Genetic regulation of the cartilage callus during large-scale bone repair

广泛骨损伤的修复仍然是一个尚未满足的临床挑战。通过开发两个 斑马鱼和小鼠大规模骨再生的新的和互补的模型 目的了解软骨骨痂在大段全厚软骨形成中的作用。 骨头。虽然骨膜在动态平衡期间会产生成骨细胞，但特定的 构建修复愈伤组织的亚群仍然没有明确的定义。使用转基因血统 追踪，我们提供了令人信服的初步证据表明，一种罕见的双功能Sox9/Runx2 骨膜群在修复过程中会产生新的软骨和骨。这一新发现的能力 标记、操纵和分离特定的干细胞群体使我们能够测试 肋骨非凡的再生能力是由于其骨膜茎的独特性质。 细胞。在目标1中，我们与骨科医生合作测试肋骨中Sox9细胞 骨膜可以在培养中扩张，用于修复临界大小的股骨缺损。这个 软骨骨痂的形成是骨修复的常见特征，但骨膜如何 只有在修复过程中才会产生软骨仍然是个谜。在初步数据中，我们发现 当斑马鱼或斑马鱼中的ihha配体缺失时，软骨骨痂严重受损 小鼠Sox9细胞中的HH受体Smo被删除。在目标2中，我们测试这反映了 IHH的修复特异性作用，与其在成骨细胞中的发育作用明显不同 分化和软骨细胞增殖。此外，我们的初步数据表明，这些 IHH诱导的修复软骨细胞与生长板中的软骨细胞有重要的不同，因为 修复软骨细胞即使在肥大前期也能共表达成骨细胞基因。这 成骨特性的增加随后与软骨细胞转化为 骨细胞。在目标3中，我们测试修复和发育的软骨细胞是否代表不同的 通过比较不同成熟阶段的全球基因表达来确定细胞类型。我们还使用 谱系追踪以量化软骨来源的骨细胞优先的程度 在修复过程中有助于形成完整的骨骼。最后，我们使用了强大的新染色质 可及性分析测试随着Sox9骨膜细胞变成软骨，它们的更大 成骨特性是保持稳定的成骨细胞增强剂的结果。我们的发现 将揭示一组稀有的骨膜干细胞如何被诱导形成软骨 在损伤期间，以及如何使用这种特殊的修复软骨来再生完整的厚度 骨头。对这些重要干细胞的更好理解将为未来的PRE奠定基础 旨在治愈身体其他部位大规模骨骼损伤的临床试验。

The repair of extensive bone injuries remains an unmet clinical challenge. By developing two new and complementary models of large-scale bone regeneration in zebrafish and mouse, we aim to understand the role of the cartilage callus in generating large segments of full thickness bone. While the periosteum generates osteoblasts during homeostasis, the specific subpopulation that builds the repair callus remains poorly defined. Using transgenic lineage tracing, we provide compelling preliminary evidence that a rare bi-potent Sox9+/Runx2+ periosteal population generates new cartilage and bone during repair. This newfound ability to label, manipulate, and isolate a specific stem cell population allows us to test whether the remarkable regenerative capacity of the rib is due to the unique properties of its periosteal stem cells. In Aim 1, we team up with an orthopaedic surgeon to test that Sox9+ cells from the rib periosteum can be expanded in culture and used to heal a critical-sized femoral defect. The formation of a cartilage callus is a common feature in bone repair, yet how the periosteum generates cartilage only during repair remains a mystery. In preliminary data, we find that the cartilage callus is severely compromised when either the Ihha ligand is deleted in zebrafish or the Hh receptor Smo is deleted from Sox9+ cells in mice. In Aim 2, we test that this reflects a repair-specific role for Ihh, which is markedly different from its developmental role in osteoblast differentiation and chondrocyte proliferation. Further, our preliminary data suggest that these Ihh-induced repair chondrocytes differ in important ways from those in the growth plate since repair chondrocytes co-express osteoblast genes even at pre-hypertrophic stages. This increase in osteogenic character subsequently correlates with a conversion of chondrocytes into osteocytes. In Aim 3, we test whether repair and developmental chondrocytes represent distinct cell types by comparing global gene expression at different stages of maturation. We also use lineage tracing to quantitate the extent to which cartilage-derived osteocytes preferentially contribute to full thickness bone during repair. Lastly, we use powerful new chromatin accessibility assays to test that as Sox9+ periosteal cells become cartilage, their greater osteogenic character results from the maintenance of poised osteoblast enhancers. Our findings will reveal how a rare population of periosteal stem cells can be induced to make cartilage during injury, and how this specialized repair cartilage can be used to regenerate full thickness bone. A better understanding of these important stem cells will form the basis of future pre- clinical trials aimed at healing large-scale skeletal lesions in other parts of the body.