| Objective :In recent years,the development of novel bone repair materials to replace traditional autologous bone grafts for the treatment of bone defects has been one of the current research hotspots.However,previous studies have focused on the influence of chemical factors in the environment in which the cells are placed,while insufficient attention has been paid to the influence of physical factors.Studies have shown that the micromorphology of the scaffold material prepared by electrostatic spinning significantly affects the fate of cells on it.Poly-L-lactic acid(PLLA)is a commonly used biodegradable medical material and is also a common material for electrostatic spinning.However,studies on the effect of PLLA scaffold material’s micromorphology on bone marrow mesenchymal stem cells(BMSC)are still incomplete.In this study,we propose to prepare PLLA scaffold materials with different fiber diameters and arrangements,and examine the changes in the biological behaviors of BMSC proliferation,migration,adhesion,travel and osteogenic differentiation after co-culture of PLLA scaffold materials with BMSC,in order to investigate the influence of microscopic morphology of PLLA scaffold materials on BMSC,and thus provide a reference for the design of bone repair materials in terms of physical factors,and in turn provide a reference for the design of bone repair materials.This will provide a reference for the design of bone repair materials in terms of physical factors,and thus provide new ideas for the treatment of bone defects and other osteogenic deficiency-related diseases.Methods:1.PLLA scaffold materials with different fiber diameters and arrangements were prepared by adjusting the technical parameters of electrostatic spinning,and four groups of qualified PLLA scaffold materials were screened by applying SEM technique to characterize the scaffold materials.Then the screened PLLA scaffold materials were tested for their hydrophilic and mechanical properties by contact angle measuring instrument and universal mechanical testing instrument.2.Four groups of PLLA scaffold materials were co-cultured with BMSC for 1,4and 7 days,respectively,and the biocompatibility of PLLA scaffold materials was examined using live-dead staining and CCK-8 proliferation assay.3.Four groups of PLLA scaffold materials were co-cultured with BMSC,and the migration area of BMSC on the four groups of PLLA scaffold materials at 48 h was observed by migration assay;the adhesion number of BMSC on the four groups of PLLA scaffold materials at 3,6,12 and 24 h was observed by adhesion assay;the travel direction and cell morphology of BMSC on the four groups of PLLA scaffold materials at 48 h was observed by travel assay and cell morphology analysis.The direction of travel and cell morphology of BMSC on the four groups of PLLA scaffold materials at 48 h were observed by walking assay and cell morphology analysis.4.The four groups of PLLA scaffold materials were co-cultured with BMSC,and the osteogenesis of BMSC on the four groups of PLLA scaffold materials was observed by performing alkaline phosphatase(ALP)staining and activity measurement on day 7 and 14,and alizarin red staining and semi-quantitative analysis on day 21;meanwhile,the osteogenesis of BMSC on the four groups of PLLA scaffold materials was detected by q RT-PCR.The expression of osteogenic genes of BMSC on the four groups of PLLA scaffold materials was measured by q RT-PCR at day 7 and day 14.Results:1.Four groups of PLLA scaffold materials with different fiber diameters and arrangements were successfully prepared by morphological characterization screening:aligned 600 nm(Aligned-600),random 600 nm(Random-600),aligned 1200 nm(Aligned-1200),and random 1200 nm(Random-1200),respectively.The SEM results showed that the fiber orientation of the aligned 600 nm group and the aligned 1200 nm group had a certain concentration trend,while the fiber orientation of the random600 nm group and the random 1200 nm group had randomness;meanwhile,the diameter comparison between the aligned 600 nm group and the aligned 1200 nm group,and the random 600 nm group and the random 1200 nm group was statistically different(P<0.05),the results indicate that the aligned/random PLLA scaffold materials with diameters of 600 nm and 1200 nm were successfully prepared in this study.The results of hydrophilic angle showed that the hydrophilic angle of the aligned 600 nm group was smaller compared to the other three groups(P<0.01).The results of the mechanical properties tests showed that the ultimate tensile strength and Young’s modulus of the aligned group were greater than those of the random group(P<0.001),with the aligned 600 nm group being greater than the aligned 1200 nm group(P<0.001);the breaking strain of the film was significantly higher in the random group than in the aligned group(P<0.001),with the aligned 600 nm group being smaller than the aligned 1200 nm group(P<0.01).2.The results of live-dead staining on days 1,4 and 7 showed that the cells cultured on all four groups showed good growth activity.the results of CCK-8proliferation assay showed no statistically significant difference in optical density(OD)values between the four groups on day 1(P>0.05);on days 4 and 7,the OD values were greater in the aligned group compared to the random group(P<0.0001).3.The results of cell migration assay showed that the cells in the aligned group migrated in the parallel direction to fill the vacancies but not in the vertical direction,while the cells in the random group basically did not migrate to fill the vacancies.The results of cell adhesion assay showed that there was no significant difference in the number of cells adhering to each group at 3,12 and 24 h(P>0.05),while the number of cells adhering to the aligned group was less than that of the random group at 6 h(P<0.0001).The results of cell walking experiments and cell morphological analysis showed that the cells on the aligned group grew in parallel directions along the fibers,and the cells showed an elongated state along the fiber direction;the cells on the random group grew randomly in different directions,and the cells showed an irregular state.4.The results of alkaline phosphatase staining and activity assay on days 7 and14 showed that the staining intensity of the aligned group was significantly higher than that of the random group,and the staining intensity of the aligned 600 nm group was the strongest among the four groups of scaffold materials.On day 7,the ALP activity was higher in the aligned group compared to the random group(P<0.0001);the ALP activity was higher in the aligned 600 nm group compared to the aligned1200 nm group(P<0.0001).The ALP activity was higher in the aligned group compared to the random group at day 14(P<0.01);the ALP activity was higher in the aligned 600 nm group compared to the aligned 1200 nm group(P<0.0001).The results of alizarin red staining and semi-quantitative analysis on day 21 showed that the staining intensity of the aligned group was significantly higher than that of the random group,and the staining intensity of the aligned 600 nm group was the strongest among the four groups of scaffold materials;the results of semi-quantitative analysis showed that the OD value of the aligned 600 nm group was higher compared with the other groups(P<0.01);the OD value of the aligned 1200 nm group was higher compared with the random 1200 nm group(P<0.05).The results of q RT-PCR showed that the expression of osteogenic-related genes was higher in the aligned 600 nm group compared with other groups at day 7,except for OCN(P<0.05);and at day14,the expression of osteogenic-related genes was higher in the aligned 600 nm group compared with other groups,except for BMP-2(P<0.05).Conclusion(s):1.PLLA scaffold materials with different fiber morphologies can be prepared by adjusting the parameters,among which the aligned 600 nm PLLA scaffold materials have the best hydrophilic and mechanical properties.2.Aligned PLLA scaffold materials can better promote BMSC proliferation and guide BMSC migration and growth in a parallel direction to the fibers.3.The aligned 600 nm PLLA scaffold material had the best effect of enabling bone differentiation compared to the other three groups. |
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