High toughness bio-inspired hydrogels for cartilage tissue engineering

描述（由申请人提供）：本申请的长期目标是设计机械上可行的软骨结构，用于治疗严重骨关节炎。本提案的总体目标是结合最近在创造高韧性互穿网络水凝胶（ipn）和聚集蛋白的成软骨能力方面的进展，创造一种重要的新型软骨再生生物材料。我们采用了软骨组织工程中常用的两种水凝胶，琼脂糖和聚乙二醇，并开发了一种新的合成方法，将这两种材料结合成一个IPN，与单个成分相比，它的机械性能大大提高。这种新型IPN水凝胶是通过琼脂糖（细胞包裹）的物理凝胶化，然后是嵌入的聚乙二醇二丙烯酸酯（PEG-DA）的光聚合而成的，在这个过程中，我们已经证明细胞保持了它们的活力（在文献中是前所未有的）。IPN具有接近天然软骨的压缩模量，最重要的是，其韧性（在压缩下断裂所需的能量）分别比单独的PEG-DA或琼脂糖大5倍和100倍。这一发现的意义在于，通过制造高韧性的ipn，我们克服了当前水凝胶支架的主要限制，因为高韧性对于在诸如人类膝盖或臀部等苛刻环境中承受骨折至关重要。另一个主要限制是不能为软骨形成提供足够的生化信号。因此，我们提出了一种新的改性支架，将聚合蛋白加入到我们的高韧性IPN中。最近在单层研究中，聚集蛋白已被用于促进和保持软骨细胞表型，但迄今为止尚未在组织工程支架中用作软骨生成信号。主要的假设是，利用IPN技术的这一突破，以及聚合蛋白作为生物活性信号，将产生具有与天然人类软骨相当的机械完整性的工程软骨结构。为了验证这一假设，我们提出了以下具体目标：(1)进一步提高琼脂糖/PEG-DA高韧性IPN的性能（最近有关IPN的文献表明，单组分网络的韧性可能增加1000倍）；(2)将生物活性分子加入琼脂糖/PEG-DA IPN中。我们将首先改变IPN的组成，以最大限度地提高其韧性（防止失效），同时将其刚度保持在天然人体软骨的范围内（提供类似的抗变形能力）。使用这种组合物，我们将聚集蛋白、粘附肽序列精氨酸-甘氨酸-天冬氨酸（RGD）或硫酸软骨素（CS）纳入IPN并包封软骨细胞，进行为期6周的研究。选择RGD和CS作为既定的比较标准，将aggrecan的疗效置于适当的背景下。该项目将材料科学与生物学和临床应用联系起来，如果成功，将为软骨组织工程提供一类新的材料，并作为未来许多研究途径的跳板。公共卫生相关性：关节炎是美国致残的主要原因，骨关节炎影响着2100万美国人，每年给美国经济造成的损失超过600亿美元。一个令人兴奋的潜在解决方案是组织工程，其目的是取代被骨关节炎破坏的关节结构。为了实现这一目标，拟议的研究将产生一种重要的新型生物材料，具有优越的软骨再生机械完整性。

DESCRIPTION (provided by applicant): The long-term objective of this application is to engineer mechanically viable cartilage constructs for treatment of severe osteoarthritis. The overall objective of this proposal is to combine recent advances in creating high toughness interpenetrating network hydrogels (IPNs) and the chondrogenic ability of aggrecan to create a significant new class of biomaterials for cartilage regeneration. We have taken two hydrogels commonly used in cartilage tissue engineering, agarose and poly(ethylene glycol), and developed a novel synthesis procedure to combine these two materials into an IPN with vastly improved mechanical properties compared to the individual constituents. This novel IPN hydrogel is created by physical gelation of the agarose (with cells encapsulated) followed by photopolymerization of embedded poly(ethylene glycol) diacrylate (PEG-DA), a process in which we have shown cells maintain their viability (unprecedented in the literature). The IPN has a compressive modulus close to that of native cartilage, and most importantly, a toughness (the energy required to fracture under compression) 5 and 100 times larger than PEG-DA or agarose alone, respectively. The significance of this discovery is that by creating IPNs of high toughness, we overcome a major limitation of current hydrogel scaffolds, as high toughness is crucial for withstanding fracture in demanding environments such as a human knee or hip. Another major limitation is the inability to provide sufficient biochemical signals for chondrogenesis. Thus we propose a novel modification to the scaffold by incorporating aggrecan into our high toughness IPN. Aggrecan has been exploited recently in monolayer studies to promote and retain chondrocytic phenotype, but heretofore has not been employed as a chondrogenic signal in a tissue engineering scaffold. The chief hypothesis is that use of this breakthrough in IPN technology, along with aggrecan as a bioactive signal, will produce engineered cartilage constructs with mechanical integrity comparable to native human cartilage. To test this hypothesis, we propose the following specific aims: (1) to further improve the performance of our high-toughness IPNs of agarose/PEG-DA (recent literature on related IPNs suggests a 1000-fold increase in toughness over the single component networks may be achievable), and (2) to incorporate bioactive molecules into the agarose/PEG-DA IPN. We will first vary the composition of the IPN to maximize its toughness (to prevent failure) while maintaining its stiffness within the range of native human cartilage (to provide similar resistance to deformation). Using this composition, we will incorporate aggrecan, the adhesion peptide sequence arginine-glycine-aspartic acid (RGD), or chondroitin sulfate (CS) into the IPN and encapsulate chondrocytes for 6-week studies. RGD and CS were chosen as established standards of comparison to place the efficacy of aggrecan in an appropriate context. This proposed project bridges materials science with biological and clinical application, and if successful, will provide a new class of materials to cartilage tissue engineering and act as a springboard to numerous avenues of future investigation. PUBLIC HEALTH RELEVANCE: Arthritis is the leading cause of disability in the United States, and osteoarthritis affects 21 million Americans at an annual cost to the United States economy exceeding $60 billion. An exciting potential solution is tissue engineering, which aims to replace joint structures ravaged from osteoarthritis. Toward that end, the proposed research will produce a significant new class of biomaterials with superior mechanical integrity for cartilage regeneration.