| Objective:To prepare NGF/Col Ⅱ-SF-CS/Col Ⅰ-SF-CS-n HAp cartilage and subchondral bone double-layer scaffold,and implant the scaffold into rabbit cartilage defect with mosaicplasty technology to observe and evaluate the effect of repairing cartilage defects,so as to provide a new treatment strategy for articular cartilage defect repair in clinic.Methods: Firstly,Col Ⅰ and Col Ⅱ solutions with concentrations of 2%,3% and4% were prepared respectively,and mixed with SF,CS and n HAp solutions with concentrations of 2% prepared by our research group in the early stage according to the same proportion,The obtained upper scaffold(Col Ⅱ-SF-CS)was divided into three groups: scaffold 1(2%),scaffold 2(3%)and scaffold 3(4%),and the lower scaffold(Col Ⅰ-SF-CS-n HAp)was divided into three groups: scaffold 4(2%),scaffold 5(3%)and scaffold 6(4%).Col Ⅱ-SF-CS/Col Ⅰ-SF-CS-n HAp cartilage and subchondral bone double-layer scaffold was prepared by vacuum freeze-drying,physical bonding and chemical crosslinking after repeated shaping.The general condition,porosity,water absorption expansion rate,hot water dissolution rate,internal shape and pore size of the scaffold,energy spectrum analysis and ion distribution measurement of the scaffold were detected.Secondly,NGF sustained-release microspheres were prepared by emulsification solvent evaporation method and polymer alloy method,and the micro-morphology,particle size,drug loading,encapsulation efficiency and sustained-release effect of the microspheres were tested.Then,the microspheres were combined with the scaffold to prepare the Col Ⅱ-SF-CS/Col Ⅰ-SF-CS-n HAp bilayer scaffold carrying NGF sustained-release.Finally,30 New Zealand white rabbits were randomly divided into 3 groups with 10 rabbits in each group(Group A: blank control group;Group B: Col Ⅱ-SF-CS/ColⅠ-SF-CS-n HAp double-layer stent was implanted;Group C: NGF/Col Ⅱ-SF-CS/ColⅠ-SF-CS-n HAp double-layer scaffold was implanted),and the corresponding scaffold was implanted into the cartilage defect of rabbits in groups B and C combined with mosaicplasty technology.The rabbits in each group were observed at 4,8 and 12 weeks after operation respectively.The repair of cartilage damage was evaluated by ICRS cartilage repair score,HE,Alixin blue,type Ⅱ collagen immunohistochemical staining and Wakitani pathological score.Results:(1)Test of bracket performance: the upper layer of each bracket is light yellow,and the lower layer is white,which looks like a cylinder.It is extremely light in weight,elastic when touched,and does not crack after bending.The porosity of the upper scaffold in each group decreased with the increase of Col Ⅱ concentration(P<0.05).There is no difference in the porosity contrast between lower scaffold 4 and scaffold 5(P>0.05),while the porosity contrast between scaffold 6,scaffold 4 and scaffold 5 is obviously reduced(P<0.05).There was no difference between groups in the water absorption expansion rate of the upper stent(P>0.05).The water absorption expansion rate of the lower stent in each group had no difference between groups(P>0.05).After 6 weeks,there was no difference in the hot water dissolution rate among the groups of the upper stents(P>0.05),and the linear regression analysis showed that the complete dissolution of the upper stents took 63.08,61.02 and 61.40 weeks respectively.There was no difference in the hot water dissolution rate of the lower stent among groups(P>0.05),and the complete dissolution took 75.63,77.87 and 77.47 weeks respectively.The whole interior of each group of brackets is honeycomb-shaped,and all pores are connected with each other.There was no difference between groups in the average pore size of the upper stent(P>0.05).There is also no difference in the average pore size of the lower stent among groups(P>0.05),but the average pore size of the upper stent is significantly larger than that of the lower stent(P<0.05).Energy spectrum analysis shows that the upper layer of the prepared scaffold is mainly composed of Ca,O and Cl elements,while the lower layer is mainly composed of Ca,O and P elements.(2)NGF sustained-release microspheres: round shape,smooth surface,average particle size of about(25.87±6.54)μm,drug loading of 0.516μg/mg,encapsulation rate of 40.5%,obviously low sudden release rate of microspheres and high bioavailability of drugs.(3)Repair of cartilage defects: The scores of ICRS cartilage repair in groups A,B and C gradually increased with the postoperative time,and there were significant differences within and between groups(P<0.05),The scores of group C were significantly higher than those of the other two groups at each time point(P<0.05).HE staining: At 12 th week after operation,the repair tissue in group A was mainly fibrous tissue,group B was mainly fibrocartilage,and the reconstruction of subchondral bone was insufficient,while the reconstruction of osteochondral bone in group C was relatively complete,showing tidal lines.Alixin blue staining: In group A,12 weeks after operation,there was still obvious cartilage loss in the defect area,and the new tissue was negative.From 4 to 12 weeks after operation in Group B and Group C,the defect area was gradually filled with new tissue with time increasing,and the surface was gradually smooth.At each time point,the staining in Group C was deeper than that in Group B,and the boundary between osteochondral layer was more obvious.Immunohistochemical staining of type II collagen: In group A,12 weeks after operation,type II collagen was still not expressed in repair tissue;From 4 to 12 weeks after operation,the expression of type II collagen in group B and group C gradually increased with time,and the expression of type II collagen in repair tissue in group C was more than that in group B at each time point.The pathological scores of Wakitani in groups A,B and C gradually decreased with the postoperative time,and there were significant differences within and between groups(P<0.05).The scores of group C were significantly lower than those of the other two groups at each time point(P<0.05).Conclusion: In this study,Col Ⅱ-SF-CS/Col Ⅰ-SF-CS-n HAp bilayer scaffold and NGF sustained-release microspheres with good physical and chemical properties were successfully prepared,and it was confirmed that NGF/Col Ⅱ-SF-CS/ColⅠ-SF-CS-n HAp bilayer scaffold can better promote the repair of rabbit cartilage defects,and it is expected to become a new bionic composite scaffold for the repair of articular cartilage defects,providing a kind of clinical treatment of articular cartilage defects. |
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