Hydrogel delivery of DBM and exosome mimetics for bone repair

摘要 先天性和后天性颅面缺损并不少见。脱矿骨基质（DBM）已被 广泛用于骨科修复。然而，DBM的更广泛使用由于其颗粒性质而受到限制 在冲洗后脱矿和快速颗粒分散后，导致不可预测的骨诱导性。 粘性赋形剂通常用于产生DBM颗粒的稳定悬浮液，但是这样的载体是不稳定的。 快速溶解在体内，以及DBM中存在的成骨成分如骨的局部作用 形态发生蛋白（BMP）可能不会出现在缺陷部位。虽然外源性骨形态发生蛋白可以与 为了增强DBM能力，其临床应用需要超生理剂量，并且已经显示出显著的 不良影响因此，有必要开发替代策略，可以提高成骨潜能 的DBM。本研究旨在通过将DBM纳入自愈合 结合了生理稳定性和促成骨特性的动态聚合物网络。在BMP上 刺激时，BMP功效大大降低，这是由于天然BMP拮抗剂的表达增强， 作为noggin。因此，本研究将通过废除BMP进一步增强DBM中存在的BMP的效力。 通过RNA干扰对抗noggin。细胞来源的外泌体模拟物（EM）将作为生物活性物质应用于生物医学领域。 载体以局部和有效的方式递送RNA干扰分子。本报告的总体目标 建议设计一种坚固的骨移植复合材料，通过整合DBM可以有效修复骨缺损 和头蛋白沉默EM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， 我们将开发一种可塑性和自我修复的水凝胶， 壳聚糖与富含二氧化硅的纳米粘土，其中修饰的植物化学物质驱动动态分子间 凝胶化和纳米粘土的相互作用充当具有骨诱导性质的物理交联剂。通过改变 通过改变植物化学物质与纳米粘土的比例和DBM颗粒的含量，设计了水凝胶/DBM复合材料 并通过评价胶凝动力学、可注射性和自修复特性来制备。所述骨诱导 将在体外和大鼠颅骨缺损中测定所开发的复合物的活性。在目标2中，我们将 从用头蛋白定向的siRNA转染的MSC收获EM，并评估EM对 DBM诱导的骨形成。我们还将通过点击交联反应将EM与水凝胶结合， 局部和长期的noggin沉默效应。最后，在目标3中，我们将集成DBM和EM， 将noggin siRNA插入由Aim 1开发的植物化学物质和纳米粘土的自修复水凝胶中，并评估 骨移植复合材料在更有挑战性的环境中使用 下颌骨缺损模型将与市售DBM产品进行比较，评价成功骨形成 和重组BMP-2。这些研究的成功完成将确定一种新的策略，以改善临床 通过在DBM介导的骨再生中最大化BMP信号传导的活性来评价当前骨移植的功效。

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.