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The goal of bone tissue engineering is to build artificial bone tissue with properties that closely resemble human bone and thereby support the optimal integration of the constructs (biografts) into the body. The development of tissues in 3D scaffolds includes several complex steps that need to be optimized and monitored. In particular, cell–material interaction during seeding, cell proliferation and cell differentiation within the scaffold pores play a key role. In this work, we seeded two types of 3D-printed scaffolds with pre-osteoblastic MC3T3-E1 cells, proliferated and differentiated the cells, before testing and adapting different assays and imaging methods to monitor these processes. Alpha-TCP/HA (α-TCP with low calcium hydroxyapatite) and baghdadite (Ca3ZrSi2O9) scaffolds were used, which had comparable porosity (~50%) and pore sizes (~300–400 µm). Cell adhesion to both scaffolds showed ~95% seeding efficiency. Cell proliferation tests provided characteristic progression curves over time and increased values for α-TCP/HA. Transmitted light imaging displayed a homogeneous population of scaffold pores and allowed us to track their opening state for the supply of the inner scaffold regions by diffusion. Fluorescence labeling enabled us to image the arrangement and morphology of the cells within the pores. During three weeks of osteogenesis, ALP activity increased sharply in both scaffolds, but was again markedly increased in α-TCP/HA scaffolds. Multiphoton SHG and autofluorescence imaging were used to investigate the distribution, morphology, and arrangement of cells; collagen-I fiber networks; and hydroxyapatite crystals. The collagen-I networks became denser and more structured during osteogenic differentiation and appeared comparable in both scaffolds. However, imaging of the HA crystals showed a different morphology between the two scaffolds and appeared to arrange in the α-TCP/HA scaffolds along collagen-I fibers. ALP activity and SHG imaging indicated a pronounced osteo-inductive effect of baghdadite. This study describes a series of methods, in particular multiphoton imaging and complementary biochemical assays, to validly measure and track the development of bone tissue in 3D scaffolds. The results contribute to the understanding of cell colonization, growth, and differentiation, emphasizing the importance of optimal media supply of the inner scaffold regions. [ABSTRACT FROM AUTHOR]