Development of 3D Printed Synthetic Bone Graft Containing Small Molecules for Sequential Activation of Hedgehog and Hypoxia Signaling for Treatment of Nonunion Fractures

开发含有小分子的 3D 打印合成骨移植物，用于顺序激活 Hedgehog 和缺氧信号，用于治疗骨不连骨折

• 批准号: 10413956
• 负责人: SUBBURAMAN MOHAN
• 金额: --
• 依托单位: VA LOMA LINDA HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10413956
• 关键词: 3D Print; Ablation; Adult; Agonist; Animal Model; Ascorbic Acid; Autologous Transplantation; Binding; Blood Vessels; Bone Development; Bone Injury; Bone Matrix; Bone Transplantation; Bone callus; Cartilage; Cell Therapy; Chondrocytes; Combined Modality Therapy; Data; Defect; Development; Diabetic mouse; Diphtheria Toxin; Disadvantaged; Drug Targeting; Electromagnetic Fields; Embryonic Development; Erinaceidae; Event; Exhibits; Failure; Fibrin; Fracture; Future; Gel; Genes; Genetic; Gold; Growth Factor; HIF1A gene; Health; Health Care Costs; Histologic; Hypertrophy; Hypothyroidism; Hypoxia; Immunohistochemistry; Impaired healing; Impairment; Invaded; Label; Lead; Measurement; Mechanics; Mediating; Mesenchymal Stem Cells; Modeling; Molecular; Morbidity - disease rate; Mus; Osteoblasts; Osteogenesis; Oxygen; Pathway interactions; Patients; Phenotype; Physiologic Ossification; Physiologic pulse; Physiological; Play; Polymers; Preparation; Process; Procollagen-Proline Dioxygenase; Proliferating; Proteins; Recombinants; Rod; Role; SHH gene; Safety; Signal Pathway; Signal Transduction; Site; Skeletal Development; Source; Testing; Therapeutic; Therapeutic Agents; Therapeutic Effect; Thyroid Hormones; Time; Tissues; Torsion; Work; aged; base; bone; bone epiphysis; bone fracture repair; bone healing; bone mass; bone repair; bone strength; cartilaginous; clinically relevant; comparative efficacy; cost effective; cost effectiveness; effective therapy; healing; inhibitor; injury and repair; intramembranous bone; long bone; mesenchymal stromal cell; microCT; mouse model; novel; novel therapeutics; repaired; scaffold; sensor; side effect; skeletal; small molecule; small molecule inhibitor; small molecule therapeutics; smoothened signaling pathway; substantia spongiosa; therapeutic effectiveness; therapeutic evaluation; transdifferentiation; tricalcium phosphate; ultrasound

ABSTRACT Bone injuries are a major health problem. There are 7.9 million bone fractures sustained annually in the U.S. Healing is impaired in about 10% of these fractures with seriously delayed union or non-union, causing morbidity for patients and enormous healthcare costs. While strategies such as bone grafting, synthetic polymers, low intensity pulsed ultrasound and electromagnetic fields, growth factors and cell therapy are currently being used or investigated to promote bone healing, each of these therapies have their own advantages and disadvantages in terms of cost, effectiveness and safety. Thus, there is a compelling need to find novel effective therapies that promote fracture healing. Vitamin C and thyroid hormone (TH) are known to play key roles in endochondral bone formation (EBF). Our recent studies on the molecular pathways for TH and vitamin C actions revealed evidence that sequential activation of hedgehog and hypoxia signaling pathways contribute to key steps involved in EBF. Our focus in this project is on the therapeutic utility and mechanisms of action of two small molecules, SAG 21k and IOX2, that activate hedgehog and hypoxia signaling pathways to promote EBF at the fracture site. In this proof of concept study, we propose to deliver SAG21K and IOX2 locally using 3D printed fibrin gel/β-tricalcium phosphate (βTCP) scaffolds at the defect site to provide mechanical strength and minimize unwanted side effects on other tissues. A clinically relevant segmental defect model in the femoral midshaft in which a 2.5-mm defect is stabilized by an intramedullary threaded rod with attached plastic spacers that does not heal over a prolonged period will be used. Three aims are proposed. In aim 1, we will 3D print fibrin gel/β-tricalcium phosphate (βTCP) scaffold preparations containing SAG21k and IOX2 and evaluate the suitability of these preparations for delivery of effective concentrations of SAG21k and IOX2 at the optimal therapeutic time window for activation of hedgehog and hypoxia signaling at the fracture site by measurement of downstream signaling targets of these signaling pathways by immunohistochemistry (IHC) and real time PCR in the fracture callus of mice at different times. In aim 2, we will test the hypothesis that sequential activation of hedgehog followed by hypoxia signaling will be effective in promoting healing of femoral segmental defects. We will compare the efficacy of bone healing with SAG21k and IOX2 with that of autografts, a gold standard used for healing of nonunion defects. We will use validated microCT, bone strength and histological measurements to evaluate the fracture healing phenotype. Therapeutic effectiveness of SAG21k/IOX2 combination therapy will be studied using aged and diabetic mice with impaired fracture healing. In aim 3, we will test the hypothesis that sequential activation of sonic hedgehog and hypoxia signaling induces bone healing by promoting direct conversion of chondrocytes-to-osteoblasts. Fracture callus chondrocytes will be labeled with TdTomato by genetic inducible fate mapping approaches and the fate of labeled chondrocytes to form osteoblasts in the bony callus will be evaluated. The role of chondrocytes in bone healing will be evaluated after chondrocyte ablation with diphtheria toxin in chondrocyte-specific Col10α1-CreER;iDTR mice. In terms of clinical relevance, we believe that the potential impact of understanding the utility of SAG21k and IOX2 in bone healing and their mechanisms of action is huge, and therefore the work proposed in this project is significant.

抽象的 骨损伤是一个主要的健康问题，美国每年有 790 万例骨折。 约 10% 的骨折存在严重延迟愈合或不愈合的情况，导致患者发病和 而骨移植、合成聚合物、低强度脉冲超声和等策略则带来巨大的医疗费用。 目前正在使用或研究电磁场、生长因子和细胞疗法来促进骨愈合， 这些疗法在成本、有效性和安全性方面都有各自的优点和缺点。 迫切需要找到促进骨折愈合的新型有效疗法。 我们最近对 TH 分子途径的研究已知在软骨内骨形成 (EBF) 中发挥关键作用。 维生素 C 的作用揭示了刺猬蛋白和缺氧信号通路的连续激活有助于 EBF 涉及的关键步骤 我们在这个项目中的重点是两种小药物的治疗效用和作用机制。 SAG 21k 和 IOX2 分子可激活刺猬信号通路和缺氧信号通路，从而促进骨折部位的 EBF。 在这项概念验证研究中，我们建议使用 3D 打印纤维蛋白凝胶/β-三钙在本地提供 SAG21K 和 IOX2 缺损部位的磷酸盐 (βTCP) 支架可提供机械强度并最大限度地减少对其他部位的不良副作用 股骨中轴的临床相关节段缺损模型，其中 2.5 毫米的缺损由一个稳定的支架固定。 将使用带有长期无法愈合的塑料垫片的髓内螺纹杆。 在目标 1 中，我们将 3D 打印含有纤维蛋白凝胶/β-磷酸三钙 (βTCP) 支架制剂。 SAG21k 和 IOX2 并评估这些制剂对于递送有效浓度的 SAG21k 的适用性 和IOX2在最佳治疗时间窗激活刺猬和骨折部位缺氧信号传导 通过免疫组织化学 (IHC) 和实时测量这些信号通路的下游信号靶点 在目标 2 中，我们将测试不同时间小鼠骨折愈伤组织中连续激活的假设。 刺猬之后的缺氧信号将有效促进股骨节段缺损的愈合。 将 SAG21k 和 IOX2 的骨愈合功效与自体移植物的骨愈合功效进行比较，自体移植物是用于骨​​愈合的金标准 我们将使用经过验证的 microCT、骨强度和组织学测量来评估骨折。 SAG21k/IOX2 联合疗法的治疗效果将使用老年人和 骨折愈合受损的糖尿病小鼠 在目标 3 中，我们将测试声波连续激活的假设。 刺猬蛋白和缺氧信号通过促进软骨细胞直接转化为成骨细胞来诱导骨愈合。 骨折愈伤组织软骨细胞将通过遗传诱导命运图谱方法用 TdTomato 进行标记，并确定其命运 将评估软骨细胞在骨愈合中的作用。 将在软骨细胞特异性 Col10α1-CreER;iDTR 小鼠中用白喉毒素消融软骨细胞后进行评估。 就临床相关性而言，我们认为了解 SAG21k 和 IOX2 在骨中的效用的潜在影响 治愈及其作用机制是巨大的，因此该项目中提出的工作意义重大。