基于微流控芯片的骨微环境模拟及成骨细胞IDG-SW3分化力生物学效应研究

The differentiation of osteoblast into mature osteocyte is critical for bone modeling and remodeling. The biochemical and mechanical microenvironment of bone tissue is the foundation for osteoblast living and differentiation. At present, researches on the response of osteoblast differentiation to mechanical cues is mainly based on the two-dimensional cell culture system, which is not appropriate for clarification the mechanical responses and mechanisms associated with the biochemical microenvironment. The development of microfluidics-based “Organ-on-a-Chip” technology has provided an efficient strategy to investigate the mechanical cues involved osteoblast differentiation and the related mechanism in the biochemical microenvironment. . In this proposal, (1) in the microfluidic devices, the bone biochemical microenvironment will be simulated by the three dimensional scaffold composed of type-I collagen and hydroxyapatite, and the bone mechanical microenvironment will be simulated by the mechanical stress and fluid shear stress control systems integrated in the devices; (2) The IDG-SW3 osteoblastic cells which replicate osteoblast-to-late-osteocyte differentiation in vitro will be used to study the effect of mechanical stress and fluid shear stress on osteoblast differentiation in simulated bone microenvironment, the changes of cell morphology, intercellular Ca2+ concentration, expression of differentiation-related biomarkers and voltage-sensitive calcium channel (VSCC) will be monitored; (3) The role of VSCC in mechanical microenvironment regulated osteoblast differentiation will be revealed. This research will provide essential experimental evidences for clarifying the mechanobiological mechanisms of osteoblast differentiation, and will also identify a potential target for maintaining bone health, prevention and treatment of disuse-induced bone loss.

成骨细胞分化为成熟骨细胞在骨塑建和骨重建平衡中起关键作用。骨组织生化微环境和力学微环境是成骨细胞生存和分化的基础。目前基于二维培养的成骨细胞分化力生物学效应研究难以在生化微环境基础上阐明力学效应机制。微流控芯片“Organ-on-a-Chip”技术的发展，为研究生化微环境中成骨细胞分化及其力学效应机制开辟了新途径。.研究内容：①在微流控芯片上利用I型胶原蛋白-羟基磷灰石三维支架模拟骨生化微环境，利用芯片机械应力/流体剪切力控制系统模拟骨力学微环境；②以具有从成骨细胞至成熟骨细胞分化功能的IDG-SW3细胞为实验对象，研究模拟微环境中机械应力和流体剪切力对细胞分化的影响，获得细胞形态、细胞游离钙、分化标志蛋白、电压敏感性钙通道(VSCC)变化规律；③揭示VSCC在力学微环境调控成骨细胞分化中的作用。为阐明成骨细胞分化力学效应机制提供新的实验依据，为骨骼健康维护、废用性骨质流失防治提供新靶点。

在项目执行的三年期间，本项目按计划顺利进行，很好地完成了研究任务和预期目标。本项目以模拟骨组织微环境为核心思想，利用微流控技术实现了bone-on-a-chip，研究了骨微环境对成骨细胞形态和功能的影响，不仅建立了利用微流控技术研究骨组织细胞功能的技术方法，其成果还有望用于骨骼疾病研究及药物筛选。具体而言，本项目首先建立了基于生化矿化原理的骨I型胶原-羟基磷灰石生化微环境构建技术，在此基础上构建了骨生化微环境模拟微流控芯片，此外还构建了能够精确控制流体剪切力大小的骨力学微环境模拟微流控芯片。利用这两种微流控芯片分别研究了骨生化微环境中不同基质蛋白对成骨细胞IDG-SW3形态和迁移特性的影响，以及成骨细胞IDG-SW3对流体剪切力的响应规律。本项目构建的bone-on-a-chip技术以及已取得的研究成果，将有望进一步用于研究因生化微环境改变或因废用导致力学刺激不足引起的骨质疏松等疾病，此外还将为基于骨微环境的骨骼疾病药物筛选提供新的技术方法。