Experimental Study On Constructing Tissue Engineering Cartilages By Binding Human Bmscs And Autologous DBM In Vitro Culture

Objective:The study aims to investigate the feasibility of constructing tissue engineering cartilage by culturing human bone mesenchymal stem cells (BMSCs) and autologous demineralized bone matrix (DBM) in vitro.Methods:1, Human BMSCs were isolated by the method of non-centrifugal whole bone marrow adherent culture from the patients’ femoral bones collected surgically, and were followed by in vitro amplification, leading to the drawing of3rd generation cells’ growth curve. Human BMSCs were identified through morphological observation and flow cytometry (FCM).2, the3rd generation of human BMSCs was used to carry out chondrogenesis by the method of high density of micelles-induced culture. The preparation was fixed in21days, and with the histopathological staining, the expressions of glycosaminoglycan (GAG) and Ⅱ collagen were observed to detect human BMSCs’ chondrogenesis inducibility.3, femoral bones collected from surgeries were made into DBM by revised Urist method. Through electron microscopy observation, degradation rate and other performance measurements, their combination with human BMSCs was divided into two groups (n=36). Experimental group was to be cultured into chondrogenesis, while control group not.4, cell scaffolds were taken for general and electron microscopy observation, H-E, alcian blue staining and Ⅱ collagen’s immunohistochemical staining in the1st,2nd and3rd month respectively after their composition. IPP was used for the measurement of IOD data by Ⅱ collagen’s immunohistochemical method, and statistical analysis was conducted.Results:1, The BMSCs isolated by the method of non-centrifugal whole bone marrow adherent culture were in long fusiforms, and their growth curves showed that5×104/ml would make the best inoculation concentration. According to FCM, CD29, CD44and CD105were highly expressed on the cell surface, while CD34and CD45were under-expressed. The existence of GAG expression was suggested after the preparation induction and alcian blue staining. Ⅱ collagen’s immunohistochemical staining showing the extracellular matrix turned brown suggested the expression of Ⅱ collagen.2, DCM was in reticulate cubic structure, with its apertures around340μm, which could be degraded almost completely in12weeks. The electron microscopy observation revealed that as DBM degraded, these cells were in the dynamic process of growth and differentiation.3, by comparing with the control group with histopathological staining, the alcian blue staining and Ⅱ collagen’s immunohistochemical staining of the experimental group were expressed positively. The IOD statistical analysis of the experimental group shows that there exists statistical meaning between these two groups in the3rd month, and that there lies statistical meaning between the comparison of the3rd month and its previous2months of experimental group, suggesting the expressions of Ⅱ collagen differ from each other.Conclusions:1, the3rd generation of seniors’BMSCs is suitable as the seed cell for cartilage tissue engineering.2, after its chondrogenesis, the composite of human BMSCs and autologous DBM cultured in vitro bears certain characteristics of tissue engineering cartilage. This paper contains45Figures,2Tables and73References.