Permanent orthopedic/dental implants should reveal good osseointegration, which is defined as an ability of the biomaterial to form a direct connection with the surrounding host bone tissue after its implantation into the living organism. Currently, biomaterial osseointegration is confirmed exclusively with the use of in vivo animal tests. This study presents for the first time ex vivo determination of osseointegration process using human trabecular bone explant that was drilled and filled with the chitosan/curdlan/hydroxyapatite biomaterial, followed by its long-term culture under in vitro conditions. Within this study, it was clearly proved that tested biomaterial allows for the formation of the connection with bone explant since osteoblasts, having ability to produce bone extracellular matrix (type I collagen, fibronectin), were detected at a bone-implant interface by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Importantly, in this research it was demonstrated by Live/Dead staining and CLSM imaging that human bone explants may stay alive for a long period of time (at least approx. 50 days) during their culture under in vitro conditions. Therefore, ex vivo bone explant, which is a heterogeneous tissue containing many different cell types, may serve as an excellent model to test biomaterial osseointegration during comparative and preliminary studies, reducing animal tests which is compatible with the principles of ‘3Rs’, aiming to Replace, Reduce and Refine the use of animals wherever possible.