The performed research explores the use of consecutive vacuum loading to control the release of biologically active substances from demineralized cancellous bone (DCB), using methylene blue (MB) as a model substance and bovine serum albumin (BSA) as a secondary coating/diffusion layer. This method aims to enhance the utility of DCBs in regenerative medicine by improving the delivery and controlled release of therapeutic agents. During the study two vacuum conditions were tested – loading the DCBs with MB at low vacuum (LV, 400 mbar) and high vacuum (HV, 100 mbar), and examining their impact on MB loading and release. Results showed that HV allowed deeper and more uniform MB penetration, resulting in a more sustained release profile. In contrast, low vacuum caused a faster initial release due to more superficial loading. Secondary loading with 10% and 20% BSA further modulated release dynamics. BSA coatings slowed initial MB diffusion but eventually supported higher sustained release especially for the group loaded under HV conditions. This effect is likely due to the film-forming and drug-binding properties of BSA, which may prevent early MB washout and enhance matrix interaction. We concluded that vacuum level and sequential loading significantly influence release behavior and can be harnessed by creating the advanced, customizable bone grafts with highly tunable release capability over a long time. These findings have important implications for designing multifunctional grafts tailored to specific therapeutic needs using tissue engineering methods.