Mesenchymal stem cells from periodontal ligament (PDL-MSCs) hold great promise for bone regeneration. Most studies regarding the osteogenic differentiation of stem cells from periodontal tissue suggest that PDL cells may have many osteoblast-like properties, including the ability to form calcified nodules in vitro. This study investigated the morphological and histochemistry aspects of human PDL-MSCs, induced for osteogenic differentiation and seeded on a xenogenic porcine bone substitute in vitro, at different times of incubation. This biomaterial seems physically identical to human bone, and it has been reported to be osteoconductive. Our results indicated that the cells had a high affinity for the three-dimensional biomaterials; in fact, cellular proliferation and colonization was evident, and after 21 days the adherent cells started to detach themselves from the substrate, and at 30 days of incubation in differentiation medium, the cells completely lost the adhesion to the Petri's disk, englobing all bioparticles. In conclusion, the in vitro behaviour of PDL-MSCs and their relationship with three-dimensional scaffold biomaterials encourage in vivo investigations for their use in dental tissue regeneration.