Developing a Bone-Mimicking Microenvironment: Surface Coating Method for Investigating Bone Remodeling in Vitro.

Abstract

To investigate bone formation and resorption in vitro, it is essential to create bone-like microenvironments on cell culture substrates. Here, we present a coating technique to create such a microenvironment on cell culture plastic (CCP) multiwell plates for studying bone remodeling in vitro. Utilizing this coating, we have developed an assay to simultaneously measure cellular mineral formation and resorption in osteoblast and osteoclast coculture models. A composite matrix of collagen type I and carbonated apatitic calcium phosphate was deposited onto CCP in a reproducible manner using a 10× simulated body fluid solution (SBF) supplemented with type I collagen. qPCR analysis and cellular imaging using fluorescence microscopy demonstrated the promotion of osteogenic differentiation, cell attachment, and proliferation of human bone-marrow-derived mesenchymal stem cells on coated substrates. Moreover, human bone-marrow-derived mononuclear cells successfully differentiated into osteoclasts and resorbed the coated substrate. Using the developed coating, an osteoblast and osteoclast coculture system was established, enabling real-time monitoring of mineral formation and resorption. By providing a controlled and physiologically relevant in vitro model, this assay facilitates the screening of therapeutic compounds, the study of bone cell interactions, and the identification of factors influencing bone remodeling, thereby enhancing translational research in bone health.