rhBMP2纳米缓释系统的制备及其对骨髓间充质干细胞增殖和分化的影响

目的：制备甲基丙烯酸缩水甘油酯右旋糖酐（dex-GMA）载重组人骨形态发生蛋白2（rhBMP2）凝胶纳米微球（NPs）缓释系统,检测其理化及生物学特性.方法：采用改良乳化溶液聚合法,制备rhBMP2纳米微球（rhBMP2-dex-NPs）;观察其形态、粒径与稳定性,检测其载药、释药特征;采用体外细胞培养技术,将rhBMP2-dex-NPs和兔骨髓间充质干细胞一起培养,MTT法检测其对细胞增殖状况的影响,碱性磷酸酶（ALP）检测试剂盒检测细胞ALP活性,以反应rhBMp2-dex-NPs对其分化状况的影响,并与单纯rhBMP2的作用进行比较;实验按照培养液中所含成分不同分为4组：培养液中加入单纯rhBMP2（A组）、加入rhBMP2-dex-NPs（B组）、加入空白NPs（C组）和对照组（D组）,其中A、B 2组均分别按照rhBMP2的不同量设置100μg/L、200μg/L、400μg/L 3个浓度组,采用SPSS 10.0对结果进行统计学分析.结果：rhBMP2-dex-NPs大小均匀,粒径分析表明,80%的纳米球粒径在20nm左右,包封率高达83%,80%的rhBMP2在前12d释放;其具有良好的生物学活性,能显著促进骨髓间充质干细胞（BMSCs）的增殖和分化,效应高于单纯施加rhBMP2的效应（P＜0.01）.rhBMP2-dex-NPs与BMSCs共培养3d内,反应BMSCs增殖和分化的OD值与A组无显著差异（P＞0.05）,但显著高于C、D组（P＜0.01）;3d后,rhBMP2-dex-NPs对细胞增殖和分化的促进作用较单纯rhBMP2明显加快;5～7d后,A、B组差异具有显著性（P＜0.01）;12d左右,B组细胞仍然有较强的增殖与分化能力,与其他3组有非常显著差异（P＜0.001）,而此时A、C、D组间差异已无统计学意义（P＞0.05）.结论：所制备的rhBMP2-dex-NPs形态良好、包封率高,理化与生物学性能稳定,可以达到对生长因子的良好缓释,比单纯rhBMP2对BMSCs有更为明显的生物学效应,可以持续促进其增殖与分化达10d以上,在组织工程领域有着广阔的应用前景.

Preparation and property of recombinant human bone morphogenetic protein-2 loaded hydrogel nanospheres and their biological effects on the proliferation and differentiation of bone mesenchymal stem cells

PURPOSE: To prepare and study the recombinant human bone morphogenetic protein-2 loaded dextran-based hydrogel nanospheres (rhBMP(2)-dex-NPs) sustained release system, and to evaluate its biological effects on cultured rabbit bone mesenchymal stem cells(BMSCs). METHODS: The rhBMP(2)-dex-NPs were prepared by improved emulsion polymerization method. Their morphology, size and size distribution, encapsulated ratio and stability were assessed by routine procedure. Dynamic dialysis method was used to determine the release characteristics of rhBMP(2)-dex-NPs in vitro. Cell culture technique and MTT colorimetric assay were used to evaluate the proliferation and differentiation of the BMSCs, ALP kit was used to evaluate the ALP activity of the BMSCs so as to show the differentiation of the cells by adding the rhBMP(2)-dex-NPs to the DMEM culture medium (B group) or rhBMP2 only (A group). Adding dex-NPs without rhBMP2 (C group) and adding nothing (D group) were taken as the controls. The results were analyzed by statistical analysis software (SPSS10.0). RESULTS: The shape of rhBMP(2)-dex-NPs was spherical, with a size distribution of 20 nm. The encapsulated ratio was 83% and rhBMP(2)-dex-NPs could be kept more than 6 months under 4 degrees C without decomposition , destruction or deposition. The release profile in vitro was in accordance with two phases kinetics law, and more than 80% of the encapsulated rhBMP(2) can be released during 12 days. Statistical analysis showed that rhBMP(2)-dex-NPs had biological activity, and could enhance both proliferation and differentiation of rabbit BMSCs significantly, the effect of the rhBMP(2)-dex-NPs was significantly higher than that of rhBMP(2) (P<0.01). During the first 3 days, the proliferation and differentiation of BMSCs between group A and B had no significance (P>0.05), but much faster than group C and D. After 5 to 7 days, rhBMP(2)-dex-NPs could enhance BMSCs proliferation and differentiation continually, but rhBMP2 had no enhancement any more. 7 days later, the difference between group A and B become much more significant (P<0.001). CONCLUSIONS: The rhBMP(2)-dex-NPs can release rhBMP2 more than 12 days and have long-drawn biological effects. To encapsulate rhBMP2 into dextran-based hydrogel nanospheres may be an effective way of growth factor controlled release in tissue engineering.