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Objectives and questions: Osteoblast (OB) senescence is considered to play a substantial role in the pathogenesis of senile osteoporosis. Senescent cells are characterized by excessive expression of cytokines and catabolic mediators, collectively referred to as the senescence-associated secretory phenotype (SASP). Through the release of these factors, senescent OB may influence other bone cells in a paracrine manner. Therefore, this study aimed to investigate the paracrine effects of senescent OB on the osteogenic differentiation (OD) of bone marrow–derived mesenchymal stem cells (MSCs).

Material and methods: Human OB were isolated from bone material obtained from osteoarthritis patients undergoing knee arthroplasty. OB senescence was induced by treatment with 0.1 µM doxorubicin for five consecutive days and confirmed by cell cycle arrest (Ki-67 and Vybrant DyeCycle staining; p16 and p21), increased expression of SASP factors (qRT-PCR), and senescence-associated β-galactosidase (SA-β-gal) staining. Conditioned media from non-senescent (CM-Ctrl) and senescent OB (CM-SASP) were supplemented with dexamethasone, β-glycerophosphate, and ascorbic acid and used to induce osteogenic differentiation in human MSCs (Lonza). After 21 days, mineralization was assessed by Alizarin Red staining, and gene expression of RUNX2, ALPL, and IBSP was analyzed. Unconditioned osteogenic differentiation medium (ODM) served as control. Data sets (n ≥5) were analyzed using one-way ANOVA.

Results: Senescent OB exhibited a pronounced cell cycle arrest, as evidenced by increased mRNA levels of p16 and p21, loss of Ki-67 positivity, and accumulation of cells in the G0/G1 phase. Expression levels of IL1B, IL6, IL8, TNF, and RANKL, as well as SA-β-gal activity, was significantly elevated, whereas OPG expression remained unchanged. After 21 days of OD, Alizarin Red staining of MSCs cultured with CM-Ctrl or CM-SASP was similarly reduced compared to ODM, while gene expression of ALPL and IBSP was increased. Notably, ALPL and IBSP expression levels were significantly lower in CM-SASP-treated MSCs compared to CM-Ctrl–treated MSCs.

Discussion and conclusions: Doxorubicin treatment effectively induced a robust senescent phenotype in human OB. The present data demonstrate that senescent OB modulate the osteogenic differentiation potential of MSCs via paracrine mechanisms. Furthermore, the shift in the RANKL/OPG balance toward RANKL suggests that senescent OB may contribute to age-related bone loss by promoting osteoclastogenesis. Overall, these findings support the hypothesis that OB senescence impairs bone homeostasis during aging through induction osteoclastogenesis and suppression of osteoblast precursor cells via paracrine mechanisms.