Experimental Study On Effect Of HUCMSCs Combined With New Walton’ Sjelly Oriented Scaffold In Repairing The Osteochondral Defect In Rat

ObjectiveWe use Wharton’s jelly in human umbilical cord as raw material,then combined it with chondroitin sulfate to create a new composite scaffold,the performance of the stent was examined and the tissue-engineered cartilage was co-cultured with isolated human umbilical cord mesenchymal stem cells.To explore the effect of the scaffold as tissue engineering cartilage,we transplanted human umbilical cord mesenchymal stem cells with new composite scaffold,and transplant them into the articular cartilage defects of SD rats.We evaluated the Wharton’s jelly chondroitin sulfate composite scaffold’s possibility in repairing articular cartilage defects at 4 weeks and 12 weeks postoperatively.Methods1.In vitroThe human umbilical cord mesenchymal stem cells were prepared by type II collagenase digestion method,and the cells were identified by immunofluorescence.The cartilage differentiation was induced for 21 days.The separated Walton glue was prepared by mechanically pulverizing,low permeability,enzymatic digestion,repeated freezing and thawing and differential centrifugation.The cells were prepared through removing the cells from the nano-sized Wharton suspension by means of pre-freezing,freeze-drying Forming,to prepare a pure Walton rubber stent.A new type of composite scaffold was prepared by crosslinking the Wharton gel and chondroitin sulfate by physical and chemical methods.The structure was observed by paraffin section,HE staining and electron scanning electron microscopy.The composition was tested by detection of toluidine blue staining.The mechanical properties were measured using an Instron biomechanical detector.2.In vivo A rat model of defective articular cartilage was made by SD rats.A bone flap with a diameter of 2 mm and a depth of 2 mm was made in the defect area.There was a blank control group,a single Wharton + hUCMSCs group,a composite scaffold + hUCMSCs group.Each group had 10 SD rats.The repairing condition was observed at 4 weeks and 12 weeks after the observation,respectively,using gross observation,HE staining,toluidine blue staining,safranine green staining,type II collagen immunohistochemical method.Results1.In vitro(1)Isolation and identification of human umbilical cord mesenchymal stem cells: Human umbilical cord mesenchymal stem cells were successfully isolated and identified by immunofluorescence to express CD44 and CD90 on mesenchymal stem cells.Cultured cell pellets were stained with alan blue.(2)Preparation of scaffold: The prepared wortan sulfate chondroitin scaffold composite scaffold has a three-dimensional orientation structure with a porosity of 90% or more,which meets the requirement of cartilage tissue scaffold and which was rich in chondrocyte extracellular matrix glycosaminoglycans,The mechanical test results show that the scaffold has anti-compression performance,and the Young’s modulus of the scaffold was close to that of normal rat cartilage.2.In vivo(1)Gross observation and scoring results: 12 weeks after operation,the colours of the composite scaffold +hUCMSCs group was very close to normal cartilage tissue,the repairing surface was flat,and had a good integration with surrounding normal tissue.The simple wharton jelly scaffold+hUCMSCs group and blank control group showed that cartilage defects were basically filled,the repairing surface was less smooth,and did which not have a good integration with surrounding cartilage tissue.(2)Histological examination: After detecting of toluidine blue staining,Safranine O-Solid Green Dyeing and Type II collagen immunohistochemistry in 4 weeks and 12 weeks,the composite scaffold +hUCMSCs group had higher content of glycosaminoglycans and typeⅡcollagen,the resulting cartilage was more consistent with normal cartilage,and the histological scores were significantly higher(P<0.05),compared with the simple wharton jelly scaffold+hUCMSCs group and blank control group.Conclusions1.Umbilical cordon is isolated from human umbilical cord mesenchymal stem cells with the potential to become cartilage.Preparation of human umbilical cord Wharton chondroitin sulfate composite scaffold for three-dimensional porous structure,physical properties and mechanical properties consistent with cartilage tissue engineering scaffold requirements.The scaffold has good biocompatibility,and the stem cells are well grown on the scaffold after growth.2.The effect of the new type of Wharton chondroitin sulfate composite scaffolds on the repair of knee osteochondral defects in SD rats was good.The scaffold provides a new idea for the selection of cartilage tissue scaffold materials.SignificanceIn this study,we examined the feasibility of the new type of Wharton chondroitin sulfate scaffold as cartilage tissue engineering scaffold in vivo and in vitro,and explored new treatments for the treatment of cartilage injury.