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

描述(申请人提供)：骨组织损伤的修复，特别是发生在颅面部的损伤，在骨科是一个巨大的挑战。目前，对这些损伤几乎没有有效的治疗方法。组织工程学寻求修复 或者通过提供必要的生化、细胞和机械线索来鼓励宿主组织的再生来替换受损组织。人骨髓间充质干细胞(HMSCs)具有向成骨组织分化的能力，易于从骨髓中获得，在培养条件下易于增殖，有可能获得足够数量的细胞用于组织工程，是一种很有前途的骨再生细胞来源。RNA干扰是一种强大的基因沉默机制，它通过靶向破坏特定的mRNA分子在翻译水平上抑制基因的表达，并有可能通过减少特定蛋白的表达来革命性地改变疾病的治疗和帮助受损组织的功能修复。此外，将短干扰RNA(SiRNA)传递到干细胞以引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运送到受损的组织部位仍然是一个挑战，需要更多的研究来确定其在hMSCs分化和促进组织形成方面的有效性。因此，这项拟议的工作旨在设计新的生物材料系统，以控制和持续地输送siRNA，并检测针对BMP拮抗剂的siRNA对hMSCs成骨分化和骨形成的影响。中心假说是，通过控制传递siRNA来沉默BMP拮抗剂的表达将促进hMSCs的成骨反应，并促进骨再生。这将通过以下具体目标来解决：(1)设计新型生物聚合物水凝胶，能够随着时间的推移以持续和可控的方式释放siRNA，(2)从生物聚合物水凝胶中针对BMP拮抗剂输送siRNA，并研究其在引导包裹和周围的hMSCs沿成骨谱系向下生长的作用，以及(3)评估含有siRNA和hMSCs的水凝胶构建体植入临界大小的骨缺损中后，该系统在体内驱动骨形成的能力。这项提案旨在展示一种新的方法的实用性，以改进骨缺损的修复，这将具有巨大的临床益处，此外，还将创建一种可用于其他治疗应用的平台技术。

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.