CAJ | 학술논문

目的探讨软骨细胞与脂肪基质细胞(adipose-derived stromal cells,ADSCs)共培养体外构建软骨的可行性,并阐明软骨细胞提供的软骨微环境能否诱导ADSCs向软骨细胞分化并形成软骨组织.方法分别培养扩增人ADSCs与猪耳软骨细胞,将2种细胞按7:3(ADSCs:软骨细胞)比例混匀,以5.0×107/ml的细胞终浓度接种于聚羟基乙酸/聚乳酸(PGA/PLA,直径8 mm,高2 mm)支架作为共培养组,以相同终浓度的单纯软骨细胞和单纯ADSCs分别接种相同支架作为阳性对照组及阴性对照组,以30％上述浓度(1.5×107/ml)的单纯软骨细胞接种作为低浓度软骨细胞对照组.每组各接种6例标本,每例接种细胞悬液200μl.全部标本均于体外培养8周时取材,通过大体观察、组织学、免疫组化及湿重、蛋白多糖定量检测等方法对新生软骨进行初步评价.多样本t检验统计分析各组湿重及蛋白多糖含量差异.结果各组细胞均与材料粘附良好.共培养组及阳性对照组体外培养8周时基本保持了复合物初始大小和形状,大体观察2组均形成了较成熟软骨组织,组织学显示大量软骨基质和软骨陷窝形成,免疫组化显示软骨特异性细胞外基质Ⅱ型胶原分泌.定量测定结果表明,共培养组的平均湿重为(174±12) mg,平均蛋白多糖含量为(7.6±0.4) mg,两者分别达到阳性对照组的75％ (P＜ 0.01)和79％ (P＜ 0.01).阴性对照组(单纯ADSCs组)明显皱缩变形,组织学未见成熟软骨陷窝.低浓度软骨细胞组明显变薄,新生软骨平均湿重为(85 ±5) mg,是阳性对照组的37％ (P＜ 0.01),只在局部形成了不连续的软骨组织.结论软骨细胞与ADSCs共培养能够在体外构建较成熟的软骨组织,软骨细胞能够诱导ADSCs成软骨分化及体外形成软骨组织.
목적 탐토연골세포여지방기질세포(adipose-derived stromal cells,ADSCs)공배양체외구건연골적가행성,병천명연골세포제공적연골미배경능부유도ADSCs향연골세포분화병형성연골조직.방법 분별배양확증인ADSCs여저이연골세포,장2충세포안7:3(ADSCs:연골세포)비례혼균,이5.0×107/ml적세포종농도접충우취간기을산/취유산(PGA/PLA,직경8 mm,고2 mm)지가작위공배양조,이상동종농도적단순연골세포화단순ADSCs분별접충상동지가작위양성대조조급음성대조조,이30％상술농도(1.5×107/ml)적단순연골세포접충작위저농도연골세포대조조.매조각접충6례표본,매례접충세포현액200μl.전부표본균우체외배양8주시취재,통과대체관찰、조직학、면역조화급습중、단백다당정량검측등방법대신생연골진행초보평개.다양본t검험통계분석각조습중급단백다당함량차이.결과 각조세포균여재료점부량호.공배양조급양성대조조체외배양8주시기본보지료복합물초시대소화형상,대체관찰2조균형성료교성숙연골조직,조직학현시대량연골기질화연골함와형성,면역조화현시연골특이성세포외기질Ⅱ형효원분비.정량측정결과표명,공배양조적평균습중위(174±12) mg,평균단백다당함량위(7.6±0.4) mg,량자분별체도양성대조조적75％ (P＜ 0.01)화79％ (P＜ 0.01).음성대조조(단순ADSCs조)명현추축변형,조직학미견성숙연골함와.저농도연골세포조명현변박,신생연골평균습중위(85 ±5) mg,시양성대조조적37％ (P＜ 0.01),지재국부형성료불련속적연골조직.결론 연골세포여ADSCs공배양능구재체외구건교성숙적연골조직,연골세포능구유도ADSCs성연골분화급체외형성연골조직.
Objective To explore the feasibility of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells (ADSCs) so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs.Methods Human ADSCs and porcine auricular chondrocytes were in vitro expanded respectively and then were mixed at the ratio of 7:3 (ADSCs: chondrocytes).200 μl mixed cells (5.0 × 107/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA)scaffold,8 mm in diameter and 2 mm in thickness,as coculture group. Chondrocytes and ADSCs with the same cell number were seeded respectively onto the scaffold as positive control group and negative control group.200 μl chondrocytes (1.5 × 107/ml) were seeded as low concentration chondrocyte group.There were 6 specimens in each group.A11 specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10％ FBS.Gross observation, histology,immunohistochemistry,wet weight measurement and glycosaminoglycan(GAG) quantification were used to evaluate the results.Multiple-sample t-test statistics analysis was done to compare the difference of wet weight and glycosaminoglycan(GAG) content between the groups.Results Cells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. In co-culture group and positive control group,cell-scaffold constructs could maintain the original size and shape during in vitro culture. At 8weeks,cartilage-like tissue formed in gross appearance and histological features,and abundant type Ⅱcollagen could be detected by immunohistochemistry.Wet weight and glycosaminoglycan(GAG) content of co-culture group were respectively( 174 ± 12) mg and(7.6 ±0.4) mg.There were respectively 75％ (P ＜0.01) and 79％ (P ＜ 0.01 )of those of positive control group.In negative control group,however,constructs shrunk gradually without mature cartilage lacuna in histology. In low concentration chondrocyte group,constructs also shrunk obviously with small amount of cartilage formation at the edge area of the construct,and wet weight was ( 85 ± 5 ) mg,which was 37％ ( P ＜ 0.01 ) of that of positive control group.Conclusions Chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs and thus promote the in vitro chondrogenesis of ADSCs.