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

摘要 骨骼损伤是一个主要的健康问题。在美国，每年有790万人持续骨折 其中约10%的骨折严重延迟愈合或不愈合，导致患者和 巨大的医疗成本。虽然骨移植、合成聚合物、低强度脉冲超声波和 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各有优劣。因此，在那里 迫切需要找到促进骨折愈合的新的有效疗法。维生素C和甲状腺激素(TH) 已知在软骨内骨形成(EBF)中起关键作用。我们在分子途径方面的最新研究进展 维生素C的作用表明，刺猬和低氧信号通路的顺序激活有助于 到EBF中涉及的关键步骤。我们在这个项目中的重点是两个小分子的治疗作用和作用机制。 SAG 21k和IOX2分子，激活Hedgehog和缺氧信号通路，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-三钙局部输送SAG21K和IOX2 缺陷部位的磷酸盐(β)支架，以提供机械强度并最大限度地减少对 纸巾。一种临床相关的股骨中段节段性缺损模型，在该模型中，2.5 mm的缺损区通过一种固定装置固定 将使用带塑料垫片的髓内螺纹棒，长时间不会愈合。三 提出了目标。在目标1中，我们将3D打印纤维蛋白凝胶/β-磷酸三钙(β)支架制剂，其中包含 SAG21k和IOX2，并评价这些制剂对于传递有效浓度的SAG21k的适宜性 和IOX2在激活Hedgehog和骨折处低氧信号的最佳治疗时间窗通过 免疫组织化学(IHC)和实时定量检测这些信号通路下游信号靶点 不同时间段小鼠骨折骨痂中的聚合酶链式反应。在目标2中，我们将检验这样的假设，即顺序激活 Hedgehog结合低氧信号转导可有效促进股骨节段性缺损区的愈合。我们会 比较SAG21k和IOX2与自体移植的骨愈合效果，自体移植是用于骨修复的金标准 不愈合缺陷。我们将使用经过验证的显微CT、骨强度和组织学测量来评估骨折 愈合表型。SAG21K/IOX2联合治疗的疗效将用老年人和 糖尿病小鼠骨折愈合受损。在目标3中，我们将检验这样一种假设，即声波的顺序激活 Hedgehog和低氧信号通过促进软骨细胞向成骨细胞的直接转化来诱导骨愈合。 骨折骨痂软骨细胞将通过遗传诱导的命运定位方法标记TdTomato和 将对标记的软骨细胞在骨痂中形成成骨细胞进行评估。软骨细胞在骨愈合中的作用 将在白喉毒素去除软骨细胞后在软骨细胞特异性COL10DTR 1-α；iDTR小鼠中进行评估。在……里面 就临床相关性而言，我们认为了解SAG21k和IOX2在骨骼中的用途的潜在影响 治疗及其作用机制是巨大的，因此本项目中提出的工作意义重大。

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.