Legg Calvé Perthes disease in the dog.

Legg Calvé Perthes disease (LCPD) is a developmental abnormality that usually affects young small breed dogs. Although the etiology is still uncertain, the pathogenesis is very similar to that described in humans. Diagnosis with physical examination and medical imaging is relatively easy. Treatments offered in dogs, mainly surgical, are also presented. Legg Calvé Perthes disease has many similarities between children and puppies. Dog is therefore an interesting spontaneous model of aseptic osteonecrosis.

Hypodynamia Alters Bone Quality and Trabecular Microarchitecture.

Disuse induces a rapid bone loss in humans and animals; hypodynamia/sedentarity is now recognized as a risk factor for osteoporosis. Hypodynamia also decreases bone mass but its effects are largely unknown and only few animal models have been described. Hypodynamic chicken is recognized as a suitable model of bone loss but the effects on the quality have not been fully explored. We have used ten chickens bred in a large enclosure (FREE group); ten others were confined in small cages with little space to move around (HYPO group). They were sacrificed at 53 days and femurs were evaluated by microcomputed tomography (microCT) and nanoindentation. Sections (4 µm thick) were analyzed by Fourier Transform InfraRed Microspectroscopy (FTIR) to see the effects on mineralization and collagen and quantitative backscattered electron imaging (qBEI) to image the mineral of the bone matrix. Trabecular bone volume and microarchitecture were significantly altered in the HYPO group. FTIR showed a significant reduction of the mineral-to-matrix ratio in the HYPO group associated with an increase in the carbonate content and an increase in crystallinity (calculated as the area ratio of subbands located at 1020 and 1030 cm-1) indicating a poor quality of the mineral. Collagen maturity (calculated as the area ratio of subbands located at 1660 and 1690 cm-1) was significantly reduced in the HYPO group. Reduced biomechanical properties were observed at the tissue level. Confined chicken represents a new model for the study of hypodynamia because bone changes are not created by a surgical lesion or a traumatic method. Animals have a reduced bone mass and present with an altered bone matrix quality which is less mineralized and whose collagen contains less crosslinks than in control chicken.

Bone mass and bone quality are altered by hypoactivity in the chicken.

Disuse induces a rapid bone loss in adults; sedentarity is now recognized as a risk factor for osteoporosis. Hypoactivity or confinement also decrease bone mass in adults but their effects are largely unknown and only few animal models have been described. We have used 10 chickens of the rapidly growing strain 857K bred in a large enclosure (FREE group); 10 others were confined in small cages with little space to move around (HYPO group). They were sacrificed at 53 days and femurs and tibias were evaluated by texture analysis, dual energy X-ray densitometry, microcomputed tomography (microCT) and histomorphometry. Hypoactivity had no effect on the length and diameter of the bones. Bone mineral density (BMD), microCT (trabecular bone volume and trabecular microarchitecture) and texture analysis were always found significantly reduced in the animals of the HYPO group. BMD was reduced at both femur and tibia diaphysises; BMD of the metaphysis was significantly reduced in the femur but not in the tibia. An increase in osteoid volume and surfaces was noted in the HYPO group. However, there was no alteration of the mineral phase as the osteoid thickness did not differ from control animals. Bone loss was much more pronounced at the lower femur metaphysis than at the upper metaphysis of the tibia. At the tibia, only microarchitectural changes of trabecular bone could be evidenced. The confined chicken represents a new method for the study of hypodynamia since these animals do not have surgical lesions.

ß-TCP granules mixed with reticulated hyaluronic acid induce an increase in bone apposition.

ß beta-tricalcium phosphate (ß-TCP) granules are suitable for repair of bone defects. They have an osteoconductive effect shortly after implantation. However, dry granules are difficult to handle in the surgical room because of low weight and lack of cohesion. Incorporation of granules in a hydrogel could be a satisfactory solution. We have investigated the use of hyaluronic acid (HyA) as an aqueous binder of the granules. ß-TCP granules were prepared by the polyurethane foam technology. Commercially available linear (LHya) and reticulated hyaluronic acid (RHyA) in aqueous solution were used to prepare a pasty mixture that can be handled more easily than granules alone. Thirteen New Zealand White rabbits (3.5-3.75 kg) were used; a 4 mm hole was drilled in each femoral condyle. After flushing, holes were filled with either LHyA, RHyA, dry ß-TCP granules alone, ß-TCP granules + LHyA and ß-TCP granules + RHyA. Rabbits were allowed to heal for one month, sacrificed and femurs were harvested and analysed by microCT and histomorphometry. The net amount of newly formed bone was derived from measurements done after thresholding the microCT images for the material and for the material+bone. LHyA and RHyA did not result in healing of the grafted area. LHyA was rapidly eluted from the grafted zone but allowed deposition of more granules, although the amount of formed bone was not significantly higher than with ß-TCP granules alone. RHyA permitted the deposition of more granules which induced significantly more bone trabeculae without inducing an inflammatory reaction. RHyA appears to be a good vehicle to implant granules of ß-TCP, since HyA does not interfere with bone remodeling.

Osteoconductive properties of poly(96L/4D-lactide)/beta-tricalcium phosphate in long term animal model.

The objective of this study was to determine the effect of calcium phosphate mineral content on the bone in-growth at the expense of composite of co-polylactide polymer charged with 2 different ratios of ß-TCP granules (10 and 24 w-% of ß-TCP). The evaluation was realized in a long term rabbit bone model. After 24, 48 and 76 weeks, the implants were examined by micro CT, scanning electron microscopy (SEM) using backscattered electron (BSE) and light microscopy (polarized and blue light microscopy). No foreign body reaction was detected during the 76 weeks follow-up in any of the test samples. Polymer hydrolysis began at approximately 24 weeks, by 76 weeks, the pure polymer implant had begun to release P(96L/4D)LA particles and show signs of peripheral localized bone resorption. A decrease in the amount of CaP was noticed between 24 and 76 weeks in both 10 wt-% and 24 wt-% ß-TCP/P(96L/4D)LA composites. The study showed that the highest bone in-growth was with 24 wt-% ß-TCP/P(96L/4D)LA composite. Bone in-growth and mineralization were evident for the composites associated with specific peripheral bone architecture. Fluorescent labelling demonstrated high bone in-growth and remodeling at the interface, while for pure co-polymer no bone remodeling or bone activity was maintained after 48 weeks. The study demonstrated the positive effect of calcium phosphate content into P(96L/4D)LA. This kind of composite is a suitable resorbable osteoconductive matrix, which provides long term stability required for ligament fixation device.

Mandibular segmental defect regenerated with macroporous biphasic calcium phosphate, collagen membrane, and bone marrow graft in dogs.

OBJECTIVE: To reconstruct segmental mandibulectomy using calcium phosphate ceramics and collagen membrane with a delayed bone marrow grafting in experimental animals. DESIGN: Defects of segmental mandibulectomy were filled with calcium phosphate granules and wrapped with a collagen membrane in 4 dogs and left empty as a control in 2 dogs. Two months later, a bone marrow graft was injected into the center of the implants. Animals were humanely killed after a 16-week delay. SUBJECTS: Six adult beagles were included in this study. INTERVENTION: Segmental mandibulectomy. MAIN OUTCOME MEASURE: Bone ingrowth and material resorption in the reconstructed segment. RESULTS: Successful osseous colonization bridged the whole length of the defects. The good new bone formation at the center and the periosteum-like formation at the periphery suggest the osteoinductive role of the bone marrow graft and the healing scaffold role of the membrane. CONCLUSIONS: This model succeeded in regenerating a large segmental defect in the mandible. An investigation with a postimplantation radiation delivery schedule is required with the use of this model, which should be considered as a preclinical study for a bone tissue engineering approach in patients with cancer-related bone defects.

A non-steroidal anti-inflammatory drug (ketoprofen) does not delay beta-TCP bone graft healing.

Beta-tricalcium phosphate (beta-TCP) is a suitable biomaterial in oral and maxillofacial surgery since it can induce a rapid proliferation of woven bone. Granules, prepared by the polyurethane foam method, were implanted in critical size defects performed in the femoral condyles of New Zealand rabbits. Animals were studied after 8 and 28 days. Ketoprofen (a non-steroidal anti-inflammatory drug (NSAID)) was given for 8 and 28 days to evaluate its effects on the healing of the graft. Before euthanasia, the rabbits received an intravenous injection of fluorescent microbeads. Bones were analyzed by microcomputed tomography. beta-TCP granules induced metaplastic bone trabeculae as early as 8 days post-surgery. At 28 days, the amount of bone was increased and the biomaterial volume decreased due to simultaneous macrophagic resorption. The amount of macrophages labeled with microbeads was less in the grafted area than in the vicinal intact marrow spaces. Ketoprofen had no effect on the amount of bone formed and on the number of labeled macrophages. The influence of small doses of NSAID, given in a short duration period, did not present deleterious effects on bone graft healing.

Alveolar ridge augmentation in irradiated rabbit mandibles.

Oral carcinomas are frequently treated with a nonsegmental mandibulectomy plus radiotherapy. Improving the quality of life of these patients depends on the possibilities for dental rehabilitation. The aim of this study was to increase the alveolar ridge height. Twelve white New Zealand rabbits underwent surgery to produce a 11 x 9 x 7 mm(3) horizontal bicortical full-thickness defect at the alveolar ridge of the left mandible. Six were implanted with a composite associating resorbable collagen membrane filled with micro-macroporous biphasic calcium phosphate granules. After a daily radiation delivery schedule for 4 weeks, a total autologous bone marrow graft was injected percutaneously into the center of the implant. All animals were sacrificed at 16 weeks. Successful osseous colonization was observed in all implants. Significant ridge augmentation was observed (p = 0.0349) in the implanted group compared with the control group. This study contributed to producing an experimental model for oncological mandible defects in rabbits.

Injectable biphasic calcium phosphate bioceramic: The HYDROS concept.

A new biphasic calcium phosphate ceramic material has been developed in our laboratory. It is composed of 60% of hydroxyapatite and 40% of beta-tricalcium phosphate, based on three granulometries (submicron, round microporous 80-200 mum and macro microporous 0.5-1 mm particles) and hydrated with water leading the formation of a putty filler for bone repair. Biocompatibility and osteogenicity were tested by filling femoral epiphyses critical size bone defect and lumbar muscles in rabbit. After 3, 6 and 12 weeks of implantation, explants were treated for histology. Results revealed the biocompatibility of the material and intensive resorption of the submicron particle fraction followed by important bone ingrowth whereas osteoconduction was provided by the larger particles.

Reconstruction of irradiated bone segmental defects with a biomaterial associating MBCP+(R), microstructured collagen membrane and total bone marrow grafting: an experimental study in rabbits.

The bone tissue engineering models used today are still a long way from any oncologic application as immediate postimplantation irradiation would decrease their osteoinductive potential. The aim of this study was to reconstruct a segmental critical size defect in a weight-bearing bone irradiated after implantation. Six white New Zealand rabbits were immediately implanted with a biomaterial associating resorbable collagen membrane EZ(R) filled and micro-macroporous biphasic calcium phosphate granules (MBCP+(R)). After a daily schedule of radiation delivery, and within 4 weeks, a total autologous bone marrow (BM) graft was injected percutaneously into the center of the implant. All the animals were sacrificed at 16 weeks. Successful osseous colonization was found to have bridged the entire length of the defects. Identical distribution of bone ingrowth and residual ceramics at the different levels of the implant suggests that the BM graft plays an osteoinductive role in the center of the defect. Periosteum-like formation was observed at the periphery, with the collagen membrane most likely playing a role. This model succeeded in bridging a large segmental defect in weight-bearing bone with immediate postimplantation fractionated radiation delivery. This has significant implications for the bone tissue engineering approach to patients with cancer-related bone defects.

Histomorphometric analysis of the osseointegration of four different implant surfaces in the femoral epiphyses of rabbits.

OBJECTIVES: The surface properties of titanium dental implants are key parameters for rapid and intimate bone-implant contact. The osseointegration of four implant surfaces was studied in the femoral epiphyses of rabbits. MATERIAL AND METHODS: Titanium implants were either grit-blasted with alumina or biphasic calcium phosphate (BCP) ceramic particles, coated with a thin octacalcium phosphate (OCP) layer, or prepared by large-grit sand blasting and acid-etched (SLA). After 2 and 8 weeks of implantation, the bone-implant contact and bone growth inside the chambers were compared. Scanning electron microscopy (SEM) and profilometry showed distinct microtopographies. RESULTS: The alumina-Ti, BCP-Ti and OCP-Ti groups had similar average surface roughness in the 1-2 mum range whereas the SLA surface was significantly higher with a roughness averaging 4.5 microm. Concerning the osseointegration, the study demonstrated a significantly greater bone-to-implant contact for both the SLA and OCP-Ti surfaces as compared with the grit-blasted surfaces, alumina- and BCP-Ti at both 2 and 8 weeks of healing. CONCLUSION: In this animal model, a biomimetic calcium phosphate coating gave similar osseointegration to the SLA surface. This biomimetic coating method may enhance the apposition of bone onto titanium dental implants.

Influence of calcium chloride and aprotinin in the in vivo biological performance of a composite combining biphasic calcium phosphate granules and fibrin sealant.

Highly bioactive biomaterials have been developed to replace bone grafts in orthopedic revision and maxillofacial surgery for bone augmentation. A mouldable, self-hardening material can be obtained by combining TricOs Biphasic Calcium Phosphate Granules and Tissucol Fibrin Sealant. Two components, calcium chloride and antifibrinolytic agents (aprotinin), are essential for the stability of the fibrin clot. The ingrowth of cells in composites combining sealants without calcium chloride or with a low concentration of aprotinin was evaluated in vivo in an experiment on rabbits. Bone colonization was compared using TricOs alone or with the composite made from TricOs and the standard fibrin sealant. Without the addition of calcium chloride, the calcium ions released by the ceramic component interacted with the components of the sealant too late to stabilize the clot. With a low concentration of aprotinin, the degradation of the clot occurred more quickly, leading to the absence of a scaffold on which the bone cells could colonize the composite. Our results indicate that a stable fibrin scaffold is crucial for bone colonization. The low calcium chloride and low aprotinin groups have shown lower bone growth. Further studies will be necessary to determine the minimal amount of antifibrinolytic agent (aprotinin) necessary to allow the same level of osteogenic activity as the TricOs-fibrin glue composite.

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 micro 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.

Hybrid composites of calcium phosphate granules, fibrin glue, and bone marrow for skeletal repair.

Synthetic bone substitutes, such as calcium phosphate ceramics, give good results in clinical applications. In order to adapt to surgical sites, bioceramics come in the form of blocks or granules, and are either dense or porous. Combining these bioceramics with fibrin glue provides a mouldable and self-hardening composite biomaterial with the biochemical properties of each component. Critical-sized defects in the femoral condyle of rabbits were filled with TricOs/fibrin glue/bone marrow hybrid/composite material. The TricOs granules (1-2 mm) were composed of hydroxyapatite and beta tricalcium phosphate (60/40 in weight). The fibrin glue was composed of fibrinogen, thrombin and other biological factors and mixed with MBCP granules either simultaneously or sequentially. Bone marrow was also added to the MBCP/fibrin composite prior to filling the defects. After 3, 6, 12, and 24 weeks of implantation, the newly-formed bone was analysed with histology, histomorphometry and mechanical tests. The newly-formed bone had grown centripetally. Simultaneous application of fibrin glue showed better results for mechanical properties than sequential application after 6 weeks. Around 40% of bone had formed after 24 weeks in the three groups. Although the addition of bone marrow did not improve bone formation, the MBCP/fibrin material could be used in clinical bone filling applications.

Osteointegration of femoral stem prostheses with a bilayered calcium phosphate coating.

Our purpose was to evaluate the osteointegration of bilayered calcium phosphate (CaP)-coated femoral hip stems in a canine model. A first layer of hydroxyapatite (HA) 20 microm thick and a superficial layer of Biphasic Calcium Phosphate (BCP) 30 microm thick were plasma-sprayed on to the proximal region of sandblasted Ti6Al4V prostheses. Bilayered CaP-coated and non-coated canine femoral stems were implanted bilaterally under general anesthesia in 6 adult female Beagle dogs. After 6 and 12 months, a significant degradation of the bilayered coating occurred with a remainder of 33.1+/-12.4 and 23.6+/-9.2 microm in thickness, respectively. Lamellar bone apposition was observed on bilayered coated implants while fibrous tissue encapsulation was observed on non-coated femoral stems. The bone-implant contacts (BIC) were 91+/-3% and 81+/-8% for coated and 7+/-8% and 8+/-12% for non-coated implants, at 6 and 12 months, respectively. Our study supports the concept of a direct relationship between the biodegradation of CaP coating and the enhanced osteointegration of titanium prostheses. A bilayered CaP coating might therefore enhance bone apposition in the early stages because of the superior bioactivity of the BCP layer while the more stable HA layer might sustain bone bonding over long periods.

In vivo biological performance of composites combining micro-macroporous biphasic calcium phosphate granules and fibrin sealant.

INTRODUCTION: Fibrin glues are currently used by surgeons and can facilitate the handling of biomaterials. Combining fibrin glue with calcium phosphate bioceramics gives a mouldable composite that cements the granules into the implantation site. In addition to the mechanical aspect of the composite, it has been suggested that the mixture also promotes wound healing. These human blood derivatives contain natural (aprotinin) or synthetic (tranexamic acid) antifibrinolytic substances. We compared the bioactivity of two composites combining calcium phosphate granules with two different types of fibrin glue, one with aprotinin and the other with tranexamic acid. MATERIALS AND METHODS: The composite was composed of fibrin glue (Tissucol) and 1 to 2 mm granules of biphasic calcium phosphate granules (MBCP) with a volume ratio of 1 for 2. Bone cavities were drilled in 12 New Zealand rabbits and filled with a composite with aprotinin-fibrin glue on the right condyle and one with tranexamic acid-fibrin glue on the left condyle. The rabbits were randomized into two groups: 3 and 6 weeks of delay. Light microscopy, scanning electron microscopy and image analysis were performed. RESULTS: No adverse reactions were observed in either sample. Bony ingrowth associated with bioceramic resorption by osteoclastic TRAP-positive cells was noted. No significant difference was observed between the two composites. The bony ingrowth and ceramic resorption were qualitatively and quantitatively similar with both composites. CONCLUSION: This study demonstrated that the choice of a natural (aprotinin) or synthetic (tranexamic acid) antifibrinolytic agent in the fibrin sealant associated with calcium phosphate granules and used as a bone substitute had no effect on the bioactivity of the composite. It remained efficient in bone reconstruction, no adverse effects were observed, and the bony ingrowth was qualitatively and quantitatively equivalent with the two types of fibrin sealant.

MBCP biphasic calcium phosphate granules and tissucol fibrin sealant in rabbit femoral defects: the effect of fibrin on bone ingrowth.

An ageing population implies an increase in bone and dental diseases, which are in turn a source of numerous handicaps. These pathologies are an expensive burden for the European health system. As no specific bioactive materials are efficient enough to cope with this burden, we have to develop an injectable, mouldable, self-hardening bone substitute to support bone tissue reconstruction and augmentation. New, highly bioactive and suitable biomaterials have been developed to replace bone grafts in orthopedic revision and maxillofacial surgery for bone augmentation. These mouldable, self-hardening materials are based on the association of MBCP Biphasic Calcium Phosphate Granules and Tissucol Fibrin Sealant. The in vivo evaluation of ingrowth in relation to the composite was made in an experiment on rabbits. The results indicate that in the presence of fibrin sealant, newly-formed bone developed at a small distance from the surface of the calcium phosphate ceramic. Two different bone apposition processes were identified. Without the fibrin component (MBCP group), bone rested directly on the surface of the granules. This observation is commonly described as osteoconduction in calcium phosphate materials. On the contrary, the presence of the fibrinogen component seemed to modify this standard osteoconduction phenomenon: the newly-formed bone essentially grew at a distance from the surface of the granules, on the fibrillar network, and could be considered as an inductive phenomenon for osteogenic cell differentiation from mesenchymal stem cells.