Effects of tensile force on the vascular lumen formation in three-dimensional printed tissue
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摘要:
目的 探讨牵张力对三维打印组织血管管腔形成的影响。 方法 采用实验研究方法。取2020年9月—2021年5月于苏州瑞华骨科医院妇产科生产的3名健康产妇（年龄22~35岁）弃用的脐带组织，提取人脐静脉内皮细胞（HUVEC）；取2020年9月—2022年9月于苏州瑞华骨科医院手外科行创面修复术的10例男性患者（年龄20~45岁）的废弃正常皮肤组织，提取人皮肤成纤维细胞（HSF）。鉴定2种细胞后取第4~6代细胞进行后续实验。以HUVEC、HSF为种子细胞，以聚己内酯、明胶、透明质酸、纤维蛋白原为支架材料，借助三维生物打印技术，构建三维打印血管化组织。将间距6、10 mm的聚己内酯支架及无聚己内酯支架的打印组织分别设为6 mm间距聚己内酯组、10 mm间距聚己内酯组和无聚己内酯组。培养4 d，取10 mm间距聚己内酯组打印组织，采用细胞活力检测试剂盒检测细胞存活情况并计算细胞存活率。培养14 d，取3组打印组织，肉眼观察组织形变情况；行免疫荧光染色，观察组织中纤丝状肌动蛋白排列，观测组织血管管腔直径、总长度与分支数。设计并打印另带有微弹簧结构的上述3组组织，培养9 d，根据力-位移曲线测量打印组织所受牵张力。以上实验样本数均为3。对数据行单因素方差分析、Tukey检验。 结果 培养4 d，10 mm间距聚己内酯组打印组织细胞存活率为（91.3±2.2）%。培养14 d，无聚己内酯组打印组织形变不明显，6 mm间距聚己内酯组和10 mm间距聚己内酯组打印组织发生明显形变。培养14 d，无聚己内酯组打印组织纤丝状肌动蛋白的排列无特定方向，而6 mm间距聚己内酯组和10 mm间距聚己内酯组打印组织中纤丝状肌动蛋白的排列具有方向性。培养14 d，6 mm间距聚己内酯组和10 mm间距聚己内酯组打印组织血管管腔直径分别为（6.0±1.3）、（10.8±1.3）μm，均明显大于无聚己内酯组的0 μm（ P<0.05），10 mm间距聚己内酯组打印组织血管管腔直径明显大于6 mm间距聚己内酯组（ P<0.05）；6 mm间距聚己内酯组和10 mm间距聚己内酯组打印组织血管总长度、分支数均明显短/少于无聚己内酯组（ P<0.05），10 mm间距聚己内酯组打印组织血管总长度、分支数均明显短/少于6 mm间距聚己内酯组（ P<0.05）。培养9 d，6 mm间距聚己内酯组和10 mm间距聚己内酯组打印组织受到的牵张力分别为（2 340±59）、（4 284±538）μN，均明显大于无聚己内酯组的0 μN（ P<0.05）；10 mm间距聚己内酯组打印组织受到的牵张力明显大于6 mm间距聚己内酯组（ P<0.05）。 结论 三维打印支架结构能对打印组织施加不同大小的牵张力，可通过调控牵张力大小调控打印组织的血管管腔直径。 Abstract:Objective To explore the effects of tensile force on vascular lumen formation in three-dimensional printed tissue. Methods The experimental research method was used. Human umbilical vein endothelial cells (HUVECs) were extracted from discarded umbilical cord tissue of 3 healthy women (aged 22 to 35 years) who gave birth in the Department of Gynaecology and Obstetrics of Suzhou Ruihua Orthopaedic Hospital from September 2020 to May 2021. Human skin fibroblasts (HSFs) were extracted from discarded normal skin tissue of 10 male patients (aged 20 to 45 years) who underwent wound repair in the Department of Hand Surgery of Suzhou Ruihua Orthopaedic Hospital from September 2020 to September 2022. After identification of the two kinds of cells, the 4 th to 6 th passage of cells were taken for the follow-up experiments. HUVECs and HSFs were used as seed cells, and polycaprolactone, gelatin, hyaluronic acid, and fibrin were used as scaffold materials, and the three-dimensional printed vascularized tissue was created by three-dimensional bioprinting technology. The printed tissue with polycaprolactone scaffold of 6 and 10 mm spacing, and without polycaprolactone scaffold were set as 6 mm spacing polycaprolactone group, 10 mm spacing polycaprolactone group, and non-polycaprolactone group, respectively. After 4 days of culture, the printed tissue in 10 mm spacing polycaprolactone group was selected to detect the cell survival by cell viability detection kit, and the cell survival rate was calculated. After 14 days of culture, the printed tissue in three groups were taken, and the shape change of tissue was observed by naked eyes; immunofluorescence staining was performed to observe the arrangement of filamentous actin, and lumen diameter, total length, and number of branches of vessel in the tissue. The tissue with micro-spring structure in the above-mentioned three groups was designed, printed, and cultured for 9 days, and the tensile force applied in the printed tissue was measured according to the force-displacement curve. The number of samples was all 3 in the above experiments. Data were statistically analyzed with one-way analysis of variance and Tukey test. Results After 4 days of culture, the cell survival rate in printed tissue in 10 mm spacing polycaprolactone group was (91.3±2.2)%. After 14 days of culture, the shape change of printed tissue in non-polycaprolactone group was not obvious, while the shape changes of printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were obvious. After 14 days of culture, the arrangement of filamentous actin in the printed tissue in non-polycaprolactone group had no specific direction, while the arrangement of filamentous actin in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group had a specific direction. After 14 days of culture, The vascular lumen diameters of the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were (6.0±1.3) and (10.8±1.3) μm, respectively, which were significantly larger than 0 μm in non-polycaprolactone group ( P<0.05), and the vascular lumen diameter of printed tissue in 10 mm spacing polycaprolactone group was significantly larger than that in 6 mm spacing polycaprolactone group ( P<0.05); the total length and number of branches of blood vessel in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were significantly shorter or less than those in non-polycaprolactone group ( P<0.05), and the total length and number of branches of blood vessel in the printed tissue in 10 mm spacing polycaprolactone group were significantly shorter or less than those in 6 mm spacing polycaprolactone group. After 9 days of culture, the tensile forces applied in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were (2 340±59) and (4 284±538) μN, respectively, which were significantly higher than 0 μN in non-polycaprolactone group ( P<0.05), and the tensile force applied in the printed tissue in 10 mm spacing polycaprolactone group was significantly higher than that in 6 mm spacing polycaprolactone group ( P<0.05). Conclusions The three-dimensional printed scaffold structure can exert different tensile force in the printed tissue, and the vascular lumen diameter of the printed tissue can be regulated by adjusting the tensile force. -
参考文献
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1 10 mm间距聚己内酯组打印组织三维生物打印主要过程。1A.打印2条聚己内酯支架；1B.打印明胶层后；1C.打印含HUVEC和HSF的水凝胶后；1D.再次打印2条聚己内酯支架后
注：HUVEC为人脐静脉内皮细胞，HSF为人皮肤成纤维细胞
2 10 mm间距聚己内酯组打印组织培养4 d HUVEC和HSF存活情况 倒置荧光显微镜×100。2A、2B.分别为活细胞、死细胞染色；2C.活细胞与死细胞染色重叠图片，细胞存活情况良好
注：HUVEC为人脐静脉内皮细胞，HSF为人皮肤成纤维细胞；活细胞染色为绿色，死细胞染色为红色
3 3组打印组织培养14 d形变情况。3A、3B、3C.分别为无聚己内酯组、6 mm间距聚己内酯组和10 mm间距聚己内酯组，图3A打印组织形变不明显，图3B、3C打印组织发生明显形变
4 3组打印组织培养14 d纤丝状肌动蛋白的排列情况 四甲基异硫氰酸罗丹明-鬼笔环肽-4′，6-二脒基-2-苯基吲哚×100。4A.无聚己内酯组打印组织纤丝状肌动蛋白排列无特定方向；4B、4C.分别为6 mm间距聚己内酯组、10 mm间距聚己内酯组打印组织，纤丝状肌动蛋白排列均具有方向性
注：纤丝状肌动蛋白阳性染色为红色，细胞核染色为蓝色
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