| Massive diaphyseal defects secondary to congenital malformations, tumors, osseous infection, and fracture nonunion present formidable clinical challenges. Current therapies, which rely on implantation of fresh autograft bone, suffer from the associated morbidity and limitation on the availability of the graft, and therefore it is indispensable to develop new strategies to solve the existing problems.More recently, great development in tissue engineering has provided a novel cue for the management of critical large bone defects. In this type of tissue engineering, culture expanded cells are seeded in some type of scaffolding to construct bioartificial osseous tissue, which is thereafter implanted into a recipient to initiate in vivo osteogenesis in the inductive microenvironment. These scaffolding materials for bioartificial tissues could provide mainly mechanical support, migration channels, and adhesive surfaces for cells and prerequisite for vessel ingrowth, and rely on the cells themselves for biological signals that regulate growth and differentiation. However, numerous obstacles as origination and biological features of seed cells, components of the potential scaffold, conjunction of cells with biomaterials and alteration of implanted cells need to be further investigated.The central process in tissue engineering is construction of the artificial tissues, in which synthesized biomaterials and seed cells play critical roles. The potential seed cells in tissue construction should meet the criteria followed. They should be easily isolated and culture-expanded, and maintain the potentialities to differentiate into osteoblasts in the process of culture. Furthermore, they should remain their molecular or cellular signalingAbstract - 6 -after implanted into the receipts and therefore, proliferate and differentiate in the presence of local microenvironment. Mesnenchymal stem cells (MSCs) derived from bone marrow are a rare population which have the capacity for differentiation into osteoblasts, adipoblasts and chondroblasts. Previous data have demonstrated that MSCs can be passaged for at least 20 times without lose of the differentiative potentials. Therefore, MSCs might be the ideal seed cells in osseous reconstruction and further investigation should be performed.In this study, an animal model of critical segmental bone defects was developed, MSCs were seeded onto biomaterials and used for the repair process. Rabbit bone marrow mononuclear cells were obtained by density centrifugation on Percoll (1.073g/ml), seeded onto plastic dishes, and cultured in the medium of low glucose Dubecco's Modified Essential Medium (LG-DMEM) with 10% fetal bovine serum from selected lots. Like human MSCs, rabbit MSCs were adherent to the culture and spindle-like in morphology. The population doubling time was around 20 hours, revealing its rapid growth status in the culture above. Cytochemistry studies showed that cultured rabbit MSCs were homogenously positive for glycogen (Periodic Acid-Schiff reaction, PAS) and negative for fatty acids (Sudan black reaction, SB)and nonspecific eterase (NSE). Rare fraction of the population was positive for alkaline phosphotase (ALP, positive percentage about 5%) and acid acetone esterase (ANAE). Flow cytometric analysis showed that the percentages of cells in G0/G1, S and G2M were 84.56%, 3.26% and 12.18% respectively, indicating that cultured cells might have the unique characteristics of selfrenewing as stem cells.To further testify the biological features, differentiation study was performed. The results revealed that the cells could be coaxed into osteoblasts and adipocytes in the presence of inductive agents, evaluated by ALP staining, calcium examination and Oil Red O staining. Cells in the osseous medium changed morphologically into polygon after 6 to 8 days of culture and the alkaline phosphotase activities were elevated gradually in the process of ind... |
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