A Bio-inspired Latent TGF-beta Conjugated Scaffold for Patient-specific Cartilage Regeneration

摘要 转化生长因子β(tgf-)已成为目前应用最广泛的促进剂之一。 组织工程(TE)应用中的软骨生长。传统上，对于体外培养阶段，转化生长因子-是 在培养基中补充，期望它能很容易地扩散到组织中并促进 健康软骨细胞外基质的生物合成。然而，越来越多的证据揭示了一个中心 这种传统的转化生长因子-给药策略的悖论：生理性转化生长因子-剂量显示有限的渗透性 进入组织，导致不希望的不均匀生长，而替代使用更高， 超生理剂量的转化生长因子-促进具有受损组织质量的软骨的形成(例如， 纤维化、肥大、增生)。与传统的TE转化生长因子-传递策略相比，自然 转化生长因子-在天然软骨中的传递过程有很大的不同，软骨细胞被 大量储存的转化生长因子-以不活跃的形式被隔离，称为潜伏性转化生长因子-(LTGF-)。软骨细胞 通过整合素或分泌酶激活ltf-存储，从而产生非常有利的、基于需求的 整个组织的活性，这允许必要的ECM生物合成，同时避免诱导 病理组织形成。 该提案通过创建受生物启发的TE策略来利用这种本地监管机制， 由此将软骨形成细胞包裹在与大量储存的转化生长因子-结合的水凝胶支架中， 与原生环境相似。这个平台允许细胞内源性地激活这些ltf-存储， 为细胞提供均匀和适度的、接近生理的转化生长因子-剂量， 它们在没有组织质量限制的情况下促进生物合成增强。 在此基础上，建立了一个新的反应扩散模型框架来预测转化生长因子的活性 暴露于构造中的细胞，同时考虑应用于 组织中的转化生长因子-。这些特定于患者的模型可以指导最佳的LTGF设计参数，从而允许 最佳活动剂量，提高TE软骨质量，缓解致病脱靶 从构建的载体上解吸转化生长因子-。 在目前的项目中，我们通过评估以下各项来检验这种受生物启发的LTGF一支架平台的有效性： 1)反应扩散模型优化患者特定细胞群体生长结果的能力 (人软骨细胞和间充质干细胞)，2)模型优化的转化生长因子-支架改善软骨细胞生长的能力 骨关节炎滑膜关节在恶劣的机械力化学环境中的软骨表现 体外滑膜关节生物反应器；3)转化生长因子-支架改善软骨性能的实验研究 在活体猪局灶性缺损区模型中。

Summary Transforming growth factor beta (TGF-) has become one of the most widely utilized mediators to promote cartilage growth in tissue engineering (TE) applications. Conventionally, for in vitro culture phases, TGF- is supplemented in the culture medium with the expectation that it will readily diffuse into tissues and promote the biosynthesis of a healthy cartilage ECM. However, a growing body of evidence brings to light a central paradox with this conventional TGF- delivery strategy: physiologic TGF- doses exhibit limited penetration into the tissue, giving rise to undesirable non-uniform growth, while the alternative use of higher, supraphysiologic TGF- doses promotes the formation of cartilage with compromised tissue quality (e.g., fibrosis, hypertrophy, hyperplasia). In contrast to conventional TE TGF- delivery strategies, the natural process of TGF- delivery in native cartilage occurs quite differently, where chondrocytes are surrounded by large stores of TGF- that are sequestered in an inactive form, termed latent TGF- (LTGF-). Chondrocytes activate LTGF- stores via integrins or secreted enzymes, leading to highly advantageous, need-based activity throughout the tissue, which allows for essential ECM biosynthesis while avoiding the induction of pathological tissue formation. This proposal capitalizes on this native regulatory mechanism by creating a bio-inspired TE strategy, whereby chondrogenic cells are encapsulated in a hydrogel scaffold conjugated with large stores of LTGF-, akin to the native environment. This platform allows cells to endogenously activate these LTGF- stores, giving rise to the highly beneficial delivery of uniform and moderated, near-physiologic TGF- doses to cells, which promote biosynthetic enhancements in the absence of tissue quality limitations. Further, a novel reaction-diffusion modeling framework is developed to predict the activity of TGF- exposed to cells in constructs while accounting for the critical patient-specific chemical reactions applied to TGF- in the tissue. These patient-specific models can guide optimal LTGF- design parameters, allowing for optimal activity doses and giving rise to improved TE cartilage quality and mitigation of pathogenic off-target desorption of TGF- from the construct. In the current project, we examine the efficacy of this bio-inspired LTGF- scaffold platform by assessing: 1) the capability of reaction-diffusion models to optimize growth outcomes in patient-specific cell populations (human chondrocytes and MSCs), 2) the capability of model-optimized LTGF- scaffolds to improve TE cartilage performance in the hostile mechanochemical environment of the OA synovial joint through use of an ex vivo synovial joint bioreactor, and 3) the capability of LTGF- scaffolds to improve TE cartilage performance in an in vivo porcine focal defect model.