Large Segmental Weight-loaded Bone Defects Repaired By Tissue Engineering Bone

Bone defect whether caused by trauma, inflammation, tumor excision or congenital malformation is a very serious problem in clinical practice. Repairing large segmental weight-loaded bone defects is much more difficult, for larger quantity of bone is lost and even the source of bone progenitors, such as periosteum, is often severely damaged. Furthermore higher mechanical strength has to be required for this kind of bone defect. Autologous bone grafts are currently considered to be the gold standard for bone tissue repair, but it is available in very limited amounts and often combined with morbidity at the donor sites. In order to avoid the problems associated with autologous bone grafting,there has been a continuous interest in the use of synthetic bone grafts (such as ceramics and biopolymers) during the past decades, but these materials are non-vital and not competent to repair large segmental bone defects perfectly.The progress in bone tissue engineering provides an innovative choice for bone defects, which combines osteogenic cells with osteoconductive scaffolds to enhance the process of bone reconstitution and remodeling. In this field, attempts have been focusing on seeding cells, suitable scaffolds and the synergistic interaction of these two key players, and much progress has been achieved. Even so, it is still unknown, which is the best way to construct tissue engineering bone in vitro and whether it is feasible to heal large segmental weight-loaded bone defects. There is even no way to expand enough seeding cells instantly and no material that can meet all the requirements wanted by bone tissue engineering. In the present study, our objective was to perform a final test in a large animal model that is as close and relevant to a particular problematic clinical situation as possible before a tissue-engineered construct can be introduced into a clinical trial, to determine the feasibility of repairing large segmental bone defects by tissue engineered bone. Firstly, the goat mesenchymal stem cells (MSCs) were harvested from bone marrow by puncture, expanded in vitro, and then induced into osteoblasts. Results showed: 1. During in vitro culture of MSCs specific colony forming units-fibroblastoid (CFU-Fs) were observed at days 4 to 6 afer seeding, which is the characteristic of stem cells; 2.It took 9～12 d for primary MSCs to be confluence with plating concentration of 3×105cells/cm2 and 4～6 d for subcultured MSCs with replating concentration of 1×104cells/cm2; 3. MSCs were positive for CD105 and negtive for CD45; 4.The cells formed calcified nodule and expressed alkaline phosphatase, typeⅠcollagen and osteocalcin after osteoinduction, which was in accordance with osteoblasts; 5. No malignant cells were found in cultured MSCs through flow cytometry analyse.Secondly, deminerilized bone matrix (DBM) was fabricated and then the interaction of DBM and MSCs was evaluated. Results suggested: 1. DBM appeared three-dimensional network structure with high porosity and interconnectivity of holes; 2. MSCs could adhere to the surface and inner walls of DBM, and spread extensively and proliferated well; 3.By normal seeding methods, adhesive cells on scaffolds did not increased when the densities of seeding cells surpassed 2×106/ml and the the adhesive rate was not more than 75%; 3. With biphasic seeding technique, the adhesive rates were kept around 80~95 % with different seeding densities, adhesive cells on scaffolds were increased following the increase of seeding densities.Thirdly, the complex of MSCs and DBM was implanted intramuscularly into the back of goats and DBM alone was implanted as control. Results showed: 1.The complex of MSCs and DBM could form bone in ectopic site but DBM along could not; 2.Endochonchal osteogenesis was observed in experimental group and no strong inflammation occurred; 3.No clear evidence of roentgenography was dectected in both groups. Finally, artificial femur defects of 3cm in goats were made and filled with the complex of the cells and scaffolds to study the osteo...