不同浓度GelMA水凝胶支架对大鼠髓核干细胞及细胞外基质相关蛋白的影响

Effect of GelMA hydrogel scaffolds with different concentrations on the rat nucleus pulposus stem cells and expression of extracellular matrix related proteins

目的探讨不同浓度的甲基丙烯酸酯(GelMA)水凝胶支架的性能,以及对大鼠髓核干细胞(NPSCs)体外增殖、分化及细胞外基质相关蛋白表达的影响。方法(1)配置浓度为5%、10%、15%GelMA水凝胶溶液,经紫外光照射固化、冷冻干燥、喷金处理,制备相应浓度的GelMA水凝胶支架。用扫描电镜观察支架并测量孔径,使用科学表面分析仪测量支架静态水接触角,使用机械测试仪测量支架压缩模量,使用精密天平测量在37℃恒温箱中放置0、2、4、6、8、10、12 h时支架的含水量。(2)取6周龄雄性清洁级SD大鼠8只,体质量为80~100 g,切开椎间盘取髓核组织,分离培养NPSCs。取第3代NPSCs分别进行成骨、成软骨及成脂分化培养,分别用茜素红、阿利新蓝及油红O对三系细胞进行染色,观察NPSCs的三系分化潜能。(3)取第3代NPSCs分为无支架的对照组和5%、10%、15%GelMA水凝胶支架组,对照组加入培养基培养3 d,不同浓度GelMA水凝胶支架组紫外光照射固化后接种NPSCs,加入培养基培养7 d。各组细胞采用免疫荧光染色,观察细胞形态的变化。(4)取第3代NPSCs分别接种于紫外光照射固化后的5%、10%、15%GelMA凝胶支架中,采用细胞计数(CCK)-8试剂盒检测细胞增殖能力,采用活细胞/死细胞双染试剂盒检测细胞活力,采用免疫荧光染色检测Agg和ColⅡ蛋白的表达情况。结果(1)5%、10%、15%GelMA水凝胶支架的孔径分别为(94.00±1.00)、(77.33±5.69)、(52.33±2.31)μm,水接触角分别为28.00°±2.65°、39.00°±2.65°、46.00°±1.00°,压缩模量分别为(45.77±8.66)、(64.63±3.06)、(86.07±10.73)kPa。不同浓度GelMA水凝胶支架随浓度的增高,支架的孔径减小,水接触角、压缩模量增大,差异均有统计学意义(F=102.40、49.40、21.51,P值均<0.05)。不同浓度GelMA水凝胶支架的含水量均随时间的增加而下降;而同一时间不同浓度GelMA水凝胶支架的含水量随浓度的增高而减少,3者间含水量比较差异均有统计学意义(P值均<0.05)。(2)NPSCs三系分化结果显示,培养的NPSCs成功分化为成骨细胞、成软骨细胞、成脂细胞,有多向分化的潜能,具备干细胞的特性。(3)水凝胶支架上NPSCs随着GelMA浓度的增加,细胞形态由正常的梭形逐渐变椭圆,细胞突伸展逐渐减弱,细胞质和细胞核比例逐渐减小,细胞聚集的集落逐渐变小。(4)NPSCs增殖实验显示:培养第1天时,不同浓度的GelMA水凝胶支架上NPSCs的光密度(OD)值差异无统计学意义(F=1.63,P=0.272);培养第4、7天时,随GelMA水凝胶支架浓度的增高,NPSCs的OD值均呈下降趋势,差异均有统计学意义(F=61.48、203.20,P值均<0.001)。NPSCs活力实验显示:培养第4天时,不同浓度的GelMA水凝胶支架上NPSCs活细胞占比差异无统计学意义(F=0.15,P=0.860);培养第7天时,随GelMA水凝胶支架浓度的增高,NPSCs活细胞占比呈下降趋势,差异有统计学意义(F=68.83,P<0.001)。(5)5%、10%、15%GelMA水凝胶支架中Agg蛋白的免疫荧光值分别为16.79±1.29、13.35±0.70、10.475±0.54,ColⅡ蛋白的免疫荧光值分别为17.17±1.49、13.32±1.65、9.53±1.01。随着GelMA水凝胶浓度的增高,Agg、ColⅡ蛋白的表达均呈下降趋势,差异均有统计学意义(F=22.10、36.64,P值均<0.001)。结论5%GelMA水凝胶支架具有较好的物理特性,适合NPSCs的生长、存活,能较好地促进细胞外基质相关蛋白的表达。

Objective To investigate the properties of hydrogel scaffolds with different concentrations of glycol methacrylate(GelMA)and their effects on the proliferation,differentiation,and expression of extracellular matrix-related proteins in rat nucleus pulposus stem cells(NPSCs)in vitro.Methods(1)Configuration of GelMA hydrogel scaffolds with concentrations of 5%,10%,and 15%;ultraviolet light curing;freeze-drying;and gold-spraying treatment.For different concentrations of GelMA hydrogel scaffolds,a scanning electron microscope was used to observe the characteristics of scaffold pore changes,a scientific surface analyzer was used to measure the scaffold static water contact angle,a mechanical tester was used to measure the scaffold compression modulus,and a precision balance was utilized to measure the water content of the scaffolds when they were placed in a constant temperature box at 37℃for 0,2,4,6,8,10,and 12 h.(2)Eight 6-week-old male clean-grade SD rats with body mass of 80-100 g were taken.Their intervertebral discs were incised to take the nucleus pulposus tissue,and the NPSCs were isolated and cultured.The third-generation NPSCs were taken for osteogenic,chondrogenic,and adipogenic differentiation cultures.The cells of the three lineages were stained with alizarin red,Alcian blue,and oil red O,respectively,to observe the three lineage differentiation potentials of the NPSCs.(3)The third-generation NPSCs were taken and divided into a control group without scaffold and 5%,10%,and 15%GelMA hydrogel scaffold groups.The control group was added to complete medium for 3 days,and the different concentrations of the GelMA hydrogel scaffold group were cured by ultraviolet light irradiation to inoculate the NPSCs and added to complete medium for 7 d.The cells in each group were stained by immunofluorescence to observe the changes in cell morphology.(4)The third-generation NPSCs were inoculated into 5%,10%,and 15%GelMA gel scaffolds cured by ultraviolet light irradiation.The cell proliferation ability was detected by using a cell countingit-8,cell viability was detected by using a live/dead cell double-staining kit,and the expression of Agg and ColⅡproteins was detected by immunofluorescence staining.Results(1)The pore diameters of 5%,10%,and 15%GelMA hydrogel scaffolds were(94.00±1.00),(77.33±5.69),and(52.33±2.31)μm,respectively;the water contact angles were 28.00°±2.65°,39.00°±2.65°,46.00°±1.00°,respectively;and the compression modulus was(45.77±8.66),(64.63±3.06),(86.07±10.73)kPa,respectively.The pore size of the scaffolds decreased,and the water contact angle and compression modulus increased with increasing concentration of GelMA hydrogel scaffolds,and the differences were statistically significant(F=102.40,49.40,21.51;all P values<0.05).The water content of GelMA hydrogel scaffolds of different concentrations showed a decreasing trend with the increase in time.Between GelMA hydrogel scaffolds of different concentrations at the same time,the water content decreased with the increase in concentration,and the differences were all statistically significant(all P values<0.05).(2)The results of the three-lineage differentiation of NPSCs showed that the cultured NPSCs successfully differentiated into osteoblasts,chondrocytes,and lipocytes,with multidirectional differentiation potential and the characteristics of stem cells.(3)The cell morphology of NPSCs on hydrogel scaffolds gradually changed from a normal shuttle shape to an oval shape with the increase in GelMA concentration,the extension of cytosolic protrusions gradually weakened,the ratio of cytoplasm to nucleus gradually decreased,and the aggregated colonies of cells gradually became smaller.(4)The proliferation experiment of NPSCs showed that the difference of OD value of NPSCs on GelMA hydrogel scaffolds with different concentrations was not statistically significant on the first day of culture(F=1.63,P=0.272),and the OD value of NPSCs showed a decreasing trend with the increase in GelMA hydrogel scaffold concentration on the fourth and seventh days of culture,with statistically significant differences(F=61.48,203.20;all P values<0.001).The NPSC viability assay showed that the difference in the percentage of live cells of NPSCs on GelMA hydrogel scaffolds with different concentrations was not statistically significant on the fourth day of culture(F=0.15,P=0.860),and the percentage of live cells of NPSCs showed a decreasing trend with the increasing concentration of GelMA hydrogel scaffolds on the seventh day of culture;the difference was statistically significant(F=68.83,P<0.001).(5)The immunofluorescence values of Agg protein in 5%,10%,and 15%GelMA hydrogel scaffolds were 16.79±1.29,13.35±0.70,and 10.475±0.54,respectively,and the immunofluorescence values of ColⅡprotein were 17.17±1.49,13.32±1.65,and 9.53±1.01,respectively.As the concentration increased,the expression of Agg and Col II proteins showed a decreasing trend,and the differences were all statistically significant(F=22.10,36.64;all P values<0.001).Conclusion The 5%GelMA hydrogel scaffold has good physical properties,which is suitable for the growth and survival of NPSCs,and can better promote the expression of extracellular matrix-related proteins.