High toughness bio-inspired hydrogels for cartilage tissue engineering

描述(申请人提供)：本申请的长期目标是设计机械可行的软骨结构，用于治疗严重的骨关节炎。这项建议的总体目标是将在创造高韧性互穿网络水凝胶(IPN)方面的最新进展与aggrecan的软骨形成能力相结合，以创建一种重要的用于软骨再生的新型生物材料。我们采用了软骨组织工程中常用的两种水凝胶，琼脂糖凝胶和聚乙二醇凝胶，并开发了一种新的合成工艺，将这两种材料组合成IPN，与单独的组分相比，IPN的力学性能有了很大的提高。这种新型的IPN水凝胶是通过琼脂糖物理凝胶化(细胞被包裹)，然后嵌入聚乙二醇二丙烯酸酯(PEG-DA)的光聚合而产生的，在这个过程中，我们已经证明了细胞保持了它们的活力(在文献中是前所未有的)。IPN的压缩模数接近天然软骨，最重要的是，韧性(压缩下断裂所需的能量)分别是聚乙二醇DA或琼脂糖凝胶的5倍和100倍。这一发现的意义在于，通过创建高韧性的IPN，我们克服了当前水凝胶支架的主要限制，因为高韧性对于在要求苛刻的环境(如人类的膝盖或髋关节)中抵御骨折至关重要。另一个主要限制是无法为软骨形成提供足够的生化信号。因此，我们提出了一种通过在我们的高韧性IPN中加入聚集素来对支架进行一种新的修饰。聚集素最近被用于单层研究以促进和保留软骨细胞的表型，但到目前为止还没有被用作组织工程支架中的软骨形成信号。主要的假设是，利用IPN技术的这一突破，以及聚集素作为生物活性信号，将生产出机械完整性可与天然人类软骨相媲美的工程化软骨结构。为了验证这一假设，我们提出了以下具体目标：(1)进一步提高我们的高韧性琼脂糖/聚乙二醇单组分互穿网络的性能(最近有关互穿网络的文献表明，韧性可能比单组分网络提高1000倍)，以及(2)将生物活性分子引入琼脂糖/聚乙二醇单组分互穿网络。我们将首先改变IPN的组成，以最大限度地增加其韧性(以防止失败)，同时将其硬度保持在天然人类软骨的范围内(以提供类似的抗变形能力)。使用这种组合物，我们将把粘附肽序列精氨酸-甘氨酸-天冬氨酸(RGD)或硫酸软骨素(CS)整合到IPN中，并将软骨细胞包裹起来进行为期6周的研究。RGD和CS被选为确定的比较标准，以将aggrecan的疗效置于适当的背景下。这个拟议的项目将材料科学与生物和临床应用联系起来，如果成功，将为软骨组织工程提供一种新的材料类别，并成为未来众多研究途径的跳板。公共卫生相关性：关节炎是美国残疾的主要原因，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学，其目标是取代骨关节炎造成的关节结构。为此，这项拟议的研究将产生一种重要的新型生物材料，具有优异的机械完整性，用于软骨再生。

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.