Purpose of the study: The mechanical and radiological course of bone allografts is often favorable but osteointegration properties could be improved. We associated a safe allograft with mesenchymatous bone marrow stem cells (MSC) with known osteogenic potential. The purpose of this preliminary study was to study the biocompatibility of the treated allograft, assess the osteoblastic differentiation properties of the MSC, and determine the optimal period for colonizing the bone matrix.

Material and methods: The support was a safe bone allograft preserved in a collagenic grid (Osteopure™). MSC harvested by adherence were seeded in a medium favoring osteoblastic differentiation by comparison with standard culture medium. Culture conditions varied to study the influence of the presence or not of support, the culture time, or the presence of human serum in the culture medium. For each culture medium, we noted: the number of cells, osteoblastic differentiation using markers: alkaline phosphate and osteocalcin. A histological study was also performed.

Results: Peak cell amplification was achieved at three weeks culture. Presence of osteoblastic differentiation markers was clearly identified in cultures grown in the presence of support material. Microscopy demonstrated that cells stimulated by the differentiation medium adhered strongly to the bone network. Histology revealed the presence of osteoblastic activity in differentiation medium with cells taking on the classical cytological aspect of osteoblasts. Cell proliferation was at least equivalent in medium with human serum as with fetal calf serum.

Discussion: This study demonstrated that the allogenic matrix does not modify the capacity of human MSC for colonization and differentiation. The cell organization is optimal compared with the absence of supporting material. Use of the patient’s own serum in the culture medium was validated enabling an autologous procedure. Use of a complex cell graft appears to be optimal after three weeks of culture. This first step proves the feasibility of the concept designed to optimize the support with the patient’s own MSC. The next step is to develop an in vivo model.

Purpose: Bone allografting appears to be optimised by in situ stromal cells which have potential to evolve into a bone line. The purpose of this study was to test the bio-compatibility of stromal cells and an allogenic human bone support treated with stromal cells as well as their evolutive potential.

Material and methods: The bone support was a human femoral head allograft harvested during total hip arthroplasty. After validation of the safety of the femoral heads by the bone bank, they were treated using the Osteopure(r) method. Human stromal cells were harvested during cardiac surgery from the sternotomy. The in vitro study was conducted in a sterile atmosphere in an incubator. Different adhesion molecules were used: collagen, gelatin, fibronectin, human serum AB, in addition to an adhesion molecule-free medium. Microscopic qualitative evaluation determined the adhesion of stromal cells and the absence of difference between the morphology of cultured stromal cells and stromal cells found in the bone marrow. Cell counts were made on days 24; 32; 48, and 64. The functional properties of the new cultured stromal cells was evaluated by seeding CD34+ cells on day0 and counting the number of CFC produced on day45 (LTCIC1). This LTCIC1 line was cultured in the different media and re-evaluated at day45 (LTCIC2).

Results: The first microscopic observations showed that the stromal cells oriented naturally in the bone architecture with no particular rejection and that they maintained their adhesion properties with each other and with the bone support. Cell counts showed increased proliferation for the stromal cells cultured on the bone support compared with cultures without bone support. Stromal cultures were favoured by the presence of bone and culture media containing collagen, gelatin, and fibronectin. But the LTCIC2 cultures demonstrated better performance with bone and gelatin.

Discussion: Proliferation of stromal cells cultured in contact with an allograft demonstrated the biocompatibility of stromal cells/treated allografts. After twelve weeks incubation, the first cell counts tended to show that stromal cells cultured in vitro on human bone substitute preserve their functional potential and allow the proliferation of certain cells participating in osteogenesis. Further research to identify the capacity of these cells to induce an osteoblastic line must be conducted to allow in situ graft osteogenesis.