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LLLT, Low Level Laser (LED- Ga -Al-As 660) Therapy –On soft Tissue Healing: Review, Mechanism, Experimental application and A case report.
LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. Research Paper Research Panel Members: 1 Dr. Md. Nazrul isla m, MBBS, M.Sc. (Biomedical Engineering). 2Professor Golam Abu Zakaria, PhD. 3Professor F. H. Sirazee, MBBS, MS. (Orthopedic). 4 Dr. Pari tosh Chandr a Debenath, MBBS, MS. (Orthopedic). 5 Dr. k azi Shami muzzama n, MBBS, MS. (Or thopedic). 6 Dr. Q uamrul Ak hter Sanj u, MBBS, F CPS, MRCS.(Surgery). 7 Dr. Ashraf Uddin Khan, MBBS, DMR D, FCPS. (Radiology). 8 Dr. Sajal Kum ar M ajumdar, MBBS, MS.(Pediatrics) 9 Dr. Subir Hossain Shuvro., MBBS. 10Sinha Abu Khalid, B. Sc. (Hons., App lied Physics & Electronics). 11Muhamma d M asud Rana , M.Sc. (Medical Physics). Correspondence to the Author: 1 Dr.Md Nazrul Islam . Shaheed Suhrawardy Medical College Hospital Department of Orthopedi c & Traumatology. Dhaka-1207, Bangladesh. E-mail: firstname.lastname@example.org, Tel: M- 880- 01196133078, Office- PA BX - 9130800-19. Contents: 1. Introduction 2. Review Skin lesions and the importance of Healing process in treatment success. Low-level laser Action of Laser on Tissues and its role In the Healing Process. 3. Mechanism of LLLT. Laser and Tissue interaction Primary Mechanism- Physical Secondary Mechanism- Biochemical Cellular signaling and response Tissue response & healing by LLLT 4. Experimental application of LLLT Experimental Studies/ application o In vitro and o In vivo studies (Animal and Human. 5. A case report: Laser Therapy on Soft Tissue. Abstract Case Study 6. Discussion 7. Conclusion 8. References. Introduction At present, cutaneous lesions represent a dilemma of global propor tions and instigate great clinical interest because of the high morbidity associated with changes in the nor mal healing process. 1 Among the clinical aspects involving this issue, we emphasize tissue repair time in an effor t to make the process quicker and more har monious, reduce possible complications in lesion resolution, and allow an adequate choice of therapy. To do this, familiarity with the pathogenesis of tissue healing is necessary, as well as an understanding of the factors a ffecting the process and the role each one plays in its progress, always seeking a clinical treatment that optimizes skin lesion care. Among the methods currently available, low-level laser therapy (LLLT) stands out. Allied health professionals regularly care for a variety of skin wounds, such as abrasions, tur f burns, surgical incisions, and ulcerations, which are perhaps the most difficult to treat. From acute wound management to augmentation of scar tissue remodeling, the clinician seeks to optimize wou nd care to promote healing. Experimental in vitro and in vivo studies have been under development since the 1960s, and in the early 1990s, LLLT was approved by the Food and Drug Administration (FDA) as an impor tant method for treating healing processes. 2-4 Recent results of a study demonstrated that LLLT is an effective method to modulate tissue repair, thus significantly contributing to a faster an d more organized healing process.5 Nevertheless, in spite of the large number of studies involving this techn ique and its wide use in clinical practice, the principles of its action in cells and tissues are still not well understood. The objective of this study is to review pathogenetic aspects of soft tissue repair to understand the major complications in skin lesion healing. In addition, it aims at for ming a concise compilation of published data from scientific literature to date to verify whether the use of low-level laser influences wound healing, since its mechanisms of action are not fully clear yet. 1 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. 1. Review A. Skin lesions and the importance of healing process in treatment success Some of the most common cutaneous wounds include excoriations, burns, surgical incisions, and acute or chronic ulcerations.2,3 Diabetes mellitus is one of the primary predisposing factor s for skin lesion development and one of the most common reasons for patients to seek health care, as it represents an impor tant cause of disability and premature death. 6 According to Pedrosa,7 serious cutaneous foot lesions in diabetic patients are the cause for hospital admission in 51% of patients in endocrinology wards of Brazilian university hospitals. When not properly healed, these lesions represent the main cause of morbidity, immobility and limb amputation, according to data from the American Diabetes Association.6 Burn injuries, a clinical condition resulting from direct or indirect action of heat on the human body that causes different degrees of skin lesions, are a significant cause of mor tality, primarily due to the infections that can evolve to septicemia. According to the Brazilian Society of Burn Injuries (Sociedade Brasileira de Queimaduras), there are 1 million cases each year in Brazil. 8, 9 Skin lesions have a great morbidity potential primarily because of complications in the normal healing process. To prevent these complications and promote cure, one needs to understand the nor mal process of soft tissue repair, as well as the factors that deter mine its nor mal healing. The normal process of soft tissue repair involves the following steps: homeostasis, inflammation ("cleaning"), demolition, proliferation, and maturing. 10 The homeostatic phase occurs immediately after the appearance of the lesion and depends on platelet activity and on blood coagulation process, which includes a complex release of vasoactive substances, adhesive proteins, and growth factors for the development of other stages.10,11 Later on, the inflammatory process sets in w ith the presence of numerous chemical mediator s and inflammatory cells (polymorphonuclear leukocytes, macrophages, and lymphocy tes).This phase is responsible for removing necrosed tissue and combating aggressive agents installed in the wound. Next, tissue proliferation, which is responsible for "closing" the wound, sets in, with re-epithelization, fibroplasia ( matrix for mation), and angiogenesis, essential for the supply of oxygen and nutrients needed for healing. Finally, there is wound contraction followed by remodeling, which takes place in the collagen of the region and has the objective of increasing tensile force and diminishing the scar size. 11,12 Therefore, tissue healing highlighted as one of the main effects of LLLT, 13, 14 is characterized by three main factors. First, there is an increment of ATP production, (as laser is considered to raise the production of ATP, 15) leading to a boost in mitotic activity and to an increase in protein synthesis by mitochondria, resulting in greater tissue regeneration in the repair process.13, 16 Second, there is a stimulus to microcirculation, which increases the delivery of nutritional elements associated with increased speed of mitosis, facilitating cell multiplication. 13, 14 Finally, new vessels are for med from preexisting vessels. 13, 14, and 17. Several factors have a direct influence on tissue healing, altering this process, making it slower, thus allowing complications associated w ith wound exposure to the external environment. The table below displays the key local and systemic factors that affect tissue wound healing. We see, then, that tissue lesions become a route for the installation of problems resulting from exposure to external agents, and therefore there is a need to accelerate the healing process by methods that shorten its duration. Laser therapy has become an important treatment for patients with cutaneous lesions, and there are ongoing studies aimed at understanding and confirming the known effects of laser application in tissue repair. B. Low-level Laser: The origin of low-intensity laser is attributed to Albert Einstein, who in his ar ticle entitled "Zur Quantum Theories der Strahlung" (1917) exposed the main physical principles of stimulated emission (laser phenomenon). This emission was later classified as "high- potency" (with destructive potential) and "low-potency" (without destructive potential).18, 19 In order to be produced, laser light needs atoms, constituted by a central nucleus, that are positively charged balanced by n egatively charged electrons that move around the nucleus in well-defined cir cular trajectories; in this rotational movement, there is no emission of energy. When the electron passes from one orbit to another, there is a release or absorption of energy called a p hoton 20 The devices that produce this beam of light are comprised of three par ts: a) an active laser medium (gain medium), b) an external energy source, and c) a resonant optical cavity. The gain medium is a gas, solid or liquid containing the atoms that enable p hotons to leap electron levels emitting photons and constituting a laser light beam.The external energy source furnishes the necessary energy to the system, so that electrons leap levels releasing, and not absorbing energy. This energy source should be able to produce high-energy or excited states. The optical or resonator cavity makes the emerging photons return to the system, producing additional stimulated emissions; this phenomenon occur s by mirrors positioned at the cavity extremities, provoking a reflection of photons back to the sample 14, 20, 21 2 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. The differences between the various types of laser beams produced are deter mined by wavelengths: the shor ter the wavelength, the greater its action and power of penetration. Additionally, lasers may be continuous or pulsed, and their potency is expre ssed in Watts ( W), varying from deciwatts to megawatts. Energy is expressed in Joules per square centimeter (J/cm2), and therefore is equal to the potency multiplied by the duration of application. 22 Knowledge of these parameters is vital for appropriate indication and therapeutic utilization of this method. 4 Table- 1 Parameters involved in determining the lllt Irradiation parameters Irradiation parameter Unit of measurement Comment Wavelength nm y Light is electromagnetic energ which tra vels in discrete ve Packets that also ha a wave-like property. Wavelength is nd Measure in nanometers (nm) a is visible in the 400-700 nm range. Irradiance W/cm2 Often called Intensity, or Power Density and is calculated as Irradiance = Power (W)/Area (cm2) Pulse Peak Power (W ) If the beam is pulsed then the Power should be the Average structure Pulse freq (Hz) Power and calculated as follows: Pulse Width (s) Average Power (W) = Peak Power (W) Duty cycle (%) pulse width (s) pulse frequency (Hz) Coherence Coherence length Coherent light produces laser speckle, which has been y Depends on postulated to pla a role in the photobiomodulation Spectral bandwidth interaction with cells and subcellular organelles. Polarization Linear polarized ve light may ha different effects than otherwise or circular identical non-polarized light (or even 90-degree rotated polarized polarized light). However, it is Known that polarized light is rapidly scra mbled in highly scattering Media such as tissue (probably in the first few hundred μm). Table- 2. Parameters involved in determining the lllt “dose” irradiation time or energy delivered ( the dose) Irradiation Unit of Parameter measurement Comment Energy (Joules) J Calculated as: Energy (J) = Power (W) x time (s) This mixes medicine and dose into a single expression and ignores Irradiance. Using Joules as an expression of dose is potentially unreliable as it assumes reciprocity (the inv erse relationship between power and time). Energy J/cm2 Common expression of LLLT “dose” is Energy Density Density This expression of dose again mixes medicine and dose into a single expression and is potentially unreliable as it assumes a reciprocity relationship between irradiance and time. Irradiation s In our view the safest way to record and prescribe LLLT is to Time define the four parameters of the medicine (see table 1.) and then define the irradiation time as “dose”. Treatment The effects of different treatment interval is underexplored Interval Hours, days or weeks at this time though there is sufficient evidence to suggest that This is an important parameter. C. Action of Laser o n Tissues and its ro le in the Healing Process: Based on the understanding of the mechanism of laser light origin, we observe that when low -intensity light is used, there is no thermal effect, i.e., the energy from the photons absorbed is not transformed into heat, but into photochemical, photophysical, and photobiological effects. According to Catão,21 this is an impor tant principle of the interaction between laser light and cell or tissue specimens. When it is a pplied at an appropriate dose, laser can stimulate cell functions that are vital for the progress and resolution of the healing process via tissue bio stimulation, such as increased mitochondrial ATP production, lymphocy te and mast cell activation, and proliferation of fibroblasts and other cells, besides promoting analgesia and anti-inflammatory effects.21,23 As previously stated, the action of laser on tissues depends on the duration of emission of the different energy densities, a nd on the application area. Therefore, if these parameters are not duly verified and/or calibrated, treatment may be ineffective, compromising therap eutic success.24 According to previously established parameters, in cutaneous lesions the tissue layer to be targeted depends on type of laser, potency used and duration of application. Kolárová et al. 25 observed that, using high potencies or application of light for fractions of a second, the power of penetration of HeNe radiation with a wavelength of 632 nm could reach up to 19 mm of depth in the der mis.25 Since the energy produced by the laser is only absorbed by a thin layer of adjacent tissue in addition to the spot targeted by the radiation, current recommen dation is to use low- intensity laser that has a low power of penetration, with wavelengths between 640 and 940 nm in a punctifor m application to the lesion. 26 Several research studies have used super ficial wounds to assess the effects of low-intensity laser on healing. Some have used clinical wounds such as ulcers of differen t sizes and depths3,27,28 and other s have developed models of super ficial wounds in animals. 19,29,30 These diverse methods have produced a variety of results and conclusions on the effects of LLLT. Cells in the wound respond to light induced reactive oxygen species (ROS) leading to the expression of growth factors, such as transfor ming growth factor beta (TGF), and platelet derived growth factor (PDGF), which encourage synthesis of more collagen, increased for mation of blood vessels, and less inflammation, all of which increase wound healing. 3 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. 2. Mechanism of Action of Laser, and Action on soft Tissue Wound Healing: Laser Tissue Interactions: 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 ph otons 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 for m of diffuse reflection. The photons that are absorbed interact with an organic molecule or chromop hore 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 orbital’s 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 ther modynamics, the energy delivered to the tissue must be conserved, and three possible pathways exist to ac count 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 fir st excited singlet state of the chromophore undergoes a transition from a higher to a lower electronic state. It is someti mes called "radiation less de-excitation", because no photons are emitted. It differ s from inter system 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 transfor med into heat. 2). The second pathway that can occur is fluorescence. Fluorescen ce 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 ab sorbed 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. Among the above three, internal conversion & fluorescence are the mechanisms those are involved in Physical Mechanism of lase r tissue interaction, and third one is recognized as Bio-chemical. PHYSI CAL MECHANISMS There are two primary for ms 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 star t a cascade of biological interaction, such as vibration, heat/light production, and o ther biological changes into tissues by increasing membrane per meability/potential (Cellular & mitochondrial). After diffuse penetration of the laser bundle in the tissue, there is absorption of polarized light in cy tochrome 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 (1). 2. Internal conversion & fluorescence of light also generates Speckle for mation, 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 si mply a phenomenon created at and limited to the tissue sur face, but is generated w ithin a volume of tissue, persisting to the total ex tent of the depth of penetration of the laser beam. Laser speckles for med deep in the tissue create temperature and pressure gradients across cell membranes, increasing the rate of diffusion across those membranes. Further, photons w ithin 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-perfor m other non-coherent light sources, especially for deeper tissue treatments). The speckle effect is a result of the inter ference of many waves, having different phases, which add together to give a resul tant wave whose amplitude, and therefore intensity, varies randomly. Each point on illuminated tissue ac ts 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 sur face. If the sur face 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 apar t 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. 4 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. BIOCHEMICAL MECHANISMS FIGURE 1.Schematic diagram showing the absorption of red and NIR light by specific Cellular chromophores photoacceptors localized in the mitochondrial respiratory chain The third pathway that can occur after the absorption of light by a ti ssue chromophore (Biochemical) represents a number of processes broadly grouped under an umbrella category of photochemistry. This is the basic mechanism by which way laser works in animal/ human cell/tissue. It has been established by thousands of research/application, and it is recognized by World Laser Association as well as Amer ican /European associations of Laser/photo-biology. Bio-chemical action of laser can be explained by “Action of photon with mitochondrial respiratory chain- Cytochrome c oxidase enzyme”. Mitochondrial respiratory chain contains five complexes of integral membrane proteins: NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), cytochrome c reductase (Complex III), cytochrome c oxidase (Complex IV), and ATP synthase (ComplexV). FIG URE 3: Structure and mode of action of cytochrome c oxidase. 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- (Sutherland 2002). A chromophore is a molecule (or part of a molecule) which imparts some decided color to the compound of which it is an ingredient. Chromophores almost always occur in one of two for ms: conjugated pi electron systems and metal complexes. Examples of such ch romophores can be seen in chlorophyll (used by plants for photosynthesis), hemoglobin, cy tochrome c oxidase (Cox), myoglobin, flavins, flavoproteins and porphyrins (Karu 1999). Figure 1 illustrates the general concept of LLLT. Although, the exact mechanism of action of LLLT is not completely understood; however, there are several theories based on ce llular research conducted over the last two decades or more. The basic premise is that LLLT stimulates cell activation processes which, in tu rn, 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 P r ize in Physiology or Medicine in 1994. 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 Kre bs cycle) to oxygen molecules to for m (with the aid of protons) water molecules harnessing the energy released by this transfer to the pumping of pr otons (H+) from the matrix to the inter membrane space. The gradient of protons formed across the inner membrane by this process of active transport for ms a miniature battery. The protons can flow back down this gradient, re-enter ing the matrix, only through another complex of integral proteins in the inner membrane, the ATP synthase complex. 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. 5 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. 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 gen er ation and metabolism. Mitochondria are sometimes described as “cellular power plants”, because they convert food molecules into e nergy 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 chain (Karu 1989). Several pieces of evidence suggest that mitochondria are responsible for the cellular response to red visible and NIR light. FIGURE 4a. Structure of the mitochondrial respiratory chain. 4b-Schematic diagram of the mitochondrial electron transport chain Peter D. Mitchell proposed the chemiosmotic hypothesis in 1961. 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". Nature 191: 144–148. doi:10.1038/191144a0. PMID 13771349.  Absorption of photons by molecules leads to electronically excited states and consequently can lead to acceleration of electr on transfer reactions and stimulate chemiomosis mechanism. Electron transfer reactions are highly impor tant 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 . 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, an d therefore the ATP level controls the level of cAMP. Both Ca2+ and cAMP are very impor tant second messengers. Ca2+ especially regulates almost every process in the human body (mu scle contraction, blood coagulation, signal transfer in nerves, gene expression, etc.). 2). Cytochrome c oxidase mediated singlet-oxygen productionm . In addition to cytochrome c oxidase mediated increase in ATP production, other mechanisms may be operating in LLLT. The first of these w e will consider is the “singlet-oxygen hypothesis.” Certain molecules with visible absorption bands like porphyrins lacking transition metal coordination centers  and some flavoproteins  can be conver ted into a long-lived tr iplet 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 for med, 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 ene rgy transfer to ground state molecular oxygen (a triplet) to for m the reactive species, singlet oxygen. Alternatively the chromop hore triplet state may undergo electron transfer (probably reduction) to for m the radical anion that can then transfer an electron to oxygen to for m superoxide. This is the same molecule utilized in photodynamic therapy (PDT) to kill cancer cells, destroy blood vessels and kill microbe s. Resear chers in PDT have known for a long time that very low doses of PDT can cause cell prol iferation and tissue stimulation instead of the killing observed at high doses  3). Cytochrome c oxidase medi ated Reactive oxygen species (ROS) formati on. FIG URE - 5. Re acti ve oxygen speci es (R OS) f ormed as a resul t of LLLT effec ts i n mi toch on dri a may ac ti vate th e red ox- sen si ti ve tran scri pti on factor NF- B (rel A-p50) vi a protei n kin aseD(PKD). The next mechanism proposed was the “redox properties alteration hypothesis” . Alteration of mitochondrial metabolism and activation ofthe 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. Other redox chains in cell s can also be activated by LLLT. NADPH-oxidase is an enzy me found on activated neutrophils and is capable of a non- mitochondrial respiratory burst and production of high amounts of R OS can be induced. They are for med as natural by-product of the normal me tabolism of oxygen. 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. . LLLT was reported to produce a shift in overall cell redox potential in the direction of greater oxidation (Karu 1999) and in creased ROS gener ation and cell redox activity have been demonstrated (Alexandratou et al. 2002; Chen et al. 2009b; Grossman et al. 1998; Lavi et al. 2003; Lubart et al. 2005; Pal et al. 2007; Zhang et al.2008).Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved and have important roles in cell signaling ( from mitochondria to nuclei, Storz 2007)), regulating nucleic acid synthesis, protein synthesis, enzy me activation and cell cycle progression (Brondon et al. 2005). 6 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. These cy tosolic responses may in turn induce transcriptional changes. Several transcription factors ar e regulated by changes in cellular r edox state. But the most important one is nuclear factor B (NF-B). Figure 5 illustrates the effect of redox-sensitive transcription factor NF-B.activated after LLLT and is instrumental in causing transcription of protec tive and stimulatory gene products. The whole process absolutely depends on the physiological status of the host organism as well as on radiation parameters. 4). Cytochrome c oxidase mediate d Photodiassociation and Cell signaling. FIG URE - 6. Wh en NO- i s rel eased from i ts bi n din g to h eme i ron an d copper cen ters i n Cytoch rome c oxi dase by th e acti on of li gh t, oxygen i s all owed to rebi n d to th ese si tes an d respi rati on i s restored to i ts forme r l evel l eadin g to i n creased A T P syn th esi s. 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 enzy me. 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 cy tochrome c oxidase (see below). The activity of cy tochrome 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 . 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”) . Data published recently by Karu et al  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) . While both observations suppor t the hypothesis of NO dependent responses to LLLT, responses to different wavelength s 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 (Shiva and Gladwin 2009). Light mediated vasodilatation was fir st 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 1998 (Mitka 1998). Later studies conducted by other researchers confir med and ex tended Furchgott’s early wor k 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. Cellular signaling and response A. Cellular signaling Figure-7 . Cell signaling pathways induced by LLLT. 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 for ms) NAD/NADH, nicotinamide adenine dinucleotide phosphate NADP/NADPH, glutathione/glutathione disulfide couple GSH /GSSG and thioredoxin/ thioredoxin disulfide couple Trx (SH) 2/TrxSS . Several impor tant regulation pathways are mediated through the cellular r edox state. Changes in redox state induce the activation of numerous intracellular signaling pathways; regulate nucleic acid synth esis, pr otein synthesis, enzy me activation and cell cycle progression . 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 for m of redox-dependent transcription factors have low DNA-binding activity. Ref-1 is an impor tant factor for the specific reduction of these transcription factors. However it was also shown that low levels of oxidants appear to stimulate proliferation and differentiation of some type of cells [25 -27].It is proposed that LLLT produces a shift in overall cell redox potential in the direction of greater oxidation . Different cells at a r ange of growth conditions have distinct redox states. Therefore, 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. 7 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. B. Cellular response 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 repor t effects on more than one of these parameters. Yu et al re por ted  on cultured keratinocy tes 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 keratin ocy tes. Medium from HeNe laser irradiated keratinocytes stimulated [3H] thy midine uptake and proliferation of cultured melanocytes. F ur thermore, melanocy te migration was enhanced either directly by HeNe laser or indirectly by the medium derived from HeNe laser treated keratinocytes. The presence of cellular responses to LLLT at molecular level was also demonstrated . Normal human fibroblasts were exposed for 3 days to 0.88J/cm2 of 628 nm light from light emitting diode. Gene expression profiles upon irr adiation 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 upregulated too. Amongst genes related to apoptosis and stress response, some genes such as JAK binding protein were upregulated, 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 re pair, and cell metabolism. Tissue response & healing by LLLT A. Tissue response LLLT can pr ov ide the following beneficial impacts in both open sur face wounds and closed con nectiv e or soft tissue injur ies as follows: 1. Enh anced leu ko cyte infilt ratio n. LLLT stimulates activ ity inv olv ing neutr ophils, monocy tes and ly mphocy tes. 2. In creased macroph ag e activity. LLLT acceler ates macrophage activity in phagocy tosis, growth factor secretion and s timulati on of collagen synthesis. 3. Increased n eo vascularizat ion . T he significant angiogenesis that occur s with laser ther apy pr omotes r evascular ization with subsequent improvement in per fusion and oxygenation. Endothelial cell r egener ation is accelerated. 18 4. In creased f ib rob last p ro liferation. LLLT stimulation in - creases fibr oblast number s and fibr oblast- mediated collagen production.19 5. Keratinocyte proliferation. T he beneficial synthesis activities and growth factor ability of keratinocy tes are enhanced by proliferation secondary to LLLT.20 6. Early ep ith elializat ion . Laser- stimulated acceler ation of epithelial cell regener ation speeds up w ound healing, minimizes scarr ing, and reduces infection oppor tunities. 7. Gro wth f acto r in creases. T wo to five fold increases in growth- phase- specific DNA synthesis in nor mal fibr oblasts, muscle cells, osteoblasts and muco sal epithelial cells irradiated w ith IR light are repor ted. Increases in vascular endothelial growth factor (VEGF) and fibroblast gr owth factor (FGF-2) secondary to IR light irr adiation hav e also been repor ted. 8. Enh anced cell p ro lif eratio n and d iff eren tiat ion . Laser- induced incr eases in NO, ATP and other compounds that stimu late higher activ ity in cell prolifer ation and differ entiation into mature cells. Increased number s of my ofibr oblasts, my ofibr ils, myotubes etc., as well as bone cell proliferation, have- been clinically documented after- LLLT. Satellite cells, the pr ecur sor cells in the process of muscle r egeneration, show significant increase in proliferation w hen irradiated w ith LLLT. 21, 22, 23 9. Great er healed wou nd tensile streng th. In both soft tissue and connectiv e tissue injuries, LLLT can incr ease the final ten sile str ength of the healed tissue. By incr easing the amount of collagen pr oduction/synthesis and by incr easing the intr a and inter - molecular hy dr ogen bonding in the collagen molecul es, laser ther apy contr ibutes to improv ed tensile strength. 24,25, 26,27. The preceding effects combine to achieve an accelerated heal ing rate ( see F igur e 3). T he time fr om onset of injury to mature he aled wound is r educed.28 The cumulative effects of (physical & Bio-chemical) laser on tissue enhances physiological activities by ion-exchange, speckle formation, singlet oxygen, redox formation, ATP production & nitric oxide formation and exerts- Enhances chemiosmosis, Enzyme & hor mone regulation Stimulates the redox activity in the mitochondria, RNA synthesis and DNA production - causing mitosis and cell proliferation Calcium- ion influx into the cytoplasm, So, Laser biostimulation may be an invaluable therapeutic modality for treating most wounds. Wound h ealing entails a) the process of inflammation during which the hematoma for med in and around the wound site is resolved; b) cellularity and protein synthesis, i.e., two processes that culminate in the formation of granulation tissue; and c) wound remodeling, a process that may continue long after the wound may be said to be well healed. 1) Accelerates the inflammatory phase of wound healing by altering the levels of those prostaglandins that influence the process. 2) Quickens protein synthesis by quickening DNA and RNA synthesis, and augments fibroplasias thereby promoting cell proliferation and for mation of granulation tissue. So far, because most of the works in this area were done on healing skin wounds and fracture s, the evidence points specifically to enhanced proliferation of fibroblasts and increased collagen synthesis. In view of the numerous complications associated with the long periods that such dense connective tissues as bones, tendons, and ligaments require to heal, it is rather a hap py coincidence that the process of repair peculiar to these tissues is specifically enhanced by laser biostimulation. 3) Increases ATP synthesis by enhancing electron transfer in the inner membrane of mitochondria, thus providing the ex tra energy required for protein synthesis and cell proliferation. 4) Enhances the ability of immune cells to combat pathogens; thus minimizing the impact of infection. Hence, chronic wounds such as those of decubitus ulcers and burns may heal faster when stimulated with laser as some h uman study has shown. 8 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. B. Tissue healing One of the tr uly unique character istics of LLLT is that it has the ability to actually pr omote and enhance healing, not just tr eat sy mptoms. The irradiation by low- level laser light acceler ates and enhances healing activities carried out by the body . Several of the unique char acter istics of LLLT that wor k to allev iate pain and inflammation also play an impor tant r ole in acceler ating the healing process; the LLLT- mediated r eduction in inflammation and pain fr ees the body ’s natur al 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 pr ogresses through the stages of inflammation, pr olifer ation, r emodeling and matur ation, laser ther apy pr esents the oppor tunity to impact each of these phases in positiv e and beneficial way s. The beneficial effect of LLLT on wound healing can be explained by considering several basic biological mechanisms including the induction of expression cy tokines and growth factor s known to be responsible for the many phases of wound healing. Firstly there is a report  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  that LLLT can upregulate cy tokines responsible for fibroblast proliferation, and migration such as bF GF, HGF and SCF. Thirdly it has been reported  that LLLT can increase growth factors such as VEGF responsible for the neovascularization necessary for w ound healing. Fourthly TGF-β is a growth factor responsible for inducing collagen synthesis from fibroblasts and has been repor ted to be upregulated by LLLT . Fifthly there are reports [52, 53] that LLLT can induce fibroblasts to undergo the transfor mation 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 (Lubart et al. 1992; Yu et al. 1994), keratinocy tes (Grossman et al. 1998), endothelial cells (Moore et al.2005), and lymphocytes (Agaiby et al. 2000; Stadler et al. 2000). The mechanism of proliferation was proposed to involve photostimulatory effects in mitochondria processes, which en hanced growth factor release, and ultimately led to cell proliferation (Bjordal et al. 2007). Kreisler et al showed (Kreisler et al. 2003) that the attachment and prolifer ation of human gingival fibroblasts were enhanced by LLLT in a dose-dependent manner. LLLT modulated matrix metalloproteinase activity and gene expression in porcine aor tic smooth muscle cells (Gavish et al. 2006). Shefer at el. showed (Shefer et al. 2002) that LLLT could activate skeletal muscle satellite cells, enhancing their proliferation, inhibiting differentiation and regulating protein synthesis. There have been a large number of both animal model and clinical studies that demonstrated highly beneficial LLLT effects on a variety of diseases, injuries, and has been w idely used in both chronic and acute conditions. LLLT may enhance neovascularisation, promote angiogenesis and increase collagen synthesis to promote healing of acute (Hopkin s et al. 2004) and chronic wounds (Yu et al. 1997). LLLT provided acceleration of cutaneous wound healing in rats with a biphasic dose response favoring lower doses (Corazza et al. 2007). LLLT can also stimulate healing of deeper structures such as nerves (Gigo-Benato et al. 2004), tendons (Fillipin et al. 2005), car tilage (Morrone et al. 2000), bones ( Weber et al. 2006) and even internal organs (Shao et al. 2005). LLLT can reduce pain (Bjordal et al. 2006a), inflammation (Bjordal et al. 2006b) and swelling (Carati et al. 2003) caused 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 vivo (Ad and Oron 2001; Oron et al. 2001a; Oron et al. 2001b; Yaakobi et al. 2001). LLLT has been used to mitigate damage after strokes (in both animals (Lapchak et al. 2008) and humans (Lampl et al. 2007)), after traumatic brain injury (Oron et al. 2007) and after spinal cord injury (Wu et al. 2009). Although the underlying mechanism of LLLT are still not completely understood, in vitro studies, animal experiments and clinical studies have all tended to indicate that LLLT delivered at low doses may produce a better result when compared to the same light delivered at high doses. LLLT can prevent cell apoptosis and improve cell proliferation; migration and adhesion at low levels of red/NIR light illumination, and there by exerts Biostimulatiive effects on cell, tissue & wound healing. 9 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. 3. Experiment al Application 42 Experimental in vitro and in vivo studie s (animals and humans) Vinck et al.31 carried out a cell culture study to observe the influence of diode light emission (LED) and LLLT in the process of wound healing, in which cultures of fibroblasts obtained from 8-day-old chicken embryos were treated for three consecutive days. Infrared laser gallium-aluminum arsenate (GaAlAs - Unilaser 301P, MDB-laser, Belgium) was applied to an area of 0.196 cm2, with an 830 nm wavelength, peak potency of 1-400 mW, and a frequency of 0-1,500 Hz LED (BIO – DIO, MDB – laser, Belgium), with three wavelengths emitted separately. All three applications covered an area of 18 cm2 and used a frequency of 0-1,500 Hz, at an application distance of 0.6 cm. LLLT was used with the following parameters: one 5-second emission with peak potency of 40 mW resulting in an exposure of 1 J/cm2. Infrared, the light spectrum with red light, has a radiation exposure of 0.53 J/cm2. Green light emits 0.1 Jcm2, corresponding to an exposure time of 1, 2, and 3 minutes, respectively, with peak potencies equivalent to 160, 80 and 10 mW, respectively. Statistical analysis showed a high proliferation of fibroblasts in vitro. Therefore, this study presupposed the possibility of stimulatory effects on in vivo wound healing when treated with LLLT. Demir et al.32 investigated the effects of electrical stimulation (ES) and LLLT in wound healing in an experimental study using 124 Swiss-albino female rats, with weights between 200 and 240 g, aged 8 to 10 months, which were divided into four groups of 30 animals each. A 6-cm linear incision was made in the dorsal region of each rat. According to the protocol used, group I was treated with ES (Endomed 582 model, Enraf-Nonius Co., Holland). For group II, a similar procedure was stipulated, including a saline solution for application, but without using current. In group III, gallium arsenate laser (Laserpet 100 model, Petas Co., Tur key) was used, with a 904 nm wavelength, average potency of 6 mW, and a 1 J/cm2 dose with the maximum frequency of 128 Hz. This dose was applied in a continuous for m for 10 minutes a day, over a 10-day period. They concluded that ES and laser treatments have positive effects on inflammation, proliferation, and maturation of wounds, and can be successfully used for decubitus ulcers and chronic wounds. An experimental study conducted by Khadra 33 observed the effects of gallium-aluminum arsenate laser (GaAlAs), a diode laser, on bone healing process in 20 rats with 2.7 mm diameter bone defects in the parietal region tr eated for 4 weeks. On the 28th day, the animals were euthanized for histological assessment of bone defects. In experimental animal tissues, there was a significant increase in calcium, phosphorus and protein. Similarly, histological analyses showed a marked growth of angiogenesis and connective tissue. They concluded, therefore, that LLLT can favor bone for mation in rats w ith bone defects. Study of LLLT on the diverse constituents of the extracellular matrix is crucial for an understanding of the healing p rocess using that agent. However, little is known about the influence of laser therapy regarding collagen and elastic fibers. Pugliese et al. 34 conducted an experimental study in 72 male and female Wistar rats weighing around 150-250 g each, which were divided into three groups. Standard cutaneous wounds were created on the back of the rats, followed by a punctifor m application of low -potency gallium- aluminum arsenate - type laser (VR-KC-610, Dentoflex, SP, Brazil) with an emission of 670 nm diode light and a potency of 9 mW of different energy densities - 4 J/cm2 for group II and 8 J/cm2 for group III. The animals were euthanized at 24, 48 and 72 hours, and 5, 7 and 14 days. The animals that underwent treatment showed a greater expression of collagen and elastic fibers. In those treated with a 4 J/cm2 flow, better results were noted than in those treated w ith an 8 J/cm 2 flow. This study led to the conclusion that laser treatment contributes to a greater expression of collagen and elastic fibers during the healing process.Over the years, different studies have been perfor med to understand the tissue repair process, as well as the possible effects of laser therapy on the process of wo und healing. Rocha Jr. et al.5 carried out a study to investigate the behavior of cutaneous wounds provoked on the dor sal region of Wistar rats (Rattus norvegicus) submitted to low-intensity treatment with a 3.8 J/cm2 dose, 15 mW of potency, and 15 s of application time. The animals (n = 12) were divided into two groups, one control and the other treated w ith laser. In the treated group, three applications were given (immediately following the surgical act, 48 hours, and 7 days after surgi cal wounds were created). Ten days after the operation, lesion samples were collected from both groups for histopathologic and histomorphologic studies. Results showed increased neovascularization and fibroblast proliferation, as well as decreased quantity of inflammatory infiltrate in the surgical lesions submitted to laser treatment. In the control group, abutting the margin of the surgical wound, a discrete epithelial proliferation was noted, and along practically the whole extension there was presence of a tissu e with a wide area of ulceration and fibrinonecrotic tissue over the granulation tissue (Figure 3). Figure - 3. Figure - 4 F igure -5) Conversely, in laser-treated group II, histopathologic study showed material with an intact epider mis lining well-developed granulation tissue with a connective tissue rich in collagen fibers parallel to the sur face of the wound, characterizing a be tter- organized tissue repair process (Figure 4). Clinical observation of skin lesion samples of the animals showed that skin lesions of group I (control) exhibited an early -phase tissue repair pattern, with for mation of a whitish crust, with slightly elevated r ims and a reddish core due to accentuated presence of blood irrigation in that area, indicating presence of granulation tissue. On the other hand, group II wounds, which were submitted to LLLT, showed complete tissue repair, showing scars w ith evident rims and a central por tion slightly unleveled, but presenting advanced morphological and functional recovery of involved tissues (Figure5). These combined results suggest that LLLT is an efficacious method of tissue repair modulation, significantly contributing to a more rapid and organized healing of tissues. 10 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. Experimental treatment in patients began in the 1970s after repor ts of positive results of radiation with LLLT of cell cultures and animal experiments.4 Nevertheless, this initial success in cell cultures and animal studies is controversial, and fur ther studies need to be conducted to analyze its usefulness in treating skin lesions in humans. 35 Gaida et al.36 conducted a study in humans with the objective of confir ming the effects of LLLT and its prophylactic use in treating burn healing, since the beneficial effects of LLLT in wound healing are still controversial. That study included 19 patients (14 men and five women, aged between 18-77 years), in which one burn from each patient was chosen for radiation, and another similar burn was defined as control. Burns were located on the face, trunk and limbs. LLLT was administered w ith a soft laser device (Helbor, Gallspach, Austria) with a circular application of a continuous diode laser using a 400 mw potency emitting a red laser light with a 670 nm wavelength. Dose used was 4 J/cm2. Treatment was given twice a week, with a minimal interval of 3 days for 8 weeks. Seventeen out of 19 lesions attained macroscopic improvement with treatment, and two did not. That study concluded that LLLT could have significa nt beneficial effects on patients during rehabilitation. Lucas et al.37 developed a study in humans with a wide sample, in which they observed the benefits of LLLT in treating stage III decubitus ulcers. The study was carried out in 86 patients; one group (n = 47) was treated w ith changes in decubitus, and the other (n = 39) also received LLLT five times a week for 6 weeks. Early results showed a relative decrease in wound area after 6 wee ks compared to initial conditions. LLLT was administered using a 904 nm infrared gallium arsenate laser (GaAs-diode) radiating an area of 12 cm 2, in a 125-s exposure. Peak potency was 12 x 70 W with 830 H z of frequency and a 150 ns pulse, with an average potency of 12 x 80 mW and radiation of 1 J/cm2, in a 125-s exposure. During treatment period, 11% of the control patients and 8% of the patients in the group treated with LLLT developed stage IV decubitus ulcers. Considering both groups in ter ms of lesion improvement, there were no significant differences between them. Therefore, this s tudy did not confir m findings that support use of LLLT as a supplement to decubitus ulcer treatment. In a clinical case presentation, Legan et al. 38 carried out a study in humans to investigate the clinical effects of LLLT in venous ulcerations when used along wound margins, as well as the relevance of this treatment on wound- debridement and measurements of affected area. Patients were seen three times a week for 8 weeks using an 830 nm emission with 9 J/cm2 in association with a process of wound sterilization at each visit. The lesions and their measurements were analyzed using videos and photos that displayed a 15% decrease in those areas at the end of the study. It was concluded that LLLT is an effective treatment for patients who suffer from venous ulcers, since LLLT favored stabilization of clinical status and increased the therapeutic effects of these doses in studied groups. Investigations on the effect of multiple exposures of LLLT on cell responses of wounded skin fibroblasts demonstrate that cor rect energy density or fluence and number of exposures can stimulate cell responses of wounded fibroblast and promote cell migration and cell proliferation by -stimulating mitochondrial activity and maintaining viability without causing additional stress or damage to wounded cells. Results indicate that the cumulative effect of lower doses determines the stimulatory effect, whereas multiple exposure at higher doses result in an inhibitory effect with more damage. 39 In an experimental study, Byrnes et al. 40 investigated the effects of photobiomodulation (PBM) on the process of skin wound healing in an animal model (Psammomy obesus rats) with type II diabetes. Results showed histological improvement in wound healing followed by wound closure with the treatment according to the protocol using 4 J/cm2, 16 mW potency, 250 s of application for 4 consecutive days. This 4 J/cm2 dose caused histological improvement in wound closure in diabetic rats compared with non-diabetic rats, with increased expression of fibroblastic growth factor. These findings showed that PBM, with an energy intensity of 4 J/cm2, is effective in- improving lesion healing processes in model animals with type II diabetes, suggesting that PBM (632 nm, 4 J/cm2) may be a promising treatment for chronic wounds in diabetic patients. Houreld et al.41 investigated in vitro exposure of wounded diabetic fibroblast cells to a helium-neon laser at 5 and 16 J/cm2. Cells exposed to 5 J/cm2 showed a higher rate or migration than cells exposed to 16 J/cm2, and there was complete wound closure by the four th day. Exposure of cells to 5 J/cm2 on two non-consecutive days did not induce additional cy totoxicity or genetic damage, whereas exposure to 16 J/cm2 did. There was a significant increase in apoptosis in exposed cells compared wi th unexposed cells. 11 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. 4. A C ASE R EPORT : Laser as an Adjunctive Modality for human Chronic W ound Healing - Place of study : Shaheed Suhraw ardy Medic al College Hospital, Dhak a-1 207, Bangladesh. Telephone: 88-9130800-19. Patient Particular: Name of the patient: Ms. Zobeda Khatun, Age: 78 Years, Gender: Female. Ward and Bed: 6 /1. Hospital stay: From October 14th /2009 to January 10th / 2110. Shaheed Suhrawardy Medical College Hospital, Sher-E-Bangla Nagor, Dhaka-1207, Bangladesh. ABSTRACT Background: Chronic wounds, par ticularly Bedsore/ Decubitus ulcerations in old age/ bedridden patients, are notor iously difficult to heal. Because current therapies are variable in their ability to induce complete healing, there remains a need to develop adjunctive treatments that can improve or accelerate the healing process. Low-level laser therapy is an impor tant method for the treatment of healing processes and several experimental studies in human & animal models an d applications has been carried out successfully in search of a greater understanding of its therapeutic possibilities. Objective The objective of this study was to review pathogenetic aspects of soft tissue repair to better understand skin lesion healin g and the role of low-intensity laser in the progression of tissue healing. Methods: We have worked on diode laser therapy on recurrent bedsore patient, made clinical observational effects of low intensity lase r therapy on constituents of the wound healing process. Results: A large number of in vivo studies on the effects of low intensity laser irradiation on wound healing show a lack of accuracy on dosimetric data and appropriate controls. Despite this fact, data from appropriately designed studies seem to indicate that this type of phototherapy should be considered a valuable (adjuvant) therapy for otherwise therapy -refractory wound-healing disorders. Conclusions: The use of low-energy lasers to stimulate wound healing has been pursued over many decades in studies of varying quality. This for m of treatment has had high appeal due to its novelty, relative ease, and low morbidity profile. However, many unanswered questions demand resear ch on the mechanism of action and on parameters of low-level laser use in different stages of wound repair to clarify how this method acts at a cell level in healing processes. High energy lasers have extensive applications in the field of surgery, ophthalmology, dermatology, medicine and oncology. The utility of low energy laser s for biostimulation, immune response mediation and wound healing is of relatively recent interest. The current communication illustrates our experience w ith low energy laser (Ga-Al-As 660 laser) as an adjunct to the conventional modes of treatment for wound healing. Many studies have ex tensively covered the effects of using laser radiation in tissues, describing its beneficial aspects in tissue healing. This study consists of a concise review of scientific literature data on the use of low -level laser and its influence on wound healing. Fur ther research aiming at elaborating optimal treatment parameters seems to be justified. Keywords: LLLT, Biostimulation, wound healing. 12 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. Case Study: Ms. Zobeda, female, Dhaka, Bangladesh, age 78, bedridden for years for spastic neurological disorder, with history of recurrent multiple large / small (approximately 1 -2cm deep, 6 -8cm in diameter) wound in the back, with inflammation and profuse purulent discharging infection was referred to this ter tiary hospital from a local hospital for long lasting (More than 6 months of local management with all effor ts, Fig. 1) non- healing chronic open wound. At the time of admission, patient’s Vital signs revealed a hear t rate of 68/min, respiratory rate of 26/min, and rectal temperature of 36.5°C. General examination- the patient is bedridden for years, all the system revealed nor mal except musculoskeletal & nervous system, which showed spastic muscular disorder and Parkinson disease. The wound was surrounded by large ulcerative skin lesion almost confluent with the spine. The surrounding areas of the lesion revealed slough and blackish necrotic debr is. The total wound area covered about 6 by 8 inches (Fig. 1). The wound details are indicated in Table I. Investigations revealed hemoglobin- 10.7g/dl; TLC - 4700 cells/cu mm; DLC- N60 and L40; absolute Neutrophil count- 2820 cell/cu mm; μ, ESR - 16 mm; and adequate platetets. Peripheral smear revealed microcytic red cells and mild leucopenia. Liver and Kidney profile revealed normal limit, Urine microscopy and cultures were positive repeatedly. Blood culture was sterile. Chest X-ray and Spine showed severe osteoporotic change.Ecg showed old MI, Echcadiography showed mild LVH. Pus culture and sensitivity was perfor med at weekly intervals which yielded Pseudomonas initially. Subsequent cultures showed Staphylococcus aureus and E-coli species which is supposed to be from contamination w ith urine . Appropriate antibiotics were given time to time, throughout the hospital stay including intravenous Ciprofloxacillin (100 mg/ kg/day) and amikacin (15 mg/kg/day) for 15 days. Antibiotics were subsequently changed both for wound and urine infection, to netilmycin (6 mg/kg/day for 7 days), cefotaxime (100 mg/kg/day for 10 days), Kenamycin (500 mg 12 hourly for 7 days) and Clindamycin (300mg 12 hourly for 10 days duration). Alternate day cleaning and dressing of the wound was done with Betadine, E-usol and Hexisol. TABLE -1 Morphology of the Wounds Before and After Therapy- Woun d Before debridement After debridement/ closure Woun d parame ters- Prior to Therapy End of Therapy Prior to End of Therapy Therapy Margin Irregular & indurated Partially Reg ular Sutured In tacked ski n Floor Unhealt hy, Almost Healthy granulation Necrotic Oozing Tissue tissue Covered Covered Base Partially Clear Spine Spine bone Exposed granulation tissue bone co vered Covered Surrounding s kin/Contraction Up to Inflamed and scar ed Partially Healthy Healthy mark Healthy Discharge Profuse purul ent Oozing pus Serous Dis charge No discharge No discharge Irradiance Parameters LED Apparatus: BioLux MD Beam source (Incoherent-Ga-Al-As) Irradiance dose:4- 8 J/cm2/min. Irradiance time: 1- 2 minutes Mode: Continuous wave Wavelengths Used: 660 nm Total session: 35 The parameter s which have been found to be most effective are in the range of 90 sec/cm2 of open wound surface, with the lase r beam set at a pulsed rate of 40-80 pulses per second (PPS), depending upon the chronicity of the lesion. The more chronic, the slower the pulse rate suggested. The optimum distance from probe tip to target surface was 1-4 mm. Probe motion during lasing was a slow, circling movement over each square centimeter of open lesion, timed to per mit the suggested dosages. As the lesion is large, i.e., 4-6 cm in diameter, a change in technique is adopted which involves a slow, traversing of the perimeter of the lesion, allowing approximately 90 sec per linear centimeter of the perimeter, at the suggested distances (1-4 cm). This technique apparently provides sufficient exposure to the laser beam to stimulate healing effectively, compared with non- treated areas and previously experienced wound management of a similar nature. Treatment Schedule (Dose duration and wound parameter) Period/ Wound Irradiation Energy Week Frequency Area/size Source Wave Fluence Point Time 2 2 1-2 week 5/ week 6.8 cm LED-660 nm (Ga-Al-As) Continuous 6 joules/cm 2 8 j oules/min. 3-5 week 3/ week 5.7 cm2 LED-660 nm (Ga-Al-As) Continuous 4 joules/cm2 2 8 j oules/min. 4-6 week 3/ week 4.4 cm2 LED-660 nm (Ga-Al-As) Continuous 4 joules/cm2 1 8 j oules/min. 2 2 7-8 week 2/week 2.2 cm LED-660 nm (Ga-Al-As) Continuous 3 joules/cm 1 8 j oules/min. 9-10 week 2/ week Closed LED-660 nm (Ga-Al-As) Continuous 3 joules/cm2 1 8 j oules/min. Low energy Ga-Al laser provides infrared rays in the wave length of around 660 nm by continuous mode. An average power of 5-8 mw was provided through a fiber optic delivery system around the wound margin for about 8-10 min at each point at a distance of one cm. Since the center of the ulcer was deep, it was decided to give laser therapy concentrating maximum irradiance there (Fig. 1). At the end of 2nd week there was an improvement in laser irradiated side with respect to ulcer size and wound margin and there was serous discharge after eight exposures (Table I). Surgical debridement was done 2 times: at 3 rd & 5th week and finally secondary closure given at the end of 8th week, within in the treatment period ( two and half months). A healthy granulation tissue appeared by 6-7th week. Fig. 6/7 reveals the post laser therapy ulcer on 6-7th week. At this stage, the center of the ulcer was still unhealthy and significant signs of healing. It was decided to irradiate the center of ulcer also with maximum per mitted irradiance dose.At the end of 8th week the wound looked pretty healthy, we decided to do secondary closure instead of skin grafting, as because of old age, we could mobiles the wound adjacent skin w ithout tension and closed the wound by four point suture. And nex t two weeks we observed the wound sur face w ith a keen observation, and continued to two days interval la ser therapy ( Total treatment 25), and medications properly. Enhancement of healing processes with open lesions is described. The effective parameters were deter mined to be a pulsed beam at 40-80 PPS, administered at a target distance of 4 mm, for 90 sec/c m2 of open lesion sur face. In addition, lasing along the perimeter of the larger wound was indicated to overcome the diminished penetration of the laser beam through the hardened eschar overlaying the lesion. No untoward reactions or side effects were reported by the patient. 13 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. Chronology of management & improvement: At the 5th day of her admission in our ward, we simultaneously star ted conservative treatment and He-Ne laser therapy. After wound cleaning, standardized digital photos were recorded weekly. At the end of 2nd week of treatment; the outlook of the wound looked better, and signs of increased vascular ity in the surrounding area noted. But serous discharge continued. At the end of 3rd week of treatment, we did wound debridement, and continued alternate day dressing and as well as laser therapy. At the 4th week of treatment, we continued to care the wound by alterna te day dressing and medications, and noticed for mation of granulation tissue at the margin of the wound. At the end of 5th week of treatment, we again debrided the center of the wound with care so that the adjacent healthy tissue could be preserved. At the end of 6th week of treatment, we observed marked improvement in the wound, and at the same time signs of vascular marking noted at the wound margin. At the 7th week of treatment, we noticed general condition of the wound healthy, and signs of healthy granulation tissue all over the wound, we continued to two days interval dressing. No discharge noted. At the end of 8th week of treatment, we did surgical toileting and secondary closure of the wound, and continued to give conservative & laser therapy twice weekly until 10th week of treatment. At the end of 9th week of treatment we observed a nice and healthy wound margin, and reddish vascular marking all over the wound sur face area. At the end of 10th week of her admission we assessed her wound and surround areas for any sign of wound dehiscence, infection, vascularity of the wound as well as adjacent areas was up to mark, and being satisfied discharged her with smile. Our purpose was to assess potential changes in healing due to LLLT over time using a human experimental wound model. Healing was measured in ter ms of wound contraction and changes in chromatic red and luminance. Chromatic red is an indication of wound healing as a wound changes in color from dark red to pale pink over time. Luminance refer s to the homogeneity of a wound as the tissue heals and becomes mo re smooth and consistent. In addition to laser therapy (Led) and surgical intervention, adjacently she was also given the following management: o Pneumatic Bed support. o Catheterization. o High protein diet, o Antibiotics (according to culture and sensivity test), o Pain killer (Non-NSID) o H2 Blocker- Pantoprazole and Ranitidine. o Tab- Perkinil, 5mg 12 hourly, o Tab- Ecosporin(Aspirin) o Vitamin B-complex, anti-oxidant, Vitamin D3 , o Iron, Folic acid and Zinc supplements, o Calcium, o Fresh blood transfusion, Chronological Picture View: 1ST DAY, 24TH OCTOBER/2009 Pretreatment photograph of wound showing irregular margins, Necrotic tissue and pus discharge. F-1: At the end of 1 st Week F-2: At the end of 2 nd week. F-3a: At the end of 3 rd week. F-3b: At the end of 3 rd week. F-4: At the end of 4 th week. F-5: At the end of 5 th week. F-6: At the end of 6 th week F-7: At the end of 7 th week F-8b: At 9th week F- 10a: At 10th week . F-10b: At The End Of 10t h Wk. F-10c: At The End Of 10t h Wk (9rd post operative day) (12rd post op. day) 14 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. Discussion Our patient demonstrated a significant benefit of Ga-Al-As 660 laser for rapid healing of skin wound. The comparison between the laser and conventionally treated previously treated wounds of the same patient at about same size clearly highlighted that despite unifor mity of host factors, local factors and systemic state, the wound healing process was stimulated on the laser ex posed side. Healing of wounds is an impor tant problem faced by general & or thopedic surgeons. The possible biostimulatory role of laser light in wound healing is of recent interes t (l). Small sub destructive repetitive doses of laser light are claimed to be useful for trophic ulcers and indolent wounds (2). The proposed mechanisms of action include local leukocyte proliferation^), neovascularization, fibroblastic proliferation and rapid epithelialization (4, 5). All these mechanisms possibly lead to more rapid closure of wounds and stronger scar for mation. In an experimental study, wounds treated with Ga-Al-As 660 laser revealed significantly more granulation tissue. This study established the biostimulatory effects of low intensity laser radi ation (6). Many repor ts now indicate benefit to non healing wounds and trophic ulcers by low-intensity laser irradiation. Out of 351 patients thus treated, 236 showed complete epithelialisation of the wound sur face (7). Twelve intractable venous ulcerations were treated by vascular surgeons in Ireland. A 44% increase in healthy granulation tissue was observed, 2/12 ulcers healed completely while 27% revealed reduction in size of the remaining ulcers indicating considerable benefit (8). Nussbaum et al. (9) in a study compared- the effect of ultraviolet-C and laser for treatment of pressure ulcers in adults with spinal cord injury. They used 660-980 nm wave length light at an energy density of 4 J/cm2. Weekly percentage changes in wound area were compared. The authors concluded exposure to UV-C decreased healing time and allowed faster return to rehabilitation programs. The UV-C light was better than the laser (9). Another nonrandomized study of laser and UV lamp on chronic skin ulcers suggested that wounds which fail to respond to topical treatments benefit from either modality (10). Evaluations of different approaches to wound healing are complicated by the large number of factors that influence wound healing. Although there are anecdotal repor ts of successful therapy, there are few well controlled studies. The use of lasers for healing wounds is becoming increasingly attractive to surgeons. A number of animal and in vitro studies (11, 12) have demonstrated that laser irradiation has a significant effect on components of tissue repair. Conclusion This study results efficacy of LLLT on wound healing in human model, and indicates that it can be a very impor tant adjective tool /modality for chronic intractable wound management, and in any way it is not har mful to human being. In the past Laser / LED were shown to be effective in wound management but in different degrees, some of those applications showed significant improvement some less effective other s no effect. Probably laser/ LED Irradiation parameters are vital for its Biostimulative effects. Inference of those results summaries that irradiation parameters are of vital to laser therapy. We used an optimal dose of irradiance which proved to be most effective biostimulation on human application Application and research of LLLT on cell responses of wounded skin fibroblasts demonstrate that correct energy density or fluence and number of exposures can stimulate cell responses of wounded fibroblast and promote cell migration and cell proliferation by stimulating mitochondrial activity and maintaining viability without causing additional stress or damage to wounded cells. Results indicate that the cu mulative effect of lower doses determines the stimulatory effect, whereas multiple exposure at higher doses result in an inhib itory effect with more damage. 39 Although various studies have ex tensively covered the effects of laser radiation on tissue, many unanswered questions remain. The mechanisms effectively responsible for cell mitotic activity has not been clarified yet, and there is no standardized ideal dose for stimulating tissue healing. Therefore, we noted that there is a need for resear ch on the action and parameters of low -intensity laser use in cutaneous lesions during the different stages of repair, as an attempt to elucidate how this method acts at a cell level in healing processes. Elucidation of these issues will enable the establishment of criteria on the true benefits of laser therapy in diseases that need healing stimulation, minimizing healing time and the complications that may occur during the clinical progress of these wounds. In addition, experimental studies indicated that the LLLT may be an impor tant therapeutic tool to stimulate wound healing in decubitus ulcer patients. In conclusion, the present repor t highlights the possible utility of Galliium-Aluminium laser at 660 wavelengths is as effective as Helium- Neon laser as an adjunctive modality for wound healing in skin/general & or thopedic practices. Acknowledgements We are grateful to Professor & director- A.K.M. Mujibu Rahman, Dr. Mir. Mahamuda Khanam- Assistant Director, Shaheed Suhrawardy Medical College Hospital. Thanks to Professor. Prof. F. H. Serazee- Ex. Head of the Department, 1Department of Orthopedic & Traumatology, Shaheed Suhrawardy Medical College Hospital, Assistant Professor. Dr. Sayed Shaheedul Islam- National Institute of Traumatology and Or thopedic Rehabilitation, Dhaka-1207, Bangladesh- for their continuous suppor t during the whole period. Also we are grateful to Md. Abdul Aziz, Ms. Farida, Fatema and Md. Abul of SSMCH for continuous services to the patient, Ms. Mrinali- in-charge, ward-6, and Ms. Shewly- operation theater in-charge for their nice co-operation. And Mr. Sinha Abu Khalid, CEO- Lab Neucleaon (Laser system provider), and Ms. Jannat, Ms. Charity(Laser operator) for their continuing effor ts to treat the patient for this long period. 15 LLLT, Low Level Laser (LED-Ga-Al- As 660) Therapy – On soft Tissue Healing: Review, Mechanism, Experimental application and A case report. 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Avevbakh MM, Sorkin MZ, Dobk VG, Kosarev II, Ostapehenko EP. Effect of Helium - Neon laser on the healing of aseptic exper imental wounds. EKSP Khir Ana esteziol 1976; 3: 56 -61. 5. Mester E, Spiry T, Szends B. Effect of laser rays on wounds healing. Bull Soc Int Chir 1973; 32:169-172. 6. Bisht D, Gupta SC, Misra V, Mittal VP, Sharma P. Effect of low intensity laser radiation on healing of open skin woundsin rats. Indian J Med Res 1 994; 100: 43- 46. 7. Georga dze AK, Karpov VI, Kuznetsov EV, Solda tov AV, Rukosuev VP. Tr eatment of non-healing wounds and trophic ulcers by low-intensity laser irradiation in 8. Sugrue ME, Careolan J, L een EJ, Feeley TM, Moore DJ, Shanik GD. The use of infrared laser therap y in the tr eatment of venous ulceration. Ann Vase Surg 1990; 179-181. nd 9. Nussbaum E L, Biemann I, Mustard B. Comparison of ultrasound/ultraviolet -c a laser for tr eatment of pr essure ulcers in patients with spinal cord injury. Physical Therapy 1994; 74: 8 12-823. 10. Crous L, Malherbe C. Laser and ultraviolet light irradia eatment of chronic ulcers. Physiotherapy 1988; 44: 73-77. tion in the tr 11. Young S. Dyson M, Bottom P. Effect of light on Calcium uptake by macrophages. Presented at a Seminar on Laser Bi omodulation at Guy's Hospital, London, 1991. l in. 12. El Sayed S, Dyson M. Comparison of the effect of multiwavelength light produced by a cluster of semiconductor diodes and of e ach individua diode onmast cell number and degranulation in intact and injured sk Laser Surge Med 1990; 10: 559-568. Research Panel Members: 1. Dr. Md. Nazrul islam, MBBS, M.Sc.(Biomedical Engineering). Resident Surgeon, Department of Orthopaedics’ & Traumatology, Shaheed Suhrawardy Medical College Hospital, Dhaka-1207. Bangladesh. 2. Professor Golam Abu Zakaria, ph. D Dept.of Medical Radiation Physics,Kreiskrankenhaus Gummersbach, Teaching Hospital of the University of Cologne, 51643 Gummersbach, Germany.Dept. of Medical Physics and Biomedical Engineering, Gono -Bishwabidyalay (Gono University), Mirzanagor, Nayarhat, Savar, Dhaka- 1344, Bangladesh. 3. Professor F. H. Sirazee, MBBS,MS Department of Orthopedic and Traumatology, Shaheed Suhrawardy Medical College Hospital, Dhaka-1207, Bangladesh. 4. Associate Prof. Paritosh Chandra Debenath, MBBS, MS Department of Orthopedic and Traumatology, Shaheed Suhrawardy Medical College Hospital, Dhaka-1207, Bangladesh. 5. Assistant Professor kazi Shamimuzzaman, MBBS, MS Department of Orthopedic and Traumatology, Shaheed Suhrawardy Medical College Hospital, Dhaka-1207, Bangladesh. 6. Assistant professor Quamrul Akhter sanju, MBBS, FCPS, MRCS. Department of Surgery, Shaheed Suhrawardy Medical College Hospital, Dhaka-1207, Bangladesh. 7. Assistant Professor Ashraf Uddin Khan, MBBS, DMRD, FCPS. Department of Radiology & Imaging, Shaheed Suhrawardy Medical College Hospital, Dhaka-1207, Bangladesh. 8. Dr. Sajal Kumar Majumdar, MBBS, MS. Department of Pediatric Surgery, Shaheed Suhrawardy Medical College Hospital, Dhaka-1207, Bangladesh. 9. Dr. Subir Hossain Shuvro. MBBS, National Institute of Traumatology & Orthopedics’ Rehabilitation, Dhaka-1207, Bangladesh. 10. Sinha Abu Khalid, B. Sc. (Hons.,) Applied Physics & Electronics, Dhaka University. Member, American Society for Laser Medicine & Surgery, CEO, LabNucelon CTS, Dhaka- 1000, Bangladesh. 11. Muhammad Masud Rana, M.Sc. (Medical Physic ist, National Institute of Cancer Research and Hospital, Dhaka, Bangladesh). 17
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