Dynamically Responsive Bioreactors for Cartilage Regeneration

描述（由申请人提供）：我们的长期目标是设计功能上胜任的软骨，用于替换受损或患病的软骨。虽然已知工程化功能胜任的软骨取决于机械环境，但选择适当的加载环境已被证明具有挑战性。这一观察结果在很大程度上部分是由于细胞所感知的生物力学线索将由 支架的机械结构和化学性质，并且随着支架的降解和新组织的发展，将是动态的。为了克服这些挑战，这项研究的总体假设是，一个动态的培养环境，检测和响应组织工程支架的变化，提高工程软骨的质量。我们的假设的核心是一种新的动态压缩生物反应器最近设计，建造和验证与合作者在NIST，它配备了在线，非破坏性的测量能力，包括单独的称重传感器，用于评估机械性能和超声换能器与视频显微镜，用于评估工程组织的发展和质量。为了检验全球假设，该项目的具体目标是：1。设计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（PEG）水凝胶。该目的测试了具有交联的水凝胶的假设，所述交联含有被细胞降解的寡肽，导致局部降解（对于局部基质加工是关键的，而不牺牲机械完整性）和通过外源性递送脂肪酶“按需”降解的聚己内酯，导致大量降解（对于宏观组织发育是关键的），从而产生改善的工程化软骨。 2.开发和验证一个动态响应的“智能”生物反应器，使用启发式控制回路来真实的修改生物化学和机械环境。该目的测试了以下假设：响应于发育中的组织的体积降解和机械载荷的真实的时间变化将导致改进的工程化软骨。我们将实现这一目标，将一个模糊控制器与一组启发式控制行动到我们目前的生物反应器中的输出变量，从超声和机械性能估计的质量指数，将与输入变量，包括应变振幅，占空比，和酶添加。在完成该探索性研究时，我们期望开发出i）基于交联聚乙二醇的新型双酶降解水凝胶，其中降解在空间和时间上与组织生长和细化更紧密地匹配，和ii）动态响应的“智能”生物反应器，其能够与组织生长雅阁地检测和响应。我们还期望已经回答了基本的问题;动态响应的培养环境是否会导致在恒定的培养环境中改善组织的精细化和功能特性？预期这样的生物反应器将使得能够容易地适应来自多个细胞来源（供体、年龄、物种）的工程化软骨，所述多个细胞来源固有地具有用于组织发育的不同动力学和时间尺度，并且可以容易地适应其他支架类型。这项资助的发现将为有竞争力地寻求NIH R 01并追求其（前）临床效用奠定基础。

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.