Microengineered platforms for bone cell mechanobiology study

骨组织工程的一大挑战是人工骨的长期维护。已有研究表明，机械载荷对骨组织的维持至关重要。在机械载荷作用下，适当的骨细胞生物力学行为对于天然骨组织和人工骨置换的长期组织维持是必不可少的。大多数骨细胞存在所谓的骨陷窝-小管网络(LCN)，这对骨细胞的正常功能和对机械载荷的反应至关重要。目前，大型生物力学测试系统已被广泛应用于基础骨细胞生物力学研究。然而，这种方法在很大程度上受到这些常规系统中细胞外环境的低生理相关性的限制。 这项研究的总体目标是开发新型的微工程平台，以研究骨细胞在生理相关环境中的生物力学行为。具体来说，我们的目标是： ·开发用于骨细胞培养和生物力学测试的微流控人工LCN； ·开发微型机械共培养系统，以允许同时对细胞进行生理上相关的机械刺激，并在不同细胞群体之间实时传输细胞信号分子；以及 ·开发基于纳米线的传感器，并将其集成到我们的微流控机械共培养系统中，以便实时表征机械刺激下细胞之间的通信。 首先，我们将设计和制作一种新型的基于微流控的人工LCN来研究基础骨细胞的生物力学。LCN中的细胞捕获将通过在细胞捕获通道和流动输送通道之间使用不同的流动阻力来实现。与生理相关的振荡流体流动将使用芯片上的气动控制系统来产生。单个骨细胞的大阵列将被种植在统一的微尺度环境中，细胞突起沿着图案化的纳米级通道形成。其次，我们将开发一种微流控多培养平台，允许同时对细胞进行机械加载，并在生理上相关的距离内实现细胞间的实时通信。系统中的共培养小室将通过水凝胶填充的微通道相互连接，以实现可溶性因子的扩散传输，而不是小室之间的对流传输。液体流将被输送到目标培养箱(S)，而不会扰乱位于其他培养箱中的细胞。最后，我们将开发基于纳米线的骨标记特异性传感器，并将其嵌入我们的微流体平台，用于实时定量机械载荷下细胞释放的蛋白质浓度。将构建一批纳米线传感器，并将与骨骼标记相对应的分析物特定受体连接到硅纳米线晶体管的表面。传感器信号将被访问并实时转换为蛋白质浓度。 在这项拟议的研究中，将尖端微制造和纳米软化技术与骨细胞生物力学研究相结合。所提出的设备将使我们能够在更具生理学意义的条件下对骨细胞进行基础生物力学研究。利用这些建议的微工程平台所获得的研究知识将为骨细胞力学和机械生物学以及骨组织工程设计提供关键信息。 这项研究的多学科性质将为学生提供各种可移植到其他研究领域和行业的技术培训，使他们对生物技术和生物医学工程的职业需求很高。

One major challenge of bone tissue engineering is the long-term maintenance of the artificial bone replacement. It has been shown that mechanical loading is critical for bone tissue maintenance. Appropriate bone cell biomechanical behaviour in response to mechanical loading is essential for long-term tissue maintenance for both native bone tissue and artificial bone replacement. The majority of bone cells inhabit a so-called lacunar-canalicular network (LCN), which is critical for bone cells' normal function and response to mechanical loading. Currently, macroscale biomechanical testing systems have been widely used to study fundamental bone cell biomechanics. However, this approach is greatly limited by the low physiological relevance of the extracellular environment in these conventional systems. The overall goal of this research is to develop novel microengineered platforms to study bone cell biomechanical behaviors in physiologically relevant environments. Specifically, we aim to: • Develop a microfluidic-based artificial LCN for bone cell culture and biomechanical testing; • Develop a microscale mechano-co-culture system to allow simultaneous physiologically relevant mechanical stimulation of cells and real-time transportation of cell signaling molecules among different cell populations; and • Develop and integrate nanowire-based sensors into our microfluidic mechano-co-culture systems to allow real-time characterization of cell-to-cell communications under mechanical stimulation. First, we will design and fabricate a novel microfluidic-based artificial LCN to study fundamental bone cell biomechanics. Cell trapping in LCN will be achieved by using differential flow resistance between cell trapping channels and flow delivery channels. Physiologically-relevant oscillatory fluid flow will be generated using an on-chip pneumatically controlled system. Large arrays of single bone cells will be seeded in a uniform microscale environment with cell processes formed along patterned nanoscale channels. Second, we will develop a microfluidic multi-culture platform to allow simultaneous mechanical loading of the cells and real-time cell-cell communication within a physiologically relevant distance. The co-culture chambers in the system will be interconnected by hydrogel-filled microchannels to enable diffusive transport of soluble factors but not convective transport among chambers. Fluid flow will be delivered to the targeted culture chamber(s) without perturbing the cells located in other culture chambers. Finally, we will develop nanowire-based bone marker specific sensors and embed them in our microfluidic platforms for real-time quantification of cell-released protein concentration under mechanical loading. Batches of nanowire sensors will be constructed and analyte-specific receptors corresponding to bone markers will be conjugated to the surface of silicon nanowire transistors. The sensor signals will be accessed and converted to protein concentration in real time. In this proposed research the cutting edge microfabrication and nanofabricaton techniques are integrated with bone cell biomechanics studies. The proposed devices will allow us to conduct fundamental biomechanics research on bone cells in more physiologically relevant conditions. Knowledge gained from the studies using these proposed microengineered platforms will provide key information in bone cell mechanics and mechanobiology and bone tissue engineering design. The multi-disciplinary nature of this research will provide students training in a variety of techniques that are transferable to other research areas and industries, making them in high demand for careers in biotechnology and biomedical engineering.