Self-assembling process in tissue engineering of articular cartilage

 描述(由申请人提供)：关节软骨中的自组装过程正在成为设计大小软骨结构的一种潜在的可靠方法。本方案的目的是评价自组装关节软骨在关节表面修复中的作用。 根据FDA的定义，通过量化生物反应(即宿主对构建物的反应)、剂量反应(即确定所需细胞的最低数量)和耐久性(即稳定性、完整性和一年以上的成熟度)来评估膝盖骨。为了改善自组装软骨结构的功能，统计优化已经产生了外部刺激的强大组合，导致构建的结构具有与天然软骨相同的生物力学和生化特性。在众多有帮助的刺激中，有三种已经变得相当有效：静水压力(10-14天，0 HZ时10 Mpa)，转化生长因子-1(30 ng/ml，持续2周)，以及软骨素酶ABC(在2周时应用)。还阐明了将这些刺激与工程组织的生物力学特性联系起来的机制，以解释它们的协同作用，并将它们整合到简单的培养方案中。功能指数(FI)允许建立量化的成功标准，并被验证用于将构建物与天然组织进行比较，表明构建物特性已达到接近天然组织值1的FI值。基于这些令人振奋的结果以及更多的在体小鼠、Leporine和绵羊数据，这项建议将通过三个目标调查全球假说，即构建物将显示出耐久性而不会引起宿主不良反应：目标1的目的是使用短期(12周)Leporine膝盖骨表面修复模型来检验假说：1)不仅构建物在体内保持稳定和完整性，而且其FI值将通过植入得到改善，2)异基因细胞和培养产品都不会引起宿主的不良反应(局部/全身)。目的2采用小鼠模型来验证种植体的放大不会改变新人工关节的生物力学特性。AIM 2还将解决不同软骨再生策略中常见的某些挑战，即初始固定、后续整合和耐磨性；这些问题将通过软骨导电胶、外源赖氨酸氧化酶和软骨调优方法来解决，该方法可产生坚固和光滑的植入物。最后，目标3将在绵羊模型中测试持久愈合可长达12个月的假设。这一目标还将确定可用于在1年内实现有效愈合的最小细胞数量。通过遵循FDA的指导文件(“为修复或替换膝关节软骨的产品准备IDES和IND”)，如果拟议的研究假设得到证实，结果将为自组装关节软骨结构的临床可翻译性提供令人兴奋的验证。

 DESCRIPTION (provided by applicant): The self-assembling process in articular cartilage is emerging as a potentially robust approach for engineering large and small cartilage constructs. The objective of this proposal is to evaluate self-assembled articular cartilage in resurfacing the patella by quantifying the biologic response (i.e., host response to the construct), dose response (i.e., identifying the minimum number of necessary cells), and durability (i.e., stability, integriy, and maturation over a year), as defined by the FDA. Statistical optimization to improve the functionality of self- assembled cartilage constructs has yielded a powerful combination of externally applied stimuli that result in constructs with biomechanical and biochemical properties on par with those of native cartilage. Among a multitude of helpful stimuli, three have emerged as quite potent: hydrostatic pressure (10MPa at 0Hz during days 10-14), TGF-ß1 (30 ng/ml for 2 wks), and chondroitinase ABC (applied at 2 wks), applied in combination. Mechanisms linking these stimuli to the engineered tissues' biomechanical properties have also been elucidated to explain their synergisms and to consolidate them into simple culture protocols. A functionality index (FI) allowing the establishment of quantitative success criteria and validated for the comparison of constructs to native tissue showed that construct properties have attained FI values approaching 1, the value of native tissue. Based on these promising results and additional in vivo murine, leporine, and ovine data, this proposal will investigate the global hypothesis that constructs will show durability without an adverse host response via three aims: The objective of Aim 1 is to use a short-term (12wks), leporine patella resurfacing model to examine the hypotheses that: 1) not only will constructs retain stability and integrity in vivo, thir FI values will be improved by implantation, and 2) neither the allogeneic cells nor the culture products will elicit adverse host responses (local/systemic). Aim 2 employs the murine model to validate that implant scale-up would not alter neocartilage biomechanical properties. Aim 2 will also address certain challenges that are common across diverse cartilage regeneration strategies, namely initial fixation, subsequent integration, and durability against wear; these issues will be tackled using a chondroconductive glue, exogenous lysyl oxidase, and the chondrotuning method that yields robust and lubricious implants. Finally, Aim 3 will test the hypothesis that durable healing can be achieved for up to 12 months in an ovine model. This aim will also identify a minimum cell number that can be employed to achieve effective healing at 1 year. By following FDA's guidance document ("Preparation of IDEs and INDs for products intended to repair or replace knee cartilage"), and if the proposed study's hypotheses are proven, the results will provide exciting validation of the clinical translatability of self-assembed articular cartilage constructs.