Hydrogel delivery of DBM and exosome mimetics for bone repair

摘要 先天性和获得性颅面部缺陷并不少见。脱钙骨基质(DBM)已被 广泛用于骨科修复。然而，由于DBM的颗粒性，其更广泛的使用受到限制 冲洗后的脱矿和快速弥散的颗粒，导致不可预测的成骨活性。 通常使用粘性赋形剂来产生DBM颗粒的稳定悬浮液，但这种载体 体内快速溶解和DBM中存在的成骨成分(如骨)的局部效应 形态发生蛋白(BMP)可能不会出现在缺陷部位。尽管外源性BMP可以结合 为了增强DBM的能力，其临床应用需要超生理剂量，并已显示出显著的 不良影响。因此，需要开发能够增强成骨能力的替代策略 对DBM的支持。这项研究试图通过将DBM结合到自我修复中来增强骨再生能力 动态聚合物网络，结合了生理稳定性和促成骨特性。在BMP上 由于天然BMP拮抗剂的表达增强，BMP的作用大大降低 就像诺金一样。因此，本研究将通过取消BMP进一步提高DBM中存在的BMP的效力 核糖核酸干扰的拮抗作用。细胞衍生的外切体模拟(EM)将作为一种生物 载体以局部化和高效的方式传递RNA干扰分子。这样做的总体目标是 建议设计一种坚固的骨移植复合材料，通过整合DBM来有效地修复骨缺损 以及将EM沉默到聚合物载体系统中。为了实现这一目标，我们提出了三个目标。在目标1中， 我们将开发一种基于植物化学接枝自组装的延展性和自愈性水凝胶 壳聚糖与富含二氧化硅的纳米粘土，其中装饰的植物化学物质驱动动态的分子间 凝胶和纳米粘土的相互作用是具有骨诱导特性的物理交联剂。通过改变 植物化学与纳米粘土的比例和DBM颗粒的含量、水凝胶/DBM复合材料的设计 并通过评价凝胶化动力学、可注射性和自愈特性来制备。骨诱导剂 所开发的复合材料的活性将在体外和大鼠颅骨缺损处进行测定。接下来在《目标2》中，我们将 从转染noggin siRNA的MSCs中获取EM，并评价EM对人脐静脉内皮细胞的协同作用 DBM诱导骨形成。我们还将通过点击交联反应将EM偶联到水凝胶上，以获得更多 局部和长期的脑部沉默效果。最后，在目标3中，我们将集成DBM和EM 将siRNA插入到目标1开发的植物化学和纳米粘土自修复水凝胶中并进行评价 骨移植复合材料在更具挑战性的环境中使用 下颌骨缺损模型。成功的骨形成将与商业DBM产品进行比较 和重组BMP-2。这些研究的成功完成将确定改善临床的新策略 最大限度提高BMP信号活性的当前骨移植在DBM介导的骨再生中的作用。

Abstract Congenital and acquired craniofacial defects are not uncommon. Demineralized bone matrix (DBM) has been widely used for the orthopedic repair. However, more extensive use of DBM is limited due to its particulate nature after demineralization and rapid particle dispersion following irrigation, resulting in unpredictable osteoinductivity. Viscous excipients are often employed to produce stable suspension of DBM particles, but such carriers are rapidly dissolved in a body and the localized effect of osteogenic components present in DBM such as bone morphogenetic proteins (BMPs) may not expect at the defect site. Although exogenous BMPs can be combined to enhance DBM capacity, its clinical application requires supraphysiological doses and has revealed significant adverse effects. Thus, there is a need to develop alternative strategies that can enhance the osteogenic potency of DBM. This study seeks to enhance bone regeneration capacity by incorporating DBM into a self-healing dynamic polymer network that combines physiological stability and pro-osteogenic properties. Upon BMP stimulation, BMP efficacy is greatly reduced due to the enhanced expression of natural BMP antagonists such as noggin. Thus, this study will further enhance the potency of BMPs present in DBM by abrogation of BMP antagonism through RNA interference for noggin. Cell-derived exosome mimetics (EM) will be applied as a bio- vector to deliver RNA interference molecules in a localized and efficient manner. The overall objective of this proposal is to devise a robust bone graft composite that can effectively repair bone defects by integrating DBM and noggin-silencing EM into polymeric carrier systems. To achieve this goal, we propose three aims. In Aim 1, we will develop a malleable and self-healing hydrogel based on the self-assembly of phytochemical-grafted chitosan with silica-rich nanoclays, where the decorated phytochemical drives dynamic intermolecular interactions for gelation and nanoclay works as physical crosslinker with osteoinductive property. By varying the ratio of phytochemical to nanoclay and the content of DBM particles, hydrogel/DBM composites will be designed and prepared by evaluating gelation kinetics, injectability and self-healing characteristics. The osteoinductive activity of the developed composite will be determined in vitro and in a rat calvarial defect. Next in Aim 2, we will harvest EM from MSCs transfected with noggin-directed siRNA and evaluate the synergistic effect of EM on DBM-induced bone formation. We will also conjugate EM to hydrogels via a click crosslinking reaction for more localized and prolonged noggin silencing effects. Finally in Aim 3, we will integrate DBM and EM loaded with noggin siRNA into self-healing hydrogels of phytochemical and nanoclay developed from Aim 1 and evaluate the ability of the bone graft composite to promote bone regeneration in more challenging environments using a mandibular defect model. Successful bone formation will be evaluated compared with commercial DBM products and recombinant BMP-2. Successful completion of these studies will identify a new strategy to improve clinical efficacy of current bone grafting by maximizing activity of BMP signaling in DBM-mediated bone regeneration.