| Objective:On the basis of pre-characterization of bone and cell interface micro-nanostructures, Polylactic Acid (PLA) fiber membranes with bone / cell interface morphology were prepared by electrostatic spindles (ES) On the proliferation, adhesion,spread and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs),and the fibroblasts with good biocompatibility and osteoinductive ability were screened, The micro-morphology of osteoblast differentiation of stem cells to guide the design of the mature bone substitute material to improve the bone-induced ability of bone substitute materials to make it more close to the ideal state of boneMethods:1. 1.Preparation, Characterization and Parameter Measurement of Bionic PLA Fiber MembraneThe polylactic acid and the chloroform were mixed and dissolved in different proportions, and the polylactic acid fiber membrane with different morphology and morphology was prepared by changing the voltage, accepting distance and receiving mode.High resolution imaging of the fiber membrane samples after drying at the critical point of dehydration was carried out by low vacuum scanning electron microscopy. Fifteen samples were randomly selected for fiber diameter and pore size measurement. Using the scanning electron microscope (SEM) and the part of the morphological parameters (collagen fiber diameter) of the bone interface, the microscopic morphology of the polylactic acid fiber membrane was selected.2. Bone marrow mesenchymal stem cells were isolated, cultured and identified.The BMSCs were isolated and purified by whole bone marrow adherence method combined with differential centrifugation. The BMSCs were isolated and cultured in vitro.The anti-rat monoclonal antibody FITC-anti-Mouse / Rat CD 44, PE-anti-Mouse / Rat CD45, APC-anti-Mouse / Rat CD90 were selected by immunofluorescence and flow cytometry respectively,to identify the molecular markers of BMSCs on behalf of BMSCs, to verify that the cells were bone marrow mesenchymal stem cells and to carry out experiments on the co-culture of cells with polylactic acid fiber membranes3. Cellular Behavior and Osteogenic Differentiation on Different topography Polylactic Acid Fiber Membranechoose the P2-P4 generation BMSCs cells, cultured on group A(with fiber diameter of 357±32nm),group B(diameter 164±13nm),group C(diameter 1311±93nm),Ctrl group (blank control with no topography), Positive group(positive blank control).Then use scanning electron microscopy (SEM) to observe the the growth, spreading and fusion of rBMSCs. Use hematoxylin staining to observe the survival of rBMSCs, and use CCK-8 kit to detect the proliferation and adhesion of rBMSCs. qRT-PCR to detecte the expression of Runx2 (runt-related transcription factor 2),collagen type ?(COLI),alkaline phosphatase (ALP) and zinc finger structural transcription factor (Osterix, Osx)mRNA. Use Western Blot to detect the expression of osteopontin (OPN) and osteocalcin (OCN). To observe the biocompatibility of polylactic acid fiber membrane and the effect of different diameter fibrous membrane on the proliferation, adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells.Results:1. By changing the polylactic acid concentration (4%-5%); Electrospinning parameter voltage (18kv-22kv); Receiving distance (12cm-15cm); Receiving time (lh-2h).A polylactic acid fiber membrane having a fiber diameter of 100 nm to 1300 nm, a porosity of 79% to 91%, and a different morphology of pore size.The 357±32nm membrane fiber that prepared by electrospinning parameters (concentration: 5%; voltage: 18kV; 15cm; accept accept distance:time: 1h) , uniform thickness, similar to the formation of network structure formation of collagen fibers in the extracellular matrix, close to the bone microstructure and interface morphology.2. P1 cells were cultured in vitro for 24 hours. Adhesive cells such as adipocytes were observed. After 48h, the number of cell colonies increased, and the cell density increased more than that before. After 72 hours, Fusiform,polygonal,more adherent cells close to the fusion layer of growth, was"fish-like",after passage of cells grow well.Fluorescence immunoassay showed that CD90 and CD44 of P2 generation BMSCs were positive and CD45 was negative.The expression rates of CD45-PE, CD44-FITC and CD90-APC were 1.65%,94.1% and 95.8%, respectively,and the typical biological characteristics of mesenchymal stem cells were observed by flow cytometry.3. hematoxylin staining showed that the cells grew well in polylactic acid fiber membrane; SEM showed that the cells attached to the climbing three polylactic acid fiber membrane surface growth is good, more than and 10 to dozens of cells that form a colony, contact between cells closely, group A(diameter 357±32nm),group B (diameter 164±13nm) fiber membrane cells with distinct cell clusters around to distribute type growth, apophysis of cells connected to each other, compared with the group C (diameter 1311±93nm) and normal culture plate group fully, a larger number of large size. The observation group A typical cell morphology ( diameter 357±32nm),group B (diameter 164±13nm) than group C (diameter 131±93nm) and normal culture plate group,cell spreading of larger and more filopodia, and the cells embedded in the fiber membrane surface, pseudopods and fibres in the A group ( diameter 357±32nm)is the most obvious; CCK-8 proliferation experiment, first, 3, 5 days normal culture plate control group and A group (diameter 357±32nm),group B(diameter 164±13nm) value was better than that of group C (diameter 1311±93nm), the differences were statistically significant (P < 0.05), the comparison between the three groups, the control group normal culture plate is better than that of A group and B group, the difference was statistically significant (P<0.05), A group, B group, the comparison between the two groups, A group than in B group,but the difference was not statistically significant (P>0.05); in the seventh, 9 days normal culture plate control group and group A( diameter 357±32nm),group B (diameter 164±13nm) value was better than that of group C (diameter 1311±93nm),the differences were statistically significant (P < 0.05), the comparison between the three groups, the control group normal culture plate is better than that of A group and B group,the difference was statistically significant (P < 0.05). A B, the comparison between the two. groups, the difference was statistically significant in A group than B. group (P < 0.05); adhesion experiment showed that in 4h, the control group was better than the ordinary plate group C (diameter 1311±93nm) the difference was statistically significant (P < 0.05), there was no significant difference among other groups (P>0.05) in the 8h,and 16h,A group (diameter 357±32nm),group B (diameter 164±13nm),C (diameter 1311±93nm) group than normal culture plate control group, the difference was statistically significant (P < 0.05),A,B,C between the three groups compared with A,B group the difference is statistically higher than that of C group Significance (P<0.05), no statistically significant A, B between the two groups difference(P>0.05); osteogenic differentiation assay showed that: A group (diameter 357±32nm),ALP,COLI,Runx2,OSX mRNA expression was significantly higher than that in group B (diameter 164±13nm), group C (diameter 1311±93nm) and the general training in the control group, lower than the positive control group; group B (diameter 164±13nm),group C (diameter 1311±93nm) and normal culture plate control group, lower than the positive control group.Conclusions:1. With the electrospinning parameters (concentration: 5%; voltage: 18kV;accept accept distance: 15cm; time: 1h) and (concentration: 4.5%; voltage: 22kv;accept accept distance: 15cm ;time: 1h) can be prepared in diameter was 357±32nm, 164±13nm fiber, uniform thickness,similar to the network the structure of collagen fibers in the extracellular matrix of the polylactic acid fiber membrane close to the microstructure and morphology of bone interface2. The morphology of the fiber membrane affects the morphology and size of the rBMSCs spreading, the diameter of the fiber membrane on the diameter of 357±32nm, the area of the cell is obvious, the growth is distributed to the surrounding area, the spreading area is larger, and the cell is more.3. Compared with the diameter of 1311±93nm fiber membrane, diameter of 357±32nm and 164±13nm fiber membrane can promote cell adhesion,proliferation.4. The micro morphology of the fiber membrane affects the osteogenic differentiation of bone marrow mesenchymal stem cells. The Bionic Membrane with a diameter of 357±32nm has obvious osteogenic induction ability. |
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