In Situ Hardening Cell-Laden Constructs for Osteochondral Tissue Engineering

项目总结/摘要 该提案的最终目标是为骨软骨组织开发创新的模块化技术 修复包括可注射、热响应、原位形成和可生物降解的水凝胶构建体 能够在空间上维持包封的软骨形成和成骨细胞群的递送， 直接的方式来促进天然组织再生。我们假设细胞相容性水凝胶系统 由非收缩的可注射水凝胶组成，具有完全可溶的降解产物， 定制的基于聚（N-异丙基丙烯酰胺）的热凝胶化大分子单体和基于赖氨酸的热凝胶化大分子单体的组合 交联大分子单体，其还含有用于硫酸软骨素（CS）共价连接的位点，以增强 所得到的结构的整合。此外，我们假设多聚赖氨酸的掺入， (PLL)将增强共包封的关节软骨形成能力 软骨细胞和间充质干细胞（AC-MSC）共培养通过诱导发育相关的 冷凝信号。最后，我们假设结合CS修饰的 具有设计矿化能力的成骨水凝胶层的成软骨水凝胶层将 以促进有效的骨软骨组织修复。提出了三个具体目标来解决这些问题 假设首先，制备包含可生物降解的聚（DL-乳酸-共-聚（DL-乳酸））的基于赖氨酸的聚酯氨基甲酸酯大分子单体。 羟基乙酸）中间嵌段和化学可交联的二胺官能团将被共价地开发 用CS改性，与热凝胶化大分子单体组合，并彻底评估以建立结构- 财产关系。第二，PLL将被并入水凝胶中，并且其对水凝胶的性能的影响是： 将评价包封的AC-MSC共培养物的软骨形成。此外，PLL的组合效应 介绍，AC-MSC共培养，和CS修饰的水凝胶结构对软骨组织整合将 也可以离体评价。第三，在Specific Aim 1中开发并针对软骨形成优化的水凝胶 Specific Aim 2中的潜力将与具有高矿化能力的水凝胶制剂合并， 包含软骨形成层和成骨层的双层水凝胶构建体 骨软骨缺损微囊化细胞在成骨细胞和成纤维细胞中的潜在协同作用， 将在体外和体内评价具有PLL递送的软骨形成层， 在良好建立的兔骨软骨缺损模型中，骨软骨组织修复的配置。的 所提出的系统将通过以下方式解决与骨软骨缺损修复相关的持续重大挑战： 使得该结构与周围的天然软骨组织通过高度的 模块化双组分设计，同时促进各自的软骨和成骨分化 分别递送以实现软骨和骨再生的细胞群。

Project Summary/Abstract The ultimate goal of this proposal is to develop an innovative and modular technology for osteochondral tissue repair comprising injectable, thermally responsive, in situ forming, and biodegradable hydrogel constructs capable of sustaining the delivery of encapsulated chondrogenic and osteogenic cell populations in a spatially directed fashion to promote native tissue regeneration. We hypothesize that a cytocompatible hydrogel system consisting of non-shrinking, injectable hydrogels with fully soluble degradation products will be formed through the combination of custom poly(N-isopropylacrylamide)-based thermogelling macromers and lysine-based crosslinking macromers that also contain sites for covalent attachment of chondroitin sulfate (CS) to enhance the integration of resultant constructs. Additionally, we hypothesize that the incorporation of poly(L-lysine) (PLL) within the thermogelling hydrogel will enhance the chondrogenic capacity of co-encapsulated articular chondrocyte and mesenchymal stem cell (AC-MSC) cocultures via the induction of developmentally relevant condensation signals. Finally, we hypothesize that a bilayered construct combining the CS-modified chondrogenic hydrogel layer with an osteogenic hydrogel layer of designer mineralizing capability will be leveraged to promote effective osteochondral tissue repair. Three Specific Aims are proposed to address these hypotheses. First, a lysine-based polyesterurethane macromer comprising a biodegradable poly(DL-lactic-co- glycolic acid) mid-block and chemically crosslinkable diamine functionalities will be developed, covalently modified with CS, combined with the thermogelling macromer and thoroughly assessed to establish structure- property relationships. Second, PLL will be incorporated into the hydrogels and its effects on the chondrogenesis of encapsulated AC-MSC cocultures will be evaluated. Further, the combined effects of PLL presentation, AC-MSC coculture, and CS-modification of hydrogel constructs on cartilage tissue integration will be also evaluated ex vivo. Third, the hydrogels developed in Specific Aim 1 and optimized for chondrogenic potential in Specific Aim 2 will be merged with hydrogel formulations with high mineralizing capability to yield bilayered hydrogel constructs comprising chondrogenic and osteogenic layers for the effective repair of osteochondral defects. The potential synergistic effects of encapsulated cells in the osteogenic and chondrogenic layers with PLL delivery will be evaluated in vitro and in vivo to determine the most effective configuration for osteochondral tissue repair in a well-established rabbit osteochondral defect model. The proposed system will address persisting significant challenges associated with osteochondral defect repair by enabling stable integration of the construct with the surrounding native cartilage tissue through a highly modular two-component design, while promoting the chondrogenic and osteogenic differentiation of respective cell populations delivered to effect both cartilage and bone regeneration, respectively.