We selected by cloning a murine periosteal cell population, which express both osteo-chondroblastic and osteoclastic lineages markers; isolated periosteal cells were differentiated in vitro in three-dimensional culture for three months and one year, as cellular aggregates and were also seeded onto acellular bone scaffolds, in presence of the same osteoblastic inducing media. Then samples were assayed, adopting molecular, histological, biochemical and biophysical analysis methods to characterize cell differentiation and bone formation. Results confirmed the presence of osteo-chondroblastic cells in periosteal murine layer, as previously reported, moreover bone scaffold support periosteal cells to maintain and mature a simil-osteoclastic phenotype as confirmed by the hydroxyapatite (HA) reabsorption after one year in vitro culture. In summary we exploited the capacity of periosteal cells to develop simil-osteoblastic–clastic phenotype, toward bone hydroxyapatite guidance, reabsorbing the underlying bone scaffold, instead three-dimensional cellular aggregates differentiate following simil-endochondral ossification mode producing carbonate hydroxyapatite, as physico-chemical analysis confirm, and does not mature osteoclastic phenotype