Experimental Study Of Tissue Engineering SF-CS/SF-CS-nHA Biphasic Scaffold Combined With PRP For Repairing Rabbit Knee Osteochondral Defect

ObjectiveThe tissue-engineered SF-CS/SF-CS-nHA scaffold and PRP gel were constructed in vitro and the animal model of femoral condylar cartilage defect was established.In vivo experiments: A total of 48 adult New Zealand white rabbits were divided into 4 groups of 12,group A: osteochondral defect model were filled with tissue engineering SF-CS/SF-CS-nHA scaffold and PRP gel;Group B : osteochondral defect model were filled with SF-CS/SF-CS-nHA scaffold and PRP gel;Group C: osteochondral defect model filled with PRP gel;Group D: osteochondral defect model were not filled any materials.At 4,8,and 12 weeks postoperatively,the micro CT scanning and immunohistochemical stainingwere performed.And to provide some theoretical basis for the preparation and osteochondral repair and more matching osteochondral repair materials.Methods: this experiment is made up of four parts1.Construction of BMP2 gene overexpression lentiviral vector and culture and identification of BMSCsBMP2 gene overexpression lentiviral vector and in vitro BMSCs isolation,culture and identification were constructed by in vitro experiments.2.Preparation of SF-CS-SF-CS-nHA Biphase Scaffold and PRP and Its Effect on BMSCsThe SF-CS / SF-CS-nHA biphasic scaffold and PRP gel were constructed in vitro.The morphology,adhesion and proliferation of BMSCs were detected by scanning electron microscopy,MTT proliferation assay,ALP assay,QPCR detection and Western Blot detection.Bone and other physical and chemical properties of the composite bone material SF-CS / SF-CS-nHA on BMSCs cell growth and osteogenesis and cartilage ability.3.Establishment and evaluation of animal model of femoral condylar cartilage defect in rabbit knee jointThe rabbit osteoarticular cartilage defect model with a diameter of 5mm and a depth of 3mm was constructed by providing a suitable method for studying the osteochondral defect model.4.Establishment of tissue engineering SF-CS/SF-CS-nHA scaffold combined with PRP in repairing rabbit knee osteochondral defectThe animal model of femoral osteochondral defect in New Zealand rabbits was successfully implanted in group A: the tissue-engineered SF-CS/SF-CS-nHA scaffold and PRP gel were filled with osteochondral defect model.Group B: osteochondral defect model were filled with SF-CS/SF-CS-nHA scaffold and PRP gel;Group C: osteochondral defect model filled PRP gel;D group: osteochondral defect model were not filled with any materials.At 4,8 and 12 weeks postoperatively,the osteochondraldefects were examined by gross observation,micro CT scanning quantitative analysis and immunofluorescence staining.Results1.Construction of BMP2 gene overexpression lentiviral vector and culture and identification of BMSCsThe lentiviral vector plasmid carrying BMP2 gene was successfully constructed in the experiment.The morphology of BMSCs was homogeneous by density gradient centrifugation and whole bone marrow culture.After induction of osteogenesis,alizarin red staining can be seen calcium nodules;type I collagen immunohistochemistry was positive;ALcian blue staining was positive,indicating that the cells have the ability to become cartilage.2.Preparation of SF-CS-SF-CS-n HA Biphase Scaffold and PRP and Its Effect on BMSCsIn this experiment,the method of preparing SF-CS/SF-CS-nHA scaffold is simple,and the two-layer single-phase stent is tightly bonded to improve the problem of easy separation of the double stent.The prepared stent can be observed Layer of white sponge-like material,scanning electron microscopy to observe the size of the stent appropriate.At the same time,the results of scanning electron microscopy and alizarin red staining showed that the biocompatibility of cells and scaffolds was good,which was beneficial to cell adsorption and proliferation,and could promote the differentiation of BMSCs into the target cells.3.Establishment and evaluation of animal model of femoral condylar cartilage defect in rabbit knee jointBy constructing a round osteochondral defect model about 5 mm in diameter and 3 mm in depth on the femoral condyles of rabbit knee,it provides a more suitable method for studying the model of osteochondral defect.4.Establishment of tissue engineering SF-CS/SF-CS-nHA scaffold combined with PRP in repairing rabbit knee osteochondral defectIn the gross observation,there was no collapse in the articular surface of the group A,and the nasopharyngeal tissue was close to the normal tissue within 12 weeks postoperatively.Most of the nasopharyngeal tissues in group B were close to normal tissue and no sag;group C had some new tissue on the joint surface,There were the existence of defects in group D.The results showed that the best effect was A group,the rest were B,C,D group.Micro CT scaningshowed a good repair of osteochondral in group A.Histological observation showed that the newborn tissues began to grow in the A,B and C groups at 4 weeks postoperatively.After 8 weeks,the scaffolds were degraded and the new tissues grew at the edge of the scaffolds and defects.There were a lot of fibrous cartilage on the 12 weeks afteroperation.There were always defects in Group D.Immunofluorescence staining showed that type?collagen staining and type ? collagen staining were positive in group A,while those in group B,C and D were weakly positive.ConclusionTissue-engineered SF-CS/SF-CS-nHA biphasic scaffolds have better and integrated repair of osteochondral defects,especially after composite BMSCs,which is ideal for repairing complexes of bone and cartilage defects material.