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

大规模骨修复过程中软骨愈伤组织的基因调控

• 批准号: 9353287
• 负责人: Francesca V Mariani
• 金额: $ 46.66万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9353287
• 关键词: Ablation; Adopted; Adult; Agonist; Biological Assay; Body part; Bone Injury; Bone Marrow; Bone Regeneration; Bone callus; Cartilage; Cells; Chondrocytes; Chromatin; Clinical; Connective Tissue; Data; Defect; Development; Engraftment; Enhancers; Epiphysial cartilage; Erinaceidae; Failure; Femur; Fishes; Future; Gene Expression; Genes; Genetic; Genomics; Goals; Gray unit of radiation dose; Homeostasis; Human; Injury; Jaw; Knock-in; Label; LacZ Genes; Lesion; Ligands; Maintenance; Mediating; Mesenchymal Stem Cells; Minerals; Modeling; Morphology; Mus; Natural regeneration; Orthopedics; Osteoblasts; Osteocytes; Pathway interactions; Periosteal Cell; Periosteum; Population; Property; Proteins; Regulation; Role; Signal Transduction; Source; Stem cells; Surgeon; Testing; Therapeutic; Thick; Transgenic Organisms; Zebrafish; bone; bone healing; cartilage cell; cartilage repair; cell type; combinatorial; epigenetic memory; healing; improved; mutant; novel; osteoblast differentiation; osteogenic; pre-clinical trial; progenitor; receptor; regenerative; repaired; response to injury; rib bone structure; skeletal; stem cell population

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.

大面积骨损伤的修复仍然是一个未解决的临床挑战。通过开发两个 斑马鱼和小鼠大规模骨再生的新补充模型，我们 旨在了解软骨愈伤组织在生成大片全厚度过程中的作用 骨。虽然骨膜在稳态过程中产生成骨细胞，但特定的 构建修复愈伤组织的亚群仍然不明确。使用转基因谱系 追踪，我们提供了令人信服的初步证据，表明罕见的双能 Sox9+/Runx2+ 骨膜群在修复过程中产生新的软骨和骨。这种新发现的能力 标记、操作和分离特定的干细胞群使我们能够测试是否 肋骨卓越的再生能力归因于其骨膜干的独特特性 细胞。在目标 1 中，我们与骨科医生合作测试来自肋骨的 Sox9+ 细胞 骨膜可以在培养物中扩张并用于治愈临界大小的股骨缺损。这 软骨愈伤组织的形成是骨修复的常见特征，但骨膜如何形成 仅在修复过程中生成软骨仍然是个谜。在初步数据中，我们发现 当斑马鱼中的 Ihha 配体被删除或 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区别，因为 即使在肥大前期，修复软骨细胞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相关 骨细胞。在目标 3 中，我们测试修复和发育软骨细胞是否代表不同的特征 通过比较不同成熟阶段的整体基因表达来确定细胞类型。我们还使用 谱系追踪以定量软骨源性骨细胞优先的程度 在修复过程中有助于形成全层骨。最后，我们使用强大的新染色质 可及性测定来测试当 Sox9+ 骨膜细胞变成软骨时，它们的更大 成骨特性是由维持平衡的成骨细胞增强剂引起的。我们的发现 将揭示如何诱导稀有的骨膜干细胞群来制造软骨 受伤期间，以及如何使用这种专门的修复软骨来再生全层软骨 骨。更好地了解这些重要的干细胞将为未来的预治疗奠定基础。 旨在治愈身体其他部位的大规模骨骼病变的临床试验。