The Research Of Decellularized Extracellular Matrix Load IPFP-SC To Repair Rabbit Osteochondral Defect

Part 1.Preparation and experimental study of rabbit osteochondral scaffolds Objective : A variety of rabbit decellularized osteochondral scaffolds were designed.Based on the decellularized effect,composition,structure and biomechanics of these scaffolds,the osteochondral complex attributed to tissue were obtained.Methods : Using the physical,chemical and enzymatic methods,the rabbit femoral trochlear joint and the rib were decellularized to obtain the integrated osteochondral scaffold.According to the hyaline cartilage,calcified cartilage,subchondral bone three layers of structure,the osteochondral tissues of the rabbit femur and the rib were separated and crushed,and each layer of particle was decellularized,re-cross-linked to prepare stratified osteochondral scaffold bio-derived from the joints and rib.The integrated osteochondral scaffolds were stained using HE,toluidine blue,Sirius red,Safranin,and the residual DNA content was determined by fluorescence colorimetry.which verified the degree of decellularization.The analysis of collagen and glycosaminoglycan(GAG)in the three layers of particles was quantitatively carried out by amino acid analyzer and fluorescent colorimetric method.The crystalline form of hydroxyapatite was characterized by XRD,compared with natural osteochondral tissue.Physical parameters such as density,porosity,pore size and water absorption of the three kinds of scaffolds were measured.Then,the mechanical properties of the three scaffolds such as elastic limit,strength limit,elastic modulus and brittleness were tested by tensile and torsion mechanical testing machine which were also in comparison with natural osteochondral tissue.Results : The decellularized level is more thorough using this method.Compared with natural osteochondral tissue,about 20-30% of the amino acids and 30-40% of the GAG are lost after the decellularized process,but the inorganic substance is contained.The density of stratified scaffolds was smaller in relation to that of integrated scaffold.While porosity,pore size and water absorption were greater.Compared with natural osteochondral tissue,the mechanical properties of both scaffolds are poor under the dry or wet conditions,while those of the stratified scaffolds were worse.Conclusions : Besides the loss of organic components,the decellularized effect of the combined method is better.Compared with natural tissue,the mechanical properties of both integrated and stratified scaffolds have declined in various degree.It is crucial that theintegrity of structure(especially the ultrastructure)and components have an impact on the biomechanical properties.Part 2.In vitro study on decellularized osteochondral scaffolds loaded with IPFP-SC Objective : The rabbit infrapatellar fat pad mesenchymal stem cells(IPFP-SC)were isolated and identified.The biocompatibility and cytotoxicity of osteochondral scaffolds were tested.And the effect of composition and structure of osteochondral matrix on the osteogenic and chondrogenic differentiation of IPFP-SC were investigated.On account of decellular technology,the bio-3D printing was preliminary researched.Methods: IPFP-SC was isolated and purified using collagenase digestion and adherent culture.Phenotypes on cell surface were identified by immunofluorescence(IF)and fluorescence-activated cell sorting(FACS),and then the ability of osteogenic,chondrogenic and adipogenic differentiation were carried out.The differences between normal and OA rabbit IPFP-SC in proliferation,migration,apoptosis and chondrogenic differentiation were shown.The biocompatibility and cytotoxicity of various kinds of decellularized osteochondral scaffolds were verified using direct inoculation,direct implantation and soak solution culture method.The biomimetic structures of bone and cartilage were measured,and the induced effects of IPFP-SC were examined on the direction of osteogenic and chondrogenic differentiation by bone and cartilage tissue composition and biomimetic structures.Using micro-CT scanning and Sirius stained serial section digital imaging,the structure data of rabbit natural osteochondral tissue were obtained.The STL format file was formed and the 3D printing using the method of decellularized matrix was preliminarily discussed.Results:The expression of CD73+?CD90+?CD105+?CD 44+?CD34-?CD45-?HLA-DRwas confirmed by FACS and IF,and the cells were differentiated into osteoblasts,chondrocytes and adipocytes.Compared with healthy IPFP-SC,there was no significant difference in proliferation of IPFP-SC from OA individuals.However,the capacity of migration and chondrogenic differentiation was weaker.There was no cytotoxicity and good biocompatibility in all kinds of scaffolds.The differentiation of IPFP-SC into osteoblasts and chondrocytes was promoted by the structure and composition of osteochondral tissue.According to the structure data of natural osteochondral tissue,ideal print was produced based on STL file.Conclusions:The isolated cells were confirmed to be MSC.The repair of cartilage was affected,attributed to the weaker abilitis of migration and chondrogenic differentiation from OA individuals.The differentiation of IPFP-SC into osteoblasts and chondrocytes were affected by both the biomimetic structures and components of the decellularized scaffolds,which was the foundation of IPFP-SC induced in vivo.KEY WORDS : mesenchymal stem cells,infrapatellar fat pad,decellularized,osteogenic,chondrogenic,in vitro osteochondral tissue.According to the structure data of natural osteochondral tissue,ideal print was produced based on STL file.Conclusions:The isolated cells were confirmed to be MSC.The repair of cartilage was affected,attributed to the weaker abilitis of migration and chondrogenic differentiation from OA individuals.The differentiation of IPFP-SC into osteoblasts and chondrocytes were affected by both the biomimetic structures and components of the decellularized scaffolds,which was the foundation of IPFP-SC induced in vivo.Part 3.In vivo study on decellularized osteochondral scaffolds loaded with IPFP-SC to repair rabbit osteochondral defects Objective:In order to investigate the effects of three tissue engineering methods on the repair of osteochondral defects,these kinds of decellularized osteochondral scaffold loaded with IPFP-SC were respectively implanted.Methods: 42 cases of standard rabbit femoral trochlear joint osteochondral defect model were established.The decellularized joint integrated osteochondral scaffolds,decellularized joint stratified osteochondral scaffolds and decellularized rib stratified osteochondral scaffolds,were divided into three groups,each groups were divided into 1,2sub-groups in presence or absence of IPFP-SC.There were 6 groups in addition to blank group,which were randomly assigned to implanted osteochondral defect model.These rabbits were sacrificed at 1 month,3 months and 6 months after operation.Generally,it was observed that the repaired surface of cartilage and subchondral bone,paraffin sections were stained with HE,Sirius red,Safranin,toluidine blue staining.The results were shown that the effect of repair was effected using the various tissue engineering methods.Results:The overall repair effect of IPFP-SC group was better than that of simple scaffold group,while the blank group was the worst.The cartilage repair effect of stratified scaffold group was better than that of the integrated scaffold group,However,the result on subchondral bone repair was opposite.After one month of operation,there was no significant difference among the groups aside from the blank group.Nevertheless,after 3and 6 months,the differences were most obvious among the groups including the blank group.Conclusions:All of three tissue engineering methods could play a certain role in the repair of osteochondral defects,of which the stratified scaffold is more capable of repairing cartilage and the integrated scaffold is more capable of repairing the subchondral bone.