Fig. 2.

Hierarchical diagram of the models for studying bone tissue. The diagram uses a pyramidal structure to show the logical progression and increasing complexity among the different approaches for studying bone tissue. The Biomaterials form the base of the pyramid. They provide essential physical and chemical support for cell growth, differentiation, and the creation of three-dimensional microenvironments. Transwell and Co-culture Systems represent an intermediate step in complexity. They facilitate cell-to-cell interactions and mutual influence through soluble and physical signals, creating a more realistic microenvironment. Bone Spheroids offer an advanced three-dimensional model, enabling the study of cell-extracellular matrix interactions and simulating the bone microenvironment in vitro. Bone Organoids are the higher level in terms of biological simulation. They replicate more complex structures and functions, such as bone formation and regeneration. Spheroids-on-chip integrate spheroids into a dynamic system that simulates in vivo conditions, such as nutrient flow and mechanical signals. Organoids-on-chip represent the most advanced level. It combines organoids with microfluidic technologies to recreate highly specific physiological microenvironments, providing a platform for complex and personalized studies. The transition from 2 to 3D models and ultimately to chip-based systems is driven by the need to better replicate the biological environment of bone, enabling more realistic models for studying bone regeneration and developing innovative therapies. Each step in the progressive evolution of techniques used to study bone—from bidimensional models to advanced technologies like organoids, spheroids, and chip systems—is based on identifying specific limitations of the previous model and addressing them through innovative approaches