| Objective:Based on the microscopic morphology parameters of the femoral head cancellous bone surface of different femurs,ages and bone states measured by the previous research group,fabricate the nano-hydroxyapatite(nHA)/polylactic acid(PLA)composite fiber membrane by electrospinning,In order to simulate the micro-topography parameters of different bone surfaces,observe the proliferation,spreading morphology and osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSCs)on fiber membrane,clarified the relationship between bone surface topography parameters and cell osteogenic differentiation,seeking for the topography parameters that beneficial to osteogenic differentiation,and provide parameter guidance for future material interface modification and surface modification.Methods:1.Fabrication,characterization and parameter measurement of nano-hydroxyapatite/polylactic acid fiber membrane,Polylactic acid,nano-hydroxyapatite,and N,N-Dimethylformamide(DMF),dichloromethane are mixed uniformly in different ratios,nHA/PLA fiber membranes with different diameter parameters were manufactured by adjusting electrospinning parameters:positive and negative voltage,acceptance distance,receiving time,and bolus speed.Characterize the dried fiber membrane samples by Scanning electron microscope(SEM),and 20 fibers were randomly selected for diameter measurement;The dried fiber membrane samples were image characterized by Scanning electron microscope(SEM),and 20 fibers were randomly selected for diameter measurement;X-ray photoelectron spectroscopy analysis of the surface element composition of the fiber membrane;water contact angle to measure the hydrophilicity of the material.2.Selction of nano-hydroxyapatite/polylactic acid fiber membraneBy adjusting the electrospinning parameters:voltage 16-24kv,receiving distance 12-18cm,bolus speed 0.2mm/min and 0.3mm/min,acceptance time 1-2h,making nHA/polylactic acid fiber membranes with different fiber diameter parameters,selecting fiber membranes with fiber diameters ranging from 100 nm to 1300 nm,choosing the coarse(diameter 900-1300 nm),medium(diameter 500-900 nm),fine(diameter 100-500 nm)set as topography group;the specific grouping parameters were A1 group:fiber diameter 1153± 151 nm,A2 group:fiber diameter 652±127 nm,A3 group:fiber diameter 271±116 nm.3.Proliferation,morphology and osteogenic differentiation of rBMSCs on different fiber diameter nHA/PLA nanofiber membranes.P5-P7 rBMSCs were cultured on the nHA/PLA nanofiber membrane of the topography group,the A1 group(fiber diameter 1153±151nm),A2 group(fiber diameter 652±127nm),A3 group(fiber diameter 271±116nm)and Ctrl group(blankcontrol plate group),observe of rBMSCs growth and spreading on fiber membrane bySEM,CCK-8 kit was used to detect the proliferation of rBMSCs,and qRT-PCR was used to detect the mRNA expression level of alkaline phosphatase(ALP),osteocalcin(OCN),and Runx-related transcription factor 2.Observe the biocompatibility of the fiber membrane and the effects of fiber membranes with different diameter parameters on the osteogenic differentiation of rBMSCs.Results:1.Results of microscopic topography parameters of cancellous bone surface of femoral headThrough the preliminary establishment of the characterization and parameter extraction analysis method of the surface topography of the cancellous bone of the femoral head by our research group,Expanding the sample size to 24,divide the osteoporosis patients’the femoral head into 3 partition:Normal area,osteopenia area,osteoporosis area,measured and analysis,we get the diameter of mineralized collagen bundles in the middle-aged male/female in the normal area was at range of 600-900nm;in the osteopenia and osteoporosis area,the average fiber diameter in is around 1200 nm,The fiber diameter is gradually increased;The fiber diameter of our previous study is the extracellular matrix collagen fiber diameter parameter obtained by consulting the literature,and the diameter is 100-500 nm.In order to compare the three,we selected the fiber diameter parameters of 100-500nm,500-900nm and 900-1300nm as comparative research objects.2.Preparation of nHA/PLA fiber membrane by electrospinning and selection of fiber membraneBy adjusting the electrospinning voltage(16kv-24kv);receiving distance(12cm-18cm);receiving time(lh-2h);pushing speed(0.2mm/min-0.3mm/min),we obtained the Polylactic acid/nano-hydroxyapatite fiber membranes with different fiber diameter in the range of 100 nm to 1300 nm,the fiber membranes with random fiber topography with better spinnability and uniform fiber and gradient distribution are selected from the following electrospinning parameters:Group Al:diameter 1153 ±151 nm(electrospinning parameters:15 cm,24 kv,1 h,0.2 mm/min);Group A2:diameter 652±127 nm(electrospinning parameters:12 cm,24 kv,2 h,0.3 mm/min);Group A3:diameter 271 ±116 nm(electrospinning parameters:15 cm,24 kv,2 h,0.3 mm/min).3.Use the SEM to observe the cell spreading,use CCK-8 kit to observe the cell proliferation,qRT-PCR to detect osteogenic differentiationThe SEM electron microscopic observation of cell spreading in the fiber membrane showed that the rBMSCs had a large area on the nHA/PLA fiber membrane of the A2 group(652±127 nm)fiber diameter,which was irregularly shaped and adhered between cells and fibers.Embedded,tightly bound,and more pseudopods.The A3 group also showed similar cell spreading morphology,and the cell spreading area of the A1 group was not as good as that of the A2 and A3 groups.The CCK-8 proliferation experiment showed that on the 1st,3rd,and 5th day,the normal culture plate control group,A2 group(652±127nm),A3 group(271±116nm)were better than A1 group(11531151nm),the difference was statistically significant(P<0.05),Compared with the A2 and A3 groups,the difference between the three groups was statistically significant(P<0.05).The A2 group were better than the A3 group,and the difference was not statistically significant(P>0.05).On the 7th and 9th day,the control group was better than the A2 and A3 groups,and the difference was statistically significant(P<0.05).The A2 group was better than the A3 group,and the difference was statistically significant(P<0.05).Osteogenic differentiation test shows:Group A2 showed excellent osteoinductive ability,the qRT-PCR detection of ALP,Runx2 and OCN showed that the expression was better than A1 and A3.Conclusions:1.Adjust the electrospinning parameters to(15cm,24kv,1h,0.2mm/min),(12cm,24kv,2h,0.3mm/min),(15cm,24kv,2h,0.3mm/min)can manufacturing the nHA/PLA fiber membranes with random fiber diameters of 1153±151 nm,652±127 nm,and 271±116 nm,it has better biocompatibility,and it’s similar to the collagen fiber network in the extracellular matrix of bone.2.The change of fiber membrane diameter parameters can affect the morphology and adhesion of rBMSCs.In the fiber membrane of A2 group(652±127nm diameter),the cells are colonized and spread,and the growth is better,showing irregular shape with more cell pseudopods.3.The proliferation of cells in the A2 group(diameter 652±127 nm)was better than that in the A1(diameter 1153±151 nm)and A3(diameter 271±116 nm).4.Osteogenic differentiation test showed that the fiber membrane with 652±127nm fiber diameter parameters had better osteoinductive ability.It was demonstrated that changes in the diameter parameters of the fiber membrane can affect the osteogenic differentiation of rBMSCs. |
