Opposing RNAi Molecule Gradient Constructs to Repair Osteochondral Defects

相反的 RNAi 分子梯度构建修复骨软骨缺损

• 批准号: 10263140
• 负责人: Eben Alsberg
• 金额: $ 34.12万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10263140
• 关键词: Address; Affect; Animal Model; Biochemical; Biocompatible Materials; Biological Assay; Biomechanics; Biopolymers; Bone Marrow; Bone Morphogenetic Proteins; Cartilage; Cell Transplantation; Cells; Charge; Chondrogenesis; Clinical; Defect; Degenerative polyarthritis; Dextrans; Dimensions; Disease; Dose; Encapsulated; Engineering; Gel; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Growth Factor; Histologic; Human; Hydrogels; Injury; Joints; Knee; Laboratories; Lead; Mechanics; Mesenchymal Stem Cells; Messenger RNA; MicroRNAs; Microfluidic Microchips; Modeling; Molecular; Morphology; Natural regeneration; Non-Viral Vector; Oryctolagus cuniculus; Osteogenesis; Pain; Pathway interactions; Patients; Polyethyleneimine; Process; Proteins; Quality of life; RNA; RNA Interference; RNA delivery; Reporter Genes; Small Interfering RNA; Source; Stains; System; Technology; Testing; Therapeutic; Tissue Engineering; Tissues; Transfection; Work; articular cartilage; bone; clinical translation; crosslink; density; design; disability; efficacious treatment; healing; implantation; improved; in vivo; knock-down; microfluidic technology; osteochondral repair; osteochondral tissue; osteogenic; public health relevance; recruit; repaired; scaffold; spatiotemporal; stem cell differentiation; subchondral bone; tissue regeneration; tool

 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.

 描述：骨软骨缺损（OCD）的治疗涉及到受影响关节的软骨下骨和关节软骨的损伤，这种使人衰弱的缺损会导致机械不稳定、疼痛和延长骨关节炎退化，目前的治疗方法无法持续修复。利用生物材料与招募和/或移植的细胞和/或生物活性因子结合的骨软骨组织工程技术已成为一种有前景的替代方法。间充质干细胞 (hMSC) 是一种有吸引力的细胞来源，因为它们可以很容易地从骨髓中分离出来，在培养中扩增而不会失去多能性，并且在适当的条件下可以分化为成骨细胞和软骨细胞谱系的细胞。它是一种强大的工具，可以通过有针对性地破坏特定的 mRNA 分子来抑制翻译后水平的基因表达，并有可能通过减少对愈合过程产生负面影响的特定蛋白质的表达或改变损伤组织的功能修复来彻底改变受损组织的功能修复。重要的是，RNAi 分子已被鉴定可以促进 hMSC 的成骨和软骨形成，然而，将 RNAi 分子有效递送至体内靶细胞仍然是限制其治疗潜力的重大挑战。局部递送生物活性 RNAi 分子，具有可定制的释放曲线，用于递送到周围和封装的细胞，并且这些凝胶已被用来在空间和时间上控制细胞基因表达和命运。因此，该应用的中心假设是。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驱动封装的 hMSC 沿相反方向成骨和软骨形成，形成骨软骨结构，从而促进强迫症的愈合。这将通过以下具体目标来解决： (1) 使用两种不同 siRNA 的竞争浓度梯度设计生物聚合物水凝胶，以实现时空控制、持续基因敲低，(2) 提供 RNAi 分子，促进生物聚合物梯度水凝胶的成骨和软骨形成，并研究其在空间上引导封装的 hMSC 成骨和软骨分化的能力，(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，以及 (4) 评估水凝胶的能力含有 hMSC 和反向 RNAi 分子梯度的构建体在植入兔强迫症模型后可驱动体内成骨和软骨形成。该申请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，这将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。