Regulated Osteochondrogenesis of Human Mesenchymal Stem Cells Using Gene Delivery

描述（由申请人提供）：疾病或损伤损伤的关节软骨的再生是一个复杂的问题，尽管有广泛的骨科研究，但仍然是一个重大的临床挑战。虽然有许多镇痛药、治疗策略和外科手术来解决这一健康问题，但大多数治疗都是短期的，而且没有补救作用。该项目的总体目标是利用人间充质干细胞（hMSCs）生成的具有空间控制的软骨和成骨特性的多层结构修复骨软骨缺损。为此，提出了以下具体目标：(1)开发3D控释系统，以适合MSCs成骨和软骨分化的速率和浓度递送质粒编码的转录因子；(2)评估在specific Aim 1中创建的控释系统产生双层软骨和成骨结构的潜力。通过基因传递转录因子Sox-5、Sox-6、Sox-9 （Sox trio）促进软骨形成和Runx-2促进成骨，MSCs在3D支架中的骨软骨分化将得以实现。每个质粒都将与我们实验室开发的新型聚合基因传递载体（支链聚乙烯亚胺和透明质酸的偶联物）络合，以提高转染效率。首先，将检查质粒浓度和暴露时间对骨软骨分化的影响，以确定适当的靶释放谱。电纺丝，同轴纤维网状支架将被制造与载体质粒复合物嵌入在聚合物纤维的核心和完善的制造变量将被利用，以实现所需的释放轮廓。对设想的最终双层结构的单个层的评估将基于种子间充质干细胞的成骨和软骨分化以及与天然组织相似的细胞外基质的产生。这些研究将涉及骨髓间充质干细胞，以评估所提出的方法在人类最终临床应用的初步可行性，同时将使用兔骨髓间充质干细胞进行平行研究，以评估兔动物模型在骨软骨组织再生新方法的转化临床前开发中的适用性。该提议的新颖之处在于，它利用(a)间充质干细胞来规避与分化细胞类型有限的可用性和维持相关的传统问题，(b)质粒编码的转录因子提供了间充质干细胞更广泛的表型诱导，(c)聚合物支架为细胞增殖/分化和基因传递载体的控制释放提供了三维支持晶格。虽然这一建议解决了特定的临床需要，但这些研究的影响并不局限于软骨甚至矫形应用。本文提出的原则提供了一种方法来规避与分离和维持分化细胞相关的问题。转录因子将潜在地促进hMSCs在许多组织工程应用中的使用。此外，新的支架制造技术允许多种基因传递载体的调控传递，同时为细胞生长提供3D支持。这些技术在组织工程中有广泛的应用，可以潜在地用于制造多层支架，模仿软骨和其他复杂组织的带状结构。

DESCRIPTION (provided by applicant): Regeneration of articular cartilage damaged either by disease or injury is a complex problem that remains a significant clinical challenge despite extensive orthopaedic research. Although there are numerous analgesics, therapeutic strategies and surgical procedures developed to address this health concern, most of the therapies are short-lived and non-remedial. The global objective of this project is to repair osteochondral defects with a multilayered construct presenting spatially controlled chondrogenic and osteogenic properties and generated from human mesenchymal stem cells (hMSCs). To this end, the following specific aims are proposed: (1) to develop 3D controlled release systems that deliver plasmid encoded transcription factors at rates and concentrations appropriate for osteogenic and chondrogenic differentiation of MSCs and (2) to evaluate the controlled release systems created in Specific Aim 1 for their potential to generate bi-layered chondrogenic and osteogenic constructs. Osteochondral differentiation of MSCs in 3D scaffolds will be achieved through gene delivery of transcription factors Sox-5, Sox-6, Sox-9 (Sox trio) for chondrogenesis, and Runx-2 for osteogenesis. Each of these plasmids will be complexed with a novel polymeric gene delivery vector developed in our laboratory (a conjugate of branched polyethylenimine and hyaluronic acid) to increase transfection efficiency. Initially, the effect of plasmid concentration and exposure duration on osteochondral differentiation will be examined to identify the appropriate target release profiles. Electrospun, co-axial fiber mesh scaffolds will then be fabricated with the vector-plasmid complexes embedded in the core of the polymer fibers and well-established fabrication variables will be utilized to achieve the desired release profiles. Evaluation of the individual layers for the envisioned final bi-layered construct will be based upon the osteogenic and chondrogenic differentiation of the seeded MSCs and the generation of extracellular matrix similar to the native tissue. The studies will involve hMSCs to evaluate the initial feasibility of the proposed approach for ultimate clinical application in humans, while parallel studies will be conducted using rabbit MSCs to evaluate the appropriateness of the envisioned application of the rabbit animal model in the translational pre-clinical development of this novel approach to osteochondral tissue regeneration. The novelty of this proposal is that it utilizes (a) MSCs to circumvent the traditional problems associated with the limited availability and maintenance of differentiated cell types, (b) transcription factors encoded in plasmids to provide a broader phenotypic induction of MSCs and (c) polymeric scaffolds to provide a three dimensional support lattice for cell proliferation/differentiaton and the controlled release of gene delivery vectors. Although this proposal addresses a specific clinical need, the impact of these studies is not limited to cartilage or even orthopaedic applications. The principles proposed herein provide a means to circumvent the problems associated with the isolation and maintenance of differentiated cells. Transcription factors will potentially facilitate the use of hMSCs in a number of tissue engineering applications. Furthermore, the novel scaffold fabrication technique permits regulated delivery of multiple gene delivery vectors while providing a 3D support for cell growth. These techniques have wide-ranging applications in tissue engineering and can potentially be used to create multilayer scaffolds that mimic the zonal architecture of cartilage and other complex tissues. PUBLIC HEALTH RELEVANCE: This project proposes an innovative technology and approach for creating osteochondral constructs for the repair of damaged articular cartilage and subchondral bone. A combination of developmental biological signals will be utilized in a novel controlled release system to deliver differential signals to induce human mesenchymal stem cells into appropriate osteochondral phenotypes. This system will increase the scope of techniques available to tissue engineers for generating anisotropic tissues with multiple cell types.