Analysis Of The Correlative Factors Influencing On The Osteogenic Differentiation And Studies Of Bone Formation Of Bone Marrow Mesenchymal Stem Cells In Vivo

Objectives: To explore the factors influencing on the osteogenic differentiation of human bone marrow mesenchymal stem cells. To observe ectopic bone formation with bone tissue engineering using human mesenchymal stem cells, and investigate the potential of tissue engineered bone to heal a segmental defect in the rabbits.Methods: MSCs are isolated from an aspirate of bone marrow harvested from the superior iliac crest of the pelvis in human. The adherent cells were culture expanded, MSCs were characterized with respect to the expression of surface antigens and the ability to differentiate into osteogenic, chondrogenic and adipogenic lineages in response to environmental stimuli. Human MSCs were treated with dexamethasone, 17β-estradiol, and cryopreservation procedures, osteoblastic related makers were detected by RT-PCR or immunohistochemical. Human MSCs were seeded on chitosan-calcium phosphate cement, the cell-scaffold constructs were cultured with complete media for 1 week. MTT test and scanning electron microscope(SEM) examination were performed. Human MSCs-loaded scaffolds were implanted intramuscularly, after 4 weeks, the samples were analyzed by histological and immunohistochemical examination. New Zealand white rabbits 15mm radius defects were created and subsequently filled with rabbit MSCs-loaded scaffolds, healing of the defect was evaluated with radiographs analysis, histological examination, and biomechanical testing.Results: During culture in monolayers, cells exhatbited a spindle shape and fibroblast like morphology. Analysis of surface antigens demonstrated that human MSCs do not express the typical hematopoietic antigens, but they express several adhesion molecules such as CD29 and CD44. Culture expanded MSCs maintained their ability to undergo osteogenic, chondrogenic and adipogenic differentiation. When Dex was 10^-8mol/L, the expression of ALP mRNA was improved but suppressed PPARγ2 gene transcription, when Dex was 10^-7mol/L, the expression of PPARγ2 was improved but suppressed ALP mRNA gene transcription. 17β-estradiol increase BMP-2 mRNA expression in human MSCs. Human MSCs cryopreserved for 6 or 9 months at-80℃using 10% dimethyl sulfoxide (DMSO) as a cryoprotective agent did not alter alkaline phosphatase and collagen typeⅠproduction. The chitosan-calcium phosphate cement possessed a fast-setting ability, SEM showed that the scaffold had a three-dimensional interconnected pore structure, human MSCs are able to adhere and proliferate on chitosan-calcium phosphate scaffold. Histological examination and immunohistochemical results revealed microscopic bone formations to be present in animals four weeks after transplantation of the human MSCs-loaded scaffolds. After 12 weeks implantation in the radius defect, there were more unions in defects with scaffold plus rabbit MSCs than in defects with scaffold alone, the radiological and biomechanical scoring was higher in MSCs-loaded group.Conclusions: Dex and 1713-estradiol can regulate the differentiation of the human MSCs into osteoblast by regulating special gene expression, the process of cryopreservation and thawing the MSCs by the simplified method had no effect on either their growth or osteogenic differentiation. Chitosan-calcium phosphate cement has good biocompatibility and osteoconduction. Tissue engineering of bone by combining human MSCs with porous scaffold revealed bone formation potential in ectopic implant. Rabbit MSCs-loaded scaffolds are revealed successfully for the repair of bone defects. These results strongly encourage the tissue engineering approach of the transplantation of MSCs within a suitable scaffold for the reconstruction of bone defect.