Meniscus Biphasic Scaffolds Prompt Successful Whole Meniscus In Situ Regeneration In A Total Meniscectomy Model

Objective:a)Fabricating the meniscus biphasic scaffolds which both mimic meniscus microenvironment and meniscus aligned microstructure,and evaluate their physicochemical properties and biocompatibility;b)constructing the meniscal fibrochondrocytes and biphasic scaffolds composites,and assess their chondrogenic differentiation capacities either in vitro or in subcutaneous implantation at nude rat;c)evaluating their in situ meniscus regeneration and knee cartilage protection abilities using a biphasic scaffolds implanted in a total medial meniscectomy model of the New Zealand white rabbit;d)evaluating their in situ meniscus regeneration and knee cartilage protection abilities using a biphasic scaffolds implanted in a sheep medial meniscectomy model.Methods:a.On the one hand,fabricating the decellularized meniscal extracellular matrix(MECM)using the waterproof pulverization and differential centrifugation approach,on the other hand,producing the aligned PCL meniscal scaffold by using the 3D printing method,then acquiring the sheep meniscus 3D images by medical imaging technologies,lastly combining the vacuum perfusion and freeze drying methods to producing meniscus biphasic scaffolds;b.Using scanning electron microscopy and biomechanical machine to evaluate meniscal micro-morphology,tensile and compressive modulus and other physicochemical properties;c.Using biphasic scaffold adhesion assay,scaffold subcutaneous implantation experiment and in vivo angiogenesis assay to further confirm the biocompatibility,immunogenicity,material degradation and angiogenesis properties of the biphasic scaffolds.d)constructing the meniscal fibrochondrocytes and biphasic scaffolds composites,assess their chondrogenic differentiation capacities using Live/Dead staining,cellular matrix specific staining and immunofluorescence staining;constructing the GFP rat meniscal fibrochondrocytes and biphasic scaffolds composites,tracing GFP rat meniscal fibrochondrocytes chondrogenic differentiation capacities using cellular matrix specific staining and immunofluorescence staining in nude rat subcutaneous implantation;e)implanting the meniscal biphasic scaffolds in a total medial meniscectomy model of the New Zealand white rabbit model,and using macroscopic observation,histological staining and some other assessment to confirm their in situ meniscus regeneration and knee cartilage protection abilities at 3 months and 6 months after implantation;f)further creating the big animal model by implanting the meniscal biphasic scaffolds in a medial meniscectomy sheep(mimicking the human knee meniscus biomechanical environment),and using macroscopic observation,histological staining,X-Ray and some other assessment to confirm their in situ meniscus regeneration and knee cartilage protection abilities at 3 months after implantation.Results:Scanning electron microscopy results showed that biphasic scaffolds possess a good pore size and porosity,3D printed PCL scaffolds can greatly mimic the native meniscal collagen microstructure,biphasic scaffolds own good biocompatibility and biomechanical capacity,rat subcutaneous implantation experiment convinces the biphasic scaffolds display very low immunogenicity,meanwhile MECM degrade faster than PCL.Rabbit meniscal fibrochondrocytes and biphasic scaffolds composites in vitro culture result showed that the biphasic scaffolds could be favorable to maintaining cell alive,and could induce the meniscal fibrochondrocytes secreting the meniscal specific cellular matrix.The GFP rat meniscal fibrochondrocytes and biphasic scaffolds composites in nude rat subcutaneous implantation experiment showed that the tracing GFP meniscal fibrochondrocytes could still keep alive after 4 weeks implantation in nude rat,and perform the ectopic cartilage differentiation and form the meniscus-like tissue.Meniscal biphasic scaffolds displayed a better meniscal regeneration results than that of the meniscectomy group in terms of the macroscopic observation results in the total medial meniscectomy model of the New Zealand white rabbit,and similar to the autograft group.The histological staining results demonstrated that there was more meniscal specific cellular matrix secretion in the biphasic scaffolds group which similar to the native meniscus,and gradually secreted more and more mature following the time.The corresponding macroscopic observation results of the knee cartilage displayed that the biphasic scaffolds could effectively protect the knee cartilage,which similar to the autograft group.The knee degeneration was slighter in the biphasic scaffolds group than that in the meniscectomy group,which similar to the autograft group,and further confirmed the macroscopic observation results and the histological results.Meniscal biphasic scaffolds group displayed an obviously better meniscal regeneration results than that of the meniscectomy group in terms of the macroscopic observation results in the medial meniscectomy sheep model.The histological staining results demonstrated that there was some meniscal specific cellular matrix secretion in the biphasic scaffolds group.The corresponding macroscopic observation results of the knee cartilage displayed that the biphasic scaffolds could effectively protect the knee cartilage,which similar to the autograft group.The knee degeneration was slighter in the biphasic scaffolds group than that in the meniscectomy group,which similar to the autograft group,and further confirmed the macroscopic observation results and X-ray images.Conclusion:The meniscus biphasic scaffolds could well mimic both meniscus microenvironment and meniscus aligned microstructure,and possess a good pore size and porosity and biocompatibility;meniscal fibrochondrocytes and biphasic scaffolds composite constructs could perform the chondrogenic differentiation both in vitro and in subcutaneous implantation of nude rat.Meniscal biphasic scaffolds showed a better meniscal regeneration results in situ in the total medial meniscectomy model of the New Zealand white rabbit,could effectively protect the knee cartilage and prevent the knee osteoarthritis development.The regenerative results of the sheep model further convinced the repairing effects and the knee cartilage protection effects.The sheep model can well mimic the human knee biomechanical environment,therefore it could lay good scientific foundation for further clinic trials.