In Situ Hardening Cell-Laden Constructs for Osteochondral Tissue Engineering

项目摘要/摘要 这项提议的最终目标是开发一种创新的、模块化的骨软骨组织技术 修复包括可注射的、热响应的、原位形成的和可生物降解的水凝胶结构 能够维持包裹的软骨和成骨细胞群体在空间上的输送 引导时尚，促进天然组织再生。我们假设一个细胞相容的水凝胶系统 由不收缩的、可注射的水凝胶和完全可溶的降解产物组成将通过 定制聚(N-异丙基丙烯酰胺)基热凝胶大分子单体与赖氨酸基大分子单体的组合 还含有硫酸软骨素(CS)共价连接部位的交联型大分子单体，以增强 合成构造物的集成。此外，我们假设多(L-赖氨酸)的掺入 热凝胶水凝胶中的PLL将增强共包裹关节的软骨形成能力 软骨细胞与间充质干细胞(AC-MSC)共培养 凝结信号。最后，我们假设一个结合了CS修饰的双层结构 具有设计矿化能力的成骨水凝胶层的软骨水凝胶层将 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，以赖氨酸为基础的聚酯氨酯大分子单体，包括可生物降解的聚(DL-乳酸-co-丙交酯) 乙醇酸)中间嵌段和化学上可交联的二胺官能团将被共价开发 用CS修饰，与热凝胶大分子单体结合，并进行彻底评估，以建立结构- 财产关系。第二，PLL将被加入到水凝胶中，它对 将对微囊化AC-MSC共培养的软骨形成进行评估。此外，锁相环的综合效应 水凝胶构建物的呈现、AC-MSC共培养和CS-修饰对软骨组织整合的影响 也可以在体外进行评估。第三，针对特定目标开发的水凝胶1，并针对软骨化进行了优化 在特定目标2中的潜力将与具有高矿化能力的水凝胶配方合并以产生 含成骨层和软骨层的双层水凝胶构建物用于有效修复 骨软骨缺陷症。微囊化细胞在成骨和骨修复中的潜在协同作用 将在体外和体内对含有PLL的软骨形成层进行评估，以确定最有效的 在已建立的兔骨软骨缺损模型中骨软骨组织修复的结构。这个 拟议的系统将通过以下方式解决与骨软骨缺陷修复相关的长期存在的重大挑战 通过高度整合，使构建物与周围的天然软骨组织稳定结合 模块化双组分设计，同时促进各自的软骨和成骨分化 细胞群分别对软骨和骨再生产生影响。

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.