3D-printed Poly(Epsilon-caprolactone)Scaffold Combined With MECM-based Hydrogel For Tissue-engineered Meniscus

Objective:Meniscus injuries are very common in the knee joint.Treating a damaged meniscus continues to be a scientific challenge in sport medicine because of its poor self-healing potential and few clinical therapeutic options.The aims of this research are to:a)investigate the ability of meniscus extracellular matrix(MECM)-based hydrogel to promote meniscus fibrochondrocytes(MFCs)proliferation and maintain cell phenotype;b)fabricate 3D-printed composite scaffolds by MECM-based hydrogel and PCL;c)evaluate the biocompatibility and biomechanical properties of 3D-printed composite scaffolds in vitro;d)study the meniscus regeneration and cartilage protection using 3D-printed composite scaffolds in rabbit meniscectomy model.Method:a)five concentrations of MECM-based hydrogel were prepared by decellularized meniscus extracellular matrix and alginate with different ratios(0%,0.5%,1%,2%,4%);b)The cell proliferation on different concentrations of MECM-based hydrogels was studied by live/dead staining,CCK-8 test and DNA quantification;redifferentiation of the passaged meniscus fibrochondrocytes was determined by biochemical assays for GAG and collagen as well as RT-PCR;c)3D-printed composite scaffolds were manufactured from PCL and MECM-based hydrogel,by fused deposition modeling and surface modification techniques.The mechanical properties and biocompatibility of composite scaffolds were evaluated by electroforce biodynamic testing system(Bose),CCK-8 test,immunological rejection experiment respectively;d)3D-printed composite scaffolds loaded with muniscus fibrochondrocytes(Group B)were implanted into the medial knee meniscus of New Zealand rabbits that had undergone total meniscectomy in comparison with sham group(group A),cell-free 3D-printed composite scaffolds group(group C),3D-printed PCL scaffolds group(group D)and the empty control group(group E).Meniscus regeneration results were assessed macroscopically,histologically and mechanically,imageologically after 3 and 6 months implantation.Results:a)The results of live/dead staining,CCK-8 test and DNA quantification showed that all concertrations of MECM-based hydrogel might promote fibrochondrocytes proliferation,with 2%group having the greatest effect.Biochemical assays and RT-PCR results indicated that all concertrations of MECM-based hydrogel could maintain fibrochondrocytes phenotype,with 2%group representing the strongest effect;b)CCK-8 toxicity test and immunological rejection results demonstrated the 3D-printed composite scaffolds possessed good biocompatibility;c)The biomechanical assessment confirmed that the compressive and tensile modulus of the composite scaffolds were closer to that of native meniscus than that of PCL scaffolds;d)Compared with group C,D and E,group B represented markedly better gross appearance and cartilage protection.Histological scores were better for regenerated meniscus as well as articular cartilage in group B than the groups C,D,E.Group B displayed a significantly better Knee MRI scores(WORMS)compared to the other three groups at both 3 and 6 months time points.Mechanical testing and biochemical assessment demonstrated better mechanical and biochenmical properties of neomeniscus tissue in group B,in comparison with the other three groups.Conclusion:MECM-based hydrogels provide a suitable microenvironment for seeded cells,and promote the MFCs proliferation and redifferentiation,with 2%group having the strongest effect.3D-printed composite scaffold has good biomechanical performance and biocompatibility,and can promote meniscus regeneration and protect articular cartilage.In conclusion,it is is a potentially good candidate for meniscal tissue engineering in the future.