Aspirin treatment induces activated T cell death <i>in vitro</i>, promotes osteogenesis of BMMSCs, and improves BMD in OVX mice.

<p>(A) Fas Ab can induce significantly reduction in number of living BMMSCs in the culture. However, the cell viability of human BMMSCs was significantly improved in aspirin (ASP)-treated groups (n = 5, [[[<i>P</i><0.005 vs. untreated control group; ###<i>P</i><0.005 vs. Fas Ab-treated group). (B) Aspirin (ASP) did not affect the cell viability of whole LN cells cultured on regular plate. (C) On the other hand, T cells activated by cultured on CD3εΑb-coated dishes showed decreased viability in 50 µg/ml ASP-treated group, but not in 2.5 µg/ml ASP-treated group. (n = 5, [<i>P</i><0.05 vs. 0 µg/ml ASP group; #<i>P</i><0.05 vs. 2.5 µg/ml ASP group). (D) The scheme indicates the experimental design on aspirin administration and OVX surgery procedure. (E) Trabecular bone structure of the distal femoral metaphysis was analyzed by microQCT. As expected OVX mice showed decreased trabecular bone (yellow circle area) and cortical bone (arrow) formation when compared to sham mice. Aspirin treated OVX mice exhibited a significant increase in trabecular bone (yellow circle area) and cortical bone (arrow) volume compared to OVX mice. (F,G) MicroQCT analysis demonstrated that BV/TV (bone volume vs. tissue volume) (F) and Tb.N (trabecular number) (G) were decreased in OVX mice as compared to sham group but significantly increased in aspirin treated OVX group (n = 5; [<i>P</i><0.01). (H) The number of CFU-F in OVX group increased significantly as compared to sham group, however, aspirin treatment (OVX+A) appeared to reduce CFU-F number to the sham group level. Error bars represent the mean±SD (n = 5, [[<i>P</i><0.01). (I) The proliferation rate of BMMSCs was assessed by BrdU incorporation assay for 24 hours. The number of BrdU-positive cells was indicated as a percentage to the total number of counted BMMSCs and averaged from 5 replicated cultures. BMMSCs derived from OVX mice showed significantly elevated BrdU-uptake rate, however, aspirin treatment (OVX+A) reduced BrdU up-take rate. Error bars represent the mean±SD (n = 5, [<i>P</i><0.01, [[<i>P</i><0.001). (J) BMMSCs from aspirin treated mice were transplanted into immunocompromised mice for eight weeks using HA/TCP (<i>HA</i>) as a carrier vehicle. Bone formation assessed by H&E staining was decreased in OVX BMMSC transplants compared to sham mouse group. Aspirin treatment (OVX+A) can improve OVX BMMSC-mediated bone formation <i>in vivo</i>. <i>B</i>; bone <i>CT</i>; connective tissue. Original magnification; ×200. The bone formation rate was calculated as the percentage of newly formed bone area per total area of transplant at the representative cross-sections. The graph represents mean±SD (sham mice, n = 4; OVX mice, n = 4; OVX+A mice, n = 4; [<i>P</i><0.01). (K) Representative images of Alizarin red staining of human BMMSCs cultured under the osteogenic inductive condition containing 2.5 and 50 µg/ml aspirin (ASP 2.5 µg/ml and ASP 50 µg/ml). Aspirin treatment can increase calcium accumulation in cultured human BMMSCs. (L) After aspirin treatment at 50 µg/ml for 1 week, <i>ex vivo</i> expanded human BMMSCs were transplanted into immunocompromised mice with HA/TCP (<i>HA</i>) as a carrier. Aspirin treated human BMMSCs exhibited significantly increased new bone formation in comparison to non-aspirin-treated control human BMMSC transplants ([ <i>P</i><0.05). (M) Human BMMSCs express low levels of telomerase activity (MSC), if there is any. Following 1-week aspirin treatment (2.5 or 50 µg/ml), human BMMSCs showed a significantly increased telomerase activity (MSC+A) ([ <i>P</i><0.01). HEK293 cells were used as a positive control (239T) and heat inactive human BMMSCs were used as negative control (H.I). (N) Aspirin treatment at 2.5 µg/ml (Asp 2.5) and 50 µg/ml (Asp 50) can slightly increased telomere length from 6.4 kb in un-treated control group (Asp 0) to 7.7 kb (Asp 2.5) and 7.6 (Asp 50) respectively. 293T cell line (12.1 kb) was used as a positive control. (O) Western blot analysis showed that aspirin treatment (ASP) at indicated dosages enables to elevate expressions of Runx2, alkaline phosphatase (ALP), and osteocalcin (OCN). β-actin was used a control for the amount of sample loading. (P) Human BMMSCs were cultured with or without aspirin at 50 µg/ml for 0, 0.5, 1, 2, 3 hours. The degradation of phospho-β-catenin was accelerated in aspirin (50 µg/ml) treated group. β-actin was used a control for the amount of sample loading.