摘要
目的探讨大鼠滑膜间充质干细胞(synovium-derived mesenchymal stem cells,SMSCs)生物学特性及诱导成骨的可行性,评估SMSCs复合羟基磷灰石/壳聚糖/聚乳酸(hydroxylapatite/chitosan/poly L-latic acid,HA/CS/PLLA)支架材料在体内异位成骨能力。方法采用酶消化法和贴壁法分离培养SMSCs,并利用流式细胞仪检测细胞表型,SMSCs成脂及成骨诱导后分别行油红O染色、ALP染色和茜素红染色鉴定。取第3代SMSCs,成骨诱导1、7、14、21、28 d后,采用实时荧光定量PCR检测骨钙素(osteocalcin,OCN)、Ⅰ型胶原、ALP以及Runx-2m RNA表达情况;成骨诱导后1、3、5、7、9、11 d,采用ELISA法检测ALP活性;成骨诱导后7、14、21、28 d,采用茜素红S法检测钙盐沉积;以正常培养SMSCs作为对照组。体内实验:取SD大鼠24只,随机分为实验组和对照组(n=12);取第3代SMSCs接种于HA/CS/PLLA支架材料,体外复合培养72 h后植入实验组大鼠右下肢肌袋,对照组大鼠植入单纯HA/CS/PLLA支架材料;术后4、8周通过X线片及组织学观察分析SMSCs体内成骨情况。结果提纯后SMSCs CD147、CD90、CD105、CD44表达超过95%,而CD117、CD34、CD14、CD45表达低于10%;油红O染色可见红色脂滴形成,茜素红染色可见红色钙化灶,ALP染色可见细胞质呈咖啡样深染。实时荧光定量PCR检测示,SMSCs成骨诱导7 d,Ⅰ型胶原、ALP、Runx-2 m RNA表达显著增加,诱导14 d OCN m RNA表达显著增加。成骨诱导后5、7、9、11 d ALP活性明显高于对照组,7 d时达峰值(P<0.05)。成骨诱导14 d,钙含量显著增加,且随诱导时间延长钙含量逐渐增加(P<0.05),且均高于对照组(P<0.05)。体内实验术后4、8周影像学和组织学观测结果显示,两组均可见新生骨形成,但实验组成骨量明显多于对照组(P<0.05)。结论 SMSCs在体内、体外均可诱导成骨,可成为骨组织工程的种子细胞。
Objective To investigate the osteogenic differentiation potential and the biological features of synovium-derived mesenchymal stem cells（SMSCs） in vitro and to observe the osteogenic capability of the composite scaffolds constructed with SMSCs and hydroxylapatite/chitosan/poly L-latic acid（HA/CS/PLLA） in vivo. Methods SMSCs were separated and cultured with adherent method and enzymatic digestion method. Specific phenotypes of SMSCs were detected by flow cytometry after purification. Then, SMSCs were identified by oil red O staining, alkaline phosphatase（ALP） staining, and alizarin red staining after adipogenic and osteogenic induction, respectively. In vitro experiments： the expressions of osteogenic related genes [osteocalcin（OCN）, collagen type I, ALP, and Runx-2] were detected by real-time fluorescent quantitative PCR at 1, 7, 14, 21, and 28 days after osteogenic induction; ALP activities were also determined by ELISA at 1, 3, 5, 7, 9, and 11 days after osteogenic induction; meanwhile, extracellular matrix calcium mineralization was detected by alizarin red S method at 7, 14, 21, and 28 days after osteogenic induction; the normal SMSCs were harvested as control group. In vivo experiments： Twenty-four Sprague Dawley（SD） rats were randomly divided into experimental group（n=12） and control group（n=12）. The 3rd passage SMSCs were seeded on HA/CS/PLLA to construct composite scaffolds, after adhesion for 72 hours in vitro, the composite scaffolds were implanted into the right thigh muscle of 12 SD rats as experimental group; HA/CS/PLLA was implanted into the right thigh muscle of the other 12 SD rats as control group. At 4 and 8 weeks after implantation, the scaffolds were harvested for X-ray film and histological examination to observe ectopic bone formation. Results The positive rates of CD147, CD90, CD105, and CD44 were more than 95%, while the positive rates of CD117, CD34, CD14, and CD45 were less than 10%. Oil red O staining demonstrated red lipid droplets in the cytoplasm, and
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2016年第1期102-109,共8页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(81341054
81171732)
皖南医学院人才引进基金资助项目(YJRC2009010)~~
关键词
滑膜间充质干细胞
成骨诱导
茜素红
ALP
新骨形成
大鼠
Synovium-derived mesenchymal stem cells
Osteogenic induction
Alizarin red
Alkaline phosphatase
New bone formation
Rat
