Experiment On The Articular Cartilage Defects Repairment By The Nanofiber Scaffold Prepared With Collagen, Hyaluronic Acid And Chondroitin Sulfate

Objective: To evaluate the physicochemical properties of the tissue-engineeredcartilage nanofiber scaffold prepared in our lab and the effects of articularcartilage defects repairment by this scaffold.Methods:(1) The nanofiber scaffold based on Collagen-Hyaluronic acid–Chondroitin blends were electrospun to mimic the componential and structuralaspects of the cartilage extracellular matrix. The electrostatic spinning solutionwas prepared by dissolved Collagen, Hyaluronic acid and Chondroitin sulfatein the mixed solvent at optimal ratio, and the mixed solvent were2,2,2-Thifluoroethanol and water. The morphology of the scaffold wasobserved by scanning electron microscopy, and water absorption, contactangle, degradation rate, adhesion rate and proliferation rate were all detected.According to these physicochemical properties, the nanofiber scaffold whichhas good morphology, excellent physicochemical and biological properties wasselected for the further study in vitro and in vivo.(2) Chondroetyes wereobtained from one week-old New Zealand rabbits costal cartilage. And the cellswere subcultured and proliferated in vitro. Then chondroetyes were implantedinto the scaffold and cultured for7days and14days. At the predetermined timepoint, the samples were fixed with4%formalin and embedded with paraffin.The growth and extracellular matrix secretion of chondroetyes on the scaffoldwere observed through Hematoxylin-eosin, safranin-O, aggrecan and type IIcollagen immunohistochemical staining.(3) The rabbit model of knee cartilagedefect was made and divided into3groups. In the experimental group: scaffoldwith chondrocytes implanted into the defects. In scaffold group: only scaffold implanted into the defects. In sham group: the defects without any treatment.Then the knee joints were harvested at8and12weeks, the repairments effectswere evaluated through gross morphology, histologically andimmunohistochemically. Results:(1) Nanofiber scaffold was prepared bycartilage extracellular matrix through electrostatic spinning successfully in ourstudy. Through the physicochemical properties of the scaffold, the opitmalpreparation parameters were selected. The temperature was25-30℃, thevoltage was15-22Kv, the needle model was the6th needle model, the distancebetween the receiving device and needle was10-12cm, the speed of theinjection pump was9ml/h-11ml/h, the concentration of the solution was80-120mg/ml and the opitmal ratio of the three materials was6-8:1:1-2.Thediameter of the scaffold was126.5±23.3nm-374.7nm±14.1nm, and expressedexcellent hydrophilicity, degradation and biocompatibility.(2) TheHematoxylin-eosin staining result showed that chondroetyes grew on thescaffold well in vitro. And the results of safranin-O staining, aggrecan and typeII collagen immunohistochemical staining were all positive, which indicatedthat chondroetyes could grow and secrete extracellular matrix.(3) The defectswere obviously repaired in the experimental group at8th week aftertransplantion. After12weeks, the repairment was excellent in the experimentgroup through the gross morphology. The histologically andimmunohistochemically results showed the regenerated tissue closelyconnecting with subchondral bone and the fuzzy boundary with normal tissue.The cartilage lacuna in the regenerated tissue was similar with normal cartilagelacuna and confirmed that the regenerate tissue was mainly hyaline-likecartilage. Compared with the experiment group, the defects in the scaffoldgroup was not been repaired incompletely, while in the sham group, the defectswere still obviously and the regenerate tissue was mainly fibrocartilage at8weeks and12weeks. Conclusions:(1) The tissue-engineered cartilagenanofiber scaffold could be prepared by cartilage extracellular matrix with electrostatic spinning method. The optimal physicochemical and biologyproperties could be adjusted through the preparation parameters.(2)Chondrocytes implanted in the scaffold could adhere, proliferate and secrete theextracellular matrix in vitro.(3)The novel tissue-engineered cartilage scaffoldcombined with chondrotyes could repair the articular cartilage defects almostcompletely.