Research Of Rat BMSCs Osteogenic And Composite Scaffolds For Tissue Engineering Bone

Objective:A kind of 3-D scaffolds was made with Nano-HA、COL、SF for bone tissue engineering, and the porosity, pore size, water absorption rate and biological mechanics performance of the scaffolds were evaluated. The BMSCs cells of the Wistar rat were extracted, and were osteoblast-directionally induced. To identify the activity of osteoblasts induction, the cell morphological changes, ALP dyeing, calcium nodules and the expression of type I collagen and osteocalcin were detected. Then, we cultivate the mixture of osteoblast-directionally induced BMSCs and the 3-D scaffolds to detect the cell biocompatibility and the feasibility of tissue engineering bone.Methods:1. The preparation of 3-D scaffolds: The 3 sets scaffolds that the mass ratio of Nano-HA、COL、SF is respectively 1:1:5(group A), 1:2:5(group B) and 1:3:5(group(C), were prepared by freeze drying. The porosity, the water absorption expansion rate were determined; Instron5865 universal mechanical testing machine was used to test mechanical properties of the three dimensional composite scaffolds. The internal microstructure was observed by the SEM.2. Extraction of BMSCs cells and cultivation: The BMSCs that were extracted under the sterile condition from the femur and tibia, were primitive cultured, and extended to the third generation with 10% fetal bovine serum DMEM/F12 medium.3. Direction induced cells and grouping: The third generation BMSCs cells grewing well that wrere inoculated into sterile within 24 orifice at 1 x 105 L- 1 cell concentration, were divided into the experimental group and control group. The alkaline phosphatase(ALP) staining, calcium nodules alizarin red staining, and type I collagen and osteocalcin expression were detected separately for each corresponding cell in the group. Interventions: the control group was cultured only with DMEM/F12 completely medium; the experimental group with DMEM/F12 containing osteogenesis revulsant completely nutrient solution culture.4. The cells after inducted was inoculated into 3-D scaffold, and the cell activity was determined by MTT after the training of 2, 4, 6, 8, 12 days; Also take the cultivation of the 8 days composite material to do a SEM and the HE slice staining.Results:1. The 3 groups of scaffold materials are 3-D porous structure, has a certain pore size and porosity. Group B support conforms to the requirement: the average pore size is 177um(P<0.05). The porosity rate was(96.72±2.78) %(P<0.05). Water absorption rate was(549.37±35.29) %(P<0.05). Mechanical performance is stable, compressive strain and modulus of elasticity index suitable for bone tissue engineering research and application.2. The most primitive BMSCs which were spherical mononuclear cells, and were spherical and elliptic adherent growth in vitro after 1~ 3days, then the number of adherent cells decreased significantly after continue to cultivate. After cultured 5 to 7 days, the cell form is given priority to with spindle. And the cell morphology were deformed to polygon after 2 ~ 3days induced, to given priority to with the polygon at 7 days. There were the most nucleus and cytoplasm staining was positive in the ALP staining and a crumb of calcium salt deposition, dark red or orange that sizes and multiple mineralization nodules can merge, is visible in calcium nodules alizarin red stain between cells. The expression of type I collagen and osteocalcin were positive.3. According to the results of SEM and after HE slice staining. Cause most cells, growth in good condition, fully extended. support internal structure is covered by a layer of cell matrix, between holes and hole cell ingrowth. Determined by MTT test results a logarithmic growth of cells in a certain period of time.Conclusion:1. The three groups of scaffolds were porous structure, and group B scaffolds are more suitable for bone tissue engineering requirements.2. Tested ALP, calcium nodules and osteocalcin indicators verify the activity of cells for osteogenesis cells, reverse validation for cells BMSCs at the same time, can be used to build bone module.3. After cell/scaffolds composite culture, cells grew well within the scaffold, and secretory cell matrix. The build complex scaffold is expected to apply the fix tissue defect.