纳米壳聚糖-胶原纤维支架的生物相容性

摘要

BACKGROUND: The structure of nanometer chitosan-sodium/collagen (nano-CS/COL) is similar to that of the extracellular matrix (ECM) in the nanometer level. Whether this can promote the adhesion and growth of bone marrow mesenchymal stem cells (MSCs) and the calcification?OBJECTIVE: To investigate the in vitro histocompatibility of nano-CS/COL. DESIGN: Single sample observation.SETTING: Department of Orthopaedics, First Hospital, Jinan University. MATERIALS: This study was performed at the Experimental Center, First Hospital Affiliated to Jinan University between March 2007 and July 2007. Ten 4-week-old female SD rats, of SPF grade, weighing 200 g, were provided by the Guangdong Provincial Laboratory Center [Permission No. SCXK (yue) 2003-0002]. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Nano-CS/COL METHODS: Bone marrow MSCs were isolated from SD rats and cultured. Cell surface antigen was detected by loss cell analytical method. Nano-CS/COL scaffold was prepared by polyelectrolyte confocal laser-scanning microscopy. The well-grown cells of the third passage were co-cultured in vitro on the nano-CS/COL scaffold. Taking simple nano-CS/COL scaffold material as control, the histocompatibility of scaffold material and cells were comprehensively evaluated by cell adherence rate, growth curve, cell activity and cycle, and scanning electron microscope observation.MAIN OUTCOME MEASURES: ① Identification of cell surface antigen marker after isolation and culture of bone marrow MSCs. ②The histocompatibility of nano-CS/COL material and bone marrow MSCs 2, 4 and 8 days after nano-CS/COL material compounded with cells. ③Determination of adherence rate of cells to nano-CS/COL material. ? Cell circle and activity detected 5 days after nano-CS/COL material compounding with cells. RESULTS: ① Detection results of cell surface antigen marker: The expression of CD29, CD106, CD44, CD34 and CD45 was 90.86%, 73.38%, 82.61%, 0.76% and 0.60%, respectively. ②Histocompatibility of bone marrow MSCs and nano-CS/COL material: It was shown under the scanning electron microscope that nano-CS/COL scaffold presented porous three-dimensional structure, and different sizes of macropoles and interconnected small pores. The interval porosity determined by quality assay was 85%-90%, and aperture averaged 150 μm (range 50 - 300 u m). Two days after bone marrow MSCs compounded to nano-CS/COL scaffold, bone marrow MSCs presented globular shape and were scattered; Four days later, bone marrow MSCs presented shuttle shape, extended and anchored on the surface of nano-CS/COL by pseudopods; Eight days later, bone marrow MSCs proliferated and fused each other, and they secreted a lot of extracellular matrix, then which covered most material particles. ③ The adherence rate of bone marrow MSCs to nano-CS/COL: Bone marrow MSCs and nano-CS/COL were co-cultured 2 and 6 hours separately. The adherence rate of bone marrow MSCs was higher to nano-CS/COL scaffold than to simple chitosan scaffold. ④ Comparison of cells and cell cycle between on nano-CS/COL scaffold and on the chitosan scaffold: On the nano-CS/COL scaffold, cell activity was 96.67%, cell cycle at G0-G1 was 90.81%, at G2-M was 0.52% and at S was 8.66%. G2/G1 was 1.81. On the simple chitosan scaffold, cell activity was 95.27%, cell cycle at G0-G1 was 87.14%, at G2-M was 9.69%, and at S was 4.16%. G2/G1 was 1.80.CONCLUSION: Nano-CS/COL scaffold can be used as tissue engineering biomaterials because bone marrow MSCs can well grow on it.%背景:新型仿生纳米壳聚糖-胶原支架在纳米水平上与细胞外基质结构相似,其是否可促进骨髓间充质干细胞的黏附及生长,并显示良好的相容性?目的:评价新型纳米壳聚糖-胶原支架与SD大鼠骨髓基质干细胞的体外相容性.设计:单一样本观察.单位:暨南大学附属第一医院骨科.材料:实验于2007-03/2007-07在暨南大学附属第一医院实验中心完成.选取10只4周龄雌性SD大鼠,SPF级,体质量200 g,由广东省实验动物中心提供(许可证号为SCXK(粤)2003-0002).实验过程中对动物的处置符合动物伦理学标准.纳米壳聚糖-胶原纤维支架由理工学院生物材料研究室提供.方法:①分离培养SD大鼠骨髓基质干细胞,流式细胞分析法对细胞表面抗原进行检测.②聚电解质共凝聚技术制作纳米壳聚糖-胶原纤维支架.③取生长良好的P3代,与纳米壳聚糖-胶原纤维支架体外联合诱导培养,以单纯纳米壳聚糖支架材料为对照,通过细胞贴壁率、生长曲线、细胞活力及周期、扫描电镜观察综合评价材料与细胞的相容性. 主要观察指标:①骨髓间充质干细胞分离培养后进行流式细胞表面抗原标志鉴定.②纳米材料及细胞复合2,4,8 d后扫描电镜观察细胞与材料相容情况.③细胞对材料黏附率的测定.④细胞与材料复合5 d检测细胞周期及活力.结果:①细胞表面抗原标志检测结果:CD29表达为90.86%,CD106表达为73.38%,CD44表达为82.61%,CD34表达为0.76%,CD45表达为0.60%.②细胞与材料相容情况:扫描电镜可见纳米壳聚糖-胶原纤维支架为多孔的三维立体结构,材料内部形成大小不一的大孔和互连的小孔,彼此相互交通.应用质量法测得的孔隙率为85%～90%,孔径为50～300 μm,平均150 μm.骨髓基质干细胞复合到纳米壳聚糖-胶原纤维支架后2 d,细胞呈球形散在分布;4 d后细胞呈梭形,延展爬行且有伪足与材料表面锚靠;8 d时细胞增殖,相互间融合,并有大量的细胞外基质分泌,大部分材料颗粒被覆盖.③细胞对材料黏附率:细胞-支架复合物共培养2及6 h,骨髓基质干细胞在纳米壳聚糖-胶原纤维支架的黏附率均高于单纯纳米壳聚糖支架.④纳米壳聚糖-胶原纤维支架与单纯纳米壳聚糖支架的细胞、细胞周期特点比较:纳米壳聚糖-胶原纤维支架细胞活力为96.67%,细胞周期G0-G1为90.81%,G2-M为0.52%,S为8.66%,G2/G1为1.81.单纯纳米壳聚糖支架细胞活力为95.27%,细胞周期G0-G1为87.14%,G2-M为9.69%,S为4.16%,G2/G1为1.80.结论:纳米壳聚糖-胶原支架与骨髓基质干细胞有良好的组织相容性,可用来做组织工程生物材料.