Wu Xiaochen, Zhang Jinbao, Yi Dinghua, Gu Chunhu, Wei Xufeng, Wu Hong, Ouyang Hui, Gao Feng
Department of Cardiovascular Surgery, Xijing Hospital, Fourth Military Medical University, 17 Changle West Road, Xi'an 710032, China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2010 Feb;27(1):80-5.
This research was carried out to investigate the effect of basic fibrous growth factor (bFGF) controlled release hydrogel nanoparticles on the proliferation and differentiation of mesenchymal stem cells. The dex-GMA-bFGF-NPs were prepared by an improved emulsion polymerization method; their morphology, size and encapsulated ratio were assessed by routine procedure. Dynamic dialysis method was used to determine the release characteristics of dex-GMA-bFGF-NPs in vitro. The secondary culture MSCs were divided into four groups according the different ingredients being added into the DMEM culture medium: free bFGF group (A), blank dex-GMA nanoparticles group (B), dex-GMA-bFGF nanoparticles group (C), nothing group (D). The proliferation of cultured MSCs was measured by using cell counting method, MTT method and flow cytometry. ALP kit was used to evaluate the ALP activity of the MSCs to show the differentiation of the cells by adding the dex-GMA-bFGF-NPs to the DMEM culture medium (C group) or bFGF only (A group). B group and D group were taken as the controls. The results were analyzed by statistical analysis software (SPSS11.0). All results showed that the shape of dex-GMA-bFGF-NPs was spherical. The encapsulated ratio was 88% and more than 85% of the encapsulated bFGF could be released during 14 days. The in vitro cellular study showed the control release of bFGF from nanoparticles could promote the proliferation of MSCs. After 12 days, the cell number in groups A, B and C was (21.97 +/- 0.25) x 10(4) cells/ml, (12.43 +/- 0.13) x 10(4) cells/ml, (27.45 +/- 0.78) x 10(4) cells/ml and (12.03 +/- 0.43) x 10(4) cells/ml, with the difference being statistically significant among them (P < 0.05). The flow cytometry revealed that the G2/M+S percentage in group C was the highest at 4-8 days after plate culture(P < 0.05). During the first 3 days, the proliferation and differentiation of BMSCs between group A and group B were of no significance (P > 0.05), but were much faster than those of group C and D. After 7 days, dex-GMA-bFGF-NPs could enhance BMSCs proliferation and differentiation continually, but bFGF had no enhancement any more, the difference between group A and group B became more significant (P < 0.05). So we made the conclusions the bFGF loading dex-GMA hydrogel nanoparticles can release bFGF more than 21 days and can promote the proliferation and differentiation of the BMSCs through a long period of controlled release of bFGF. Dex-GMA-bFGF-NPs may be an ideal controlled release carrier for bioactive growth factors.
本研究旨在探讨碱性成纤维细胞生长因子（bFGF）控释水凝胶纳米粒对间充质干细胞增殖和分化的影响。采用改进的乳液聚合法制备了右旋糖酐-甲基丙烯酸缩水甘油酯-bFGF纳米粒（dex-GMA-bFGF-NPs）；通过常规方法评估其形态、大小和包封率。采用动态透析法测定dex-GMA-bFGF-NPs的体外释放特性。根据向DMEM培养基中添加的不同成分，将传代培养的间充质干细胞分为四组：游离bFGF组（A组）、空白右旋糖酐-甲基丙烯酸缩水甘油酯纳米粒组（B组）、dex-GMA-bFGF纳米粒组（C组）、空白组（D组）。采用细胞计数法、MTT法和流式细胞术检测培养的间充质干细胞的增殖情况。通过碱性磷酸酶（ALP）试剂盒评估间充质干细胞的ALP活性，以显示向DMEM培养基中添加dex-GMA-bFGF-NPs（C组）或仅添加bFGF（A组）后细胞的分化情况。B组和D组作为对照。结果采用统计分析软件（SPSS11.0）进行分析。所有结果表明，dex-GMA-bFGF-NPs呈球形。包封率为88%，且在14天内超过85%的包封bFGF能够释放。体外细胞研究表明，纳米粒中bFGF的控释可促进间充质干细胞的增殖。12天后，A组、B组、C组和D组的细胞数量分别为（21.97±0.25）×10⁴个细胞/ml、（12.43±0.13）×10⁴个细胞/ml、（27.45±0.78）×10⁴个细胞/ml和（12.03±0.43）×10⁴个细胞/ml，它们之间的差异具有统计学意义（P<0.05）。流式细胞术显示，平板培养后4-8天，C组的G2/M+S百分比最高（P<0.05）。在最初3天，A组和B组骨髓间充质干细胞的增殖和分化无显著差异（P>0.05），但比C组和D组快得多。7天后，dex-GMA-bFGF-NPs可持续增强骨髓间充质干细胞的增殖和分化，但bFGF不再有增强作用，A组和B组之间的差异变得更加显著（P<0.05）。因此，我们得出结论，负载bFGF的dex-GMA水凝胶纳米粒可在21天以上释放bFGF，并可通过bFGF的长期控释促进骨髓间充质干细胞的增殖和分化。dex-GMA-bFGF-NPs可能是生物活性生长因子的理想控释载体。
