Cdkn1a deletion or suppression by cyclic stretch enhance the osteogenic potential of bone marrow mesenchymal stem cell-derived cultures

CDKN1A/P21 is a potent inhibitor of cell cycle progression and its overexpression is thought to be associated with inhibition of normal bone regenerative osteogenesis during spaceflight. To test whether CDKN1A/P21 regulates osteogenesis in response to mechanical loading we studied cyclic stretch versus static culture of Cdkn1a-/-(null) or wildtype primary mouse bone marrow osteoprogenitors during 21-day ex-vivo mineralization assays. Cyclically stretched Cdkn1a-/- cells are 3.95-fold more proliferative than wildtype, while static Cdkn1a-/- cells show a 2.50-fold increase. Furthermore, stage-specific single cell RNAseq analyses show expression of Cdkn1ais strongly suppressed by cyclic stretch in early and late osteoblasts, and minimally in the progenitor population. Lastly, both stretch and/or Cdkn1a deletion cause population shift from osteoprogenitors to osteoblasts, also indicating increased differentiation. Collectively, our results support the hypothesis that Cdkn1aconstitutively plays a mechano-reversible anti-proliferative role during osteogenesis and suggests a new molecular target to counter regenerative deficits caused by disuse.