and metaphysis of long bones. At the tissue level, it is thought that the two bone
types are identical.




                                                                                                             19 | P a g e
Types of Bone (Microscopic)




         Lamellar Bone                                      Woven Bone
    Figure 2.2.8a - Lamellar Bone               &           2.28b- Woven Bone

       Lamellar Bone: Lamellar bone is stronger and filled with many collagen fibers parallel to other fibers in the same
       layer (these parallel columns are called osteons).
       Woven Bone (non-lamellar): Woven bone is weaker, with a small number of randomly oriented fibers, but forms
       quickly. It is replaced by lamellar bone, which is highly organized in concentric sheets with a low proportion of
       osteocytes.

Bone Composition




       Table: 2.2.9 – Schematic diagram of bone composition


       Cells
     o Osteoprogenitor cells
     o Osteoclasts
     o Osteocytes
     o Osteoclasts

      Extracellular Matrix
       Organic (35%)
     o Collagen (type I) 90%
     o Osteocalcin, osteonectin, proteoglycans, glycosaminoglycans, lipids (ground substance)
       Inorganic (65%)
     o Primarily hydroxyapatite Ca5(PO4)3(OH)2



                                                                                                         20 | P a g e
Cells of Bone:




There are 4 types of cells constituting the bone-

             Osteoprogenitor cells
The osteoprogenitor cell is a primitive cell derived from the mesenchyme. It forms in the inner layer of the periosteum
and lines the marrow cavity as well as Haversian and Volkmann‘s canals of compact bone. During periods of growth
and remodeling these cells are stimulated to differentiate into osteoblasts that lay down new bone. They can also
differentiate into other cell types such as fibroblasts, chondroblasts and adipose cells during bone loss 7. In mature
bone that is not actively remodeling, these cells are quiescent and are called bone- lining cells. Bone lining cells are
generally inactive and have very few cytoplasmic organelles. Their processes extend through canaliculi to
neighboring cells which suggests that they may be involved in mechano-transduction and cellular communication.


     Osteoblasts
Osteoblasts are cells which are responsible for the production of organic bone matrix. These cells synthesize and
secrete small vesicles into the existing bone. These matrix vesicles are formed by pinching off portions of the
plasmalemma and contain enzymes, including alkaline phosphatase, which load the vesicle with calcium 8. Rupture
of these vesicles initiates local mineralization by releasing calcium and by- negating local inhibiting mechanisms.
Deposition of mineral makes the bone matrix stiffer, impermeable and more capable of bearing loads.
     Osteocytes
Osteocytes are the most abundant cells in the bone matrix and are mature osteoblasts that have been ‗walled-in‘ by
the bone tissue which has been laid down around them.
Approximately 10% of all active osteoblasts are converted into osteocytes. The full role of these cells is still not
known however, they are thought to have mechanosensory and chemosensory regulatory roles. Osteocytes are a
candidate mechanosensory cell type because- they are ideally situated to sense mechanical stimulation such as
strain or interstitial fluid- flow.These actions are caused by mechanical loading and thus osteocytes are thought to be
in some way responsible for bone adaptation and remodeling9. Osteocytes maintain healthy tissue by secreting
enzymes and controlling the bone mineral content, they also control the calcium release from the bone tissue to the
blood.
     Osteoclasts
Osteoclasts are large multinucleate cells that break down bone tissue. They are derived from the mononuclear
phagocytic lineage of the haemopoietic system. They are formed by monocytes either by the fusion of several cells or
by DNA replication without cell division in response to stimuli from osteoblasts, osteocytes and hormones. When
these cells are active- they rest directly on the bone surface in a resorption bay or a Howship‘s lacuna. They are
characterized by two easily identifiable features; the ‗ruffled border‘ which is an infolded plasma membrane where the
resorption takes place, and the ‗clear zone‘ which is the point of attachment of the osteoclast to the underlying bone
matrix 10.




                                                                                                        21 | P a g e
Extracellular Matrix


Matrix comprises a major constituent of bone. It consists of living cells embedded in a calcium carbonate matrix that
makes up the main bone material. The majority of bone is made of the bone matrix & bone cell. In mature bone, 10-
20% by weight of the matrix is water. Dried bone consists of about 70% inorganic matrix and 30% organic matrix by
weight. The organic matrix is 90-95% collagen fibers with the remainder being a homogenous ground substance.




                  Figure: 2.210-Picture showing Bone composition & extracellular-
                   matrix.




1. Organic
The organic part of matrix is mainly composed of Type I collagen. This is synthesized intracellularly as tropocollagen
and then exported, forming fibrils. The organic part is also composed of various growth factors, the functions of which
are not fully known.
Factors present include glycosaminoglycans, osteocalcin, osteonectin, bone sialo protein, osteopontin and Cell
Attachment Factor. One of the main things that distinguishes the matrix of a bone from that of another cell is that the
matrix in bone is hard. In the event of a broken bone, the cells are brought out of semi-stasis to repair the matrix.
2. Inorganic
The inorganic part is mainly composed of crystalline mineral salts and calcium, which is present in the form of
hydroxyapatite. The matrix is initially laid down as unmineralised osteoid (manufactured by osteoblasts).
Mineralization involves osteoblasts secreting vesicles containing alkaline phosphatase. This cleaves the phosphate
groups and acts as the foci for calcium and phosphate deposition. The vesicles then rupture and act as a centre for
crystals to grow on.
Osteoblasts, cells that take up calcium compounds from the blood and secrete sturdy bone matrix, live on the surface
of existing matrix. Cells gradually become embedded in their own matrix, forming uncalcified bone matrix (osteoid).
The addition of calcium phosphate forms the calcified bone matrix, which surrounds the mature bone cells
(osteocytes).




                                                                                                          22 | P a g e
Anatomy of bone




             Figure: 2.2.11- Parts of a long bone.



Long bones are characterized by a shaft, the diaphysis that is much greater in length than width. They are comprised
mostly of compact bone and lesser amounts of marrow, which is located within the medullary cavity, and spongy bone.
Most bones of the limbs, including those of the fingers and toes, are long bones. The exceptions are those of the wrist,
ankle and kneecap. Short bones are roughly cube-shaped, and have only a thin layer of compact bone surrounding a
spongy interior.
The bones of the wrist and ankle are short bones, as are the sesamoid bones. Flat bones are thin and generally curved,
with two parallel layers of compact bones sandwiching a layer of spongy bone. Most of the bones of the skull are flat
bones, as is the sternum. Irregular bones do not fit- into the above categories. They consist of thin layers of compact bone
surrounding a spongy interior. As implied by the name, their shapes are irregular and complicated. The bones of the spine
and hips are irregular bones. Sesamoid bones are bones embedded in tendons. Since they act to hold the tendon further
away from the joint, the angle of the tendon is increased and thus the leverage of the muscle is increased. Examples of
sesamoid bones are the patella and the pisiform. The point where two or more bones come together is called a joint, or
articulation. Different kinds of joints enable different ranges of motion. Some joints barely move, such as those between
the interlocking bones of the skull. Other bones, held together by tough connective- tissues called ligaments, form joints
such as the hinge joint in the elbow, which- permits movement in only one direction. The pivot joint between the first and
second vertebrae allows the head to turn from side to side.


Intimately associated with bone is another type of connective tissue called cartilage. Cartilage is softer, more elastic, and
more compressible than bone. It is found in body parts that require both stiffness and flexibility, such as the ends of bones,
the tip of the nose, and the outer part of the ear. Bone is not a uniformly solid material, but rather has some spaces
between its hard elements.




                                                                                                              23 | P a g e
 Figure: 2.2.12- a, b, c, d- Appendicular upper extremity bones




Figure: 2.2.12- e, f, g- Appendicular lower extremity bones




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Histology of bone




          Figure: 2.2.13 a-Schematic histological structure of bone.


Haversian system (osteon), functional unit of bone-




          Figure: 2.2.13b: HaversianCanal: A higher magnification view of slide clearly shows the concentric circles.
          After osteoclasts remove old bone, osteoblasts deposit bone in this circular arrangement beginning with
          the outer ring and working inward. As the osteoblasts become trapped in their own calcified deposits, they
          are known as osteocytes.




Haversian canal carries blood vessel through center of osteon lamellae "little layer" of matrix between concentric rings of
osteocytes lacunae "pools" which house osteocytes osteocytes "bone cells" which- maintain bone- Volkmann's canal
feeder cross connecting vessel for blood supply- canaliculi protoplasmic extensions from osteocytes by which
maintenance of bone is performed, interstitial lamellae layers between adjacent Haversian systems. Osteoblasts form the
lamellae sequentially, from the most external inward toward the Haversian canal. Some of the osteoblasts develop into
osteocytes, each living within its own small space, or lacuna. Osteocytes make contact with the cytoplasmic processes of
their counterparts via a network of small canals, or canaliculi. This network facilitates the exchange of nutrients and
metabolic waste.
Collagen fibers in a particular lamella run parallel to each other but the orientation of collagen fibers within other lamellae
is oblique. The collagen fiber density is lowest at the- seams between lamellae, accounting for the distinctive microscopic
appearance of a transverse section of osteons.


                                                                                                                    25 | P a g e
2.3 Bone Fracture

A bone fracture is a break in a bone. Most people fracture at least one bone during their lifetime. The
severity of fractures increase with age. Children's bones are more flexible and less likely to break. Falls or
other accidents that do not harm children can cause complete fractures in older- adults. Older adults suffer
from fractures more than children because their bones are more likely to be brittle.



Causes of fracture

Fractures occur when a bone can't withstand the physical force exerted on it. Fractures happen because an area of bone
is not able to support the energy placed on it (quite obvious, but it -becomes more complicated). Therefore, there are two
critical factors in determining why a fracture occurs:
          The energy of the event
          The strength of the bone
The energy can being acute, high-energy (e.g. car crash), or chronic, low-energy (e.g. stress fracture). The bone strength
can either be normal or decreased (e.g. osteoporosis). A very simple problem, the broken bone, just became a whole lot
more complicated!



Mechanism of Bone fracture

               a.   Tension,
               b.   Compression,
               c.   Bending, shear,
               d.   Torsion &
               e.   Combined effects
                                                                Types of Fractures




Figure: 2.3.14-Mechanism of bone fracture.                            Figure: 2.3.15-Different types of fracture.




                                                                                                          26 | P a g e
Symptoms of a fracture

          The most common symptoms of a fracture are:
             Swelling around the injured area
             Loss of function in the injured area
             Bruising around the injured area
             Deformity of a limb.


     2.4- Healing of fracture




                      Figure: 2.4.16-Schematic diagram of fracture healing



The natural process of healing a fracture starts when the injured bone and surrounding tissues bleed, forming what's
called fracture Hematoma. The blood coagulates to form a blood clot situated between the broken fragments. Within a few
days blood vessels grow into the jelly-like matrix of the blood clot. The new blood vessels bring phagocytes to the area,
which gradually remove the non-viable material.
The blood vessels also bring fibroblasts in the walls of the vessels and these multiply and produce collagen fibers. In this
way the blood clot is replaced by a matrix of collagen. Collagen's rubbery consistency allows bone fragments to move only
a small amount unless severe or persistent force is applied.
At this stage, some of the fibroblasts begin to lay down bone matrix (calcium hydroxyapatite) in the form of insoluble
crystals. This mineralization of the collagen matrix stiffens it and transforms -it into bone. In fact, bone is a mineralized
collagen matrix; if the mineral is dissolved out of bone, it becomes rubbery.
Healing bone callus is on average sufficiently mineralized to show up on X-ray within 6 weeks in adults and less in
children. This initial "woven" bone does not have the strong mechanical- properties of mature bone. By a process of
remodeling, the woven bone is replaced by mature "lamellar" bone. The whole process can take up to 18 months, but in
adults the strength of the healing bone is usually 80% of normal by 3 months after the injury.




                                                                                                             27 | P a g e
Phases of fracture healing




                     Table: 2.4.17- schematic diagram of Phases of fracture healing.

Bone Healing
There are three major phases of fracture healing, two of which can be further sub-divided to make a total of five phases;
1. Reactive Phase
i. Impact, Induction and inflammation.
ii. Granulation tissue formation
2. Reparative Phase
iii.Callus formation
iv.Lamellar bone deposition
3. Remodeling Phase
v. Remodeling to original bone contour.
Reactive
After fracture, the first change seen by light and electron microscopy is the presence of blood cells within the tissues which
are adjacent to the injury site. Soon after fracture, the blood vessels constrict, stopping any further bleeding.[1] within a
few hours after fracture, the extra-vascular- blood cells, known as a "hematoma", form a blood clot. All of the cells within
the blood -clot degenerate and die.[2] Some of the cells outside of the blood clot, but adjacent to the injury site, also
degenerate and die.[3] Within this same area, the fibroblasts survive and replicate. They form a loose aggregate of cells,
interspersed with small blood vessels, known as granulation tissue.[4].
Reparative
Days after fracture, the cells of the periosteum replicate and transform. The periosteal cells proximal to the fracture gap
develop into chondroblasts and form hyaline cartilage. The periosteal cells distal to the fracture gap develop into
osteoblasts and form woven bone.
The fibroblasts within the granulation tissue also develop into chondroblasts and form hyaline cartilage. These two new
tissues grow in size until they unite with their counterparts from- other pieces of the fracture. This process forms the
fracture callus.[6] Eventually, the fracture gap is bridged by the hyaline cartilage and woven bone, restoring some of its
original strength.
The next phase is the replacement of the hyaline cartilage and woven bone with lamellar bone. The replacement process
is known as endochondral ossification with respect to the hyaline cartilage and "bony substitution" with respect to the
woven bone. Substitution of the woven bone- with lamellar bone precedes the substitution of the hyaline cartilage with
lamellar bone.
The lamellar bone begins forming soon after the collagen matrix of either tissue becomes mineralized. At this point,
"vascular channels" with many accompanying osteoblasts penetrate the mineralized matrix.
                                                                                                              28 | P a g e
The osteoblasts form new lamellar bone upon the recently exposed surface of the mineralized matrix. This new lamellar
bone is in the form of trabecular bone.[7] Eventually, all of the woven bone and cartilage of the original fracture callus is
replaced by trabecular bone, restoring most of the bone's original strength.

Remodeling
The remodeling of bone requires the coordinated activity of two types of cells:
Osteoclasts that demineralise bone in their vicinity
Osteoblast that secretes collagen and mineral to lay down new bone.
Stages / Cycle of Bone Remodeling:
                                    1.   Resting,
                                    2.   Resorption,
                                    3.   Reversal,
                                    4.   Bone formation




Figure: 2.4.18-Schematic diagram of Bone Remodeling

Normal bone remodeling. (i) Resorption: stimulated osteoblast precursors release factors that induce osteoclast
differentiation and activity. Osteoclasts remove bone mineral and matrix, creating an erosion cavity. (ii) Reversal:
mononuclear cells prepare bone surface for new- osteoblasts to begin forming bone. (iii) Formation: successive waves of
osteoblasts synthesize an organic matrix to replace resorbed bone and fill the cavity with new bone. (iv) Resting: bone
surface is covered with flattened lining cells. A prolonged resting period follows with little cellular activity until a new
remodeling cycle begins. Bone remodeling is a lifelong process where old bone is removed from the skeleton (a sub-
process called bone resorption) and new bone is added (a sub-process called ossification or bone formation). In the first
year of life, almost 100% of the skeleton is replaced. In adults, remodeling proceeds at about 10% per year.[1] Histological
analysis of secondary fracture healing in bone showing the progression of repair on days 1, 3, 14, 21, and 28.
Fractured bone appears denser than the surrounding tissue. On day 7, extensive soft callus is seen forming around the
injured bone. At day 14, the soft callus becomes mineralized to form new bone and achieve union by day 21 and 28 (H&E
stain, x40)20.
In the process of fracture healing, several phases of recovery facilitate the proliferation and protection of the areas
surrounding fractures and dislocations. The length of the process depends on the extent of the injury, and usual margins
of two to three weeks are given for the reparation of most upper bodily fractures; anywhere above four weeks given for
lower bodily injury.
The process of the entire regeneration of the bone can depend on the angle of dislocation or fracture. While the bone
formation usually spans the entire duration of the healing process, in some instances, bone marrow within the fracture
having- healed two or fewer weeks before the final remodeling phase. While immobilization and surgery may facilitate-
healing, a fracture ultimately heals through physiological processes. The healing process is mainly determined by the-
periosteum (the connective tissue membrane covering the bone).

                                                                                                             29 | P a g e
 The periosteum is the primary source of precursor cells which develop into- chondroblasts and osteoblasts that are
 essential to the healing of bone. The bone marrow (when present), endosteum, small blood vessels, and fibroblasts are
 secondary sources of precursor cells.
 Factors which affects fracture healing
 Fracture treatment is not purely a question of effective fracture reduction and fixation but a complex biological process.
 The natural tendency for a fracture is to unite. When delay or failure of union occurs, the causes are either local factors at
 the site of fracture or defects in the methods employed in treatment.




               Figure: 2.4.19- Schematic diagram of bone healing factors.
 General Causes
a) Imperfect immobilization:
   (I) Too little extent of immobilization. And
   (ii) Too short a period of immobilization.
b) Distraction: Too heavy a pull of the distal fragment by skeletal traction.
c) Surgical intervention: This empties the fracture hematoma and strips the periosteum, interfering with the blood supply and slowing the
 healing process.
 Local causes
a) Infection: This is the commonest cause for delayed union or non-union in open fractures.
b) Inadequate blood supply to one fragment: Certain sites are notorious for slow union or non-union e.g. (I) Fracture neck of femur. The
  blood supply to the head of the femur is poor.
(ii) Fracture scaphoid. The blood supply to the proximal fragment is poor.
c) Interposition of soft tissues between the fragments prevents bony apposition and interferes with healing.
d) Type of fracture: Transverse fractures unite slowly compared to oblique or spiral fractures.
e) Type of bone: Fracture at the cancerous ends of bone unites better than those in the mid shaft of long bones where cancellous bone is
  minimal.
  Fractures in children unite very rapidly whereas delayed union is common in the aged. Other factors like protein and vitamin
 deficiencies, general diseases like syphilis and diabetes play only a small part in influencing the rate of healing.
 Bio-Compression at the fracture site through protected weight bearing at the proper time promotes healing of the fractures.


 How to Speed up fracture healing!
 Although there are no magical ways to fix a bone fracture, but there are ways to help speed up
 the healing process, and help fracture to heal properly/ faster.
  Proper medical management.
  Nutrition Support.
  Osteoblast cell injections
  Electrical stimulation.
  Magnetic stimulation.
  Ultrasound therapy.
  Gene-therapy
  Low Level Laser therapy.

                                                                                                                         30 | P a g e
    Chapter-3
    Laser & Laser System


    3.1   Laser
    3.2   Laser principle
    3.3   Components of a laser system
    3.4   Laser Machine
    3.5   Measurement, Parameter & Protocol of Laser


    3.1 Laser
    “Any device which can be made to produce or amplify electromagnetic radiation in the
    wavelength range from 180nm to 1mm primarily by process of controlled stimulated
    emission” European Standard 1EC 601.
    Criteria of Low Level Laser

          Coherent-referring to the wave nature of light, the peaks and troughs of the waves occur synchronously in time (i.e., a
          fixed phase relationship between the electric fields of the electromagnetic field)
          Collimated-exhibiting minimal divergence (increase in the beam diameter) as the beam propagates
          Monochromatic-of a single or very limited spectral line width, i.e., a single color
          High intensity-displaying a high optical power per unit area for a given amount of energy compared to broadband
          sources


    Types and Classification of Lasers Lasers have been classified with respect to their hazards based on power,
    wavelength, and pulse duration. These definitions are wordy and cumbersome to read out of context, but when given the
    specifications of a laser or laser systems are not difficult to apply.
     Types
   According to their sources:
       Gas Lasers
       Crystal Lasers
       Semiconductors Lasers
       Liquid Lasers
   2. According to the nature of emission:
      Continuous Wave
      Pulsed Laser
     Q-switched lasers
     while most often laser types are discussed in terms of what they can treat, it is important to
     recognize the broader categories of lasers.
 Continuous wave (cw) lasers
    A continuous beam and include these emit the CO2 and krypton lasers. Pseudo-continuous wave lasers emit a beam in
    such close pulses that the effect on tissue is similar to that of a continuous wave laser. These lasers are used to coagulate
    tissue, as for example in the treatment of moles and warts.
   Pulsed lasers
    Lasers that emit a beam in short pulses usually separated by 0.1-1 second. Pulsed lasers are more selective in their
    destructive effect than continuous wave lasers, and are used in selective photothermolysis.



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   Q-switched lasers
    Q-switching refers to the process of storing up laser energy in the laser cavity and releasing it in one single very short and
    extremely powerful pulse. This results in power outputs in the megawatt to gig watt range, and allows for mechanical (vs.
    thermal) destruction of the target. Such lasers are often used in the removal of tattoos.
     3. According to their wavelength:
       Visible Region
       Infrared Region
       Ultraviolet Region
       Microwave Region
        X-Ray Region




                          Table:3.1.1- Types of laser according to wavelengths




    Classes of Lasers (adoptedfromANSIZ-136.1-2000)
    Class Levels 1-4-
    •    1 = incapable of producing damaging radiation levels (laser printers & CD players)
    •    2 = low-power visible lasers (400-700 nm wavelength, 1 mW)
    •    3 = medium-power lasers - needs eye protection
                    • 3a – up to 5 mW
                    • 3b** – 5 mw-500 mW
    •    4 = high-power lasers– presents fire hazard (exceeds 500 mW).




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     3.2 Laser principle-

     Stimulated emission is fundamental to light amplification and thus to the operation of the laser. To understand it, it must
     be placed in the context of interactions between light and matter. Here, the matter is composed of optically active
     elements in ―solution‖ in a gas, plasma, solid or liquid medium. These elements can be atoms, ions, molecules, free
     radicals or electrons (for simplicity, we consider ―atoms‖ in the following). Their energy levels are quantified and are
     such that light of a certain frequency can interact with the population found in these levels. More precisely, let us
     consider two energy levels E1 and E2 (E1 is less than E2) whose atoms can interact with light of frequency

                       . The group E1-E2 is called radiative transition if atoms can only pass from E1 to E2 (or from E2 to

     E1) by interacting with light. E1 is called the lower energy level and E2 the upper energy level.

     The emission-absorption principle

     The three different mechanisms are shown below (Figure 3.2.2):
1.   Absorption: An atom in a lower level absorbs a photon of frequency hν and moves to an upper level.
2.   Spontaneous emission: An atom in an upper level can decay spontaneously to the lower level and emit a photon of
     frequency hν if the transition between E2 and E1 is radiative. This photon has a random direction and phase.
3.   Stimulated emission: An incident photon causes an upper level atom to decay, emitting a ―stimulated‖ photon whose
     properties are identical to those of the incident photon. The term ―stimulated‖ underlines the fact that this kind of
     radiation only occurs if an- incident photon is present. The amplification arises due to the similarities between the
     incident and emitted photons.




               Figure 3.2.2: Mechanism of the interaction between an atom and a photon (The photon has
               an energy hν equal to the difference between the two atomic energy levels).


     Competition between the three mechanisms
     For a radiative transition, these three mechanisms are always present at the same time. To make a laser medium,
     conditions have to be found that favour stimulated emission over absorption and spontaneous emission. Thus, both the

     right medium and the right conditions must be chosen to produce the laser effect. An incident photon of energy
     has an equal chance of being absorbed by a ground-state atom as being duplicated (or amplified!) by interacting with
     an excited-state atom. Absorption and stimulated emission are really two reciprocal processes subject to the same
     probability. To favour stimulated emission over absorption, there need to be more excited-state atoms than ground-
     state atoms. Spontaneous emission naturally tends to empty the upper level so this level has to be emptied faster by
     stimulated- emission. It has been proved that stimulated emission is much more likely to happen if the medium used is-



                                                                                                                   33 | P a g e
flooded with light (i.e. with a large number of photons). A good way to do this is to confine the photons in an optical
cavity.
Population inversion and pumping
If there are more atoms in the upper level (N2) than in the lower level (N1), the system is not at equilibrium. In fact, at

thermodynamic equilibrium, the distribution of the atoms between the levels can be given by Boltzmann's Law.
N2= N1x exp – {(E2-E1)/ KT}.
In this case, N2 is always less than N1. A situation not at equilibrium must be created by adding energy via a process

known as ―pumping‖ in order to raise enough atoms to the upper level. This is known as population inversion and is

given by                      . Light is amplified when the population inversion is positive. Pumping may be electrical,
optical or chemical.
Spectroscopic systems used to create a laser
Not all atoms, ions and molecules, with their different energy levels, are capable of creating a population inversion and
a laser effect. Only radiative transitions (where the atoms are excited due to light absorption) should be used and non-
radiative transitions should be avoided. Some transitions have both a radiative and a non-radiative part. In this case,
the upper level empties as a result of a non-radiative effect as well as spontaneous emission. This leads to additional
problems for achieving a population inversion because it is difficult to store atoms in the upper level under these
conditions. Thus, this type of transition should also be avoided.
Next, the relative energy levels specific to each type of atom must be considered. For example, choosing a lower level
with more energy than the ground state will greatly limit the population N 1, which may even be zero (Figure 3). This

means that only one atom would have to be excited to achieve population inversion.




                       Figure 3.2.3: Laser transition with the lower level far above the ground state.


The population at thermodynamic equilibrium is defined by Boltzmann's Law.
In addition, pumping must be able to move atoms to a higher level. Every pumping system (particularly optical or
electrical) corresponds to a certain energy, which must be transferable to the atoms of the medium. The difference in
energy between the excited state and the ground state must match the pumping energy. In optical pumping, there must
be at least three different- energy levels to create a population inversion. Figure 4 illustrates such a system. It shows
the pumping transition- (between E1 and E3) and the laser transition (between E2 and E1). The objective is to store

atoms in level E2 by absorbing ―pumping‖ radiation whose wavelength is shorter than that of the laser transition. This

means that the excited atoms must quickly decay from level 3 to level 2 only, a condition that limits the choice of
systems that will work. Figure 4 also shows an ideal cycle for an atom: it rises into level 3 by absorbing a photon from-

                                                                                                              34 | P a g e
the pumping light. It then falls very- rapidly into level 2. Finally, it decays by stimulated emission to level 1. Despite its
simplicity, this is not a very easy system to implement as the ground state of the laser transition has a large population
at thermodynamic equilibrium and at least half of this population must be excited to level 2 to obtain population-
inversion. Moreover, level 2 must be able to store these atoms so spontaneous emission must be- very unlikely. This
affects the choice of the system. A large pumping energy is also needed. The first ever laser was of this -type and used

a ruby (Cr3+:Al2O3). Ruby is composed of an aluminium crystal matrix and a doped ion (Cr 3+) whose energy levels

are used to create the laser effect. The medium is strongly pumped by discharge lamps.




                     Figure 3.2.4: Example of a three-level system with optical pumping.

Another example of a spectroscopic system is the four-level laser (Figure 5). Here, the pumping transition (optical
pumping) and the laser transition occur over a pair of distinct levels (E0 to E3 for the pump and E1 to E2 for the laser).

E1 is chosen to be sufficiently far from the ground state E0 so that the thermal population at thermodynamic

equilibrium is negligible. Similarly, atoms do not stay in level 3 or level 1. Figure 5 represents an ideal four-level
system. Unlike- the three-level system, as soon as one atom moves to level 2, a population inversion occurs and the
medium becomes amplifying. To maintain the population inversion, atoms must not accumulate in level 1 but must

rapidly decay to level 0. One of the best known mediums operating in this way is neodymium YAG (Nd3+:Y3Al5O12).




               Figure3.2 5: Example of a four-level system with optical pumping.

A final example of a spectroscopic system providing a laser effect is the helium-neon gas system (Figure 6). In this
case the pumping method is electrical. Neon transitions are used for the laser transitions: there are several but the
most well-known is the coloured one at 632.8 nm. Helium is used as an intermediary gas, capable of transferring
energy from the electrons to the neon particles via collisions. Helium is also unique in having two excited states said to-
                                                                                                                 35 | P a g e
be ―metastable‖ i.e-. atoms can stay there a long time before falling to the ground state. Helium atoms are carried into-
the excited state by collisions with electrons.


Energy is easily transferred to neon when the atoms collide because these metastable levels coincide with the excited
states of neon. This process is given by the equation: He* + Ne -> He +Ne*
An excited helium atom meets a ground-state neon atom and transfers its energy while decaying.Figure 3.2.6 also
shows that the lower levels of the laser transitions are far from the ground state, which favours population inversion (no
thermal population).




          Figure 3.2.6: A Helium-Neon System.




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3.3. Components of a laser system

 Laser-Components




                     Figure: 3.3.7: Diagram of Components of laser System

All lasers are composed of four basic components:
    o The lasing medium
    o The optical cavity
    o The pumping system
    o The delivery system

There are three different vital parts to a Ga-Al-As laser: 1. an energy source, 2. a laser material that absorbs this energy
emits it as light, and 3. a cavity that makes the light resonate and channels it in to narrow beam. Within the cavity very
high circulating photon densities stimulate the emission of light from the energized laser material. This design creates a
powerful beam of billions of photons, unlike to laser and differentiating them from lower intensity light sources like LEDs.




3.4 Laser Machine




     Figure: 3.4.8: schematic diagram of interior of a laser machine.




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There are five (5) basic components that make up the laser system, the control panel, the motherboard, the DC power
supply, the laser tube assembly, and the motion system.

    A. DC Power Supply.
    B. Motherboard
    C. Control Panel
    D. Laser Tube Assembly
    E. Motion System




Figure: 3.4.9: schematic diagram of the control panel of a laser machine




                                                                                                       38 | P a g e
3.5 Measurement, Parameter & Protocol of Laser

3.5.1 Calculating Laser and Treatment Parameters-
Laser Therapy devices are generally specified in terms of the average output power (milliwatts) of the laser diode,
and the wavelength (nanometers) of light they emit. This is necessary information, but not enough with which to
accurately define the parameters of the laser system. To do this, one must also know the area of the laser beam
(cm2) at the treatment surface (usually the tip of the hand piece when in contact with the skin).
If the output power (mW) and beam area (cm 2) are known, it is a reasonably straight-forward exercise to calculate
the remaining parameters which allow the precise dosage measurement and delivery.The output power of a laser,
measured in milliwatts, refers to the number of photons emitted at the particular wavelength of the laser diode.
Power Density measures the potential thermal effect of those photons at the treatment area. It is a function of
Laser Output Power and Beam area, and is calculated as:




                                                  Laser Output Power (W)
           1) Power Density (W/cm2) =
                                                  Beam area (cm2)
Beam area can be calculated by either:
           2) Beam Area (cm2) =                   Diameter(cm)2 x 0.7854



           or: Beam Area (cm2) =                  Pi x Radius(cm)2

The total photonic energy delivered into the tissue by a laser diode operating at a particular output power over a certain period is measured
in Joules, and is calculated as follows:


                                                   Laser Output Power (Watts) x Time (Secs)
           3) Energy (Joules) =



It is important to know the distribution of the total energy over the treatment area, in order to accurately measure
dosage. This distribution is measured as Energy Density (Joules/cm 2). "For a given wavelength of light, energy
density is the most important factor in determining- the tissue reaction"(Baxter, 1994). Energy Density is a function
of Power Density and Time in seconds, and is calculated as:

                                                  Laser Output Power (Watts) x Time (Secs)
           4) Energy Density (Joule/cm2) =
                                                  Beam Area (cm2)
           OR: Energy Density (Joule/cm2) = Power Density (W/cm2) x Time (Secs)


To calculate the treatment time for a particular dosage, you will need to know the Energy Density (J/cm 2) or Energy (J), as well as the Output
Power (mW), and Beam Area (cm 2 ). First, calculate the Output Power Density (mW/cm 2) as per Equation 1, then:

                                                  Energy Density (Joules/cm2)
           5) Treatment Time (Seconds) =          Output Power Density (W/cm2)


                                                  Energy (Joules)
           or: Treatment Time (Seconds) =
                                                  Laser Output Power (Watts)
Finally:

                                                  Laser Output Power (mW)
           Laser Output Power (Watts) =
                                                  1000




                                                                                                                             39 | P a g e
         Output          Beam          Treatment        Energy        Energy Density
         Power           Spot Size     Time (Secs)      (Joules)      (Joules/cm 2 )
         (mW)            (cm 2 )

         5               0.1           8.0              0.04          0.4


         50              0.1           8.0              0.4           4.0


         125             0.2           8.0              1.0           5.0


         250             0.2           8.0              2.0           10.0


         500             0.2           8.0              4.0           20.0


       Table: 3. 5.10: Various Laser Parameters v Dosage/Time: Illustrates the
       difference in Joules and Joules/cm 2 dosages for differing output parameters.
       The calculation of these parameters is explained above.

3.5.2 Laser Parameters for Effective Treatment


"For a given wavelength of light, energy density is the most important factor in determining the tissue reaction"
(Baxter, 1994). Research indicates that Energy Densities in the range 0.5 to 4 Joules/cm 2 are most effective in
triggering a photobiological response in tissue (e.g. Mester & Jaszagi-Nagy, 1973; Mester & Mester, 1989;
Mashiko et al, 1983; Haina, 1982), with 4 Joules/cm 2 having the greatest effect on wound healing (Mester et al,
1973; Mester et al, 1989).
Australian research suggests that this 'therapeutic window' of biostimulation may be extended to include
10/Joules/cm 2 (Laakso et al, 1994), and has applications in other areas of practice, such as Myofascial Trigger
Point therapy and pain control and tissue healing. Dosages above 10J/cm 2 is proved to be bioinhibitive, and the
resulting bioinhibition, may also have therapeutic applications, such as in the treatment of keloid scarring and pain
management.
Many practitioners have found straight Joules dosages - up to 20 J/cm 2 in some cases 94.7J/cm2, to be effective

in the treatment of a number of common musculoskeletal disorders. This is possibly due to the combined action of

the pain attenuating properties of laser bioinhibition at high dosages, and the biostimulatory effect of the lower -

powered 'halo' around the target treatment point. However, the same effect may not be e licited from a different

laser unit, due to differences in laser parameters (esp. Power Density) and configuration.

It is the Output Power Density which determines the time required delivering a particular Energy Density

(Joules/cm 2) dosage, and the Output Power which determines the corresponding Energy (Joules) delivered during

that time. Results obtained from particular dosages and treatments are likely to vary between individual

practitioners and patients, therefore, practitioner discretion is recommende d in determining the applicable

wavelength and dosage parameters for- each patient. It is important to note- that the appropriate- configuration of

a laser unit will depend primarily upon the types of conditions most commonly treated, and so specific

requirements will generally differ between practitioners.


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3.5.3 Treatment Protocol, Frequency, and Response




To maximize irradiance at the target tissue, the laser probe should be held in contact with, and perpendicular to,
the tissue surface. When treating open wounds, the probe should be held slightly away from the tissue surface,
whilst still maintaining a 90 o angle. The probe tip may be covered with plastic cling film, in order to reduce the
likelihood of cross-contamination.
In treating musculoskeletal conditions, laser therapy should be carried out following cryotherapy as the
vasoconstriction caused by cooling the tissue will increase the penetration depth of the laser irradiation. Laser
therapy helps to relax muscles, and so manipulations should be carried out following laser irradiation. Heat
therapies and various creams and lotions can be applied after laser therapy.
Laser treatments can be carried out by irradiating daily for the first week, then gradually increasing the interval
between treatments over successive weeks, according to the progression of the condition being treated. The total
dosage should not exceed 100-200 J in any single treatment session.
Laser dosage is cumulative, and so overtreatment causing a degradation of LLLT effectiveness can come from
overly-high dosages in one treatment session, or too many treatment sessions in close succession. Individual
practitioner discretion is to be used to determine the appropriate maximum session dosage, and the frequency of
treatment, for each particular patient.
Patients may report a number of sensations, such as localized feelings of warmth, tingling, or an increase or
decrease in symptoms, within the period immediately following laser therapy. Other sensations that may be
experienced in response to laser therapy are nausea or dizziness. It is good practice to advice patients of this
possibility.
Treatment reactions, if they occur, are often reported after initial laser treatments, however, they generally
diminish after the second or third treatment. If a severe reaction is experienced during treatment, stop
immediately.
To reiterate, optimal biostimulation is affected by the application of smaller dosages-per-point to more points at the
treatment site. Optimal bioinhibition is achieved through applying higher dosages-per-point, but to less treatment
points.
When treating acute musculoskeletal injuries, the initial desired outcome of laser therapy is the reduction of pain
and inflammation. It is very effective when used in conjunction with cryo-therapy, rest and elevation of the injury
site. Ideally treatment will begin as soon as possible after the injury occurs, with relativel y high, inhibitory dosages
(8-12 Joules per- point, up to 10 points) being used to attenuate the pain and reduce the initial inflammatory
response. A treatment frequency of 1-2 sessions per day may be used for the first 2-4 days post-injury.
As the time post-injury progresses, dosages and treatment frequency may be reduced. In the period 5 -10 days
post-injury, dosages of 6-8 Joules per point may be useful in promoting the rate of the inflammatory process and in
clearing its products from the injury site, thus allowing healing to begin sooner.
Moving into the healing phase, dosages are lowered and treatment frequency is reduced further. Throughout the
healing and rehabilitation phase of an injury, biostimulatory dosages (1-4 Joules per point) are used to promote
tissue repair and- reduce scarring and adhesions. Higher doses may be used as required to alleviate any pain that
results from over-working the injured body part during rehabilitation.
When treating chronic injuries or pain, it is best to start with lower doses and then work up to the most effective
dose for that particular patient, as a high initial dose may cause an unpleasant exacerbation of symptomatic pain.

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Chapter-4


Biophysical Aspects, , Laser- Tissue Interaction, Mechanisms and Bone Regeneration.

 4.1 Biophysical Aspects & light transport theory
4.2 Laser - Tissue Interaction
4.3 The Mechanisms of Low Level Laser Therapy
4.4.Effects of Laser on Biological Cell/Tissue healing
     Laser on hard tissue & Bone stimulation/ Regeneration
4.5 Medical application of Low Level Laser

4.1a- Biophysical Aspects of Low Level Laser Therapy (By Courtesy of-Herbert Klima, Atomic Institute of the Austrian
Universities, Vienna, Austria)


Biophysical aspects of low level laser therapy will be discussed from two points of view:
     1. The electromagnetic and
     2. The thermodynamical point of view.

From electromagnetic point of view,
Living systems are mainly governed by the electromagnetic interaction whose interacting particles are called photons.
Each interaction between molecules, macromolecules or living cells is basically electromagnetic and governed by photons.
For this reason, we must expect that electromagnetic influences like laser light of proper wavelength will have remarkable
impact on the regulation of living processes. An impressive example of this regulating function of various wavelengths of
light is found in the realm of botany, where photons of 660 nm are able to trigger the growth of plants which leads among
other things to the formation of buds. On the other hand, irradiation of plants by 730 nm photons may stop the growth and
the flowering. Human phagocyting cells are natively emitting light which can be detected by single photon counting
methods. Singlet oxygen molecules are the main sources of this light emitted at 480, 570, 633, 760, 1060 and 1270 nm-
wavelengths. On the other hand, human cells (leukocytes, lymphocytes, stem cells, fibroblasts, etc) can be stimulated by
low power laser light of just these wavelengths.


From thermodynamical point of view,
Living systems - in contrast to dead organisms - are open systems which need metabolism in order to maintain their
highly ordered state of life. Such states can only exist far from thermodynamical equilibrium thus dissipating heat in order
to maintain their high order and complexity. Such nonequilibrium systems are called dissipative structures proposed by the
Nobel laureat I. Prigogine. One of the main feature of dissipative structures is their ability to react very sensibly on weak
influences, e.g. they are able to amplify even very small stimuli.
Therefore, we must expect that even weak laser light of proper wavelength and proper irradiation should be able to
influence the dynamics of regulation in living systems. For example, the transition from a cell at rest to a dividing one will
occur during a phase transition already influenced by the tiniest fluctuations. External stimuli can induce these phase
transitions which would otherwise not even take place. These phase transitions induced by light can be impressively
illustrated by various chemical and- physiological reactions as special kinds of dissipative systems. One of the most
important biochemical reaction localized in mitochondria is the oxidation of NADH in the respiratory chain of aerobic cells.




                                                                                                              42 | P a g e
A similar reaction has been found to be a dissipative process showing oscillating and chaotic behavior capable to absorb
and amplify photons of proper wavelength.
A great variety of experimental and clinical results in the field of low level laser therapy supports these two biophysical
points of view concerning the interaction between life and laser light. Our former, but also our recent experimental results
on the effects of low level- laser light on human cells are steps in this direction.
By using cytometric, photometric and radiochemical methods it is shown that the increase or decrease of cells growth
depends on the applied wavelengths (480, 570, 633, 700, 760, 904, 1060, 1270 nm), on the irradiance (100 - 5000 J/m2),
on the pulse sequence modulated to laser beams (constant, periodic, chaotic pulses), on the type of cells- (leukocytes,
lymphocytes, fibroblasts, normal and cancer cells) and on the density of the cells in tissue cultures.
Our experimental results support our hypothesis which states that triplet oxygen molecules are able to absorb proper laser
light at wavelength at wavelengths 480, 570, 633, 700, 760, 904, 1060, 1270 nm thus producing singlet oxygen molecules.
Singlet oxygen takes part in many metabolic processes, e.g. catalytic oxidation of NADH which has been shown to be a
dissipative system far from thermodynamical equilibrium and sensitive even to small stimuli. Therefore, laser light of
proper wavelength and irradiance in low level laser therapy is assumed to be able- to excite oxygen molecules thus
influencing or amplifying metabolism and consequently influencing and supporting fundamental healing processes.




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4.1b- Light transport theory

When light is sent into biological tissue, different processes can occur. While most of the light enters the tissue, a small
part of it can be reflected off the tissue surface. The amount of reflected light depends on the angle of incidence and the
index of refraction. Inside the tissue, the light can be absorbed or scattered. Both processes are highly wavelength
dependent. In the lower part of the visible wavelength region, the scattering probability is comparable to the absorption. In
the red and near-infrared wavelength region light penetrates tissue better. This region is called the optical window. Based
on measurements of optical properties, physiological or structural information about the probed tissue can be extracted.
Here will outline the mathematical basis of light transport in tissues and describe the tissue features that affect this
transport.
The process of light transport in turbid media may be described mathematically either by analytical theory, based directly
on Maxwell's equations, or by transport theory. Maxwell's equations can describe the interaction between light and tissue
as an electromagnetic wave propagating through a medium with random dielectric fluctuations. However, due to the
complex structure of tissue it is in principle impossible to obtain a formulation that takes all its dielectric properties into
account (A. Ishimaru 1978). Transport theory, on the other hand, treats the problem as a flow of power through a
scattering medium. Transport theory is less mathematically- rigorous than electromagnetic theory and does not in itself
include effects such as diffraction or interference. However, it has proven useful for calculating photon transport in tissue.
It is usually expressed for the radiance L(r,s,t) [Wm-2sr-1], which is the radiant power per unit area and unit solid angle in
direction s, at a position in space r. It is obtained by multiplying the light distribution function, N(r,s,t) [m-3sr-1], with the
speed and energy of the photons in the medium. Radiance is the quantity used to describe the propagation of photon
power. The transport equation can be formulated as




Where c is the speed of light in the tissue, c = c0/n/ is the speed of light in vacuum and n is the refractive index of the
medium. The scattering and absorption coefficients μs and μa describe the probability of a scattering or absorption event
per unit length. The phase function, p(s, s‘) denotes the probability that a scattered photon initially travelling in direction s'
continues in direction s after the scattering event. The integral of the probability density function over all- solid angles dΩ'
is equal to one. The transport equation describes the energy balance in an infinitesimal volume element in the tissue.
The left-hand side of Equation (8) is the change in number of photons at position r, with direction s at time t. On the right-
hand side, the first two terms describe the loss of- photons, due to escape over the volume boundaries, scattering into
other directions or- absorption. The third term represents the gain through photons that are scattered from- other
directions into direction s, while the last term is gain due to a light source. It is assumed that all photons have the same
energy and that all scattering is elastic.
It is further assumed that the scattering is symmetric about the incident wave, which means that the phase function is a
function of the scattering angle alone, such that p(s,s')= p(θ), where θ is the angle- between the incident and the scattered
photon. It is often useful to have an analytic expression for the phase function.




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The most popular phase function for light transport in biological tissue is the Henyey-Greenstein function(L.G Henyey
,J.LGreenstein 1941)




where g, the anisotropy factor, is the mean cosine of the scattering angle θ. For nearly isotropic scattering, the value of g
is close to zero, while a g close to unity indicates a strongly forward directed scattering. Tissue is in general highly forward
scattering. Analytic solutions to the transport equation are only known for a few special cases (K.M Case andP.F Zwiefel
1978). In practise, either numerical methods such as Monte Carlo simulations or expansion in spherical harmonics. The
Monte Carlo method simulates a migration of photon packages in a scattering and absorbing medium. The simulated
interaction events of these photon packages are based on random samplings from probability distributions of the step size
between interaction events and scattering angles. For each scattering event, the light is also attenuated due to absorption.
The trajectory of the photon package is followed until it exits through a boundary or is totally lost by absorption. The Monte
Carlo method is useful in that it can be used for any geometry, including layered and inhomogeneous media, and for any
optical properties.
The main disadvantage is that, due to the statistical nature of the method, a large number of photon packages have to be
simulated, and requiring long computation times.




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4.2 Laser - Tissue Interaction




                                       Figure-4.2.1-Schematic diagram of laser-tissue interaction



Laser follows Lambert Beer law when it inter-acts with biological tissues.

Lambert Beer law

I = Io 10-aX
α= absorption coefficient
X = thickness of material/tissue
Io = incident intensity
I = transmitted intensity
Extinction length = 1/α = L; where 90% of the intensity is absorbed, when light energy is reflected,
transmitted, absorbed and scattered by interacting with biological tissues.


Laser light can have the following effects with biological tissue:
    1) Photochemical /Photodynamic effects
    2) Photothermal effects
    3) Mechanical effects
    4) Photoablative effect.


     Photochemical

Laser energy can interact directly or indirectly with chemical structures within tissue. Photobiomodulation (laser
biostimulation, "cold laser" therapy): Low level laser or narrowband light has been used with varying success to modulate
cellular activity to achieve a biological effect such as stimulation of hair growth, collagen remodeling, accelerated wound
healing, etc. In most cases the mechanism of action remains unclear, although changes in mitochondrial activity or cell
membrane permeability may be responsible.




                                                                                                           46 | P a g e
    Summary of the Photochemical Process:
   Photons



   Absorbed in Mitochondria and Cell Membrane within cytochroms and porphyrins



   Singlet Oxygen is produced



   Changes in Membrane Permeability



   ATP Synthesized and DNA Produced



   Increase in Cell Metabolism from a Depressed Rate to a Normal Level



   Selective Bio-Stimulatory Effect on Impaired Cells
   (Note: Cells and tissues functioning normally are not affected)



 Photothermal-

Photothermal effects: Most medical applications involve the selective absorption of light energy using a longer (micro to
millisecond domain) pulse width to cause rapid but selective heating of the target tissue with thermal action. Thermal
effects occur when photon absorption by the outer electrons or molecular vibrations produces enough temperature rises to
denature the biomolecules and results- hyperthermia: meaning a- moderate rise in temperature of several ºC,
corresponding to temperatures of 41º to 44º for some tens of minutes and resulting in cell death due to changes in
enzymatic processes.
Coagulation: refers to an irreversible necrosis without immediate tissue destruction. The temperature reached (50º to 100º
C) for around a second, produces desiccation, blanching, and a shrinking of the tissues by denaturation of proteins and
collagen. Volatilization: means a loss of material. The various constituents of tissue disappear in smoke at above 100º C,
in a relatively short time of around one tenth of a second. At the edges of the volatilization zone -there is a region of
coagulation necrosis: there is a gradual transition between the volatilization and healthy zones.
 The photo-ablative effect
This effect is defined by as a pure ablation of material without thermal lesions at the margins, such as one would get with a
scalpel. It occurs because of the principle of dissociation. With very short wavelengths (190 to 300 nm); the electric field
associated with the light is higher than the binding energy between molecules. The molecular bonds are broken and the
tissue components are vaporized, without generation of any heat at the edges. This effect is obtained with lasers of very
energetic- wavelengths. The action is very superficial because light at these wavelengths is very strongly absorbed by
tissue. The depth of photoablation can vary from several tens to about 150 microns. In Bone surgery it can be used to
perform osteotomy.
 Mechanical effects
Mechanical effects can result from the creation of plasma, an explosive vaporization, or the phenomenon of cavitations,
each of which is associated with the production of a shock wave. With nano- or pico-second pulsed Nd: YAG lasers, a
very high intensity of luminous flux over a small area (between 1010 and 1012 W/cm2) ionizes atoms and creates plasma.
At the boundary of the ionized region, there is a very high pressure- gradient which causes the propagation of a shock
wave. It is the expansion of this shock wave which causes the destructive effect.
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Mechanical Cavitations- If a mechanical containment is added to a thermal containment, the explosive vaporization does
not occur, and a gas bubble is created which will implode when the laser beam is interrupted, creating the phenomenon of
cavitations. This is the mechanism which is used for fragmentation of urinary stones by a laser emitting micro-second
pulses.


Laser action on different biological tissues

           Absorption
The probability of light absorption by tissue molecules is described by the absorption coefficient μa [m-1].The absorption of
light in tissue is strongly wavelength dependent, since different absorbing molecules, called chromophores, absorb in
different wavelength regions. The absorbed energy is most often converted to heat, but can also be reemitted as
fluorescence or be used for a photochemical reaction. The wavelength dependence of the absorption coefficient for typical
breast tissue is illustrated in Fig. 4.2.2. The spectrum in the figure was calculated by adding the absorption spectra of the
contributing chromophores at realistic concentrations. The curve shape of absorption spectra measured in vivo on female
breast tissue corresponds well to in this figure.




                                                              Fig 4.2.2: Absorption spectrum of typical breast     tissue
Between approximately 600 nm and 1300 nm, the absorption is relatively low. At shorter wavelengths, hemoglobin
absorption is large, and at higher wavelengths the strong water absorption drastically reduces light penetration. Light in
this wave length region, often referred to as the tissue optical (or therapeutic) window, is used for many diagnostic and
therapeutic purposes, offering a possibility to reach targets deep into the tissue.
Water
Water is present in most soft tissues, in varying abundances. In skeletal muscle, for example, the water concentration is
on an average 75 %, and in adipose (fatty) tissue it is approximately 20 %. Water has a high absorption in the ultraviolet
(UV) region up to about 200 nm wavelength. Further up in the UV, the absorption is low. At visible wavelengths up to
approximately 600 nm, the absorption coefficient is even lower, less than 0.0001 mm-1. Above that, it starts rising, with-
absorption peaks at approximately 975 nm, 1200 nm, 1440 nm and several more peaks further out in the infrared (IR)
(G.M Hale and M.R Querry 1973). Above approximately 950 nm, water dominates the absorption spectrum of most soft
tissues.


Fig 4.2.3: Absorption spectrum of pure water




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Haemoglobin
Haemoglobin, which is the iron-containing pigment serving as the oxygen-carrier in the red blood cells, has a very high
absorption in the visible wavelength region. The binding of oxygen to the hemoglobin molecule markedly changes its
three-dimensional structure- and this, in turn, affects the light absorption. Hemoglobin is the strongest absorber in human
tissue. However, the concentrations in soft tissues is usually only in the order of a few percent of volume. The absorption
of the oxy-genated (HbO2) and deoxygenated (Hb) forms have different spectral features (Fig.). Oxy-hemoglobin has its
highest peak at approximately 414 nm, and two lower peaks at 542 and 577 nm. The absorption peak of deoxy-
haemoglobin is shifted towards a longer wavelength, 433 nm, and a single peak at 556 nm replaces the double peaks in
the green wavelength region of the oxy-hemoglobin spectrum. Deoxy-haemoglobin also has a small absorption peak at
760 nm not found in oxy-haemoglobin. These differences in spectral features between oxy- and deoxy-haemoglobin can
be used to measure changes in haemoglobin saturation, as is now routinely done in clinical practice by pulse-oximetry.




4.2.4: Absorption spectra of oxy- and deoxy-haemoglobin,
(both at a concentration of 150g/liter of blood).




Melanin
Melanin is a dark pigment which accounts for most of the light absorption in (and thus gives the colour to) hair, skin, and
the iris in the eye. Two groups of melanin exist, eumelanin, which is a black-to-dark-brown pigment, and pheomelanin,
which is a yellow-to-reddish-brown pigment found in red hair. Melanin is synthesized in the melanosome, an organelle of
roughly 1 μm diameter. Melanosomes may contain a variable amount of melanin, and variation in absorption values of
melanin can be tenfold. The spectrum shows no sharp features, and the attenuation decreases exponentially with the
wavelength.
This attenuation spectrum may be due to a combination of Rayleigh scattering and absorption. The measurement of light
extinction in melanin is difficult, since it is insoluble and one cannot extract it from the skin without changing its properties.
However, an approximate attenuation spectrum of melanosomes in skin can be seen in Fig 4.2.5 (S.L Jacques, D.T Mc
Auliffe 1991).




                                                        Fig 4.2.5:   Attenuation spectrum of melanosome in skin.



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          Scattering

The wavelength dependence of the scattering properties of tissue is not as strong as for the absorption. The scattering
coefficient μs [m-1], which is the probability of scattering per unit length, decreases with light wavelength, and the spectrum
has no sharp features as can be seen in Fig 4.2.6. In soft tissue μs is typically in the order of 10 mm-1, so that the
scattering mean free path is in the order of 0.1 mm. As mentioned above, tissue scattering is not isotropic, but strongly
forwardly directed, and typical values of the anisotropy factor g measured in vitro in mammalian tissues are in the range
0.7–0.95( G.J Tearney and M.E Brezinski 1995). A typical value of g in the wavelength region of the optical window is 0.9.
Hence, the reduced scattering coefficient, μs' is usually in the order of 1 mm-1.




Fig: 4.2.6: Reduced scattering coefficient
of typical tissue as calculated by linear extrapolation.




Optical properties of biological tissue
The optical principle behind the measurement of blood volume changes in the skin by means of PPG is the fact that
hemoglobin in the blood absorbs infrared light many times more strongly than the remaining skin tissue.




Light reflection (R) and absorption (A)




It is know that in the range of nonvisible infrared light around 900 nm is a particularly favorable ―measurement window ―for
optical sensing. Only a small proportion (approximately 15%) of the entering light is absorbed by the epidermis. There is
also a large difference between the reflection of the bloodless skin and the reflection from the vessels filled with blood.
As blood pressure in the skin vessel decreases, the surface area of the vessel will reduce. This increases the average the
reflection in the measuring window, so it will be recorded as an increase in PPG signal. The optoelectronic measuring
principle of the PPG thus depends on detecting the changes in reflection of the sub-epidermal layers of- skin during and
after a defined movement or occlusion routine, which causes variations in the volume of the vessel plexuses in the skin.




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Light distribution in Biological tissue




Concerning electromagnetic radiation in the visible and near-infrared spectrum, biological tissue can be viewed as a highly
opaque and nonhomogeneous material. Especially the wavelength-dependent spreading of the photons emitted into the
tissue is of high interest in applications of PPG sensors. The register static and dynamic blood volume changes in skin and
can detect early stages of arterial and venous diseases in that way. The optical radiation in tissue , part of the photons will
be reflected directly by skin surface, another fraction will be distributed in the tissue by absorption or scattering , while the
remaining photons will travel into the tissue either straight -through (ballistic photon) or with a- number of collisions. About
120 of 1 million injected photons are reflected, if the distance between emitter and detector is e.g 5 mm. The shortest flight
time of the photons in fig is 25 ps.The absorption and scattering coefficient of biological tissue varies- with skin depth and
colour but some typical values are about 0.05-0.15 mm-1 ( μa) and 3-10 mm--1(μs).
Mean absorption to scattering probability ration is about 50 and the free photon path between two collisions is about 0.2
mm Monte carlo Simulation of different photon paths in a realistic skin model is shown below.




Monte-carlo Simulation of different
Photon paths in a realistic skin model




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4.3 The Mechanisms of Low Level Laser Therapy




Figure: 4.3.8: Schematic diagram of a cell & site of laser action

The primary (physical) mechanisms relate to the interaction between photons and molecules in the tissue, while the
secondary mechanisms relate to the effect of the chemical (Bio-chemical) changes induced by primary effects.
According to quantum mechanical theory, light energy is composed of photons or discrete packets of electromagnetic
energy. The energy of an individual photon depends only on the wavelength. Therefore, the energy of a "dose" of light
depends only on the number of photons and on their wavelength or color (blue photons have more energy than green
photons that have more energy than red, that have more energy than NIR, etc). Photons that are delivered into living
tissue can either be absorbed or scattered. Scattered photons- will eventually be absorbed or will escape from the tissue
in the form of diffuse reflection. The photons that are absorbed interact with an organic molecule or chromophore located
within the tissue.
Because these photons have wavelengths in the red or NIR regions of the spectrum, the chromophores that absorb these
photons tend to have delocalized electrons in molecular orbitals that can be excited from the ground state to the first
excited state by the quantum- of energy delivered by the photon. According to the first law of thermodynamics, the energy
delivered to- the tissue must be conserved, and three possible pathways exist to account for what happens to the
delivered light energy when low level laser therapy is delivered into tissue.
1).The first and commonest pathway that occurs when light is absorbed by living tissue is called internal conversion.This
happens when the first excited singlet state of the chromophore- undergoes a transition from a higher to a lower electronic
state. It is sometimes called "radiation less de-excitation", because no photons are emitted. It differs from intersystem
crossing in that, while both are radiationless methods of de-excitation, the molecular spin state for internal conversion
remains the same, whereas it changes for intersystem crossing.
The energy of the electronically excited state is given off to vibration modes of the molecule, in other words, the excitation
energy is transformed into heat.
2).The second pathway that can occur is fluorescence. Fluorescence is a luminescence or re-emission of light, in which
the molecular absorption of a photon triggers the emission of another photon with a longer wavelength.
The energy difference between the- absorbed and emitted photons ends up as molecular vibrations or heat. The
wavelengths involved depend on the absorbance curve and Stokes shift of the particular fluorophore.
  3).The third pathway that can occur after the absorption of light by a tissue chromophore (Biochemical) represents a
number of processes broadly grouped under an umbrella category of photochemistry.

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Among the above three, internal conversion & fluorescence are the mechanisms those are involved in Physical
Mechanism of laser tissue interaction, and third one is recognized as Bio-chemical.




      PHYSICAL MECHANISMS

     There are two primary forms of physical effects generated by laser irradiation of biological tissues:



1.   Photon-absorption (the basis of photobiological action, and generated by all forms of light):
As stated above photon is absorbed by means of internal conversion &fluorescence, these procedures exerts photon
absorption into biological tissues. Following absorption they start a cascade of biological interaction, such as vibration,
heat/light production, and other biological changes into tissues by increasing membrane permeability/potential (Cellular &
mitochondrial). After diffuse penetration of the laser bundle in the tissue, there is absorption of polarized light in
cytochrome molecules (e.g. porphyrins), the electrical field across the cell membrane creates a dipole moment on the bar
shaped lipids, and finally local differences in intensity creates temperature and pressure gradients across cell membranes.
2.   Internal conversion &fluorescence of light also generates Speckle formation, which is unique to laser therapy:
The speckle field is created when coherent laser radiation is reflected, refracted and scattered. The speckle field is not
simply a phenomenon created at and limited to the- tissue surface, but is generated within a volume of tissue, persisting to
the total extent of the depth of penetration of the laser beam. Laser speckles formed deep in the tissue create temperature
and pressure gradients across cell membranes, increasing the rate of diffusion across those membranes. Further, photons
within each speckle are highly polarized, leading to an increased probability of photon absorption (one possible reason for
why laser therapy has been shown to consistently out-perform other non-coherent light sources, especially for deeper
tissue treatments).
The speckle effect is a result of the interference of many waves, having different phases, which add together to give a
resultant wave whose amplitude, and therefore intensity, varies randomly. Each point on illuminated tissue acts as a
source of secondary spherical waves. The light at any point in the scattered light field is made up of waves that have been
scattered from each point on- the illuminated surface. If the surface is rough enough to create path-length differences
exceeding one wavelength, giving rise to phase changes greater than 2         , the amplitude (and hence the intensity) of the
resultant- light varies randomly. It is proposed that the variation in intensity between speckle spots that are about 1 micron
apart can give rise to small but steep temperature gradients within sub cellular organelles such as mitochondria without
causing photochemistry. These temperature gradients are proposed to cause some unspecified changes in mitochondrial
metabolism.




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      BIOCHEMICAL MECHANISMS




                                        FIGURE: 4.3.9: Schematic diagram showing absorption side of Laser
                                        by cellular membrane & mitochondria.

The third pathway that can occur after the absorption of light by a tissue chromophore (Biochemical) represents a number
of processes broadly grouped under an umbrella category of photochemistry. The first law of photobiology states that for
low- power visible light to have any effect on a living biological system, the photons must be absorbed by electronic
absorption bands belonging to some molecular photoacceptors, or chromophores 1. A chromophore is a molecule (or part
of a molecule) which imparts some decided color to the compound of which it is an- ingredient. Cellular chromophores,
photoacceptors are localized in the mitochondrial respiratory chain.
Chromophores almost always occur in one of two forms: conjugated pi electron systems and metal complexes. Examples
of such chromophores can be seen in chlorophyll (used by plants for photosynthesis), hemoglobin, cytochrome c oxidase
(Cox), myoglobin, flavins, flavoproteins and porphyrins2.

Bio-chemical action of laser can be explained by “Action of photon with mitochondrial
respiratory chain- Cytochrome c oxidase enzyme”.




                                        FIGURE: 4.3.10a -  Schematic diagram showing the absorption of red and NIR
                                        light by specific Cellular chromophores photoacceptors localized in the
                                        mitochondrial respiratory chain,
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                                         FIGURE4.3.10b- Structure and mode of action of cytochrome c oxidase.

Although, the exact mechanism of action of LLLT is not completely understood; however, there are several theories based
on cellular research conducted over the last two decades or more. The basic premise is that LLLT stimulates cell
activation processes which, in turn, intensify physiologic activity. Healing is essentially a cellular process and light energy
initiates a cascade of reactions, from the cell membrane to the cytoplasm, to the nucleus and DNA. This is called cellular
amplification; a phenomenon whose demonstration earned the Nobel Prize in Physiology or Medicine in 1994.



1)   Cytochrome c oxidase mediated increase in ATP production.
2)   Cytochrome c oxidase mediated singlet-oxygen production.
3)   Cytochrome c oxidase mediated Reactive oxygen species (ROS) formation.
4)   Cytochrome c oxidase mediated Photodiassociation and Nitric Oxide Production.

1). Cytochrome c oxidase mediated increase in ATP production.

 Current research about the mechanism of LLLT effects inevitably involves mitochondria. Mitochondria play an important
role in energy generation and metabolism. Mitochondria are sometimes described as ―cellular power plants‖, because they
convert food molecules into energy in the form of ATP via the process of oxidative phosphorylation.
The mechanism of LLLT at the cellular level has been attributed to the absorption of monochromatic visible and NIR
radiation by components of the cellular respiratory chain3. Several pieces of evidence suggest that mitochondria are
responsible for the cellular response to red visible and NIR light.




          FIGURE 4.3.11 & 4.3.12: Schematic Structure of the mitochondrial respiratory chain & mitochondrial
           electron transport chain




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Absorption of photons by molecules leads to electronically excited states and consequently can lead to acceleration of
electron transfer reactions and stimulate chemiomosis mechanism.
Peter D. Mitchell proposed the chemiosmotic hypothesis in 1961.[1] The theory suggests essentially that most ATP
synthesis in respiring cells come from the electrochemical gradient- across the inner membranes of mitochondria by using
the energy of NADH and FADH2 formed from the breaking down of energy rich molecules such as glucose.^Peter Mitchell
(1961). "Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism".
( N.B: The inner mitochondrial membrane contains 5 complexes of integral membrane proteins: NADH dehydrogenase
(Complex I), succinate dehydrogenase (Complex II), cytochrome c reductase (Complex III), cytochrome c oxidase
(Complex IV), ATP synthase (Complex V), and two freely diffusible molecules, ubiquinone and cytochrome c, which
shuttle electrons from one complex to the next (Figure 3). The respiratory chain accomplishes the stepwise transfer of
electrons from NADH and FADH2 (produced in the citric acid or Krebs cycle) to oxygen molecules to form (with the aid of
protons) water molecules harnessing the energy released by this transfer to the pumping of protons (H+) from the matrix
to the intermembrane space. The gradient of protons formed across the inner membrane by this process of active
transport forms a miniature battery.


The protons can flow back down this gradient, re-entering the matrix, only through another complex of integral proteins in
the inner membrane, the ATP synthase complex). Electron transfer reactions are highly important in the mitochondrial
respiratory chain, where the principal chromophores involved in laser therapy are thought to be situated. More electron
transport necessarily leads to increased production of ATP [4].
Light induced increase in ATP synthesis and increased proton gradient leads to an increasing activity of the Na+/H+ and
Ca2+/Na+ antiporters and of all the ATP driven carriers for ions, such as Na+/K+ ATPase and Ca2+ pumps. ATP is the
substrate for adenylcyclase, and therefore the ATP level controls the level of cAMP. Both Ca2+ and cAMP are very
important second messengers. Ca2+ especially regulates almost every process in the human body (muscle contraction,
blood coagulation, signal transfer in nerves, bone metabolism, gene expression, etc.).


2). Cytochrome c oxidase mediated singlet-oxygen production.

In addition to cytochrome c oxidase mediated increase in ATP production, other mechanisms may be operating in LLLT.
The first of these we will consider is the
―singlet-oxygen hypothesis.‖ Certain molecules with visible absorption bands like porphyrins lacking transition metal
coordination centers   [5]   and some flavoproteins   [6]   can be converted into a long-lived triplet state after photon absorption.
Because of the energy of the photons involved, covalent bonds cannot be broken. However, the energy is sufficient for the
first excited singlet state to be formed, and this can undergo intersystem crossing to the long-lived triplet state of the
chromophore. The long life of this species allows reactions to occur, such as energy transfer to ground state molecular
oxygen (a triplet) to form the reactive species, singlet oxygen. Alternatively the chromophore triplet state may undergo
electron transfer (probably reduction) to form the radical anion that can then transfer an electron to oxygen to form
superoxide. This is the same molecule utilized in photodynamic therapy (PDT) to kill cancer cells, destroy blood vessels
and kill microbes. Researchers in PDT have known for a long time that very low doses of PDT can cause cell proliferation
and tissue stimulation instead of the killing observed at high doses [7]..




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3). Cytochrome c oxidase mediated Reactive oxygen species (ROS) formation.




                                        FIGURE: 4.3.13: 5.Reactive oxygen species (ROS) formed as a result
                                         of LLLT effects in mitochondria may activate the redox-sensitive
                                        transcription factor NF-B (relA-p50) via protein kinaseD (PKD).



The next mechanism proposed was the ―redox properties alteration hypothesis‖ [8]. Alteration of mitochondrial metabolism
and activation of the respiratory chain by illumination would also increase production of superoxide anions O2 •- It has
been shown that the total cellular production of O2 •- depends primarily on the metabolic state of the mitochondria. They
are formed as natural by-product of the normal metabolism of oxygen.
Other redox chains in cells can also be activated by LLLT. NADPH-oxidase is an enzyme found on activated neutrophils
and is capable of a non-mitochondrial respiratory burst and production of high amounts of ROS can be induced. Reactive
oxygen species (ROS) are very small, highly- biological molecules such as proteins, nucleic acids and unsaturated lipids
molecules that include oxygen ions such as superoxide, free radicals such as hydroxyl radical, and hydrogen peroxide,
and organic
peroxides. [9].LLLT was reported to produce a shift in overall cell redox potential in the direction of greater oxidation and
increased ROS generation and cell redox activity have been demonstrated10.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved and have important roles in cell
signaling 11, regulating nucleic acid synthesis, protein synthesis, enzyme activation and cell cycle progression 12.These
cytosolic responses may in turn induce transcriptional changes. Several transcription factors are regulated by changes in
cellular redox state. But the most important one is nuclear factor B (NF-B). Figure: 4.3.13- illustrates the effect of redox-
sensitive transcription factor NF-B, activated after LLLT and is instrumental in causing transcription of protective and
stimulatory gene products. The whole process absolutely depends on the physiological status of the host organism as well
as on radiation parameters.




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4). Cytochrome c oxidase mediated Photodissociation and Cell signaling-




                                         FIGURE: 4.3.14: When NO is released from its binding to heme iron
                                         and copper centers in Cytochrome c oxidase by the action of light,
                                         oxygen is allowed to rebind to these sites and respiration is restored
                                         to its former level leading to increased ATP synthesis.

A third photochemistry pathway that can occur after the absorption of a red or NIR photon is the dissociation of a non-
covalently bound ligand from a binding site on a metal containing cofactor in an enzyme. The most likely candidate for this
pathway is the binding of nitric oxide to the iron-containing and copper-containing redox centers in unit IV of the
mitochondrial respiratory chain, known as cytochrome c oxidase.
The activity of cytochrome c oxidase is inhibited by nitric oxide (NO). This inhibition of mitochondrial respiration by NO can
be explained by a direct competition between NO and O2 for the reduced binuclear center CuB/a3 of cytochrome c
oxidase and is reversible [13]. It was proposed that laser irradiation could reverse the inhibition of cytochrome c oxidase by-
NO and thus may increase the respiration rate (―NO hypothesis‖)       [14].   Data published recently by Karu et al   [14]   indirectly
support this- hypothesis. Another piece of evidence for NO involvement in responses to LLLT is an increase in inducible
nitric oxide synthase production after exposure to light (635 nm) [15].
While both observations support the hypothesis of NO dependent responses to LLLT, responses to different wavelengths
of light in different models may be governed by distinct mechanisms.
In addition to NO being photodissociated from Cox as described, it may also be photo-released from other intracellular
stores such as nitrosylated hemoglobin and nitrosylated myoglobin 16(Shiva and Gladwin 2009).
Light mediated vasodilatation was first described in 1968 by R F Furchgott, in his nitric oxide research that lead to his
receipt of a Nobel Prize thirty years later in 199817. Later studies conducted by other researchers confirmed and extended
Furchgott‘s early work and demonstrated the ability of light to influence the localized production or release of NO and
stimulate vasodilatation through the effect NO on cyclic guanine monophosphate (cGMP). This finding suggested that
properly designed illumination devices may be effective, noninvasive therapeutic agents for patients who would benefit
from increased localized NO availability.




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4.4: Effects of Laser on Biological Cell / Tissue healing
     Laser on hard tissue & Bone stimulation/Regeneration

4.4a: Effects of Laser on Biological Cell / Tissue healing


Cellular signaling and response

Cellular signaling




                                                   Figure-4.4.15: schematic diagram of laser induced cell signaling
                                                   pathway


It is proposed that LLLT produces a shift in overall cell redox potential in the direction of greater oxidation   [18].   Different
cells at a range of growth conditions have distinct redox states.The combination of the products of the reduction potential
and reducing capacity of the linked redox couples present in cells and tissues represent the redox environment (redox
state) of the cell. Redox couples present in the cell include: nicotinamide adenine- dinucleotide (oxidized/ reduced forms)
NAD/NADH,
nicotinamide adenine dinucleotide phosphate NADP/NADPH, glutathione/glutathione disulfide couple GSH/GSSG and
thioredoxin/ thioredoxin disulfide couple Trx (SH) 2/TrxSS [19].
Several important regulation pathways are mediated through the cellular redox state. Changes in redox state induce the
activation of numerous intracellular signaling pathways; regulate nucleic acid synthesis, protein synthesis, enzyme
activation and cell cycle progression [20]. These cytosolic responses in turn induce transcriptional changes.
Several transcription factors are regulated by changes in cellular redox state. Among them redox factor –1 (Ref-1) -
dependent activator protein-1 (AP-1) (FOS and Jun), nuclear factor κB (NF-κB), p53, activating transcription factor/cAMP-
response element–binding protein (ATF/ -CREB), hypoxia inducible- factor (HIF)-1α, and HIF-like factor. As a rule, the
oxidized form of redox-dependent transcription factors have low DNA-binding activity.




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Figure: 4.4.15b: LLLT induced cellular changes

However it was also shown that low levels of oxidants appear to stimulate proliferation and differentiation of some type of
cells   [21-23].The   effects of LLLT can vary considerably. Cells being initially at a more reduced state (low intracellular pH)
have high potential to respond to LLLT, while cells at the optimal redox state respond weakly or do not respond to
treatment with light.

                           Cellular response
―Red light aids in the production of ATP in cells, which increases cellular health and energy. The radiation (energy) in
normal cells stimulates adjacent cells to divide in the normal process of regeneration and healing. Laser light stimulates
abnormal tissue to activate normal inter-cellular radiation, thus stimulating the normal healing process to start again. The
photons produced by laser light normalize tissue by activating enzymes within cells. Once activated, enzymes within a-
cell trigger a chemical reaction in which more enzymes are activated in a domino-type effect. Low Level Laser Therapy
has no effect on normal tissue. Photons will only be absorbed by cells that need them.‖
The cellular responses observed in vitro after LLLT can be broadly classed under increases in metabolism, migration,
proliferation, and increases in synthesis and secretion of various proteins. Many studies report effects on more than one of
these parameters. Yu et al reported         [24]   on cultured keratinocytes and fibroblasts that were irradiated with 0.5-1.5 J/cm2
HeNe laser. They found a significant increase in basic fibroblast growth factor (bFGF) release from both keratinocytes and
fibroblasts and a significant increase in nerve growth factor release from keratinocytes.
Medium from He-Ne laser irradiated keratinocytes stimulated [3H] thymidine uptake and proliferation of cultured
melanocytes. Furthermore, melanocyte migration was enhanced either directly by He-Ne laser or indirectly by the medium
derived from He-Ne laser treated keratinocytes.
The presence of cellular responses to LLLT at molecular level was also demonstrated [25]. Normal human fibroblasts were
exposed for 3 days to 0.88J/cm2 of 628 nm light from light emitting diode. Gene expression profiles upon irradiation were
examined using a cDNA microarray containing 9982 human genes. 111 genes were found to be affected by light. All
genes from- antioxidant related category and genes related to energy metabolism- and respiratory chain were
upregulated. Most of the genes related to cell proliferation were up-regulated too.
Amongst genes related to apoptosis and stress response, some genes such as JAK binding protein were up-regulated;
others such as HSP701A, caspase 6 and stress-induced phosphoprotein were down regulated. It was suggested that
LLLT stimulates cell growth directly by regulating the expression of specific genes, as well as indirectly by regulating the
expression of the genes related to DNA synthesis and repair, and cell metabolism.

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             Tissue healing

One of the truly unique characteristics ofLLLT is that it has the ability to actually promote and enhance healing,
not just treat symptoms. The irradiation by low-level laser light accelerates and enhances healing activities
carried out by the body. Several of the unique characteristics of LLLT that work to alleviate pain and
inflammation also play an important role in acceler ating the healing process; the LLLT-mediated reduction in
inflammation and pain frees the body‘s natural ability to repair and heal itself.
The effects of LLLT can vary considerably. Cells being initially at a more reduced state (low intracellular pH) have high
potential to respond to LLLT, while cells at the optimal redox state respond weakly or do not respond to treatment with
light.
As wound healing progresses through the stages of inflam mation, proliferation, remodeling and maturation,
laser therapy presents the opportunity to impact each of these phases in positive and beneficial ways. The
beneficial effect of LLLT on wound healing can be explained by considering several basic biological mechanisms including
the induction of expression cytokines and growth factors known to be responsible for the many phases of wound healing.
Firstly there is a report [26] that laser increases both protein and mRNA levels of IL-1α and IL-8 in keratinocytes. These are
cytokines responsible for the initial inflammatory phase of wound healing. Secondly there are reports            [27]   that LLLT can
upregulated cytokines responsible for fibroblast proliferation, and migration such as bFGF, HGF and SCF. Thirdly it has
been reported [28] that LLLT can increase growth factors such as VEGF responsible for the neovascularization necessary
for wound healing. Fourthly TGF-β is a growth factor responsible for inducing collagen synthesis from fibroblasts and has
been reported to be upregulated by LLLT [29].
Fifthly there are reports [30, 31]that LLLT can induce fibroblasts to undergo the transformation into myofibloblasts, a cell type
that expresses smooth muscle α-actin and desmin and has the phenotype of contractile cells that hasten wound
contraction.
LLLT at low doses has been shown to enhance cell proliferation in vitro in several types of cells: fibroblasts 32,
keratinocytes 33, endothelial cells 34(Moore et al.2005), and lymphocytes35.The mechanism of proliferation was proposed to
involve photostimulatory effects in mitochondria processes, which enhanced growth factor release, and ultimately led to
cell proliferation    36.   Kreisler et al showed37 that the attachment and proliferation of human gingival fibroblasts were
enhanced by LLLT in a dose-dependent manner.
LLLT modulated matrix metalloproteinase activity and gene expression in porcine aortic smooth muscle cells. Shefer at el.
Showed38 that LLLT could activate skeletal muscle satellite cells, enhancing their proliferation, inhibiting differentiation and
regulating protein synthesis.
LLLT may enhance neo-vascularisation, promote angiogenesis and increase collagen synthesis to promote healing of
acute    39and    chronic wounds    40.LLLT    provided acceleration of cutaneous wound healing in rats with a biphasic dose
response favoring lower doses          41.   LLLT can also stimulate healing of deeper structures such as nerves 42, tendons 43,
cartilage   44,   bones45 and even- internal organs46. LLLT can reduce pain         47,   inflammation 47and swelling     48caused   by
injuries, degenerative diseases or autoimmune diseases. Oron reported beneficial effect of LLLT on repair processes after
injury or ischemia in skeletal- and heart muscles in multiple animal models in vivo49. LLLT has been used to mitigate
damage after strokes (in both animals 50and humans 51, after traumatic brain injury 52 and after spinal cord injury53.




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4.4b- Laser on hard tissue & Bone stimulation/ Regeneration



Laser action on biological hard tissue




Figur: 4.4.16 LLLT mechanism and application.
Incoming red and NIR photons are absorbed in
cell Mitochondria, producing reactive oxygen
species (ROS) and releasing nitric oxide (NO),
whic     to gene transcription via activation of
transcription factors (NF-κB And AP1).




Laser action on biological hard tissue can be sub-divided into-
1. Photochemical/Biostimulative
2. Photo-Ablation.

Photochemical/Biostimulative

The anecdotal and researched evidence for the effects of Low Level Laser Therapy (LLLT) on the stimulation of bone/
hard tissue have been reported for over 20 years. This has been in the form of local as well as systematic effects –
including contra-lateral effects. Over the following decade and a half, further studies have also investigated and
demonstrated that LLLT is effective for the stimulation of bone tissue. Reports of stimulation of rabbit radii fractures / mice
femurs were made as early as 1986 and 1987 and on human bone as of 1974, with irradiated- bones healing faster than
controls and contra-lateral non-treated fractures similarly demonstrating faster healing times.
The process of wound healing involves several types of cells; enzymes; growth factors and other substances. Bone
healing differs from that of soft tissues because of its morphology and- composition. The natural course of bone healing
comprises of consecutive phases, which differs according to the type and intensity of trauma and the extent of damage to
the bone.The use of LLLT (Low Level Laser Therapy) for wound healing has been shown to be effective in modulating
both local and systemic response. In order to observe the biomodulating effects of LLLT, some level of tissue deficiency
seems necessary [2]. LLLT seems ineffective when used on normal tissues1 .
The reasons for which bone regenerates faster than normal, have been attributed to the general effects of LLLT and its
ability to increase the rates of healing through mitochondrial ATP production and alteration in the cellular lipid bi-
layer.Additional hypothesis include the subsequent capacity of irradiated cells to alter their ion exchange rate and thus
influence the- catalytic effects of the specific enzymes and substrates. These in turn initiate and promote the healing
process completing the cascading cycle of events.These aspects would increase both the release of mediators and micro-
vascularization, which in turn would accelerate bone healing. It was suggested that PGE2 activates wound- healing [3], and
increased- level of PGE2 was observed by Messer et al. 4.




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There is evidence that PGE2 is also produced by osteoblast and that its effects may be therapeutic or adverse                       [5].


However, some reports [6], suggested that LLLT would improve bone matrix production due to improved vascularization
and anti-inflammatory effect.
Some previous reports do recognize that LLLT has positive effects on bone            [7, 8].   These studies reflect the idea that non-
differentiated mesenchymal cells could be biomodulated positively to osteoblast that would more rapidly change to
osteocytes [7].It is also known that the osteogenic potential of mesenchymal cells depends on several genetic factors and
also on systemic and local inducer factors         [7]..    LLLT may act as an inducer factor. This- aspect may be possibly
corroborated by several previous studies in which LLLT was used- in fractures [9], bone defects [10], tooth extraction [11, 12, 13]
and after the placement of dental implants [14].
It is possible that LLLT effect on bone regeneration depends not only on the total dose of irradiation, but also on the
irradiation time and the irradiation mode (Continuous or Pulsed). Most importantly, a recent study has suggested that the
threshold energy density and intensity are biologically independent from each other. This independence accounts for the
success and- the failure of LLLTachieved at low energy density levels as described previously by Sommer et al [15].
Some previous reports, which found no biomodulating effects of LLLT on bone [16], did not consider the systemic effect of
LLLT[6, 10]. They used the contra-lateral side of the same subject as controls. On the other hand, the findings of this
investigation is very close to a study which found intense activity and high numbers of osteoblast 5-6 days after the
procedure was performed on bone defects in a similar model.



Photo-Ablation




                                                           Figure-4.4.17: Ablation and melting are the two basic modalities by
                                                           which the effect of lasers on the hard tissues.

When discussing the effect of laser on hard tissues, the energy absorption in the hydroxyapatite plays a major role in
addition to its absorption in water.When laser energy- is absorbed in the water of the hard tissues, a rapid volume
expansion of the evaporating- water occurs as a result of a substantial- temperature elevation in the interaction site. Micro
explosions are produced causing- hard tissue disintegration. If temperatures are raised beyond 5 degrees C level, damage
to the tissue is irreversible.
Histological, after laser ablation, presence of odontoblastic nuclei is important. Consistency and composition of the
intracellular tissue is another factor influencing cell viability. If heat is intensive and exists for an extended time, the
consistency of the intracellular ground substance may not be preserved. Accordingly, the application of excessive energy-
densities has been shown to result in significant damage to hard tissue and in particular structures. Studies showed that
the use- of Er.-YAG laser to treat hard tissues is both safe and effective for Incision, Coagulation and removal/osteotomy.

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On the other hand Co2 laser has been widely used over the past few years in orthopaedic surgery to do osteotomy/
amputation, and it has been proved that in recommended method & procedure post surgery follow-up has no negative/
untoward effect on cut margin/ or on surrounding tissues.




                                                 Figure: 4.4.18: Ablation curves of fresh and dried bone obtained
                                                 with a CO2 laser (pulse duration: 250 μs, wavelength: 10.6 μm).
                                                 Due to its higher water content, freshbone is ablated more
                                                 efficiently.Data according to Forrer et al. (1993).




                                                 Figure: 4.4.19: Ablation curve of bone obtained with an Er:YAG
                                                 laser (pulse duration: 180 μs, wavelength:2.94 μm).
                                                 Data according to Scholz and Grothves-Spork (1992).




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Bone Regeneration by Infra Red Laser Therapy:

Infrared laser radiation enhances bone regeneration/ formation by two consecutive phases of cellular, intra
cellular and tissue modulating cascades of inter-depended process-

1. Directly by simulation of osteoblast formation, inhibition of osteoclast activities, proliferation/
differentiation of fibroblast and enhancement bone growth factors/ modulation of cytokines.
2. Indirectly by enhancing some specific bone formation modulation, and creating a friendly environment
that fascinates bone formation /regeneration.


           Directly by simulative and inhibiting of osteoblast, osteoclast,
           and bone growth factors enhancement.

Laser irradiation play two principal roles in stimulating bone formation. One is stimulation of cellular proliferation, especially
proliferation of nodule-forming cells of osteoblast lineage, and the other is stimulation of cellular differentiation, especially
to committed precursors, resulting in an increase in the number of more differentiated osteoblastic cells and an increase in
bone formation(1).
Studies of bone healing response to infrared light show acceleration of osteoblast formation as well as calcium salt
deposition under the influence of infrared light    (2, 3, 4, 5, 6, 7).   In osteoblastic cells, the osteoglycin/mimecan gene was
upregulated 2.3-fold at 2 h after exposure to infrared light (8). Osteoclast Inhibition Prevents Bone Mineral Resorption (9).
Low-Level Laser Irradiation on Osteoglycin Gene Expression in Osteoblasts noted in experimental methods. Studies have
also demonstrated that bone growth factors are stimulated by IR light. Osteoglycin is a small leucine-rich proteoglycan
(SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in
the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell- proliferation, differentiation and
adhesion in close association with collagen and many other growth factors. Study also proved the stimulatory effect of-
infrared light on bone formation during the early proliferation stage of cultured osteoblastic cells.


           Indirectly by enhancing some specific bone formation modulation and
           creating a friendly environment that fascinates bone formation.

     o    Ion Exchange and Bone Mineralization (10, 11)
Light affects the mitochondrial respiratory chain by changing the electric potential of cell membranes and, consequently,
their selective permeability for sodium, potassium and calcium ions, or by increasing the activity of certain enzymes such
as cytochrome oxidase and adenosine triphosphatase.1
Mitochondrial ATP production and alteration in the cellular lipid bi-layer. Additional hypotheses include the subsequent
capacity of irradiated cells to alter their ion exchange rate and thus influence the catalytic effects of the specific enzymes
and substrates. These in turn initiate and promote the healing process completing the cascading cycle of events. 2


     o    Role of Nitric Oxide in Bone Formation (12, 13)
Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. Bone metabolism involves the balance
between formation and resorption. In this process, known as bone remodeling, mineralized bone is continuously resorbed
by osteoclasts and new bone is formed by osteoblast. Nitric Oxide produces rapid osteoclast detachment and contraction
in-vitro, and this effect is accompanied by a profound inhibition of bone resorption.
Inhibition of NO synthase (resulting in absence of NO) in normal rats is followed by increased bone resorption reflected by
a marked loss in bone mineral density. Further investigation shows that inhibition of the inducible Nitric Oxide

                                                                                                                  65 | P a g e
Bone resorption depends on both osteoclast precursor replication and on the activity of mature osteoclast cells. Addition of
an NO donor results in depressed replication in human -preosteoclast lines. Taken together, these results strongly suggest
that NO maintains a central control of bone- resorption by exerting a powerful tonic restraint of osteoclast- numbers and
activity. Since NO also influences behavior of the osteoblast, the bone-forming cell, invitro, a similar effect in-vivo might
imply a general influence on bone remodeling.


     o    Lymphatic Circulation and Bone regeneration (14.15, 16)
The majority of fluid transfer and exchange within the living bone is predominantly influenced by the lymphatic rather than
the vascular circulation. This is justified through studies on bone fluid input and output levels that have demonstrated that
the venous and arterial aspect of circulation alone cannot account for the demonstrated levels of output nor the presence
of free radical molecules which exceed those of the vascular input.Furthermore, the diameter of large (globular) proteins
within the bone exceed the diameter of the vessels that form the terminal- aspects of the circulatory system making it
impossible for them to have been delivered via this system. Infrared light is well documented and known as having effects
that influence the lymphatic circulation and wound healing process.
Presence of infrared light by increasing lymphatic circulation does so by virtue of an increase in the diameter of the
lymphatic vessels, not just by increased flow rates within the vessel at an unchanged diameter.Infrared light, with its
known general effects and specific direct effects on the lymphatic system, would act to stimulate mitochondria ATP that
increases cellular and circulatory motility as well as directly influencing lymphatic flow. It also promotes increased
permeability in interstitial tissue and facial layers (Gabel 1995) reducing stagnation and blockage. These actions would
assist the increase in lymphatic flow and consequently the circulation within the affected bone.




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4.5 Medical application of Low Level Laser

            Therapeutic:

 Low-Level Laser has been shown to be effective in, but not limited to, the treatment of the following
indications:
 780-830nm Infra-Red Wavelengths - Deep Tissue Penetration:
Sprains & strains
Wounds and abrasions
Ligament & tendon injuries, bowed tendon
Inflammation
Joint injuries
Pain points and deep-tissue acupuncture points
Chronic & acute pain
Hematomas
Enhancement of bone fractures union.
630-700nm Visible Red Wavelengths - Shallow Tissue Penetration:
Mucous membranes
Post-surgical wounds
Wounds & abrasions
Superficial acupuncture points


          LLLT for pain relief, inflammation and healing.




           Figure: 4.5.16- Schematic diagram of pain, inflammation & tissue healing by LLLT.

LLLT have been reported to have a significant effect on pain, inflammation and tissue healing. In recent years, there has
been growing interest in the use of laser biostimulation as a therapeutic modality for- pain management. Alterations in
neuronal activity have been suggested to play a role in pain relief by laser therapy. Many published reports document- the
positive -findings for laser biostimulation in pain management. This level of evidence- relates to chronic neck pain-
tendonitis, chronic joint disorders, musculoskeletal pain, and chronic pain. Randomized controlled trials provide evidence
for the efficacy of laser therapy in chronic low back pain.
LLLT has been used clinically since 1981 for the treatment of patients with inflammatory pathology. The beneficial effect of
LLLT on wound healing can be explained by considering several basic biological mechanisms, including the induction of
the expression of cytokines and growth factors known to be responsible for the many phases of wound healing. Above
schematic diagram shows pathways & steps of laser action on pain inflammation and tissue healing.



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      Medical Research

                                        Laser hyperthermia photodynamic studies
                                        DNA analysis
                                        Contact cutting, ablation
                                        Coagulation necrosis
                                        Tissue welding/fusion
      Diagnostic:




                                   Figure: 4.5.17-Schematic diagram of diagnostic application of Laser.

In one sense, all traditional (old-fashioned) diagnostic medicine involved light and optics. The doctor would examine
his patient under a bright light or a colored light, or- use a hand lens to improve upon what he could see unaided.
Later-on, the advent of various imaging- technologies such as X-ray, computed X-ray tomography, magnetic
resonance imaging, positron-emission tomography, single photon emission computed tomography developed.
Now things have come full circle with researchers developing optical imaging technologies relying on visible and
near-infrared (NIR) light. Visible light penetrates into biological tissues more than one might think. Red and NIR light
penetrate deeper than green, blue or violet light. We can visualize this phenomenon by shining a white flashlight
through hand, observing a red glow on the other side (the blue and green wavelengths having been absorbed). Red
light penetrates more because it is not strongly absorbed by blood and because it tends to scatter more.
Therefore, red or NIR light is generally used to "see" deeper into the body. Optical imaging techniques could be
cheaper, less invasive and less toxic, because light is non-ionizing compared with the previous techniques mentioned
above. Of course, tissue is far from transparent to visible and NIR wavelengths when compared too much shorter-
wavelengths (X-rays), or too much longer wavelengths (radio waves). Therefore extraordinary measures must be
taken to derive useful diagnostic imaging information from these wavelengths of light (Grier, 2003).
Information may be acquired from photons that are scattered, from photons that are absorbed, and from photons that
are re-emitted after being absorbed in tissue. Examples of the first class of techniques are optical coherence
tomography in vivo confocal microscopy, and light scattering spectroscopy. Examples of the second class are the
pulse oximeter for measuring blood oxygenation, diffuse optical- tomography and photoacoustic imaging and
spectroscopy. Examples of the third class include auto fluorescence imaging, in vivo confocal fluorescence
microscopy, and Raman spectroscopy.



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Chapter-5



Materials & Methods
5.1 Materials
5.2 Methods
Duration of study:
The duration of the study was two years (from April- 2008 to March- 2010)
Type of study: Prospective Randomized case control study.
Place of study: the study wass conducted at Shaheed Suhrawardy Medical College Hospital
in the Department of Orthopedics and Traumatology , Dhaka-1207, Bangladesh.



5.1 Materials
5.1.1: Machine used in this work: The laser machine used on this research was BioLux MD (Ga-Al-As-830 nm) Diode
Laser.




         830 bone probe                                                               Tip of 830 probe

          Figure: 5.1.1-BioLux MD Ga-Al-As Laser (830 nm) Machine with Probe.




                                                                                                  69 | P a g e
5.1.2 Sample collection & distribution
The sample was collected randomly from admitted patients with bones fracture in superior and inferior extremity in
Shaheed Suhrawardy Medical College Hospital in the Department of Orthopedics and Traumatology, Dhaka-1207,
Bangladesh. A total of 40 patients randomly collected; among which 20 were in the Laser group (L1,L2) and 20 were in the
control group (C1,C2). The patients were briefed about the study and written consent (Informed consent) was obtained
from all patients/ medico legal guardian for other patients.
5.1.2:. A. Inclusion Criteria
1. Patient suffering from recent (1-7 days old) axial bone fracture of male and female patients,
2. Age between 15- 95 years,
3. Are not taking any pain medications,
4. Whether taking any bone supplement or not,
5. Aren’t pregnant, haven’t any previous fracture history,
6. Have not systemic or psychological disorder.

5.1.2. B: Exclusion Criteria
1. Bone fracture with open wound were excluded
2. Bone fracture with any active infection
3. Bone fracture with previous Surgery.


               Patient distribution: General

                                                                           Study design

                                                    15
                                                    10
                                                        5
                                                                                                     Total Pt.40
                                                        0




                                              Table: 5.1.2- Patient distribution according to study design.

                                                            Patient distribution: By age-
              Laser Group (L1&L2)

                                                  20

                                                  15

                                                  10                                         Total

                                                    5                                                     Male

                                                    0                                                   Female




                                               Table: 5.13-Bar-graph showing-patient distribution according to age
                                              in study (laser) group.



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 Control Group (C1&C2)

                             20

                             15

                             10                                                  Total
                                                                                              Male
                              5

                              0                                                             Female




                          Table: 5.1.4- Bar-graph showing-patient distribution according to age in non-
                         study (Control)
                         group.


Patient distribution: By bone
                        Laser Group (L1&L2)

                             10
                              8
                              6
                              4                                                          Up. Limb
                              2                                                          Low. Limb
                              0




                          Table: 5.15-Bar-graph showing patient distribution according to type of bone
                         in study (laser) group.

Control Group(C1&C2)

                             12
                             10
                              8
                              6
                              4                                                          Up. Limb
                              2                                                          Low. Limb
                              0




                         Table: 5.1.6- Bar-graph showing patient distribution according to type
                         of bone in control group.



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5.1.3: Treatment Protocols


Laser Treatment Protocols used in this work:

o   4- 8 joules /cm2
o   4 points/ session
o   Power-500mw
o   Point spacing is every 2-4 sq. cm.
o   Treatment schedule: daily for the first week, followed by alternate day in the second week (9
    days total).




                               Treatment Chart maintained in this work:
                               Laser Treatment/Therapy schedule-
                               Group       Days       Dose                  Duration          Observation
                               Adult                  Joule                 Minutes


                               Ø
                               Laser:      1                  32            5.33
                               L1,L2
                                           2                  32            5.33
                               (Adult)
                                           3                  32            5.33
                                           4                  32            5.33
                                           5                  32            5.33
                                           6                  32            5.33
                                           7                  24            4
                                           8                  24            4
                                           9                  24            4
                               Table: 5.1.7- Laser Treatment Protocol in adult group used in this work.




                               Laser Treatment/Therapy schedule-
                               Group              Days         Dose                Duration        Observation
                               Child                           Joule               Minutes



                               Ø
                               Laser:
                                                  1                    16          2.66
                               L1,L2
                                                  2                    16          2.66
                               (Child)
                                                  3                    16          2.66
                                                  4                    16          2.66
                                                  5                    16          2.66
                                                  6                    16          2.66
                                                  7                    12          2
                                                  8                    12          2
                                                  9                    12          2
                               Table: 5.1.8- Laser Treatment Protocol in child group used in this work.




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 5.2 Methods




          Figure: 5.2.9-A patient on therapy.

This randomized clinical trial (RCT) has been done at Shaheed Suhrawardy Medical College Hospital, Sher-E-Bangla
Nagor, Dhaka-1207, Bangladesh. Fourty (40) patients with appendicular bone fracture in the range of 15- 95 years of age
were randomly divided to the laser treatment group (Group L1 & L2) and non-laser/control group (Group C1 & C2). The
laser group went under treatment for 6 times in a week for the first week and three times/ week (alternate day) for the next
week of total 9 sessions
At first, demographic data such as age and sex and subsequently pain and functional specifications were assessed and
documented. Pain functional assessments were based on- Visual- Analogue Scale, and bone union was assessed on
clinical point of view and by radiological assessment of callus formation.
Applied laser in laser treatment group was continuous infrared laser with BioLux MD with 830 nm wavelength and 8 J/cm2
dose (energy) of total dose 8*4*9 J/cm2 for adult & 4 J/cm2 dose- (energy) of total dose 4*4*9 J/cm2 for child, and was
irradiated on the fracture- side in pointing- method, in 4 anatomical locations at 500 mW; 0.5 centimeter away from
radiological fracture line, two points in each site of line/ day. The irradiation was performed transcutaneously and the first
session was performed on the 5th day after surgery/ incidence; based on previous research work which proved that laser
works best on the proliferative stage of tissue healing.
The data‘s were routinely processed, by measuring the callus/ new bone formation. The best sets of weekly x-ray images
of each patient from each group were selected for this analysis, and data‘s are also shown in datasheets and bar graphs.
Efficacies of treatment were evaluated with pain questionnaire, clinical assessment and serial weekly radiograph starting
from 1st up to 4th week and on the 6th week.




                                                                                                                73 | P a g e
The patients were analyzed by-



             Clinical assessment
             Assessments of Clinical parameters were:
             a. Pain & inflammation level.
             b. Stability of fracture side.
             c. Movement of fracture side.
             d. Immobilization duration.
             e. Patient compliance.

             Radiological assessment

                  a. Radiographic Scoring System (by Lane and Sandhu) of fracture site, done
                     weekly.
                  b. Densitometer assessment of Callus in the radiograph of fracture site, taken
                     weekly.

        o    Radiographic Scoring System (by Lane and Sandhu) of fracture site:
             Bone formation (Periosreal)                          Score           Radiographic Scoring System by
             No evidence of bone formation                        0               Lane and Sandhu:
             Bone formation occupying 25% of defect               1               Based on Bone formation
             Bone formation occupying 50% of defect                2              Union and Remodeling.
             Bone formation occupying 75% of defect                3
             Full gap bone formation                               4
             Bone Union
             Full fracture line                                    0
             Partial fracture line                                 2
             Absent fracture line                                  4
             Bone Remodeling
             No evidence of remodeling                             0
             Remodeling of intramedullary canal                    2
             Remodeling of cortex                                  4

             Table: 5.2.10-Radiographic Scoring System (by Lane
             and Sandhu) of fracture site, taken weekly.


             o   Densitometer assessment of Callus in the
                 radiograph of fracture site, taken weekly.




                                                                                    .
            Figure: 5.2.11-Optical densitometer used in this work.


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Chapter-6
Observation & Result


The aim of this work was to asses comprehensive (Both subjective & Objective) study/ evaluation of LLLT effect (Ga-Al-As
830nm@ Pw 500) on human bone.
Objective parameters were: Clinical (Qualitative) assessment included fracture line/margins, fracture gap, external callus
appearance, callus-to-cortex ratio, bridging, and radiologic union/nonunion. Stability/ movement of the fracture site,
functional ability, Pain scale, subsiding of inflammation, compliance of patient and onwards follow-up of patient, in
comparison to controlled group. And Subjective parameters were: Quantitative assessment- done by weekly serial
radiograph, and analyzed by Radiographic Scoring System by Lane and Sandhu, callus density measurement by optical
densitometer, taken in-between treatment period & continued up to 6th week.


         C1-1. Patient Name: Halima Begum
         Age: 55 Years
         Sex: Female
         Diagnosis: Fracture Rt. Shaft of Femur
         Study group: C1

                                                     Patient Name: Ms Halima Begum
                                    Week Vs Bone Density: Non-fracture side(Cortex,Medulla) & fracture Side
                                                             ( Cortex,Medulla).

                                        Non-fracture site, Medulla        fracture site, Medulla
                                        Non-fracture site, Cortex         fracture site,Cortex



                                                                                        2.0033
                                                                        1.7767 1.8233
                                                                                  1.8133
                                                                                     1.7567
                                                                                                               1.43
                                                     1.2867
                                                        1.2267
                                           1.1667 1.1033                                                 1.14
                  0.9267   1.0167
                        1.0033                                                                       0.93331.0167
                      0.87           0.8367
                                        0.8333
                                  0.5633




                   At the end of         At the end of        At the end of       At the end of       At the end of
                   the 1st week          the 2nd week         the 3rd week        the 4th week        the 6th week

         Table: 6.1-Dose Vs density bar-graph showing change of bone density in the fracture & non-fracture (two
         centimeter away from fracture line) cortex & medulla weekly in group C1(control group, operated).


Clinically the laser group showed better stable fracture site, earlier movement of limb and removal of cast/plaster was
performed. Pain & inflammation also subsided much earlier in the laser group (L1 &L2) than the control group (C1&C2).
Radio-logically, this study compared degree of callus formation, callus density changes by weeks, and assessment of
union, pain & inflammation parameters changes, with and without laser radiation (LLLT) in the post laser therapy period,
starting from 2nd week up to 6th.




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C2-1. Patient Name: Md Hafez Kamal
Age: 42 Years
Sex: Male
Diagnosis: Fracture Rt. Colles
Study group: C2
                                    Patient Name: Mr. Hafez Kamal
                 Week Vs Density Change: non-fracture side (Cortex, Medulla) & fracture side
                                           (Cortex, Medulla).


                         Non-fracture site, Medulla        fracture site, Medulla
                         Non-fracture site, Cortex         fracture site, Cortex

                                                                     1.4933 1.52
                                                                   1.44 1.38
                                                                                               1.33
                                                    1.2    1.2                           1.2
                                                                                     1.0333 1.07
                                                0.9 0.85
                         0.74
                      0.72
            0.57
        0.5533 0.5667         0.63
              0.4267        0.5




         At the end of     At the end of       At the end of      At the end of       At the end of
         the 1st week      the 2nd week        the 3rd week       the 4th week        the 6th week

Table: 6.2- Dose Vs density bar-graph showing change of bone density in the fracture & non-fracture (two
centimeter away from fracture line) cortex & medulla weekly in group C2 (Control group, non-operated).



L1-1. Patient Name: Ms Anowara
Age: 55 Years
Sex: Female
Diagnosis: Fracture Rt. Shaft of Radius
Study group: L1

                                        Patient Name: Ms Anowara
                Week Vs Bone Density Change: Non-fracture side (Cortex, Medulla) & fracture side (
                                              Cortex, Medulla)

                          Non-fracture site, Medulla        fracture site, Medulla
                          Non-fracture site, Cortex         fracture site, Cortex


                                                1.39 1.4267
                                                  1.3067
                                                     1.2333
                              1.1733                                        1.1667 1.09671.1767
                        1.0933
                 0.9667
           0.9033
        0.8967              0.88 0.9067                               0.8367
                                                                         0.8333        0.88 0.9033
               0.78
                                                                  0.5633




         At the end of     At the end of       At the end of       At the end of       At the end of
         the 1st week      the 2nd week        the 3rd week        the 4th week        the 6th week

Table: 6.3- Dose Vs density bar-graph showing change of bone density in the fracture & non-fracture (two
centimeter away from fracture line) cortex & medulla weekly in group L1 (Study group, non-operated).




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L2-2. Patient Name: Mr Enamul Haque
Age: 55 Years.
Sex: Male
Diagnosis: Fracture Rt. Colles.
Study group: L2

                                  Patient Name: Mr Enamul Haque
              Week Vs Bone Density Change: Non- fracture side (Cortex, Medulla) & fracture
                                        side(Cortex, Medulla).

                         Non-fracture site, Medulla     fracture site, Medulla
                         Non-fracture site, Cortex      fracture site,Cortex


                                                     2.0033
                                            1.8233
                                               1.8133
                                                  1.7567                                  1.8333
                 1.6033              1.68
            1.38           1.4467                                       1.46
              1.2233    1.21331.2567                                1.1933       1.3
        1.0567                                                   0.9967           1.1033
                                                                             0.9333
                                                              0.7633




         At the end of     At the end of     At the end of     At the end of     At the end of
         the 1st week      the 2nd week      the 3rd week      the 4th week      the 6th week

Table: 6.4- Dose Vs density bar-graph showing change of bone density in the fracture & non-fracture (two
centimeter away from fracture line) cortex & medulla weekly in group L2 (Study group, operated).



L1-2. Patient Name: Md Dalil Uddin
Age: 95 Years
Sex: Male
Diagnosis: Fracture Lt. Humerus (Shaft)
Study group: L1




Figure: 6.5 a-Before starting of treatment.




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Serial weekly x-rays of post-laser therapy of a L1 group patient –




          Figure: 6.5b-At 1st week            Figure: 6.5 c-At 2nd week            Figure: 6.5 d-At 3rd week




           Figure: 6.5 e-At 4th week           Figure: 6.5 f-At 6th week            Figure: 6.5g-At 12th week

          Figure: 6.5 Stages of union/ callus formation by week (from 2nd to 12th week) of a study group
          (L1) patient.



In this study, at the end of 3rd week following laser treatment, the presence of callus/ new bone formation in fracture
defects was more advanced in laser group( Groups L1 and L2 ,Table 6.3 and 6.4, respectively) than in the control group (
C1 & C2). In the control group (C1 & C2) similar callus/new bone formation was observed at the end of 4 th week (Table 6.1
& 6.2). There was a significant difference in the degree of callus formation and in the degree of bone union between study
groups to which values were assigned to the different degrees of new bone formation. So, it can be inferred from this
study that LLLT enhances bone regeneration/ callus formation in the early stages (Proliferative and reparative stages) of
bone union which is compatible to earlier studies. Again, L2 group (Laser non-operated) showed earlier clinical &
radiological union/ callus formation than L1 (Laser operated), which also infers that bone regeneration is faster in non-
operated patient by LLLT. Within our study period, after 4th week, from 5th up-to 6th week in both study group (Laser &
control) showed almost same degree of clinical & radiological bone healing parameters. Moe-over, there was no side
effect/ negative effect of laser on the targeted bone site or on surrounding soft tissue
The Mann whitny and Kruskal-Wallis test were used for statistical analysis. In this analysis the control group had lower
values of callus density / new bone formation in 2nd-3rd week, than the infrared laser groups (p ≤ 0.01).




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Chapter-7



Discussion

The result of this study reveals a better bone healing after irradiation with 830nm diode laser (Ga-Al-As).This study result
also concludes that better bone healing after irradiation with Ga-Al-As, 830nm diode laser in human model as an
adjunctive to regular fracture management that accelerates bone union significantly and enhances patient compliances.
The results observed are similar to previous reports, which demonstrated increased vascularization activates cytokines,
growth factors, necessary hormonal activities for tissue healing enhancement in the proliferative stage, thereby reduction
of pain & inflammation, and increased fibroblast, chondrocyte and osteoblast proliferation that activities bone regeneration
Some previous reports do recognize that LLLT has positive effects on bone                    [1, 2].   These studies reflect the idea that non-
differentiated mesenchymal cells could be biomodulated positively to osteoblasts that would more rapidly change to
osteocytes [1]. This aspect may be possibly corroborated by several previous studies in which LLLT was used in fractures
[3],   bone defects [4], tooth extraction   [5, 6, 7]   and after the placement of dental implants [8]. On the other hand, LLLT seems
ineffective when used on normal tissues [9]. In order to observe the biomodulating effects of LLLT, some level of tissue
deficiency seems necessary 10].
 It is known that the osteogenic potential of mesenchymal cells depends on several genetic factors and also on systemic
and local inducer factors [11]. LLLT may act as an inducer factor. However, some reports                       [12],   suggested that LLLT would
improve bone matrix production due to improved vascularization, anti-inflammatory effect and enhanced Collagen
synthesis.
These aspects would increase both the release of mediators and micro-vascularization, which in turn would accelerate
bone healing. It was suggested that PGE2 activates wound healing                  [13],   and increased level of PGE2 was observed by
Messer et al.     [14].   There is evidence that PGE2 is also produced by osteoblast and that its effects may be therapeutic or
adverse [15].
Collagen is an important component of the extracellular matrix of bone. It is significantly increases by LLLT.The
mechanism by which LLLTinterferes in collagen synthesis is not fully understood; however, it may be because of
alterations in the genetic regulation or in the modulation of enzymatic activity involved in the metabolism of the collagen as
suggested previously (16).
Studies of bone healing response to infrared light show acceleration of osteoblast formation as well as calcium salt
deposition under the influence of infrared light. (17, 18) Studies have- demonstrated that bone growth factors are stimulated
by IR light. Osteoglycin is a small leucine rich proteoglycan (SLRP) of the extracellular matrix which was previously called
the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and
are thought to regulate cell proliferation, differentiation and adhesion in close association with collagen and many other
growth factors.
In addition to the above mentioned references and application, Bone regeneration/ healing by Laser therapy has been
proved in In-Vitro/ vivo models in hundreds of researches / applications over the past few decades both in animal & human
model. Bone healing and bone engraftment by Infrared Low Level- Laser treatment is thought to be the cumulative
coordinated effects of some specific physiological changes (in locally & systematically).
They are -Ion Exchange and Bone Mineralization (19, 20), Nitric Oxide in Bone Formation (21, 22), Lymphatic Circulation (23, 24,
and 25)   and BoneOsteoblast Proliferation Increases Bone Formation              (26, 27, 28, and 29),   Osteoblast Gene Expression      (30),   and
Osteoclast Inhibition Prevents Bone Mineral Resorption (31).



                                                                                                                                  79 | P a g e
It is acknowledged that the controversy observed in the literature are due to different protocols used in which different
wavelengths, association of wavelengths, different modes of emission and several doses were utilized in different animal
or cell models. It is recognized that each method has its advantage and disadvantage.
The finding of this study is not consistent with some other research groups, which did not show positive effects of LLLT on
healing bone [31, 32, 33, 34, 35 and37.], they did not consider the systemic effect of LLLT [23, 26]. They used the contra-lateral side
of the same subject as controls. On the other hand, the findings of this investigation is very close to a study which found
intense activity and high numbers of osteoblast 5-6 days after the procedure was performed on bone defects in a similar
model. Previous work using 790nm laser at a similar dose used in the present investigation, demonstrated a 10% increase
on the amount of mineralized bone at seven days following irradiation. Another study examined bone consolidation,
increased formation of trabecular bone, and the number of osteoblast after the use of He-Ne laser (633nm, 1mW, f
~1.1mm). The experimental period was seven days and doses per treatment were 3.15, 31.5 and 94.7J/cm2. Positive
responses were found at 31.5 and 94.7J/cm2 but not at lower doses. These values were higher than that used in this
work. This may indicate a more effective effect of 830nm laser light in comparison to lasers emitting 632.8 or 790nm, since
830nm penetrates to a deeper level.
The doses used in this study are in agreement with several previous reports that suggested that 8-10 J/cm2 induces
positive effects on both bone and soft tissues      (38, 39, and 40).   A total dose (adult) of 32 J/ session is in accordance with the
clinical parameters recommended- by Pinheiro et al. (41).
The literature shows that biomodulatory effects are dose dependent (42). It is also recognized that other factors such as the
phase of cell growth (43) and the frequency and number of sessions (44) also influence the final result of the use of LLLT.
It is concluded that the use of LLLT at 830nm significantly improves bone healing during the early stages. Further studies
are needed on the effects LLLT on growth factors, BMPs, prostaglandin and bone forming genes.




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Chapter- 8


Conclusion

Laser therapy is a standard therapeutic procedure, with unambiguous indications and
contraindications. Among the reasons for this are: positive Clinical experiences,
scientifically verified objective changes in tissue equilibrium caused by laser, and above
all, better understanding of the mechanisms of laser effects. Clinical and experimental
experience shows that laser therapy has its greatest effects on cells/tissue/organs®
affected by a generally deteriorated condition with the ph. value lower than normal.
During the last decade, it was discovered that low-power laser irradiation has
stimulatory effects on bone cell proliferation and gene expression. The purposes of this
review are to analyze the effects of low- power laser irradiation on bone cells and bone
fracture repair, to examine what has been done so far, and to explore the additional
works needed in this area. The studies reviewed show how laser therapy can be used to
enhance bone repair at cell and tissue levels. As noted by researchers, laser properties,
the combinations of wavelength and energy dose need to be carefully chosen so as to
yield bone stimulation. With better study designs, the results will be more credible,
allowing for greater recognition of advances in bone repair using laser therapy. Many
studies on the effects of laser therapy on bone healing and fracture repair have used
biochemical and histological methods, but those subjective assessments are not enough
for practical use rather objective assessment (clinical) should be preferred. However, in
final sentence, in order to establish the effects of laser treatment on bone healing/
regeneration, additional studies need to be performed using clinical, radiological,
biomechanical point of view, the ultimate evidences of bone repair.
Recommendations for Future Studies
This study has demonstrated the potential of low level laser therapy in treatment of
enhancement of human bone fracture union. A large multicentric study pointing
important -subjective (i.e. mechanical, biochemical and histological) as well as objective
(clinical) parameters in addition to- laser protocol (dose, duration, type of laser & mode
of operation),patient selection criteria and procedure of therapy is highly desirable to
make this non-invasive method of bone stimulation applicable in medical science.




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Chapter-9


References

Chapter-1
1.1 Introduction
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1.2 Background
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     13. Gresh MR. Microcurrent electrical stimulation: Putting it in perspective. Clinical Management 1987, 9(4): 51-54.
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     18. Trelles MA, Mayayo E. Bone fracture consolidates faster with low power laser.Lasers Surgical Medicine 1987, 7(1): 36-45.
     19. Yamada K. Biological effects of low power laser irradiation on clonal osteoblastic cells (MC3T3-E1).The Journal of the Japanese
     Orthopedic Association 1991, 65(9): 101-114.
     20. Gordjestani M, Dermaut L, Thierens H. Infrared laser and bone metabolism: A pilot study.International Journal of Oral and
     Maxillofacial Surgery 1994,23(1): 54-56.
     21. Tang XM, Chai BP. Effect of CO2 laser irradiation on experimental fracture healing: A transmission electron microscopic study. Lasers
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     25. David R, Nissan M, Cohen I, Soudry M. Effect of low power He-Ne laser on fracture healing in rats.Lasers in Surgery and Medicine
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     1.3 Literature review
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    influence of laser radiation. Procedings of the 2nd Thematic Symposium of Scientific Practical Papers on the Problem of Physical Self
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    2. Loc. Cit. 18 (Gatev, S. Helium-Neon laser radiation in the rehabilitation of fracture patients. Voprosy Kurortologii Fizioterapii i
    Lechebnoi Fizicheskoi Kultury 2; 28-30. (1989).)
    3. Glinkowski, W. Delayed Union Healing with Diode Laser Theapy. Case Report and Review of Literature. John Wiley & Sons, Ltd. 0898-
    5901/90/030107-03$05.00. (1990).
    4. Silva, A. MD, Physical Medicine Associates, Naperville, IL. Personal communication.
    5. Ibid.
    6. Ibid.
7. Maawan Khadraet al,. Effect of laser therapy on attachment, proliferation anddifferentiation of human osteoblast-like cells cultured on
    titanium implant material. Elseviers, Biomaterials 26 (2005) 3503–3509.
8. Chuahan A, Sarin P. Low Level Laser Therapy in Treatment of Stress Fractures Tibia: A Prospective Randomized Trial. MJAFI 2006; 62 : 27-
    29.
9. Teixeira S. et al,. Osteoblast Proliferation and Morphology Analysis on Laser Modified Hydroxyapatite Surfaces: Preliminary Results.
    Trans Techpublications, Switzerland, Key Engineering Materials vols. 309-311(2006) pp.105-108.
10. Dimitrov Sl, Dogandzhiyska V, Ishkitiev N. Irradiation with different wavelength on the proliferation activity of human pulp fibroblast
    cells, depending on irradiatin parameters and hard tissue thickness.Journal of IMAB - Annual Proceeding (Scientific Papers) 2009, book
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    Chapter-2
    1
     Eriksen et al. 1994b, Marks & Hermey 1996, An & Bell 1999. 2 Väänänen 1996. 3 Rodan et al. 1996. 4 Heaney et al. 2000. 5Pocock et al.
    1987, Selmenda 1991, 6. Martin and Burr, 1998. 7. Ross et al, 1994. 8.Junqueira and Carneiro, 1998. 9 Hazenberg, 2007. 10Ross and
    Romrell, 1989.

     Chapter-3
     1(Bjordal et al., 2006),2(Bjordal et al., 2003), 3(Gerber et al., 2001, 4(Aronoff, 1999). 5 (Lucas et al., 2002). 6(Grier, 2003), 7 (Brezinski,
     2006) ,8 (Selkin et al., 2001), 9 (Perelman, 2006). 10 (Trivedi et al., 1997), 11(van de Ven et al., 2009) 12(Wang, 2009). 13(Schmitz-
     Valckenberg et al., 2008), 14(Goldman et al., 2005), 15(Smith et al., 2005).


     Chapter-4
1.  Sutherland 2002,
2.  Karu 1999
3.  Karu 1989
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5.  H. Friedmann, R. Lubart, I. Laulicht and S. Rochkind, A possible explanation of laser-induced stimulation and damage of cell cultures, J
    Photochem Photobiol B 11 (1991) 87-91.
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7. K. Plaetzer, T. Kiesslich, B. Krammer and P. Hammerl, Characterization of the cell death modes and the associated changes in cellular
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11. Mitochondria to nuclei, Storz 2007,

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    22 W.G. Kirlin, J. Cai, S.A. Thompson, D. Diaz, T.J. Kavanagh and D.P. Jones, Glutathione redox potential in response to differentiation
    and enzyme inducers, Free Radic Biol Med 27 (1999) 1208-18.
    23 S. Alaluf, H. Muir-Howie, H.L. Hu, A. Evans and M.R. Green, Atmospheric oxygen accelerates the
    induction of a post-mitotic phenotype in human dermal fibroblasts: the key protective role of glutathione,
    Differentiation 66 (2000) 147-55.
    24 H.S. Yu, C.S. Wu, C.L. Yu, Y.H. Kao and M.H. Chiou, Helium-neon laser irradiation stimulates migrationand proliferation in melanocytes
    and induces repigmentation in segmental-type vitiligo, J Invest Dermatol120 (2003) 56-64.
    25 Y. Zhang, S. Song, C.C. Fong, C.H. Tsang, Z. Yang and M. Yang, cDNA microarray analysis of gene expression profiles in human
    fibroblast cells irradiated with red light, J Invest Dermatol 120 (2003) 849-57.
    26 H.S. Yu, K.L. Chang, C.L. Yu, J.W. Chen and G.S. Chen, Low-energy helium-neon laser irradiation stimulates interleukin-1 alpha and
    interleukin-8 release from cultured human keratinocytes, J Invest Dermatol 107 (1996) 593-6.
    27 V.K. Poon, L. Huang and A. Burd, Biostimulation of dermal fibroblast by sublethal Q-switched Nd:YAG
    532 nm laser: collagen remodeling and pigmentation, J Photochem Photobiol B 81 (2005) 1-8.
    28 N. Kipshidze, V. Nikolaychik, M.H. Keelan, L.R. Shankar, A. Khanna, R. Kornowski, M. Leon and J.
    Moses, Low-power helium: neon laser irradiation enhances production of vascular endothelial growthfactor and promotes growth of
    endothelial cells in vitro, Lasers Surg Med 28 (2001) 355-64.
    29 A. Khanna, L.R. Shankar, M.H. Keelan, R. Kornowski, M. Leon, J. Moses and N. Kipshidze,
    Augmentation of the expression of proangiogenic genes in cardiomyocytes with low dose laser irradiationin vitro, Cardiovasc Radiat
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    30 A.R. Medrado, L.S. Pugliese, S.R. Reis and Z.A. Andrade, Influence of low level laser therapy on woundhealing and its biological action
    upon myofibroblasts, Lasers Surg Med 32 (2003) 239-44.
    31 E.J. Neiburger, Rapid healing of gingival incisions by the helium-neon diode laser, J Mass Dent Soc 48
    (1999) 8-13, 40.
    32. Lubart et al. 1992; Yu et al. 1994. 33. Grossman et al. 1998, 34. Moore et al.2005, 35. Agaiby et al. 2000; Stadler et al. 2000. 36.
    Bjordal et al. 2007- 37. Kreisler et al. 2003- 38. Shefer et al. 2002- 39. Hopkins et al. 2004-
    40. (Yu et al. 1997). 41. Corazza et al. 2007. 42. Gigo-Benato et al. 2004 , 43. Fillipin et al. 2005 , 44. Morrone et al. 2000, 45. Weber et al.
    2006- 46. Shao et al. 2005 . 47. Bjordal et al. 2006a, Bjordal et al. 2006b 48. Carati et al. 2003- 49. Ad and Oron 2001; Oron etal. 2001a;
    Oron et al. 2001b; Yaakobi et al. 2001- 50. Lapchak et al. 2008-
    51. Lampl et al. 2007, 52. Oron et al. 2007- 53. Wu et al. 2009.
    4.4a- Effects of Laser on Biological Hard Tissue and Bone stimulation/ Regeneration
    1.Saito S and Shimizu N (1997): Stimulatory effects of low-power laser irradiation on bone regeneration in midpalatal suture during
    expansion in the rat. American Journal of Orthodontics & Dentofacial Orthopedics, 111:525 - 532.
    2.Pinheiro ALB, Nascimento SC, Vieira ALB, Rolim, AB, Silva, OS, Brugnera Jr, A, Zanin, FA. (2001): Effects of LLLT on malignant Cells:
    Study in Vitro. In (Rechman P, and Fried D,Haning P, ed). Lasers in Dentistry VII. Billingham: SPIE. pp 56 - 60.
    3.Hight WB (1985) In: Trelles MA, and Mayayo E (1987): Bone fracture consolidates faster with lowpower laser. Lasers in Surgery and
    Medicine, 7:36 - 45.
    4.Mester E, Mester AF, and Mester A (1985): The biomedical effects of laser application. Lasers in Surgery and Medicine, 5: 31 - 39.
    5.Valcanaia TC (1999): A influência do uso do antiinflamatório não hormonal, o diclofenaco potássico, no reparo ósseo. Tese de
    Doutorado. Porto Alegre: Pontifícia Universidade Católica do Rio Grande do Sul.99p.
    6.Trelles MA, and Mayayo E (1987): Bone fracture consolidates faster with low-power laser. Lasers in
    Surgery and Medicine, 7:36 - 45.
    7.Katchburian E, and Arana-Chavez VE (1999): Histologia e Embriologia Oral: Texto-AtlasCorrelações Clínicas. São Paulo: Panamericana.
    pp 41 -70.
    8.Kucerová H, Dostálová T, Himmlová L, Bártová J, and Mazánek J (2000): Low-level laser therapy after molar extraction. Journal of
    Clinical Laser in Medicine and Surgery, 18:309 - 315.
    9.Luger EJ, Rochkind S, Wollman Y, and Kogan G (1998): Effect of low-power laser irradiation on the mechanical properties of bone
    fracture healing in rats. Lasers in Surgery and Medicine, 22: 97 - 102.
    10.Yaakobi T, Maltz L, and Oron U (1996): Promotion of bone repair in the cortical of the tibia in rats by low energy laser (He-Ne)
    irradiation. Calcified Tissue International. 59:297 - 300.
    11.Freitas AC (1998): Avaliação do efeito antiinflamatóriodo laser Diodo Infravermelho de 830nm através damonitorização da proteína
    C-Reativa. Recife:Universidade Federal de Pernambuco. 74pp
    12. Mester E, Mester AF, and Mester A (1985): The biomedical effects of laser application. Lasers in Surgery and Medicine, 5: 31 - 39.
     13. Takeda Y (1988): Irradiation effect of Low-energy laser on alveolar bone after tooth extraction:
    Experimental study in rats. International Journal of Oral Maxillofacial Surgery, 7:388 - 391.
    14. Oliveira MAM (1999): Efeito da Radiação Laser Não Cirúrgica na Bioestimulação Óssea Pós-Implante: Ánálise com Microscopia
    Eletrônica de Varredura. Monografia de Especialização; Recife:Faculdade de Odontologia da Universidade Federal de Pernambuco. 88p
    15. Sommer AP, Pinheiro ALB, Mester A, Franke RP, and Whelan, HT (2001): Biostimulatory windows in low intensity laser activation:
    Lasers, Scanners and NASA's Light Emitting Diode Array System. Journal Clinical Laser Medicine and Surgery,19:29 - 34.
    16. Gordjestani M, Dermaut, M, and Thierens, H. (1994): Infrared laser and bone metabolism: A pilot study. International Journal of Oral
    and Maxillofacial Surgery, 23:54 - 56.

                                                                                                                                   84 | P a g e
4.4. b- Bone Regeneration by Infra Red Laser Therapy:
1. Bone fracture consolidates faster with low-power laser
Dr. M. A. Trelles, M.D. 1 *, E. Mayayo, M.D. 2
Instituto Médico Vilafortuny, Cambrils/Tarragona, and Department of Pathological Anatomy of the John XXIII Hospital in
Tarragona2Department of Histology of the Medical School in Reus/University of Barcelona, Barcelona, Spain
2. Stein A, Benayahu D, Maltz L, Oron U.Low-level laser irradiation promotes proliferation and differentiation of human
osteoblasts in vitro. Photomed Laser Surg. 2005 Apr; 23(2):161-6.
3. E. Fukuhara, T. Goto, T. Matayoshi, S. Kobayashi, And T. Takahashi Stimulatory Effects of Low energy Laser Irradiation
on the Initial Proliferation of Rat Calvarial Osteoblasts . Kyushu Dental College, Kitakyushu, Japan.
4. Guzzardella, G A et al (2002). Laser stimulation on bone defect healing: An in vitro study. Lasers Med Sci. 17(3): 216-
220.
5. Outná M., Janisch R., Veselská R. Effects of Low-Power Laser Irradiation on CellProliferation. Scripta Medica (Brno) – 76
(3): 163–172, June 2003
6. ibid
7. E. Fukuhara, T. Goto, T. Matayoshi, S. Kobayashi, And T. Takahashi Stimulatory Effects ofLow-energy Laser Irradiation
on the Initial Proliferation of Rat Calvarial Osteoblasts. KyushuDental College, Kitakyushu, Japan.
8. Hamajima S, Hiratsuka K, Kiyama-Kishikawa M, Tagawa T, Kawahara M, Ohta M, Sasahara H, Abiko Y. Effect of Low-
Level Laser Irradiation on Osteoglycin Gene Expression in Osteoblasts. Nihon University School of Dentistry at Matsudo,
Chiba, Japan. Lasers Med Sci. 2003; 18(2):78-82.
I MacIntyre, M Zaidi, A S Alam, H K Datta, B S Moonga, P S Lidbury, M Hecker, and J R Vane.Osteoclastic inhibition: an
action of nitric oxide not mediated by cyclic GMP. Proc Natl Acad Sci U S A. 1991 April 1; 88(7): 2936–2940.
10. Koutná M., Janisch R., Veselská R. Effects of Low-Power Laser Irradiation on Cell Proliferation.Scripta Medica (BRNO) –
76 (3): 163–172, June 2003
11. Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects. Philip Gable, B App Sc P.T. G Dip Sc Res
(LLLT) MSc, Australia, Jan Tunér, D.D.S., Sweden
12. Jose´ Aguirre, Lee Buttery, et.al. Endothelial Nitric Oxide Synthase Gene-Deficient Mice Demonstrate Marked
Retardation in Postnatal Bone Formation, Reduced Bone Volume, and Defects in Osteoblast Maturation and Activity.
American Journal of Pathology, Vol. 158, No. 1, January 2001.
13. Brandi, M.L. et.al. Bidirectional regulation of osteoclast functions by nitric oxide synthase isoforms. Proceedings of the
National Academy of Science, Vol. 92, pp. 2954-2958, March 1995.
14. Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects. Philip Gable, B App Sc P.T. G Dip Sc Res
(LLLT) MSc, Australia, Jan Tunér, D.D.S., Sweden.
15. Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects. Philip Gable, B App Sc P.T. G Dip Sc Res
(LLLT) MSc, Australia, Jan Tunér, D.D.S., Sweden.
16. Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects. Philip Gable, B App Sc P.T. G Dip Sc Res
(LLLT) MSc, Australia, Jan Tunér, D.D.S., Sweden.



Chapter-7
1 Pinheiro ALP, and Frame JF (1992): Laser em Odontologia: Seu Uso Atual e Perspectivas Futuras.
Revista Gaucha de Odontologia. 40: 327 - 332.
2 Saito S, and Shimizu N (1997): Stimulatory effects of low-power laser irradiation on bone regeneration in midpalatal suture during
expansion in the rat. American Journal of Orthodontics & Dentofacial Orthopedics, 111:525 - 532.
3 Luger EJ, Rochkind S, Wollman Y, and Kogan G (1998): Effect of low-power laser irradiation on the mechanical properties of bone
fracture healing in rats.Lasers in Surgery and Medicine, 22: 97 - 102.
4 Yaakobi T, Maltz L, and Oron U (1996): Promotion of bone repair in the cortical of the tibia in rats by low energy laser (He-Ne)
irradiation. Calcified Tissue International. 59:297 - 300.
5 Freitas AC (1998): Avaliação do efeito antiinflamatório do laser Diodo Infravermelho de 830nm através da monitorização da proteína
C-Reativa. Recife: Universidade Federal de Pernambuco. 74pp.
6 Kucerová H, Dostálová T, Himmlová L, Bártová J, and Mazánek J (2000): Low-level laser therapy after molar extraction. Journal of
Clinical Laser in Medicine and Surgery, 18:309 - 315.
7 Takeda Y (1988): Irradiation effect of Low-energy laser on alveolar bone after tooth extraction: Experimental study in rats.
International Journal of Oral Maxillofacial Surgery, 7:388 - 391.
8 Oliveira MAM (1999): Efeito da Radiação Laser Não Cirúrgica na Bioestimulação Óssea Pós-Implante: Ánálise com Microscopia
Eletrônica de Varredura. Monografia de Especialização; Recife:Faculdade de Odontologia da Universidade Federal de Pernambuco. 88p
9 Saito S, and Shimizu N (1997): Stimulatory effects of low-power laser irradiation on bone regeneration in midpalatal suture during
expansion in the rat. American Journal of Orthodontics & Dentofacial Orthopedics, 111:525 - 532.
10 Pinheiro ALB, Nascimento SC, Vieira ALB, Rolim, AB, Silva, OS, Brugnera Jr, A, Zanin, FA. (2001): Effects of LLLT on malignant Cells:
Study in Vitro. In (Rechman P, and Fried D,Haning P, ed). Lasers in Dentistry VII. Billingham: SPIE. pp 56 - 60.
11 Katchburian E, and Arana-Chavez VE (1999): Histologia e Embriologia Oral: Texto-Atlas Correlações Clínicas. São Paulo: Panamericana.
pp 41 - 70.
12 Trelles MA, and Mayayo E (1987): Bone fracture consolidates faster with low-power laser. Lasers in Surgery and Medicine, 7:36 - 45.
13 Hight WB (1985) In: Trelles MA, and Mayayo E (1987): Bone fracture consolidates faster with lowpower laser. Lasers in Surgery and
Medicine, 7:36 - 45.


14 Mester E, Mester AF, and Mester A (1985): The biomedical effects of laser application. Lasers in Surgery and Medicine, 5: 31 - 39.
15 Valcanaia TC (1999): A influência do uso do antiinflamatório não hormonal, o diclofenaco potássico, no reparo ósseo. Tese de
Doutorado. Porto Alegre: Pontifícia Universidade Católica do Rio Grande do Sul. 99p.
16 Reddy GK, Stehno-Bittel L, Enwemeka CS. Laser photostimulation of collagen production in healing rabbit achilles tendons. Lasers
Surg Med 1998;22:281-287.


                                                                                                                           85 | P a g e
17 Stein A, Benayahu D, Maltz L, Oron U.Low-level laser irradiation promotes proliferation and differentiation of human osteoblasts in
vitro. Photomed Laser Surg. 2005 Apr; 23(2):161-6.
 18 Fukuhara, T. Goto, T. Matayoshi, S. Kobayashi, And T. Takahashi Stimulatory Effects of Low-energy Laser
Irradiation on the Initial Proliferation of Rat Calvarial Osteoblasts. Kyushu Dental College, Kitakyushu, Japan.
19 Koutná M., Janisch R., Veselská R. Effects of Low-Power Laser Irradiation on Cell Proliferation. Scripta Medica (BRNO) – 76 (3): 163–
172, June 2003
 20 Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects. Philip Gable, B App Sc P.T. G Dip Sc Res (LLLT) MSc,
Australia, Jan Tunér, D.D.S., Sweden
21 Jose´ Aguirre, Lee Buttery, et.al. Endothelial Nitric Oxide Synthase Gene-Deficient Mice Demonstrate Marked Retardation in Postnatal
Bone Formation, Reduced Bone Volume, and Defects in Osteoblast Maturation and Activity. American Journal of Pathology, Vol. 158,
No. 1, January 2001.
22 Brandi, M.L. et.al. Bidirectional regulation of osteoclast function by nitric oxide synthase isoforms. Proceedings of the National
Academy of Science, Vol. 92, pp. 2954-2958, March 1995.
23 Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects. Philip Gable, B App Sc P.T. G Dip Sc Res (LLLT) MSc,
Australia, Jan Tunér, D.D.S., Sweden.
24 Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects. Philip Gable, B App Sc P.T. G Dip Sc Res (LLLT) MSc,
Australia, Jan Tunér, D.D.S., Sweden.
25 Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects. Philip Gable, B App Sc P.T. G Dip Sc Res (LLLT) MSc,
Australia, Jan Tunér, D.D.S., Sweden.
26 Guzzardella, G A et al (2002). Laser stimulation on bone defect healing: An in vitro study. Lasers Med Sci. 17(3): 216-220.
27 Koutná M., Janisch R., Veselská R. Effects Of Low-Power Laser Irradiation On Cell Proliferation. Scripta Medica (Brno) – 76 (3): 163–
172, June 2003
28 ibid
29 E. Fukuhara, T. Goto, T. Matayoshi, S. Kobayashi, And T. Takahashi Stimulatory Effects of Low-energy Laser Irradiation on the Initial
Proliferation of Rat Calvarial Osteoblasts . Kyushu Dental College, Kitakyushu, Japan.
 30 Hamajima S, Hiratsuka K, Kiyama-Kishikawa M, Tagawa T, Kawahara M, Ohta M, Sasahara H,Abiko Y. Effect Of Low-Level Laser
Irradiation On Osteoglycin Gene Expression In Osteoblasts. Nihon University School of Dentistry at Matsudo, Chiba, Japan. Lasers Med
Sci. 2003;18(2):78- 82.
31 I MacIntyre, M Zaidi, A S Alam, H K Datta, B S Moonga, P S Lidbury, M Hecker, and J R Vane.
Osteoclastic inhibition: an action of nitric oxide not mediated by cyclic GMP. Proc Natl Acad Sci U
S A. 1991 April 1; 88(7): 2936–2940.
32 Anneroth G, Hall G, Ryden H, and Zetterquist Lb (1988): The effect of low-energy infrared laser radiation on wound healing in rats.
Journal of Oral and Maxillofacial Surgery, 26:12 - 17.
33 Bisht D, Mehrotra, R, Singh, PA, Atri, SC, Kumar, A. (1994): Effect of low laser radiation on healing of open skin wounds in rats. Indian
Journal MedicalResearch, 100:43 - 46.
34 Freitas AC (1998): Avaliação do efeito antiinflamatório do laser Diodo Infravermelho de 830nm através da monitorização da proteína
C-Reativa. Recife: Universidade Federal de Pernambuco. 74pp.
35 Gordjestani M, Dermaut, M, and Thierens, H. (1994): Infrared laser and bone metabolism: A pilot study. International Journal of Oral
and Maxillofacial Surgery, 23:54 - 56.
36 Hall G, Anneroth, G, Schennings, T, Zetterquist L, Ryden H (1994): Effect of low energy laser irradiation on wound healing. An
experimental study in rats. Swedish Dental Journal, 18: 29 - 34.
37 Gordjestani M, Dermaut, M, and Thierens, H. (1994): Infrared laser and bone metabolism: A pilot study. International Journal of Oral
and Maxillofacial Surgery, 23:54 - 56.
38 Pinheiro ALB, Oliveira MAM, Martins PPM. Biomodulação da cicatrização óssea pós implantar com o uso da laserterapia
nãocirúrgica: Estudo por microscopia eletrônica de varredura. Rev FOUFBA 2001;22:12-19.
39 Pinheiro ALB. Low-level laser therapy in management of disorders of the maxillofacial region. J Clin Laser Med Surg 1997;15:181-183.
40. Reddy GK, Stehno-Bittel L, Enwemeka CS. Laser photostimulation of collagen production in healing rabbit achilles tendons. Lasers
Surg Med 1998;22:281-287.
 41 Pinheiro ALB. Low-level laser therapy in management of disorders of the maxillofacial region. J Clin Laser Med Surg 1997;15:181-183.
 42 Kipshidze N, Nikolaychik V, Keelan MH, Shankar LR, Khanna A, Komowsky R, Leon M, Moses J. Low-power helium:neon laser
irradiation enhances production of vascular endothelial growth factor and promotes growth of endothelial cells in vitro. Lasers Surg Med
2001;28:355-364.
43 Osawa Y, Shimizu N, Kariya G, Abiko Y. Low-power laser irradiation stimulates bone nodule formation at early stages of cell culture in
rat calvarial cells. Bone 1998;22:347-354.
44 Silva Júnior AN, Pinheiro ALB, Oliveira MG, Weismann R, Ramalho LM, Nicolau RA. Computerized morphometric assessment of the
effect of low-level laser therapy on bone repair: an experimental animal study, J Clin Laser Med Surg 2002; 20:83- 87.




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Chapter-10
Appendices

10.1 Figure & Table list-


Chapter-1
Figure1.3.1: Before & after laser therapy.
Chapter-2
Figure: 2. 1.1- Eukaryotic cells
Figure: 2 1.2-Cell membrane
Figure: 2.1.3- Simplified structure of mitochondrion
Figure: 2 1.4-Main pathways of cellular and mitochondrial energy metabolism.
Figure: 2. 1.5: Mitochondrial respiratory chain.
Table: 2.2.6-Schemic diagram of bone structure
Figure: 2.2 .7- A femur head with a cortex of compact bone and medulla of trabecular bone.
Figure 2.2.8a Figure2.28b- Lamellar Bone & Woven Bone
Table: 2.2.9 – Schematic diagram of bone
Figure: 2.210-Picture showing Bone composition & extracellular matrix
Figure: 2.2.11- Parts of a long bone
Figure: 2. 2.12- Appendicular upper & lower extremity bones
Figure: 2.2.13 a-Schematic histological structure of bone
Figure: 2.3.14-Mechanism of bone fracture
Figure: 2.3.15-Types of fracture
Figure: 2.4.16-Schematic diagram of fracture healing
Table: 2.4.17- schematic diagram of Phases of fracture healing
Figure: 2.4.18-Schematic diagram of Bone Remodeling
Figure: 2.4.19- Schematic diagram of bone healing factors.

Chapter-3
Figure: 3.2.1-Components of laser
Figure 3.2.2: Mechanism of the interaction between an atom and a photon (The photon has an energy hν equal to the
difference between the two atomic energy levels).
Figure 3.2.3: Laser transition with the lower level far above the ground state. The population at thermodynamic
equilibrium is defined by Boltzmann's Law
 Figure 3.2.4: Example of a three-level system with optical pumping
 Figure3.2 5: Example of a four-level system with optical pumping.
 Figure 3.2.6: A Helium-Neon System.
 Figure: 3.3.7: Diagram of Components of laser System
 Figure: 3.4.8: schematic diagram of interior of a laser machine.
 Figure: 3.4.9: schematic diagram of the control panel of a laser machine
Table: 3.5.10: Various Laser Parameters v Dosage/Time: Illustrates the difference in Joules
 and Joules/cm2 dosages for differing output parameters.


Chapter-4
Figure-4.2.1-Schematic diagram of laser-tissue interaction
FIGURE: 4.2.2: Absorption spectrum of typical breast tissue
FIGURE: 4.2.3:Absorption spectrum of pure water
FIGURE 4.2.4: Absorption spectra of oxy- and deoxy-haemoglobin, both at a concentration of 150g/liter of blood.
FIGURE: 4.2.5: Attenuation spectrum of melanosome in skin.
FIGURE: 4.2.6: Reduced scattering coefficient of typical tissue as calculated by linear extrapolation.
FIGURE: 4.2.7: shows the light reflection (R) and absorption (A) of light
Figure-4.3.8:Schematic diagram of a cell & site of laser action
Figure- 4.3.9: Schematic diagram showing absorption of Laser by cellular membrane & mitochondria.
FIGURE: 4.3.10a- Schematic diagram showing the absorption of red and NIR light by specific Cellular chromophores
photoacceptors localized in the mitochondrial respiratory chain, FIGURE4.3.10b- Structure and mode of action of
cytochrome c oxidase
FIGURE 4.3.11: Structure of the mitochondrial respiratory chain.
FIGURE: 4.3.12: Schematic diagram of the mitochondrial electron transport chain
FIGURE: 4.3.13: 5.Reactive oxygen species (ROS) formed as a result of LLLT effects in mitochondria
may activate the redox-sensitive transcription factor NF-B (relA-p50) via protein kinaseD (PKD).
FIGURE: 4.3.14: When NO is released from its binding to heme iron and copper centers in Cytochrome c oxidase by the
action of light, oxygen is allowed to rebind to these sites and respiration is restored to its former level leading to
increased ATP synthesis.

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Figure: 4.4.15a: 4.4.15: schematic diagram of laser induced cell signaling pathway/ 4.4.15b: / LLLT induced cellular
changes.
Figur:4.4.16 LLLT mechanism and application. Incoming red and NIR photons are absorbed
 in cell Mitochondria, producing reactive oxygen species (ROS) and releasing nitric oxide
(NO), whic to gene transcription via activation of transcription factors (NF-κB And AP1).
Figure-4.4.17: Ablation and melting are the two basic modalities by which the effect of lasers on the hard tissues.
Figure: 4.4.18: Ablation curves of fresh and dried bone obtained with a CO2 laser (pulse duration: 250 μs, wavelength: 10.6
μm). Due to its higher water content, freshbone is ablated more efficiently.Data according to Forrer et al. (1993).
Figure:4.4.19: Ablation curve of bone obtained with an Er:YAG laser (pulse duration: 180 μs,
wavelength:2.94 μm). Data according to Scholz and Grothves-Spork (1992).
Figure: 4.5.20: Schematic diagram of pain, inflammation & tissue healing by LLLT.
Figure: 4.5.21: Schematic diagram of diagnostic application of Laser.

Chapter-5
Figure: 5.1.1-BioLux MD Ga-Al-As Laser (830 nm) Machine with Probe & probe tip.
Table: 5.1.2- Patient distribution according to study design.
Table: 5.1.3-Bar-graph showing-patient distribution according to age in study (laser) group.
Table: 5.1.4-Bar-graph showing-patient distribution according to age in non-study (Control) group.
Table: 5.15- Bar-graph showing patient distribution according to type of bone in study (laser) group.
Table: 5.1.6- Bar-graph showing patient distribution according to type of bone in control group.
Table: 5.1.7- Laser Treatment Protocol in adult group used in this work.
Table: 5.1.8- Laser Treatment Protocol in child group used in this work.
Figure: 5.2.9-A patient on therapy
Table: 5.2.10- Radiographic Scoring System (by Lane and Sandhu) of fracture site, taken weekly.
Figure: 5.2.11- Optical densitometer used in this work.


Chapter-6
Table: 6.1- Table: 6.1-Dose Vs density bar-graph showing change of bone density in the fracture & non-fracture (two
centimeter away from fracture line) cortex & medulla weekly in group C1 (control group, operated).
Table: 6.2- Dose Vs density bar-graph showing change of bone density in the fracture & non-fracture (two centimeter
away from fracture line) cortex & medulla weekly in group C2 (Control group, non-operated)
Table: 6.3- Dose Vs density bar-graph showing change of bone density in the fracture & non-fracture (two centimeter
away from fracture line) cortex & medulla weekly in group L1 (Study group, non-operated)
Table: 6.4- Dose Vs density bar-graph showing change of bone density in the fracture & non-fracture (two centimeter
away from fracture line) cortex & medulla weekly in group L2 (Study group, operated).
Figure: 6.5 Stages of union/ callus formation by week of a study group (L1) patient.




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10.2 Laser Books & Articles

Some laser Books which enriched this research-

     o   Hand Book Of Lasers, writer: Marvin J. Weber
     o   Biomedical Photonics Handbook: by Tiina I. Karu
     o   Institute of Laser and Information Technologies Russian Academy of Sciences Troitsk,
     o   Moscow Region, Russian Federation.
     o   Laser Tissue interaction by Markolf H. Niemz.
     o   Fundamentals of Laser Dynamics by Ya I Khanin
     o   Frequency Standards by Basics and Applications by Fritz Riehle
     o   Fundamentals of Light Sources And Lasers by Mark Csele
     o   Laser Medicine and Surgery by Gregory T Absten
     o   Introduction to laser-tissue interactions by Ben cox
     o   Welding of Skin using Nd:YAG Laser with Bipolar Contact Applicators
     o   by Lynette M Brodie


Some important internationally published articles on Laser & Laser bio-stimulation

1. Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects- Philip Gable, B App Sc P.T. G
Dip Sc Res (LLLT) MSc, Australia, Jan Tunér, D.D.S., Sweden
2. Mechanisms of Low Level Light Therapy. Michael R Hamblin a,b,c,* and Tatiana N Demidova a,d
3. Laser photons and Pharmacological treatments In wound healing
farouk A.H. Al-Watban, MSc, PhD, and Bernard L. Andres, MT(AMT) Laser Medicine Research Section,
Biological and Medical Research Department, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi
Arabia.
4. Advances in laser therapy for bone repair- A. Barber 1, JE. Luger 1, A. Karpf 1 , Kh. Salame 2 B. Shlomi 3, G.
Kogan 3, M. Nissan 4, M. Alon 5, and S. Rochkind 2,6., 1Foot & Ankle Unit, Departments of Orthopedic
Surgery "B", Departments of 2Neurosurgery, 3Oral and Maxillofacial Surgery, and 5Rehabilitation, 6Division
of Peripheral Nerve Reconstruction, Tel Aviv Sourasky Medical Center, Tel Aviv University; 4Ben Gurion
University, Israel.
5. Biophotonics and bone biology- Gregory Zimmerli and David Fischer NASA Glenn Research Center,
Cleveland, OH, Marius Asipauskas, Chirag Chauhan, Nicole Compitello, and Jamie Burke National Center for
Microgravity Research, Cleveland, OH Melissa Knothe Tate Cleveland Clinic Foundation, Lerner Research
Institute, Cleveland, OH
6. Basic photomedicine-          Ying-Ying Huang, Pawel Mroz, and Michael R. Hamblin Department of
Dermatology, Harvard Medical School, BAR 414, Wellman Center for Photomedicine, Massachusetts General
Hospital
7. Diode laser treatment for osteal and osteoarticular panaritium- Valery A. Privalov, Ivan V. Krochek,
Alexander V. Lappa*, Andrew N. Poltavsky, Andrew A. Antonov Medical Physics Center at Chelyabinsk State
University and Chelyabinsk State Medical Academy Br. Kashirinykh 129, Chelyabinsk, 454021, Russia
8. Laser Biostimulation of Healing Wounds: Specific Effects and - Mechanisms of Action
Chukuka s. Enwemeka, phd*
9.Mechanisms of Low Level Light Therapy.Michael R Hamblin a,b,c,* and Tatiana N Demidova a, d.
10. Biphasic dose response in low level light therapy
Ying-Ying Huang _ Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA;
Department of Dermatology, Harvard Medical School, Boston, MA; Aesthetic and Plastic Center of Guangxi
Medical University, Nanning, P.R. China,
11. Co2 laser interaction with biological tissue, J. E. Juliá1, V. Aboites2, M. A. Casillas2
12. Advances in laser therapy for bone repair- A. Barber 1, JE. Luger 1, A. Karpf 1 , Kh. Salame 2 , B. Shlomi 3,
G. Kogan 3, M. Nissan 4, M. Alon 5, and S. Rochkind 2,6
13. Effects of low-power laser irradiation on Cell proliferation- KOUTNA M., JANISCH R., VESELSKA R.
Department of Biology, Faculty of Medicine, Masaryk University, Brno
14. The biological effects of laser therapy and other physical modalities on connective Tissue repair
processes- Chukuka S. Enwemeka, P.T., Ph.D., FACSM, G. Kesava Reddy, Ph.D., Department of Physical
Therapy and Rehabilitation Sciences, University of Kansas Medical Center, Kansas City, KS 66160-7601, USA
15. Laser Physics and Laser-Tissue interaction. J. Welch, PhD Jorge H. Torres, MS Wai-Fung Cheong, MS, MD
16. Lasers and their therapeutic applications in chiropractic- Don Fitz-Ritson, DC, FCCRS(C), DACRB*

                                                                                                   89 | P a g e
10.3 Datasheet


      C1-1.. Patient Name: Halima Begum
      Age: 55 Years
      Sex: Female
      Diagnosis: Fracture Rt. Shaft of Femur
      Study group: C1
                      Non-Fracture Side,     Fracture Side, Cortex    Non-Fracture Side,      Fracture Side,
      Week.           Cortex                                          Medulla                 Medulla

                      Reading      Mean      Reading      Mean        Reading        Mean     Reading          Mean

      At the end of   0.98                   1.02                     0.92                    0.88
        1st week.     0.96         1.0033    1.04         1.0167      0.97           0.9267   0.87             0.87
                      1.07                   0.99                     0.89                    0.86
      At the end of   0.90                   1.13                     0.71                    0.82
        2nd week.     0.78         0.8333    1.19         1.1667      0.57           0.5633   0.85             0.8367
                      0.82                   1.18                     0.41                    0.84
      At the end of   1.09                   2.19                     1.08                    1.36
        3rd week.     1.39         1.2267    1.28         1.7767      1.02           1.1033   1.22             1.2867
                      1.26                   1.86                     1.21                    1.28
      At the end of   1.56                   2.07                     1.98                    1.80
       4th week.      1.80         1.7567    2.01         2.0033      1.83           1.8233   1.83             1.8133
                      1.91                   1.93                     1.66                    1.81
      At the end of   0.98                   1.48                     0.99                    1.21
       6th week.      1.14         1.0167    1.53         1.43        0.93           0.9333   0.97             1.14
                      0.93                   1.28                     0.88                    1.24




      C1-2. Patient Name: Mr. Shohel
      Age: 26 Years
      Sex: Male
      Diagnosis: Fracture Lt Tibia (Shaft)
      Study group: C1
                       Non-Fracture Side,     Fracture Side, Cortex    Non-Fracture Side,     Fracture Side,
      Week.            Cortex                                          Medulla                Medulla

                       Reading      Mean      Reading       Mean       Reading       Mean     Reading          Mean
      At the end of
                       0.78                   0.64                     0.53                   0.57
      1st week.
                       0.71         0.73      0.64          0.6267     0.53          0.54     0.55             0.56
                       0.70                   0.6                      0.56                   0.56
      At the end of    1.26                   1.14                     0.78                   0.99
      2nd week.        1.10         1.16      1.05          1.0367     0.67          0.7167   1.20             1.1633
                       1.12                   0.92                     0.70                   1.30
      At the end of    1.33                   1.40                     0.99                   1.20
      3rd week.                     1.33                    1.3733                   1.0367                    1.2467
                       1.30                   1.42                     1.08                   1.28
                       1.36                   1.30                     1.04                   1.26
      At the end of    1.36                   1.70                     1.24                   1.37
      4th week.                     1.38                    1.6067                   1.17                      1.3633
                       1.40                   1.54                     1.12                   1.33
                       1.38                   1.58                     1.15                   1.39
      At the end of    1.09                   1.00                     0.88                   1.12
      6th week.        1.07         1.1033    1.04          0.9967     0.92          0.90     1.06             1.1067
                       1.15                   0.95                     0.90                   1.14




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C2-1. Patient Name: Md Hafez Kamal
Age: 42 Years
Sex: Male
Diagnosis: Fracture Rt. Colles
Study group: C2
                Non-Fracture Side,    Fracture Side, Cortex   Non-Fracture Side,    Fracture Side,
Week.           Cortex                                        Medulla               Medulla

                Reading      Mean     Reading      Mean       Reading      Mean     Reading          Mean

At the end of   0.55                  0.62                    0.47                  0.55
1st week.       0.58         0.5533   0.51         0.57       0.41         0.4267   0.62             0.5667
                0.53                  0.58                    0.40                  0.53
At the end of   0.70         0.72     0.74         0.74       0.50         0.50     0.64
2nd week.       0.74                  0.68                    0.48                  0.60             0.63
                0.72                  0.80                    0.52                  0.65
At the end of   0.88         0.90     1.22         1.20       0.85         0.85     1.18             1.20
3rd week.       0.92                  1.20                    0.86                  1.20
                0.90                  1.18                    0.84                  1.22
At the end of   1.46                  1.45                    1.37                  1.49
4th week.       1.47         1.44     1.56         1.4933     1.38         1.38     1.53             1.52
                1.39                  1.47                    1.39                  1.54
At the end of   0.99         1.0333   1.22         1.20       1.09         1.07     1.34             1.33
6th week.       1.09                  1.15                    1.12                  1.36
                1.02                  1.23                    1.0                   1.29




C2-2. Patient Name: Md Sayed Koraishi
Age: 32 Years
Sex: Male
Diagnosis: Fracture Rt. Ulna
Study group: C1
                Non-Fracture Side,    Fracture Side, Cortex   Non-Fracture Side,    Fracture Side,
Week.           Cortex                                        Medulla               Medulla

                Reading      Mean     Reading      Mean       Reading      Mean     Reading          Mean

At the end of   0.44         0.4567   0.66         0.6467     0.42         0.42     0.50             0.4767
1st week.       0.45                  0.63                    0.41                  0.45
                0.48                  0.65                    0.43                  0.48
At the end of   0.50         0.4833   0.65         0.66       0.48         0.4567   0.52             0.4733
2nd week.       0.46                  0.69                    0.44                  0.44
                0.49                  0.64                    0.45                  0.46
At the end of   0.56         0.55     0.70         0.68       0.57         0.55     0.47             0.48
3rd week.       0.55                  0.66                    0.53                  0.49
                0.54                  0.68                    0.55                  0.48
At the end of   0.63         0.62     0.71         0.6967     0.54         0.5733   0.47             0.4833
4th week.       0.61                  0.70                    0.60                  0.46
                0.62                  0.68                    0.58                  0.52
At the end of   0.52         0.5467   0.56         0.57       0.47         0.45     0.46             0.46
6th week.       0.54                  0.57                    0.45                  0.48
                0.58                  0.58                    0.43                  0.44




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L1-1. Patient Name: Ms Anowara
Age: 55 Years
Sex: Female
Diagnosis: Fracture Rt. Shaft of Radius
Study group: L1
                Non-Fracture Side,     Fracture Side, Cortex     Non-Fracture Side,     Fracture Side,
Week.           Cortex                                           Medulla                Medulla

                Reading      Mean      Reading       Mean        Reading      Mean      Reading          Mean

At the end of   0.89         0.78      0.98          0.9667      0.83         0.8967    0.90             0.9033
  1st week.     0.73                   0.95                      0.96                   0.93
                0.72                   0.97                      0.90                   0.88
At the end of   1.17         1.1733    0.94          0.9067      1.09         1.0933    0.90             0.88
  2nd week.     1.18                   0.90                      1.12                   0.86
                1.17                   0.88                      1.07                   0.88
At the end of   1.30         1.2333    1.41          1.4267      1.42         1.39      1.34             1.3067
  3rd week.     1.24                   1.47                      1.34                   1.33
                1.16                   1.40                      1.41                   1.25
At the end of   0.90         0.8333    1.13          1.1667      0.71         0.5633    0.82             0.8367
 4th week.      0.78                   1.19                      0.57                   0.85
                0.82                   1.18                      0.41                   0.84
At the end of   1.23         1.1767    0.89          0.9033      1.09         1.0967    0.80             0.88
 6th week.      1.10                   0.90                      1.13                   0.93
                1.20                   0.92                      1.07                   0.91




L1-2. Patient Name: Md Dalil Uddin
Age: 95 Years
Sex: Male
Diagnosis: Fracture Lt. Humerus (Shaft)
Study group: L1
                Non-Fracture Side,    Fracture Side, Cortex    Non-Fracture Side,      Fracture Side, Medulla
Week.           Cortex                                         Medulla

                Reading      Mean     Reading      Mean        Reading      Mean       Reading           Mean

At the end of   0.89         0.8767   1.19         1.2100      0.98         0.9667     1.12              1.0767
1st week.       0.97                  1.22                     0.98                    1.10
                0.77                  1.22                     0.94                    1.01
At the end of   1.00         1.1167   0.88         0.9433      1.05         1.0033     0.76              0.7500
2nd week.       1.15                  1.13                     0.98                    0.83
                1.20                  0.82                     0.98                    0.66
At the end of   1.49         1.4400   1.33         1.4067      1.39         1.2667     0.97              0.9933
3rd week.       1.40                  1.39                     1.33                    0.97
                1.43                  1.50                     1.08                    1.04
At the end of   0.84         0.7833   1.09         0.9633      0.83         0.7267     0.64              0.6333
4th week.       0.71                  0.90                     0.71                    0.65
                0.80                  0.90                     0.64                    0.61
At the end of   0.83         0.8533   1.23         1.0300      0.77         0.6267     0.71              0.6733
6th week.       0.56                  0.88                     0.56                    0.69
                1.17                  0.98                     0.55                    0.62




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L1-3. Patient Name: Mr. Sagir Ahmed
Age: 24 Years
Sex: Male
Diagnosis: Fracture Lt. Humerus
Study group: L1
                Non-Fracture Side,     Fracture Side, Cortex   Non-Fracture Side,       Fracture Side, Medulla
Week.           Cortex                                         Medulla

                Reading       Mean     Reading      Mean       Reading       Mean       Reading       Mean
At the end of
                0.87                   0.89                    0.48                     0.59
  1st week.
                0.96          0.79     0.84         0.8567     0.46          0.51       0.56          0.5767
                0.81                   0.84                    0.59                     0.58
At the end of   0.91                   1.01                    0.64                     0.65
  2nd week.     0.96          1.00     0.94         0.95       0.49          0.55       0.70          0.69
                1.13                   0.89                    0.52                     0.72
At the end of   1.80                   1.77                    1.35                     1.55
  3rd week.     1.75          1.7233   1.91         1.7867     1.23          1.3167     1.34          1.4733
                1.62                   1.68                    1.37                     1.53
At the end of   1.64                   1.89                    1.26                     1.39
 4th week.      1.59          1.64     1.65         1.70       1.45          1.34       1.47          1.45
                1.69                   1.56                    1.31                     1.49
At the end of   0.99                   1.08                    1.12                     1.03
 6th week.      0.89          0.9433   0.98         1.0667     1.03          1.07       1.18          1.05
                0.95                   1.14                    1.06                     0.94




L1-4. Patient Name: Md. Aktaruzzaman
Age: 26 Years
Sex: Male
Diagnosis: Fracture Lt. Humerus (Shaft)
Study group: L2
                Non-Fracture Side,     Fracture Side, Cortex    Non-Fracture Side,         Fracture Side,
Week.           Cortex                                          Medulla                    Medulla


                Reading     Mean       Reading      Mean        Reading        Mean        Reading      Mean

                0.97        0.98       1.00         0.9533      0.65           0.7467      0.73         0.7233
At the end
                1.03                   0.95                     0.73                       0.75
of
1st week.       0.94                   0.91                     0.86                       0.69
At the end      0.99        0.9433     1.08         1.0667      1.12           1.07        1.03         1.05
of              0.89                   0.98                     1.03                       1.18
2nd week.       0.95                   1.14                     1.06                       0.94
At the end      1.29        1.31       1.63         1.67        1.46           1.53        1.53         1.4567
of              1.30                   1.74                     1.65                       1.45
3rd week.       1.34                   1.64                     1.48                       1.39
At the end      0.95        0.8867     1.04         1.0267      0.94           0.9367      0.93         0.92
of              0.82                   0.99                     1.02                       0.94
4th week.       0.89                   1.05                     0.85                       0.89
At the end      0.44        0.80       0.60         0.90        0.46           0.88        0.39         0.85
of              0.44                   0.61                     0.54                       0.40
6th week.       0.50                   0.68                     0.54                       0.44




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L2-1. Patient Name: Rashidun Nabi
Age: 17 Years
Sex: Male
Diagnosis: Fracture Rt. Colles
Study group: L2
                Non-Fracture Side,    Fracture Side, Cortex   Non-Fracture Side,    Fracture Side, Medulla
Week.           Cortex                                        Medulla

                Reading      Mean     Reading      Mean       Reading      Mean     Reading      Mean
At the end of   0.72                  0.63                    0.49                  0.51
1st week.       0.54         0.6367   0.63         0.63       0.41         0.45     0.52         0.53
                0.65                  0.63                    0.46                  0.56
At the end of   0.70                  0.66                    0.49                  0.55
2nd week.       0.65         0.6667   0.68         0.6633     0.46         0.47     0.52         0.5433
                0.65                  0.65                    0.46                  0.56
At the end of   0.98                  1.48                    0.99                  1.21
3rd week.       1.14         1.0167   1.53         1.43       0.93         0.9333   0.97         1.14
                0.93                  1.28                    0.88                  1.24
At the end of   1.09                  2.19                    1.08                  1.36
4th week.       1.39         1.2267   1.28         1.7767     1.02         1.1033   1.22         1.2867
                1.26                  1.86                    1.21                  1.28
At the end of   1.1                   1.01                    0.82                  1.01
6th week.       1.01         1.12     1            1.0767     0.75         0.82     0.90         0.98
                1.25                  1.22                    0.89                  1.04




L2-2. Patient Name: Enamul Haque
Age: 55 Years.
Sex: Male
Diagnosis: Fracture Rt. Colles.
Study group: L2
                Non-Fracture Side,    Fracture Side, Cortex   Non-Fracture Side,    Fracture Side, Medulla
Week.           Cortex                                        Medulla

                Reading      Mean     Reading      Mean       Reading      Mean     Reading      Mean
At the end of
                1.21                  1.96                    1.09                  1.34
1st week.
                1.20         1.2233   1.90         1.6033     1.02         1.0567   1.26         1.3800
                1.26                  0.95                    1.06                  1.54
At the end of   1.28                  1.88                    1.22                  1.37
2nd week.       1.22         1.2567   1.74         1.6800     1.25         1.2133   1.52         1.4467
                1.27                  1.42                    1.17                  1.45
At the end of   1.56                  2.07                    1.98         1.8233   1.80
3rd week.       1.80         1.7567   2.01         2.0033     1.83                  1.83         1.8133
                1.91                  1.93                    1.66                  1.81
At the end of   1.22                  1.49                    0.74                  0.93
4th week.       0.98         1.1933   1.55         1.46       0.78         0.7633   0.94         0.9967
                1.38                  1.34                    .77                   1.12
At the end of   1.04                  1.75                    0.95                  1.12
6th week.       1.13         1.1033   1.83         1.8333     0.87         0.9333   1.31         1.30
                1.14                  1.92                    0.98                  1.30




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10.4 Publications & Presentation:


PUBLICATIONS

Work arising from this thesis has been published (international) as follows:
1. "Basics of Laser "
http://www.scribd.com/full/16351752?access_key=key-1t6hx2qg83t1c47sgfly
http://www.slideworld.org/slideshow.aspx/Laser-Basics-ppt-2842839#
http://www.slideshare.net/abbirr/basics-of-laser
http://www.docstoc.com/docs/7435870/Basics-of-Laser
http://www.slideworld.com/slideshow.aspx/Laser-ppt-2766960
2. "Mode of Action of Laser in Tissues"
http://www.scribd.com/full/16351317?access_key=key-1kwgngizgl4udvhx8ali
http://www.slideworld.com/slideshow.aspx/LASER-ppt-2766961
http://www.docstoc.com/docs/7488218/Mode-of-action-of-laser-Tissue
http://www.slideshare.net/abbirr/mode-of-action-of-laser-in-tissues
http://www.slideworld.org/slideshow.aspx/Laser-mode-of-action-ppt-2842841
3. "Laser on Soft Tissue/Orthopedic view"
http://www.scribd.com/full/16510047?access_key=key-1j3v9h6i7bbe5447e58e
http://www.slideworld.org/slideshow.aspx/Laser-on-Soft-Tissue-ppt-2842842
http://www.authorstream.com/Presentation/abbirr-203225-laser-soft-tissue-ortopedic-view-fc
http://www.docstoc.com/docs/7487977/Laser-on-Soft-Tissue-Orthopedic-View.
4. "Laser on Hard Tissue"
http://www.scribd.com/full/17429815?access_key=key-23mwlu6kzxaq7vmed42y
http://www.docstoc.com/docs/10644250/Laser--on-Hard-tissue(-Orthopedic-View)
http://www.slideworld.com/slideshow.aspx/Laser-ppt-2766903
http://www.slideworld.org/slideshow.aspx/Laser-on-Hard-Tissue-ppt-2842905
5. Laser: Fundamentals & Medical Application.
6. Cold Laser Therapy(LED-660) On Soft Tissue Healing and On Chronic Wound:
    Review, Experiment & application and a Caser report.

PRESENTATIONS

 Work arising from this thesis has been presented at the following national / Institutional conferences
(Shaheed Suhrawardy Medical Hospital College, Department of Orthopedic & Traumatology)-
o "Basics of Laser”
o "Mode of Action of Laser in Tissues"
o "Laser on Soft Tissue/ Orthopedic view"
o "Laser on Hard Tissue/ Orthopedic view "
o “Laser: Fundamentals & Medical Application”.
o “Cold Laser Therapy (LED-660) On Soft Tissue Healing and On Chronic Wound:
     Review, Experiment & application and a Caser report”.




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10. 5 Biography & pictures




            Biography:

             Name: Md Nazrul Islam, MBBS, M.Sc.
             Born at Barisal Sadar.1st October 1964.
             Designation: Resident Surgeon
             Department of Orthopaedics & Traumatology
             Shaheed Suhrawardy Medical College Hospital.
     Present Address:
             HD-14, Doctors Quarter, SSMCH Complex.
             Phone: M-88-02-01196133078, Off. –9130800-19.
             E-mail: abbirr@gmail.com, abbirr@yahoo.com
     Permanent address:
             Jolly View, 4D(4th Fl.).22/13A Khilzee Rd.
             Mohammadpur, Dhaka-1207, Bangladesh.
     Education:
              Secondary School Certificate, Barisal Zilla School-1979
             Higher Secondary Government B.M. University College-
             1981.
             Bachelor of Medicine & Surgery,Sher-e-Bangla Medical College,
             Barisal (University of Dhaka)-1989.
             M.Sc., Biomedical Engineering, Gono-Biswabidyalay.
     Work Placement:
             Dhaka Shishu(Children) Hospital, Sher-E-Bangla Nagor, Dhaka-1207,
             1991-94.
             Shaheed Suhrawardy Medical Hospital,Sher-E-Bangla Nagor,
             Dhaka-1207-1997-2005.
             Carnegie Hill Institute, 86 Manhattan, NewYork, USA-2005-2007.
             Shaheed Suhrawardy Medical College Hospital,,Sher-E-Bangla Nagor,
             Dhaka-1207, 2007-Present.




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    Thesis supervisor, Professor Golam Abu Zakaria, Ph D. and Dr. Md Nazrul Islam are
in a clinical discussion about this work at Shaheed Suhrawardy Medical College Hospital
in the Department of Orthopaedic & Traumatology, Sher-E-Bangla Nagor, Dhaka-1207,
Bangladesh.




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Description: EFFECTS OF LOW LEVEL LASER THERAPY ON HUMAN BONE REGENERATION