As a strategy to optimise osteointegration of biomaterials by inducing proper extracellular matrix synthesis, and specifically angiogenic growth factor production and storage, we tested the effects of cyclic mechanical compression on 3D cultures of human osteoblast-like cells. MG-63 cells were seeded into 3D porous hydroxyapatite ceramics under vacuum to enable a homogenous cellular distribution. A four-day culture period allowed cell proliferation throughout the scaffolds. Low amplitude cyclic compressions were then applied to the scaffolds for 15 min with different regimens generated by the ZetOS system. A 3 Hz sinusoidal (sine) signal increased slightly collagen and fibronectin expression. When 50 Hz or 100 Hz vibrations were superimposed to the 3 Hz signal, matrix protein expression was down-regulated. In contrast, adding a 25 Hz vibration up-regulated significantly collagen and fibronectin. Moreover, expression of a matrix-bound variant of vascular endothelial growth factor-A (VEGF-A) was specifically stimulated compared to control or 3 Hz sine, and non-soluble VEGF protein was increased. Our study enabled us to identify low-amplitude, high-frequency strain regimen able to increase major matrix proteins of bone tissue and to regulate the expression of VEGF variants, showing that an appropriate combined loading has the potential to functionalise cellularized bone-like constructs.