Jianxi Lu

ROLE OF INTERCONNECTIONS IN POROUS BIOCERAMICS ON BONE RECOLONIZATION IN VITRO AND IN VIVO

The interconnections in a porous biomaterial are the pathways between the pores. They conduct cells and vessels between pores. Thus they favour bone ingrowth inside ceramics. The aim of our study was to determine the effect on bone ingrowth of interconnections in two ceramics: hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) with the same porosity of about 50% and a mean pores size of 100–300 μm and a mean interconnection size of 30–100 μm. In vitro, four discs for osteoblast culture were studied after 14 and 28 days of incubation. The results show that human osteoblasts can penetrate interconnections over 20 μm in size, and colonize and proliferate inside macropores, but the most favourable size is over 40 μm. In vivo, eight cylinders were implanted in the middle shaft of both rabbit femurs for 12 or 24 weeks. The histomorphometric results show that interconnections in porous ceramics favour bone ingrowth inside the macropores. In the HA group the rate of calcification and bone ingrowth do not differ, and chondroid tissue is observed inside pores. But in β-TCP, the calcification rate and the bone ingrowth increased significantly. At week 12 significant correlation between new bone ingrowth and the size of the interconnections is observed between new bone ingrowth and the density of pores. In conclusion we notice that in vivo a 20 μm interconnection size only allows cell penetration and chondroid tissue formation; however the size of the interconnections must be over 50 μm to favour new bone ingrowth inside the pores. We propose the concept of “interconnection density” which expresses the quantity of links between pores of porous materials. It assures cell proliferation and differentiation with blood circulation and extracellular liquid exchange. In resorbable materials, pore density and interconnection density are more important than their size, contrary to unresorbable materials in which the sizes and the densities are equally important.

Comparative study of tissue reactions to calcium phosphate ceramics among cancellous, cortical, and medullar bone sites in rabbits

In order to understand the influence of the implantation site on bone biomaterial evaluation, we implanted cylinders of HA and beta-TCP ceramics in the femoral diaphysis and condyle of rabbits. After 3, 8, 12, and 24 weeks of implantation, histological investigation and histomorphometry were performed on undecalcified samples. Our results show that spontaneous bone healing in the empty cavities is significantly different (p < 0.05) between cortical (SBH > 80%) and cancellous bone sites (SBH < 31%) and that no new bone is formed in marrow tissue. For both porous ceramics, the highest osteogenesis was obtained in the cortical site. Osteogenesis was intermediate in the cancellous site and weak in the medullar site. The material biodegradation was the strongest in the medullar site and higher in the cancellous site than in the cortical site. Both activities were better in the beta-TCP than in the HA (p > 0.05). The marrow tissue presents a foreign-body reaction more reliable, sensitive, and durable than other bone tissues. Therefore, the cancellous bone site is a good site for evaluation of the biofunctionality of biomaterials because of the equilibrium of the osteogenesis and the biodegradation activities, but marrow tissue seems to be better for testing material biocompatibility in vivo.

Relationship between bioceramics sintering and micro-particles-induced cellular damages

We performed experimental studies to confirm the hypothesis that cellular damages occurring around implanted biphasic bioceramics could be related to a micro-particles release because of an insufficient sintering. First, an in vitro cytotoxicity study was performed on four biphasic ceramic (BCP) samples. Without treatment of the extraction medium, a cytotoxicity was observed, although after centrifugation this cytotoxicity disappeared in all samples. Second, micro-particles of hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) and 40% β-TCP/60%HA mixture were used for a cell inhibition study. A decrease of cell viability was observed with the increase in particles concentration. At 10 000 particles per cell, the viability and proliferation were completely inhibited. Third, HA, β-TCP and BCP ceramic granules were implanted in rabbit femoral cavities for 12 weeks. No degradation of HA granules was observed. The degradation was higher for β-TCP (40%) than for BCP (5%). On the other hand, new bone formation was significantly higher for β-TCP (21%) and HA (18%) than for BCP (12%). More micro-particles were formed around BCP granules than around β-TCP, and phagocytised by macrophages. The release of ceramic micro-particles could be related to the sintering process. BCP ceramic have to be sintered at only 1160 °C. Consequently, HA micro-particles of BCP ceramic are incompletely sintered and easily released after immersion or implantation. The micro-particles could be at the origin of local inflammation and cell damage and could perhaps modify osteogenesis. Attention must be paid to this problem especially with BCP ceramics because of the sintering difficulties of this bioceramic.

Clinical, radiological and histological study of the failure of cervical interbody fusions with bone substitutes

Few histological studies on bone substitutes in human cervical spine are available and the biological processes of bone substitutes are not well documented. The authors studied four failure cases of cervical interbody fusion: two cases with hydroxyapatite (HA), one case with β-tricalcium phosphate ceramic (β-TCP) and one case with xenograft (bovine bone). Clinical data showed that all the patients experienced neck pain with or without numbness of upper extremity due to fusion failure. Successful fusions were achieved after the salvage surgeries in which autograft were used. Radiographs showed that radiolucent lines were present in all cases. Two HA substitutes fractured without complications. One of them sank into the vertebral body. Some small β-TCP fragments were found under the microscope. Histological study demonstrated only a few newly formed bones at the interface of the substitutes. The fragments of HA were encapsulated by fibrous tissue. The degradation process and bone regeneration were more active in β-TCP than in HA. The intertrabecular spaces of bovine bone were filled with fibrous tissue. The results suggest that a porous calcium phosphate ceramic with special design might assure bone ingrowth and meet the mechanical requirements in cervical interbody fusion. The complications of these materials in the cervical spine should be highlighted.

Histological aspects in bone regeneration of an association with porous hydroxyapatite and bone marrow cells.

The osteogenic potential of an association of two kinds of hydroxyapatite (HA) porous ceramics with autologous bone marrow cells cultured with or without dexamethasone (10-8M) addition in the culture medium and non-cultured rabbit marrow stromal stem cells (MSCs) was tested after 4 weeks of implantation in the dorsal muscles of spine in rabbit. A significantly higher number of rabbits with implants containing bone tissue inside pores were obtained with 107 cells ml-1 cultured treated with Dex. In the HA porous ceramic using naphtalen as porogen agent, the bone recolonization remains only at the periphery of implants and in the second row of pores, while in the HA porous ceramic using polymethacrylate (PMMA) microbeads as porogen agent, the bone recolonization is observed in the depth of implants. In the PMMA HA group, the Kru¨skal–Wallis variance analysis between the rabbits is significantly different with the percentage of number of occupied pores and occupied pores with bone tissue is different (p < 0.05).