Opposing RNAi Molecule Gradient Constructs to Repair Osteochondral Defects

 产品说明：骨软骨缺损（OCD）的治疗涉及受影响关节的软骨下骨和关节软骨的损伤，具有挑战性。这种使人衰弱的缺陷导致机械不稳定、疼痛和骨关节炎变性恶化。目前的疗法不能持续地修复和恢复组织功能。骨软骨组织工程技术利用生物材料结合募集和/或移植的细胞，和/或生物活性因子已成为一个有前途的替代方法。人间充质干细胞（hMSC）是一种有吸引力的细胞来源，因为它们可以容易地从骨髓中分离，在培养中扩增而不失去多能性，并且在适当的条件下可以分化成成骨和软骨谱系的细胞。RNA干扰（RNAi）是一种通过靶向破坏特定mRNA分子来抑制翻译后水平的基因表达的有力工具，并且具有通过降低对愈合过程产生负面影响的特定蛋白质的表达或通过改变干细胞分化途径来彻底改变受损组织的功能修复的潜力。重要的是，已经鉴定了可以促进hMSC的成骨和软骨分化的RNAi分子。然而，在体内将RNA干扰分子有效递送至靶细胞仍然是一个重大挑战，限制了其治疗潜力。我们已经设计了能够局部递送生物活性RNAi分子的生物聚合物水凝胶，其具有可定制的释放曲线，用于递送到周围和封装的细胞，并且这些凝胶已用于在空间和时间上控制细胞基因表达和命运。因此，本申请的中心假设是生物聚合物水凝胶中双重相反RNAi分子梯度的受控空间和时间呈现将在相反方向上驱动包封的hMSC的骨生成和软骨生成，以形成可促进OCD愈合的骨软骨构建体。这将通过以下具体目标来解决：（2）从生物聚合物梯度水凝胶递送促进骨生成和软骨生成的RNAi分子，并研究它们在空间上引导包封的hMSC的成骨和软骨生成分化的能力，（3）使用微流体技术开发具有可定制尺寸的反向RNAi分子梯度水凝胶，和（4）评估含有hMSC和反向RNAi分子梯度的水凝胶构建体在植入兔OCD模型后体内驱动骨生成和软骨生成的能力。该应用旨在证明一种新的组织工程方法在增强骨软骨组织再生方面的实用性，该方法通过改善强迫症患者的生活质量而具有巨大的临床实用性。

 DESCRIPTION: The treatment of osteochondral defects (OCDs), which involve damage to both the subchondral bone and articular cartilage in the affected joint, is challenging. Such debilitating defects lead to mechanical instability, pain and worsening osteoarthritic degeneration. Current therapies fail to consistently repair and restore tissue function. Osteochondral tissue engineering technology utilizing biomaterials in combination with recruited and/or transplanted cells, and/or bioactive factors has emerged as a promising alternative approach. Human mesenchymal stem cells (hMSCs) are an attractive cell source as they can easily be isolated from bone marrow, expanded in culture without losing multipotency, and under appropriate conditions can differentiate into cells of the osteogenic and chondrogenic lineages. RNA interference (RNAi) is a powerful tool permitting inhibition of gene expression at the post-translational level by the targeted destruction of specific mRNA molecules, and has the potential to revolutionize the functional repair of damaged tissue by decreasing the expression of specific proteins that negatively impact healing processes or by altering stem cell differentiation pathways. Importantly, RNAi molecules have been identified that can promote the osteogenic and chondrogenic differentiation of hMSCs. However, effective delivery of RNAi molecules to target cells in vivo remains a significant challenge limiting its therapeutic potentia. We have engineered biopolymer hydrogels capable of locally delivering bioactive RNAi molecules with tailorable release profiles for delivery to surrounding and encapsulated cells, and these gels have been used to spatially and temporally control cell gene expression and fate. Therefore, the central hypothesis of this application is that the controlled spatial and temporal presentation of dual opposing RNAi molecule gradients in a biopolymer hydrogel will drive osteogenesis and chondrogenesis of encapsulated hMSCs in opposite directions to form osteochondral constructs that can promote the healing of OCDs. This will be addressed by the following specific aims: (1) Engineer biopolymer hydrogels with opposing concentration gradients of two different siRNAs for spatiotemporally controlled, sustained gene knockdown, (2) Deliver RNAi molecules that promote osteogenesis and chondrogenesis from biopolymer gradient hydrogels and investigate their capacity to spatially guide the osteogenic and chondrogenic differentiation of encapsulated hMSCs, (3) Develop opposing RNAi molecule gradient hydrogels with tailorable dimensions using microfluidic technology, and (4) Assess the ability of the hydrogel constructs containing hMSCs and opposing RNAi molecule gradients to drive osteogenesis and chondrogenesis in vivo upon implantation into a rabbit OCD model. This application aims to demonstrate the utility of a new tissue engineering approach for enhanced osteochondral tissue regeneration, which would have great clinical utility by improving the quality of life of patients suffering from OCDs.