Investigating the synergistic effects of spatially resolved biochemical, physicochemical, and physical key stimuli to generate biomimetic niches in perfusion bioreactor and their proficiency to derive large bone-like constructs.

生物反应器越来越多地用于骨组织工程方法，但现有的同质生态位应用通常不足以实现/支持骨样结构的形成。在生理上，多个骨软骨细胞高密度存在，它们之间的动态相互作用对骨发育和再生过程至关重要。这也补充了支持参数，如氧张力（pO2）分布、生化刺激和物理/机械刺激，以建立naïve器官（即软骨和骨骼）生长所必需的动态生态位并实现其功能。在这个提议中，我们的目标是研究5个不同参数的协同潜力，以概括灌注生物反应器中的骨发育过程。通过建立动态仿生生态位，将评估对体外构建体发育和获得骨样组织能力的影响。这些参数包括：1)介质pH；2)警察乙;3) 3d打印支架杨氏模量；4)多步骨软骨分化机制和5)类似变压器的感应电场（TLC-EF）刺激模拟运动过程中的压电刺激。人骨髓间充质干细胞（hMSC）和人诱导多能干细胞（hiPSC）将被用来定义仿生生态位的熟练程度。它们在仿生壁龛中衍生不同骨软骨细胞类型的潜力将被严格评估。特别是对于TLC-EF刺激，描述EF/Bone结构相互作用的多尺度建模/模拟以及对成骨的影响将与实验验证相结合。这样做的目的是为骨组织工程应用定义一个有效的EF参数范围。最终，这项研究将产生一组特定的条件/刺激，可以概括体外的生理样生态位。所应用的方法也将为组织工程领域引入一个新的概念，并可以进一步开发在生物反应器中获得具有接近生理特性的类骨结构。

Bioreactors are used increasingly in bone tissue engineering approaches, but the existing homogenous niche applied is generally insufficient to achieve/support formation of bone-like constructs. In physiology, multiple osteochondral cells are present in high cell density and their dynamic interactions are critical for bone development and regeneration processes. This is also complemented with supporting parameters such as oxygen tension (pO2) distribution, biochemical stimuli, and physical/mechanical stimuli to establish the dynamic niches essential for the growth of naïve organs (i.e. cartilage and bone) and attaining their functionalities.In this proposal, we aim to investigate the synergistic potentials of 5 distinct parameters to recapitulate bone developmental processes in a perfusion bioreactor. Through the established dynamic biomimetic niches, the effects on construct development and capability to achieve bone-like tissues in vitro will be assessed. These parameters include 1) medium pH; 2) pO2; 3) Young´s modulus of 3D-printed scaffolds; 4) multi-step osteochondral differentiation regimes and 5) Transformer-like induced electric field (TLC-EF) stimulation to emulate piezoelectric stimuli during movement.Human bone marrow-derived mesenchymal stem cells (hMSC) and human induced pluripotent stem cells (hiPSC) will be used to define the proficiency of biomimetic niches. Their potential to derive different osteochondral cell-types in the biomimetic niches will be critically assessed. Specifically for TLC-EF stimulations, multiscale modeling/simulations that describe EF/Bone construct interactions and the effects on osteogenesis will be combined with experimental validations. This is done with the aim to define an effective range of EF parameters for bone tissue engineering applications. Ultimately, this study will result in a specific set of conditions/stimuli that can recapitulate physiological-like niches in vitro. The approaches applied will also introduce a novel concept to the field of tissue engineering and can be further exploited to derive bone-like constructs with near-physiological properties in a bioreactor.