Recapitulation of the natural healing process is receiving increasing recognition as a strategy to induce robust tissue regeneration. Endochondral ossification has been recognized as an essential reparative approach in natural jawbone defect healing. However, such an approach has been overlooked in the recent development of cell-based therapeutics for jawbone repair. Therefore, this study aimed to explore a bioinspired stem cell-based strategy for jawbone repair by mimicking the mesenchymal condensation of progenitor cells during the early endochondral ossification process. For this purpose, passage 3 of jawbone periosteum-derived cells (jb-PDCs) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 96.4 ± 5.8 µm after 48 h. Compared to monolayer culture, the jb-PDC spheroids showed a significant reduction of stemness marker expression evidenced by flow cytometry. Furthermore, a significant upregulation of chondrogenic transcription factor SOX9 in both gene and protein levels was observed in the jb-PDC spheroids after 48 h of chondrogenic induction. RNA sequencing and Western blotting analysis further suggested that the enhanced SOX9-mediated chondrogenic differentiation in jb-PDC spheroids was attributed to the activation of the p38 MAPK pathway. Impressively, inhibition of p38 kinase activity significantly attenuated chondrogenic differentiation jb-PDC spheroids, evidenced by a significant decline of SOX9 in both gene and protein levels. Strikingly, the jb-PDC spheroids implanted in 6- to 8-wk-old male C57BL/6 mice with critical-size jawbone defects (1.8 mm in diameter) showed an evident contribution to cartilaginous callus formation after 1 wk, evidenced by histological analysis. Furthermore, micro-computed tomography analysis showed that the jb-PDC spheroids significantly accelerated bone healing after 2 wk in the absence of exogenous growth factors. In sum, the presented findings represent the successful development of cell-based therapeutics to reengineer the endochondral bone repair process and illustrate the potential application to improve bone repair and regeneration in the craniofacial skeleton.

中文翻译：

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使用骨膜细胞球体进行颌骨缺损的软骨内修复。

自然愈合过程的重现作为诱导强健组织再生的策略越来越受到认可。软骨内骨化已被认为是自然颌骨缺损愈合的重要修复方法。然而，在最近开发的基于细胞的颌骨修复疗法中，这种方法被忽视了。因此，本研究旨在通过模拟早期软骨内骨化过程中祖细胞的间充质凝结，探索基于生物干细胞的颌骨修复策略。为此，在我们之前报道的非贴壁微孔（直径 200 µm，深度 148 µm，间距 100 µm）中培养第 3 代颌骨骨膜衍生细胞 (jb-PDC)，并自组装成球体48 小时后直径为 96.4 ± 5.8 µm。与单层培养相比，流式细胞术证明 jb-PDC 球体的干性标记表达显着减少。此外，在软骨形成诱导 48 小时后，在 jb-PDC 球体中观察到软骨形成转录因子 SOX9 在基因和蛋白质水平上显着上调。RNA测序和Western印迹分析进一步表明，SOX9介导的jb-PDC球体中软骨分化的增强归因于p38 MAPK通路的激活。令人印象深刻的是，抑制 p38 激酶活性显着减弱 jb-PDC 球体的软骨形成分化，SOX9 基因和蛋白质水平的显着下降证明了这一点。引人注目的是，组织学分析证明，植入具有临界大小颌骨缺损（直径 1.8 毫米）的 6 至 8 周龄雄性 C57BL/6 小鼠中的 jb-PDC 球体在 1 周后对软骨愈伤组织形成有明显贡献。此外，微型计算机断层扫描分析表明，在没有外源生长因子的情况下，jb-PDC 球体在 2 周后显着加速骨愈合。总之，所提出的研究结果代表了基于细胞的疗法的成功开发，以重新设计软骨内骨修复过程，并说明了改善颅面骨骼骨修复和再生的潜在应用。