Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Guangxi Medical University, Nanning, P. R. China.
Guangxi Key Laboratory of Oral and Maxillofacial Rehabilitation and Reconstruction, Nanning, P. R. China.
J Dent Res. 2023 Aug;102(9):1058-1068. doi: 10.1177/00220345231176522. Epub 2023 Jun 30.
Bone defect (BD) caused by trauma, infection, congenital defects, or neoplasia is a major cause of physical limitation. Distraction osteogenesis (DO) is a highly effective procedure for bone regeneration, while the concrete mechanism remains unknown. In this study, canine DO and BD models of the mandible were established. The results of micro-computed tomography and histological staining revealed that DO led to an increased mineralized volume fraction and robust new bone formation; in contrast, BD demonstrated incomplete bone union. Mesenchymal stem cells (MSCs) from DO and BD calluses were isolated and identified. Compared with BD-MSCs, DO-MSCs were found to have a stronger osteogenic capability. Single-cell RNA sequencing analysis was further performed to comprehensively define cell differences between mandibular DO and BD calluses. Twenty-six clusters of cells representing 6 major cell populations were identified, including paired related homeobox 1-expressing MSCs (MSCs), endothelial cells (ECs), T cells, B cells, neutrophils, and macrophages. Interestingly, 2 subpopulations in MSCs in the DO group were found to express the marker of neural crest cells (NCCs) and were associated with the process of epithelial-mesenchymal transition. The immunofluorescence assay was performed to further corroborate these results in vivo and in vitro, experimentally validating that continuous distraction maintained the MSCs in an embryonic-like state. Finally, we used CRISPR/Cas9 to knock out (KO) in the context of DO, which significantly blunted the capability of jawbone regeneration, resulting in a diminished NCC-like program and reduction of new bone volume. In addition, the ability of osteogenesis, cell migration, and proliferation in cultured MSCs was inhibited. Taken together, this study provides a novel, comprehensive atlas of the cell fates in the context of DO regeneration, and MSCs act essential roles.
骨缺损（BD）由创伤、感染、先天缺陷或肿瘤引起，是身体受限的主要原因。牵张成骨（DO）是一种非常有效的骨再生方法，但其具体机制尚不清楚。本研究建立了犬下颌骨 DO 和 BD 模型。微计算机断层扫描和组织学染色的结果表明，DO 导致矿化体积分数增加和强健的新骨形成；相比之下，BD 表现为不完全骨融合。分离并鉴定了来自 DO 和 BD 骨痂的间充质干细胞（MSCs）。与 BD-MSCs 相比，DO-MSCs 具有更强的成骨能力。进一步进行单细胞 RNA 测序分析，以全面定义下颌骨 DO 和 BD 骨痂之间的细胞差异。鉴定出 26 个细胞簇，代表 6 种主要细胞群，包括成对相关同源盒 1 表达 MSC（MSCs）、内皮细胞（ECs）、T 细胞、B 细胞、中性粒细胞和巨噬细胞。有趣的是，DO 组中 MSC 的 2 个亚群被发现表达神经嵴细胞（NCCs）的标志物，与上皮-间充质转化过程相关。免疫荧光检测进一步在体内和体外证实了这些结果，实验验证了连续牵张使 MSCs 保持胚胎样状态。最后，我们使用 CRISPR/Cas9 在 DO 背景下敲除（KO），这显著削弱了颌骨再生能力，导致 NCC 样程序减少和新骨体积减少。此外，培养的 MSC 中的成骨能力、细胞迁移和增殖能力受到抑制。总之，这项研究提供了 DO 再生背景下细胞命运的全新、全面图谱，MSCs 发挥了重要作用。
