Deng Rongrong, Kang Ran, Jin Xiaoyu, Wang Zihan, Liu Xin, Wang Qing, Xie Lin
Third School of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
Department of Orthopedics, Nanjing Lishui Hospital of Traditional Chinese Medicine, Nanjing, Jiangsu, China.
Front Bioeng Biotechnol. 2023 Feb 10;11:1137199. doi: 10.3389/fbioe.2023.1137199. eCollection 2023.
Mesenchymal stem cells (MSCs) and scaffolds offer promising perspectives for annulus fibrosus (AF) repair. The repair effect was linked to features of the local mechanical environment related to the differentiation of MSCs. In this study, we established a Fibrinogen-Thrombin-Genipin (Fib-T-G) gel which is sticky and could transfer strain force from AF tissue to the human mesenchymal stem cells (hMSCs) embedded in the gel. After the Fib-T-G biological gel was injected into the AF fissures, the histology scores of intervertebral disc (IVD) and AF tissue showed that Fib-T-G gel could better repair the AF fissure in caudal IVD of rats, and increase the expression of AF-related proteins including Collagen 1 (COL1), Collagen 2 (COL2) as well as mechanotransduction-related proteins including RhoA and ROCK1. To clarify the mechanism that sticky Fib-T-G gel induces the healing of AF fissures and the differentiation of hMSCs, we further investigated the differentiation of hMSCs under mechanical strain . It was demonstrated that both AF-specific genes, including Mohawk and SOX-9, and ECM markers (COL1, COL2, aggrecan) of hMSCs were up-regulated in the environment of strain force. Moreover, RhoA/ROCK1 proteins were also found to be significantly up-regulated. In addition, we further -demonstrated that the fibrochondroinductive effect of the mechanical microenvironment process could be significantly blocked or up-regulated by inhibiting the RhoA/ROCK1 pathway or overexpressing RhoA in MSCs, respectively. Summarily, this study will provide a therapeutic alternative to repair AF tears and provide evidence that RhoA/ROCK1 is vital for hMSCs response to mechanical strain and AF-like differentiation.
间充质干细胞（MSCs）和支架为纤维环（AF）修复提供了有前景的前景。修复效果与与MSCs分化相关的局部力学环境特征有关。在本研究中，我们建立了一种纤维蛋白原-凝血酶-京尼平（Fib-T-G）凝胶，其具有粘性，能够将应变力从AF组织传递至嵌入凝胶中的人间充质干细胞（hMSCs）。将Fib-T-G生物凝胶注入AF裂隙后，椎间盘（IVD）和AF组织的组织学评分表明，Fib-T-G凝胶能够更好地修复大鼠尾侧IVD中的AF裂隙，并增加包括胶原蛋白1（COL1）、胶原蛋白2（COL2）在内的AF相关蛋白以及包括RhoA和ROCK1在内的机械转导相关蛋白的表达。为了阐明粘性Fib-T-G凝胶诱导AF裂隙愈合和hMSCs分化的机制，我们进一步研究了机械应变下hMSCs的分化。结果表明，在应变力环境下，hMSCs的AF特异性基因，包括莫霍克基因和SOX-9，以及细胞外基质标志物（COL1、COL2、聚集蛋白聚糖）均上调。此外，还发现RhoA/ROCK1蛋白也显著上调。此外，我们进一步证明，分别通过抑制RhoA/ROCK1途径或在MSCs中过表达RhoA，机械微环境过程的纤维软骨诱导作用可被显著阻断或上调。总之，本研究将为修复AF撕裂提供一种治疗选择，并提供证据表明RhoA/ROCK1对于hMSCs对机械应变的反应和AF样分化至关重要。
