CAJ | 학술논문

目的利用BMSC细胞外基质(extracellular matrix,ECM)制备新型可塑形生物支架材料,探讨其应用于关节软骨组织工程的可行性. 方法取新西兰幼兔骨髓分离培养获得第2或3代BMSC,高密度培养后收集ECM,采用冷冻干燥技术制备ECM三维多孔状支架.采用组织学、免疫组织化学、扫描电镜、孔隙率、吸水率及生物力学测定等方法对材料进行物理化学性能观察.取第3代成年新西兰兔膝关节软骨细胞,以5.0 ×104个/ml分别与达尔伯克改良伊格尔培养液(Dulbecco's modified Eagle's medium,DMEM)(对照组)及体积分数50％(实验Ⅰ组)、体积分数100％(实验Ⅱ组)的材料浸提液进行培养,于培养后1,3,5,7d采用MTT法测定吸光度(A)值并分析其对细胞的毒性. 结果制备的ECM支架呈白色、银白色、象牙色的三维多孔状.HE染色及扫描电镜示支架呈交联的多孔状结构,支架内孔洞相互连通,孔径为100 ～400 μm,孔隙率为(91.50±2.84)％,吸水率为(2 014±224)％,压缩强度为(6.8±1.5)kPa; MTT检测软骨细胞在各组均生长良好,A值随时间延长而增大,同组各时相点比较,差异均有统计学意义(P＜0.05).结论 BMSC来源的ECM支架是一种新型三维多孔状支架,无须进行脱细胞处理,且安全无毒,具备合适的孔径和孔隙率,细胞相容性良好,是软骨组织工程良好的支架载体之一.
목적 이용BMSC세포외기질(extracellular matrix,ECM)제비신형가소형생물지가재료,탐토기응용우관절연골조직공정적가행성. 방법 취신서란유토골수분리배양획득제2혹3대BMSC,고밀도배양후수집ECM,채용냉동간조기술제비ECM삼유다공상지가.채용조직학、면역조직화학、소묘전경、공극솔、흡수솔급생물역학측정등방법대재료진행물이화학성능관찰.취제3대성년신서란토슬관절연골세포,이5.0 ×104개/ml분별여체이백극개량이격이배양액(Dulbecco's modified Eagle's medium,DMEM)(대조조)급체적분수50％(실험Ⅰ조)、체적분수100％(실험Ⅱ조)적재료침제액진행배양,우배양후1,3,5,7d채용MTT법측정흡광도(A)치병분석기대세포적독성. 결과 제비적ECM지가정백색、은백색、상아색적삼유다공상.HE염색급소묘전경시지가정교련적다공상결구,지가내공동상호련통,공경위100 ～400 μm,공극솔위(91.50±2.84)％,흡수솔위(2 014±224)％,압축강도위(6.8±1.5)kPa; MTT검측연골세포재각조균생장량호,A치수시간연장이증대,동조각시상점비교,차이균유통계학의의(P＜0.05).결론 BMSC래원적ECM지가시일충신형삼유다공상지가,무수진행탈세포처리,차안전무독,구비합괄적공경화공극솔,세포상용성량호,시연골조직공정량호적지가재체지일.
Objective To fabricate a novel biological scaffold using bone marrow stem cells (BMSCs)-derived extracellular matrix and investigate the feasibility of the scaffold used in cartilage tissue engineering.Methods Bone marrows were isolated from immature New Zealand rabbits to further harvest second-or third-generation BMSCs.ECM derived from BMSCs was collected after high-density culture and was fabricated into a three-dimensional porous scaffold using a freeze-dying technique.Physicochemical properties of the scaffolds were determined by histology,immunohistochemistry,scanning electron microscope,porosity and water absorption measurement,and biomechanical study.Third-generation chondrocytes harvested from knee joints of adult New Zealand rabbits were seeded at a density of 5.0 ×104 cells/ml with Dulbecco' s modified Eagle' s medium (DMEM) (control group),scaffold lixivium at volume fraction of 50％ (study group Ⅰ),and scaffold lixivium at volume fraction of 100％ (study group Ⅱ),respectively.At days 1,3,and 7 after co-culture,MTT test was done to assess the absorbance and cytotoxicity of the scaffold.Results Scaffolds appeared white/silver white/ivory color.HE staining and scanning electron microscopy demonstrated that the scaffolds were cross-linked and pore interconnected with pore size of 100-400 μm,porosity of (91.50 ±2.84) ％,water absorption rate of (2 014 ± 224) ％,and compression strength of (6.80 ± 1.50) kPa.MTT test showed that the chondrocytes were proliferated well in each group and that the absorbance value was increased with time.Besides,the absorbance value was significantly different within groups at each time point (P ＜ 0.05).Conclusion Novel three-dimensional porous scaffolds made from BMSCs-derived ECM are acellular,safe and non-toxic,exhibit favorable pore size and porosity and good biocompatibility,and hence can be good scaffolds for cartilage tissue engineering.