Li Ting, Hou Juedong, Wang Ling, Zeng Guanjie, Wang Zihan, Yu Liu, Yang Qiao, Yin Junfeiyang, Long Meng, Chen Lizhi, Chen Siyuan, Zhang Hongwu, Li Yanbing, Wu Yaobin, Huang Wenhua
Guangdong Engineering Research Center for Translation of Medical 3D Printing Application, Guangdong Provincial Key Laboratory of Medical Biomechanics, Department of Human Anatomy, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.
Biomaterials Research Center, School of Biomedical Engineering, Southern Medical University, Guangzhou Guangdong 510515, China.
Acta Biomater. 2023 Jan 15;156:21-36. doi: 10.1016/j.actbio.2022.08.037. Epub 2022 Aug 21.
Viscoelastic hydrogels can enhance 3D cell migration and proliferation due to the faster stress relaxation promoting the arrangement of the cellular microenvironment. However, most synthetic photocurable hydrogels used as bioink materials for 3D bioprinting are typically elastic. Developing a photocurable hydrogel bioink with fast stress relaxation would be beneficial for 3D bioprinting engineered 3D skeletal muscles in vitro and repairing volumetric muscle loss (VML) in vivo; however, this remains an ongoing challenge. This study aims to develop an interpenetrating network (IPN) hydrogel with tunable stress relaxation using a combination of gelatin methacryloyl (GelMA) and fibrinogen. These IPN hydrogels with faster stress relaxation showed higher 3D cellular proliferation and better differentiation. A 3D anisotropic biomimetic scaffold was further developed via a printing gel-in-gel strategy, where the extrusion printing of cell-laden viscoelastic FG hydrogel within Carbopol supported gel. The 3D engineered skeletal muscle tissue was further developed via 3D aligned myotube formation and contraction. Furthermore, the cell-free 3D printed scaffold was implanted into a rat VML model, and both the short and long-term repair results demonstrated its ability to enhance functional skeletal muscle tissue regeneration. These data suggest that such viscoelastic hydrogel provided a suitable 3D microenvironment for enhancing 3D myogenic differentiation, and the 3D bioprinted anisotropic structure provided a 3D macroenvironment for myotube organization, which indicated the potential in skeletal muscle engineering and VML regeneration. STATEMENT OF SIGNIFICANCE: The development of a viscoelastic 3D aligned biomimetic skeletal muscle scaffold has been focused on skeletal muscle regeneration. However, a credible technique combining viscoelastic hydrogel and printing gel-in-gel strategy for fabricating skeletal muscle tissue was rarely reported. Therefore, in this study, we present an interpenetrating network (IPN) hydrogel with fast stress relaxation for 3D bioprinting engineered skeletal muscle via a printing gel-in-gel strategy. Such IPN hydrogels with tunable fast stress relaxation resulted in high 3D cellular proliferation and adequate differentiation in vitro. Besides, the 3D hydrogel-based scaffolds also enhance functional skeletal muscle regeneration in situ. We believe that this study provides several notable advances in tissue engineering that can be potentially used for skeletal muscle injury treatment in clinical.
由于更快的应力松弛促进了细胞微环境的排列，粘弹性水凝胶可以增强三维细胞迁移和增殖。然而，大多数用作三维生物打印生物墨水材料的合成光固化水凝胶通常是弹性的。开发一种具有快速应力松弛的光固化水凝胶生物墨水将有利于体外三维生物打印工程化三维骨骼肌以及体内修复体积性肌肉损失（VML）；然而，这仍然是一个持续存在的挑战。本研究旨在通过明胶甲基丙烯酰（GelMA）和纤维蛋白原的组合开发一种具有可调应力松弛的互穿网络（IPN）水凝胶。这些具有更快应力松弛的IPN水凝胶显示出更高的三维细胞增殖和更好的分化。通过凝胶包凝胶打印策略进一步开发了一种三维各向异性仿生支架，即在卡波姆支撑凝胶内挤压打印载有细胞的粘弹性FG水凝胶。通过三维排列的肌管形成和收缩进一步构建了三维工程化骨骼肌组织。此外，将无细胞的三维打印支架植入大鼠VML模型中，短期和长期修复结果均表明其能够增强功能性骨骼肌组织再生。这些数据表明，这种粘弹性水凝胶为增强三维成肌分化提供了合适的三维微环境，而三维生物打印的各向异性结构为肌管组织提供了三维宏观环境，这表明其在骨骼肌工程和VML再生方面具有潜力。重要性声明：具有粘弹性的三维排列仿生骨骼肌支架的开发一直聚焦于骨骼肌再生。然而，很少有报道将粘弹性水凝胶和凝胶包凝胶打印策略相结合来制造骨骼肌组织的可靠技术。因此，在本研究中，我们通过凝胶包凝胶打印策略展示了一种用于三维生物打印工程化骨骼肌的具有快速应力松弛的互穿网络（IPN）水凝胶。这种具有可调快速应力松弛的IPN水凝胶在体外导致了高三维细胞增殖和充分分化。此外，基于三维水凝胶的支架还能原位增强功能性骨骼肌再生。我们相信，本研究在组织工程方面取得了一些显著进展，有可能用于临床骨骼肌损伤治疗。
