CAJ | 학술논문

目的探讨大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架的优越性.方法选用6周龄健康Wistar大鼠,分离出脂肪干细胞后行体外培养.将Ⅰ型胶原溶液与壳聚糖溶液混合后冷冻干燥,交联硫酸软骨素后再冷冻干燥得到复合三维支架,检测支架的孔径值、含水量及孔隙率.将接种的脂肪干细胞消化后分别接种到平面、微球和支架,软骨方向诱导培养.MTT检测细胞增殖情况,3周后倒置显微镜及扫描电镜观察细胞形态及在支架上的生长及黏附情况,并分析成软骨分化的情况.结果 5 d后MTT检测显示三维支架组及微球组细胞增殖速度较平面组快;三维支架组14 d后仍有细胞增殖.组织学分析显示细胞在支架上密集重叠生长,内层仍有残留支架结构.Ⅱ型胶原免疫组化检测结果显示,三维支架组及微球组表达呈强阳性,而平面组表达呈弱阳性.RT-PCR结果显示各组均有软骨特异性mRNA的表达.但平面组一直表达X型胶原,微球组培养至21 d时也表达X型胶原,而三维支架组则一直未表达.结论复合胶原-壳聚糖-硫酸软骨素三维支架能促进细胞的增殖、分化,并能更好地维持软骨细胞的表型,可以作为组织工程构建软骨的最佳选择.
목적 탐토대서지방간세포복합효원-각취당-류산연골소삼유지가적우월성.방법 선용6주령건강Wistar대서,분리출지방간세포후행체외배양.장Ⅰ형효원용액여각취당용액혼합후냉동간조,교련류산연골소후재냉동간조득도복합삼유지가,검측지가적공경치、함수량급공극솔.장접충적지방간세포소화후분별접충도평면、미구화지가,연골방향유도배양.MTT검측세포증식정황,3주후도치현미경급소묘전경관찰세포형태급재지가상적생장급점부정황,병분석성연골분화적정황.결과 5 d후MTT검측현시삼유지가조급미구조세포증식속도교평면조쾌;삼유지가조14 d후잉유세포증식.조직학분석현시세포재지가상밀집중첩생장,내층잉유잔류지가결구.Ⅱ형효원면역조화검측결과현시,삼유지가조급미구조표체정강양성,이평면조표체정약양성.RT-PCR결과현시각조균유연골특이성mRNA적표체.단평면조일직표체X형효원,미구조배양지21 d시야표체X형효원,이삼유지가조칙일직미표체.결론 복합효원-각취당-류산연골소삼유지가능촉진세포적증식、분화,병능경호지유지연골세포적표형,가이작위조직공정구건연골적최가선택.
Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.