gms | German Medical Science

Objectives: Cellular senescence drives age-related bone loss. 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 and whether their genetic clearance modulates fracture healing 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.

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.

In addition, we performed a weekly x-ray analysis and an in-depth bone formation rate analysis with 4 different fluorescent dyes.

Results and conclusion: The GFP+ (i.e., p21-enriched) cells had higher expression not only of p21 but also Dcst2 (DC-STAMP domain-containing protein 2) and multiple other pre-osteoclastic as well as senescent-associated secretory phenotype (SASP) markers, indicating that the senescent cells in the fracture callus were, at least in part, pre-osteoclastic cells. Interestingly, the enriched SASP genes in p21+ cells were also enriched in publicly available microarray data from human fracture non-unions.

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.

The weekly x-ray analysis demonstrated a significant improvement of the callus healing area beginning in the first week as well as increased callus bone volume upon completion of fracture healing.

We showed accelerated bone formation rates in the AP20187-treated mice.

Thus, we demonstrate the presence of p21+ senescent cells in the murine fracture callus and identify them as pre-osteoclastic. In addition, genetic clearance of these 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.