The Study Of A Novel Decellularized Cartilage Scaffold Constructed With Wharton’s Jelly To Promote Cartilage Injury Repair

Objective: To prepare a novel decellularized cartilage scaffold based on umbilical Wharton’s jelly,investigating its effect in constructing tissueengineered cartilage in vitro after loading human bone marrow mesenchymal stem cells(h BMSCs)and repairing cartilage defects in knee joints of SD rats.Methods :(1)To prepare a novel decellularized wharton’s jelly extracellular matrix(d WJECM)using chemical freeze-thaw(CFT)method and to observe the gross morphology and microstructure.The general physical properties such as pore size,porosity,density,swelling rate,and degradation rate of the scaffolds were determined and analyzed in comparison with d WJECM prepared by digestion freeze-thaw(DFT)method.(2)The h BMSCs were used as seed cells and inoculated in d WJECM prepared by different methods to construct tissue-engineered cartilage in vitro.The biocompatibility of the scaffolds was tested using CCK8 cell proliferation and cell live-dead staining experiments.The growth and chondrogenic differentiation of h BMSCs in the scaffold were evaluated by HE Staining,Safranine O-Fast Green Staining,Toluidine Blue Staining,Immunohistochemistry of Type II Collagen and q RT-PCR.(3)An animal model of cartilage defects in the knee joint of SD rats was constructed,and the scaffold materials prepared by different methods were implanted.The cartilage defect specimens of SD rats were taken at 6 and12 weeks after surgery for gross observation,Micro-CT,HE Staining,Safranine O-Fast Green Staining,Toluidine Blue Staining,Immunohistochemistry of Type II Collagen to evaluate the rehabilitation of cartilage defect areas.Results:(1)Both d WJECMs presented a three-dimensional porous,reticulated interlaced structure,the pores were loosely arranged,and the CFT-d WJECM was more tightly arranged.The histological staining showed that the original cells of the Wharton’s jelly were basically removed,and the staining was positive in all the Safranine O-Fast Green Staining,Toluidine Blue Staining,and Sirius-red staining,which indicated that the scaffolds prepared by both methods contained glycosaminoglycan and collagen.In terms of general physical properties,CFT-d WJECM showed smaller pore size and porosity,higher density,swelling rate and degradation rate.(2)The results of tissue engineered cartilage constructed in vitro showed that the cell proliferation in both complex systems was excellent,and the cell viability rate was above 90%,which indicated that d WJECM has a great biocompatibility.Histological staining(HE Staining,Safranine O-Fast Green Staining,Toluidine Blue Staining,Immunohistochemistry of Type II Collagen)showed that the CFTd WJECM group presented stronger positive expression than the DFTd WJECM group with the increase of time,q RT-PCR results showed that COL2A1,SOX9,and ACAN expression in the CFT-d WJECM group at day 7 and 21 were higher than those in the DFT-d WJECM group(P <0.05),which indicated that CFT-d WJECM loaded h BMSCs produced more glycosaminoglycan,type II collagen during chondrogenesis.(3)After implantation of d WJECM into the SD rat knee cartilage defect model,the defect area gradually acquired regenerative cartilage repair over time.Micro-CT results showed that all three groups had some degree of subchondral bone repair,with the CFT-d WJECM group being the best,and the morphology was close to the surrounding normal bone trabeculae.The results of histological staining(HE Staining,Safranine O-Fast Green Staining,Toluidine Blue Staining,Immunohistochemistry of Type II Collagen)showed that the defect model implanted with d WJECM could produce more type II collagen and glycosaminoglycan during the repair process,and the CFT-d WJECM group had the best regeneration and repair.Conclusion :(1)The d WJECM scaffolds are equipped with the general performance requirements of tissue-engineered cartilage scaffolds,which can promote the chondrogenic differentiation of h BMSCs in vitro and the repair of knee cartilage defects in SD rats in vivo,and provide a novel idea for the selection of cartilage tissue engineering scaffold materials.(2)In comparison,the d WJECM scaffold prepared by chemical freeze-thaw method can achieve better cartilage repair,which is a more suitable method for decellularization of d WJECM scaffold.