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工程化大小软骨结构的潜在稳健方法。本研究的目的是评估自组装关节软骨在关节表面重建中的作用。 通过量化生物反应（即，宿主对构建体的反应），剂量反应（即，识别所需单元的最小数量），和耐久性（即，稳定性、完整性和一年以上的成熟度），如FDA所定义。用于改善自组装软骨构建体的功能的统计优化已经产生了外部施加的刺激的强大组合，其导致具有与天然软骨的生物力学和生物化学性质相当的生物力学和生物化学性质的构建体。在众多有益的刺激中，有三种已经显现为相当有效的：静水压（10 MPa，0 Hz，在第10-14天期间）、TGF-β 1（30 ng/ml，持续2周）和软骨素酶ABC（在2周时施用），组合施用。将这些刺激与工程化组织的生物力学性质联系起来的机制也已阐明，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），允许建立定量的成功标准，并验证了结构与天然组织的比较表明，结构性能已达到FI值接近1，天然组织的值。基于这些有希望的结果和额外的小鼠、兔和绵羊体内数据，本提案将通过三个目标研究构建体将显示耐久性而无不良宿主反应的总体假设：目标1的目标是使用短期（12周）兔髌骨表面置换模型来检查以下假设：1）不仅构建体在体内保持稳定性和完整性，其FI值将通过植入而改善，和2）同种异体细胞和培养产物都不会引起不利的宿主反应（局部/全身）。目的2采用小鼠模型来验证植入物按比例放大不会改变新软骨的生物力学特性。目标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;这些问题将使用软骨传导胶、外源性赖氨酰氧化酶和软骨调节方法来解决，从而产生坚固和润滑的植入物。最后，目标3将检验在绵羊模型中可实现长达12个月的持久愈合的假设。该目标还将确定可用于实现1年有效愈合的最小细胞数量。通过遵循FDA的指导文件（“用于修复或置换膝关节软骨的产品的IDE和IND的准备”），如果拟议研究的假设得到证实，结果将为自体关节软骨结构的临床可平移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.