Controlled, Sustained Delivery of siRNA to hMSCs for Enhanced Bone Regeneration

描述（由申请人提供）：骨组织损伤的修复，尤其是发生在颅面区域的骨组织损伤的修复，是骨科中的一个重大挑战。目前对这些损伤的有效治疗方法很少。组织工程试图修复 或通过提供必要的生化、细胞和机械信号促进宿主组织再生来替换受损组织。人骨髓间充质干细胞（human mesenchymal stem cells，hMSC）是一种有前途的骨再生细胞来源，因为它们能够分化成骨组织，容易从骨髓中获得，并且可以在培养中容易地增殖，从而可以获得足够数量的细胞用于组织工程方法。RNA干扰是一种强大的基因沉默机制，通过靶向破坏特定mRNA分子来抑制翻译水平上的基因表达，并且具有通过降低特定蛋白质的表达来彻底改变疾病治疗和辅助受损组织的功能修复的潜力。此外，向干细胞递送短干扰RNA（siRNA）以指导其分化以促进所需组织生长的潜力是令人兴奋的。然而，有效地将生物活性siRNA递送到受损组织部位仍然是一个挑战，需要更多的研究来确定其在hMSCs分化和增强所得组织形成中的有效性。因此，这项工作旨在设计新的生物材料系统，用于控制和持续递送siRNA，并研究针对BMP拮抗剂递送siRNA对hMSC的成骨分化和骨形成的影响。中心假设是通过siRNA的受控递送沉默BMP拮抗剂的表达将促进hMSC的成骨反应，并增强骨再生。这将通过以下具体目标来解决：（1）设计能够随时间以持续和可控的方式释放siRNA的新型生物聚合物水凝胶，（2）从生物聚合物水凝胶递送针对BMP拮抗剂的siRNA，并研究其对引导包封的和周围的hMSC向下成骨谱系的作用，以及（3）评估该系统在将含有siRNA和hMSC的水凝胶构建体植入临界尺寸的骨缺损中后体内驱动骨形成的能力。该提案旨在证明一种新方法的实用性，以改善骨缺损的修复，这将具有很大的临床效益，除了创建一个平台技术，然后可用于其他治疗应用。

DESCRIPTION (provided by applicant): The repair of bone tissue injuries, especially those occurring in the craniofacial region, is a significant challenge in orthopaedics. There are currently few effective treatments available for these injuries. Tissue engineering seeks to repair or replace the damaged tissue by encouraging regeneration of the host tissue through provision of the necessary biochemical, cellular, and mechanical cues. Human mesenchymal stem cells (hMSCs) are a promising cell source for bone regeneration as they are capable of differentiating into osteogenic tissue, are easily obtainable from bone marrow, and can proliferate in culture readily so that it is possible to obtain a sufficient number of cells for tissue engineering approaches. RNA interference is a powerful gene silencing mechanism that inhibits gene expression at the translational level by the targeted destruction of specific mRNA molecules, and has the potential to revolutionize disease treatment and aid in the functional repair of damaged tissue by decreasing the expression of specific proteins. Additionally, the potential for delivering short interfering RNA (siRNA) to stem cells to direct their differentiation to promote the desired tissue growth is exciting. However, effectively delivering bioactive siRNA to damaged tissue sites remains a challenge, and more research is needed to determine its effectiveness in the differentiation of hMSCs and enhancement of resultant tissue formation. Thus, this proposed work seeks to engineer novel biomaterial systems for controlled and sustained delivery of siRNA and to examine the effect of delivering siRNA against BMP antagonists on the osteogenic diferentiation of hMSCs and bone formation. The central hypothesis is that silencing the expression of BMP antagonists via controlled delivery of siRNA will promote the osteogenic response of hMSCs, and enhance bone regeneration. This will be addressed by the following specific aims: (1) engineer novel biopolymer hydrogels capable of releasing siRNA in a sustained and controllable manner over time, (2) deliver siRNA against a BMP antagonist from biopolymer hydrogels and investigate its effect on guiding encapsulated and surrounding hMSCs down the osteogenic lineage and (3) assess the ability of the system to drive bone formation in vivo upon implantation of hydrogel constructs containing siRNA and hMSCs into a critical-size bone defect. This proposal aims to demonstrate the utility of a new approach to improve the repair of bony defects, which would have great clinical benefit, in addition to creating a platform technology that could then be used for other therapeutic applications.