Osteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the sca...Osteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells （BMSCs） in the repair of a large, high-load-bearing, osteochondral defect in a canine model. Methods The biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy （SEM） and micro-computed tomography （micro-CT）. Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects （4.2 mm （diameter） ×6 mm （depth）） were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold （control group）. The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation. Results The osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group （6 months） was （6.95±0.79） N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was （158.16±24.30） N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was （218±21.6） tJg/mg （dry weight）, 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, wi展开更多
Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derive...Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and 展开更多
Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, hut cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydro...Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, hut cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhe-sion stage. Moreover, seeded cells were distributed throughout the material.展开更多
基金the grants from the National Science Foundation of China，the Research Foundation of the Tianjin Health Bureau
文摘Osteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells （BMSCs） in the repair of a large, high-load-bearing, osteochondral defect in a canine model. Methods The biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy （SEM） and micro-computed tomography （micro-CT）. Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects （4.2 mm （diameter） ×6 mm （depth）） were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold （control group）. The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation. Results The osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group （6 months） was （6.95±0.79） N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was （158.16±24.30） N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was （218±21.6） tJg/mg （dry weight）, 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, wi
基金This study was funded by the National Natural Science Foundation of China （Nos. 31000432, 30930092 and 81272046） and National Technology Research and Development Program of China （No. 2012AA020502, 2012CB518106）.Acknowledgments： We thank HUANG Jing-xiang, T1AN Yue, and SUI Xiang for kind assistance in cell culture and histology.
文摘Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and
基金supported by the National Natural Science Foundation of China,No.31071222Jilin Province Science and Technology Development Project in China,No.20080738the Frontier Interdiscipline Program of Norman Bethune Health Science Center of Jilin University in China,No.2013106023
文摘Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, hut cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhe-sion stage. Moreover, seeded cells were distributed throughout the material.
