Luo Wen, Fan Jiannan, Ye Chuan
Graduate School of Guiyang Medical University, Guiyang Guizhou, 550004, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Dec;26(12):1505-11.
To construct a new type of self-assembling peptide nanofiber scaffolds-RGDmx, and to study the cell compatibility of the new scaffolds and the proliferation and chondrogenic differentiation of precartilaginous stem cells (PSCs) in scaffolds.
PSCs were separated and purified from newborn Sprague Dawley rats by magnetic activated cell sorting and indentified by immunohistochemistry and immunofluorescent staining. The RGDmx were constructed by mixing KLD-12 and KLD-12-PRG at volume ratio of 1:1. PSCs at passage 3 were seeded into the KLD-12 scaffold (control group) and RGDmx scaffold (experimental group). The proliferation of PSCs in 2 groups were observed with the method of cell counting kit (CCK)-8 after 1, 3, 7, and 14 days after culture. The RGDmx were constructed by mixing KLD-12-PRG and KLD-12 at different volume ratios of 0, 20%, 40%, 60%, 80%, and 100% and the proliferation of PSCs was also observed. The complete chondrogenic medium (CCM) was used to induce chondrogenic differentiation of PSCs in different scaffolds. The differentiation of PSCs was observed by toluidine blue staining and RT-PCR assay.
PSCs were separated and purified successfully, which were identified by immunohistochemistry and immunofluorescent staining methods. The results of CCK-8 showed that the absorbance (A) value in the experimental group increased gradually and reached the highest at 7 days; the A value in the experimental group was significantly higher than that in the control group at 7 days and 14 days (P < 0.05). Meanwhile, the A value in the RGDmx scaffold with a volume ratio of 40% was significantly higher than those in others (P < 0.05). After 14 days of induction culture with CCM, the toluidine blue staining results were positive in 2 groups; the results of RT-PCR showed that the expression levels of collagen type II and the aggrecan in the experimental group were significantly higher than those in the control group (P < 0.05).
The self-assembling peptide nanofiber scaffold-RGDmx is an ideal scaffold for tissue engineer because it has good cell compatibility and more effective properties of promoting the differentiation of PSCs to chondrocytes.
构建一种新型自组装肽纳米纤维支架-RGDmx，并研究该新型支架的细胞相容性以及软骨前体细胞（PSCs）在支架中的增殖和软骨分化情况。
通过磁激活细胞分选从新生Sprague Dawley大鼠中分离并纯化PSCs，采用免疫组织化学和免疫荧光染色进行鉴定。将KLD-12与KLD-12-PRG按体积比1:1混合构建RGDmx。将第3代PSCs接种到KLD-12支架（对照组）和RGDmx支架（实验组）中。培养1、3、7和14天后，采用细胞计数试剂盒（CCK）-8法观察两组中PSCs的增殖情况。还通过将KLD-12-PRG与KLD-12按0、20%、40%、60%、80%和100%的不同体积比混合构建RGDmx，并观察PSCs的增殖情况。使用完全软骨形成培养基（CCM）诱导不同支架中PSCs的软骨分化。通过甲苯胺蓝染色和RT-PCR检测观察PSCs的分化情况。
成功分离并纯化了PSCs，通过免疫组织化学和免疫荧光染色方法进行了鉴定。CCK-8结果显示，实验组的吸光度（A）值逐渐升高，在7天时达到最高；实验组在7天和14天时的A值显著高于对照组（P<0.05）。同时，体积比为40%的RGDmx支架中的A值显著高于其他组（P<0.05）。用CCM诱导培养14天后，两组甲苯胺蓝染色结果均为阳性；RT-PCR结果显示，实验组中Ⅱ型胶原蛋白和聚集蛋白聚糖的表达水平显著高于对照组（P<0.05）。
自组装肽纳米纤维支架-RGDmx具有良好的细胞相容性和更有效的促进PSCs向软骨细胞分化的特性，是一种理想的组织工程支架。
