Abstract Traditionally, bone tissue engineering has been used for creating implants for bone regeneration, but recently, it is increasingly applied to create three-dimensional (3D) in vitro bone models to study bone physiology and pathology. For 3D in vitro bone models, the engineered extracellular matrix organization should resemble the in vivo physiological bone structure because this is often the hallmark in skeletal pathologies — an issue that has not been solved yet. In this review article, we define the extracellular matrix requirements for an optimal 3D in vitro model based on the most recent advances on bone structure. To meet these requirements, osteoclasts, osteocytes, and mature osteoblasts should work together under physiological conditions, and the formed extracellular matrix should be analyzed and optimized at multiple length scales.
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