脱细胞真皮基质/生物矿化胶原一体化骨软骨支架的制备及其生物相容性的研究
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摘要
目的成功制备脱细胞真皮基质/生物矿化胶原一体化骨软骨支架,并进行理化性质、生物力学与生物相容性评估,为骨软骨缺损修复提供理论依据。方法以物理和化学方法制备牛源脱细胞真皮基质,利用磷酸三钙和胶原,按照不同比例混合,利用自组装和冻干技术,制备以脱细胞真皮基质支架为软骨层,生物矿化胶原为骨层的骨软骨一体化双相支架。通过大体观察、扫描电镜观察、生物力学等检测,对骨软骨一体化支架进行理化性质和力学性能评价。原代培养小鼠软骨细胞和成骨细胞,并分别种植在脱细胞真皮基质和生物矿化胶原双相支架上,利用细胞毒、死/活细胞染色和增殖实验等观察细胞生长情况,测定骨软骨一体化支架的生物相容性。结果大体观察表明支架各层间结合紧密,未出现明显不连续和相互分离。扫描电镜各层内的孔隙结构相互连通且均具有立体多维性,其中脱细胞真皮基质、生物矿化胶原和双相支架的孔径分别为(121.6±8.65)、(98.40±5.56)和(103.2±3.94)μm。同时三组支架的压缩模量分别为(41.05±11.69)、(108.0±12.71)和(84.98±8.51)k Pa,弹性模量分别为(17.24±3.93)、(28.98±3.31)和(21.74±2.92)k Pa。MTT法表明骨软骨一体化支架无细胞毒性。荧光显微镜观察显示,纵切的支架薄片上细胞生长分布均匀,表明支架生物相容性较好,CCK8实验表明支架可以维持良好的细胞活性。结论脱细胞真皮基质/生物矿化胶原骨双相支架中上下层间的理化性能展示了骨软骨组织结构和力学方面的双重仿生,为动物体内实验奠定了基础,有望成为治疗骨软骨缺损修复的一种新手段。
Objective To manufacture an acellular dermal matrix/biomineralized collagen diphasic osteochondral scaffold and then evaluate the physical and chemical properties, biomechanics and biocompatibility of the scaffold to provide theoretical basis for the repair of osteochondral defects.Methods Bovine acellular dermal matrix was prepared by physical and chemical methods such as acellular, pepsin and cross-linking. Bone-cartilage integrated biphasic scaffolds with acellular dermal matrix(ADM) as cartilage layer and biomineralized collagen(DCS) as bone layer were prepared by mixing tricalcium phosphate and collagen in different proportions and using self-assembly and freeze-drying technology. We constructed the acellular dermal matrix scaffold(ADM), biomineralized collagen scaffold(DCS), and ADM/DCS scaffold(ADM/DCS), respectively, and then the physical and chemical properties and mechanical properties of osteochondral integrated scaffolds were evaluated by gross observation, scanning electron microscopy and biomechanical testing. The primary cultured chondrocytes and osteoblasts cells of mice were implanted into acellular dermal matrix and biomineralized collagen, respectively. The scaffold was observed by cytotoxicity, dead/living cell staining and proliferation experiments.Results Gross observation showed that the scaffolds were closely interlaminar, without obvious discontinuity and separation. The pore structures in each layer of the scanning electron microscope were interconnected and multidimensional. The pore sizes of ADM,DCS and biphasic scaffolds were(121.6 ± 8.65),(98.40 ± 5.56) and(103.2 ± 3.94) μm, respectively. At the same time, the compression modulus of the three groups of scaffolds were(41.05 ± 11.69),(108.0 ± 12.71) and(84.98 ± 8.51) kPa, and the elastic modulus were(17.24 ± 3.93),(28.98 ± 3.31) and(21.74 ± 2.92) kPa. The result from MTT assay showed that the osteochondral scaffold had no cytotoxicity. Cells grew evenly on the scaffold, indicating that the scaffolds had good biocompatibility. The data from CCK8 experiments showed that the scaffold possessed excellent biocompatibility.Conclusion The physical and chemical properties between the upper and lower layers of the ADM/DCS biphasic scaffolds demonstrate the dual biomimetic structure and mechanics of osteochondral tissue, which lays a foundation for further research in vivo experiments. Meanwhile, it becomes a new method for osteochondral defect repair.
Objective To manufacture an acellular dermal matrix/biomineralized collagen diphasic osteochondral scaffold and then evaluate the physical and chemical properties, biomechanics and biocompatibility of the scaffold to provide theoretical basis for the repair of osteochondral defects.Methods Bovine acellular dermal matrix was prepared by physical and chemical methods such as acellular, pepsin and cross-linking. Bone-cartilage integrated biphasic scaffolds with acellular dermal matrix(ADM) as cartilage layer and biomineralized collagen(DCS) as bone layer were prepared by mixing tricalcium phosphate and collagen in different proportions and using self-assembly and freeze-drying technology. We constructed the acellular dermal matrix scaffold(ADM), biomineralized collagen scaffold(DCS), and ADM/DCS scaffold(ADM/DCS), respectively, and then the physical and chemical properties and mechanical properties of osteochondral integrated scaffolds were evaluated by gross observation, scanning electron microscopy and biomechanical testing. The primary cultured chondrocytes and osteoblasts cells of mice were implanted into acellular dermal matrix and biomineralized collagen, respectively. The scaffold was observed by cytotoxicity, dead/living cell staining and proliferation experiments.Results Gross observation showed that the scaffolds were closely interlaminar, without obvious discontinuity and separation. The pore structures in each layer of the scanning electron microscope were interconnected and multidimensional. The pore sizes of ADM,DCS and biphasic scaffolds were(121.6 ± 8.65),(98.40 ± 5.56) and(103.2 ± 3.94) μm, respectively. At the same time, the compression modulus of the three groups of scaffolds were(41.05 ± 11.69),(108.0 ± 12.71) and(84.98 ± 8.51) kPa, and the elastic modulus were(17.24 ± 3.93),(28.98 ± 3.31) and(21.74 ± 2.92) kPa. The result from MTT assay showed that the osteochondral scaffold had no cytotoxicity. Cells grew evenly on the scaffold, indicating that the scaffolds had good biocompatibility. The data from CCK8 experiments showed that the scaffold possessed excellent biocompatibility.Conclusion The physical and chemical properties between the upper and lower layers of the ADM/DCS biphasic scaffolds demonstrate the dual biomimetic structure and mechanics of osteochondral tissue, which lays a foundation for further research in vivo experiments. Meanwhile, it becomes a new method for osteochondral defect repair.
引文
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[12]Shu X,Zheng R,Jie YS,et al.Effect of adipose-derived stem cells combine with acellular dermal matrix in repair of rabbit articular cartilage defects.Chin Med Biotechnol,2017,12(2):143-148.(in Chinese)舒雄,郑蕊,杰永生,等.脂肪干细胞复合真皮脱细胞基质修复兔关节软骨缺损的实验研究.中国医药生物技术,2017,12(2):143-148.
[13]Qu D,Li J,Li Y,et al.Ectopic osteochondral formation of biomimetic porous PVA-n-HA/PA6 bilayered scaffold and BMSCs construct in rabbit.J Biomed Mater Res B Appl Biomater,2011,96(1):9-15.
[14]Panseri S,Russo A,Cunha C,et al.Osteochondral tissue engineering approaches for articular cartilage and subchondral bone regeneration.Knee Surg Sports Traumatol Arthrosc,2012,20(6):1182-1191.
[15]Ma A,Jiang L,Song L,et al.Reconstruction of cartilage with clonal mesenchymal stem cell-acellular dermal matrix in cartilage defect model in nonhuman primates.Int Immunopharmacol,2013,16(3):399-408.
[16]Zha G,Li X,Niu X,et al.Application of collagen-β-tricalcium phosphate complex for repairing articular cartilage defects.J Biomaterials Tissue Eng,2017,7(3):241-247.
[17]Wu JY,Chen H,Yang L.Current status of intra-articular injection of drugs and biological agents in the treatment of osteoarthritis.J Clin Orthop Res,2019,4(2):113-119.(in Chinese)吴江怡,陈昊,杨柳.骨关节炎的关节腔内注射药物及生物制剂治疗现状.骨科临床与研究杂志,2019,4(2):113-119.
[2]Huey DJ,Hu JC,Athanasiou KA.Unlike bone,cartilage regeneration remains elusive.Science,2012,338(6109):917-927.
[3]Erggelet C,Vavken P.Microfracture for the treatment of cartilage defects in the knee joint-A golden standard?J Clin Orthop Trauma,2016,7(3):145-152.
[4]Emre TY,Atbasi Z,Demircioglu DT,et al.Autologous osteochondral transplantation(mosaicplasty)in articular cartilage defects of the patellofemoral joint:retrospective analysis of 33 cases.Musculoskelet Surg,2017,101(2):133-138.
[5]Schroeder JH,Hufeland M,Sch Tz M,et al.Injectable autologous chondrocyte transplantation for full thickness acetabular cartilage defects:early clinical results.Arch Orthop Trauma Surg,2016,136(10):1445-1451.
[6]de Windt TS,Vonk LA,Slaper-Cortenbach ICM,et al.Allogeneic MSCs and recycled autologous chondrons mixed in a one-stage cartilage cell transplantion:a first-in-man trial in 35 patients.Stem Cells,2017,35(8):1984-1993.
[7]Hung KC,Tseng CS,Dai LG,et al.Water-based polyurethane 3Dprinted scaffolds with controlled release function for customized cartilage tissue engineering.Biomaterials,2016,83:156-168.
[8]Gugjoo MB,Amarpal,Sharma GT,et al.Cartilage tissue engineering:Role of mesenchymal stem cells along with growth factors&scaffolds.Indian J Med Res,2016,144(3):339-347.
[9]Levingstone TJ,Matsiko A,Dickson GR,et al.A biomimetic multi-layered collagen-based scaffold for osteochondral repair.Acta Biomater,2014,10(5):1996-2004.
[10]Nonoyama T,Wada S,Kiyama R,et al.Double-network hydrogels strongly bondable to bones by spontaneous osteogenesis penetration.Adv Mater,2016,28(31):6740-6745.
[11]Choi B,Kim S,Lin B,et al.Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering.ACS Appl Mater Interfaces,2014,6(22):20110-20121.
[12]Shu X,Zheng R,Jie YS,et al.Effect of adipose-derived stem cells combine with acellular dermal matrix in repair of rabbit articular cartilage defects.Chin Med Biotechnol,2017,12(2):143-148.(in Chinese)舒雄,郑蕊,杰永生,等.脂肪干细胞复合真皮脱细胞基质修复兔关节软骨缺损的实验研究.中国医药生物技术,2017,12(2):143-148.
[13]Qu D,Li J,Li Y,et al.Ectopic osteochondral formation of biomimetic porous PVA-n-HA/PA6 bilayered scaffold and BMSCs construct in rabbit.J Biomed Mater Res B Appl Biomater,2011,96(1):9-15.
[14]Panseri S,Russo A,Cunha C,et al.Osteochondral tissue engineering approaches for articular cartilage and subchondral bone regeneration.Knee Surg Sports Traumatol Arthrosc,2012,20(6):1182-1191.
[15]Ma A,Jiang L,Song L,et al.Reconstruction of cartilage with clonal mesenchymal stem cell-acellular dermal matrix in cartilage defect model in nonhuman primates.Int Immunopharmacol,2013,16(3):399-408.
[16]Zha G,Li X,Niu X,et al.Application of collagen-β-tricalcium phosphate complex for repairing articular cartilage defects.J Biomaterials Tissue Eng,2017,7(3):241-247.
[17]Wu JY,Chen H,Yang L.Current status of intra-articular injection of drugs and biological agents in the treatment of osteoarthritis.J Clin Orthop Res,2019,4(2):113-119.(in Chinese)吴江怡,陈昊,杨柳.骨关节炎的关节腔内注射药物及生物制剂治疗现状.骨科临床与研究杂志,2019,4(2):113-119.