Production of an autologous bone tissue using cell-derived extracellular matrix as a natural biomaterial

摘要

Regenerative medicine has become an increasingly important field in medical research. Several therapeutic approaches are exploited in orthopedic and maxillofaciai reconstruction to achieve bone repair, such as using prosthetics, biomaterials and autografts. Tissue engineering strategies targeting the production of graftable bone substitutes using a patient's own cells are promising. For example, adipose tissue is an abundant source of highly multipotent cells that can be harvested with a minimally-invasive lipoaspiration procedure. We hypothesized that extracellular matrix deposition and calcification could occur simultaneously during osteogenic differentiation of adipose mesenchymal stem cells resulting into a graftable bone tissue. In this context, our goal was to promote the osteogenic differentiation of human adipose-derived stromal/stem cells (ASCs) to produce an entirely autologous three-dimensional bone tissue made of cell-secreted extracellular matrix that could be used for the reconstruction of mandibular continuity defects. Using a modified self-assembly method of tissue engineering, ASCs directed towards osteogenesis were capable of producing bone-forming cells encompassed in a three-dimensional, calcified matrix. In vitro studies compared ASCs cultured in DMEM-10% fetal calf serum in the presence and absence of additives promoting osteogenic differentiation (SS-glycerophosphate, dexamethasone and vitamin D3). Additionally, freshly prepared ascorbic acid at 50 μg/ml was added in both conditions to stimulate the production and deposition of extracellular matrix. After four weeks, three sheets of tissue were superimposed and cultured for 7 additional days. The resulting constructs were analyzed by histology, immunofluorescence of bone marker expression, and quantitative calcium assays. Preliminary results showed that calcium content in the osteogenically-induced ASC-derived tissues was significantly greater than in the non-induced control tissues (n=9, ~(***) p＜0.0001). The presence of hydroxyapatite matrix mineralization within induced ASC substitutes was 7.9 fold higher than non-induced ASCs and 9.4 fold above induced fibroblasts when applying a quantitative fluorescent analysis using an IVIS Lumina Ⅱ imaging system. Masson's trichrome, H&E, and indirect immunofluorescence staining for collagen Ⅰ, osteoadherin, and osteocalcin supported the development of a three-dimensional ECM produced via the secretory actions of functional, osteogenically differentiated ASCs. Quantitative analysis of osteocalcin by ELISA further supported the terminal osteogenic differentiation of induced ASC-derived tissues compared to non-induced controls (n=9, ~(***) p＜0.0001). Herein, we have demonstrated our ability to create a three-dimensional, calcified human tissue derived from ASC-secreted ECM under osteogenic conditions using a self-assembly method. This model produced a mineralized matrix surrounding osteoblast cells. These methods have the potential to produce a tissue-engineered product that may be useful for mandibular continuity defect reconstruction.