Opposing RNAi molecule gradient constructs to repair osteochondral defects

 描述：骨软骨缺陷(OCDs)的治疗是具有挑战性的，它涉及到受影响关节的软骨下骨和关节软骨的损害。这种衰弱的缺陷会导致机械不稳定、疼痛和恶化的骨关节退行性变。目前的治疗方法未能始终如一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与招募和/或移植的细胞和/或生物活性因子相结合，已成为一种有前途的替代方法。人骨髓间充质干细胞(HMSCs)是一种很有吸引力的细胞来源，因为它可以很容易地从骨髓中分离出来，在不丧失多能性的情况下进行培养，并且在适当的条件下可以分化为成骨和软骨样细胞。RNA干扰(RNAi)是一种强大的工具，可以通过靶向破坏特定的mRNA分子在翻译后水平上抑制基因的表达，并有可能通过减少对修复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。重要的是，RNAi分子已经被确定可以促进hMSCs的成骨和成软骨分化。然而，有效地将RNAi分子运送到体内的靶细胞仍然是一个巨大的挑战，限制了其治疗潜力。我们设计了生物聚合物水凝胶，能够局部运送具有生物活性的RNAi分子，并将其释放到周围和包裹的细胞，这些凝胶已被用于在空间和时间上控制细胞的基因表达和命运。因此，这一应用的中心假设是，在生物聚合物水凝胶中控制相反的RNAi分子梯度的时空呈现，将推动包裹的hMSCs以相反的方向成骨和成软骨，形成骨软骨结构，从而促进OCDs的愈合。具体目标如下：(1)设计具有相反浓度梯度的两种不同siRNAs的生物聚合物水凝胶，用于时空控制、持续的基因敲除；(2)从生物聚合物梯度水凝胶中携带促进成骨和软骨形成的RNAi分子，并研究其在空间上指导包裹的hMSCs成骨和向软骨分化的能力；(3)利用微流控技术开发尺寸可调的相反的RNAi分子梯度水凝胶；以及(4)评估含有hMSCs和相反的RNAi分子梯度的水凝胶载体在植入兔OCD模型后在体内推动成骨和软骨形成的能力。本申请旨在展示一种新的组织工程学方法用于增强骨软骨组织再生的实用性，这将通过改善强迫症患者的生活质量而具有很大的临床实用价值。

 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.