Effects of immune responses mediated by topological structures of three-dimensional bioprinted scaffolds on hair follicle cycle in mice
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摘要:
目的 探讨具有不同拓扑结构的三维生物打印支架介导的免疫反应对小鼠毛囊周期的影响。 方法 该研究为实验研究。将海藻酸钠-明胶复合水凝胶用三维生物打印机打印成3种支架，并按照支架的3种拓扑结构（打印时打印喷头的旋转角度分别为45°、60°、90°），分别命名为T45支架、T60支架、T90支架，肉眼观察3种支架交联后的形态。取9只8周龄雌性C57BL/6J小鼠，按随机数字表法分为T45组、T60组、T90组，每组3只，分别于背部皮下埋植T45、T60、T90支架。于植入后7 d，观察小鼠背部脱毛区毛发生长情况，行苏木精-伊红染色观测支架周围的纤维囊厚度，行免疫荧光染色检测支架周围组织中CD68、骨形态发生蛋白2（BMP-2）、肿瘤坏死因子（TNF）蛋白的表达水平。以上实验样本数均为3。 结果 3种支架交联后均拓扑结构明显，保真度高。植入后7 d，T45组、T90组小鼠背部脱毛区毛发均生长明显；T60组小鼠支架植入区毛发生长缓慢，与未植入区差别明显。植入后7 d，与T90组［（18±4）μm］相比，T45组、T60组小鼠支架周围的纤维囊厚度［（39±4）、（55±8）μm］均明显增加（ P<0.05）；与T45组相比，T60组小鼠支架周围的纤维囊厚度明显增加（ P<0.05）。植入后7 d，T60组小鼠支架周围组织中CD68蛋白的表达水平明显高于T45组和T90组（ P值均<0.05）；T60组小鼠支架周围组织中BMP-2蛋白的表达水平明显高于T45组、T90组（ P值均<0.05），T45组小鼠支架周围组织中BMP-2蛋白的表达水平明显高于T90组（ P<0.05）；T60组小鼠支架周围组织中TNF蛋白的表达水平明显低于T45组和T90组（ P值均<0.05）。 结论 具有不同拓扑结构的三维生物打印支架植入小鼠体内后会介导不同程度的免疫反应。适度的免疫反应可促进小鼠脱毛区毛发生长，过强的免疫反应抑制毛囊进入生长期。 Abstract:Objective To explore the effects of the immune responses mediated by topological structures of three-dimensional bioprinted scaffolds on hair follicle cycle in mice. Methods The study was an experimental research. The alginate-gelatin composite hydrogels were printed into scaffolds using a three-dimensional bioprinter and named T45 scaffolds, T60 scaffolds, and T90 scaffolds according to the 3 topological structures of the scaffolds (the rotation angles of the printhead during printing were 45°, 60°, and 90°, respectively), and the morphology of the three scaffolds was observed after cross-linking by naked eyes. Nine 8-week-old female C57BL/6J mice were divided into T45 group, T60 group, and T90 group, according to the random number table, with three mice in each group, and the T45, T60, and T90 scaffolds were subcutaneously implanted on the back of mice, respectively. On post implantation day (PID) 7, the hair growth in the dorsal depilated area of mice was observed, the thickness of the fiber capsule around the scaffolds was observed by hematoxylin-eosin staining, and the expression levels of CD68, bone morphogenetic protein-2 (BMP-2), and tumor necrosis factor (TNF) protein in the tissue surrounding the scaffolds were observed by immunofluorescence staining. The samples of the above experiments were all 3. Results The topological structures of the three scaffolds were all clear with high fidelity after cross-linking. On PID 7, the hair growth was obvious in the dorsal depilated area of mice in T45 group and T90 group, while hair growth was slow in the scaffold implantation area of mice in T60 group, which was significantly different from that of the unimplanted area. On PID 7, compared with (18±4) μm in T90 group, the thickness of both the fiber capsule around the scaffolds ((39±4) and (55±8) μm) of mice in T45 group and T60 group was significantly increased ( P<0.05); the thickness of the fiber capsule around the scaffolds of mice in T60 group was also significantly increased compared with that in T45 group ( P<0.05). On PID 7, the expression level of CD68 protein in the tissue surrounding the scaffolds of mice in T60 group was significantly higher than the levels in T45 group and T90 group (with both P values <0.05). The expression level of BMP-2 protein in the tissue surrounding the scaffolds of mice in T60 group was significantly higher than the levels in T45 group and T90 group (with both P values <0.05), and the expression level of BMP-2 protein in the tissue surrounding the scaffolds of mice in T45 group was significantly higher than that in T90 group ( P<0.05). The expression level of TNF protein in the tissue surrounding the scaffolds of mice in T60 group was significantly lower than the levels in T45 group and T90 group (with both P values <0.05). Conclusions Three-dimensional bioprinted scaffolds with different topological structures mediate different degrees of immune responses after being implanted in mice. A moderate immune response promotes hair growth in depilated area of mice, while an excessive immune response results inhibits the hair follicle entering into the anagen phase. -
Key words:
- Tissue engineering /
- Printing, three-dimensional /
- Immunity /
- Tissue scaffolds /
- Hair follicle /
- Topology
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1 3种拓扑结构的三维生物打印海藻酸钠-明胶复合水凝胶支架形态。1A、1B、1C.分别为交联后的T45、T60、T90支架，形态均与模型一致
注：T45、T60、T90中T指拓扑结构，45、60、90指三维生物打印支架时打印喷头的旋转角度，各图中右上角为模型图像
2 3组小鼠皮下埋植具有不同拓扑结构的三维生物打印海藻酸钠-明胶复合水凝胶支架7 d后的大体形态。2A、2B、2C.分别为T45组、T60组、T90组支架植入后的大体形态，图2A、2C中支架植入区与未植入区的毛发生长情况相似，图2B中支架植入区毛发生长缓慢、皮肤呈粉红色
注：T45、T60、T90中T指拓扑结构，45、60、90指三维生物打印支架时打印喷头的旋转角度；黄色虚线内区域为支架植入区
3 3组小鼠皮下埋植具有不同拓扑结构的三维生物打印海藻酸钠-明胶复合水凝胶支架7 d后支架周围组织病理变化 苏木精-伊红×100。3A、3B、3C.分别为T45组、T60组、T90组支架未降解部分及其周围的皮肤组织，各组支架周围均形成纤维囊，相对于图3A、3C，图3B中支架周围的纤维囊较厚
注：T45、T60、T90中T指拓扑结构，45、60、90指三维生物打印支架时打印喷头的旋转角度；红色箭头指示纤维囊，蓝色箭头指示未降解的支架
4 3组小鼠皮下埋植具有不同拓扑结构的三维生物打印海藻酸钠-明胶复合水凝胶支架7 d后支架周围组织中CD68、BMP-2、TNF蛋白的表达水平 Alexa Fluor 488-4′，6-二脒基-2-苯基吲哚×200。4A、4B、4C.分别为T45组、T60组、T90组支架周围组织中CD68蛋白的表达水平，图4B中CD68蛋白的表达水平高于图4A、4C；4D、4E、4F.分别为T45组、T60组、T90组支架周围组织中BMP-2蛋白的表达水平，图4E中BMP-2蛋白的表达水平高于图4D、4F；4G、4H、4I.分别为T45组、T60组、T90组支架周围组织中TNF蛋白的表达水平，图4H中TNF蛋白的表达水平低于图4G、4I
注：BMP-2为骨形态发生蛋白2、TNF为肿瘤坏死因子；CD68、BMP-2、TNF蛋白阳性表达均为绿色；T45、T60、T90中T指拓扑结构，45、60、90指三维生物打印支架时打印喷头的旋转角度
表1 3组小鼠皮下埋植具有不同拓扑结构的三维生物打印海藻酸钠-明胶复合水凝胶支架7 d后支架周围组织中3种蛋白的表达比较（
表1. Comparison of the expressions of 3 proteins in the tissue surrounding the scaffolds of subcutaneously implanted three-dimensional bioprinted scaffolds of alginate-gelation composite hydrogels with different topological structures in 3 groups of mice after 7 days
组别 样本数 CD68 BMP-2 TNF T45组 3 23.9±2.0 6.1±0.5 15.4±3.5 T60组 3 63.8±12.5 17.5±0.8 8.0±0.9 T90组 3 25.4±2.2 2.4±0.9 15.4±2.3 F值 27.58 177.60 8.95 P值 0.001 <0.001 0.016 P 1值 0.002 <0.001 0.032 P 2值 >0.999 0.003 >0.999 P 3值 0.002 <0.001 0.031 注：BMP-2为骨形态发生蛋白2，TNF为肿瘤坏死因子；T45、T60、T90中T指拓扑结构，45、60、90指三维生物打印支架时打印喷头的旋转角度； F值、 P值为3组间各指标总体比较所得； P 1值、 P 2值分别为T45组与T60组、T90组比较所得， P 3值为T60组与T90组比较所得 
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