gms | German Medical Science

Objectives: Age-related bone loss is driven by a process called cellular senescence. We recently demonstrated the transient appearance of senescent cells during fracture healing. These cells within the callus upregulate hallmarks of senescence, including expression of Cdkn1a (p21) and Cdkn2a (p16), acquire DNA damage at sites of telomeres (telomere-associated foci, TAF), and produce a bioactive secretome - i.e., senescence-associated secretory phenotype (SASP). The identity of these senescent cells remains unknown.

Methods: We first induced tibial fractures in a novel transgenic mouse where the p21 promoter drives GFP and performed scRNAseq on GFP+ vs GFP- cells harvested from the callus. The GFP+ (i.e., p21-enriched) cells had higher expression not only of p21 but also Dcst2 (DC-STAMP domain-containing protein 2) and senescent-associated secretory phenotype (SASP) markers. Interestingly, the enriched SASP genes in p21+ cells were also enriched in publicly available microarray data from human fracture non-unions. Next, we used a newly validated p21-ATTAC mouse to genetically clear these p21-expressing cells in 4-month-old mice using the synthetic drug AP20187. By using FISH probes, we demonstrated a reduction of TAF (a definitive marker of senescence)-positive cells in the callus in the AP20187-treated mice.

Results and conclusion: Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. An in-depth bone formation rate analysis with 4 different fluorescent dyes showed accelerated bone formation rates in the AP20187-treated mice. Interestingly and following fracture, p21+ neutrophils were also enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ osteochondroprogenitors (OCHs) were highly enriched in SASP factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells are key regulators of tissue repair in bone and potentially across tissues. In summary, genetic clearance of p21+ cells accelerates fracture healing by enhancing bone formation in the callus. Combined with our data, the presence of a p21-related SASP in human fracture non-unions indicates that clinical trials using senolytic compounds may have therapeutic benefit in treating this debilitating disorder.