Dynamically Responsive Bioreactors for Cartilage Regeneration

描述(由申请者提供)：我们的长期目标是设计出具有功能的软骨，用于替代受损或患病的软骨。虽然众所周知，设计一个功能良好的软骨依赖于机械环境，但事实证明，选择合适的加载环境是具有挑战性的。这种观察在很大程度上是由于这样一个事实，即细胞感受到的生物力学线索将由 支架的机械结构和化学成分将随着支架的降解和肿瘤组织的发展而动态变化。为了克服这些挑战，这项研究的全球假设是，动态的培养环境可以检测到组织工程支架中的变化并做出反应，从而提高工程软骨的质量。我们假设的核心是最近与NIST的合作者设计、建造和验证的一种新型动态压缩生物反应器，它配备了在线、无损测量能力，包括用于评估机械性能的单个称重单元和用于评估工程组织的发育和质量的超声换能器和视频显微镜。为了验证全球假说，该项目的具体目标是：1.设计一种具有细胞介导的局部降解和按需整体降解能力的双酶降解聚乙二醇水凝胶。这一目标验证了这样一种假设，即具有交联性的水凝胶含有寡肽，可由细胞降解，导致局部降解(对局部基质形成至关重要，而不牺牲机械完整性)和聚己内酯，可通过外源性脂肪酶的传递按需降解，导致整体降解(对宏观组织发育至关重要)，从而产生改进的工程软骨。2.利用启发式控制回路实时改变生化和机械环境，开发并验证了一种动态响应的智能生物反应器。这一目标验证了这样一种假设，即随着组织的发育，实时改变为体积降解和机械载荷将导致改进的工程化软骨。我们将通过将模糊控制器和一组启发式控制操作结合到我们目前的生物反应器中来实现这一目标，其中输出变量，即从超声波和机械特性估计的质量指数，将与输入变量相关，包括应变幅度、占空比和酶的添加。在这项探索性研究完成后，我们预计将开发出i)一种新型的基于交联型聚乙二醇的双酶降解水凝胶，其中降解在空间和时间上与组织的生长和细化更紧密地匹配，以及ii)能够根据组织生长进行检测和响应的动态响应的“智能”生物反应器。我们还希望回答一个基本问题：动态响应的培养环境是否会在持续的培养环境中带来组织精致程度和功能特性的改善？预计这样的生物反应器将能够方便地适应来自多种细胞来源(供体、年龄、物种)的工程软骨，这些细胞来源固有地具有不同的组织发育动力学和时间尺度，并且可以很容易地适应其他支架类型。这项拨款的发现将为寻求具有竞争力的NIH R01并追求其(前期)临床实用奠定基础。

DESCRIPTION (provided by applicant): Our long-term goal is to engineer functionally competent cartilage for replacing damaged or diseased cartilage. While it is known that engineering a functionally competent cartilage depends on the mechanical environment, choosing the appropriate loading environment has proven challenging. This observation is largely in part due to the fact that the biomechanical cues sensed by the cells will be dictated by the mechanical structure and chemistry of the scaffold and will be dynamic in time as the scaffold degrades and neotissue develops. To overcome these challenges, the global hypothesis for this research is that a dynamic culture environment that detects and responds to changes in a tissue-engineered scaffold improves the quality of the engineered cartilage. Central to our hypothesis is a novel dynamic compressive bioreactor recently designed, constructed and validated with collaborators at NIST, which is equipped with online, nondestructive measurement capabilities comprised of individual load cells for assessing mechanical properties and an ultrasonic transducer coupled with a video microscope for assessing development and quality of the engineered tissue. To test the global hypothesis, the specific aims of the project are to: 1. Design a dual enzyme degrading polyethylene glycol (PEG) hydrogel with cell-mediated local degradation and 'on demand' bulk degradation capabilities. This aim tests the hypothesis that a hydrogel with crosslinks containing oligopeptides that are degraded by cells, leading to local degradation (critical for local matrix elaboration without sacrificing mechanical integrity) and polycaprolactone that is degraded 'on demand' by exogenous delivery of lipase, leading to bulk degradation (critical for macroscopic tissue development) yields improved engineered cartilage. 2. Develop and validate a dynamically responsive 'smart' bioreactor using a heuristic control loop to modify the biochemical and mechanical environment in real time. This aim tests the hypothesis that real time changes to bulk degradation and mechanical loading in response to the developing tissue will lead to improved engineered cartilage. We will achieve this aim by incorporating a fuzzy controller with a set of heuristic control actions into our current bioreactor where the output variables, the quality index estimated from ultrasound and mechanical properties, will be related to input variables that include strain amplitude, duty cycle, and enzyme addition. At the completion of this exploratory research, we expect to have developed i) a new class of dual enzyme degrading hydrogels based on cross-linked polyethylene glycol where degradation is more closely matched spatially and temporally to tissue growth and elaboration and ii) a dynamically responsive 'smart' bioreactor that is capable of detecting and responding in accord with tissue growth. We also expect to have answered the fundamental question; does a dynamically responsive culture environment lead to improved tissue elaboration and functional properties over a constant culture environment? It is anticipated that such a bioreactor would enable facile adaption to engineering cartilage from multiple cell sources (donor, age, species), which inherently have different dynamics and timescales for tissue development, and can readily be adapted to other scaffold types. Findings from this grant will lay the foundation for seeking competitively a NIH R01 and to pursue their (pre)clinical utility.