Fabrication And Cartilage Tissue Engineering Research Of Biomimetic Structured PLGA/Articular Cartilage ECM Composite Scaffold

Background: As a new method to get native ECM, decellularization technique has been adopted to remove cellular antigens and preserve most of structural and functional proteins that constitute ECM. ECM from various decellularized tissues have been studied and applied for tissue engineering and regenerative medicine applications. Using an improved method of combining physical and chemical decellularization procedure in our study, the ACECM derived porous scaffold was developed with a freeze drying method. In vitro and in vivo studies demonstrated that the ACECM scaffold reserved main components of cartilage glycosaminoglycans (GAGs) and type II collagen, provided adequate space structure and had good biocompatibility for supporting MSCs attachment, proliferation and differentiation into chondrocytes. However, the scaffolds made of natural biomaterials are insufficient in mechanical strength especially in hydrous status. ACECM scaffold is the same with lower tensile and compressive strength. An ideal cartilage scaffold seeded with cells should possess structural integrity to temporarily withstand functional loading in vivo as natural cartilage does.The aim of tissue engineering and regenerative medicine is to regain structure and function of original tissue architecture. The biochemical composition and structure-function relationship of the scaffold are critical for optimizing mechanical and biological performance. It is significant that the morphological structure of scaffold is biomimetic to the repaired natural tissue. The organization of articular cartilage collagen network has most notable zonal variations, which principally responsible for tensile and compressive properties of cartilage. The alignment of collagen fibers in deep zone cartilage is oriented vertical to the subchondral bone. So the oriented scaffold with vertical microtubules structure would be closely related to physiological and biomechanical function of cartilage.Synthetic polymers (PCL, PLGA, PLA and so on) have been used widely in tissue engineering with the characteristics of satisfactory biocompatibility and good mechanical property. However, the surface of PLGA is hydrophobic and low intrinsic bioactive for cells adhesion. Although synthetic and naturally derived polymeric materials have their own advantages and drawbacks, the effects of these advantages and drawbacks on properties of the composite scaffold could be respectively enhanced and reduced. The composite scaffolds derived naturally and synthetic biomaterials with good cell affinity and enough mechanical strength to serve as an initial support have widely applied in cartilage tissue engineering. It is remaining significant challenge in fabrication functional and biomimetic scaffold.In this study, the oriented ACECM/PLGA composite scaffold was fabricated. The cell affinity and mechanical property of this scaffold were evaluated in vitro. Meanwhile, the composite scaffolds loaded with autologous MSCs were implanted in to the defect of rabbits’knee joint. The regeneration of articular cartilage was evaluated and observed by histological stainings. It is significant that whether the oriented structure scaffold can direct the cartilage regeneration with vertical orientation and induce the MSCs differentiate into chondrocytes.Part One: Fabrication and exosyndrome of biomimeticarticular cartilage ECM oriented scaffoldObjective: 1. Fabrication nanofibrous articular cartilage ECM. 2. Fabrication of ACECM oriented scaffold. 3. Evaluation of nanofibrous ACECM and oriented ACECM scaffold.Methods: To shatter and decellularize with combining physical and chemical procedure was illustrated as follows. The porcine cartilage slices were suspended in sterile PBS, then homogenized under powerfully shearing force using a tissue disintegrator to form suspension slurry and centrifugated by differential centrifugation method. The ACECM precipitation was digested in 3% TritonX-100 and 0.25% trypsin successively with gentle agitation for decellularization then in deoxyribonuclease I and ribonuclease A for removing nuclear materials. The oriented ACECM scaffold was fabricated by improved thermal induced phase separation technique. Morphology of ACECM and scaffold was observed by SEM. The chemical composition of ACECM was analyzed by histological stainings.Results: The decellularized ACECM was fibrous shape and nanometer size. Immunofluorescent staining revealed the presence of collagen II, while safranin O positive staining indicated that GAGs were existed in oriented scaffold. The oriented ACECM scaffold possessed homogeneous interconnected porous structure and the pores distributed uniformly. The vertical microtubules interconnected and aligned congruously.Conclusion: The nanofibrous ACECM was fabricated by the foregoing procedure. The ACECM derived oriented scaffold was developed by improved TIPS technique. The scaffold was biomimetic to the biochemical composition and morphological structure of deep zone articular cartilage.Part Two: The evaluation of oriented ACECM scaffold loaded with MSCs in vitro and ectopic chondrogenesis in nude miceObjective: 1. Evaluation of the biocompatibility of ACECM. 2. Observing the influence of oriented structure on the MSCs alignment in vitro. 3. Ectopic chondrogenesis in nude mice by the ACECM loaded with chondrogenic differentiation-induced MSCsMethods: Isolation, culture and chondrogenic induction of MSCs was seeded into the oriented ACECM scaffold and observed by SEM. The cell proliferation was quantitative evaluated by the CCK-8. Cell viability in cells/scaffold structures was evaluated using a Live/Dead assay kit. The chondrogenic induced MSCs were labeled with fluorescent dye PKH26 and implanted subcutaneously in dorsa of nude mice. After implantation 4W, the imaging of implants was displayed by the system of In-Vivo Imaging Systems. The implants were evaluated by histological stainings.Results: The cells spreaded along vertical microtubules and aggregated each other and filled the pore space. It displayed that MSCs proliferated rapidly. There were many live cells adhered on the scaffold. Besides, there were a few dead cells dispersed sporadicly in scaffold. The PKH26 was effective for the labeling of chondrogenic differentiation induced MSCs. The fluorescent imaging indicated that the implants come from the PKH26 labeled MSCs-ACECM scaffold structures. The staining results showed that the chondrocyte-like cells proliferated, new cartilage ECM was secreted, the ACECM retained the phenotype of differentiated stem cells and MSCs-scaffold structure formed tissue engineering cartilage in the ACECM microenvironment in vivo.Conclusion: The ACECM scaffold provided a microenvironment for MSCs adherence and proliferation in vitro. The seeding cells aligned along the oriented structure. In the meanwhile, the PKH26 fluorescent labeled MSCs was traced in nude mice and the ability of MSCs chondrogenic differentiation in vivo was maintained in the scaffold effectively.Part Three: Fabrication and evaluation in vitro of biomimetic structured PLGA/articular cartilage ECM composite scaffoldObjective: 1. Fabrication of PLGA/ACECM composite oriented scaffold. 2. Mechanical analysis of oriented scaffolds. 3. Evaluation the cell viability on the composite oriented.Methods: The method for fabrication oriented PLGA/ACECM composite scaffold was same as fabrication ACECM scaffold. The mechanical properties of oriented composite scaffolds in both dry and hydrous status were determined respectively by measuring compressive modulus. Cell proliferation and viability assay was evaluation by CCK-8 and Live/Dead assay kit respectively.Results: The oriented structure of PLGA/ACECM scaffold was similar with ACECM oriented scaffold. In dry status, the compressive modulus of composite scaffold was 4.20±0.17 MPa, while ACECM scaffold were 2.20±0.17 MPa. In the hydrous status, the compressive modulus of ACECM scaffold was only 0.11±0.01 MPa, while the composite scaffold was 1.03±0.1 MPa. Cell proliferation and viability assay displayed that the composite possessed good cell affinity than PLGA scaffold.Conclusion: A biomimetic composite scaffold from nanofibrous ACECM and PLGA was fabricated by improved TIPS technique. Comparing with oriented PLGA scaffold, the composite scaffold possesses preferable hydrophilicity and cell affinity for cells adhesion and proliferation. The composite scaffold possesses even higher mechanical strength than ACECM scaffold especially in the hydrous status.Part Four: The restoration of full-thickness cartilage defects with MSCs loaded PLGA/ACECM oriented constructsObjective: 1. The composite oriented scaffold was used to repair cartilage defect. 2. The oriented scaffold can direct cartilage regeneration with oriented structure. 3. The ACECM can induce MSCs differentiate in to chondrocytes in vivo.Methods: Oriented PLGA/ACECM composite scaffold loading MSCs was implanted to the articular cartilage defects in the rabbit model, while the only scaffold and defect was used as control. After both 12W and 24W, the repaired cartilage was analyzed by immunohistochemically and histologically.Results: After implantation 24W, the cartilage defect was repaired completely by hyaline cartilage. The regenerated cartilage was aligned along the vertical orientation.Conclusion: The PLGA/ACECM composite scaffold can repair rabbit’cartilage defect. Oriented PLGA/ACECM composite scaffold can direct oriented regeneration of articular cartilage. The ACECM can induce the MSCs into chondrocyte effectively in vivo.