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

这种在培养物中维持骨细胞的新能力代表了骨研究领域的一个重大突破，该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，该模型包含骨重建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。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，并将其应用于药物筛选。现在，我们将使用这种独特的器官培养技术来开发一种体外模型，该模型再现了骨重建的更多生理学和医学相关场景。这将需要除了骨细胞之外的成骨细胞和破骨细胞的结合和维持，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的活性以及所产生的骨的完整性和组成。我们将使用我们的SSBM作为活性的活基质来支持骨重塑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重塑器官型模型，并走向生物学，以产生一个筛选平台，可用于测试新药的疗效。假设：1.2.3.4. SSBM构建体将通过与骨细胞小管的细胞相互作用提供生理环境和刺激来支持成骨细胞存活、成熟和骨基质产生和矿化。SSBM将支持破骨细胞存活，成骨细胞、破骨细胞和骨细胞之间通过直接细胞接触和分泌细胞因子/生长因子进行交流（如RANKL和OPG）将调节骨吸收率，从而产生骨重建的器官型模型。该器官型模型将用作测试新疗法的筛选平台。该项目涉及我们突破性的骨细胞培养方法发展成为一个更医学相关的模型，其验证作为一个准确和有用的模型的生理过程和其应用作为一个药物筛选工具。

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.