3D Co-culture with Micropatternable Hydrogels to Promote Fibrous Tissue Formation

描述（由申请人提供）：自体间充质干细胞（MSC）已被提议作为韧带损伤的潜在治疗方法，因为它们通过降低供体部位发病率同时引起最小免疫反应而提供传统治疗的优势。然而，迄今为止，由于缺乏关于MSC和常驻成纤维细胞之间的相互作用如何导致两种细胞类型的表型改变的知识，因此在设计用于基于MSC的疗法的最佳递送时机、剂量和载体材料方面存在很大困难，所述表型改变最终导致基质产生和组织修复。该项目的长期目标是产生改进的基于MSC的疗法，包括组织工程结构，以帮助韧带损伤的再生。作为实现这一目标的第一步，本申请的目的是开发一种可微图案化的生物材料系统，用于在精确控制的环境中3D共培养MSC和韧带成纤维细胞，并使用该技术来确定周围细胞的存在如何影响每种细胞类型中的增殖和细胞外基质产生。这些研究的中心假设是，共培养将促进MSC和前交叉韧带（ACL）成纤维细胞的增殖和细胞外基质（ECM）的产生，在培养物中每种细胞类型的数量相等时，增殖和ECM的产生最大。我们将通过以下两个具体目标来测试该假设：1）设计可图案化的水凝胶、可图案化的水凝胶和图案化方法，其允许两个细胞群体的共培养和容易分离以用于培养后生物测定; 2）确定共培养对增殖和细胞外基质产生的影响（通过基因表达和免疫染色确定）。完成这些研究后，我们希望开发一种具有细胞释放能力的空间控制的3D共培养系统，以更好地了解可溶性因子对体外ACL模型中细胞分化和组织产生的影响。我们的方法是创新的，因为这里开发的技术可以在许多情况下实现组织再生的共培养和细胞工程。该项目意义重大，因为它代表了首次尝试使用受控的三维环境来了解可溶性因子如何在多种细胞类型存在下影响组织形成，例如韧带组织再生过程中存在的细胞类型。公共卫生相关性：我们希望这些研究的结果能够改善涉及MSC的再生医学策略，用于韧带修复。此外，我们预计在这个提案中开发的模式化和三维共培养技术将使在许多生物学背景下的组织工程的发展和转化研究的基础研究。

DESCRIPTION (provided by applicant): Autologous mesenchymal stem cells (MSCs) have been proposed as a potential therapy for ligament injury because they offer advantages to traditional treatments by reducing donor site morbidity while invoking minimal immune response. However, to date, there has been great difficulty in designing the optimal delivery timing, dosage and carrier material for MSC-based therapies due to the dearth of knowledge about how interactions between MSCs and resident fibroblasts cause alterations in the phenotype of both cell types that eventually lead to matrix production and tissue repair. The long-term goal of this project is to generate improved MSC-based therapies, including tissue- engineered constructs, to aid regeneration of ligament injuries. As a first step toward this goal, the objective of this application is to develop a micro-patternable biomaterial system for 3D co-culture of MSCs and ligament fibroblasts in a precisely controlled environment, and to use this technology to determine how the presence of surrounding cells affects proliferation and extracellular matrix production in each cell type. The central hypothesis of these studies is that co-culture will promote proliferation and extracellular matrix (ECM) production in both MSCs and anterior cruciate ligament (ACL) fibroblasts, with maximal proliferation and ECM production at equal numbers of each cell type in culture. We will test this hypothesis through the following two specific aims: 1) Engineer photopatternable hydrogels, digestable hydrogels, and patterning methodologies that allow co-culture of two populations of cells and easy separation for post-culture bioassays; 2) Determine the effect of co-culture on proliferation and extracellular matrix production (determined by gene expression and immunostaining) by encapsulated rabbit MSCs and ACL fibroblasts over 15 days. Upon completion of these studies, we expect to develop a spatially-controlled 3D co-culture system with cell-release capabilities to better understand the effects of soluble factors on cell differentiation and tissue production in an in vitro ACL model. Our approach is innovative because the technology developed here enables co-culture and cellular engineering for tissue regeneration in many contexts. This project is significant because it represents a first attempt to use a controlled, three-dimensional environment to understand how soluble factors influence tissue formation in the presence of multiple cell types such as those present during ligament tissue regeneration. PUBLIC HEALTH RELEVANCE: We expect results from these studies to lead to improved regenerative medicine strategies involving MSCs for ligament repair. In addition, we expect the patterning and three-dimensional co-culture techniques developed in this proposal will enable both fundamental studies in development and translational research in tissue engineering in many biological contexts.