Chen Lin, Xu Mingqing, Liu Yang, Duan Hao, Hua Wenda, He Fei
Department of Orthopedics, First Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650000, P. R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2016 Dec 8;30(12):1524-1531. doi: 10.7507/1002-1892.20160315.
To investigate the effect of tissue interface stiffness change on the spreading, proliferation, and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), and to find the suitable stiffness range for stem cell differentiation.
Bone marrow of male Sprague Dawley rats (4 weeks old) were selected to isolate and culture BMSCs by whole bone marrow cell adherent method. The third generation BMSCs (1×10 cells/mL) were inoculated into the ordinary culture dishes covered with polyacrylamide hydrophilic gel (PA) which elastic modulus was 1, 4, 10, 40, and 80 kPa (cells seeded on PA), and ordinary culture dish (75 MPa extreme high elastic modulus) as control. Spreading of cells in different stiffness of PA was observed under light microscope. The elastic modulus values of 4, 10, and 40 kPa PA were selected as groups A, B, and C respectively; the ordinary culture dish (75 MPa extreme high elastic modulus) was used as control group (group D). Cell counts was used to detect the growth conditions of BMSCs, alkaline phosphatase (ALP) kit to detect the concentration of ALP, alizarin red staining technique to detect calcium deposition status, and real-time quatitative PCR technique to detect the expressions of bone gla protein (BGP), Runx2, and collagen type I mRNA.
With increased PA stiffness, BMSCs spreading area gradually increased, especially in 10 kPa and 40 kPa. At 1 and 2 days after culture, the growth rate showed no significant difference between groups (>0.05); at 3-5 days, the growth rate of groups B and C was significantly faster than that of groups A and D (<0.05), but difference was not statistically significant between groups A and D (<0.05); at 5 days, the proliferation of group C was significantly higher than that of group B (<0.05). ALP concentrations were (53.69±0.89), (97.30±1.57), (126.60±14.54), and (12.93±0.58) U/gprot in groups A, B, C, and D respectively; groups A, B, and C were significantly higher than group D, and group C was significantly higher than groups A and B (<0.05). Alizarin red staining showed that the percentages of calcium nodules was 20.07%±4.24% in group C; group C was significantly higher than groups A, B, and D (<0.05). The expression levels of BGP and collagen type I mRNA were significantly higher in groups A, B, and C than group D, and in group C than groups A and B (<0.05). The expression level of Runx2 mRNA was significantly higher in groups B and C than group D, and in group C than group B (<0.05), but no significant difference was found between groups A and D (>0.05).
PA elastic modulus of 10-40 kPa can promote the proliferation and osteogenic differentiation of BMSCs, and the higher the stiffness, the stronger the promoting effect.
研究组织界面硬度变化对大鼠骨髓间充质干细胞（BMSCs）铺展、增殖及成骨分化的影响，寻找适合干细胞分化的硬度范围。
选取4周龄雄性Sprague Dawley大鼠的骨髓，采用全骨髓细胞贴壁法分离培养BMSCs。将第三代BMSCs（1×10⁴个细胞/mL）接种到覆盖有弹性模量分别为1、4、10、40和80 kPa的聚丙烯酰胺亲水凝胶（PA）的普通培养皿（接种于PA上的细胞）以及普通培养皿（75 MPa极高弹性模量）作为对照。在光学显微镜下观察细胞在不同硬度PA上的铺展情况。分别选取弹性模量值为4、10和40 kPa的PA作为A、B、C组；普通培养皿（75 MPa极高弹性模量）作为对照组（D组）。采用细胞计数法检测BMSCs的生长情况，碱性磷酸酶（ALP）试剂盒检测ALP浓度，茜素红染色技术检测钙沉积情况，实时定量PCR技术检测骨钙素（BGP）、Runx2和I型胶原mRNA的表达。
随着PA硬度增加，BMSCs铺展面积逐渐增大，尤其是在10 kPa和40 kPa时。培养1天和2天时，各组生长速率差异无统计学意义（>0.05）；培养3 - 5天时，B组和C组生长速率显著快于A组和D组（<0.05），但A组和D组之间差异无统计学意义（<0.05）；培养5天时，C组增殖显著高于B组（<0.05）。A、B、C、D组ALP浓度分别为（53.69±0.89）、（97.30±1.57）、（126.60±14.54）和（12.93±0.58）U/gprot；A、B、C组显著高于D组，C组显著高于A组和B组（<0.05）。茜素红染色显示C组钙结节百分比为20.07%±4.24%；C组显著高于A、B、D组（<0.05）。A、B、C组BGP和I型胶原mRNA表达水平显著高于D组，C组高于A组和B组（<0.05）。B组和C组Runx2 mRNA表达水平显著高于D组，C组高于B组（<0.05），但A组和D组之间差异无统计学意义（>0.05）。
10 - 40 kPa的PA弹性模量可促进BMSCs的增殖和成骨分化，硬度越高，促进作用越强。
