Systematic treatment with the senolytic drug DQ prevented bone loss in middle-aged postmenopausal osteoporotic rats (PMO rats)
We first sought to determine whether the senolytic drug DQ, which has been in clinical trials for many age-related diseases, could alleviate PMO systematically36,37. Considering that most postmenopausal women are in middle age, we chose 12-month-old (12-mo) female rats as the animal model to match the pathological age to the maximum extent. The middle-aged rats were subjected to a sham operation (M-SHAM), or ovariectomy with (M-DQ) or without (M-OVX) DQ treatment afterwards, and the femurs were dissected at the 16th month after surgery for analysis (Fig. 1a). Systematic DQ treatment significantly increased the enrichment and density of trabecular bone in the M-DQ group compared to that in the M-OVX group (Fig. 1b). To investigate the trabecular bone microarchitecture, areas in the distal femur were further analyzed. The 3D obtained micro-computed tomography (µCT) images demonstrated that DQ treatment substantially resulted in better femur trabecular bone microarchitecture (Fig. 1c), with elevated bone volume fraction (BV/TV, Fig. 1d) and trabecular number (Tb.N, Fig. 1e) by approximately 2 fold, as well as the declined trabecular separation (Tb.Sp, Fig. 1f) and structure model index (SMI, Fig. 1g). Consistent with these results, increased trabecular number and growth plate amelioration were also observed in the DQ-treated postmenopausal osteoporotic rats (PMO rats) (Fig. 1h). In addition, the possible side effects of DQ on other organs were examined (Fig S1). Significant tissue damage or pharmacological toxicity was not observed in the kidney, lung, spleen, liver and heart tissues, suggesting the combination of DQ could be a favorable candidate for PMO treatment. In summary, these results demonstrated that, for the first time to our knowledge, systematic treatment with the senolytic drug DQ ameliorated PMO in middle-aged rats.
Accumulation of SnCs accelerated bone loss in middle-aged PMO rats
We next investigated whether PMO was related to SnCs in bone tissues. SnCs, which appear throughout the lifespan, are suspected to be cleared by the immune system at a young age but accumulate rapidly in old age, probably due to the decreased clearance capacity of the aged immune system38,39. Although a steep increasing number of senescent cells in bone tissue was accounted in individuals of senile OP (type Ⅱ)22, whether SnCs were accumulated during PMO (type Ⅰ) was unclear. . A previous study has reported that SnCs were not remarkably accumulated in the bone tissue of young OVX mice, and found that eliminating SnCs did not rescue the bone loss40. However, our results showed a therapeutic effect of DQ treatment in middle-aged rats, indicating a probable accelerated SnC accumulation in middle-aged PMO rats. Hence, to thoroughly reveal the relationship between SnCs and PMO, both 6-mo (young, Y) and 12-mo (middle-aged, M) rats were subjected to a sham operation (Y-SHAM or M-SHAM) or ovariectomy (Y-OVX or M-OVX), with 19-mo (old, O-SHAM) rats as naturally aged control for comparison analysis (Fig. 2a). Deteriorated skeletal architecture and remodeling were observed in the OVX groups compared with SHAM groups at both young and middle age stages (Fig. 2b)- including reduced BV/TV (Fig. 2c) and Tb.N (Fig. 2d), as well as increased Tb.Sp (Fig. 2e) and SMI (Fig. 2f). Notably, the Tb.Sp of the M-OVX rats was significantly higher (3-fold) than that of the Y-OVX rats, but was equivalent to that of the O-SHAM rats (Fig. 2e), indicating the deterioration of microenvironment in the M-OVX rats. Although the BV/TV and Tb.N of the M-OVX rats and Y-OVX rats showed no significant differences, consistent with observations reported in a previous study41, the more significant value reduction was observed in middle-aged OVX rats (Fig. 2c-d). Furthermore, a more serious destruction of trabecular bone structure was observed in hemoxylin and eosin (H&E) stained images (Fig. 2g), simultaneously with a decreased number and more structural disorders in trabecular bone, similar to O-SHAM rats. These results showed that middle-aged PMO rats exhibited more severe bone structure deterioration equivalent to that of naturally aged rats.
To examine whether SnCs contribute to PMO, p16 (also known as cyclin-dependent kinase inhibitor 2a and highly expressed in SnCs) and p53 (another typical biomarker of SnCs) levels were measure to identify SnCs in bone tissue. A remarkable accumulation of both p16 and p53 positive SnCs was observed in the M-OVX rats compared with that in the Y-OVX rats and was equivalent to that in the O-SHAM rats (Fig. 2g-k). In summary, we suggest that SnC accumulation accelerated bone loss in middle-aged PMO rats, and established correlation of the pathogenesis of type Ⅰ and type Ⅱ osteoporosis.
DQ eliminated SnCs in middle-aged PMO rats
Considering that SnCs accumulated in middle-aged PMO rats, we next investigated the effect of DQ on SnCs in PMO rats at different age stages. Both Y-OVX and M-OVX rats were administrated DQ orally for two months (Y-DQ and M-DQ) and then the lumbar vertebrae were dissected for analysis. Treatment with DQ reduced the SnC number in the M-DQ rats compared with the M-OVX rats, with the decreased expression of p16 and p53 by approximately twofold. However, no significant difference was observed among young rats (Fig. 3a, b). Consistent with the lumbar vertebrae results, DQ reduced the number of p16-positive and p53-positive SnCs in the femur, nearly a twofold reduction compared with that in the M-OVX group (Fig. 3c-f). Our results showed that SnCs accumulated in middle-aged PMO rats, as characterized by increased expression of p16 and p53, and DQ effectively eliminated SnCs, as indicated by decreased p16 and p53 expression in middle-aged PMO rats.
MSCs exhibited senescence-associated characteristics in middle-aged PMO rats
Stem cells, especially MSCs, are key contributors to bone formation and regeneration due to their excellent self-renewal and osteogenic capacities. However, MSC senescence, characterized by exhaustion and inappropriate adipogenesis, is induced by the pro-inflammatory microenvironment during aging, and is considered the culprit of osteoporosis and degeneration21,42-44. To further probe the performance of MSCs in middle-aged PMO rats, we first examined the frequency and self-renewal capacity of the MSCs. Femurs and tibias from middle-aged SHAM or OVX group rats were dissected, and cells were isolated for flow cytometry analysis and colony-forming unit (CFU) assay (Fig. 4a). CD45-negative, CD44-positive and CD29-positive cells were identified as MSCs. The frequency of MSCs in OVX group rats decreased by 4.9% compared with that in the SHAM group rats (Fig. 4b-d). Consistent with the flow cytometry results, the CFU assay showed a decreased self-renewal capacity, by approximately fivefold, in the OVX group (Fig. 4e). The exhaustion of MSCs strongly suggested MSC senescence in middle-aged PMO rats.
Based on aforementioned findings, the relationship between MSC exhaustion and senescence was further investigated through a transcriptome analysis. We hypothesized that MSC behavior was regulated by estrogen-deficiency induced microenvironment. To mimic the microenvironment in vitro, serum extracted from OVX rats were mixed with cell culture medium to prepare the conditioned medium (CM). Normal MSCs (isolated from one-month-old rats) were exposed for 3 days to CM containing serum from M-SHAM (SHAM CM), M-OVX (OVX CM) and O-SHAM (Old CM) rats for 3 days, and the transcriptome of the different CM-treated MSCs was analyzed by RNA sequencing. The transcriptomic profiles of each group clustered distinctly, as demonstrated by principal component analysis (PCA, Fig. S2a), and the differentially expressed genes (DEGs) between the SHAM and OVX groups were defined with a cutoff of adjusted p value < 0.05 and │log fold change│ ≥1 (Fig. S2b). The DEGs downregulated in the OVX group were enriched in the cell cycle, DNA repair (homologous recombination, Fanconi anemia pathway, mismatch repair and base excision repair), and DNA replication pathways (Fig. 4f), which all have been correlated with cellular senescence. Common DEGs identified in both OVX vs. SHAM and Old vs. SHAM analyses accounted for 22.8% of total DEGs identified and were hierarchically clustered following the same trend in the OVX and Old groups compared with that in the SHAM group (Fig. 4g, h), in which DEGs were also enriched in pathways similar to those identified in OVX vs. SHAM analysis (Fig. 4i). Gene-set enrichment analysis (GSEA) showed similar regulatory effects between MSCs in the OVX and Old groups, with downregulation of cell cycle pathway and upregulation of steroid biosynthesis pathway (Fig. 4j). To determine whether the features of MSC exhaustion in middle-aged PMO rats were similar to those of senescent MSC exhaustion, we further investigated the cell cycle pathway. MSCs in OVX group exhibited G1/S downregulation which was also observed in senescent MSCs, with lower levels of cyclin E (CycE), cyclin A (CycA), cell division cycle 6 (Cdc6), cell division cycle 45 (Cdc45), and minichromosome maintenance complex component 5 (MCM5) (Fig. 4k). These results demonstrated that MSCs in the middle-aged PMO rats exhibited senescence-associated hallmarks and presented transcriptomic profiles similar to that of senescent MSCs in CM containing old rat serum.
DQ rescued MSC exhaustion and restored MSC function via attenuating SASP
To further investigate the effect of DQ treatment on the MSC fate, we evaluated the cellular senescence, proliferation and osteogenesis capability of MSCs after treatment with CM composed of serum extracted from M-DQ rats (DQ CM). First, MSC senescence was delayed by DQ as proven by decreased senescence-associated β-galactosidase (SA-β-gal) positive cell numbers (Fig. 5a) and lower gene expression of p16, p21 and p53 in the DQ CM group (Fig. 5b). To account for possible cell cycle arrest, we conducted the cell cycle test by performing flow cytometry, and the results showed a decreased percentage of cells in the DQ CM group in the G1 phase compared with that in the OVX CM group, suggesting reestablished cell cycle progression after DQ treatment, not arrest in the G1 phase (Fig. 5c, d). Furthermore, the expression of SASP genes was strikingly downregulated by DQ; in contrast, these genes were highly expressed in OVX CM group, indicating that DQ attenuated the development of a pro-inflammatory microenvironment in the PMO context (Fig. 5e). MSC exhaustion was also rescued by DQ, demonstrated by an increased number of colonies (Fig. 5f, g) and increased proliferative ability (Fig. 5h). In addition, DQ improved MSC morphology, showing diminished lipofuscin formation (Fig. S3). To further investigate the effect of DQ on MSCs in PMO rats, Alizarin red S (ARS) staining was performed. The results showed that the impaired osteogenesis ability of the MSCs in OVX CM, characterized by decreased calcium nodules, was significantly restored by DQ (Fig. 5i).
Transcriptomes of the MSCs in SHAM CM, OVX CM, or DQ CM were compared by RNA sequencing to determine the regulation of senescence-associated processes in the PMO context after treatment with DQ. The transcriptomic profiles of the DQ group showed distinct clusters (Fig. S2a), and the DEGs between the OVX and DQ groups were identified on the basis of a cutoff adjusted p value < 0.05 (Fig. S2c). Compared with the effect on the MSCs in OVX CM, DQ upregulated pathways related to DNA replication, DNA repair and cellular senescence; the DEGs in these pathways were found to be significantly downregulated in the OVX vs. SHAM group analysis (Fig. 6a). Common DEGs identified in both the DQ vs. OVX and DQ vs. SHAM analyses accounted for 31.5% of the total DEGs, showing the same trend in the DQ and SHAM groups, and these DEGs were enriched in some of the same pathways identified in the OVX vs. SHAM analysis (Fig. 6b-d). The GSEA showed the upregulation of the cell cycle-related genes and downregulation of steroid biosynthesis-related genes in DQ-treated MSCs, in contrast to the findings in the Old or OVX MSCs (Fig. 6e). In addition, DQ also upregulated homologous recombination and glutathione metabolism pathways (Fig. S2d), which contribute to anti-senescence effects. These results demonstrated shifted transcriptome profiles of MSCs in OVX CM toward a profile similar to that of SHAM MSCs after DQ treatment (Fig. 6b-e). The ten most active hub genes in the MSCs in DQ vs. OVX analysis were predicted using Cytoscape software (Fig. 6f, Table S1). Interestingly, these genes all intersected in the OVX vs. SHAM, Old vs. SHAM and DQ vs. OVX analysis (Fig. 6g) and were all downregulated in the OVX and Old groups but upregulated in the DQ group (Fig. 6h), indicating that DQ mainly regulated senescence-associated processes in PMO. In summary, DQ regulated a transcriptome shift toward the SHAM phenotype, rescued MSC exhaustion and restored MSC function by attenuating the SASP.
Local implantation of DQ-encapsulated and BMP2-fixed hydrogel rejuvenated bone regeneration
The aforementioned findings demonstrated that DQ effectively ameliorated PMO in a systematic way, but notably, PMO leads to many complications with serious consequences, for which DQ may also exert a positive effect. Local osteoporotic fracture is a typical complication, and the risk in women is as high as 40%, making it a major cause of death, disability, and worldwide healthcare costs45. Here, we investigated the effect of DQ on bone regeneration in a PMO defect model through the combination and local administration of DQ and human recombinant bone morphogenetic protein 2 (BMP2), a commonly used osteo-inductive agent. To improve the microenvironment for local bone defect regeneration, DQ was expected to be released prior to BMP2, resulting in a negatable influence of the SASP. Therefore, we fabricated a DQ-encapsulated and BMP2-fixed injectable hydrogel for implantation (DQ+BMP2) (Fig. 7a). To realize sequential administration of DQ and BMP2 locally in vivo, bioglass microspheres with mesopores (MBG) of 8 nm, which was compatible with the BMP2 size (7.5 nm), were fabricated as previously described for sustained release of BMP246 (Fig. S4a, b). DQ was directly mixed into the gelatinous methacryloyl (GelMA) hydrogel and was released earlier than BMP2 (Fig. S4c). The encapsulated amount of D and Q were 685 μg mL-1 and 68.5 μg mL-1, respectively. The micro-CT analysis demonstrated that local treatment with DQ had a synergistic effect with BMP2 on bone regeneration, as proven by significantly increased newly formed bone, especially cortical bone, in the DQ+BMP2 group (Fig. 7b). Compared with the effect of BMP2 released separately, DQ elevated the BV/TV (Fig. 7C), Tb.Th (Fig. 7d) and Tb.N (Fig. 7e) by approximately twofold and decreased the Tb.Sp ((Fig. 7f) in the DQ+BMP2 group. Consistent with these results, the H&E- and Masson-stained images showed orderly arranged trabeculae accompanied by abundant infiltration of bone marrow cells in the cavities (Fig. 7g, S5a, S5b). Less cell infiltration was observed between bone trabeculae in the BMP2 group.
Next, we investigated the mechanism creating of BMP2 and DQ synergism in bone regeneration by dissolving DQ into OVX CM. The concentration of DQ for inducing MSC senescence was tested using an oxidative stress-induced senescence model (Fig. S6a). The optimal concentrations of DQ in combination with quercetin (Q10) was 10 μM, and it was 10 nM with dasatinib (D10) (Fig. S6b-e). Senescent MSCs (Sen) were selectively eliminated, while proliferating non-senescent MSCs (Pro) survived. After 3 days of DQ-treatment, MSCs (DQ) exhibited decreased proliferation by approximately 27% compared to Sen MSCs due to the clearance. However, when DQ was removed on the 4th day, the MSCs restored the proliferation capability and increased approximately 23% viability compared to that under the treatment of DQ, throughout the following three days (Fig. S6e). In a Transwell experiment, decreased p16, p21, p53 and SASP expression was found in proliferating MSCs cocultured with DQ MSCs (Fig. S6g).
To investigate the synergistic effect of DQ and BMP2, MSCs were sequentially treated with conditioned medium (SHAM, OVX, or DQ CM) for 7 days and complete medium containing a low dose of BMP2 (SHAM+BMP2, OVX+BMP2, or DQ+BMP2) for another 7 days (Fig. 7h). p16 expression on the 7th day was downregulated by local administration of DQ, while both pSmad1/5/9 (a signal transduction molecule involved in BMP2 pathway activation) and col Ⅰ expression was upregulated (Fig. 7i-k). ARS and Oil red O staining on the 14th day revealed that compared with MSCs in OVX+BMP2 CM, MSCs in DQ+BMP2 CM showed restored osteogenic differentiation and decreased adipogenic differentiation, which may have led to bone loss as reported elsewhere43,47 (Fig. 7l, m). However, when treated of DQ and BMP2 simultaneously (DQ/BMP2), senescent MSCs exhibited inferior osteogenesis compared with that treated with BMP2 only (Fig. S7). We proposed that the improved osteogenesis after sequential treatment with DQ and BMP2 (DQ+BMP2) might be attributed to the improved microenvironment and recovered proliferating ability, while the inferior osteogenesis after the simultaneous treatment of DQ and BMP2 (DQ/BMP2) was ascribed to the decreased amount of MSCs (Fig. S6e). These results solidified our design of the hydrogel with sequential releasing of DQ to regulate the microenvironment and followed by BMP2 to induce higher osteogenesis. The in vivo immunofluorescence staining analysis further confirmed the increase in osteogenesis, by revealing higher expression of Runt-related transcription factor 2 (Runx2, Fig. 7n, S8a), decreased expression of p16 and p53 (Fig. 7o-p, S8b-c) and diminished matrix metallopeptidase 9 (MMP9, a typical protease in the SASP, Fig. 7q, S8d). Taken together, local administration of DQ combined with BMP2 showed a synergistic effect on bone regeneration by inhibiting cellular senescence and promoting pSmad1/5/9 pathway activation.