Bone In-growth Induced by Biphasic Calcium Phosphate Ceramic in Femoral Defect of Dogs by materialresearch


									              Journal of Biomaterials

 Bone In-growth Induced by Biphasic Calcium Phosphate Ceramic in
                     Femoral Defect of Dogs
                 I. Manjubala, T. P. Sastry and R. V. Suresh Kumar
                           J Biomater Appl 2005; 19; 341
                         DOI: 10.1177/0885328205048633

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       Bone In-growth Induced by
       Biphasic Calcium Phosphate
        Ceramic in Femoral Defect
                 of Dogs

                     I. MANJUBALA1,2,* AND T. P. SASTRY2
                              Materials Science Centre
                           Department of Nuclear Physics
                       University of Madras, Guindy Campus
                             Chennai – 600 025, India
                         Central Leather Research Institute
                          Adyar, Chennai – 600 020, India

                                      R. V. SURESH KUMAR
                       Department of Surgery and Radiology
                           College of Veterinary Science
                            Tirupati – 517 502, India

ABSTRACT: Biphasic calcium phosphate (BCP) ceramics consisting of
hydroxyapatite (HA) and tricalcium phosphate (TCP) has been used as a bone
graft material during the last decade. In this paper, we report the bone
in-growth induced by BCP ceramic in the experimentally created circular defects
in the femur of dogs. This BCP ceramic consists of 55% hydroxyapatite (HA) and
45% b-tricalcium phosphate (TCP) prepared in situ by the microwave method.
The defects were created as 4-mm holes on the lateral aspect of the femur
of dogs and the holes were packed with the implant material. The defective
sites were radiographed at a period of 4, 8, and 12 weeks postoperatively. The
radiographical results showed that the process of ossification started after

*Author to whom correspondence should be addressed. Present address: Department
of Biomaterials, Max-Planck Institute of Colloids and Interfaces, Potsdam, Germany.

JOURNAL OF BIOMATERIALS APPLICATIONS Volume 19 — April 2005                                         341
        0885-3282/05/04 0341–20 $10.00/0   DOI: 10.1177/0885328205048633
                             ß 2005 Sage Publications

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342                                        I. MANJUBALA        ET AL.

4 weeks and the defect was completely filled with new woven bone after 12 weeks.
Histological examination of the tissue showed the formation of osteoblast
inducing the osteogenesis in the defect. The collageneous fibrous matrix and
the complete Haversian system were observed after 12 weeks. The blood serum
was collected postoperatively and biochemical assays for alkaline phosphatase
activity were carried out. The measurement of alkaline phosphatase activity
levels also correlated with the formation of osteoblast-like cells. This microwave-
prepared BCP ceramic has proved to be a good biocompatible implant as well as
osteoconductive and osteoinductive materials to fill bone defects.

KEY WORDS: biphasic calcium phosphate, bioceramic, bone filling,
osteogenesis, osteoblast, osteoinduction.


I  n recent years, many efforts have been directed toward the
   development of osteoconductive materials composed of various
calcium phosphate compounds, because of their close resemblance to
the body’s hard tissue mineral component. The calcium phosphates
are not only biocompatible and osteoconductive that stimulate new bone
growth to creep along the implant from the interface contacting the host
bone, but also forms bone bonding with the host bone [2,10,14,15,22].
The absence of inflammatory reactions and bone bonding capacity were
considered to be the basic profile of calcium phosphate implants as bone
substitutes. Thus, osteoconductive calcium phosphate materials would
aid in wound healing of bone defect by acting, preferably temporarily, as
a scaffold for bone in-growth and bridge between the host bone and
the implant. An ideal biomaterial for hard tissue repair should be
biocompatible, osteoconductive, resorbable, and osteoinductive [7,29].
Thus in recent years, attention has been directed to the development of
resorbable and osteoinductive biomaterials from calcium phosphates.
Therefore, for ideal bone substitutes, the biological and biomechanical
properties of the implant should resemble bone as much as possible.
For a material to be used as a bone graft or bone-filling material, the
material has to resorb or dissolve in a controlled way, and the result is
the new remodeled bone.
  The most widely investigated calcium phosphate compounds are
hydroxyapatite [HA, Ca10(PO4)6(OH)2] and tricalcium phosphate [TCP,
Ca3(PO4)2]. They differ not only in their composition but also in their
rate of resorption [16]. The mechanical strength of HA is higher than
TCP, while the biodegradation of TCP is more than HA at physiological
pH [6,8,20]. TCP degrades in an unpredictable way, i.e., it dissolves
12.3 times faster than HA in acidic medium and 22.3 times faster in basic
medium and so it may not provide a scaffold for new bone to grow [18].

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A controlled biodegradable osteoconductive material consisting of
a mixture of hydroxyapatite and b-tricalcium phosphate in different
ratios called as biphasic calcium phosphate (BCP) ceramic was developed
which will not produce a permanent indwelling foreign body and will
eventually provide greater room for bone in-growth [23]. The BCP is
soluble and gradually dissolves at the implant site, seeding new bone
formation as it releases calcium and phosphorus ions into the biological
medium. It appeared that resorption of calcium and phosphate is
beneficial to bone formation and that free Ca2þ ions could be considered
as the origin of bioactivity. This is the case when resorption rate of
calcium phosphate is within a certain limit, achieved by BCP with lower
TCP content. Too much of dissolved Ca2þ and PO4 ions leading to a

sharp change of the microenvironment, may disturb the activity of host
cells and create an adverse effect on tissues. Thus, the high dissolution
rate of TCP ceramics was found to be harmful for bone formation and
detrimental to the newly formed bone [28].
   It is well known that the mineral constituent of human tissue shows
a biphasic nature after sintering at high temperature. The development
of BCP ceramics has provided an ideal calcium phosphate whose
bioactivity and biodegradability is controlled by the HA/TCP ratio that
promotes material resorption/bone substitute events [6,12,18,23].
The bioperformance of biphasic calcium phosphate ceramics is related to
the dissolution–deposition of apatite calcium phosphate at the implant
interface and may be influenced by physical and chemical factors [6].
Many researchers have reported and proved the osteoinductive behavior
of calcium phosphate ceramic, i.e., to induce bone tissue formation in the
nonosseous site or environment [25,26]. The osteoinductive behavior
of calcium phosphate ceramics is considered as the intrinsic property of
the materials when implanted in the bone defect site as well in the
nonbony site [5,17,24,25]. Ripamonti and Yuan et al. [24,30] have
proved the osteoinduction in porous HA when implanted in dogs and
baboons, respectively and Cong et al. [4] has shown osteoinduction in
BCP ceramics swathed with periosteum. Zhang et al. [31,32] have ex-
plained the mechanism of osteoinduction in calcium phosphate ceramics.
   The in vivo and clinical studies using BCP implant material have
shown that bone in-growth into BCP particles is rapid [1,26]. Later, it
was reported that not only bone in-growth occurred but also osteo-
induction behavior of BCP was noticed at the bone-defect site in dogs.
However, the earlier reports on BCP studies were based on BCP powder
prepared by a mere mixture of HA and TCP powders.
   In our earlier report, we have focused on the development of in situ
BCP using the novel method of microwave processing in which HA

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344                                        I. MANJUBALA        ET AL.

and TCP are mixed at the atomic level and physicochemical properties
were reported [19]. The BCP thus prepared was formed in situ and not
by partial decomposition of HA, and there were no other phases except
HA and TCP. In the present work, we report the mechanism of bone
tissue formation induced by BCP ceramic implant in the experimentally
created femoral defect of dogs.

                            MATERIALS AND METHODS

Preparation of Implant Materials

  Analytical grade calcium hydroxide Ca(OH)2 (Loba Chemie, India)
and diammonium hydrogen orthophosphate (NH4)2HPO4 (Merck,
India) were used as the starting materials. The conditions for the
formation of pure HA using microwave irradiation were initially
optimized and reported earlier [19]. The Ca/P ratio of BCP has to be
optimized in between that of HA and TCP, and preparation conditions
are according to the phase diagram of CaO and P2O5 as shown in Figure 1.
For BCP preparation, equivolume of 0.252 mol/L diammonium phos-
phate solution was added very slowly to 0.4 mol/L calcium hydroxide

Figure 1. Equilibrium phase diagram of different calcium phosphates. The shaded region
shows the phases of interest for BCP formation (T1 ¼ 1360 C and T2 ¼ 1475 C).
(HA – Hydroxyapatite, TCP – Tricalcium phosphate, TTCP – Tetracalcium phosphate,
CaO – Calcium oxide).

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suspension under stirring conditions so that the Ca/P ratio is equal to
1.58. The solution mixture was immediately transferred to a domestic
microwave oven (BPL India, microconvention system, 2.45 GHz, 800 W
power) and irradiated with microwaves for 40–45 min. The operating
power of the oven was adjusted to be less than 400 W to avoid the
overflow of the solution from the reacting vessel during the reaction
inside the microwave oven. Care was taken to release the ammonia
gas evolved during the reaction. The irradiation was carried out until
the reaction was completed forming a thick solid gel, which was subse-
quently removed and dried in an oven. The dried solid was then ground
to fine powder using mortar and pestle.


  The ceramic powder was heated to 600 C so that the powder is neither
highly crystalline nor amorphous. The BCP powder was examined using
a high resolution X-ray powder diffractometer ( ¼ 1.540598 A, Seifert
XRD3000, Germany). The X-ray diffraction patterns were recorded in
steps of 0.02 /s.
  The BCP powder was sterilized in an autoclave at 114 C for 20 min.
The cytotoxicity was tested using fibroblast L929 cells. The BCP powder
was mixed with physiological saline to form a thick paste prior to
implantation in animals.

In Vivo Experiment

Surgical Procedure
   Three holes were created in the femur to evaluate the osteogenicity
of the material in 16 adult mongrel dogs with an average weight of
10–15 kg, divided into two groups of eight animals each. One group of
animals were treated as control without filling the defect and the other
group as BCP material-implanted group. The animals were anesthetized
using intramuscular injection containing xylazine and ketamine
(2 mg/kg body weight). A longitudinal incision of about 6 cm was created
on the lateral aspect of femur and the femur was exposed by the blunt
dissection method. Three holes of about 4 mm diameter were created
about 1 cm apart using a stainless steel drill and the holes were flushed
with saline solution to remove blood clots. The implant material was
filled in the holes and the surgical procedure is shown in Figure 2. After
implantation, the incision was closed in layers with resorbable sutures.
In the control group, the holes were left unfilled. The animals were

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346                                         I. MANJUBALA        ET AL.

                                                (a)                                                   (b)

                                                (c)                                                    (d)

Figure 2. Photographs showing the surgical procedure (a) lateral incision, (b) blunt
dissection, (c) created drill holes and (d) suturing after implantation in experimentally
created defective sites.

treated with antibiotics for five postoperative days and maintained
ad libitum diet.

Radiographic Analysis
  Sequential plain X-ray radiograph of the femur were taken at 4, 8, and
12 weeks after surgery. All radiographs were recorded using Tungsten
source (GE Medical Systems, USA) to follow the variation in density of
the external callus formation around the implant.

Histologic Analysis
  The animals were euthanized after 4, 8, and 12 weeks of surgery by
injecting an overdose of sodium barbitopental. Harvested implant sites
were cleaned off from adhering tissue, fixed in 10% buffered formalde-
hyde solution and decalcified using formic–hydrochloric acid mixture.
The tissue pieces were processed and embedded in paraffin wax. Serial
sections of 5 mm thickness were cut perpendicular to the bone axis
using a microtome (Leica 2055 microtome, Germany) and stained with
hemotoxylin and eosin. Photomicrographs of the histological sections
were taken using a light transmission microscope (Reichert Polyvar 2
Met) with an attached camera (Yashica, Japan).

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Serum Biochemical Analysis
  Serum collected after a period of 2, 4, 8, and 12 weeks were analyzed
for inorganic calcium, phosphorus content, and alkaline phosphatase
activity. Serum calcium and inorganic phosphorus content, and the
alkaline phosphatase (ALP) activity levels were measured using the
modified Lowry method provided by Raichem, Division of Hemagen
Diagnostics, Inc., USA. The reagents and the ready kits were purchased
from Raichem. The calcium, phosphorus content, and alkaline phos-
phatase activity levels were recorded spectrophotometrically at 650, 340,
and 405 nm, respectively.

Tissue Mineral Content Analysis
  To evaluate the extent of mineralization, the calcium content
of tissues at the implant sites was determined after 4, 8, and 12 weeks.
The tissue taken out from the implant sites was heated for 4 h at
700 C. The ash of each sample was dissolved in 3 mL of concentrated
hydrochloric acid and diluted to 100 mL. Later, the samples were
assayed for calcium and phosphorus content by comparison to standard
calcium chloride solution using a Varion Techtron Atomic Absorption


Material Characterization

   The observed X-ray diffraction pattern of the microwave prepared
BCP powder is shown in Figure 3. The pattern shows both the HA and
TCP phases formed in situ. The BCP powder was prepared using
wet chemical method by the following reaction and no other phases of
calcium phosphate was present in the final product except HA and TCP
(x ¼ 0.33 in this case).

10CaðOHÞ2 þ ð6 þ xÞðNH4 Þ2 HPO4 ! ð1 À xÞCa5 ðPO4 Þ3 OH
                                                      þ xCa3 ðPO4 Þ2 þ H2 O þ NH3"                ð1Þ

The mass fraction of the TCP phase present in the BCP ceramic is
calculated by the simple height law equation

                                                              I100 ðTCPÞ
        % mass fraction of TCP ¼                                              Â 100               ð2Þ
                                                       I100 ðTCPÞ þ I100 ðHAÞ

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Figure 3. X-ray diffraction pattern of biphasic calcium phosphate ceramic showing both
the phases of HA and TCP. HA peaks are indexed and b-TCP peaks are marked as (f).

where I100(TCP) and I100(HA) are the normalized integrated intensities
of the main peak of TCP and HA, respectively. The ratio of HA/TCP in
this BCP was calculated to be 60/40 approximately [19]. Since calcium
hydroxide and diammonium phosphate were used as precursors, there
was no formation of other products in the reaction except for HA
and TCP.

In Vivo Results

Macroscopic Observation
   The results of macroscopic visual observation of the animals
postoperatively indicated that the sites healed uneventfully with no
clinical evidence of inflammatory response of the ceramic implant and
no toxic signs during the healing period.

Radiographic Observation
  Radiographic observations are of importance in evaluating any
bone graft or implant material in any biological system. The series of
radiographs taken at different intervals provide continuous information

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Figure 4. Radiographs of defect after 4 weeks postoperatively (a) in control group
showing clearly visible radiolucent hole defects and (b) in BCP implanted group showing
union of material with host bone giving uniform radiographic density. No periosteal
reaction was observed.

about the fate of the implant in the system. In the control group, after
4 weeks of surgery, the defect showed uniform bone density with
radiolucent circular holes (Figure 4a). After 8 weeks, clearly visible
radiolucent holes in the femoral shaft was noticed (Figure 5a), with
fibrous tissue proliferation in the defect. After 12 weeks postoperatively,
less dense area suggesting fibrous tissue occupying the holes was noticed
with incomplete ossification (Figure 6a).
   In the BCP ceramic-implanted group, the radiographs taken after
a period of 4, 8, and 12 weeks are shown in Figures 4b, 5b, and 6b,
respectively. The implant material was strongly fit into holes and was
intact. There was no spill over of material from the defect site and
the process of ossification had started. The photographs show radiopaque
nature of the defect site and radiographic evidence of callus formation
indicating bone defect healing after 4 weeks. There was evidence of
establishment of clear union of implant materials with the host bone
giving uniform radiological density. There was also increased radio-
density around the implanted material which had filled the bony defect
and the complete bridging of the defective site was observed after

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350                                        I. MANJUBALA        ET AL.



Figure 5. Radiographs of defect after 8 weeks postoperatively (a) in control group
showing clearly visible radiolucent holes and (b) in BCP implanted group showing the
ossification process.



Figure 6. Radiographs of defect after 12 weeks postoperatively (a) in control group
showing fibrous tissue formation in holes with incomplete ossification and (b) in BCP
implanted group showing perfect union of material with the host bone in the defect.

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12 weeks. There was no periosteal reaction or host rejection observed. The
implants were exactly merged with the experimentally created defects.

Histologic Observation
  In the control group, after 2 weeks postoperatively, an area of necrotic
bone as well as proliferation of fibroblasts with abundant collagen at
the level of defect were found over the periosteum (Figure 7a). It was
observed that the bony trabeculae was yet to bridge the defect. After
12 weeks postoperatively, woven bone seems to be filling up the gap and
the margin of the defect was clearly visible and the new bone was formed
at the level of defect (Figure 7b).
  The photomicrographs of the sections of BCP implanted area are
shown in Figure 8a–d after a period of 2, 4, 8, and 12 weeks. After
2 weeks of implantation, trabecular bone formation was observed at the
level of defect. Osteogenic activity had started and the periosteum was


                                                                                 x 100


                                                                                x 100
Figure 7. Photomicrographs of decalcified section of the defect site in the control
group (H&E stain) (a) 4 weeks postoperatively showing trabecular bone formation and
(b) 12 weeks postoperatively showing new woven bone formation in the defect
(Magnification 100Â).

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                                               (a)                                                      (b)


                                           x 320                                                      x 320

                                               (c)                                                      (d)

                                           x 320                                                      x 100

Figure 8. Photomicrographs of histological decalcified section of BCP implanted defect
site (H&E stain): (a) at 2 weeks postoperatively showing collagen rich fibrous tissue with
the presence of osteogenesis (Magnification 320Â), (b) at 4 weeks postoperatively showing
osteoblast activity along with new bone formation, (c) at 8 weeks postoperatively showing
fibroblast and osteoblast in the new bone region (Magnification 320Â) (Insert shows the
magnified image of (c)) and (d) at 12 weeks postoperatively showing complete bone
growth of periosteum restoring the Harvesian system (Magnification 100Â). (O – Osteoblast,
F – Fibroblast, H – Haversian system, C – Collageneous fibrous tissue.)

partially bridged. The defect was filled with new bone growth and the
osteoblastic activity with osteogenesis was prompt. After 4 weeks,
the osteoblastic activity along with new bone formation was seen. The
gap was bridged by collagenous fibrous tissue and newly formed vessel
inducing osteogenesis was evident. The presence of osteoid tissue and
the active osteoblast confirms the bone growth activity. After 8 weeks,
the defect was completely bridged at the level of the periosteum and the
new bone formation was evident. The outer layer of the defect was
entirely filled up by new bone tissue. The fibroblasts found at the level
of the periosteum were transformed into osteoblasts. After 12 weeks of
implantation, there was complete bridging of the defect with the
restoration of the Haversian canal at the level of defect. The histological
observation indicated that after 12 weeks of implantation, the bone
tissue had completely filled the defect and a considerable amount of
mineralized tissue was observed. The presence of osteoid tissue and

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active osteoblast along with the Haversian system indicated that the
bone tissue apposition was almost complete.

Serum Analysis
   The calcium content is a direct measure of the extent of mineraliza-
tion during bone healing and fracture. The changes in serum calcium
levels are shown in Figure 9a. The serum inorganic phosphorus content
was observed to increase initially and then decrease to the normal level
as seen from Figure 9b. Alkaline phosphatase (ALP) was considered
to play an important role in bone formation. ALP is an enzyme that is
tightly bound to the cell membrane and can be used as a marker of
osteoinductive cells. The variation in the ALP activity is shown in
Figure 9c.

Tissue Mineral Analysis
  Calcium content is a direct measure of the extent of mineralization
during the bone fracture healing. Calcium and phosphorus content
was determined in the implanted site after 2, 4, 8, and 12 weeks post-
operatively and the values are shown in Figure 10a and b. The calcium
and phosphorus content in the materials implanted group was higher
than the control group showing that the calcium and phosphate ions
are released from the implant material after implantation. After the
formation of the new bone, during the remodeling process, the calcium
and phosphorus values decrease to the normal value.


   Calcium phosphate ceramics are known to be osteocompatible and
osteoconductive. Osseointegration of calcium phosphate ceramics has
been proved to depend on the chemical composition of the ceramic.
The frequently used calcium phosphates as implant materials are HA
and TCP, of which TCP is biodegradable. Therefore, the dissolution
rate of BCP depends on the ratio of HA to TCP. Bone regeneration
was reported in BCP ceramic with various ratios of HA and TCP
varying from 85/15 HA/TCP [13] to 20/80 HA/TCP [21] and the best
ratio of HA/TCP is not yet known. The low sintering temperature
is beneficial to calcium phosphate-induced osteogenesis, since amor-
phous is highly soluble and the more sintered body is highly dense
and nonresorbable.
   The b-TCP ceramic has been reported to dissolve more quickly than
HA in physiological environment. This property of TCP interferes
with the ossification process at the point of contact with the implant.

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354                                                           I. MANJUBALA        ET AL.

                                     (a)                                                                         BCP

          Calcium mg/dl


                                       0           2            4           6            8           10          12        14

                                                                    Period (weeks)

         Phosphorus (mg/dl)




                                           0           2            4         6            8          10          12       14
                                                                        Period (weeks)
Figure 9. The observed variation in the serum (a) calcium, (b) inorganic phosphorous
content and (c) serum alkaline phosphatase activity levels in both the control and the BCP-
implanted groups.

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                                                                Bone In-growth Induced by BCP                                               355

                                                          (c)                                                            BCP
          Activity levels (mU/ml)



                                                           0             2        4           6          8          10          12     14
                                                                                   Period (weeks)

                                                                             Figure 9. Continued.


                                    Calcium wt %




                                                               0             2        4           6          8        10         12
                                                                                 Period (weeks)

Figure 10. The observed variation in the tissue mineral (a) calcium and (b) phosphorous
content in both the control and the BCP-implanted groups.

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356                                                   I. MANJUBALA        ET AL.

                           30           (b)                                                         Control
         Phosphorus wt %






                                    0          2          4           6          8         10         12        14
                                                          Period (weeks)
                                                   Figure 10. Continued.

The calcium released from TCP, being secondary messenger for cells,
interferes with cell differentiation at the point of contact. Therefore, the
partial dissolution of TCP in BCP ceramics produces an increased local
concentration of calcium and phosphorus ions with subsequent
precipitation of the apatite crystals that makes the collagen fibrils
mineralization easier. The BCP ceramics have intermediate degradation
behavior so that their progressive resorption and ultimate replacement
are adapted to bone growth. Therefore, it was suggested that the
observed osteoconductive potential of the BCP may be related to this
degradation behavior. The resorption of calcium phosphate ceramics
occurs because of either chemical dissolution due to the circulation of
biological fluids or due to cellular degradation by both macrophagic cells
and osteoclasts. The ceramic solubility influences the osteoclast
resorption activity and a very high solubility with pure TCP can inhibit
this activity because too much of calcium is released in the cell
resorption microenvironment [11]. BCP ceramics having intermediate
composition thus apparently undergoes cellular resorption activity
involving osteoclasts [27]. The fibroblasts differentiated into osteoblast
produces suitable environment for osteogenesis. As reported by
Ducheyne and Qiu, the osteogenesis event can take place as a sequence

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                           Bone In-growth Induced by BCP                                           357

of the following reactions: dissolution from the ceramic, precipitation
from solution onto ceramic, ionic exchange, deposition of mineral phase
with or without integration of ceramic, cell attachment, proliferation,
differentiation, and extracellular matrix formation [9].
  The calcium phosphate ceramics may possess osteoconductivity
and osteoinductivity in proper biological environment, and the bone
formation can be analyzed in terms of osteoinductivity [3,32]. The
formation of bone cells and the trabecular bone within 4 weeks is
an indication of increased osteoinductivity of the material. It may be
inferred that new fibroblast and osteoblast cells directly proliferating,
differentiating, and creeping into the surface of the material filling the
defect as an indication of osteoinduction and bonding them chemically
to complete biological integration between materials and the existing
bone creating osteoconduction. There is no formation of chondrocytes
and therefore, there is induced ossification and new bone formation.
The bone tissue induced by calcium phosphate ceramic is not due to
pathological calcification around the surface of calcium phosphate
  The enhancement of phosphorus level in blood may be considered as
the releasing of the phosphate ions from BCP due to degradation. The
increase in the ALP levels is overlapped with the appearance of active
osteoblast cells as evident from histological results with the activity level
achieving a peak at 4 weeks postoperatively. During fracture healing,
increased osteoblastic activity accounts for serum enzyme because the
rate of release of enzyme into serum will exceed its rate of inactivation.
Thus, the integration between the bone and the BCP ceramics is not
merely a simple connection, but a series of biochemical and physiological


   The biphasic calcium phosphate ceramic prepared by microwave
irradiation demonstrates good tissue biocompatibility and has pro-
duced no adverse effects. The radiographic and histologic evaluation
confirms progressive growth of new bone into the defect. The BCP
ceramic has controlled biodegradability, thus producing calcium ions
in the microenvironment inducing the bone formation and gradual
remodeling of the new bone. The histological results showed the
induced osteogenic process as a regular bone formation process. The
new bone formation process is not due to pathologic calcification but
due to the formation and resorption of cells inducing the remodeling
of the bone.

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358                                       I. MANJUBALA        ET AL.


  The authors gratefully acknowledge the help rendered by
Dr. Makeena Sreenu, Dr. P. Veena, and Mr. Vishwanath Reddy during
the animal experiments. The histologic analysis from Dr. Murali
Manohar, Madras Veterinary College is gratefully acknowledged.


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