| Background:The treatment of articular cartilage and osteochondral defects has been a problem in orthopedic surgery up to now.The defects may be caused by trauma,osteoarthritis and other related diseases.Because cartilage has a limited capacity for self-repair and numerous strategies currently in clinical treatment inculding microfracture techniques,subcondral drilling, periosteal or perichondrial grafts,transplantation of osteochondral autografts or allografts,many shortcomings limit the repair procedure and compromise long-term results.Tissue engineering of cartilage and osteochondral constructs might be regarded as one with the biggest potential to make a significant clinical impact in the future.Tissue engineering include three main fators:cells,scaffolds and cytokine.Bone mesenchymal stem cells have exhibit wide differentiation potential including cartilage and bone differentiation.Because of the removal of the xenogeneic or allogenic cellular antigens and preservation of most of the structural and functional proteins that constitute the ECM,biologic scaffolds derived from decellularized tissue and organs have been successfully used in tissue engineering.Furthermore,many kinds of decellularized scaffolds,including human dermis,porcine SIS,procine heart valves,and porcine urinary bladder, have received approval for use in humans.However,to our knowledge,little research has investigated cartilage ECM-derived biomaterials for use as scaffolds for cartilage tissue engineering.Also,cartilage decellularization seems difficult, because cartilage is too compact to allow efficient penetration of solutions used in the decellularisation procedure.The goal of present study was to develop scaffold that can be used in tissue-engineered cartilage and osteochondral constructs,and to investigate the feasibility of tissue-engineered cartilage and osteochondral constructs in vitro and in vivo using BMSCs and new scaffold.The study was divided into three main parts.The first part includes development and characteristic of a cartilage ECM-derived scaffold,and tissue-engineered cartilage with BMSCs in vitro and in vivo.The second part was to develop bilayered(or biphasic)osteochondral scaffold using this cartilage ECM, and tissue-engineered osteochondral constructs in vitro.The third part was aimed at implantation of the cells/scaffold complex for repairing osteochondral defects in canine model,and assessing the reconstituted defects grossly,histologically, Micro-CT,biochemically and biomechanically.Methods:(1)Human cartilage was physically shattered,then decellularized sequentially and made into a suspension.The scaffold was fabricated by simple freeze drying and cross-linking technique.Scaffolds was evaluated using histology,quantitive biochemical measure of glycosaminoglycan,collagen,and DNA content,and physicochemical characterization BMSCs was isolated from bone marrow of canine,and for assessment the cytotoxicity and biocompatibility of the scaffold.(2)A composite chitosan/cartilage ECM 3-D porous scaffold was fabricated and its physicochemical characterization was explored.(3)After induced by medium containing TGF-β1 at 10ng/ml,bFGF at 25ng/ml and dexamethasone at 10-7TM,BMSCs were seeded into the CEDPS scaffolds,cell attachment was confirmed by SEM and the viability of attached cells on the scaffold was confirmed by a live/dead assessment.After cultured in vitro for 1 and 3 weeks,cell-scaffolds were evaluated by histology,immunohistochemistry examination.(4)Chondrogenically induced BMSCs labeled with fluorescent dye PKH26 were then grown on scaffolds in vitro for 3 days and implanted subcutaneously into nude mice.Then in vivo fluorescent imaging system was used for evaluating the cell-scaffold constructs.After 4 weeks,the constructs was analyzed by histology,immunohistochemistry and immunofluorescence examnation.(5)A acellular bone matrix scaffold was developed using a novel decellularization methods,and characterized.(6)Two kinds of bilayered scaffolds were fabricated named CEDPS/ACBM bilayered scaffold and PLGA/ACBM bilayered scaffold,and characterized.(7)Osteochondral complex was engineered by chondrogenic BMSCs and CEDPS/ACBM bilayered scaffold, and evaluated by histology,SEM.(8)An osteochondral defects(4.2mm in diameter,6mm in depth)was created in the high-bearing area of femoral condyle of canine.The canine femoral condyles were divided into two groups: Control Group,CEDPS/ACBM bilayered scaffold only;Experimental Group, BMSCs-CEDPS/ACBM scaffold.Specimens were harvested at 3,6 months postoperatively,and assessed the reconstituted defects grossly,histologically, biochemically,biomechanically and by Micro-CT.Results:(1)On histology,scaffolds showed most of the ECM components after removal of the cell fragments with positive staining for Safranin O, tuoluidine blue and collagenⅡ,quantitive biochemical analysis also showed most of the GAG and Collagen was remained,Micro-CT and scanning electron microscopy revealed a 3-D interconnected porous structure.The biomechanical results the scaffolds have the similar biomechanical character as the cartilage. Cellular viability assay revealed no cytotoxic effects on the cells and the novel scaffold could provide a suitable 3-D environment to support the cell adheration and proliferation.(2)The composite chitosan/cartilage ECM scaffold has the good 3-D interconnected porous structure and water absorption ability, biomechanical analysis show its has similar biomechanical character as the cartilage.(3)Cartilage-like tissue formed after in vitro cultrue,with positive staining for Safranin O,tuoluidine blue and collagenⅡ.Dead/Live staining in the confocal microscopy of cell-scaffold constructs revealed cells with green fluorescence(live cell).Histology and SEM showed a large mount of extracellular matrix around the cells as time grown.(4)Four weeks later,cartilage-like tissue formed in nude mice,with positive staining for Safranin O,tuoluidine blue and collagenⅡ.Cells in the samples seemed to confirm that they originated from the labeled BMSCs,as confirmed by in vivo fluorescent imaging and immunofluorescence examination.(5)The novel ACBM has the similar microstructure and biomechanical character as the normal bone.(6)The two kinds of bilayered scaffolds both have good microstructure and the two layer (bone layer and cartilage layer)integrated well.(7)In vitro engineered osteochondral constructs formed osteochondral like tissue,and its cartilage layer integrated well with bone layer,with positive staining for Safranin O,tuoluidine blue and collagenⅡ.(8)In animal experimental,results indicated that every group have been repaired by fibrocartilage or hyaline cartilage in different degree. By gross and histological grading scale,the experimental group has better result than control group."The stiffness of the repaired cartilage of experiment group at 6 month reached 70.1%of the normal cartilage,which has the same tendency with the GAG content in biochemical analysis.Micro-CT revealed that there was no statistically difference in the restoration of subchondral bone between the experimental group and control group.Conclusions:(1)Cartilage ECM-derived 3-D porous scaffold retains most of the cartilage ECM components after removal of the cell fragments and has good structure,biomechanical character and biocompatibility, which makes it a suitable candidate for cartilage tissue engineering.(2)The composite chitosan/cartilage ECM scaffold has the good 3-D interconnected porous structure,water absorption ability,similar biomechanical character as the cartilage,and may serve as an alternative scaffold for cartilage tissue engineering..(3)The chondrogenic BMSCs-CEDPS constructs cultured in vitro can form cartilage-like tissue.CEDPS scaffold can provide appropriate3-D support for cell proliferation and differention of BMSCs into chondrocytes.(4)PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo(5)Chondrogenic BMSCs-CEDPS constructs implanted in nude mice can form cartilage-like tissue.(6)The novel acellular bone matrix with decellularization using a new method has good microstructure and similar biomechanical character,which make it a appropriate candidate for bone tissue engineering.(7)The novel two kinds of bilayered scaffolds(CEDPS/ACBM, PLGA/ACBM)have two layer(bone layer and cartilage layer), which integrated well.These two scaffolds can serve as good cell-carrier for osteochondral tissue engineering.(8)The chondrogenic BMSCs-CEDPS/ACBM complex cultured in vitro can form osteochondral like tissue.(9)CEDPS/ACBM biphasic scaffolds with chondrogenic BMSC may be an alternative treatment for large osteochondral defects in high-loading sites. |
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