Aspirin treatment inhibits osteoclast activities.

<p>(A) OVX mice have increased levels of RANKL and type I collagen C-terminal telopeptides and decreased levels of OPG in blood serum as compared to sham mice. Aspirin treated OVX mice (OVX+A) showed a significant decreased levels of RANKL and type I collagen C-terminal telopeptides along with increased levels OPG in blood serum. The graph represents mean±SD (n = 5; [<i>P</i><0.01 vs. Sham; #<i>P</i><0.05 vs. OVX). (B) TRAP staining confirmed that OVX mice had increased number of TRAP positive cells in epiphysis and trabecular bone areas of the distal femurs as compared to sham mice. Administration of aspirin for three months resulted in a significant decrease in number of TRAP positive cells (OVX+A) in the epiphysis and trabecular bone areas (n = 6; [[[<i>P</i><0.005 vs. Sham; ###<i>P</i><0.005 vs. OVX). (C) <i>Ex vivo</i> co-culture bone marrow cells or spleen cells with osteoblastic cells revealed that <i>in vitro</i> aspirin treatment inhibits the formation of TRAP-positive multinuclear cells (MNCs) in a dose dependent manner (2–200 µg/ml). The graph represents mean±SD (n = 5; [[[<i>P</i><0.005 vs. ASP 0 µg/mL). (D) RANKL-induced osteoclastogenesis was partially blocked by aspirin treatment at 50 µg/ml as seen a significantly decreased number of TRAP-positive osteoclasts in aspirin treated group (ASP 50). The graph represents mean±SD (n = 5; [[[<i>P</i><0.005 vs. ASP 0 µg/mL).</p

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.</p

Adoptive transfer of CD4⁺CD25⁻CD45RB^+hi T cells derived from C3H mice restores bone loss phenotype in OVX-immunocompromised mice.

<p>(A) Estrogen-deficiency failed to induce osteoporosis in 15-week-old bg-nu/nu-xid immunocompromised mice. At one-month post-OVX, there is no BMD difference in femurs between OVX and sham group (n = 4), assessed by Dual x-ray absorptiometry (DEXA) analysis. The graph represents mean±SD. (B) Isolation of CD4⁺CD25⁻CD45RB^+hi (open arrow) and CD4⁺CD25⁻CD45RB^−/low (triangle arrow) T cells from spleen of C3H mice by flow cytometry for systemic infusion. (C) At one month post-OVX, DEXA analysis revealed a significant decreased BMD in the femurs of CD4⁺CD25⁻CD45RB^+hi OVX mice (OVX+T^RB+hi) when compared to the OVX mice (OVX) or CD4⁺CD25⁻CD45RB^−/low OVX mice (OVX+T^RB−/low; n = 4; [[<i>P</i><0.01). (D) The number of CFU-F decreased significantly in CD4⁺CD25⁻CD45RB^+hi OVX mice (OVX+T^RB+hi) compared to OVX and CD4⁺CD25⁻CD45RB^−/low OVX mice (OVX+T^RB−/low). Totally 10⁶ all nuclear cells (ANC) were used in each group. Error bars represent the mean±SD (n = 5; [[<i>P</i><0.01). (E) The proliferation of BMMSCs from CD4⁺CD25⁻CD45RB^+hi OVX mice (OVX+T^RB+hi) was significantly increased in compared to CD4⁺CD25⁻CD45RB^−/low OVX mice (OVX+T^RB−/low) and OVX mice as 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. Error bars represent the mean±SD (n = 5; [[<i>P</i><0.001). (F) Alizarin red staining showed that BMMSCs derived from CD4⁺CD25⁻CD45RB^+hi OVX mice (OVX+T^RB+hi) had lower calcium accumulation than that of BMMSCs from OVX and CD4⁺CD25⁻CD45RB^−/low OVX mice (OVX+T^RB−/low) when cultured under the osteogenic inductive conditions. The graph represents mean±SD (OVX mice, n = 4; CD4⁺CD45RB^+hi OVX mice, n = 5; CD4⁺CD45RB^−/low OVX mice, n = 4; [<i>P</i><0.001). (G) BMMSCs were transplanted into immunocompromised mice using HA/TCP (<i>HA</i>) as a carrier for eight weeks. Bone formation assessed by H&E staining was decreased in transplants of BMMSCs derived from CD4⁺CD25⁻CD45RB^+hi OVX mice (OVX+T^RB+hi) compared to BMMSC transplants of OVX and CD4⁺CD25⁻CD45RB^−/low OVX mice (OVX+T^RB−/low). <i>B</i>; bone <i>CT</i>; connective tissue. Original magnification; ×200. The graph represents mean±SD (OVX mice, n = 4; CD4⁺CD45RB^+hi OVX mice, n = 5; CD4⁺CD45RB^−/low OVX mice, n = 4; [<i>P</i><0.001). (H) TRAP staining showed that 15-week old OVX mice received CD4⁺CD45RB^hi cells (OVX+T^RB+hi) contain elevated number of TRAP-positive osteoclastic cells (white arrows) in epiphysis and trabecular bone areas of femurs as compared to Sham, OVX, and OVX received CD4⁺CD45RB^−/low cells (OVX+T^RB−/low). The graph represents mean±SD (n = 4; [<i>P</i><0.05, [[<i>P</i><0.01). BM: bone marrow, TB: trabecular bone.</p

Lymph node (LN) cells activated by an anti-CD3 antibody induce BMMSC apoptosis <i>in vitro</i>.

<p>(A) Mouse BMMSCs co-cultured with LN cells indicated that anti-CD3 antibody activated LN cells were capable of inducing BMMSC death as shown a blank well without BMMSC staining (blue). When co-cultured BMMSCs (MSC) and LN cells were separated by a transwell culture system, anti-CD3 antibody treated LN cells failed to induce BMMSC death. (B) It is known that immunocompromised mice have no T lymphocytes. Thus, LN cells derived from immunocompromised failed to induce BMMSC death following anti-CD3 antibody activation in the co-culture system. (C) TUNEL staining showed that BMMSC death caused by anti-CD3 antibody-activated LN cell is through an apoptotic pathway. (D) Condition medium (CM) derived from naïve LN cells and anti-CD3 antibody activated LN cells were not able to induce cell death of BMMSCs. (E) Neutralizing anti-TNF-α and IFN-γ antibodies were not able to inhibit BMMSC death induced by anti-CD3 antibody-activated LN cells. (F) Neutralizing Fas ligand antibodies and brefeldin A, but not concanamycin A, were capable of blocking BMMSC death induced by anti-CD3 antibody-activated LN cells. (G) Western blot analysis showed that mouse and human BMMSCs (mMSC and hMSC) express Fas. (H) Fas antibody can induce significant reduction in number of living BMMSCs in culture. (I) Anti-CD3 antibody-activated LN cells were not able to induce cell death of BMMSCs derived from <i>CD95</i>-deficient mice (<i>lpr</i>). (n = 5; [<i>P</i><0.05 and [[[<i>P</i><0.005).</p