基质细胞衍生因子-1/壳聚糖/β-甘油磷酸钠复合生物膜体外生物学特性的实验研究

doi:10.13591/j.cnki.kqyx.2023.03.004

摘要/Abstract

摘要：

目的利用温敏凝胶制备基质细胞衍生因子-1(stromall cell derived factor-1, SDF-1)/壳聚糖(chitosan, CS)/β-甘油磷酸钠(β-sodium glycerphosphate, β-GP)复合生物膜并观察其体外生物学特性。方法制备CS/β-GP溶液并添加SDF-1混匀,观察其37 ℃时凝胶时间的变化。将凝胶铺膜、干燥形成SDF-1/CS/β-GP复合生物膜,扫描电镜观察其形貌特征。将复合生物膜置于含细胞培养液的六孔板,静置6、12、24、36、48、60 h后提取上清液(n=3),测定溶液SDF-1浓度变化。将Transwell小室上室中加入骨髓间充质干细胞(BMSCs),下室置入复合生物膜,分为4组(n=3):M0组即对照组(CS/β-GP膜);M1、M2、M3组均为SDF-1/CS/β-GP膜组,分别含100、200、400 ng/mL SDF-1。分别于6、12、24 h对膜下细胞Giemsa染色观察和MTT法测定光密度(OD)值计算细胞增殖率。采用SPSS 19.0进行多因素方差分析。结果 CS/β-GP溶液加入一定量的SDF-1后其凝胶时间无明显变化(P>0.05);SDF-1/CS/β-GP溶液在37 ℃条件下铺膜、干燥后形成多孔隙结构的半透明生物膜;SDF-1/CS/β-GP复合生物膜缓释SDF-1的体积质量随时间延长而逐渐增加(P<0.01);Transwell细胞迁移实验,BMSCs定向迁移的数量随着时间延长而增加(P<0.01),随着复合膜负载SDF-1体积质量增高而增加(P<0.01);MTT实验,迁移细胞OD值随着时间延长而增加(P<0.01),随着复合膜负载SDF-1体积质量增高而增加(P<0.01)。结论 SDF-1/CS/β-GP复合生物膜具有多孔隙结构并能趋化BMSCs定向迁移,是一种有应用潜力的引导组织再生膜。

关键词: 壳聚糖, 温敏凝胶, 基质细胞衍生因子-1, 引导组织再生膜

Abstract:

Objective To prepare a composite membrane by chitosan/β-sodium glycerophosphate(CS/β-GP) thermosensitive hydrogel combined with stromal cell derived factor-1(SDF-1) and observe its biological characteristics in vitro. Methods Different doses of SDF-1 were added into CS/β-GP solution and then the thermosensitive gel time was measured. The SDF-1/CS/β-GP solution was membrane paved and dried to prepare composite membranes. The morphological characteristics were observed by scanning electron microscope(SEM). Composite membranes were placed into cell culture medium, and the supernatant(n=3) was extracted after standing at 6, 12, 24, 36, 48, 60 h, respectively. The concentration of SDF-1 in the solution was measured. Bone mesenchymal stem cells(BMSCs) were cultured in the Transwell room, and the composite membranes containing different concentrations of SDF-1 were placed in the lower chamber. There were four groups(n=3): Group M0 used CS/β-GP membrane(control group), Group M1, M2, M3 used SDF-1/CS/β-GP membrane(SDF-1 was 100, 200, 400 ng/mL respectively). After culture for 6, 12 and 24 h, the cells under the membrane were preserved and Giemsa stained and counted. The absorbance(OD) value was measured by MTT method to calculate the cell proliferation rate. SPSS 19.0 was used for multi-factor analysis of variance. Results After adding a certain amount of SDF-1 into CS/β-GP solution, the gel time did not change significantly(P>0.05). The SDF-1/CS/β-GP membrane was translucent and porous at 37 ℃. In this experiment, the volumic mass of SDF-1 released by SDF-1/CS/β-GP composite membrane increased gradually with the experimental time(P<0.01). Transwell cell chemotaxis test showed that the number of BMSCs cells with directional migration increased with the prolongation of observation time(P<0.01) and the increase of SDF-1 volumic mass(P<0.01). In MTT test, the OD value of migration cell solution increased with the prolongation of time(P<0.01) and the increase of SDF-1 volumic mass(P<0.01). Conclusion The SDF-1/CS/β-GP composite membrane has a porous structure and biological activity of chemotactic BMSCs directional migration. It is a potential membrane for guided tissue regeneration.

Key words: chitosan, thermosensitive gel, stromal cell derived factor-1(SDF-1), guided tissue regeneration membrane

中图分类号:

R783.1

张凯奇, 董建勇, 张雅杰, 何彦亭, 霍媛媛, 李肇元, 崔军. 基质细胞衍生因子-1/壳聚糖/β-甘油磷酸钠复合生物膜体外生物学特性的实验研究[J]. 口腔医学, 2023, 43(3): 212-217.

ZHANG Kaiqi, DONG Jianyong, ZHANG Yajie, HE Yanting, HUO Yuanyuan, LI Zhaoyuan, CUI Jun. An experimental study on the biological characteristics of SDF-1/CS/β-GP composite bio-membrane in vitro[J]. Stomatology, 2023, 43(3): 212-217.

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