Ronzoni Flavio L, Aliberti Flaminia, Scocozza Franca, Benedetti Laura, Auricchio Ferdinando, Sampaolesi Maurilio, Cusella Gabriella, Redwan Itedale Namro, Ceccarelli Gabriele, Conti Michele
Department of Public Health, Experimental and Forensic Medicine, Human Anatomy Unit, University of Pavia, Pavia, Italy.
Department of Biomedical Sciences, Humanitas University, Pieve Emanuele, Italy.
J Tissue Eng Regen Med. 2022 May;16(5):484-495. doi: 10.1002/term.3293. Epub 2022 Mar 4.
Skeletal muscle regeneration is one of the major areas of interest in sport medicine as well as trauma centers. Three-dimensional (3D) bioprinting (BioP) is nowadays widely adopted to manufacture 3D constructs for regenerative medicine but a comparison between the available biomaterial-based inks (bioinks) is missing. The present study aims to assess the impact of different hydrogels on the viability, proliferation, and differentiation of murine myoblasts (C2C12) encapsulated in 3D bioprinted constructs aided to muscle regeneration. We tested three different commercially available hydrogels bioinks based on: (1) gelatin methacrylate and alginate crosslinked by UV light; (2) gelatin methacrylate, xanthan gum, and alginate-fibrinogen; (3) nanofibrillated cellulose (NFC)/alginate-fibrinogen crosslinked with calcium chloride and thrombin. Constructs embedding the cells were manufactured by extrusion-based BioP and C2C12 viability, proliferation, and differentiation were assessed after 24 h, 7, 14, 21, and 28 days in culture. Although viability, proliferation, and differentiation were observed in all the constructs, among the investigated bioinks, the best results were obtained by using NFC/alginate-fibrinogen-based hydrogel from 7 to 14 days in culture, when the embedded myoblasts started fusing, forming at day 21 and day 28 multinucleated myotubes within the 3D bioprinted structures. The results revealed an extensive myotube alignment all over the linear structure of the hydrogel, demonstrating cell maturation, and enhanced myogenesis. The bioprinting strategies that we describe here denote a strong and endorsed approach for the creation of in vitro artificial muscle to improve skeletal muscle tissue engineering for future therapeutic applications.
骨骼肌再生是运动医学以及创伤中心关注的主要领域之一。如今，三维（3D）生物打印（BioP）被广泛应用于制造用于再生医学的3D构建体，但目前尚缺乏对现有基于生物材料的墨水（生物墨水）的比较。本研究旨在评估不同水凝胶对封装在3D生物打印构建体中、有助于肌肉再生的小鼠成肌细胞（C2C12）的活力、增殖和分化的影响。我们测试了三种不同的市售水凝胶生物墨水，它们分别基于：（1）通过紫外线交联的甲基丙烯酸明胶和藻酸盐；（2）甲基丙烯酸明胶、黄原胶以及藻酸盐 - 纤维蛋白原；（3）与氯化钙和凝血酶交联的纳米纤化纤维素（NFC）/藻酸盐 - 纤维蛋白原。通过基于挤出的生物打印制造嵌入细胞的构建体，并在培养24小时、7天、14天、21天和28天后评估C2C12的活力、增殖和分化。尽管在所有构建体中均观察到活力、增殖和分化，但在所研究的生物墨水中，在培养7至14天期间，使用基于NFC/藻酸盐 - 纤维蛋白原的水凝胶获得了最佳结果，此时嵌入的成肌细胞开始融合，并在第21天和第28天在3D生物打印结构内形成多核肌管。结果显示，在水凝胶的线性结构中广泛存在肌管排列，表明细胞成熟且肌生成增强。我们在此描述的生物打印策略为创建体外人工肌肉以改善骨骼肌组织工程用于未来治疗应用提供了一种强大且可靠的方法。
