New antibacterial microporous CaP materials loaded with phages for prophylactic treatment in bone surgery.

Hydroxyapatite and beta-tricalcium phosphate (ß-TCP) are materials commonly used in bone repair. The most important problem occurring in bone repair surgery is bacterial infection which is usually overcome by treatment with antibiotics. Currently, emergence of multidrug resistant strains has led to development of alternative treatments such as phage therapy. Phages are bacterial viruses with several advantages over chemotherapy such as specificity of bacterial strain, no side effects and fast response. This study evaluates the possibility of loading hydroxyapatite and ß-tricalcium phosphate ceramics used as bone substitutes with phages and their antibacterial activity against Escherichia coli K12. The majority of phages were retained in dense and microporous HA and ß-TCP samples during at least 6 days suggesting the occurrence of strong interaction between phages and ceramics, which did not prevent bacterial attachment and lysis. This study has shown for the first time that phage loaded ceramics could be used in prophylactic treatments.

Biomimetic evaluation of ß tricalcium phosphate prepared by hot isostatic pressing.

Two types of completely densified ß-TCP tablets were synthesized from a stoichiometric ß-TCP powder. The first ones (TCP) were conventionally sintered, while the second ones (TCP-T) were sintered and treated by hot isostatic process (HIP). The HIP produced completely densified materials with relative densities greater than 99.9% and a transparent appearance of tablets. Samples were immersed in culture medium with (CM) or without serum (NCM) in static and dynamic conditions for a biomimetic evaluation. Similarly, SaOs-2 cells were cultured on samples in a static or dynamic flow perfusion system. The results of surface transformation in absence of cells showed that the dynamic condition increased the speed of calcium phosphate precipitations compared with the static condition. The morphology of precipitates was different with nature of tablets. The immersion in CM did impede this precipitation. XPS analysis of TCP-T tablets showed the presence of hydroxyapatite (HA) precipitates after incubation in NCM while octacalcium phosphate (OCP) precipitates were formed after incubation in CM. The analysis of the response of SaOs-2 cells on surfaces showed that the two types of materials are biocompatible. However, the dynamic mode of culture stimulated the differentiation of cells. Finally, it appears that the HIP treatment of TCP produces highly densified and transparent samples that display a good in vitro biocompatibility in static and dynamic culture conditions. Moreover, an interesting result of this work is the relationship between the presence of proteins in the immersion medium and the quality of precipitates formed on hipped TCP surface.

Prolonged local antibiotics delivery from hydroxyapatite functionalised with cyclodextrin polymers.

Per-operative infection is a common complication for bone-graft surgery. Combining antiseptic agents with graft materials may offer a solution by increasing local drug concentration at target sites. Aiming to achieve a sustained local antibiotic (ATB) delivery for a widely applied bone substitute material - hydroxyapatite (HA), we attempted incorporating hydroxypropyl-beta-cyclodextrin polymer (polyHPbetaCD) into microporous HA via impregnating either in a CD monomers mixture solution or a pre-synthesized CD polymer solution, followed by thermal fixation processing. In such functionalised material (CD-HA), polyHPbetaCD could entrap ATBs and release them progressively. Infrared-spectroscopic analysis confirmed the presence of polyHPbetaCD in functionalised HA via both processing pathways; polyHPbetaCD functionalisation yields were quantitated by thermogravimetric analysis for optimising the processing regime. Ciprofloxacin (CFX) and vancomycin (VCM), commonly applied in orthopaedics, have been respectively loaded on CD-HA by dip-coating. For both ATBs, kinetic release test in phosphate buffered saline showed significantly increased initial-burst amount and prolonged release from CD-HA compared with those from non-functionalised HA. Encouragingly, ATBs loaded CD-HA also revealed a prolonged bacteriostatic activity against Staphylococcus aureus and progressively increased cytocompatibility to osteoblasts (MC3T3-E1). Overall, polyHPbetaCD functionalisation on HA could be an effective drug-delivery model for loading different drug molecules in prevention of infection.

beta-TCP microporosity decreases the viability and osteoblast differentiation of human bone marrow stromal cells.

Association of osteoprogenitor cells to calcium phosphate ceramics is currently under intense investigation, for its considered ability to induce bone formation and therefore to allow the successful repair of large bone defects. However, if the first experimental and clinical studies provided promising results, lack of new bone formation has been reported in a large number of animal experiments. In this context and since it has been reported that in some conditions, calcium phosphate ceramic microstructure induces ectopic bone formation, we investigated the effects of ceramic microporosity on the behavior of osteoprogenitor cells for the development of hybrid materials. Human bone marrow stromal cells (BMSCs) were seeded on beta-tricalcium phosphate (TCP) ceramics with 0, 25, or 45% microporosity and cell adhesion, viability, and osteoblastic differentiation have been studied for three weeks. Cell counts, measurement of MTS conversion, and LDH activity indicated that microporosity decreased the viability of BMSCs in a time and rate-dependent manner. In addition, microporosity inhibited osteoblastic differentiation as compared with nonmicroporous ceramics, as revealed by decreased alkaline phosphatase activity and osteocalcin secretion. Results of this in vitro study therefore highlight a negative role for beta-TCP microporosity in the behavior of human osteoprogenitor cells.

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), beta-tricalcium phosphate (beta-TCP) and 40% beta-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 10000 particles per cell, the viability and proliferation were completely inhibited. Third, HA, beta-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 beta-TCP (40%) than for BCP (5%). On the other hand, new bone formation was significantly higher for beta-TCP (21%) and HA (18%) than for BCP (12%). More micro-particles were formed around BCP granules than around beta-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 degrees C. Consequently, HA micro-particles of BCP ceramic are incompletely sintered and easily released after immersion or implantation. The microparticles 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.

Repair of osteochondral defects with autologous chondrocytes seeded onto bioceramic scaffold in sheep.

At present, the most popular biomaterials used in cartilage tissue engineering are synthetic polymers. However, problems-such as acidic by-product accumulation and side effects in local or systemic inflammatory reactions during in vivo degradation-are drawing much attention. The polymers are also highly hydrophobic and degrade within 4 weeks, allowing insufficient time to support neocartilage formation. All these have made polymers less promising in clinical application. In this study, we tested a new bioceramic scaffold made of artificial synthesized powder of beta-tricalcium phosphate (beta-TCP) in a sheep model. Osteochondral defects were filled with a bioceramic-chondrocyte construct and neocartilage tissue completely resurfaced the cartilage defects after 24 weeks. Typical hyaline cartilage structure was generated in the engineered cartilage. Biodegradation of bioceramic was notable, leading to bioceramic fragmentation and particle formation. Numerous ceramic particles (size, 0.5-1.9 microm) and numerous macrophages were observed at the ceramic-tissue interface as well as in the marrow tissue. No macrophages were visible in the neocartilage tissue. Although long-term in vivo study is needed to further determine the pathological sequences of the beta-TCP-based cartilage construct, this study suggests that this bioceramic might be used to repair chondral or osteochondral defects and could be used as a scaffold for cartilage tissue engineering.

The biodegradation mechanism of calcium phosphate biomaterials in bone.

This study was undertaken to understand the biodegradation mechanisms of calcium phosphate (Ca-P) biomaterials with different crystallization. Two types of sintered Ca-P porous ceramic (HA and beta-TCP) and a Ca-P bone cement (CPC) were implanted into cavities drilled in rabbit femoral and tibiae condyles. The results have shown that a material biodegradation was rapid in the beta-TCP and the CPC, but very weak in the HA. This biodegradation presented a decrease of material volume from the periphery to the center as well as a particle formation causing phagocytosis by numerous macrophages and multinucleated giant cells in the CPC. In the beta-TCP, there was a peripheral and central decrease of material volume as well as an absence of particle formation or visible phagocytosis. The process of biodegradation is considered to be directly influenced by the type of material crystallization. The sintered bioceramics processed at a high temperature exhibit good crystallization and are primarily degraded by a process dependent on interstitial liquids. However, the bone cement is formed by physicochemical crystallization and is degraded through a dissolution process associated with a cellular process.