gms | German Medical Science

Problem: To evaluate the feasibility of an intraoperative approach to engineer cell-based bone grafts with tissue harvest, cell-isolation, cell-seeding in the scaffold and subsequent implantation within a few hours. Mesenchymal and endothelial lineage cells isolated from the stromal vascular fraction (SVF) of human adipose tissue have proven osteogenic and vasculogenic capacity in vivo when seeded and expanded inside 3D hydroxyapatite scaffolds in a perfusion system for five days. However, compliance and practicability in the clinical field might be hampered by the requirement of a GMP-facility and the need for two surgical procedures.

Methods: SVF cells were isolated from lipoaspirates and excised fat samples of 4 healthy donors by enzymatic digestion. 1–2mm silicated calcium-phosphate granules (Actifuse^®, Apatech) where mixed with A) 1mio or B) 4mio fibrin embedded nucleated SVF-cells with or without addition of 250ng BMP-2. Cell-fibrin-scaffold-constructs were immediately implanted ectopically in nude mice for eight weeks. In parallel, cells were analysed by cytofluorimetry and assessment of clonogenicity. Upon explantation, constructs were processed histologically.

Results and conclusion: Clonogenicity of the freshly isolated SVF and subsequently implanted cells was 13%±3.1% resulting in a density of 2.12mio±0.6mio (A) and 12.2mio (B) CFU-f/ml. Mesenchymal markers (CD105 and CD90) were expressed by 33±18% and 68±15%, endothelial markers (CD31 and CD34) were expressed by 40±14% and 62±13% of the cells. After eight weeks of implantation, we observed dense matrix but no true bone tissue with and without addition of BMP-2 in condition (A). In condition (B), typical bone tissue with compact collagen matrix containing osteocytes could be observed only when BMP-2 has been added. In situ hybridisation for human specific ALU sequences confirmed that the osteocytes and lining osteoblasts were human cells and thus originated from the implanted SVF cells.

To our knowledge, this is the first report providing preliminary evidences and proof-of-principle that intraoperative engineering of autologous cell-based bone graft substitutes could be achieved by mixing adipose tissue stromal cells embedded in a fibrin matrix with ceramic-based scaffolds. Further studies will determine whether the engineered constructs, implanted in a relevant orthotopic model, would be able to induce de novo formation of bone tissue also in the absence of BMP.