In vitro lifespan and senescent behaviour of human periosteal derived stem cells.

Periosteum Derived Progenitor Cells (PDPCs) represent promising Mesenchymal Stem Cells (MSCs) for skeletal regeneration and to test bone cell based tissue engineering strategies. Most of regenerative medicine approaches based on MSCs require a noteworthy amount of cells that must be expanded in vitro prior to their use. As culture expansion method may impact on cell behaviour, we assessed the replicative and metabolic capacity (nitric oxide production and glucose consumption), senescence hallmarks of PDPCs serially passaged as well as the expression of selected genes specifically related to early osteoblastic differentiation, bone remodelling and stemness during PDPC sequential passaging. We also scouted a Systems Biology approach to examine and elucidate the experimental results through mathematical modelling and in silico simulations. PDPC subculture led to a progressive proliferative decline but, despite this, PDPCs maintained almost constant their metabolic activity. In vitro, senescent PDPCs displayed the typical “replicative senescence” features, involving p16 and not p53 in the regulation of this phenomenon. Gene expression analysis evidenced the tendency of sub-cultured PDPCs to increase the expression of genes involved in bone resorption. The mathematical analysis of the experimental results showed a strict similarity between replicative senescence and age-related changes, enabling the definition of an in silico model mimicking PDPC behaviour in terms of Nitric Oxide (NO) production. The relationship between NO production and subculture passages could represent a cutting edge “replicative senescence index”. Overall, our findings suggest the possibility to use early-passage PDPCs for bone regenerative approaches based on the local recruitment of stem cells, whilst the later cell passages could be a suitable in vitro tool to validate scaffolds intended for bone regeneration in elderly subjects.