Preparation And Properties Study Of Gradual Gradient Aperture Osteochondral Scaffold

Objective: Preparation of SF-CS-nHAp gradual gradient aperture OC scaffold and complete its performance testing,in order to provide a more suitable bionic composite scaffold for the repair of OC defects.Methods: SF solution,CS solution and nHAp suspension with 2%,3%,4%concentration were prepared respectively.The same concentration of materials were mixed in equal proportion and divided into three groups: scaffold-1(2%),scaffold-2(3%)and scaffold-3(4%).The gradual gradient aperture OC scaffolds with different concentrations were prepared by centrifugation,vacuum freeze-drying and chemical cross-linking after three times of shaping.The general situation,porosity,water swelling rate,compression water swelling rate,hot water dissolution rate,water swelling rate after dissolution,mechanical properties,internal structure observation and pore size of the scaffolds in each group were detected,and the scaffolds with better overall performance were analyzed.ADSCs were extracted by collagenase digestion and identified by immunofluorescence and flow cytometry.The proliferation activity of ADSCs was detected by CCK-8 method.The effect of scaffold leach liquor on the proliferation of ADSCs was detected by CCK-8method.ADSCs were implanted into the scaffold to construct the scaffold-ADSCs complex.The proliferation of cells in each layer of the scaffold-ADSCs complex was detected by CCK-8 method.The distribution and morphology of cells in each layer of the scaffold-ADSCs complex were observed by nuclear fluorescence staining and scanning electron microscopy.Results: The scaffolds in each group are white,regular in shape,approximate to a cylinder,very light in weight,with a certain degree of compression resistance and elasticity.The porosity of scaffold-1,scaffold-2 and scaffold-3 were(91.30±3.35)%,(89.50±3.11)%,(82.58±1.47)%.The porosity of scaffold-3 was lower than that of the other two groups(P<0.05).The water swelling rates were(3218.53 ±84.37)%,(2573.83±132.51)%,(1969.50±90.01)%.There were significant differences among the groups(P<0.05).Compressed water swelling rates were(1587.27±63.09)%,(2379.53±77.04)%,(1989.53±118.90)%.Compared with the water swelling rate before compression,scaffold-1decreased significantly(P<0.05),while scaffold-2 and scaffold-3 did not change significantly(P>0.05).The hot water dissolution rates were(16.57±3.18)%,(15.03±2.17)%,(15.65±0.68)%.There was no significant difference among the groups(P>0.05).Regression analysis showed that the scaffold was completely dissolved in vitro,which required 65.85,60.92 and 73.86 weeks,respectively.The water swelling rates after dissolution were(4267.15±132.31)%,(3192.30±223.41)%,(2807.70±235.16)%.Compared with the water swelling rate before dissolution,the water swelling rate after dissolution of the scaffolds in the three groups increased significantly(P<0.05).The modulus of elasticity were(0.0955,0.1762,0.3468)MPa,respectively.SEM showed that the scaffolds in each group were honeycomb-shaped with regular pore shape and interflow.The pore distribution was gradually dense from the Chondrogenic side to the osteogenic side of the scaffolds,and the pore diameter was gradually reduced(P<0.05).The ADSCs extracted by collagenase digestion have a uniform morphology,a spindle-like shape,a vortex-like growth,and good cell proliferation activity.Flow cytometry showed that the positive rates of CD29,CD90 and CD44 were over 90%,while the positive rates of CD34,HLA-DR,IgG-Isotype control and Blank were less than 5%.The results of immunofluorescence were consistent with those of flow cytometry.The results of cytotoxicity test showed that the scaffold leach liquor had no significant effect on the proliferation of ADSCs in different time periods.The RGR values were above 90%,and the toxicity ratings were between 0 and I.The cells in the scaffold-ADSCs complex began to exhibit logarithmic proliferation after the third day of culture,and entered the plateau phase after the 11 th day.On the 5th-15 th day of culture,the cell proliferation activity of the complex into the Chondrogenic side was higher than that of the osteogenic side(P<0.05).After DAPI staining,the cells in each layer of the scaffold were evenly distributed,the shape and size of the nucleus were uniform,and there was no obvious abnormal staining and fragmentation of the nucleus.SEM observation showed that a large number of cells adhered to the pore walls of each layer of the scaffold-ADSCs complex.The cells were uniform in shape and size,closely arranged in long spindle shape,and no obvious cell disruption or morphological abnormalities were observed.Conclusion: In this study,a gradual gradient aperture OC scaffold was successfully prepared.The physical properties of the scaffold were good,consistent with the gradualgradient structure of the normal OC from the cartilage layer to the bone layer.In this study,we successfully extracted high-purity ADSCs and successfully constructed a scaffoldADSCs complex.By detecting the scaffold was non-cytotoxic and biocompatible.In summary,the SF-CS-nHAp gradual gradient aperture OC scaffold and the scaffoldADSCs complex prepared in this study are expected to be new biomimetic composite scaffolds for repairing OC defects.