聚乙丙交酯支架的细胞相容性特点

背景:聚乳酸及其衍生物具有良好的生物相容性、降解产物无毒性、易加工、具备一定强度等优点在骨组织工程中得到越来越广泛的应用。目的:观察骨髓基质干细胞与聚乙丙交酯类支架的细胞相容性及体外贴附情况,为制备负载多种细胞因子的聚乙丙交酯类支架提供研究基础。设计:对比观察。单位:南方医科大学南方医院创伤骨科。材料:实验于2004-09/2005-06在广东省组织构建与检测重点实验室完成。选择健康2月龄雄性新西兰兔1只。实验用主要材料:聚乙丙交酯支架(中山大学高分子研究所),β-磷酸三钙(瑞士AO公司)。方法:抽取新西兰兔骨髓,用全骨髓培养法获取单核细胞,经条件培养液体外诱导、扩增。骨髓基质干细胞以1×109L-1接种于聚乙丙交酯和β-磷酸三钙上,另设不加材料的空白对照组。通过倒置相差显微镜、扫描电镜观察细胞生长及细胞与材料的附着情况。以MTT法、流式细胞仪等手段检测各组细胞增殖和细胞周期变化情况。主要观察指标:①相差显微镜下每日定期观察细胞生长及与材料贴附情况。②培养1,3,6d扫描电镜下观察细胞生长情况。③MTT法检测细胞增殖情况。④采用化学比色法测定碱性磷酸酶活性。⑤流式细胞仪检测2种材料对骨髓基质干细胞的细胞周期、DNA含量及倍体水平的影响,并计算样品细胞的DNA指数。结果:①细胞培养后相差显微镜观察:空白对照组培养7～10d时细胞多呈梭形,未发现接触抑制现象。聚乙丙交酯组细胞开始贴壁时间明显晚于β-磷酸三钙组,随培养时间延长,细胞在材料周围与材料表面密集生长,细胞形态为多角形,两材料组细胞生长状态及细胞形态与空白对照组相似。②细胞培养后扫描电镜观察:空白对照组培养7d的细胞仍为单层并融合成片,但多角形细胞增多,细胞表面存在颗粒状物,细胞间以微丝相连。聚乙丙交酯组培养7d时,细胞数量大为增加,胞体扁平,跨越孔隙表面,形成细胞间连结,细胞连接成片,有大量基质形成。β-磷酸三钙组培养7d时,细胞数量增加,并相互连接,表面呈单层排列,可有细胞外基质形成,细胞长入孔隙内。③细胞增殖情况:随培养时间的延长各组细胞数量都有所增加,但聚乙丙交酯组与空白对照组及与β-磷酸三钙组间差异无显著性意义(P>0.05)。④碱性磷酸酶活性:随细胞培养时间延长,检测到各组细胞的碱性磷酸酶活性均增加,但聚乙丙交酯组与空白对照组及与β-磷酸三钙组间细胞碱性磷酸酶活性差异无显著性意义(P>0.05)。⑤细胞周期情况:两种材料对兔骨髓基质干细胞的细胞周期影响不大,各组细胞皆为正常的二倍体细胞,未见异倍体细胞形成。结论:聚乙丙交酯类支架的细胞相容性较好,并可进一步作为多种细胞因子载体构建缓释型支架用于骨组织工程。

Cellular compatibility of poly (lactic-co-glycolic acid)scaffold

BACKGROUND:The poly-lactic acid and its ramifications have many advantages, such as eligible biocompatibility,nontoxicity of degradation product, easy procession and suitable intensity. Thus, they have been widely used in bone tissue engineering.OBJECTIVE: To study the cellular biocompatibility and in vitro adhesion of poly(lactic-co-glycolic acid) (PLGA) scaffold and bone marrow stromal stem cells (BMSCs) so as to provide a basis for preparation a PLGA scaffold that can load many cytokines.DESIGN: Contrast observation.SETTING: Department of Orthopaedics and Traumatology in Nanfang Hospital of Southern Medical University.MATERIALS: The experiment was carried out in Key Laboratory of Tissue Construction and Detection of Guangdong Province from September 2004 to June 2005. One New Zealand healthy male rabbit (2 months old, 1.5-2.0 kg weight)was adopted in this study. Experimental materials: PLGA scaffold was obtained from Institute of Polymer Science in Sun Yat-sen University); beta-tricalcium phosphate (β-TCP) was supplied by AO Company (Switzerland).METHODS: Bone marrow was aspirated from the New Zealand rabbit. Mononuclear cells were harvested using whole bone marrow culturing, then were induced and amplified in the conditional culture medium. BMSCs were inoculated onto the PLGA and β-TCP at a concentration of 1 ×109 L-1, while those in the medium without materials were taken as blank control group. The development of implanted cells and the adhesion between cells and materials were observed with phase contrast microscope and scanning electron microscope. The proliferation and cycle of cells were tested with MTT method and flow cytometry.MATN OUTCOME MEASURES: ①Phase contrast microscope was used to observe the development of cells and the adherence between cells and materials at a fixed time every day. ②Cellular development on days 1, 3, 6 was observed by scanning electron microscope. ③Cellular proliferation was detected by MTT method. ④Alkaline phosphatase (ALP) activity was determined by chemical colorimetry.⑤Flow cytometer test: The effects of PLGA and β-TCP on the cellular cycle, content of DNA and polyploid levels of BMSCs were investigated. The DNA index of the candidate cells was also calculated.RESULTS: ①Phase contrast microscope observation: In the blank control group, cells culture for 7-10 days presented a larger number of shuttle-shaped, and no contact inhibition effect was observed. The time of cells beginning adherence in PLGA group was obviously later than that in β-TCP group. Cells began to develop on the circumambience and surface of the materials, with the prolonging of culture time. Most of the cells were multangular. The cells in both PLGA and β-TCP groups were similar to those in control group in items of cellular development and shape. ②Scanning electron microscope observation: On the seventh day of culture, the cells of control group remained a monolayer and amalgamated to be a patch with multangular-shaped ones increasing. There were substances in granule shape on the surface of the cells and micro-silk links between cells. In PLGA group, the cells After 7 days' cultivation proliferated sharply, and the compressed-shaped ones were inosculated to be a patch through integrations among cells, resulting in a large number of matrixes. While in β-TCP group, the number of cells increased from the 7th day. The cells were combined together to be a monolayer and moved to pores with matrixes creating out of cells.③Cellular proliferation: The number of the cells in each group all increased to some extents. However, there was no significant difference between the PLGA group and the control group, as well as the PLGA group and the β-TCP group (P ＞ 0.05).④ALP activity: The　content of ALP in all the groups enhanced without exception, while the activities between the PLGA group and the Both control group, as well as the PLGA group and the β-TCP group had no significant difference (P ＞ 0.05).⑤Cellular cycle:Both PLGA and β-TCP had slight effects on cellular cycle of BMSCs. The cells in each group were all normal diploid,and no heteroploid cells were discovered.CONCLUSION: This type of PLGA scaffold possesses good cellular biocompatibility, and can be used as a carrier of many factors in bone tissue engineering.