Application Of The Stent In Cartilage Tissue Engineering Autologous Bone Marrow Mesenchymal Stem Cells Extracellular Matrix (ECM)

Part one:Preparation of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold and a study of its propertiesObjective This study was designed to develop a novel autologous bone marrow mesenchymal stem cell-derived extracellular matrix (aBMSC-dECM) scaffold, and evaluate the properties of this scaffold.Methods The autologous bone marrow mesenchymal stem cells were harvested from two weeks old rabbits. The aBMSC-dECM membrane was collected after4weeks primary culture, and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques.The scaffold was investigated by HE staining, scanning electron microscope and compression strength test.Results The aBMSC-dECM scaffold was three-dimensional and spongy, with uniform porosity and a highly interconnected structure.The scaffold possessed pore size304.4±108.2μm, porosity of93.3±4.5%, density of28.7±1.4mg/ml, and a compressive modulus of6.8±1.5KPa.Conclusion aBMSC-dECM scaffold can be produced and demonstrate a good microstructure and biomechanical strength. It could be a novel candidate scaffold for tissue engineering. Part two:Application of aBMSC-dECM scaffold for cartilage regenerationObject To evaluate the feasibility of aBMSC-dECM scaffold for tissue engineering cartilageMethods The autologous bone marrow mesenchymal stem cells were harvested from two weeks old rabbits. The aBMSC-dECM membrane was collected after4weeks primary culture, and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes was seeded into the aBMSC-dECM scaffold. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The atelocollagen scaffold was used as the control group. Live-Dead staining was analyzed at48h after seeding. The engineered cartilage was harvested after1,2and4weeks in vitro culture, and analyzed by histological analysis, Real-Time PCR and Western blotting assay. The engineered cartilage was harvested after1,2and3weeks after in vivo implantation, and analyzed by gross morphological and volume measurement, histological analysis, biochemical assay and compression strength test.Results Live-Dead staining result showed that the aBMSC-dECM scaffold had a good biocompatibility. After4weeks in vitro culture, the engineered cartilage in aBMSC-dECM scaffold group formed thicker cartilage tissue with more homogeneous structure, higher expression of cartilaginous gene and protein compared to the atelocollagen scaffold group. Furthermore, the engineered cartilage based on aBMSC-dECM scaffold showed better cartilage formation in terms of volume and homogeneity, higher cartilage matrix content and stronger compressive modulus after3weeks in vivo.Conclusion The aBMSC-dECM scaffold could enhance the viability and biological function of chondrocytes, thus promote engineered cartilage regeneration. It could be a novel candidate scaffold for cartilage tissue engineering. Part three:The effect of aBMSC-dECM scaffold on chondrogenic differentiation of autologous BMSCsObject To evaluate the effect of aBMSC-dECM scaffold on chondrogenic differentiation of autologous BMSCsMethods The autologous bone marrow mesenchymal stem cells were harvested from two weeks old rabbits. The aBMSC-dECM membrane was collected after4weeks primary culture, and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes was seeded into the aBMSC-dECM scaffold. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on chondrogenic differentiation. The in vitro study was divided at random into four groups:①C+group:BMSCs/atelocollagen scaffold constructs were cultured in a defined medium with TGF-β3.②E+group:BMSCs/aBMSC-dECM scaffold constructs were cultured in a defined medium with TGF-β3.③C-group:BMSCs/atelocollagen scaffold constructs were cultured in a defined medium without TGF-β3.④E-group:BMSCs/aBMSC-dECM scaffold constructs were cultured in a defined medium without TGF-β3. The regenerated tissue was harvested after3,10and21days in vitro culture, and analyzed by histological analysis, Real-Time PCR and biochemical assay. For implanted subcutaneously in nude mice, BMSCs/scaffold constructs were first cultured for1week in a defined medium without TGF-β3. The regenerated tissue was harvested after1,2and3weeks after in vivo implantation, and analyzed by gross morphological and volume measurement, chondrogenic and osteogenic differentiation assay.Results During the in vitro culture, increasing expression of proteoglycan and type Ⅱ collagen, increasing expression of the COL2A1,ACAN and SOX9gene, high levels of DNA content and increasing GAG content and GAG/DNA ratio were observed in the regenerated tissue in E+and E-group compared to C+and C-group. During the vivo implantation, the regenerated tissue based on aBMSC-dECM scaffold has stronge expression proteoglycan and type II collagen in the early stage, but chondrogenic phenotypes was gradually lost and accompanied by the calcification of matrix. However, the volume of in vivo regenerated tissue gradually decreased in atelocollagen scaffold group. No chondrogenic phenotype can be found, and bone formation was observed in most area of the regenerated tissue at the late stage.Conclusion These results suggest that the aBMSC-dECM scaffold can support chondrogenic differentiation of autologous BMSCs, and maintain its phenotype. It could be a promising tool for cartilage tissue engineering. Part four:Enhanced Bone Marrow Stimulation technique with aBMSC-dECM Scaffold for Articular Cartilage RepairObject To investigate the effect of implanting aBMSC-dECM scaffold after bone marrow stimulation on articular cartilage repair.Methods Twenty-four New Zealand white rabbits were used in this study. The autologous bone marrow mesenchymal stem cells were harvested. The aBMSC-dECM membrane was collected after4weeks primary culture, and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Full osteochondral defects were performed on the trochlear groove of both knees. The treatments for defects were divided into two groups:the performance of BMS only in the right knee (the BMS group); BMS followed by implantation of the aBMSC-dECM scaffold in left knee (the BMS+Scaffold group). The regenerated cartilage of both groups was harvested at6and12weeks after surgery, and analyzed by gross morphology, histological analysis and biochemical assay.Results At12weeks, the regenerated cartilage covered most area of defect and showed a better integration with surrounding cartilage in the BMS+Scaffold group compared to the BMS group. The content and distribution of proteoglycan and type II collagen in the BMS+Scaffold group were similar to normal hyaline cartilage.Lots of cells resembling chondrocytes were embedded in the regenerated cartilage of the BMS+Scaffold group. The score in BMS+Scaffold group was significantly higher than BMS group. Collagen fibers of the repaired tissue in the BMS+Scaffold group were well aligned vertically similar to normal hyaline cartilage. The GAG and DNA contents of the repaired tissue in the BMS+Scaffold group were compatible to normal hyaline cartilage.Conclusion Implanting aBMSC-dECM scaffold after BMS showed a positive therapeutic effect on articular cartilage repair. It has a high value of clinical practice.