Effects of three-dimensional macroporosity and matrix elasticity on the outcomes of stem cells based cartilage tissue regeneration

提案#:1605604干细胞的软骨再生为数百万软骨损伤（如骨关节炎）的患者提供了巨大的希望。该项目的总体目标是了解三维环境中孔隙大小和基质弹性如何调节基于干细胞的软骨再生。新型3D支架的构建模块是带状和微尺寸的水凝胶，可以交联来封装人类间充质干细胞，并提供具有独立可调孔径、基质弹性和化学性质的环境。提出的研究将为实现完全干细胞软骨修复所需的环境提供有价值的信息。教育和推广影响将通过增强参与学生的研究经验，新的研究生水平课程，以及涉及高中教师和代表性不足的K-12学生的活动来实现。该奖项由材料研究部的生物材料项目通过BioMaPs项目共同资助。关节软骨损伤作为创伤和退行性疾病的结果是一个严重的健康问题。在美国，仅骨关节炎就影响了2700多万人，预计每2个人中就有1人受其影响。软骨中的软骨细胞再生能力较差，大多数关节损伤不经开放手术或其他侵入性干预无法愈合。自体软骨植入（ACI）和微骨折技术是软骨修复的既定治疗方法，但这些方法往往受到软骨供应不足、软骨持续退变和纤维化软骨形成的限制，这些软骨不能提供足够的机械强度来承受身体负荷。以干细胞为基础的软骨再生为软骨损伤患者带来了巨大的希望。干细胞软骨形成的成功依赖于一个理想的细胞生态位，提供适当的生态位特性，形成对软骨形成至关重要的关键元素，包括生化和生物物理线索，以促进所需的干细胞生物活性。大孔是一种不小于典型细胞的孔隙空间，是一种极具潜力的控制干细胞成软骨分化的机械传感调节剂。然而，三维生态位特性调控干细胞软骨形成的机制仍不清楚。目前缺乏支持生态位特性与干细胞生物活性之间复杂相互作用的基础研究平台。考虑到干细胞在我们体内对生态位特性的反应的复杂性，一个可以轻松控制大孔隙度、基质弹性和细胞形态的三维生态位模型将有助于对生态位对软骨形成作用的机制研究，并可能有一天导致理想的细胞生态位实现软骨完全修复的最终目标。该项目将通过使用可交联的微带（过去几年出现的带状和微米大小的水凝胶）作为构建模型细胞龛的基石，为实现这一目标迈出第一步，以提供孔隙大小、细胞形状和弹性的巨大变化。微带提供了以下功能，以促进对生态位成分如何影响干细胞软骨形成的全面研究：1)在三维上直接封装细胞，2)通过可调节的大孔大小来控制细胞形状，3)独立调节的生化和机械线索，以及4)相互连接的大孔来保留细胞产生的ECM成分。利用巨带，研究人员实现了三个目标：1)开发一个将人间充质干细胞（MSC）暴露于各种大孔大小和基质弹性的3D模型，并评估MSC在细胞形状、局点粘附、细胞骨架组织和转录因子活性方面的细胞特性；2)确定大孔大小、基质弹性及相关细胞特性对人间充质干细胞成软骨分化的影响；3)：了解大孔隙率和基质弹性如何影响软骨基质的生成和软骨细胞表型的稳定性。如果成功，许多患有软骨损伤的患者可以从开发的3D大孔细胞龛中受益。拟议的项目涉及多个学科，包括工程、生物学和医学。学生进行拟议的研究将受益于接触各种各样的实验，包括有机合成，纳米压痕，干细胞培养和细胞分析。新的研究生水平的课程将纳入从拟议的研究元素，将研究成果从实验室长凳转化为课堂。项目中使用的技术，如湿纺和脚手架制造，将用于PI和工程学院主办的外展项目，为高中教师和代表性不足的K-12学生提供简短的讲座、夏令营和动手实验经验。PI和联合PI将积极招募女性和少数民族学生进行拟议的研究。

PI: Han, Li-Hsin Proposal #: 1605604Cartilage regeneration by stem cells provides a tremendous hope for the millions of people who suffer from cartilage injuries, e.g., osteoarthritis. The overall objective of this project is to understand how pore size and matrix elasticity in a 3D environment regulate stem cell-based cartilage regeneration. The building blocks for the novel 3D scaffold are ribbon-like and microsized hydrogels that can be cross linked to encapsulate human mesenchymal stem cells and provide an environment with independently tunable pore size, matrix elasticity and chemical properties. The proposed studies will provide valuable information about the environment needed to achieve complete stem cell based cartilage repair. Educational and outreach impact will be achieved through enhanced research experiences for involved students, new graduate level courses, and activities involving high school teachers and underrepresented K-12 students. This award is co-funded by the Biomaterials program in the Division of Materials Research through the BioMaPs program.Articular cartilage injuries as a result of trauma and degenerative diseases present a serious health problem. Osteoarthritis alone affects more than 27 million people in the U.S. and is predicted to affect 1 in 2 people in their lifetime. Chondrocytes in the cartilage have a poor regenerative capacity, and most articular injuries cannot heal without open surgery or other invasive intervention. Autologous cartilage implantation, or ACI, and the microfracture technology are the established treatments for cartilage repair, but these methods are often limited by insufficient cartilage supply, continual cartilage degeneration, and the formation of fibrosis cartilages that do not provide sufficient mechanical strength to sustain body loads. Stem cell-based cartilage regeneration provides tremendous hopes for people suffering from cartilage injuries. The success of stem cell chondrogenesis relies on an ideal cell niche that provide properly orchestrated niche properties, forming the key elements that are crucial for chondrogenesis, including biochemical and biophysical cues, to promote the desired stem cell bioactivities. Macropores, the pore space no smaller than the typical cells, is a highly potential mechanosensing regulator to control the stem cell chondrogenic differentiation. However, the mechanisms by which niche properties in three-dimensions regulate stem cells chondrogenesis remain largely unclear. A platform to support the fundamental study on the complex interaction between niche properties and stem cell bioactivities is currently lacking. Given the complex nature of how stem cells respond to niche properties in our body, a three-dimensional niche model that can easily control macroporosity, matrix elasticity and cell morphology will facilitate a mechanistic study on the niche effect on chondrogenesis, and may one day lead to an ideal cell niche to realize the ultimate goal of complete cartilage repair. This project will conduct the first step to attempt such goal by using the crosslinkable microribbons, which are ribbon-like and micron-sized hydrogels that emerged in the past couple years, as the building blocks to construct the model cell niches to provide a vast variation of pore size, cell shape, and elasticity. The microribbons provide the following functions to facilitate a comprehensive study on how niche compositions impact stem cells chondrogenesis: 1) direct cell encapsulation in three dimensions, 2) control of cell shape by tunable macropore size, 3) independently tunable biochemical and mechanical cues, and 4) interconnected macroporosity to retain the ECM components produced by cells. Using macroribbons, the investigators address 3 aims: 1) To develop a 3D model that exposes human mesenchymal stems cells (MSC) to various macropore sizes and matrix elasticity, and to evaluate the cellular properties of MSC with regard to cell shape, focal adhesion, cytoskeleton organization, and transcription factor activity; 2) To determine how macropore size, matrix elasticity and the associated cellular properties influence the chondrogenic differentiation of human MSC; and 3): To understand how macroporosity and matrix elasticity influence the production of cartilage matrix and the stability of chondrocyte phenotypes. If successful, numerous patients suffering from cartilage injuries can benefit from the 3D macroporous cell niche developed. The proposed project spans multiple disciplines including engineering, biology and medicine. Students carrying out proposed research will benefit greatly by being exposed to a variety of experiments including organic synthesis, nanoindentation, stem cell cultivation and cellular assays. New graduate-level courses incorporating the elements from the proposed research will be developed to translate the research outcomes from lab benches to the classroom. Technology used for the proposed project, such as wet-spinning and scaffold fabrication, will be contributed to the outreaching programs hosted by the PI and the college of engineering, providing short lectures, summer camps, and hands-on lab experience to high school teachers and underrepresented K-12 students. The PI and co-PI will actively recruit female and minority students to conduct the proposed research.