| The reconstruction of severe facial bone defects that caused by trauma, inflammation, cancer and other diseases has been the major goal both at home and abroad. From a biological perspective, the fully restoration of the structure and function of bone tissue depends upon, (i) adequate stem cells, and (ii) the appropriate microentironment to promote the excretion, maturation and calcification of the extracellular matrix. At present, only autogenous bone graft can approach the functional recovery, and it is still the gold standard in bone transplantation. However, the clinical application of autogenous bone graft is limited because of the secondary trauma in donor site. The development of tissue engineered bone graft substitutes provides a promising strategy to overcome the current predicament.Traditional bone tissue engineering strategy is seeding the plenty cells onto the scaffolds and through the effects of the scaffolds or growth factors to induce the cells differentiation and calcification. Previous studies demonstrated that the characteristics between the cells cultured on the scaffolds and grew in vivo have obvious differentiation. And it is limited that nutrition and waste metabolism of the cells all depends on infiltration, especially for the cells located in the internal porous scaffolds. Cell sheet composed of the cells and extracellular matrix derived from the cells, and remained the relative protein and biological activity factors. Consequently, cell sheet can provide a suitable microenvironment for the bone regeneration in vivo. However, only cell sheet is not suitable for bone tissue regeneration which comprises less cell and rich matrix. The similarity between the process of bone defects repair and normal bone development suggests that we can ameliorate the bone tissue engineering strategy of bone regeneration through the imitation of the bone development process. In theory, osteogenic culture medium induced bone marrow mesenchymal stem cells (BMSCs) combined with discal scaffolds may provide stem cells an appropriate microenvironment similar to in vivo condition in development process.To find a novel strategy of bone tissue engineering, the purposes of current study were, (i) to develop the procedure of culturing, harvesting and transplantion of BMSCs cell sheet, and in this course, we try to use osteogenic culture medium to stimulate the maturation and calcification of BMSCs, (ii) and on this basis, to fabricate three-dimensional culture system with cell sheet and discal coral scaffolds, (iii) to repair the ectopic and critical-sized in situ bone defects in vivo with the cell sheet/coral combinations.The main results of this research:1?The formation and differentiation of BMSCs cell sheet in vitroIn order to verify the capability of BMSCs cell sheet and discal coral in bone regeneration, this study established the BMSCs/coral co-culture system, and observed the osteogenic differentiation potential of BMSCs and the osteogenic induced BMSCs cell sheet. The results showed that there was no significant difference between the cells growth curves after they were cultured in coral extracts and normal medium. The osteogenic culture medium induced BMSCs cell sheet can differentiate into osteoblast lineage effectively, and in the early early stage of induction, they can express bone and vessel relative genes.The results indicated that BMSCs cell sheet cultured in vitro expressed bone relative genes, which is important to early angiogenesis and the transportation of oxygen and nutrients for internal osteoblasts. The cell sheet comprises rich extracellular matrix, and that is important to the signal communication between the cells in the process of bone foramation in vivo. The results of in vitro examinations demonstrated that our hypothesis that use the BMSCs cell sheet combined with discal coral based on the mechanism of auxanology to construct tissue engineered bone is feasible.2?The preparation of BMSCs cell sheet/discal coral combination and the in vivo study of using the combination for bone regenerationIn this study, a novel strategy of bone tissue engineering using discal coral and cell sheet cultivation system was investigated based on the technology of cell sheet culture. At first we fabricated the BMSCs cell sheet, and then processed the coral material into uniform discs. Then the discal coral was enwrapped with the cell sheet to form the cell sheet/discal coral combination. The DMEM medium containing the ascorbic acid can stimulate BMSCs to secrete the extracellular matrix. And with the interaction of the cells and extracellular matrix, the cell sheet was formed. Cell sheet has a certain strength and operability that can be folded or wrapped carefully.In ectopic bone formation experiment, the animals were separated into two groups (cell sheet group and cell sheet/discal coral combination group) in random. The results of ectopic bone formation experiment showed that there was no evident for bone-like structure formation in the BMSCs cell sheet group which was induced by osteogenic medium. And in the cell sheet/discal coral combination group, we found bone-like structure. The results of in situ bone formation experiment showed that in coral group, a little bone-like tissue was occurred adjacented to the defects verge. And in the group of osteogenic induced BMSCs seeded onto discal coral before transplantion, woven bone-like tissue was occurred. In the cell sheet/discal coral combination group, we found lamellar bone-like tissue.This study demonstrated that because of their capability of secrete biological activity factors and extracellular matrix, BMSCs cell sheet combined with discal coral could simulate the process of intramembranous ossification. Although the results were not perfect and the bone defects were not repaired ideally, it has been proved that our strategy is reasonable and feasible. However, further studies are needed to ameliorate the details of this technology and observed the feasibility of this strategy in large animal models.In conclusion, this study demonstrated that using cell sheet for bone tissue engineering has advantages and our novel strategy to construct bone tissues is practicable. |
PDF Full Text Request