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

基本信息

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
项目类别：
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

项目摘要

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790万骨骨折这些骨折中约有10％受到严重延迟的联合或非工会的损害，导致患者的发病率巨大的医疗费用。而诸如骨移植，合成聚合物，低强度脉冲超声和等策略目前正在使用或研究电子领域，生长因子和细胞疗法以促进骨骼愈合，这些疗法中的每一种都在成本，有效性和安全性方面都有自己的优势和缺点。那，那里是找到促进骨折愈合的新型有效疗法的迫切需要。维生素C和甲状腺马（Th）已知会在软骨骨形成（EBF）中扮演关键角色。我们最近关于TH分子途径的研究维生素C的作用揭示了证据表明，刺猬和缺氧信号通路的顺序激活有助于涉及EBF的关键步骤。我们在这个项目上的重点是两个小型的治疗效用和作用机制分子，SAG 21K和IOX2，可激活刺猬和缺氧信号通路，以在断裂部位促进EBF。在这项概念研究证明中，我们建议使用3D印刷纤维蛋白凝胶/β-三核心在本地提供SAG21K和IOX2 缺陷部位处的磷酸盐（βTCP）支架，以提供机械强度并最大程度地减少对其他的副作用组织。股骨中层中与临床相关的节段缺陷模型，其中2.5毫米缺陷被稳定将使用带有连接的塑料垫片的髓内螺纹杆，该杆将在长时间内无法愈合。三提出了目标。在AIM 1中，我们将3D打印纤维蛋白凝胶/β-三磷酸磷酸盐（βTCP）支架制剂 SAG21K和IOX2并评估这些准备的适用性，以提供有效浓度的SAG21K 和IOX2在最佳治疗时间窗口中，用于通过折断部位激活刺猬和缺氧信号的窗口。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传导目标在不同时间的小鼠断裂愈伤组织中的PCR。在AIM 2中，我们将测试以下假设刺猬随后进行缺氧信号传导将有效促进股骨分段缺陷的愈合。我们将将骨骼愈合的效率与SAG21K和IOX2与自体移植的效率进行比较，这是一种用于愈合的黄金标准骨不足。我们将使用经过验证的Microct，骨骼强度和组织学测量来评估断裂治愈表型。 SAG21K/IOX2组合疗法的治疗有效性将使用老化和糖尿病小鼠骨折愈合受损。在AIM 3中，我们将测试Sonic顺序激活的假设刺猬和缺氧信号传导通过促进软骨细胞到骨细胞的直接转化来诱导骨骼愈合。骨折愈伤组织软骨细胞将通过遗传诱导命运映射方法和命运标记为TDTOMATO 将评估标有软骨细胞在骨愈伤组织中形成成骨细胞的标记。软骨细胞在骨骼愈合中的作用在软骨细胞特异性COL10α1-CREER中，软骨细胞消融后，将评估软骨细胞。在临床相关性的术语，我们认为了解SAG21K和IOX2在骨骼中的实用性的潜在影响康复及其作用机制是巨大的，因此该项目提出的工作很重要。