Experimental Study On Reconstruction Of Rabbit Medial Meniscus With PCL/PE-m-PAAs-GO Hybrid Scaffold

Objective:This study aimed to fabricate the Poly ε-polycaprolactone/phosphate-mpoly(amino acids)-graphene oxide(PCL/PE-m-PAAs-GO)meniscus scaffold by 3D-printing the blending polycaprolactone(PCL)with the powder of poly(phosphate amino copolymer)-graphene oxide composite in dichloromethane.The biocompatibility and fibrocartilage induction activity of the polymer scaffold were evaluated,and the ability of the polymer scaffold to promote the regeneration of medial meniscus was evaluated by loaded with BMSCs.Materials and Methods:(1)Preparation and characterization of scaffolds:PAAs were prepared by melt copolymerization of aminocaproic acid,aminobutyric acid and caprolactam.After the ring opening of cyclic phosphate(CP),the above amino acid monomers were polymerized to fabricate PE-m-PAAs.During the process of PAAs fpolymerization,0.1%,0.5%and 1.0%graphene oxide were added to prepare the composite.The polymer was characterized by infrared spectroscopy,X-ray photoelectron spectroscopy and thermogravimetric analysis.Then the artificial composite meniscus scaffold was prepared by blending the polymer with polycaprolactone in the dichloromethane and for later 3D printing.The morphology and hydrophilicity of the composite meniscus scaffold were evaluated by scanning electron microscopy and water contact angle test.(2)The biological properties of scaffold materials in vitro:BMSCs were extracted,isolated and amplified,and identified by immunofluorescence staining of CD44 and CD45.The multi-directional differentiation of BMSCs was identified by three-line induction and staining.The cytotoxicity was detected by CCk-8.The biocompatibility of BMSCs was evaluated by Live/Dead staining,phalloidin/DAPI staining and scanning electron microscopy.The materials were co-cultured with BMSCs and were induced to differentiate into fibrocartilage,and RT-qPCR was used to detect the expression level of fibrocartilage related genes on the cell scaffold complex,and enzyme linked immunosorbent assay(ELISA)was used to evaluate the biological performance of the scaffold in promoting the differentiation of BMSCs into fibrochondrocytes.The best mass fraction of PCL/PE-m-PAAs-GO meniscus scaffold was defined through the above experiments.(3)Biocompatibility and regeneration of scaffold materials in vivo:New Zealand white rabbits were used as experimental animals to establish the model of medial meniscus defect.They were divided into sham operation group,PCL/PE-m-PAAsGO+BMSCs group,PCL/PE-m-PAAs-GO group,PCL group and blank group.The animals were sacrificed at 12 and 24 weeks after operation.The meniscus of each group was analyzed by gross specimen,histological staining,immunohistochemical staining and imaging analysis,and the corresponding semi-quantitative scores explore the feasibility of repairing the damaged meniscus structure and function with PCL/PE-m-PAAs-GO scaffold.Results:(1)The results of FTIR and XPS analysis showed that the phosphoramide bond was formed by hydrolysis of cyclophosphate reacted with PAAs,which indicated that the phosphate bond was successfully introduced into the main chain of PAAs.The results of thermogravimetric analysis indicated that the addition of graphene oxide improved the thermal stability of the copolymer.Raman spectrum analysis shows that D(Alg)and G(E2g)bands of graphene structure exist in the polymer.The results of SEM showed that the surface roughness increased with the introduction of PE-mPAAs-GO.The results of water contact angle test showed that the hydrophilicity increased with the introduction of PE-m-PAAs-GO,but decreased with the increase of graphene oxide polymerization.(2)Fluorescence staining of CD44 and CD45 confirmed that BMSCs were successfully extracted by whole bone marrow adherent method and could differentiate into osteogenic,chondrogenic and adipogenic cells under suitable conditions.The results of live death staining and CCK-8 showed that BMSCs could adhere to the surface of composite scaffolds well,indicating its good cell safety;the cell adhesion degree of PCL/PE-m-PAAs-GO polymer was proportional to the mass fraction of GO,which was better than that of pure PCL scaffolds.The results of phalloidin/DAPI staining and scanning electron microscope showed that BMSCs could adhere to the scaffold surface better than PCL.The BMSCs adhered to the scaffold in PCL/PE-mPAAs-0.5%-GO group showed better cell morphology.RT-qPCR results showed that the gene expression levels of Col Ⅰ,Col Ⅱ,Aggrecan and Sox9 in PE-m-PAAs-GO group were higher than those in PCL group on the 7th and 14th day(P<0.05).PCL/PE-m-PAAs-1.0%GO had the highest gene expression level on day 7(P<0.05),while PCL/PE-m-PAAs-0.5%GO group had the best gene expression level on day 14(P<0.05).ELISA results showed that the secretion of Col Ⅰ,COL Ⅱ and GAG in PCL group was the lowest at each time point(P<0.05).PCL/PE-m-PAAs-1.0%GO on the 7th day was higher than that in PCL/PCL/PE-m-PAAs-0.5%GO group,but only CoLⅡ had significant difference(P<0.05).On 14th day,the secretion of Col I and GAG in PCL/PE-m-PAAs-0.5%GO group was higher than that in other groups(P<0.05),while the secretion of COL Ⅱin PCL/PE-m-PAAs-1.0%GO group was lower than that in PCL/PE-m-PAAs-1.0%go group,but the difference was not statistically significant(P>0.05).Therefore,PCL/PE-m-PAAs-0.5%go group was selected as the follow-up meniscus repair experimental group.(3)PCL/PE-m-PAAs-GO composite scaffolds have good biocompatibility and no observed adverse reactions.The results of gross specimen,histology and biomechanics of regenerated meniscus confirmed that PCL/PE-m-PAAs composite scaffold can promote the repair and regeneration of medial meniscus defect.The repair effect of PCL/PE-m-PAAs-0.5%GO combined with BMSCs is better than that of single scaffold,which has better ability of meniscus tissue integration.Conclusion:PCL/PE-m-PAAs-GO scaffolds can be successfully prepared by melt copolymerization combined with PCL blending 3D printing.The hydrophilicity and surface morphology of PCL/PE-m-PAAs-GO scaffolds are better than those of PCL scaffolds.In this study,PCL/PE-m-PAAs-GO scaffold combined with BMSCs under certain induction conditions can promote the differentiation of BMSCs into fibrocartilage and secrete meniscus related extracellular matrix.PCL/PE-m-PAAs0.5%GO group has the best effect.The repair experiment of medial meniscus defect in rabbits showed that the scaffold constructed by PCL/PE-m-PAAs-0.5%GO and BMSCs could effectively promote the repair and regeneration of injured meniscus and delay the degeneration of articular cartilage,and it was better than PCL/PE-m-PAAs0.5%GO and PCL scaffold alone.