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790万人骨折。 在这些骨折中，约有10%的骨折严重延迟愈合或不愈合，导致患者发病， 巨大的医疗费用。虽然诸如骨移植、合成聚合物、低强度脉冲超声和 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， 这些疗法中的每一种在成本、有效性和安全性方面都有其自身的优点和缺点。因此 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。近年来我们对TH的分子途径的研究 和维生素C的作用揭示的证据表明，刺猬和缺氧信号通路的顺序激活有助于 参与EBF的关键步骤。我们在这个项目中的重点是两个小的治疗效用和作用机制， 分子SAG 21 k和IOX 2，激活刺猬和缺氧信号通路，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打印的纤维蛋白凝胶/β-三钙局部递送SAG 21 K和IOX 2。 磷酸盐（βTCP）支架在缺损部位提供机械强度，并尽量减少对其他不必要的副作用 组织中股骨中段的临床相关节段性缺损模型，其中2.5 mm缺损通过 将使用在长时间内不会愈合的具有附接的塑料间隔物的髓内螺纹杆。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（βTCP）支架制剂， SAG 21 k和IOX 2，并评价这些制剂用于递送有效浓度的SAG 21 k的适用性 和IOX 2在骨折部位激活hedgehog和缺氧信号的最佳治疗时间窗， 通过免疫组织化学（IHC）和真实的时间测量这些信号传导途径的下游信号传导靶标 小鼠骨折后不同时间骨痂中的PCR。在目标2中，我们将检验以下假设： hedgehog随后缺氧信号传导将有效地促进股骨节段性缺损的愈合。我们将 将SAG 21 k和IOX 2的骨愈合效果与自体移植物的骨愈合效果进行比较，自体移植物是用于骨愈合的金标准， 骨不连缺陷。我们将使用经验证的microCT、骨强度和组织学测量来评估骨折 愈合表型SAG 21 k/IOX 2组合疗法的治疗有效性将使用老年人和 骨折愈合受损的糖尿病小鼠。在目标3中，我们将测试假设， hedgehog和缺氧信号传导通过促进软骨细胞向成骨细胞的直接转化来诱导骨愈合。 骨折愈伤组织软骨细胞将通过遗传诱导的命运作图方法用TdTomato标记，并且 将评价标记的软骨细胞在骨痂中形成成骨细胞的能力。软骨细胞在骨愈合中的作用 将在软骨细胞特异性Col 10 α1-CreER;iDTR小鼠中用白喉毒素进行软骨细胞消融后进行评价。在 就临床相关性而言，我们认为了解SAG 21 k和IOX 2在骨中的效用的潜在影响 愈合和他们的行动机制是巨大的，因此，在这个项目中提出的工作是重要的。

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.