Fabrication and mechanical testing of porous calcium phosphate bioceramic granules

Porous hydroxyapatite/tricalcium phosphate (HA/TCP) granules were fabricated by a novel technique of vacuum impregnation of reticulated polyurethane (PU) foams with ceramic slip. The resultant granules had 5-10% interconnected porosity with controlled pore sizes necessary to allow bone ingrowth combined with good mechanical properties. Using PU foams with a different number of pores per inch (ppi), porous HA/TCP granules in the size range of 2-8 mm were successfully manufactured. Dieplunger tests were used to compare the compression and relaxation properties of the granules with those of a commercially available bone graft product, BoneSave. The results of the die-plunger testing showed that the experimental granules were stiffer than the BoneSave materials and had less of a tendency to crumble to powder after testing. This therefore suggests that these experimental granules would be useful for impaction grafting and space filling applications.     

Evaluation of collagen/heparin coated TCP/HA granules for long-term delivery of BMP-2

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. However, a delivery system is essential to take advantage of the osteoinductive effect of BMPs. The purpose of this study was to develop a sustained delivery system for recombinant human bone morphogenetic protein-2 (BMP-2). We covalently attached heparin to a cross-linked collagen type I coated tricalciumphosphate/hydroxyapatite (TCP/HA) bone substitute and subsequently loaded it with BMP-2. To systematically evaluate the contribution of each component with respect to the binding and release of BMP-2, six constructs were prepared and characterized: TCP/HA, TCP/HA with collagen (TCP/HACol), and TCP/HA with collagen and heparin (TCP/HAColHep) with and without BMP-2 (B). More BMP-2 bound to the TCP/HAColHep + B (92.9 ± 4.8 ng BMP-2/mg granule) granules as compared to the TCP/HACol + B (69.0 ± 9.6 ng BMP-2/mg granule) and TCP/HA + B granules (62.9 ± 5.4 ng BMP-2/mg granule). No difference in release pattern was found between the TCP/HA + B and TCP/HACol + B granules. Up to day 14, BMP-2 was still bound to the TCP/HAColHep + B granules, whereas most BMP had been released from TCP/HACol + B and TCP/HA + B granules at that time. After 21 days most BMP-2 also had been released from the TCP/HAColHep + B granules. The local and sustained delivery system for BMP-2 developed in this study may be useful as a carrier for BMP-2 and could possibly enhance bone regeneration efficacy for the treatment of large bone defects.     

Effects of porous beta-tricalcium phosphate-based ceramics used as an E. coli-derived rhBMP-2 carrier for bone regeneration

Recombinant human bone morphogenetic protein-2 (rhBMP-2) requires carriers for clinical effectiveness. In this study, whether porous beta-tricalcium phosphate (β-TCP)-based ceramics are ideal carriers for rhBMP-2 was investigated. Hydroxyapatite (HA), β-TCP, TCP/HA (80 %/20 %), HA with rhBMP-2, TCP with rhBMP-2, and TCP/HA (80 %/20 %) with rhBMP-2 were manufactured by a sponge method with a pore size of 300 μm or more and macro-porosity of 83 %. The alkaline phosphatase (ALP) activity and ALP expression of the cells with 100 % β-TCP granules were more increased than the those of cells with 100 % HA and TCP/HA (80 %/20 %) at the baseline or when treated with 15 ng/ml of rhBMP-2. In an SD rat calvarial defect model, new bone formation was evidently shown in the TCP 100 %-rhBMP-2 and TCP/HA (80 %/20 %)-rhBMP-2 groups, showing that the most affected area was filled with newly-formed bone, that the percent bone volume and trabecular number were larger when compared to the groups without rhBMP-2 treatment at both 4 and 8 weeks after surgery using micro-CT and histology. Porous TCP-based ceramic granules enhanced the osteoblastic differentiation in the hMSC system when treated with 15 ng/ml of rhBMP-2 and accelerated bone-healing by trabecular number in a rat calvarial defect model. Thus, in this study it was proposed that TCP-based ceramics might be useful carriers of rhBMP-2.     

In vivo behaviour of two different biphasic ceramic implanted in mandibular bone of dogs

Alloplastic calcium phosphate bone substitutes such as hydroxyapatite (HA) and tricalcium phosphate (TCP) have been studied extensively due to their composition closely resembling the inorganic phase of bone tissue. On the same way, by manipulating the HA/TCP ratio it may be possible to change the substitution rate and the bioactivity of these materials, an advantage which has brought them to clinical use in oral and orthopaedic surgery. In this work, we evaluated the histological response in bone of two biphasic calcium phosphate ceramics by varying the proportion of their components. All premolars of 6 beagle dogs were removed from both sides of the mandible. Three months later, four cylinders composed of 85% HA and 15% β-TCP (BCP 1) were implanted in the right side of mandible and other four cylinders composed of 15% HA and 85% β-TCP (BCP 2) were implanted in the left side of mandible of dogs for 4, 12 ad 26 weeks, respectively. Two dogs were used in each time point. The histological study indicated that both biphasic ceramic were biocompatible. The earlier and more quantity of bone formed in BCP 2 than in BCP 1 suggested that the first one had a higher osteoinductive potential than the second one in mandibular bone. The resorption of the phosphate phase and the subsequent migration of bone into the resorbed portions were detected in both biphasic ceramics although two processes appeared faster in BCP 2 than in BCP 1. These dates conclude that varying the components of our biphasic ceramic we improve its osteoinductive potential.     

The copolymer of ε-caprolactone-lactide and tricalcium phosphate does not enhance bone growth in mandibular defect of sheep

In the field of craniomaxillofacial and orthopaedic surgery there is a constant need for bone or bone substitute. At the present, the most effective way to enhance bone healing clinically is to use autogenous bone grafts. The problems associated with the use of these autografts are donor site morbidity, limited supply and need for a second operative site. Currently there are several different synthetic products commercially available in the market; nevertheless, none of them is ideal for filling bone defects. Therefore, search for new synthetic materials for bone replacement is necessary. A mixture of tricalcium phosphate (TCP) and ε-caprolactone-lactide copolymer P(ε -CL/DL-LA) was prepared and implanted in critical size mandibular bone defects in twelve sheep. Contralateral side was used as a control. Follow-up times for histological and radiological studies were 9, 14, 24 and 52 weeks. We found that the implanted material did not enhance bone formation compared to control site. We also confirmed that defect size was of critical size, since there was no complete healing of the control site either. The results do not encourage us to continue our studies with the mixture of TCP and P(ε-CL/DL-LA) as a filling material for bone defects. Therefore the search for the ideal material is still ongoing.     

Testing bone substitutes in a small animal model of revision arthroplasty

This study evaluated a modification of the rat-pin model to enable testing of bone substitute materials. The model was characterized using the ceramic, -tricalcium phosphate (TCP) as a filler. A 1 mm wide, 3.6 mm deep defect was created around a stainless steel (SS) implant in the proximal tibia of a rat. This defect was filled with a ceramic powder. Large particles (90–312m) of TCP were mixed with Gelfoam® to form a paste which was then molded around the proximal end of either an uncoated SS pin or a pin coated with hydroxyapatite (HA). The pin with its ceramic collar was then implanted into the proximal tibia of 16 male Sprague Dawley rats. Two animals with coated implants and two with uncoated implants were sacrificed at 3, 6, 14 and 26 weeks. Longitudinal sections of each tibia were stained with toluidine blue and labeled for tartrate resistant acid phosphatase (TRAP). There was initial fibrous tissue interposition around the implants which was completely remodeled around the HA coated pins but which persisted in apposition to the SS pins. The remodeling process peaked at 3 weeks around the HA coated pins and at 6 weeks around the uncoated implants. There was little remodeling around either implant by 26 weeks. There was considerable residual TCP present which was well tolerated as the particles were often encased in bone. The model has several characteristics of revision arthroplasty and the results demonstrate the suitability of this model for testing bone substitutes.An erratum to this article can be found at     

Biofunctionality of MBCP ceramic granules (TricOs™) plus fibrin sealant (Tisseel®) versus MBCP ceramic granules as a filler of large periprosthetic bone defects: an investigative ovine study

We aimed to quantify bone colonization toward an untreated titanium implant with primary stability following filling of the defect with micromacroporous biphasic calcium phosphate (MBCP) granules (TricOs?) or MBCP granules mixed with fibrin sealant (Tisseel^®). Medial arthrotomy was performed on the knees of 20 sheep to create a bone defect (16 mm deep; 10 mm diameter), followed by anchorage of a titanium screw. Defects were filled with TricOs or TricOs–Tisseel granules, a perforated MBCP washer, a titanium washer and titanium screw. Sheep were euthanized at 3, 6, 12 and 26 weeks. From Week 12 onwards, the percentage of bone in contact with the 8 mm anchorage part of the screw increased in both groups, confirming its primary stability. At 26 weeks, whereas bone colonization was similar in both groups, biodegradation of ceramic was more rapid in the TricOs–Tisseel group (P = 0.0422). The centripetal nature of bone colonization was evident. Bone contact with the titanium implant surface was negligible. In conclusion, the use of a model that reproduces a large metaphyseal bone defect around a titanium implant with primary stability, filled with a mixture of either TricOs ceramic granules or TricOs granules mixed with Tisseel fibrin sealant, suggests that the addition of fibrin to TricOs enhances bone filling surgical technology.     

Long term results after implantation of tissue engineered cartilage for the treatment of osteochondral lesions in a minipig model

In present study we determined the long term in vivo integration and histological modeling of an in vitro engineered cartilage construct. Tissue engineered autologous cartilagenous tissue was cultured on calcium phosphate cylinders and implanted into osteochondral defects into the femoral condyles in minipigs. Radiological follow-up was performed at 2, 8, 26 and 52 weeks, condyles were harvested 26 and 52 weeks post-implantation. Thickness of cultivated tissue (1.10 ± 0.55 mm) was comparable to in situ cartilage and cells produced in vitro cartilage specific proteins. In vivo, 26 and 52 weeks post-implantation defects were resurfaced with hyaline-like tissue, the implants were well integrated with no gap at the interface between the engineered neocartilage and the adjacent articular cartilage. Synthesis of type II collagen was detected 26 and 52 weeks after implantation. The modified ICRS score increased from 26 to 52 weeks. Histomorphometric evaluation revealed a decrease in cellularity in tissue engineered cartilage from 2.2-fold of native cartilage after 26 weeks to 1.5-fold after 52 weeks. In conclusion, these findings demonstrate the integration and maturation of tissue engineered cartilage pellets attached on a bone substitute carrier implanted in osteochondral defects over a long time. J. P. Petersen, P. Ueblacker, C. Goepfert have contributed equally to this study.     

Comparative evaluation of a biomimic collagen/hydroxyapatite/β-tricaleium phosphate scaffold in alveolar ridge preservation with Bio-Oss Collagen

Bone scaffolds are critical in current implant and periodontal regeneration approaches. In this study, we prepared a novel composite type-I collagen and hydroxyapatite (HA)/β-tricaleium phosphate (TCP) scaffold (CHTS) by incorporating type-I collagen and bovine calcined bone granules, prepared as a mixture of 50% HA and 50% TCP, by freeze drying. We then characterized the CHTS and determined its cytotoxic effects. Additionally, ridge preservation experiments were carried out to evaluate the clinical effects of the CHTS. The results demonstrated that the composite scaffolds had good surface morphology and no cytotoxicity. Additionally, an in vivo experiment in an animal model showed that the CHTS performed equally as well as Bio-Oss Collagen, a widely used bone graft in ridge preservation. These findings revealed that the CHTS, which contained natural constituents of bone, could be used as a scaffold for bone regeneration and clinical use.     

Osteogenic properties of calcium phosphate ceramics and fibrin glue based composites

Calcium phosphate (Ca-P) ceramics are currently used in various types of orthopaedic and maxillofacial applications because of their osteoconductive properties. Fibrin glue is also used in surgery due to its haemostatic, chemotactic and mitogenic properties and also as scaffolds for cell culture and transplantation. In order to adapt to surgical sites, bioceramics are shaped in blocks or granules and preferably in porous forms. Combining these bioceramics with fibrin glue provides a mouldable and self-hardening composite biomaterial. The aim of this work is to study the osteogenic properties of this composite material using two different animal models. The formation of newly formed bone (osteoinduction) and bone healing capacity (osteconduction) have been study in the paravertebral muscles of sheep and in critical sized defects in the femoral condyle of rabbits, respectively. The different implantations sites were filled with composite material associating Ca-P granules and fibrin glue. Ca-P granules of 1–2 mm were composed with 60% of hydroxyapatite and 40% of beta tricalcium phosphate in weight. The fibrin glue was composed of fibrinogen, thrombin and other biological factors. After both intramuscular or intraosseous implantations for 24 weeks and 3, 6, 12 and 24 weeks, samples were analyzed using histology and histomorphometry and mechanical test. In all cases, the newly formed bone was observed in close contact and around the ceramic granules. Depending on method of quantification, 6.7% (with BSEM) or 17% (with μ CT) of bone had formed in the sheep muscles and around 40% in the critical sized bone rabbit defect after 24 weeks. The Ca-P/fibrin material could be used for filling bone cavities in various clinical indications.     

Immunohistochemical evaluation after Sr-enriched biphasic ceramic implantation in rabbits femoral neck: comparison of seven different bone conditions

Strontium (Sr) has shown effectiveness for stimulating bone remodeling. Nevertheless, the exact therapeutic values are not established yet. Authors hypothesized that local application of Sr-enriched ceramics would enhance bone remodeling in constant osteoporosis of rabbits’ femoral neck bone. Seven different bone conditions were analyzed: ten healthy rabbits composed a control group, while other twenty underwent ovariectomy and were divided into three groups. Bone defect was filled with hydroxyapatite 30% (HAP) and tricalcium phosphate 70% (TCP) granules in 7 rabbits, 5% of Sr-enriched HAP/TCP granules in 7, but sham defect was left unfilled in 6 rabbits. Bone samples were obtained from operated and non-operated legs 12 weeks after surgery and analyzed by histomorphometry and immunohistochemistry (IMH). Mean trabecular bone area in control group was 0.393 mm2, in HAP/TCP – 0.226 mm2, in HAP/TCP/Sr – 0.234 mm2 and after sham surgery – 0.242 mm2. IMH revealed that HAP/TCP/Sr induced most noticeable increase of nuclear factor kappa beta 105 (NFkB 105), osteoprotegerin (OPG), osteocalcin (OC), bone morphogenetic protein 2/4 (BMP 2/4), collagen type 1α (COL-1α), interleukin 1 (IL-1) with comparison to intact leg; NFkB 105 and OPG rather than pure HAP/TCP or sham bone. We concluded that Sr-enriched biomaterials induce higher potential to improve bone regeneration than pure bioceramics in constant osteoporosis of femoral neck bone. Further studies on bigger osteoporotic animals using Sr-substituted orthopedic implants for femoral neck fixation should be performed to confirm valuable role in local treatment of osteoporotic femoral neck fractures in humans.

Open image in new window

     

Ultrastructural study of the effects of coral skeleton on cultured human gingival fibroblasts in three-dimensional collagen lattices

Natural coral skeleton has recently been introduced as a bone graft substitute which enhances bone formation in man and animals. The effects of NCS on cultured human cells has not previously been investigated. In the present study we report these effects as studied by light microscopy, transmission and scanning electron microscopy in three-dimensional culture. The results showed that natural coral skeleton does not inhibit the normal function of fibroblasts in contracting collagen lattices. After 8 weeks, the cells maintained a healthy ultrastructural morphology. At the collagen/coral interface, the cells were well-spread and attached to the surface by numerous adhesion plaques. Evidence for biosynthetic activity was also observed; the cells showed numerous ribosomes, mitochondria and prominent rough endoplasmic reticulum. Extracellularly, a perigranular dense matrix, appearing as nodules in the SEM, was deposited on the coral surface. This matrix was made of highly organized fibrils lacking periodicity, and a ground substance. The present study shows that coral was well-tolerated by human gingival fibroblasts, and that it provided a surface for cell spreading, attachment and deposition of the special extracellular matrix.     

Porous calcium phosphate ceramic granules and their behaviour in differently loaded areas of skeleton

Two kinds of calcium phosphate ceramic (CPC) granules of high porosity (50 ± 5%) and improved (for such materials) compressive strength (10–25 MPa) consisted of hydroxyapatite (PHA) and a mixture of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) in 60 HA/40 β-TCP composition (PCPC) were developed. A comparative study of in vivo behavior of the materials implanted into an almost unloaded (greater trochanter of femur) and loaded (distal methaphysis of femur) zones in the skeleton of rabbits was performed. Significant activating influence of loading on the processes of new bone formation and reconstruction in macropores of both materials during all periods of implantation (up to 6 months) was observed. The role of relevant cells in the processes in the unsoluble PHA and the degradable PCPC (in which the processes was observed to intensify due to dissolution of the material) was studied and is discussed. Great disturbance in pore structure of the BCPC was revealed in more late periods of implantation. After 6 months, presence of large composite fragments located in intertrabecula spaces of greater trochanter was a characteristic feature of the PCPC crushing. The developed CPC materials seems to have good perspective for using in bone defect plasty in some loaded areas of the skeleton.     

Effects of strain rate on the mechanical properties of tricalcium phosphate/poly(l-lactide) composites

Bioactive ceramic/bioresorbable plastic composites have been expected as materials for the bone fracture fixations which have more biocompatibility than monolithic bioresorbable plastics. Many studies have been conducted on these materials. Most studies, however, focused on the mechanical properties under static loading. In the actual usage, these materials are loaded dynamically. In this study, effects of strain rate on the mechanical properties of tricalcium phosphate/poly(l-lactide) (TCP/PLLA) composites were investigated experimentally and analytically. The TCP/PLLA composites containing three different TCP contents (5, 10 and 15 wt.%) were prepared by injection molding. In order to characterize the mechanical properties, tensile and compressive tests were conducted. The results of tensile tests indicated that the Young’s moduli of composites increased with increasing TCP contents. For each TCP contents, tensile Young’s modulus kept constant up to strain rate of 10⁻¹/s. On the other hand, tensile strength increased with increasing strain rate. The effect of strain rate became larger with decreasing TCP contents, which means the strain rate dependency of the PLLA is more effective than that of TCP. From the results of compressive tests, similar results with tensile tests were obtained. That is, compressive Young’s modulus kept constant up to strain rate of 10⁻¹/s and the 0.2% proof stress increased with increasing strain rate. In order to predict the mechanical behavior of TCP/PLLA composites, the micro-damage mechanics was proposed. In this analysis, 3-phases particle reinforced composites, which include the intact particles, damaged particles and matrix, are assumed. The elastic constants are calculated with micromechanics based on the analyses by Eshelby and Mori and Tanaka. Only the debonding between particle and matrix are assumed as the damage. The nonlinearity in the stress-strain behavior of matrix PLLA is also considered. The debonding particles are assumed as voids. Void formation is calculated based on the energy criterion. The energy release rate associated with void formation was estimated by fitting the analytical results with the experimental results of the composites with 15 wt.% TCP contents for each strain rate. Then the analytical results for the composites with 5 and 10 wt.% TCP contents were compared with the experimental results. The analytical tensile stress-strain curves are in good agreement with experimental results. It is also clarified that the energy release rate associated with void formation increased with increasing strain rate.     

Tissue reaction of hydroxyapatite sol to rat molar pulp

Hydroxyapatite sol was prepared by dispersing unheated hydroxyapatite microcrystals into distilled water. A suspension of hydroxyapatite heated at 800 °C (HAp-800°C) in distilled water was used as a comparative material. These were characterized by X-ray diffractometry, SEM and electroconductivity measurements. 0.001 ml of each material was injected into the upper first molar pulp of rats. After 7–14 days post-operatively, the group with hydroxyapatite sol showed slight inflammation in the pulp cavities and a highly calcified tubular dentine bridge was observed. Osteodentine had developed and adhered to the surface of the hydroxyapatite microcrystals. Matrix vesicles were observed in the extracellular matrix between collagen fibrils and electron-dense granules. After 28 days, the inflammation completely disappeared and tubular dentine was observed below the osteodentine. Acid phosphates (ACPase) activity was detected along all biosynthesizing pathways in odontoblast-like cells. Localized dehydrogenate (LDH) activity indicated phagocytosis of hydroxyapatite microcrystals by odontoblasts. In the group with HAp-800°C after 7–14 days post-operatively, the inflammation was moderate. After 28 days, relatively fewer calcified dentine tubules were observed. ACPase activity was detected only along the membranes bounding cytoplasmatic vacuoles. It was concluded that hydroxyapatite sol is a safe biomaterial for use as dental pulp and induces early formation of dentine-bridge more so than HAp-800 °C. © 1998 Kluwer Academic Publishers     

Stimulation of healing within a rabbit calvarial defect by a PCL/PLGA scaffold blended with TCP using solid freeform fabrication technology

The purpose of this study was to investigate the healing capacity within an 8-mm rabbit calvarial defect using a polycaprolactone (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffold blended with tri-calcium phosphate (TCP) that was constructed using solid freeform fabrication (SFF) technology. The PCL/PLGA/TCP scaffold showed a 37?% higher compressive strength and rougher surface than the PCL/PLGA scaffold. In animal experiments, new bone formation was analyzed using microcomputed tomography (micro-CT) and histological and histometric analyses. The PCL/PLGA/TCP groups had significantly greater neo-tissue areas as compared with the control groups at 4 and 8 weeks (P?<?0.05). The PCL/PLGA/TCP group had significantly greater bone density as compared with the control and PCL/PLGA groups at 4 and 8 weeks (P?<?0.005). The results of this study suggest that the PCL/PLGA/TCP scaffold fabricated using SFF technology is useful for recovering and enhancing new bone formation in bony defects in rabbits.     

“In vitro” behaviour of adult mesenchymal stem cells of human bone marrow origin seeded on a novel bioactive ceramics in the Ca2SiO4–Ca3(PO4)2 system

This work describes the evaluation of three ceramic materials as potential osteogenic substrate for bone tissue engineering. The capacity of adult human mesenchymal stem cells cultured under experimental conditions known to adhere, proliferate and differentiate into osteoblasts was studied. Two types of culture medium: growth medium and osteogenic medium were evaluated. The materials were pure ??-tricalcium phosphate and also ??TCP doped with either 1.5 or 3?wt% of dicalcium silicate. The results showed that the hMSCs cultured adhered, spread, proliferated and produced mineralized extracellular matrix on all the ceramics studied. They showed an osteoblastic phenotype, especially in the ??TCP doped with 1.5?wt% C₂S, indicating osteoblastic differentiation as a result of the increased concentration of silicon in solid solution in TCP. Ceramics evaluated in this work are bioactive, cytocompatible and capable of promoting the differentiation of hMSCs into osteoblast.     

Preliminary<Emphasis Type="Italic">in vitro</Emphasis> and<Emphasis Type="Italic">in vivo</Emphasis> characterizations of a sol-gel derived bioactive glass-ceramic system

This study investigates quantitatively and qualitatively the sol-gel derived bioactive glass-ceramic system (BGS)—apatite-wollastonite (AW) type granules in the size range of 0.5–1 mm, as an effective graft material for bone augmentation and restoration. Scanning electron micrographs (SEM) of the sintered granules revealed the rough material surface with micropores in the range 10–30 μm. X-ray diffraction (XRD) pattern of the granules revealed the presence of crystalline phases of the hydroxyapatite and wollastonite, and the functional groups of the silicate and phosphates were identified by Fourier transform infrared spectroscopy (FT-IR). Thein vitro cell culture studies with L929 mouse fibroblast cell line showed very few cells adhered on the BGS disc after 24 h. This could be due to the highly reactive surface of the disc concomitant with the crystallization but not due to the cytotoxicity of the material, since the cellular viability (MTT assay) with the material was 80‰ Cytotoxicity and cytocompatibility studies proved that the material was non-toxic and biocompatible. After 12 weeks of implantation of the BGS granules in the tibia bone of New Zealand white rabbits, the granules were found to be well osteointegrated, as observed in the radiographs. Angiogram with barium sulphate and Indian ink after 12 weeks showed the presence of microcapillaries in the vicinity of the implant site implicating high vascularity. Gross observation of the implant site did not show any inflammation or necrosis. SEM of the implanted site after 24 weeks revealed good osteointegration of the material with the newly formed bone and host bone. New bone was also observed within the material, which was degrading. Histological evaluation of the bone healing with the BGS granules in the tibial defect at all time intervals was without inflammation or fibrous tissue encapsulation. After 2 weeks the new bone was observed as a trabeculae network around the granules, and by 6 weeks the defect was completely closed with immature woven bone. By 12 weeks mature woven bone was observed, and new immature woven bone was seen within the cracks of the granules. After 24 weeks the defect was completely healed with lamellar bone and the size of the granules decreased. Histomorphometrically the area percentage of new bone formed was 67.77% after 12 weeks and 63.37% after 24 weeks. Less bone formation after 24 weeks was due to an increased implant surface area contributed by the material degradation and active bone remodeling. The osteostimulative and osteoconductive potential of the BGS granules was established by tetracycline labelling of the mineralizing areas by 2 and 6 weeks. This sol-gel derived BGS granules proved to be bioactive and resorbable which in turn encouraged active bone formation.     

Fabrication of transparent hydroxyapatite and application to bone marrow derived cell/hydroxyapatite interaction observation in-vivo

Transparent hydroxyapatite (HA), 99.6% relative density, was prepared by hot isostatic pressing. Optical transmittance of 1 mm thick HA was greater than 60% at 700 nm. Twenty transparent HA plates (10×7×1 mm) were implanted into the medullary cavities of two dogs' femora. At 1 and 2 weeks after implantation the bone marrow derived cells that adhered to the HA surfaces were histochemically examined by light microscopy and scanning electron microscopy. Four types of enzymatic reactions were employed to identify cell types. Alkaline phosphatase was used as a marker for osteoblasts, acid phosphatase and tartrate-resistant acid phosphatase as a marker for osteoclasts and non-specific esterase as a marker for macrophages. Cells adhered to the HA surfaces were a mixed population of osteoblasts, osteoclasts and macrophages. By 2 weeks, the number of osteoblasts increased and formed a bone-like matrix. A small number of osteoclasts formed resorption lacunae on the HA after 2 weeks. By utilizing the transparency of the HA, whole-view of bone matrix formation by osteoblasts and simultaneous resorption of HA by osteoclasts was observed simply and dynamically by light microscopy.