Experimental Study On Biomimetic Mineralization Of PLGA And The Effects On Attachment, Proliferation And Differentiation Of Rat BMSCs On The Mineralized Scaffolds

Part One Experimental Study on BiomimeticMineralization of Poly Lactide-co-GlycolideObjective:To construct a mineralized poly (lactide-co-glycolide) by biomimetic approach in order to improve the property of PLGA surface,and to develop novel biomaterials for orthopaedic tissue regeneration.Methods: PLGA films and 3-D porous PLGA were subjected to a hydrolysis treatment with alkaline solution prior to mineralization. The wettability of PLGA films were measured with High Temperature Microscope. PLGA hydrolyzed for 60 minutes were mineralized in 0.5 fold of the standard SBF for 20 days,in SBF for 14 days or in 1.5 fold of SBF for 9 days. The morphology,composition,and phase of mineral grown on PLGA were analyzed with SME,FTIR and XRD. The porosity was detected by using liquid displacement method. The biomechanics of PLGA scaffolds were detected by using Shimadzu universal mechanic tester.Results: The wettability of PLGA films enhanced after being hydrolyzed by alkaline solution. The morphology of the mineral dependents on the solution characteristics. The main component of mineral was hydroxyapatite containing a little of CO32- group which the Ca/P ratio is 1.53, similar to the major mineral component of bone tissue. The porosities of the mineralized and unmineralized 3-D porous PLGA were (84.86±8.52)% and(79.70±7.70) % respectively. The biomechanical strength was 0.784±0.156 N/mm2 in unmineralized PLGA and 0.858±0.145N/mm2 in mineralized PLGA. There is no significant difference of porosity and biomechanics between the mineralized and unmineralized scaffolds (p>0.05).Conclusion:Mineralization of PLGA by biomimetic approach is a suitable way to construct bioactive artificial bone.Part TwoIsolation,Culture and Identification of Rat Bone Marrow-derived Mesenchymal Stem CellsObjective: To acquire the rat bone marrow-derived mesenchymal stem cells (BMSCs) and identify them. This work will ultimately be used to study the characterization of biomaterials.Methods: BMSCs were isolated from the bone marrow of the young SD rats according to a protocol modified from our laboratory. Light microscopy was used to study the morphologic features and growth status. Immunocytochemistry technique was employed to examine the cell surface antigens. Histochemistry technique was employed to examine whether the cells can be induced to differentiate along the osteoblastic pathways or not.Results: The purity of the obtained cells was higher. The cells we acquired was founded that both growed stably and proliferated rapidly. The positive expression of CD44,CD54 and CD71 was detected while CD34,CD45 were negative. Culturing confluent BMSCs was induced along osteoblastic differentiation for 2 weeks in conditional medium,alkaline phosphatase was found positive expression in most of the cells. Calcium deposited on the extracellular matrix was detected after being induced for 3 weeks. It is proved that the obtained cells can differentiate into osteoblast. The cells were proved to be BMSCs.Conclusion:The obtained cells were demonstrated to be the undifferentiated BMSC by the detection of cell surface antigens and the characteristics which could differentiate along osteoblastic pathways.Part Three The Attachment, Proliferation and Differentiation Behavior of Rat BMSCs on Mineralized PLGA ScaffoldsObjective:To investigate the attachment, proliferation and differentiation behavior of rat BMSCs on mineralized PLGA scaffolds , which will provide experimental basis for the construction of PLGA artificial bone.Methods: BMSCs were co-cultured with mineralized PLGA, unmineralized PLGA and heterogeneous sintered bone. The cell-material complex was observed in order to evaluate the interaction between cells and materials. After being co-cultured for 24 hours the cell adhersion rate of each materials was calculated according to the method of indirect cell count. The proliferation of cells was determined by the fluorometric quantification of cell DNA when cells were seeded 2,4,6,8,10,12,14 days,respectively. The growth curves of the cells were analyzed for the use of cell proliferation. We analysed by RT-PCR the expression of osteoblast-specific genes of the cells grown on each group scaffolds. Expression of these osteoblast-specific genes can be greatly explained by BMSCs differentiation along the osteoblastic pathways.Results:The cell adhersion rate of the unmineralized PLGA group,mineralized PLGA group and sintered bone group are (36.7±5.4)%, (66.2±8.9)% and (71.5±6.4)%,respectively. There is significantly different between mineralized PLGA group and unmineralized PLGA group(p<0.05),while there is no significantly different between mineralized PLGA group and sintered bone group(p>0.05). The cell growth curves showed that the cells of sintered bone group grew exponentially from the start of subculture and reached the stationary phase after 10 days. The cells of mineralized PLGA group grew slowly within 6 days and grew exponentially from then on. At the 14 days,there were no differences in cell number between the mineralized PLGA group and sintered bone group. The cells number of the unmineralized PLGA group has no change for 6 days,and then the cell grew slowly until the14 days. The cell growth speed of mineralized PLGA group is higher than that of unmineralized PLGA group,but lower than that of sintered bone group. The expression level of osteoblast-specific genes is the highest in the group of sinered bone and the lowest in the unmineralized PLGA group.Conclusion:These results indicate that biomimetic mineralization of PLGA presented here can improve the function of BMSCs's attachment,proliferation and differentiation along the osteoblastic pathways.