Influence of porosity and Young's modulus of gelatin methacrylate anhydride hydrogels on the biological behavior of mouse bone marrow mesenchymal stem cells
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摘要:
目的 探究甲基丙烯酸酐化明胶（GelMA）水凝胶的孔隙率与杨氏模量对小鼠骨髓间充质干细胞（BMSC）生物学行为的影响,为开发用于皮肤创面修复的组织工程支架提供理论基础。 方法 该研究为实验研究。将由50 g/L的GelMA溶液光交联30、60 s及150 g/L的GelMA溶液光交联30、60 s制成的GelMA水凝胶分别设为低浓度短时组、低浓度长时组及高浓度短时组、高浓度长时组,使用Image J 1.54软件测量并计算4组水凝胶冷冻干燥48 h后的孔隙率,采用杨氏模量测定仪检测4组水凝胶的杨氏模量,评估前述孔隙率与杨氏模量的相关性并建立二者之间的预测模型,计算4组水凝胶在磷酸盐缓冲液中浸泡7 d后的剩余质量百分比。从10只雌雄不明1 d龄C57BL/6小鼠股骨分离BMSC,将由BMSC与50 g/L的GelMA溶液混合后光交联30、60 s及BMSC与150 g/L的GelMA溶液混合后光交联30、60 s制成的载细胞GelMA水凝胶分别设为低浓度短时载细胞组、低浓度长时载细胞组、高浓度短时载细胞组、高浓度长时载细胞组,采用细胞活/死试剂盒检测4个载细胞组水凝胶培养7 d后细胞存活情况并计算细胞存活率；将4个载细胞组水凝胶成骨诱导培养10 d后,采用实时荧光定量反转录PCR法检测细胞中成骨相关因子Runt相关转录因子2（Runx2）、碱性磷酸酶（ALP）及干性相关因子SRY盒转录因子2（Sox2）、Nanog的mRNA表达,采用免疫荧光法检测细胞中Runx2与Nanog的蛋白表达。除蛋白表达（定性观察）外,其余指标每组样本数均为3。 结果 低浓度短时组、低浓度长时组、高浓度短时组、高浓度长时组水凝胶冷冻干燥48 h后的孔隙率依次降低,分别为（92.7±0.9）%、（85.0±1.8）%、（68.6±1.2）%、（56.3±5.8）%；杨氏模量依次升高,分别为（5.933±0.020）、（7.803±0.089）、（20.772±0.106）、（22.498±0.060）kPa。除高浓度长时组与高浓度短时组间水凝胶杨氏模量（P>0.05）外,其余组间水凝胶的孔隙率或杨氏模量两两比较,差异均有统计学意义（P<0.05）；水凝胶的孔隙率与杨氏模量呈显著负相关（R2=0.91,P<0.05）,并据此建立了二者之间的线性方程预测模型。浸泡7 d后,低浓度长时组、高浓度短时组、高浓度长时组水凝胶剩余质量百分比均显著高于低浓度短时组（P<0.05）,高浓度长时组水凝胶剩余质量百分比显著高于低浓度长时组（P<0.05）。培养7 d后,4个载细胞组水凝胶中均可见大量活细胞,组间细胞存活率总体比较,差异无统计学意义（P>0.05）。成骨诱导培养10 d后,高浓度长时载细胞组水凝胶的细胞中Runx2与ALP的mRNA表达均显著高于其余3个载细胞组（P<0.05）,低浓度短时载细胞组水凝胶的细胞中Sox2与Nanog的mRNA表达均显著高于其余3个载细胞组（P<0.05）；高浓度长时载细胞组水凝胶的细胞中Runx2的蛋白表达最高,低浓度短时载细胞组水凝胶的细胞中Nanog的蛋白表达最高。 结论 GelMA水凝胶的孔隙率与杨氏模量通过协同作用调控小鼠BMSC的生物学行为,其中高孔隙率-低杨氏模量利于干性维持,而低孔隙率-高杨氏模量驱动成骨分化。 Abstract:Objective To investigate the influence of porosity and Young's modulus of gelatin methacrylate anhydride (GelMA) hydrogels on the biological behavior of mouse bone marrow mesenchymal stem cells (BMSCs), and to provide a theoretical basis for the development of tissue engineering scaffolds for skin wound repair. Methods This study was an experimental study. GelMA hydrogels that were prepared by photo-crosslinking 50 g/L GelMA solution for 30 and 60 seconds and 150 g/L GelMA solution for 30 and 60 seconds, were designated respectively as low-concentration short-time group, low-concentration long-time group, high-concentration short-time group, and high-concentration long-time group. The porosity of the 4 groups of hydrogels after freeze-drying for 48 hours was measured and calculated using Image J 1.54 software. The Young's modulus of the 4 groups of hydrogels was detected using a Young's modulus tester. The correlation between the porosity and Young's modulus was evaluated and a predictive model between them was established. The remaining mass percentages of the 4 groups of hydrogels after soaking in phosphate buffered saline for 7 days were calculated. BMSCs were isolated from the femurs of 10 C57BL/6 mice aged 1 day of unknown sex. Cell-loaded GelMA hydrogels were prepared by mixing BMSCs with 50 g/L GelMA solution and photo-crosslinking for 30 and 60 seconds, and by mixing BMSCs with 150 g/L GelMA solution and photo-crosslinking for 30 and 60 seconds, and were designated respectively as low-concentration short-time cell-loaded group, low-concentration long-time cell-loaded group, high-concentration short-time cell-loaded group, and high-concentration long-time cell-loaded group. The cell survival in the 4 cell-loaded groups of hydrogels after 7 days of culture was detected using a live/dead cell kit, and the cell survival rate was calculated. After 10 days of osteogenic induction culture of the hydrogels in the 4 cell-loaded groups, the mRNA expressions of osteogenic-related factors Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP), and stemness-related factors SRY-box transcription factor 2 (Sox2) and Nanog in the cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction; the protein expressions of Runx2 and Nanog in the cells were detected by immunofluorescence method. Except for the protein expression (qualitative observation), the sample size of each group for the other indicators was three. Results The porosity of hydrogels in low-concentration short-time group, low-concentration long-time group, high-concentration short-time group, and high-concentration long-time group after freeze-drying for 48 hours decreased successively, being (92.7±0.9)%, (85.0±1.8)%, (68.6±1.2)%, and (56.3±5.8)%, respectively; the Young's modulus increased successively, being (5.933±0.020), (7.803±0.089), (20.772±0.106), and (22.498±0.060) kPa, respectively. Except for the Young's modulus of hydrogels between the high-concentration long-time group and high-concentration short-time group (P>0.05), the pairwise comparisons of hydrogel porosity or Young's modulus among the remaining groups showed statistically significant differences (P<0.05). The porosity and Young's modulus of the hydrogels were significantly negatively correlated (R2=0.91, P<0.05), and a linear equation predictive model between them was established accordingly. After soaking for 7 days, the remaining mass percentages of hydrogels in low-concentration long-time group, high-concentration short-time group, and high-concentration long-time group were significantly higher than that in low-concentration short-time group (P<0.05), and the remaining mass percentage of hydrogels in high-concentration long-time group was significantly higher than that in low-concentration long-time group (P<0.05). After 7 days of culture, a large number of live cells was observed in the 4 cell-loaded groups of hydrogels, and there was no statistically significant difference in the overall comparison of cell survival rate among the groups (P>0.05). After 10 days of osteogenic induction culture, the mRNA expressions of Runx2 and ALP in the cells of hydrogels in high-concentration long-time cell-loaded group were significantly higher than those in the other three cell-loaded groups (P<0.05), and the mRNA expressions of Sox2 and Nanog in the cells of hydrogels in low-concentration short-time cell-loaded group were significantly higher than those in the other three cell-loaded groups (P<0.05); the protein expression of Runx2 in the cells of hydrogels in high-concentration long-time cell-loaded group was the highest, and the protein expression of Nanog in the cells of hydrogels in low-concentration short-time cell-loaded group was the highest. Conclusions The porosity and Young's modulus of GelMA hydrogel synergistically regulate the biological behavior of mouse BMSCs, where high porosity and low Young's modulus are conducive to maintaining stemness, while low porosity and high Young's modulus drive osteogenic differentiation. -
参考文献
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图 1 2种浓度GelMA溶液在紫外线照射前后的形态变化。1A.37 ℃下50 g/L的GelMA溶液（左）和150 g/L的GelMA溶液（右）倾斜时均流动；1B.50 g/L的GelMA溶液（左）和150 g/L的GelMA溶液（右）经紫外线照射30 s后形成凝胶状态的GelMA水凝胶倾斜时可保持稳定不流动；1C.50 g/L的GelMA溶液（左）和150 g/L的GelMA溶液（右）经紫外线照射60 s后形成凝胶状态的GelMA水凝胶倾斜时可保持稳定不流动
注：GelMA为甲基丙烯酸酐化明胶
图 2 4组GelMA水凝胶冷冻干燥48 h后的微观形貌 扫描电子显微镜×100。2A、2B、2C、2D.分别为低浓度短时组、低浓度长时组、高浓度短时组、高浓度长时组水凝胶,均呈多孔网状结构且孔径大小不一,图2A与图2B中大孔径较多,图2C与图2D中小孔径较多
注：低浓度短时组、低浓度长时组及高浓度短时组、高浓度长时组甲基丙烯酸酐化明胶（GelMA）水凝胶分别由50 g/L的GelMA溶液光交联30、60 s及150 g/L的GelMA溶液光交联30、60 s制成
图 3 4组GelMA水凝胶的孔隙率与杨氏模量的相关性（总样本数为12）
注：低浓度短时组、低浓度长时组及高浓度短时组、高浓度长时组甲基丙烯酸酐化明胶（GelMA）水凝胶分别由50 g/L的GelMA溶液光交联30、60 s及150 g/L的GelMA溶液光交联30、60 s制成；孔隙率与杨氏模量呈显著负相关,R2=0.91,P<0.001
图 4 4组载小鼠BMSC的GelMA水凝胶中细胞培养7 d后存活情况 钙黄绿素-乙酰甲酯-碘化丙啶×20。4A、4B、4C、4D.分别为低浓度短时载细胞组、低浓度长时载细胞组、高浓度短时载细胞组、高浓度长时载细胞组,均可见大量活细胞、少量死细胞
注：低浓度短时载细胞组、低浓度长时载细胞组及高浓度短时载细胞组、高浓度长时载细胞组的载细胞甲基丙烯酸酐化明胶（GelMA）水凝胶分别由骨髓间充质干细胞（BMSC）与50 g/L的GelMA溶液混合后光交联30、60 s及BMSC与150 g/L的GelMA溶液混合后光交联30、60 s制成；活细胞阳性染色为绿色,死细胞阳性染色为红色
图 5 4组载小鼠BMSC的GelMA水凝胶中细胞成骨诱导培养10 d后Runx2和Nanog的蛋白表达情况 Alexa Fluor 488-Alexa Fluor 647-4',6-二脒基-2-苯基吲哚×40。5A、5B、5C、5D.分别为低浓度短时载细胞组、低浓度长时载细胞组、高浓度短时载细胞组、高浓度长时载细胞组Runx2蛋白表达情况,图5D中Runx2蛋白表达明显高于图5A、5B、5C；5E、5F、5G、5H.分别为低浓度短时载细胞组、低浓度长时载细胞组、高浓度短时载细胞组、高浓度长时载细胞组Nanog蛋白表达情况,图5E中Nanog蛋白表达明显高于图5F、5G、5H
注：低浓度短时载细胞组、低浓度长时载细胞组及高浓度短时载细胞组、高浓度长时载细胞组的载细胞甲基丙烯酸酐化明胶（GelMA）水凝胶分别由骨髓间充质干细胞（BMSC）与50 g/L的GelMA溶液混合后光交联30、60 s及BMSC与150 g/L的GelMA溶液混合后光交联30、60 s制成；细胞核、Runt相关转录因子2（Runx2）、Nanog阳性染色分别为蓝色、绿色、红色
Table 1. 4组载小鼠BMSC的GelMA水凝胶中细胞成骨诱导培养10 d后成骨与干性相关因子的mRNA表达比较（
组别 样本数 Runx2 ALP Sox2 Nanog 低浓度短时载细胞组 3 1.01±0.16 1.000±0.086 1.002±0.090 1.000±0.017 低浓度长时载细胞组 3 1.13±0.10 1.014±0.058 0.758±0.022 0.188±0.035 高浓度短时载细胞组 3 1.52±0.13 1.098±0.073 0.650±0.047 0.153±0.040 高浓度长时载细胞组 3 1.89±0.10 1.647±0.019 0.547±0.010 0.083±0.012 P1值 0.542 0.990 0.002 <0.001 P2值 0.002 0.220 <0.001 <0.001 P3值 <0.001 <0.001 <0.001 <0.001 P4值 0.013 0.324 0.110 0.408 P5值 <0.001 <0.001 0.004 0.005 P6值 0.015 <0.001 0.132 0.042 注：低浓度短时载细胞组、低浓度长时载细胞组及高浓度短时载细胞组、高浓度长时载细胞组的载细胞甲基丙烯酸酐化明胶（GelMA）水凝胶分别由骨髓间充质干细胞（BMSC）与50 g/L的GelMA溶液混合后光交联30、60 s及BMSC与150 g/L的GelMA溶液混合后光交联30、60 s制成；Runt相关转录因子2（Runx2）、碱性磷酸酶（ALP）、SRY盒转录因子2（Sox2）、Nanog的浓度因素主效应,F值分别为95.59、127.50、94.90、1 017.00,P值均<0.001；时间因素主效应,F值分别为14.89、75.55、36.09、874.10,P值分别为0.005、<0.001、<0.001、<0.001；两者交互作用,F值分别为3.62、68.32、6.00、618.50,P值分别为0.094、<0.001、0.040、<0.001；P1值、P2值、P3值分别为低浓度长时载细胞组、高浓度短时载细胞组、高浓度长时载细胞组与低浓度短时载细胞组各指标比较所得；P4值、P5值分别为高浓度短时载细胞组、高浓度长时载细胞组与低浓度长时载细胞组各指标比较所得；P6值为高浓度短时载细胞组与高浓度长时载细胞组各指标比较所得 
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