三维生物打印结合海藻酸钠和纤维蛋白原构建仿生尿道修复补片的实验研究

Experimental study on the reconstruction of biomimetic urethral flap through three-dimensional bioprinting with sodium alginate and fibrin

目的将三维(3D)生物打印技术应用于尿道修复补片的构建,并评估其力学性能、细胞活力和临床应用可行性。方法以兔尿道组织和脱细胞基质作为模板,分离兔尿道成纤维细胞,应用3D生物打印技术结合海藻酸钠和纤维蛋白原水凝胶共同制备两种负载兔成纤维细胞的仿生尿道修复补片:仿生尿道修复补片1(单纯海藻酸钠水凝胶构建的尿道补片)和仿生尿道修复补片2(在海藻酸钠水凝胶基础上使用纤维蛋白原水凝胶构建的尿道补片)。通过生物降解实验和生物力学测试分别评估仿生尿道修复补片1的生物降解性和力学性能。通过细胞活性染色,观察并计算0、3、6 d仿生尿道修复补片1和2内细胞的存活率。通过Cell Counting Kit-8(CCK-8)实验检测仿生尿道修复补片2内纤维蛋白原水凝胶中细胞的扩增速率,以分光光度计吸光度(A值)表示。结果生物降解实验结果显示,仿生尿道修复补片1在Ⅱ型胶原酶溶液和灭菌水中均有明显的降解,10 d内其在Ⅱ型胶原酶溶液中的降解速率显著快于在灭菌水中,10～20 d时的降解速率较在灭菌水中平稳,>20～<30 d时与在灭菌水中同步,30 d时其在两种溶液中的降解率均接近50%。生物力学测试结果显示,仿生尿道修复补片1的力学性能表现为线性增加,最大牵拉强度为6 N。细胞活性染色结果显示,仿生尿道修复补片1内的细胞为点状分布,无明显的舒展,细胞死亡率较高,细胞在培养0、3、6 d的存活率分别为62.91%±3.39%、41.92%±8.86%和57.11%±6.12%;仿生尿道修复补片2内细胞形态舒展,死亡率较低,在培养第6天时即有明显的细胞扩增,细胞在培养0、3、6 d的存活率分别为95.12%±1.37%、79.46%±4.77%和84.70%±8.62%。CCK-8实验结果显示,仿生尿道修复补片2内纤维蛋白原水凝胶中的细胞,在培养1、4、7、10、15 d的扩增率的A值分别为0.012 752 083、0.131 895 833、0.208 958 333、0.430 608 333、0.623 875 000,有明显的扩增表现。结论生物打印技术能够制备力学性能充足、细胞活性良好的3D立体尿道结构,证实了生物打印技术在尿道修复领域应用的可行性,为生物打印尿道的体内研究奠定了研究基础。

Objective To prepare urethral flap by using three-dimensional(3D) bioprinting technology, and to evaluate the mechanical property, cellular activity and feasibility in clinic. Methods Urethral tissue and acellular matrix of rabbit were used as the module. Fibroblasts of urethra were separated. Sodium alginate and fibrinogen were used to prepare two kinds of urethral flaps by 3 D bioprinting technique. Flap 1 was fabricated with only sodium alginate hydrogel. Flap 2 was made with fibrin hydrogel combined with sodium alginate hydrogel.The biodegradable property and mechanical strength of Flap 1 were detected with biodegradation experimentand biomechanical test. Survival rate of cells in Flap 1 and 2 were observed by vital staining on day 0,3 and 6. Cell proliferation in Flap 2 was analyzed by Cell Counting Kit-8(CCK-8) and optical density(OD) value.Results The biodegradation experiment showed obvious degradation rate of sodium alginate in both type 2 collagen and sterilized water. Compared with that in sterilized water, the degradation rate in type 2 collagen was significantly faster within 10 days, more stable between 10 and 20 days, and synchronous between 20 and 30 days. The hydrogel weight dropped to 50% within 30 days in both solutions. The mechanical strength test showed a linear increase, and the max tensile strenghth reached 6 Nmm in Flap 1. The cellular activity staining located as dots with high death rate in Flap 1, and survival rate on day 0,3 and 6 were 62.91%±3.39%, 41.92%±8.86% and 57.11%±6.12%, respectively. The cells in Flap 2 stretched out with low death rate, and the cells proliferated well on day 6;survival rate of cells on day 0,3 and 6 were 95.12%±1.37%, 79.46%±4.77% and 84.70%±8.62%. CCK-8 results showed a OD value of 0.012 752 083, 0.131 895 833, 0.208 958 333, 0.430 608 333 and 0.623 875 000 on day 1, 4, 7, 10 and 15 with good proliferation. Conclusion The experiments have proven that 3 D bioprinting technique can produce urethral flap with sufficient mechanical property and good cellular activity. The flap could be used in the urethral reconstruction.