The repair of segmental bone defects with porous bioglass: An experimental study in goat

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Research in Veterinary Science 86 (2009) 162–173 www.elsevier.com/locate/rvsc

The repair of segmental bone defects with porous bioglass: An experimental study in goat Samit K. Nandi a, Biswanath Kundu b,*, Someswar Datta b, Dipak K. De a, Debabrata Basu b a

West Bengal University of Animal and Fishery Sciences, Department of Veterinary Surgery and Radiology, Kolkata 700037, India b Central Glass and Ceramic Research Institute, Bioceramics and Coating Division, Kolkata 700032, India Accepted 18 April 2008

Abstract This study was exclusively conducted to evaluate healing of surgically created defects on the radius of adult Black Bengal goat after implantation of porous bioglass blocks and compare the process kinetics with normal healing. Twelve Black Bengal goats were divided randomly into two groups: control and experimental group implanted with bioglass blocks. Unicortical bone defects in radius were generated in all animals under aseptic condition. Local inflammatory reaction and healing of wound, radiological investigations, histological studies, oxytetracycline leveling and angiographic studies were performed up to 90th day post-operatively and compared with normal healing. It has been found that extensive new bone formation originating from host bone towards the implant whereas in control, the process was active from both the ends; the defect site appeared as homogenous nonfluorescent area. Thus, porous bioglass promoted bone formation over the entire extension of the defect independent of size of block in comparison to control group. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Black Bengal goats; Bioglass; Radiology; Histology; Oxytetracycline leveling study; Angiography

1. Introduction The ideal bone graft substitute should be osteogenic, biocompatible, bioresorbable, easy to use material with specific properties to provide structural support and deliver drugs of prescribed dosage for a stipulated period of time (Lane et al., 1999; Shors, 1999). The preferred treatment of segmental bone defect by autologous bone graft has its limitation due to generation of additional surgical trauma to the patient. Further, often sufficient amount of bone tissue to fill the whole defect is not available from the otherwise wounded patients and it may not be always possible to salvage donor material from the iliac crest, tibia or skull. To overcome these problems, bone allografts have been chosen but chances of adverse immune response and disease transmission has restricted its widespread acceptance (Gazdag et al., 1995; Chapman et al., 1997). Application *

Corresponding author. Tel.: +91 33 24733469; fax: +91 33 24730957. E-mail address: biswa_kundu@rediffmail.com (B. Kundu).

0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.04.008

of the biological principle of the guided bone regeneration has rendered interesting results in treatment of minor bone defect (Macedo et al., 1993; Buser et al., 1995; Jovanovic et al, 1995). Nevertheless, membrane exposition can occur and its consequent contamination may lead to local infection, thus, hindering the desired new bone formation. In the last three decades, many glass and glass–ceramic compositions were studied to assess their biocompatibility and bioactivity. In 1971, Hench first claimed of certain silicate glass compositions that can form a bond with bone tissue (Hench et al., 1971). It has also been observed that these bioactive glasses can bond with certain types of connective tissue through attachment of collagen to the glass surface (Hench, 1991). Bioactive materials with interconnected porosity in their structure have added advantages in hard tissue prosthesis (De Groot, 1988; Tencer et al., 1990; Gauthier et al., 1998). The porous structure of bioactive materials support tissue in/on growth and are generally effected for supplementing the implant stability by biological fixation (Cook et al., 1991; Engh et al., 1995).

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Nevertheless, porous materials have certain deficiencies as a result of their low fracture strength or fatigue resistance. However, these materials despite of their poor mechanical strength could be used successfully in load free situation or as coating on metal substrates (De Groot, 1988; Gauthier et al., 1998). In the present investigation, a porous biomaterial made of bioactive glass has been developed with high interconnecting porosity (38%) with a regular pore shape and size. The materials after thorough characterization for physical and mechanical properties were inserted in the radius of six Black Bengal goats to study the extent and rate of bone ingrowth in segmental bone healing and were compared with that of the controls in which the created bone defects were kept empty. 2. Materials and methods 2.1. Preparation of bioactive glass Bioactive glass was prepared through conventional glass melting procedure. The appropriate amounts of reagents viz. silica (SiO2), calcium carbonate (CaCO3), dry soda ash (Na2CO3), decahydrated borax (Na2B4O7
10H2O), titania (TiO2), and di-ammonium hydrogen ortho-phosphate (DAP) were mixed together thoroughly in water. The batch composition of the glass is shown in Table 1. The mixture was sufficiently dried at 120 °C to remove the water and the residue was melted in air at temperature 1400 °C for 3 h in a platinum-crucible. The melt was quenched into water to obtain flaky glass particles which were dried, subsequently milled to fine powders in aqueous medium using alumina balls in an alumina lined ball mill for 48 h. Thorough chemical analysis was performed to obtain the final composition used subsequently for fabrication. X-ray diffraction (XRD) (Philips Analytical, X’Pert, 1830, Netherlands) was performed to assess its amorphous nature. 2.2. Fabrication of porous bioactive glass In the present study, porous (35–40% by volume) bioactive glass blocks were fabricated by using b-naphthalene and polyvinyl alcohol (both G.R. grade, SDFine-Chem, India) as combustible organic materials. For the purpose, bioactive glass powders were milled with oleic acid surfactant and pre-calculated amount of b-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

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for 3 days. Finally, bioactive glass specimens were sintered at 600 °C/1 h. Archimedes’ principle using water as medium was used to calculate the apparent porosity of the sintered specimens, pore morphology were checked by scanning electron microscopy (SEM) (Leo 430 Steroscan, U.K.), whereas pore size and its distribution were analyzed by mercury intrusion porosimetry technique (Quantachrome Poremaster, version 4.01, USA). The porous struts were initially pasteurized using distilled water and subsequently autoclaved at 121 °C for 30 min before implantation. 2.3. Experimental study In the present study, 12 Black Bengal goats weighing 10–12 kg were randomly divided into two groups of six animals each: The Group I were kept as control while in Group II, bioactive glass implants were inserted. The animals were placed in shade, in standard conditions, water ad libitum, and without restriction of movement according to the guidelines of Institutional 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 2% Lignocaine hydrochloride. Analgesia was maintained by intramuscular injection of Meloxicam (0.5 ml) and antibiotic prophylaxis was adopted by means of two injections of Cefotaxime (250 mg). By using a motorized dental drill, a bone defect (12  5  3 mm3) in all the animals, were performed in the lateral aspect of diaphysis of radius bone. In control (Group I) the defects were left as such without any implant while in Group II, porous bioactive glass blocks were placed in the defects. In all the animals, implants were secured in position by suturing periosteum, muscle, subcutaneous tissue and skin in layers. All the animals were administered Cefotaxime sodium 250 mg IM twice daily and Injection Meloxicam @ 0.5 ml once daily for 5 days. Surgical wounds were dressed daily with Povidone Iodine and antibiotic cream for 5 days postoperatively. 2.4. Local inflammatory reaction and healing of wound Lameness, weight bearing, 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

Table 1 Batch composition of the glass (in wt%) SiO2

Na2B4O7
10H2O

Na2CO3

CaCO3

(NH4)2HPO4

TiO2

43–44

6–7

11–12

29–30

8–9

1–2

Anterio/posterior (A/P) radiographs were taken immediately after implantation and subsequently on day 21, 30, 60 and 90 postoperatively of the operated fore limb. Radiographs were minutely examined for the status of

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implant, host-bone reaction to implant and new bone formation. 2.6. Histological study The implanted bone/ceramic implants were collected from the animals at day 90 postoperatively. The sections were cut (3–4 mm thick) using hacksaw including both normal and implanted area of bone. The bone pieces were washed thoroughly with normal saline and were fixed in 10% formalin for 7 days. Subsequently bones were decalcified in Goodling and Stewart’s fluid containing formic acid 15 ml, formalin 5 ml and distilled water 80 ml solution and it was stirred daily and changed once in three days. The sections were checked regularly for the status of decalcification. They were considered as completely decalcified when sections became flexible, transparent and easily penetrable by pin. The decalcified tissues were processed in a routine manner and 4 lm 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 dihydrate 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 section (2–3 mm) thickness including the implanted area was cut with the help of hacksaw. Undecalcified ground sections were prepared as described by Parasnali (1988). The sections were then ground to 20 lm thickness using different grade sand papers. Final grinding was done over the bone under moderate pressure using slow circular motions. During grinding, ground sections were examined repeatedly under microscope (10) for transparency of the sections and presence of structural details of the bone. The specimens were kept wet by dipping in water during the entire procedure. The ground-undecalcified sections were observed under ultraviolet incidental light by a Leitz Orthoplan Universal Widefield Microscope, USA (Excitation filter, BP-400 range) for tetracycline labelling to find out the amount and source of newly formed bone.

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 (ConrayÒ) 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 thereafter released. Both 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 euthanising the animal at the end of the experiment and perfused with lead oxide suspension (20% W/V). The angiograms were observed for vascular response of the host bone and surrounding tissue in the implanted area and visualization of the implant. 3. Results 3.1. Properties of the powders and fabricated porous struts The X-ray diffraction pattern clearly indicative of the absence of any crystalline region as seen in Fig. 1. Final composition of the glass powder obtained after milling is shown in Table 2, whereas the physical properties of the porous blocks after processing at 600 °C is given in Table 3. The mercury intrusion porosimetry results revealed that the specimens had an open porosity about 38% with a pore sizes ranging between 4 and 165 lm (Fig. 2). But, it may be noted that the distribution of pores was quite different viz. about 30 vol% pore fall within 20–30 lm, another 27% fall within 10–20 lm. The SEM photomicrograph also (Fig. 3) revealed that bioactive glass surface contained agglomerates and pores of bigger size varied from meso- (>20 lm) to micropores (<5 lm) range only with less variation.

2.8. Angiographic study Radial angiography was performed by making a 4–5 cm skin incision aseptically on the medial aspect of the region below elbow under Xylazine hydrochloride sedation and local 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

Fig. 1. X-ray diffraction pattern of the bioactive glass powders used for the fabrication of porous blocks.

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Table 2 Final chemical composition of the powders used for fabrication of porous struts SiO2

Na2O

CaO

P2O5

B2O3

TiO2

58.60

9.32

23.66

3.38

3.78

1.26

3.2. Local inflammatory reactions and healing of wound

Fig. 2. Histogram of pore size distribution patterns of bioactive glass blocks used for implantation purpose.

No marked inflammatory reactions were observed in both the control and experimental groups following placement of bioactive glass implant up to 90th day post-operatively. Weight bearing capacity in each animal subject gradually improved as signs of inflammation subsided (within 10 days). There was no adverse local effect such as marked hematoma or edema during the early postoperative period. Wound healing was uneventful in all the cases and sutures were removed on the 10th postoperative day. 3.3. Radiological observations: Group I (control study) On 0th day, radiographs showed the cortical defect devoid of any implant resulting to radiolucent gap (Fig. 4A). On day 21, radiographs revealed with minimal periosteal reaction and smoothing of edges of cortical bone defect. The shape of the defect was observed to have changed and the corners of rectangular defect became oval suggesting initiation of bone healing (Fig. 4B). 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 of similar density to that of host bone (Fig. 4C). On day 60, it was observed that the defect was not further obliterated by newly grown bony tissue (Fig. 4D). 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, the defect site was observed to remain more or less equivalent except that the fractured end become smooth and round (Fig. 4E). 3.4. Radiological observations: Group II (bioactive glass) On the 0th day, radiographs immediately after implantation of bioactive glass block in animal showed that the rectangular bioactive glass implant completely occupied the mid shaft radial defect (Fig. 5A). On day 21, severe periosteal reaction surrounding the implant was observed which was not associated with any soft tissue reaction. The edge of the implant noticed to be smoothening and the sharp corners rounding off which indicated initiation of resorption of bioactive glass implant by the host tissue

Fig. 3. SEM of porous specimen before implantation in goat for bioactive glass.

(Fig. 5B). At this stage the implant looked more radiodense than that of earlier days suggesting the process of mineralization over the implant. On day 30, radiographic observations revealed that the radiodense implants changed their shape, size and radiodensity. The implants became oval shaped with indifferentiable callus along with longitudinal axis of the bone defects (Fig. 5C). The extent callus formation was so high that excess of them remained elevated from the host implant surface. The reduction of size of the implants indicated initiation of their resorption process. On day 60, radiographs showed the presence of firm organized callus surrounding the implant. The radiodensity of the implant was markedly reduced as well as shape and size was changed indicating the implant in advance stage of resorption (Fig. 5D). On day 90 post-operatively, radiograph showed that the rectangular shaped radial diaphyseal defect was completely surrounded by well organized thin callus with negligible radiographic impression for presence of implant. The implant was completely resorbed keeping the unfilled defects (Fig. 5E).

Table 3 Physical characteristics of porous specimens Specimens 3

Bioglass (12  5  3 mm )

Sintering temperature/time

Apparent porosity (%)

Pore size distribution (lm)

600 °C/1 h

38.6

14–160

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Fig. 4. Radiograph of the control after (A) ‘0’ day (B) on day 21st, (C) day 30th, (D) day 60th and (E) on day 90th.

Fig. 5. Radiographs after implantation of bioactive glass block in animals after (A) ‘0’ day (B) on day 21st (C) day 30th (D) day 60th and (E) on day 90th.

3.5. Histological studies: Group I (control)

3.6. Histological studies: Group II (bioactive glass)

Section showed mild inflammatory reaction with moderate fibro-collagenisation. Cortex showed lamellar appearance of the bone along with presence of wooven bone at places. Marrow space showed good amount of marrow material, fat cells and blood vessels. No unabsorbed material was seen (Fig. 6A and B).

Section showed picture of well developed lamellar bone containing fair number of havarsian canals. Periostium appeared mildly thickened. Medullary space contained marrow element with a few fat cells and fair number of blood vessels. No foreign material was found to be present at any region (Fig. 7A and B).

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Fig. 6. Histopathological section of the bone (A) in cortical area of control bone (HE X 10) [1. fat cell, 2. marrow material and 3. lamellar bone] and (B) indicating presence of woven bone at cortex (HE X 45) [1. Havarsian canal and 2. Havarsian system].

Fig. 7. Histopathological section in bioactive glass implanted bone (A) HE X 10 [1. Havarsian canal and 2. lamellar bone] and (B) HE X 45 [1. blood vessels and 2. fat cells].

3.7. Oxytetracycline labelling study: 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 was 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 linear zone near the periosteum revealed a golden yellow fluorescence suggestive of new bone formation in the area (Fig. 8). Union in the defect site of bone was complete in most of the experimental species. 3.8. Oxytetracycline labelling study: Group II (bioactive glass) Extensive new bone formation was seen originating from the host bone towards the implant. The amount of new bone formation was more on periosteal surface as compared to endosteal surface. The defect area was

Fig. 8. Oxytetracycline labelling study at the defect site of the control. Photomicrograph showing the presence of homogenous nonfluorescent area of cancellous bone at the same site.

replaced by golden yellow fluorescence of newly formed bone originated from periosteal and endosteal surface.

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Fig. 9. Fluorochrome labelled photomicrograph showing (A) crossing over and union of new bone trabeculae at the defect site of bioactive glass and (B) the presence of well organized homogeneous fluorescent for newly formed bony tissue at day 90th.

The bone defect was completely filled with newly formed cancellous bone showing osseous activity by marked golden yellow fluorescence. Cavities formed due to resorption were lined by newly formed osteoid tissue (Fig. 9A and B). 3.9. Angiographic study: Group I (control) Angiogram of the animals in control group showed that there was uniform angiogenesis in the defect site. There is evidence of capillary network on and around the defect site containing radiodense contrast material. The angiogram also revealed well establishment of uniform medullary cavity (Fig. 10). 3.10. Angiographic study: Group II (bioactive glass) Angiogram of the animals treated with bioactive glass at day 90 postoperatively revealed that there was marked reduction in the size of transplant material. The non

Fig. 11. Angiograph showing the remnants of unabsorbed graft at the defect site with trans-transplant angiogenesis for bioactive glass.

resorbable material had reduced to approximately 20% of its original size. There was evidence of new angiogenesis surrounding the transplant which was suggestive of well acceptance of implant by the host tissue and implant in the process of osteoconduction (Fig. 11). 4. Discussion and conclusions

Fig. 10. Angiograph at day ‘90’ showing well established medullary cavity and uniform capillary network containing radiodense dye adjacent to the created defects (control).

Bone grafts are often necessary to provide support, fill voids and enhance biologic repair of skeletal defects. Strategies for the development of biological substitutes capable of mimicking the homeostasis are based on a better understanding of the basic events in the healing of the fractures. The biological approach aims to provide the key components which play a pivotal role in the repair of the bone (Bruder et al., 1998; Vacanti and Bonassar, 1999). In the recent years, considerable strides have been made on the use of ceramics/polymers in orthopedic surgery, particularly as permanent implants or joint replacement. These materials include natural coral, hydroxyapatite, tricalcium

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phosphate, bioactive glasses and synthetic polymers. They have been used as filling material in bone defects in experimental animals and also in number of clinical applications (Guillemin et al., 1987; Heise et al., 1990; Elsinger and Leal, 1995; Peltola, 2001). The incorporation of these materials in the host bone is clearly inferior to autogenous bone grafts. However, they enhance osteoconduction, which is a three dimensional process of the growth of the capillaries, perivascular tissue and osteoprogenitor cells of the host into the graft (Goldberg and Stevenson, 1987). Recently, clinical application of bioactive glass in dentistry has become a common practice. On the initial implantation stage, they bond to the bone tissue, inhibiting fibroblastic growth. Schepers et al. studied the osteogenesis stimulated by bioactive glass and showed that the ionic exchange on the interface between particles and tissue fluids forms a silica gel layer which is rich in Ca and P (Schepers et al., 1991). 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 suggest that even the resulted mild 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 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). Radiography evaluation of implant has a number of qualities that make it useful for imaging. These include high spatial resolution and image contrast for cortical and trabecular bone, a capacity for broad anatomical coverage with a single image, low cost and wide availability. But its inability to discriminate among non-calcified, non-fatty soft tissues, and its projectional viewing perspective made it disadvantageous also. However, we have discussed the following on the combinations of histological findings too. In animals of Group II, on day 0, bioactive glass blocks maintained their original shape in the created defect site and showed less radiodensity than the surrounding host bones. Radiograph on day 21 revealed smoothen and rounded corners of the implant indicating initiation of resorption of implant by the host tissue. At this stage, the implant looked more radiodense than that of earlier days

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indicating initiation of mineralization process over the implant which corroborated the findings of Gil-Albarova et al. using sol–gel glass and glass–ceramic during critical diaphyseal bone defects healing in rabbits (Gil-Albarova et al., 2005). On day 30, radiograph revealed formation of bony callus along with longitudinal axis of the bone defects. Yu et al. also observed initiation of new bone formation as early as 4 weeks using sol–gel bioactive glass composite porous scaffold in rabbits (Yu et al., 2005). On day 60, radiograph revealed presence of firm organized callus surrounding the implant and markedly reduced radiodensity as well as size, shape indicating advanced stage of resorption of implant. On day 90, diaphyseal radial defect was completely surrounded by well organized thin callus with negligible radiographic impression for presence of implant. Yu et al. observed similar findings with formation of new bone 8 weeks after the implantation of sol–gel bioactive glass composite and the defect was found to be completely repaired 12 weeks after operation. Gil-Albarova et al. reported good implant stability with periosteal growth and remodelling around and over the bone defect in a 4 month radiographic study with sol–gel glass and glass–ceramic in rabbit (Gil-Albarova et al., 2004). A positive effect of bioactive glasses on attachment, proliferation, differentiation and mineralization of bone cells, compared to non-reactive surfaces has been demonstrated in vitro (Loty et al., 2001). Some bioactive glass compositions (<55 mol% of SiO2) exhibit a high bioactive index and osteoconductivity, and are able to induce osteoproduction by stimulating the proliferation of osteoprogenitor cells (Hattar et al., 2002). In the present study, bone growth and remodelling of the implant were observed through the original pores in the bioactive glass caused by gradual dissolution of the structure which is in conformity with the observations of De Aza et al. (2003). Histologically, in Group I, there was moderate fibro-collagenisation with presence of woven bone at places. The new bone formation was not sufficient to fill the entire defect although marrow space showed good amount of marrow materials and blood vessels. This observation supported the findings of different authors (Singh, 1998; Bolander and Balian, 1986; Gil-Albarova et al., 2004). Bolander and Balian reported that ungrafted ulnae did not successfully heal across the defect and limited amount of new bone formation in the vicinity of the cut end of defect (Bolander and Balian, 1986), which is in conformity of the observation of this study. In Group II, the histological section showed well developed lamellar bone containing fair number of havarsian canals and evidence of fair number of blood vessels with marrow element in medullary space. These results are suggesting a process of mesenchymal cells recruitment of surrounding tissues and their subsequent transformation to bone forming cells (Gil-Albarova et al., 2004). The bioactive glass blocks showed osteoconductive and osteointegration properties, as documented in the present study by the close contact between the material and newly formed bone,

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as well as bone growth around and inside of them. The histological material also showed areas with osteoid tissue (bone tissue being formed), which would call for a longer time for bone maturation and complete resorption of the material with bone replacement. Similar findings are also observed by Macedo et al. using bioactive glass in rat tibias (Macedo et al., 2004). No bioactive glass material was seen at any region suggesting quick reabsorption of this material than hydroxyapatite, thus allowing a much more precocious new bone formation in the repair of bone defects (Gil-Albarova et al., 2005; Gil-Albarova et al., 2004; Moore et al., 2001). Besides, the material could not be seen in the present study might be due to the fact that they were completely incorporated into the newly formed bone tissue which is in conformity with the observations of Oonishi et al. (1997). However, porosity within the implants also highly enhance their bone bonding ability due to the following reasons: (a) it has got a large surface area resulting in a high tendency to bioresorb, and induce high bioactivity, (b) interconnected pores can provide a framework for bone growth into the matrix of the implant, and thus anchor them with the surrounding bone, preventing micro-motion that in turn increases further bone growth, (c) interconnected porosity acts like an organization of vascular canals, which can ensure the blood and nutrition supply for the bone. A broad guideline exists regarding the level of total porosity (>50–60%), minimal interconnection size (>50– 100 lm channels) and level of strut porosity (>20% with an upper limit typical for resorbable materials). But it is proved that physical barrier can impair the in-growth of bone tissue in dense implants, which limits the proliferation of blood vessels essential for bone repair. Recent studies on in vitro and in vivo have shown that not only macroporosity of the implants has an influence on integration and volume of regenerated bone but microporosity also has an influence on biological sensitivity for bone formation (Boyde et al., 1999; Bignon et al., 2003). This is assumed to be through intervention of cell attachment and/or selective binding of adhesion proteins and Growth Factors (Ripamonti, 1996). Studies on the influence of bioactive glass microporosity on the rate and quality of bone healing in vivo demonstrated that faster apposition in microporous scaffolds with microporosity levels of >20% was linked to the rate of development of the vascular network. In the present study, the resorption occurred through combined chemical and cellular action, enabling complete restoration of new bone tissue to the defect site within 90 postoperative days. There are several methods to examine newly formed bone using specific bone markers and labelling techniques (Turner, 1994; Konig et al., 1998). The tetracycline labelling method was introduced to measure the exact quantity of newly formed bone as tetracycline molecule is having fluorescence property in ultraviolet light. Oxytetracycline follows the ionized calcium and is fixed by a process of adsorption which is restricted to areas where active deposi-

tion of mineralized tissue is taking place (Gibson et al., 1978). The labelled new bone and old bone emitted bright golden yellow and dark sea green fluorescence respectively, when viewed under UV light and were useful in assessing the amount of new bone formation and fracture healing (Frost, 1958; Ricci et al., 1986; Maiti and Singh, 1995). This characterization technique is important to study gross bone architecture and histological mapping of new bone formation using fluorescent bone markers (Tam and Anderson, 1980; Maiti, 1990). In the present study also, oxytetracycline labelling @ 50 mg/kg body weight (2-6-2 pattern) before end of the study was found sufficient to quantify the extent of new bone formation at the implanted site of bone. In animals of Group I, most of the bone defects were occupied by homogenous nonfluorescent area suggesting little amount of new bone formation although the process was active from both the ends which corroborated the findings of Singh (1998). Newly formed bony tissue was originated more from periostial surface as compared to endosteal side indicating bony union at the defect site through normal healing process. These findings simulated the observations of Suryawanshi et al. where two anabolic hormones were used in tibial fracture healing in dogs (Suryawanshi et al., 1999). However, golden yellow fluorescence was seen in a narrow linear zone near periosteum suggestive of new bone formation in the area. In animals of Group II, under UV light golden yellow fluorescence were seen at the defect site suggesting the presence of good amount of newly formed osteoid tissue. These observations suggest that bioactive glass bonds to bone fairly without an intervening fibrous connective tissue interface (Meffert et al., 1985; Schepers et al., 1991). Numerous resorption cavities lined with fluorescent yellow newly formed osteoid tissue were indicative of remodelling phenomenon. Sodium iothalmate, as a contrast media, has been successfully used by various scientists for the visualization of different vascular pattern (Singh, 1978; Singh et al., 1978; Varshney et al., 1994;). Since the contrast material recommended for angiography procedures is drained out very quickly, it is difficult to visualize minute vascular branches. An immediate radiographic exposure was necessary with Conray-420 to minimize the venous drainage when the procedure was delayed. Lead oxide soap suspension (20%), as a contrast media, was found satisfactory for the visualization of major arteries and also minute vascular branches. Other workers also supported this contrast media for angiographic evaluation of fracture healing (Maiti and Singh, 1996). This material is toxic, cannot be drained out by the venous system and as such, the animal was to be sacrificed before performing angiography. Critical evaluation of angiographic results of the present study revealed varying degrees of vascularization in these two groups. There was well organized trans transplant angiogenesis and establishment of vascular supply across the bone defects in bioactive glass (Group II). The minute

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vessels of the periosteal and endosteal origin invaded the implant bed and this phenomenon 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. Further, the periosteal and surrounding soft tissue vessels play an important role in the early stage of fracture healing (Wray and Lynch, 1959). The soft tissue vessels near the fractured bone were found to be immediate source to the early periosteal callus formation (Gothman, 1961). Stripping of periosteum from its bony attachment resulted in greater dilatation of periosteal vessels (Zucman, 1960). The development of new vessels during fracture healing passes through five stages: hyperemia, proliferation of periosteal and medullary vessels, differentiation of proliferated vessels and their organization (Koekenberg, 1963). The slow mineralization process in case of control animals might be due to the absence of any scaffold material in the defect site. Presence of bridging callus with irregularly arranged osteon in the edge and fibro-cartilaginous tissues in center of the defect were suggestive of normal healing, similar findings were also observed by Singh et al. (2000) and Bolander and Balian (1986). The results of bioactive glass suggested the process of mesenchymal cells recruitment of surrounding tissues and subsequent transformation of bone forming cells (Gil-Albarova et al., 2004). Vogel et al. (2001), De Aza et al. (2003) and Fujibayashi et al. (2003) also examined bone formation around bioactive glass particles. It was reported that the interfacial gap between the bioactive glass and neighboring bone was least suggestive of new bone formation in peripheral and central porosities of the implant (De Aza et al., 2003). In the present study the implanted bioactive glass was porous in nature, which allow fibrovascular and bone tissue ingrowth, making direct integration with a neighboring bone possible (Klawitter and Hulbert, 1971). Also well-formed vascularisation towards the implant block was observed which is required to unite the implant with the host during the osseous healing process (Lin et al., 1997; Chang et al., 1999). This vascularisation is very important for successful bone healing and development of blood vasculature, as it is of critical importance for providing blood-bone factors in the osteogenetic process. De Aza et al. (2003) observed similar finding where anatomical arrangement of a well developed vascular system directly juxtaposed to the surface of glass implanted Rabbit tibia after 12 weeks. Hench and Paschall (1974) reported that bone formation mechanism in bioglass and surrounding bone tissues are different and in those cases bioglass bond to bone chemically by forming a Ca–P rich layer at the interface. When glass with a low ratio of network former and alkali ion content is implanted in tissue, a sequence of reactions occurs, starts with surface dissolution, together with CaO it leads to the formation of a strong interface. All the alkali ions leach out, yielding the formation of a silicon-rich

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layer, on top of which a Ca–P-rich layer is formed. Both diffusion from the glass as well as deposition from the extracellular fluids around the implant create this Ca–Prich layer (Hench and Paschall, 1974; Nakamura et al., 1985; Neo et al., 1992). Mineralization of bone involves a concentration enhancement of silicon at the mineralization front. This biologically active SiO2 in combination with apatite is responsible for inhibiting the proliferation of fibroblasts at a bioactive implant interface. It is reported that presence of silicon at a critical concentration is an essential prerequisite to trigger bone formation. On the other hand in bioactive glass/glass–ceramics the concurrence of SiO2 hydrolysis and condensation with biological hydroxyapatite mineralization promotes bone growth, which is also the natural process of bone repair (Hench and Paschall, 1974). In the present case, presence of titania in the bioactive glass also plays key role. Since, the glass releases Ca2+, Na+, or K+ ions from its surfaces via an exchange with the H3O+ ion in the surrounding body fluid to form Si–OH or Ti–OH groups on their surfaces. The same water molecules simultaneously react with the Si–O–Si or Ti–O–Ti bond to form additional Si–OH or Ti–OH groups. These groups formed induce apatite nucleation, and the released Ca2+, Na+, or K+ ions accelerate apatite nucleation. Once the apatite nuclei are formed, they can grow spontaneously by taking the calcium and phosphate ions from the surrounding fluid since it is highly supersaturated with respect to this new apatite (Neuman and Neuman, 1958). Considering the results obtained, we conclude that porous bioactive glass promoted bone formation over the extension of the defect and offers interesting potential for orthopedic reconstructive procedures and that further studies are warranted. 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 Bignon, A., Chouteau, J., Chevalier, J., Fantozzi, G., Carret, J.P., Chavassieux, P., Boivin, G., Melin, M., Hartmann, D., 2003. Effect of micro- and macroporosity of bone substitutes on their mechanical properties and cellular response. Journal of Materials Science: Materials in Medicine 14, 1089–1097. Bolander, M.E., Balian, G., 1986. The use of demineralized bone matrix in the repair of segmental defects. Journal of Bone and Joint Surgery 68A, 1264–1274. Boyde, A., Corsi, A., Quarto, R., Cancedda, R., Bianco, P., 1999. Osteoconduction in large macroporous hydroxyapatite ceramic implants: evidence for a complementary integration and disintegration mechanism. Bone 24, 579–589. Bruder, S.P., Kurth, A.A., Shea, M., Hayes, W.C., Jaiswal, N., Kadiyala, S., 1998. Bone regeneration by implantation of purified, culture-

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