Developing, characterising and testing a humanised 3D in vitro bone model, capable of long-term osteocyte culture

这种在培养中维持骨细胞的新能力代表了骨研究领域的重大突破，这项技术现在为我们提供了一个独特的机会来开发一种体外模型，包含骨重塑所需的所有三种关键细胞类型，可以取代目前使用的一些体内模型。目的建立由人破骨细胞、成骨细胞和骨细胞组成的骨重塑器官型模型。验证模型重现骨重塑和对现有药物治疗反应的能力。将分析方法小型化，使其适合作为药物筛选工具应用。我们的自结构骨形成模型（SSBM）首次实现了骨细胞的分化和长期培养。现在，我们将使用这种独特的器官培养技术来开发一个体外模型，以概括更生理和医学上相关的骨重塑场景。除了骨细胞外，这将需要整合和维持成骨细胞和破骨细胞，并且需要更复杂的读数来评估存在的三种细胞类型中的每一种细胞的活性以及所产生的骨的完整性和组成。我们将使用SSBM作为活性的活基质来支持骨模型所需的其他细胞类型：成骨细胞和破骨细胞。然后，我们将验证由此产生的骨模型的器官型模型，并朝着小型化的方向发展，以产生一个可用于测试新药疗效的筛选平台。假设:为1.2.3.4。SSBM结构将通过与骨细胞小管相互作用提供生理环境和刺激，支持成骨细胞的存活、成熟、骨基质的产生和矿化。SSBM将支持破骨细胞存活、成熟和骨吸收活性（如上所述）。成骨细胞、破骨细胞和骨细胞之间通过直接细胞接触和细胞因子/生长因子（如RANKL和OPG）的分泌进行交流，将调节骨吸收率，从而形成骨重塑的器官型模型。未来3Rs影响：该项目涉及将我们突破性的骨细胞培养方法发展为更具有医学相关性的模型，其作为生理过程的准确和有用模型的验证，以及其作为药物筛选工具的应用。

This new ability to maintain osteocytes in culture represents a substantial breakthrough forthe bone research field and the technology now provides us with a unique opportunity to develop an in vitromodel, containing all three of the key cell types required for bone remodelling, that could replace some of thecurrently used in vivo models.ObjectivesDevelop an organotypic model of bone remodelling comprising human osteoclasts, osteoblasts and osteocytes.Validate the ability of the model to recapitulate bone remodelling and response to existing drug treatments.Miniaturise the assay into a format suitable for application as a drug screening tool.Our self-structuring bone formation model (SSBM) has, for the first time, enabled differentiation and long-termculture of osteocytes. Now we will use this unique organ culture technology to develop an in vitro modelthat recapitulates the more physiologically and medically relevant scenario of bone remodelling. Thiswill require the incorporation and sustainment of osteoblasts and osteoclasts in addition to the osteocytes andwill necessitate substantially more complicated readouts to assess the activity of each of the three cell typespresent and the integrity and composition of the bone produced.We will use our SSBM as an active, living substrate to support the additional cell types required for boneremodelling: Osteoblasts and osteoclasts. We will then validate the resulting organotypic model of boneremodelling and move towards miniaturisation to produce a screening platform that can be used to test noveldrugs for efficacy.Hypotheses: 1.2.3.4.The SSBM constructs will support osteoblast survival, maturation and bone matrix productionand mineralisation by providing a physiological environment and stimulation through cellularinteractions with osteocyte canaliculi.The SSBM will support osteoclast survival, maturation and bone resorption activity (asabove).Communication between osteoblasts, osteoclasts and osteocytes via direct cell contact andsecretion of cytokines/growth factors (e.g. RANKL and OPG) will regulate bone resorptionrates resulting in an organotypic model of bone remodelling.The organotypic model will be useful as a screening platform to test novel therapeuticsFuture 3Rs Impact: This project involves the development of our ground-breaking osteocyte-culturemethod into a more medically relevant model, its validation as an accurate and useful model of aphysiological process and its application as a drug screening tool.