- Email: [email protected]
Evaluation of new porous β-tri-calcium phosphate ceramic as bone substitute in goat model
Available online at www.sciencedirect.com
Small Ruminant Research 75 (2008) 144–153
Evaluation of new porous -tri-calcium phosphate ceramic as bone substitute in goat model Samit K. Nandi a,∗ , Samir K. Ghosh b,1 , Biswanath Kundu b,1 , Dipak K. De a,2 , Debabrata Basu b,1 a
West Bengal University of Animal and Fishery Sciences, 37 & 68, Kshudiram Bose Sarani, Kolkata 700037, India b Central Glass and Ceramic Research Institute, 196, Raja S.C. Mullick Road, P.O.: Jadavpur University, Kolkata 700032, India Received 8 February 2007; accepted 13 September 2007 Available online 14 November 2007
Abstract The present study was carried out to evaluate the porous -tri-calcium phosphate (TCP) (prepared by aqueous solution combustion technique) as bone substitute and compared with normal healing in 12 adult Black Bengal goats on the basis of clinical and radiographic findings, histological studies, oxytetracycline labeling, angiography studies (on day 90). Bone defects created in the diaphysis of radius were left unfilled in control animals (group I); while in treated (group II) animals the defects were filled with porous TCP blocks. The three months study showed no marked acute inflammatory reactions in all animals, wound healing was uneventful and the implants were clinically stable in the bone. Radiological studies showed presence of unabsorbed implants which acted as a scaffold for new bone growth across the defect whereas in control animals the defect was more or less same except that the newly formed bony tissue was less organized. Histological section showed moderately differentiated lamellar bone in the cortical part with presence of woven bone at peripheral cortex whereas control animals showed moderate fibro-collagenisation and good amount of marrow material, fat cells and blood vessels. Oxytetracycline labeling study showed crossing over of new bony trabeculae along with presence of resorption cavities within the new osteoid tissues whereas in group I, the process of new bone formation was active from both the ends; the defect site appeared as a homogenous non-fluorescent area. Angiogram of the animals in control showed uniform angiogenesis in the defect site with establishment of trans transplant angiogenesis, whereas in group II there was complete trans transplant shunting of blood vessels communication. The results of this study pointed out that the porous TCP promoted extensive bone formation over the entire extension of the defect in comparison to control group, thus conforming their biological osteoconductive property. © 2007 Elsevier B.V. All rights reserved. Keywords: -Tricalcium phosphate; Bone defects; Radiography; Histology; Oxytetracycline labeling; Angiography
∗ Corresponding author. Tel.: +91 33 25569234; fax: +91 33 25571986. E-mail addresses: [email protected] (S.K. Nandi), [email protected] (S.K. Ghosh), biswa [email protected] (B. Kundu), professor [email protected] (D.K. De), [email protected] (D. Basu). 1 Tel.: +91 33 24733469/96/76/77; fax: +91 33 24730957. 2 Tel.: +91 33 25569234; fax: +91 33 25571986.
0921-4488/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2007.09.006
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
1. Introduction Bone grafts are often necessary to provide support, fill voids, and enhance biologic repair of osseous defect. Although autogenous bone is considered the gold standard, to which other methods are compared, autograft has significant limitations, including donor site morbidity, inadequate availability in inappropriate form (Summers and Eisenstein, 1989; Banwart et al., 1995). Allograft bone has been widely used, which of course have several problems encountered when used, including immunogenicity and disease transmission such as hepatitis and HIV (Friedlander, 1983), loss of biologic and mechanical properties secondary to its processing and increased cost. Consequently significant strides have been made to search ideal bone graft substitutes. Various synthetic materials have been developed as bone substitute which include marine coral, hydroxyapatite (HAp), bioactive glasses and synthetic polymers with chemical composition and crystallinity equivalent to that of the mineral phase of natural bone (Heise et al., 1990; Peltola, 2001). Tri-calcium phosphate offers excellent bioresorbable and biocompatible properties. The small particle size and interconnected microporosity are believed to improve osteoconductive properties of the material and promote timely resorption concomitant with the process of remodeling (Erbe et al., 2001). The porous structure of bioactive materials support tissue in/on-growth and are generally affected for supplementing the implant stability by biological fixation (Engh et al., 1995). However, depending on the preparation technique the material exhibits grossly different powder characteristics, microstructure and associated mechanical and biological properties. A biomaterial made of -tri-calcium phosphate has been prepared by a novel aqueous solution combustion technique using calcium nitrate tetrahydrate and di-ammonium hydrogen orthophosphate as the starting raw materials and glycine/urea as the fuel. The aim of this present study was to evaluate the porous -TCP, having an interconnecting porosity (apparent porosity of ∼37%) and regular pore shape and size as bone substitute in segmental bone healing.
145
materials. Glycine (C2 H5 NO2 ) (Glaxo, Qualigens, India) was used as fuel. Aqueous stock solutions of calcium nitrate tetrahydrate (2.72 M) and DAP (2.20 M) were mixed in the ratio of 1.67 and 1.50, which are required for the formation of calcium phosphates. A few drops of concentrated nitric acid (Merck, India) were added to dissolve the resulting white precipitate to make a clear homogeneous solution. A predetermined amount of fuel was added to the clear solution which was homogenized by stirring with a magnetic stirrer for 30 min at room temperature. A glass–ceramic coated mild steel wide container containing the solution was introduced into a muffle furnace preheated to the desired temperature. A stainless steel wire mesh was put on the reaction container to reduce particle loss through aerosol formation and helped in attaining uniformity of temperature. When placed in the furnace, the mixed solution soon started to boil, underwent dehydration, decomposition and a large volume of gases containing oxides of nitrogen and traces of ammonia were evolved. The mass then frothed and swelled to yield foam, where a flame appeared and produced incandescence. The entire process required less than 10 min with flame duration of nearly 1 min. The as-synthesized products were fluffy foam-like mass that occupied a large volume. The resulting soft-agglomerated powders were readily ground manually in an agate mortar/pestle into fine powder and were thoroughly characterized for its phase composition, crystallinity, etc. 2.2. Fabrication of porous -TCP In the present study, porous (35–40% by volume) -TCP was fabricated by using -naphthalene and polyvinyl alcohol (both from S.D. Fine-Chem. Ltd., India) as combustible organic materials. TCP powder was milled separately with oleic acid surfactant and pre-calculated amount of -naphthalene. Rectangular shaped (12 × 5 × 3 mm3 ) blocks were uniaxially cold compacted with low pressure, which were subsequently cold iso-statically pressed at 100 MPa for homogeneous densification. All specimens were slowly dried at 80 ◦ C for 3 days. Finally, -TCP specimen was sintered at 1050 ◦ C with holding time of 2 h at that temperature. Archimedes’ principle using water as the immersing medium was used to calculate the density and apparent porosity of sintered specimens. The porous struts were initially pasteurized using distilled water and subsequently autoclaved at 121 ◦ C for 30 min before implantation. The physical properties thus calculated are shown in Table 1. 2.3. Animal experimentation
2. Materials and methods 2.1. Preparation of -TCP powder by aqueous solution combustion technique In this study, calcium nitrate tetrahydrate (Ca(NO3 )2 ·4H2 O) (A.R. grade, S.D. Fine-Chem. Ltd., India), and di-ammonium hydrogen ortho-phosphate (DAP, (NH4 )2 HPO4 ) (A.R. grade, S.D. Fine-Chem. Ltd., India) were used as the starting raw
In the present study, 12 Black Bengal goats of either sex, weighing 10–12 kg were randomly distributed into two groups of six animals each: control group I (in which the created defects were left as such) and the test animals, group II (in which porous -TCP blocks were inserted within the created defects). The animals were group housed indoor in standard conditions, given water ad libitum and were without restriction of movement according to the guidelines of Institutional
146
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
Table 1 Physical characteristics of porous specimens Specimens
Sintering temperature/time
Bulk density (g/c.c.)
Apparent porosity (%)
Pore size distribution (m)
-TCP (12 × 5 × 3 mm3 )
1050 ◦ C/2 h
1.934
37
8–150
Animal Ethical Committee of the West Bengal University of Animal and Fishery Sciences, India. Surgery was performed under aseptic conditions and sedation by injection of xylazine hydrochloride (0.05 mg/kg) followed by local infiltration of 2% lignocaine hydrochloride. Analgesia was maintained by intramuscular injection of meloxicam (0.5 ml) and antibiotic prophylaxis was done by means of cefotaxime (250 mg) injections 12 h interval for 2 days. A bone defect (10 × 5 mm2 ) in all the animals was performed in the lateral aspect of diaphysis of radius bone with the help of a motorized dental drill. In control (group I) the defect was left as such and in group II, porous -TCP blocks were placed in the defect sites. In all the animals, implants were secured in position by suturing muscle, subcutaneous tissue and skin in layers. All the treated animals were administered cefotaxime sodium at a dose rate 250 mg intramuscularly, 12 h interval daily and injected meloxicam at 0.5 ml once daily for 5 days. Surgical wounds were dressed daily with povidone iodine and antibiotic ointment (soframycin ointment) for 5 days postoperatively. 2.4. Local inflammatory reaction and healing of wound Lameness, weight bearing, capability fracture repair in terms of palpable callus, swelling, seroma formation, hematoma, edema and associated signs of local inflammatory reactions were observed from the day of operation up to 90th day postoperatively and changes were evaluated by visual and manual examinations. 2.5. Radiological examination Radiographs were taken immediately after implantation and subsequently on days 21, 30, 60 and 90 postoperatively of the operated fore limb. Radiographs were observed for the status of implant, host-bone reaction to implant and new bone formation. 2.6. Histological study The implanted ceramic implants along with the surrounding bones were collected from the animals at day 90 postoperatively. The sections from both normal and implanted area were cut (3–4 mm thick) using hacksaw and washed thoroughly with normal saline and were fixed in 10% formalin for 7 days. Subsequently bones were decalcified in Gooding and Stewart’s fluid containing formic acid 15 ml, formalin 5 ml and distilled water 80 ml solution. The decalcified tissues were processed in a routine manner and 4 m sections were cut and stained with haematoxylin and eosin. The stained sections were observed
for status of the bone implants and cellular response of host bone to the implants. 2.7. Oxytetracycline labelling study Fluorochrome, viz., oxytetracycline di-hydrate, at a dose rate 50 mg/kg body weight was given on days 77, 78 and later after 6 days interval on days 85 and 86 (2-6-2) postoperatively for double toning of new bone. The implanted segments of the bone were collected and transverse sections (2–3 mm thickness) including the implanted area were cut with the help of hacksaw. Undecalcified ground sections were prepared as described by Parasnalli (1988). The sections were then ground to 20 m thickness using different grade sand papers. Final grinding was done over the bone under moderate pressure using slow circular motions. The ground-undecalcified sections were observed under ultraviolet incidental light by a Leitz Orthoplan Universal Widefield Microscope, USA (Excitation filter, BP400 range) for tetracycline labeling to find out the amount and source of newly formed bone. 2.8. Angiographic study Radial angiography was performed by making a 4–5 cm skin incision aseptically on the medial aspect of the thigh under xylazine hydrochloride sedation and local infiltration analgesia with 2% lignocaine hydrochloride at day 90 postoperatively. The radial arteries were located and exteriorized and catheterized using polyethylene catheters. The catheter was then pushed downwards into the artery and a tight ligature was applied around the catheter so as to prevent any leakage or backflow of the blood/contrast materials from the artery. An elastic tourniquet was applied at the proximal part of the thigh to prevent retrograde flow of the contrast medium. A syringe containing 15 ml sodium iothalmate (Conray® ) was connected to the catheter. The contrast material was infused with a regular gentle digital pressure and radiographs were taken at 14 mA-s, 50 kVP and 90 cm FFD. The catheter was removed and the puncture of the artery was sutured with the help of 4-0 chromic cat-gut. The tourniquet was released and subsequently both the ligature and clamp placed earlier in the radial artery were removed and the skin wound was closed. For better visualization of the arteries, one test limb from each group was collected after euthanizing the animal with overdoses of thiopental sodium (20 mg/kg body weight) at the end of the experiment and perfused with lead oxide suspension (20% w/v) in the similar manner. The angiograms were critically examined for vascular response of the host bone and surrounding tissue in the implanted area and visualization of the implants.
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
3. Results 3.1. Local inflammatory reactions and healing of wound
147
ful in all the cases and sutures were removed on the 10th postoperative day. The implants were clinically stable in the bone. 3.2. Radiological observations
No marked inflammatory reactions were observed in both the control and experimental group following placement of bioceramic implant up to 90th day postoperatively. Weight bearing capacity in each animal subject was gradually improved. There was no adverse local effect such as marked hematoma or edema during the early postoperative period. Wound healing was unevent-
3.2.1. Group I (control study) On “day 0”, radiographs showed the cortical defect devoid of any implant resulting into a radiolucent gap (Fig. 1A). On the day 21, radiographs revealed with minimal periosteal reaction and smoothing edges of the cortical bone defects. The shape of the defects changed
Fig. 1. Radiographs of the control site after (A) ‘0’ day (B) on day 21, (C) on day 30, (D) on day 60 and (E) on day 90.
148
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
and the sharp corners of rectangular defect became oval indicating initiation of bone healing (Fig. 1B). On day 30, radiographs showed that there was substantial reduction in the gap size which was in the process of obliteration by hard tissue material (Fig. 1C). On day 60, radiographs showed that the defect was not obliterated by newly grown bony tissue (Fig. 1D). However, in one animal in lateral view, the radiograph showed the presence of dent at the defect site which indicated that the defect was yet to be completely filled in by the host bone. On day 90, radiographs showed that the defect was more or less same except that the newly formed bony tissue was more organized and the fractured end become smooth and round (Fig. 1E).
3.2.2. Group II (tri-calcium phosphate) On “day 0”, anterio-posterior radiographs immediately after implantation of -TCP block in animals showed well positioning of block in the mid-shaft bone defect of the radius (Fig. 2A). On day 21, radiographs showed severe periosteal reaction surrounding the implants. The gap between the implant and the host tissue was noticed to be reduced (Fig. 2B) in comparison to that of the “0th” day which seemed to be new bony growth from the edge of the defect. On day 30, radiographs showed unaltered implant in its shape, size and radiodensity. The gap between the host and the implant was almost filled-up with newly grown host tissues which exhibited similar radiodensity to the adjacent
Fig. 2. Radiographs after implantation of -TCP block in animals after (A) ‘0’ day (B) on day 21 (C) on day 30 (D) on day 60 and (E) on day 90.
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
149
bone (Fig. 2C). On day 60, radiographs revealed that there was complete obliteration of gap between implant and host tissue without any alteration of shape, size and radiodensity of implant. The callus adjoining the cortex of the defect was organized, consolidated, which was of similar density to that of host bone. There was no sign of resorption of implant which looked to be more radiodense than the host bones (Fig. 2D). On day 90, radiographs showed that the implant was intact in shape and size with unchanged radiodensity. The bridging of the cortex along the axis of the bone became complete without any detectable callus at the defect site which indicated for the completion of healing process without any noticeable change in the implant (Fig. 2E). 3.3. Histological studies 3.3.1. Group I (control) Section showed no marked inflammatory reaction with moderate fibro-collagenisation. Cortex showed lamellar appearance of the bone along with presence of woven bone at places. Marrow space showed good amount of marrow material, fat cells and blood vessels. The cellular components of bony tissue and matrix showed a proportional relation. Marrow materials in medullary regions showed hyperactiveness of formation of bony matrix. No unabsorbed material was seen (Fig. 3A and B). 3.3.2. Group II (tri-calcium phosphate) Section showed picture of moderately differentiated lamellar bone in the cortical part containing a few granular foreign body material. Surrounding the foreign materials, new osteogenesis was moderate to normal. Peripheral part of the cortex showed presence of woven bone. Marrow space showed evidence of angiogenesis with focal development of marrow material in the midst of unabsorbed foreign granular debris. The periostium appeared mildly thickened and fibro-collagenous. In between cortical and medullary boundary, the existence of osteoids was quite normal (Fig. 4A and B). 3.4. Oxytetracycline labeling study 3.4.1. Group I (control) In this group, the process of new bone formation was active from both the ends. Newly formed osseous tissues originating from periosteal as well as endosteal surface of bone were also seen, however, its intensity was more on periosteal side. The defect was completely filled with newly formed cancellous bone and appeared as homogenous non-fluorescent area. However, a narrow
Fig. 3. Histopathological section of the bone (A) in cortical area of control bone (HE × 10) and (B) indicating presence of woven bone at cortex (HE × 45).
linear zone near the periosteum revealed a golden yellow fluorescence suggestive of new bone formation in the area (Fig. 5A). Union in the defect site of bone was complete in most of the animals. 3.4.2. Group II (tri-calcium phosphate) Microphotographs viewed under fluorescent light (6.3 × 10 magnification) imparted a double tone golden yellow fluorescence in wider zone in the defect site and the host bone evinced dark sea green homogenous color.
150
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
Fig. 4. Histopathological section in TCP implanted bone (A) HE × 10 and (B) HE × 45.
However, at some places, the host bone showed newly formed woven bone, which was cancellous in nature. Further, in the defect area, some light sea green homogenous color appeared amongst the trabeculae suggesting newly formed bony tissue became older. The implant was seen as fragmented homogenous mass between trabeculae of newly formed bone (Fig. 5B). 3.5. Angiographic study 3.5.1. Group I (control) Angiogram of the animals in control group showed that there was uniform angiogenesis in the defect site. Establishment of trans transplant angiogenesis was evidenced by the presence of capillary network on and
Fig. 5. Oxytetracycline labelling study at the defect site of the control as well as the TCP implanted site. Photomicrograph showing the presence of homogenous nonfluoroscent area of cancellous bone at the same site for control (A) and crossing over and union of new bone trabeculae at the TCP site (B).
around the defect site containing radiodense contrast material (Fig. 6A). 3.5.2. Group II (tri-calcium phosphate) Angiogram of the animals implanted with -TCP at day 90 postoperatively revealed that establishment of newly grown capillaries had already been initiated over and surrounding the transplant. Angiogram also revealed that there was intact transplant material without any radiographically detectable changes which was indicative of transplant non-rejection (Fig. 6B). Vascularity of both the control and TCP implanted site has been comparatively shown in Table 2.
Table 2 Comparative evaluation of vascularity in different groups
Control Tricalcium phosphate
Cross over circulation
Pooling/stagnation of contrast medium
Leakage of contrast material
Intensity of vascularization
Bone healing/new bone formation
+++ ++
− −
+ −
+++ ++
+ +
(+) mild, (++) moderate, (+++) marked, (−) negative.
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
151
Fig. 6. Angiograph on day ‘90’ showing well-established medullary cavity and uniform capillary network containing radiodense dye adjacent to the created defects of control (A) and intact unabsorbed rectangular graft at the defect site with well appreciable penetrating capillaries containing radiodense contrast into the graft along the axis of TCP (B).
4. Discussion Clinically, in all the surgically created defect areas, the implants were well placed, well accepted and tolerated by the animals, causing no serious inflammation in the surrounding tissue. Healing was uneventful in all animals and there was no evidence of rejection of implant in any case which corroborated with the findings of Holmes et al. (1986). Lameness disappeared gradually, which indicated that inflammation was subsided and fracture was getting stable. This finding was in agreement with the observations of ulnar fracture in dog by Shukla (1989) and in rabbit by Singh (1998). In the present study no foreign body response or toxicity was elicited and hence it was confirmed that the implant was accepted as a suitable alternative bone graft to fill the defect. Critical evaluation of radiographs taken at different intervals in animals of group I revealed no appreciable evidence of fracture union as compared to other group. However, at the initial stages, minimal periosteal reaction and smoothing edges of cortical bone defects were
noticed. Subsequently, there was substantial reduction of gap size by newly formed osseous tissue, making the defect more round and smooth. Similar finding was also reported by Bolander and Balian (1986). Radiologically, on “day 0”, -TCP blocks were well positioned in the bone defect in all the animals of group II. The radiodensity of -TCP block was found to be higher than that of the host bone as also reported by Murphey et al. (1992) and Singh (1998). On day 21, radiograph revealed severe periosteal reaction surrounding the implants. The gap between the implant and the host tissue was reduced which seems to be new bony growth from the edge of the defect. There was no untoward reaction in the TCP implanted bone which corroborated the findings of Cameron et al. (1977) in dogs and Singh (1998) in rabbits. On day 30, radiograph revealed presence of TCP implant without any resorption. The gap between the host bone and implant was almost filled up with newly grown bony tissue and bridging callus was found occupying the space surrounding the implant. On day 60, radiograph revealed complete obliteration of host implant gap and callus adjoining the
152
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
defect was organized and consolidated. On day 90, bridging of the cortex had been completed which indicated completion of healing process. There was no sign of resorption of implant which was however contradictory to the findings of Cutright et al. (1972) who reported 95% resorption of TCP ceramic implants in rat tibias 48 days postoperatively with extensive bone formation. Renooij et al. (1985) found 25–30% bioresorption of TCP in 22 weeks whereas Shimazaki and Mooney (1985) observed 46.4% implant biodegradation in 24 weeks when implanted into rabbit tibia. Bhaskar et al. (1971) observed early bone formation within a week in TCP implanted rat tibias. Histologically, the bone defect was almost repaired with newly formed osseous tissue with well-developed blood vessels in haversian channels and little amount of marrow materials sparingly present at places. Bits of non-absorbed biodegradable materials were seen in lamellar cortical bone. These observations corroborated the findings of Uchida et al. (1984) and Singh (1998) where they implanted HAp in skull of rat and skull and ulnae in rabbits. Oxytetracycline labeling study demonstrated that the defect site in -TCP implanted bone exhibited some light sea green homogenous color within bright golden yellow matrix of newly formed trabeculae suggesting existence of early formed bony tissue which became matured during the study. These findings suggest that TCP did not possess osteogenic activity which was in accordance with the findings of Duquette (1973). Resorption cavities were present in TCP implanted bone, suggesting that the resorption and replacement of the bone was well under progress. Besides, the resorption cavities were indicative of initiation of bone remodeling. Critical evaluation of angiographic results of the present study revealed varying degrees of vascularization. There was bridging of newly budded blood vessels through the implant in animals of group II (-TCP). Further, the evidence of trans transplant angiogenesis was evident in animals implanted with TCP. Angiogram study also revealed that transplant materials (-TCP) remained intact in the defect site indicating non-rejection of the transplant which was in the process of osteoconduction. This could be attributed to the fact that all the transplants used in the present study were biocompatible and subsequently had low or no inflammatory response after transplantation. Similar observations were also noted by Burwell (1963) and Singh (1978). The minute vessels of the periosteal and endosteal origin invaded the implant bed which supports the view that the vascularization in fracture healing is directly related to the amount of new bone formation (Cavadias and
Trueta, 1965). Angiogram of control animals (group I) revealed comparatively lesser uniform and trans transplant angiogenesis although medullary cavity was well established. 5. Conclusions Considering the results obtained, it can be concluded that porous -tricalcium phosphate based implants promoted bone formation over the extension of the defect and offered interesting potential for orthopedic reconstructive procedures although further in-depth studies are warranted to establish the exact mechanism. Acknowledgements The authors wish to express their thanks to the Dean, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, Kolkata, India for his kind permission to use the facilities for the experimentation. References Banwart, J.C., Asher, M.A., Hassanein, R.S., 1995. Iliac crest bone graft harvest donor site morbidity: a statistical evaluation. Spine 20, 1055–1060. Bhaskar, S.N., Brady, J.M., Getter, L., 1971. Biodegradable ceramics implants in bone: electron and light microscopic analysis. Oral Surg. 32, 336–346. Bolander, M.E., Balian, G., 1986. The use of demineralized bone matrix in the repair of segmental defects. J. Bone Joint Surg. 68A, 1264–1274. Burwell, R.G., 1963. Studies in the transplantation of bone. V. The capacity of fresh and treated homografts of bone to evoke transplantation immunity. J. Bone Joint Surg. 45B, 386–401. Cameron, H.U., Macnab, I., Pilliar, R.M., 1977. Evaluation of biodegradable ceramics. J. Biomed. Mater. Res. 11 (2), 179–186. Cavadias, A.X., Trueta, J., 1965. An experimental study of vascular contribution to the callus of fracture. Surg. Gynaecol. Obstet. 120, 737–747. Cutright, D.E., Bhaskar, S.M., Brody, J.M., 1972. Reaction of bone to tricalcium phosphate pellets. Oral Surg. 33, 850–856. Duquette, P., 1973. An evaluation of the osteogenic potential of the porous single-phase tricalcium phosphate ceramic. Indiana University Thesis. Engh, C.A., Hooten Jr., J.P., Zettl Schaffer, K.F., et al., 1995. Evaluation of bone in-growth in proximally and extensively porous-coated anatomic medullary locking prostheses retrieved at autopsy. J. Bone Joint Surg. 77A, 903–910. Erbe, E.M., Marx, J.G., Clineff, T.D., Bellincampi, L.D., 2001. Potential of an ultraporous -tricalcium phosphate synthetic cancellous bone void filler and bone marrow aspirate composite graft. Eur. Spine J. 10 (Suppl. 2), 141–146. Friedlander, G.E., 1983. Immune responses to osteochondral allografts: current knowledge and future directions. Clin. Orthop. 174, 58–68.
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153 Heise, U., Osborn, J.F., Duwe, F., 1990. Hydroxyapatite ceramic as a large substitute. Int. Orthop. 14, 329–338. Holmes, R.E., Buchloz, R.W., Mooney, V., 1986. Porous hydroxyapatite as a bone graft substitutes in metaphyseal defects. A histometric study. J. Bone Joint Surg. 68A, 904–911. Murphey, M.D., Sartoris, D.J., Bramble, J.M., 1992. Radiographic assessment of bone grafts. In: Habal, M.B., Hari Reddi, A. (Eds.), Bone Grafts and Bone Substitutes. W.B. Saunders, Philadelphia, pp. 9–36. Parasnalli, B., 1988. A comparative study on the osteoinductive property, healing process and fate of decalcified autoclaved, deep frozen and organic allogenic cortical bone grafts in bovines. M.V. Sc Thesis, I.V.R.I., Izatnagar, India. Peltola, M., 2001. Bioactive glass in frontal sinus and calvarial bone defect obliteration. Experimental and Clinical studies. Thesis, Annales Universitatis Turkuensis, D 435. Renooij, W., Hoogendoorn, H.A., Visser, W.J., Lentferink, R.H., Schmitz, M.G., Vanleperen, H., Oldenburg, S.J., Janssen, W.M., 1985. Bioresorption of ceramics strontium-85 labeled calcium phosphate implants in dogs femora. A pilot study to qualitative bioresorption. Clin. Orthop. Relat. Res. 197, 272–285.
153
Shimazaki, K., Mooney, V., 1985. Comparative study of porous hydroxyapatite and tricalcium phosphate as bone substitute. J. Orthop. Res. 3 (3), 301–310. Shukla, B.P., 1989. A comparative evaluation of fresh autogenous vis-`a-vis freeze dried and decalcified freeze dried segmental xenogenous bone grafts in dogs. M.V. Sc. Thesis, I.V.R.I., Izatnagar, India. Singh, H., 1978. Surgical studies on grafting materials for better osteogenesis in animals. Final ICAR Report, Deptt. of Surgery and Radiology, C.V. Sc., GBPAUT, Pantnagar, India. Singh, S., 1998. Reconstruction of segmental ulnar defect with tricalcium phosphate, calcium hydroxyapatite and calcium hydroxyapatite-bone matrix combination in rabbit. M.V. Sc. Thesis, Submitted to I.V.R.I., Izatnagar, India. Summers, B.N., Eisenstein, S.M., 1989. Donor site pain from the ileum: a complication of lumbar spine fusion. J. Bone Joint Surg. Br. 71, 677–680. Uchida, A., Nade, S.M.I., McCartney, E.R., Ching, W., 1984. A comparative study of three different porous ceramics. J. Bone Joint Surg. 66B (2), 269–275.
Small Ruminant Research 75 (2008) 144–153
Evaluation of new porous -tri-calcium phosphate ceramic as bone substitute in goat model Samit K. Nandi a,∗ , Samir K. Ghosh b,1 , Biswanath Kundu b,1 , Dipak K. De a,2 , Debabrata Basu b,1 a
West Bengal University of Animal and Fishery Sciences, 37 & 68, Kshudiram Bose Sarani, Kolkata 700037, India b Central Glass and Ceramic Research Institute, 196, Raja S.C. Mullick Road, P.O.: Jadavpur University, Kolkata 700032, India Received 8 February 2007; accepted 13 September 2007 Available online 14 November 2007
Abstract The present study was carried out to evaluate the porous -tri-calcium phosphate (TCP) (prepared by aqueous solution combustion technique) as bone substitute and compared with normal healing in 12 adult Black Bengal goats on the basis of clinical and radiographic findings, histological studies, oxytetracycline labeling, angiography studies (on day 90). Bone defects created in the diaphysis of radius were left unfilled in control animals (group I); while in treated (group II) animals the defects were filled with porous TCP blocks. The three months study showed no marked acute inflammatory reactions in all animals, wound healing was uneventful and the implants were clinically stable in the bone. Radiological studies showed presence of unabsorbed implants which acted as a scaffold for new bone growth across the defect whereas in control animals the defect was more or less same except that the newly formed bony tissue was less organized. Histological section showed moderately differentiated lamellar bone in the cortical part with presence of woven bone at peripheral cortex whereas control animals showed moderate fibro-collagenisation and good amount of marrow material, fat cells and blood vessels. Oxytetracycline labeling study showed crossing over of new bony trabeculae along with presence of resorption cavities within the new osteoid tissues whereas in group I, the process of new bone formation was active from both the ends; the defect site appeared as a homogenous non-fluorescent area. Angiogram of the animals in control showed uniform angiogenesis in the defect site with establishment of trans transplant angiogenesis, whereas in group II there was complete trans transplant shunting of blood vessels communication. The results of this study pointed out that the porous TCP promoted extensive bone formation over the entire extension of the defect in comparison to control group, thus conforming their biological osteoconductive property. © 2007 Elsevier B.V. All rights reserved. Keywords: -Tricalcium phosphate; Bone defects; Radiography; Histology; Oxytetracycline labeling; Angiography
∗ Corresponding author. Tel.: +91 33 25569234; fax: +91 33 25571986. E-mail addresses: [email protected] (S.K. Nandi), [email protected] (S.K. Ghosh), biswa [email protected] (B. Kundu), professor [email protected] (D.K. De), [email protected] (D. Basu). 1 Tel.: +91 33 24733469/96/76/77; fax: +91 33 24730957. 2 Tel.: +91 33 25569234; fax: +91 33 25571986.
0921-4488/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2007.09.006
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
1. Introduction Bone grafts are often necessary to provide support, fill voids, and enhance biologic repair of osseous defect. Although autogenous bone is considered the gold standard, to which other methods are compared, autograft has significant limitations, including donor site morbidity, inadequate availability in inappropriate form (Summers and Eisenstein, 1989; Banwart et al., 1995). Allograft bone has been widely used, which of course have several problems encountered when used, including immunogenicity and disease transmission such as hepatitis and HIV (Friedlander, 1983), loss of biologic and mechanical properties secondary to its processing and increased cost. Consequently significant strides have been made to search ideal bone graft substitutes. Various synthetic materials have been developed as bone substitute which include marine coral, hydroxyapatite (HAp), bioactive glasses and synthetic polymers with chemical composition and crystallinity equivalent to that of the mineral phase of natural bone (Heise et al., 1990; Peltola, 2001). Tri-calcium phosphate offers excellent bioresorbable and biocompatible properties. The small particle size and interconnected microporosity are believed to improve osteoconductive properties of the material and promote timely resorption concomitant with the process of remodeling (Erbe et al., 2001). The porous structure of bioactive materials support tissue in/on-growth and are generally affected for supplementing the implant stability by biological fixation (Engh et al., 1995). However, depending on the preparation technique the material exhibits grossly different powder characteristics, microstructure and associated mechanical and biological properties. A biomaterial made of -tri-calcium phosphate has been prepared by a novel aqueous solution combustion technique using calcium nitrate tetrahydrate and di-ammonium hydrogen orthophosphate as the starting raw materials and glycine/urea as the fuel. The aim of this present study was to evaluate the porous -TCP, having an interconnecting porosity (apparent porosity of ∼37%) and regular pore shape and size as bone substitute in segmental bone healing.
145
materials. Glycine (C2 H5 NO2 ) (Glaxo, Qualigens, India) was used as fuel. Aqueous stock solutions of calcium nitrate tetrahydrate (2.72 M) and DAP (2.20 M) were mixed in the ratio of 1.67 and 1.50, which are required for the formation of calcium phosphates. A few drops of concentrated nitric acid (Merck, India) were added to dissolve the resulting white precipitate to make a clear homogeneous solution. A predetermined amount of fuel was added to the clear solution which was homogenized by stirring with a magnetic stirrer for 30 min at room temperature. A glass–ceramic coated mild steel wide container containing the solution was introduced into a muffle furnace preheated to the desired temperature. A stainless steel wire mesh was put on the reaction container to reduce particle loss through aerosol formation and helped in attaining uniformity of temperature. When placed in the furnace, the mixed solution soon started to boil, underwent dehydration, decomposition and a large volume of gases containing oxides of nitrogen and traces of ammonia were evolved. The mass then frothed and swelled to yield foam, where a flame appeared and produced incandescence. The entire process required less than 10 min with flame duration of nearly 1 min. The as-synthesized products were fluffy foam-like mass that occupied a large volume. The resulting soft-agglomerated powders were readily ground manually in an agate mortar/pestle into fine powder and were thoroughly characterized for its phase composition, crystallinity, etc. 2.2. Fabrication of porous -TCP In the present study, porous (35–40% by volume) -TCP was fabricated by using -naphthalene and polyvinyl alcohol (both from S.D. Fine-Chem. Ltd., India) as combustible organic materials. TCP powder was milled separately with oleic acid surfactant and pre-calculated amount of -naphthalene. Rectangular shaped (12 × 5 × 3 mm3 ) blocks were uniaxially cold compacted with low pressure, which were subsequently cold iso-statically pressed at 100 MPa for homogeneous densification. All specimens were slowly dried at 80 ◦ C for 3 days. Finally, -TCP specimen was sintered at 1050 ◦ C with holding time of 2 h at that temperature. Archimedes’ principle using water as the immersing medium was used to calculate the density and apparent porosity of sintered specimens. The porous struts were initially pasteurized using distilled water and subsequently autoclaved at 121 ◦ C for 30 min before implantation. The physical properties thus calculated are shown in Table 1. 2.3. Animal experimentation
2. Materials and methods 2.1. Preparation of -TCP powder by aqueous solution combustion technique In this study, calcium nitrate tetrahydrate (Ca(NO3 )2 ·4H2 O) (A.R. grade, S.D. Fine-Chem. Ltd., India), and di-ammonium hydrogen ortho-phosphate (DAP, (NH4 )2 HPO4 ) (A.R. grade, S.D. Fine-Chem. Ltd., India) were used as the starting raw
In the present study, 12 Black Bengal goats of either sex, weighing 10–12 kg were randomly distributed into two groups of six animals each: control group I (in which the created defects were left as such) and the test animals, group II (in which porous -TCP blocks were inserted within the created defects). The animals were group housed indoor in standard conditions, given water ad libitum and were without restriction of movement according to the guidelines of Institutional
146
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
Table 1 Physical characteristics of porous specimens Specimens
Sintering temperature/time
Bulk density (g/c.c.)
Apparent porosity (%)
Pore size distribution (m)
-TCP (12 × 5 × 3 mm3 )
1050 ◦ C/2 h
1.934
37
8–150
Animal Ethical Committee of the West Bengal University of Animal and Fishery Sciences, India. Surgery was performed under aseptic conditions and sedation by injection of xylazine hydrochloride (0.05 mg/kg) followed by local infiltration of 2% lignocaine hydrochloride. Analgesia was maintained by intramuscular injection of meloxicam (0.5 ml) and antibiotic prophylaxis was done by means of cefotaxime (250 mg) injections 12 h interval for 2 days. A bone defect (10 × 5 mm2 ) in all the animals was performed in the lateral aspect of diaphysis of radius bone with the help of a motorized dental drill. In control (group I) the defect was left as such and in group II, porous -TCP blocks were placed in the defect sites. In all the animals, implants were secured in position by suturing muscle, subcutaneous tissue and skin in layers. All the treated animals were administered cefotaxime sodium at a dose rate 250 mg intramuscularly, 12 h interval daily and injected meloxicam at 0.5 ml once daily for 5 days. Surgical wounds were dressed daily with povidone iodine and antibiotic ointment (soframycin ointment) for 5 days postoperatively. 2.4. Local inflammatory reaction and healing of wound Lameness, weight bearing, capability fracture repair in terms of palpable callus, swelling, seroma formation, hematoma, edema and associated signs of local inflammatory reactions were observed from the day of operation up to 90th day postoperatively and changes were evaluated by visual and manual examinations. 2.5. Radiological examination Radiographs were taken immediately after implantation and subsequently on days 21, 30, 60 and 90 postoperatively of the operated fore limb. Radiographs were observed for the status of implant, host-bone reaction to implant and new bone formation. 2.6. Histological study The implanted ceramic implants along with the surrounding bones were collected from the animals at day 90 postoperatively. The sections from both normal and implanted area were cut (3–4 mm thick) using hacksaw and washed thoroughly with normal saline and were fixed in 10% formalin for 7 days. Subsequently bones were decalcified in Gooding and Stewart’s fluid containing formic acid 15 ml, formalin 5 ml and distilled water 80 ml solution. The decalcified tissues were processed in a routine manner and 4 m sections were cut and stained with haematoxylin and eosin. The stained sections were observed
for status of the bone implants and cellular response of host bone to the implants. 2.7. Oxytetracycline labelling study Fluorochrome, viz., oxytetracycline di-hydrate, at a dose rate 50 mg/kg body weight was given on days 77, 78 and later after 6 days interval on days 85 and 86 (2-6-2) postoperatively for double toning of new bone. The implanted segments of the bone were collected and transverse sections (2–3 mm thickness) including the implanted area were cut with the help of hacksaw. Undecalcified ground sections were prepared as described by Parasnalli (1988). The sections were then ground to 20 m thickness using different grade sand papers. Final grinding was done over the bone under moderate pressure using slow circular motions. The ground-undecalcified sections were observed under ultraviolet incidental light by a Leitz Orthoplan Universal Widefield Microscope, USA (Excitation filter, BP400 range) for tetracycline labeling to find out the amount and source of newly formed bone. 2.8. Angiographic study Radial angiography was performed by making a 4–5 cm skin incision aseptically on the medial aspect of the thigh under xylazine hydrochloride sedation and local infiltration analgesia with 2% lignocaine hydrochloride at day 90 postoperatively. The radial arteries were located and exteriorized and catheterized using polyethylene catheters. The catheter was then pushed downwards into the artery and a tight ligature was applied around the catheter so as to prevent any leakage or backflow of the blood/contrast materials from the artery. An elastic tourniquet was applied at the proximal part of the thigh to prevent retrograde flow of the contrast medium. A syringe containing 15 ml sodium iothalmate (Conray® ) was connected to the catheter. The contrast material was infused with a regular gentle digital pressure and radiographs were taken at 14 mA-s, 50 kVP and 90 cm FFD. The catheter was removed and the puncture of the artery was sutured with the help of 4-0 chromic cat-gut. The tourniquet was released and subsequently both the ligature and clamp placed earlier in the radial artery were removed and the skin wound was closed. For better visualization of the arteries, one test limb from each group was collected after euthanizing the animal with overdoses of thiopental sodium (20 mg/kg body weight) at the end of the experiment and perfused with lead oxide suspension (20% w/v) in the similar manner. The angiograms were critically examined for vascular response of the host bone and surrounding tissue in the implanted area and visualization of the implants.
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
3. Results 3.1. Local inflammatory reactions and healing of wound
147
ful in all the cases and sutures were removed on the 10th postoperative day. The implants were clinically stable in the bone. 3.2. Radiological observations
No marked inflammatory reactions were observed in both the control and experimental group following placement of bioceramic implant up to 90th day postoperatively. Weight bearing capacity in each animal subject was gradually improved. There was no adverse local effect such as marked hematoma or edema during the early postoperative period. Wound healing was unevent-
3.2.1. Group I (control study) On “day 0”, radiographs showed the cortical defect devoid of any implant resulting into a radiolucent gap (Fig. 1A). On the day 21, radiographs revealed with minimal periosteal reaction and smoothing edges of the cortical bone defects. The shape of the defects changed
Fig. 1. Radiographs of the control site after (A) ‘0’ day (B) on day 21, (C) on day 30, (D) on day 60 and (E) on day 90.
148
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
and the sharp corners of rectangular defect became oval indicating initiation of bone healing (Fig. 1B). On day 30, radiographs showed that there was substantial reduction in the gap size which was in the process of obliteration by hard tissue material (Fig. 1C). On day 60, radiographs showed that the defect was not obliterated by newly grown bony tissue (Fig. 1D). However, in one animal in lateral view, the radiograph showed the presence of dent at the defect site which indicated that the defect was yet to be completely filled in by the host bone. On day 90, radiographs showed that the defect was more or less same except that the newly formed bony tissue was more organized and the fractured end become smooth and round (Fig. 1E).
3.2.2. Group II (tri-calcium phosphate) On “day 0”, anterio-posterior radiographs immediately after implantation of -TCP block in animals showed well positioning of block in the mid-shaft bone defect of the radius (Fig. 2A). On day 21, radiographs showed severe periosteal reaction surrounding the implants. The gap between the implant and the host tissue was noticed to be reduced (Fig. 2B) in comparison to that of the “0th” day which seemed to be new bony growth from the edge of the defect. On day 30, radiographs showed unaltered implant in its shape, size and radiodensity. The gap between the host and the implant was almost filled-up with newly grown host tissues which exhibited similar radiodensity to the adjacent
Fig. 2. Radiographs after implantation of -TCP block in animals after (A) ‘0’ day (B) on day 21 (C) on day 30 (D) on day 60 and (E) on day 90.
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
149
bone (Fig. 2C). On day 60, radiographs revealed that there was complete obliteration of gap between implant and host tissue without any alteration of shape, size and radiodensity of implant. The callus adjoining the cortex of the defect was organized, consolidated, which was of similar density to that of host bone. There was no sign of resorption of implant which looked to be more radiodense than the host bones (Fig. 2D). On day 90, radiographs showed that the implant was intact in shape and size with unchanged radiodensity. The bridging of the cortex along the axis of the bone became complete without any detectable callus at the defect site which indicated for the completion of healing process without any noticeable change in the implant (Fig. 2E). 3.3. Histological studies 3.3.1. Group I (control) Section showed no marked inflammatory reaction with moderate fibro-collagenisation. Cortex showed lamellar appearance of the bone along with presence of woven bone at places. Marrow space showed good amount of marrow material, fat cells and blood vessels. The cellular components of bony tissue and matrix showed a proportional relation. Marrow materials in medullary regions showed hyperactiveness of formation of bony matrix. No unabsorbed material was seen (Fig. 3A and B). 3.3.2. Group II (tri-calcium phosphate) Section showed picture of moderately differentiated lamellar bone in the cortical part containing a few granular foreign body material. Surrounding the foreign materials, new osteogenesis was moderate to normal. Peripheral part of the cortex showed presence of woven bone. Marrow space showed evidence of angiogenesis with focal development of marrow material in the midst of unabsorbed foreign granular debris. The periostium appeared mildly thickened and fibro-collagenous. In between cortical and medullary boundary, the existence of osteoids was quite normal (Fig. 4A and B). 3.4. Oxytetracycline labeling study 3.4.1. Group I (control) In this group, the process of new bone formation was active from both the ends. Newly formed osseous tissues originating from periosteal as well as endosteal surface of bone were also seen, however, its intensity was more on periosteal side. The defect was completely filled with newly formed cancellous bone and appeared as homogenous non-fluorescent area. However, a narrow
Fig. 3. Histopathological section of the bone (A) in cortical area of control bone (HE × 10) and (B) indicating presence of woven bone at cortex (HE × 45).
linear zone near the periosteum revealed a golden yellow fluorescence suggestive of new bone formation in the area (Fig. 5A). Union in the defect site of bone was complete in most of the animals. 3.4.2. Group II (tri-calcium phosphate) Microphotographs viewed under fluorescent light (6.3 × 10 magnification) imparted a double tone golden yellow fluorescence in wider zone in the defect site and the host bone evinced dark sea green homogenous color.
150
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
Fig. 4. Histopathological section in TCP implanted bone (A) HE × 10 and (B) HE × 45.
However, at some places, the host bone showed newly formed woven bone, which was cancellous in nature. Further, in the defect area, some light sea green homogenous color appeared amongst the trabeculae suggesting newly formed bony tissue became older. The implant was seen as fragmented homogenous mass between trabeculae of newly formed bone (Fig. 5B). 3.5. Angiographic study 3.5.1. Group I (control) Angiogram of the animals in control group showed that there was uniform angiogenesis in the defect site. Establishment of trans transplant angiogenesis was evidenced by the presence of capillary network on and
Fig. 5. Oxytetracycline labelling study at the defect site of the control as well as the TCP implanted site. Photomicrograph showing the presence of homogenous nonfluoroscent area of cancellous bone at the same site for control (A) and crossing over and union of new bone trabeculae at the TCP site (B).
around the defect site containing radiodense contrast material (Fig. 6A). 3.5.2. Group II (tri-calcium phosphate) Angiogram of the animals implanted with -TCP at day 90 postoperatively revealed that establishment of newly grown capillaries had already been initiated over and surrounding the transplant. Angiogram also revealed that there was intact transplant material without any radiographically detectable changes which was indicative of transplant non-rejection (Fig. 6B). Vascularity of both the control and TCP implanted site has been comparatively shown in Table 2.
Table 2 Comparative evaluation of vascularity in different groups
Control Tricalcium phosphate
Cross over circulation
Pooling/stagnation of contrast medium
Leakage of contrast material
Intensity of vascularization
Bone healing/new bone formation
+++ ++
− −
+ −
+++ ++
+ +
(+) mild, (++) moderate, (+++) marked, (−) negative.
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
151
Fig. 6. Angiograph on day ‘90’ showing well-established medullary cavity and uniform capillary network containing radiodense dye adjacent to the created defects of control (A) and intact unabsorbed rectangular graft at the defect site with well appreciable penetrating capillaries containing radiodense contrast into the graft along the axis of TCP (B).
4. Discussion Clinically, in all the surgically created defect areas, the implants were well placed, well accepted and tolerated by the animals, causing no serious inflammation in the surrounding tissue. Healing was uneventful in all animals and there was no evidence of rejection of implant in any case which corroborated with the findings of Holmes et al. (1986). Lameness disappeared gradually, which indicated that inflammation was subsided and fracture was getting stable. This finding was in agreement with the observations of ulnar fracture in dog by Shukla (1989) and in rabbit by Singh (1998). In the present study no foreign body response or toxicity was elicited and hence it was confirmed that the implant was accepted as a suitable alternative bone graft to fill the defect. Critical evaluation of radiographs taken at different intervals in animals of group I revealed no appreciable evidence of fracture union as compared to other group. However, at the initial stages, minimal periosteal reaction and smoothing edges of cortical bone defects were
noticed. Subsequently, there was substantial reduction of gap size by newly formed osseous tissue, making the defect more round and smooth. Similar finding was also reported by Bolander and Balian (1986). Radiologically, on “day 0”, -TCP blocks were well positioned in the bone defect in all the animals of group II. The radiodensity of -TCP block was found to be higher than that of the host bone as also reported by Murphey et al. (1992) and Singh (1998). On day 21, radiograph revealed severe periosteal reaction surrounding the implants. The gap between the implant and the host tissue was reduced which seems to be new bony growth from the edge of the defect. There was no untoward reaction in the TCP implanted bone which corroborated the findings of Cameron et al. (1977) in dogs and Singh (1998) in rabbits. On day 30, radiograph revealed presence of TCP implant without any resorption. The gap between the host bone and implant was almost filled up with newly grown bony tissue and bridging callus was found occupying the space surrounding the implant. On day 60, radiograph revealed complete obliteration of host implant gap and callus adjoining the
152
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153
defect was organized and consolidated. On day 90, bridging of the cortex had been completed which indicated completion of healing process. There was no sign of resorption of implant which was however contradictory to the findings of Cutright et al. (1972) who reported 95% resorption of TCP ceramic implants in rat tibias 48 days postoperatively with extensive bone formation. Renooij et al. (1985) found 25–30% bioresorption of TCP in 22 weeks whereas Shimazaki and Mooney (1985) observed 46.4% implant biodegradation in 24 weeks when implanted into rabbit tibia. Bhaskar et al. (1971) observed early bone formation within a week in TCP implanted rat tibias. Histologically, the bone defect was almost repaired with newly formed osseous tissue with well-developed blood vessels in haversian channels and little amount of marrow materials sparingly present at places. Bits of non-absorbed biodegradable materials were seen in lamellar cortical bone. These observations corroborated the findings of Uchida et al. (1984) and Singh (1998) where they implanted HAp in skull of rat and skull and ulnae in rabbits. Oxytetracycline labeling study demonstrated that the defect site in -TCP implanted bone exhibited some light sea green homogenous color within bright golden yellow matrix of newly formed trabeculae suggesting existence of early formed bony tissue which became matured during the study. These findings suggest that TCP did not possess osteogenic activity which was in accordance with the findings of Duquette (1973). Resorption cavities were present in TCP implanted bone, suggesting that the resorption and replacement of the bone was well under progress. Besides, the resorption cavities were indicative of initiation of bone remodeling. Critical evaluation of angiographic results of the present study revealed varying degrees of vascularization. There was bridging of newly budded blood vessels through the implant in animals of group II (-TCP). Further, the evidence of trans transplant angiogenesis was evident in animals implanted with TCP. Angiogram study also revealed that transplant materials (-TCP) remained intact in the defect site indicating non-rejection of the transplant which was in the process of osteoconduction. This could be attributed to the fact that all the transplants used in the present study were biocompatible and subsequently had low or no inflammatory response after transplantation. Similar observations were also noted by Burwell (1963) and Singh (1978). The minute vessels of the periosteal and endosteal origin invaded the implant bed which supports the view that the vascularization in fracture healing is directly related to the amount of new bone formation (Cavadias and
Trueta, 1965). Angiogram of control animals (group I) revealed comparatively lesser uniform and trans transplant angiogenesis although medullary cavity was well established. 5. Conclusions Considering the results obtained, it can be concluded that porous -tricalcium phosphate based implants promoted bone formation over the extension of the defect and offered interesting potential for orthopedic reconstructive procedures although further in-depth studies are warranted to establish the exact mechanism. Acknowledgements The authors wish to express their thanks to the Dean, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, Kolkata, India for his kind permission to use the facilities for the experimentation. References Banwart, J.C., Asher, M.A., Hassanein, R.S., 1995. Iliac crest bone graft harvest donor site morbidity: a statistical evaluation. Spine 20, 1055–1060. Bhaskar, S.N., Brady, J.M., Getter, L., 1971. Biodegradable ceramics implants in bone: electron and light microscopic analysis. Oral Surg. 32, 336–346. Bolander, M.E., Balian, G., 1986. The use of demineralized bone matrix in the repair of segmental defects. J. Bone Joint Surg. 68A, 1264–1274. Burwell, R.G., 1963. Studies in the transplantation of bone. V. The capacity of fresh and treated homografts of bone to evoke transplantation immunity. J. Bone Joint Surg. 45B, 386–401. Cameron, H.U., Macnab, I., Pilliar, R.M., 1977. Evaluation of biodegradable ceramics. J. Biomed. Mater. Res. 11 (2), 179–186. Cavadias, A.X., Trueta, J., 1965. An experimental study of vascular contribution to the callus of fracture. Surg. Gynaecol. Obstet. 120, 737–747. Cutright, D.E., Bhaskar, S.M., Brody, J.M., 1972. Reaction of bone to tricalcium phosphate pellets. Oral Surg. 33, 850–856. Duquette, P., 1973. An evaluation of the osteogenic potential of the porous single-phase tricalcium phosphate ceramic. Indiana University Thesis. Engh, C.A., Hooten Jr., J.P., Zettl Schaffer, K.F., et al., 1995. Evaluation of bone in-growth in proximally and extensively porous-coated anatomic medullary locking prostheses retrieved at autopsy. J. Bone Joint Surg. 77A, 903–910. Erbe, E.M., Marx, J.G., Clineff, T.D., Bellincampi, L.D., 2001. Potential of an ultraporous -tricalcium phosphate synthetic cancellous bone void filler and bone marrow aspirate composite graft. Eur. Spine J. 10 (Suppl. 2), 141–146. Friedlander, G.E., 1983. Immune responses to osteochondral allografts: current knowledge and future directions. Clin. Orthop. 174, 58–68.
S.K. Nandi et al. / Small Ruminant Research 75 (2008) 144–153 Heise, U., Osborn, J.F., Duwe, F., 1990. Hydroxyapatite ceramic as a large substitute. Int. Orthop. 14, 329–338. Holmes, R.E., Buchloz, R.W., Mooney, V., 1986. Porous hydroxyapatite as a bone graft substitutes in metaphyseal defects. A histometric study. J. Bone Joint Surg. 68A, 904–911. Murphey, M.D., Sartoris, D.J., Bramble, J.M., 1992. Radiographic assessment of bone grafts. In: Habal, M.B., Hari Reddi, A. (Eds.), Bone Grafts and Bone Substitutes. W.B. Saunders, Philadelphia, pp. 9–36. Parasnalli, B., 1988. A comparative study on the osteoinductive property, healing process and fate of decalcified autoclaved, deep frozen and organic allogenic cortical bone grafts in bovines. M.V. Sc Thesis, I.V.R.I., Izatnagar, India. Peltola, M., 2001. Bioactive glass in frontal sinus and calvarial bone defect obliteration. Experimental and Clinical studies. Thesis, Annales Universitatis Turkuensis, D 435. Renooij, W., Hoogendoorn, H.A., Visser, W.J., Lentferink, R.H., Schmitz, M.G., Vanleperen, H., Oldenburg, S.J., Janssen, W.M., 1985. Bioresorption of ceramics strontium-85 labeled calcium phosphate implants in dogs femora. A pilot study to qualitative bioresorption. Clin. Orthop. Relat. Res. 197, 272–285.
153
Shimazaki, K., Mooney, V., 1985. Comparative study of porous hydroxyapatite and tricalcium phosphate as bone substitute. J. Orthop. Res. 3 (3), 301–310. Shukla, B.P., 1989. A comparative evaluation of fresh autogenous vis-`a-vis freeze dried and decalcified freeze dried segmental xenogenous bone grafts in dogs. M.V. Sc. Thesis, I.V.R.I., Izatnagar, India. Singh, H., 1978. Surgical studies on grafting materials for better osteogenesis in animals. Final ICAR Report, Deptt. of Surgery and Radiology, C.V. Sc., GBPAUT, Pantnagar, India. Singh, S., 1998. Reconstruction of segmental ulnar defect with tricalcium phosphate, calcium hydroxyapatite and calcium hydroxyapatite-bone matrix combination in rabbit. M.V. Sc. Thesis, Submitted to I.V.R.I., Izatnagar, India. Summers, B.N., Eisenstein, S.M., 1989. Donor site pain from the ileum: a complication of lumbar spine fusion. J. Bone Joint Surg. Br. 71, 677–680. Uchida, A., Nade, S.M.I., McCartney, E.R., Ching, W., 1984. A comparative study of three different porous ceramics. J. Bone Joint Surg. 66B (2), 269–275.