Co-administration of aspirin and allogeneic adipose-derived stromal cells attenuates bone loss in ovariectomized rats through the anti-inflammatory and chemotactic abilities of aspirin

Abstract

Introduction: Osteoporosis is a syndrome of excessive skeletal fragility characterized by the loss of mass and deterioration of microarchitecture in bone. Single use of aspirin or adipose-derived stromal cells (ASCs) has been recognized recently to be effective against osteoporosis. The goal of the study was to evaluate the osteogenic effects of the co-administration of aspirin and allogeneic rat adipose-derived stromal cells (rASCs) on ovariectomized (OVX)-induced bone loss in rats. The underlying mechanisms were investigated in vitro and in vivo.

Methods: Firstly, allogeneic rASCs were isolated and cultured, and the conditioned medium (CM) from the maintenance of rASCs was collected. Secondly, the OVX rats were administrated CM, rASCs, aspirin (ASP) or rASCs + ASP, respectively. Twelve weeks later, the anti-inflammatory and osteogenic effects were assessed by micro-CT, undecalcified histological sections, dynamic histomorphometric analyses and serologic assays for biochemical markers. Finally, a Transwell migration assay in vitro and cell-trafficking analyses in vivo were used to explore the effects of aspirin on rASC migration.

Results: Systemic administration of aspirin and rASCs attenuated OVX-induced bone loss better than single use of aspirin or ASCs (p < 0.05, respectively). Next, we analyzed the underlying mechanisms of the anti-inflammatory and chemotactic abilities of aspirin. Aspirin suppressed serum levels of the pro-inflammatory cytokines on tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and the anti-inflammatory ability was positively associated with bone morphometry. Also, aspirin exhibited excellent chemotactic effects in vitro and accelerated the homing of allogeneic rASCs into bone marrow during early in vivo stages.

Conclusions: Co-administered aspirin and allogeneic ASCs can partially reverse OVX-induced bone loss in rats. This effect appears to be mediated by the anti-inflammatory and chemotactic abilities of aspirin.

Fig. 1

Representative images of changes in bone microarchitecture in rats. Tibias of F344 rats were dissected and evaluated ex vivo by micro-CT (a) and slicing of undecalcified tissues (H & E staining); (b) eight weeks after treatment

Fig. 2

Dynamic histomorphometric analyses of rat tibias. a Representative fluorescence images obtained from tibias after double labeling with alizarin red and calcein. Scale bar = 50 μm. b Bone formation rate (BFR) was measured from the tibia using Bio-quant software. Data are the mean ± SD. *p < 0.05 vs. Sham, #p < 0.05 vs. OVX, &p < 0.05 vs. ASC CM, %p < 0.05 vs. ASC, black triangle p < 0.05 vs. ASP. OVX ovariectomized, ASC adipose-derived stromal cells, ASP aspirin

Fig. 3

Serum levels of biochemical markers of bone turnover. Serum levels of P1NP (a) and ALP (b) (marker of bone formation), and TRAP 5b (marker of bone resorption) (c) eight weeks after treatment. Data are the mean ± SD of experiments carried out in triplicate. *p < 0.05 vs. Sham, #p < 0.05 vs. OVX, $p < 0.05 vs. ZOL, &p < 0.05 vs. ASC CM, %p < 0.05 vs. ASC, black triangle p < 0.05 vs. ASP. P1NP, procollagen 1 N-terminal peptide, ALP alkaline phosphatase, TRAP, tartrate-resistant acid phosphatase, OVX ovariectomized, ZOL zoledronate, ASC adipose-derived stromal cells, ASP aspirin

Fig. 4

Serum levels of calcium eight weeks after treatment. Data are the mean ± SD.*p < 0.05 vs. Sham, #p < 0.05 vs. OVX, $p < 0.05 vs. ZOL, &p < 0.05 vs. ASC CM, black triangle p < 0.05 vs. ASP. ALP alkaline phosphatase, OVX ovariectomized, ZOL zoledronate, ASC CM Adipose-derived stromal cells conditioned medium, ASP aspirin

Fig. 5

Serum levels of TNF-α and IFN-γ by ELISA eight weeks after treatment. Serum levels of TNF-α (a) and IFN-γ (b) among the seven groups. Data are the mean ± SD of experiments undertaken in triplicate. *p < 0.05 vs. Sham, #p < 0.05 vs. OVX, $p < 0.05 vs. ZOL, &p < 0.05 vs. ASC CM, %p < 0.05 vs. ASC. OVX ovariectomized, ZOL zoledronate, ASC CM adipose-derived stromal cells conditioned medium

Fig. 6

The in vitro chemotactic ability of aspirin by transwell assays. a Representative images of ASC migration at different concentrations of aspirin. Scale bar = 50 μm. b The number of migrating cells at different concentrations of aspirin. Data are the mean ± SD of experiments undertaken in triplicate. *p < 0.05 vs. control, #p < 0.05 vs. 0.0001 mM, $p < 0.05 vs. 0.001 mM, &p < 0.05 vs. 0.01 mM. ASC adipose-derived stromal cells

Fig. 7

Effect of aspirin on homing of rASCs by cell-trafficking assays in vivo. BM cells were flushed 6, 12, 24, 48, and 72 h after injection of CM-DiL-labeled cells in the lateral tail vein and primary culture carried out. Thereafter, numbers of CM-DiL-positive cells were measured by flow cytometry. The percentage of rASCs/BMSCs in the BM at different time points (a) and representative images of flow cytometry (b) are shown. Data are the mean ± SD. *p < 0.05 vs. OVX-vehicle at 6 h, #p < 0.05 vs. OVX-vehicle at 12 h, $p < 0.05 vs. OVX-vehicle at 24 h. rASCs rat adipose-derived stromal cells, BM bone marrow, CM conditioned medium, BMSCs bone marrow stromal cells, OVX ovariectomized.