| BACKGROUND:Repairing the large weight-bearing bone defects has been a problem faced by orthopedic surgeons.The research and development of bone tissue engineering provides a good way to repair large weight-bearing bone defects.Experimental studies reported that large bone defect model can be effectively repaired by using the scaffold materials with growth factors,seed cells and appropriate mechanical environment.But the new bone tissue ingrowth rate is different when the scaffolds with different structure and different composition implantation into the bone defect.Numerous studies have showed that scaffold structure can significantly affect the rate of bone tissue ingrowth,The porosity and pore size is one of the most important factor.Therefore, the research on the pore size of the scaffolds of different materials on the treatment of large bone defects is very important.In this study, three kinds of pore size of calcium phosphate bone cement (200-300μm ,300-450μm ,450-600μm) and bone marrow mesenchymal stem cells were co-cultured in vitro,Observed the different among the three groups of cell surface adhesion, proliferation and differentiation.analyze the role of the mechanism to the pore structure of scaffolds to promote fracture healing.1. The effect of pore size on cell adhesion in CPC scaffoldsOBJECTIVE: To study the effect of CPC scaffolds with different pore sizes on cell adhesion. METHOD: Primary cultured rat bone mesenchymal stem cells were used in the present study. Three kinds of CPC scaffolds with different sizes of aperture (200-300μm, 300-450μm, 450-600μm) were divided into three groups named A, B and C group, respectively. Micro-CT was used to analyze the average pore size, porosity. The three kinds of CPC scaffold were placed in 24-well plates and 2×105 cells were incubated per-well. The cells were harvested 12 and 24 hours after the incubation, and then cell numbers and cell adhesion rate were counted. 24 hours after the incubation, apoptosis and cell necrosis were detected by flow cytometry, and cell morphology and cell adhesion were observed by scanning electron microscope.RESULTS: pore size of CPC scaffold used in this study interconnected. Porosity of the three groups was greater than 67% with the average pore size of 240μm, 410μm and 510μm, respectively. The amount of cell adhesion in A group was significantly less than that of B and C groups. The number of cell necrosis in A group was significantly more than that of B and C groups. CONCLUSIONS: The pore size may affect cell adhesion and cell necrosis. Along with the increase of the pore size, cell adhesion rate increased gradually and cell necrosis rate decreased gradually. The present study provides the foundation for further study on the effect of pore size structure on cell viability. 2. The Effect of Pore size on Cell Proliferation in CPC ScaffoldsOBJECTIVE: To study the effect of CPC material with different pore sizes on cell proliferation. METHOD: Primary cultured rat bone mesenchymal stem cells were used in the present study. Three kinds of CPC scaffolds with different pore sizes (200-300μm, 300-450μm, 450-600μm) were placed in 24-well plates and 5×104 cells were incubated per-well. The cells were harvested 1,4,7,14 and 21 days after the incubation, and then use the PicoGreen dsDNA Quantitation Reagent kit to test the cell proliferation on the three group of scaffolds. The cell morphology and cell condition were observed by scanning electron microscope in 7,21days. RESULTS: The amount of cell in the 3 groups has no significant in day 1.In the 4th day the amount of cell in the 450 - 600μm group more than that of B and C groups. In the 7th day,with the increase of the pore size, the amount of cell increased gradually.In the 14th and 21th day, the amount of cell in 200-300μm group was significantly less than the other two groups. CONCLUSIONS: The pore size may affect cell proliferation. Along with the increase of the spore size, cell proliferation rate increased gradually. The present study provides the foundation for further study on the effect of aperture structure on cell viability.3. The Effect of Pore size on Cell Proliferation in CPC ScaffoldsOBJECTIVE: To study the effect of CPC material with different pore sizes on cell differentiation. METHOD: Three kinds of CPC scaffolds with different pore sizes (200-300μm, 300-450μm, 450-600μm) were placed in 24-well plates and 5×104 cells were incubated per-well. The cells were harvested 4,7,14 and 21 days after the incubation, and then use the ALP activity kit to test the cell osteogenic differentiation on the three group of scaffolds and use the real-time reverse transcriptase polymerase chain reaction to test 5 osteogenic markers expression.RESULTS: found that the 200-300μm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the other two groups as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for osteogenic markers. CONCLUSIONS: The pore size may affect cell differentiation. Along with the increase of the spore size, cell proliferation rate decreased gradually. The study shown that the 200-300μm pore scaffolds exhibited a faster rate of osteogenic differentiation ,The 450-600μm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by PicoGreen dsDNA Quantitation Reagent kit, scanning electron microscopy)... |
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