The Experimental Research Of The HUCMSC’ Chondrogenic Induction On The Type Ⅱ Collagen Fiber Composite Glycosaminoglycan Scaffold

ObjectiveThe main purpose of this study is to assess the human umbilical cord derived mesenchymal stem cells (HUCMSCs) as the feasibility of cartilage tissue engineering seed cells, and assess weather the scaffolds with type Ⅱ collagen fiber and glycosaminoglycan could creat conditions for the tissue engineering. The study includes three parts:the first part was aimed to evaluated their biological characteristics and the culture of HUCMSCs, expected it to provide experiment foundation for the study of seed cell of tissue engineering; The second part was manufacture the type Ⅱ collagen fiber composite glycosaminoglycan scaffold and evaluated its physical and chemical properties; the third part was culture the human umbilical cord derived mesenchymal stem cells on the scaffold, and evaluated the cartilage tissue.Methods1. The cells were provided by the cell compartment of the clinical laboratory department of Kunming General Hospital. The cell suspensions were plated and cultured in DMEM/F12 medium congtaining 10% FBS、100U/mL penicillin、 100mg/L streptomycin. While a cell growth to 80%-90% fusion,1:3spread generation culture.Observed the morphologic changes of the cells by the inverted microscope. The immunophnotypic characterization of the primary cells and P3 cells were analyzed by flow cytometry. The cells were digestioned by trypsin/EDTA suspension, then put them in the centrifuge under 1000rmp for five minutes, and reset the cells in the bottom of the centrifugal bottle with dimethyl sulfoxide dilution by FBS. Stored the cells in liquid nitrogen tank after stored in the refrigerator at the temperature of -80℃ for 24 hours.Revived the cells for culture、subculture and flow cytometry after three months.2. Smash the cartilage tissue of pigs joint with cell-free processing. manufacture the type II collagen fiber composite glycosaminoglycan scaffold, observed under a scanning electron microscope,to determine the pore diameter and porosity of the scaffold,detect its hydrophilic、physical and chemical properties。3. Dissociate the cells of the third passage,transplanted them on the type II collagen fiber composite glycosaminoglycan scaffold with disinfect under irradiation,cultured the cells in DMEM/F12 medium congtaining 10%FBS.Three weeks later,pick out the scaffold with toluidine blue、safranin O staining and type II collagen immunohistochemical staining.Results1. Three hours after inoculating, a small number of umbilical cord mesenchymal stem cells were begun to grow with adherent in culture flasks. The cells morphology with fusiform, spindle, a small colony growth can be visible presented, and a most cells adherent growth has been completed after inoculation in the 24 hours. Covering the extent of visible adherent cells reached 80-90% in 3 days to 4 days. Uniform spindle cells were grown like the fibroblasts and showed a typical swirling arrangement of growth observed by microscope. The cells were passaged no significant change in the process. But spread to the rest of single cell morphology in the third generation. Flow cytometric analysis showed that primary cultured HUCMSCs has begun expression CD44, CD90, CD 105, but there is still a certain degree of expression of CD34, CD45, and the third generation of HUCMSCs is highly expressed CD29, CD44, CD90, CD 105 mesenchymal cell marker, no expression with CD34, CD45 endothelial cells, hematopoietic cell markers and human major histocompatibility leukocyte antigen DR antigen. The cells show full shape, well cell activity, good growth passage under the microscope compared with the pre-frozen, its surface markers is similar with not frozen HUCMSC by flow cytometry after the HUCMSCs frozen three months to recovery2. Type II collagen fiber composite glycosaminoglycan scaffold is white, porous foam cylinder with many pores on the surface. Under the scanning electron microscope, the scaffold is porous foam which is homogeneous distribution and interconnection. The scaffold porosity is 91.8%±2.17%, and the average pore diameter is between 110-230μm. The scaffold processed good hydrophilicity with water absorption expansion rate of 213.71%±1.31%. Toluidine blue, safranin O and type II collagen staining of the scaffolds is positive.3. The surface of Type Ⅱ collagen fiber composite glycosaminoglycan scaffolds becomes smooth gradually along with cell culture. As the same time, the texture becomes toughness. The cartilage-like tissue is observed, and gradually increased. Toluidine blue, safranin O and type Ⅱ collagen staining of the scaffolds is positive.Conclusion1. The human umbilical cord derived mesenchymal stem cells were in line with the basic biological characteristics could be the ideal seed cell of tissue engineering.2. The type Ⅱ collagen fiber composite glycosaminoglycan scaffold had favorable pore diameter、porosity and hydrophilic,was suitable for the cells to grow and proliferate, could be the favorable cell-carrier for the tissue engineering.3. The human umbilical cord derived mesenchymal stem cells could construct tissue-engineering cartilage on the type Ⅱ collagen fiber composite glycosaminoglycan scaffold without inducer,provided the experimental basis for the repair of cartilage damage.