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

总结 转化生长因子β（TGF-β）已成为促进肿瘤生长的最广泛使用的介质之一。 组织工程（TE）应用中的软骨生长。常规地，对于体外培养阶段，TGF-β 1是 补充在培养基中，期望其将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，越来越多的证据表明， 这种常规TGF-β递送策略的矛盾之处在于：生理TGF-β剂量表现出有限的渗透性 进入组织，引起不希望的不均匀生长，而可选择使用更高， 超生理的TGF-β剂量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 TGF-β 1递送策略相反， TGF-β 1在天然软骨中的传递过程完全不同，其中软骨细胞被 大量TGF-β以非活性形式被隔离，称为潜伏性TGF-β（LTGF-β）。软骨细胞 通过整联蛋白或分泌酶激活LTGF储存，从而产生非常有利的、基于需求的 在整个组织中的活性，这允许必要的ECM生物合成，同时避免诱导 病理组织形成。 该提案通过创建生物启发的TE策略来利用这种天然调节机制， 由此软骨形成细胞被封装在与大量储存的LTGF-β缀合的水凝胶支架中， 类似于原生环境。该平台允许细胞内源性激活这些LTGF-β储存， 产生了均匀和适度的、接近生理的TGF-β剂量到细胞的高度有益的递送， 其在没有组织质量限制的情况下促进生物合成增强。 此外，开发了一种新的反应扩散模型框架来预测TGF-β的活性 暴露于构建体中的细胞，同时考虑应用于 组织中的TGF-β。这些患者特异性模型可以指导最佳的LTGF-CABG设计参数， 最佳活性剂量，并提高TE软骨质量和减轻致病性脱靶 TGF-β 1从构建体解吸。 在目前的项目中，我们通过评估来检查这种生物启发的LTGF-100支架平台的功效： 1)反应扩散模型优化患者特异性细胞群生长结果的能力 （人软骨细胞和MSC），2）模型优化的LTGF-TGF-β支架改善TE的能力， 软骨性能在OA滑膜关节的不利机械化学环境中，通过使用 离体滑膜关节生物反应器，和3）LTGF-TGF-β支架改善TE软骨性能的能力 在体内猪局灶性缺损模型中。

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.