Regulated Osteochondrogenesis of Human Mesenchymal Stem Cells Using Gene Delivery

描述(申请人提供)：关节软骨的再生，无论是疾病或损伤，是一个复杂的问题，仍然是一个重大的临床挑战，尽管广泛的骨科研究。虽然有许多止痛药、治疗策略和外科手术程序被开发来解决这一健康问题，但大多数治疗方法都是短暂的和不可补救的。该项目的总体目标是用多层结构修复骨软骨缺损，该结构呈现空间可控的软骨和成骨特性，并由人骨髓间充质干细胞(HMSCs)生成。为此，提出了以下具体目标：(1)开发3D控制释放系统，以适合MSCs成骨和成软骨分化的速率和浓度递送编码的转录因子；(2)评估在特定目标1中创建的控制释放系统产生双层软骨和成骨构建物的潜力。通过转录因子SOX-5、SOX-6、SOX-9(SOX Trio)和RUNX-2的基因传递，实现MSCs在三维支架中的骨软骨分化。每一种载体都将与我们实验室开发的新型聚合物基因传递载体(支化的聚乙烯亚胺和透明质酸的结合物)复合，以提高转染率。一开始，我们将研究质粒浓度和暴露时间对骨软骨分化的影响，以确定合适的靶向释放谱。然后，电纺同轴纤维网状支架将被嵌入到聚合物纤维的核心中的载体-质粒复合体中，并将利用公认的制造变量来实现所需的释放曲线。对于设想的最终双层结构的各个层的评估将基于种子MSCs的成骨和软骨分化以及类似于天然组织的细胞外基质的产生。这些研究将涉及hMSCs来评估所提出的方法在人类身上最终临床应用的初步可行性，同时将使用兔MSCs进行平行研究，以评估在这种新的骨软骨组织再生方法的翻译临床前开发中，所设想的兔动物模型的适宜性。这一建议的创新之处在于，它利用(A)骨髓间充质干细胞(MSCs)绕过了与分化细胞类型有限和维持相关的传统问题，(B)编码在质粒中的转录因子提供了更广泛的MSCs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撑网格。尽管这项建议针对的是特定的临床需求，但这些研究的影响并不局限于软骨甚至矫形外科的应用。这里提出的原则提供了一种方法来绕过与分化细胞的分离和维持相关的问题。转录因子可能会促进hMSCs在许多组织工程应用中的应用。此外，新的支架制造技术允许多个基因递送载体的受控递送，同时为细胞生长提供3D支持。这些技术在组织工程中有着广泛的应用，并有可能被用来创建模仿软骨和其他复杂组织的带状结构的多层支架。 与公共健康相关：该项目提出了一种创新的技术和方法，用于创建骨软骨构建物，用于修复受损的关节软骨和软骨下骨。在一种新型的控制释放系统中，将利用发育生物学信号的组合来传递差异信号，以诱导人骨髓间充质干细胞分化为合适的骨软骨表型。该系统将扩大组织工程师可用于产生具有多种细胞类型的各向异性组织的技术范围。

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.