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. 2016 Feb 25:6:22005.
doi: 10.1038/srep22005.

Akermanite bioceramics promote osteogenesis, angiogenesis and suppress osteoclastogenesis for osteoporotic bone regeneration

Lunguo Xia  1 , Zhilan Yin  2 , Lixia Mao  1 , Xiuhui Wang  2 , Jiaqiang Liu  1 , Xinquan Jiang  3 , Zhiyuan Zhang  4 , Kaili Lin  2   5 , Jiang Chang  2 , Bing Fang  1
Affiliations

Akermanite bioceramics promote osteogenesis, angiogenesis and suppress osteoclastogenesis for osteoporotic bone regeneration

Lunguo Xia et al. Sci Rep. .

Abstract

It is a big challenge for bone healing under osteoporotic pathological condition with impaired angiogenesis, osteogenesis and remodeling. In the present study, the effect of Ca, Mg, Si containing akermanite bioceramics (Ca2MgSi2O7) extract on cell proliferation, osteogenic differentiation and angiogenic factor expression of BMSCs derived from ovariectomized rats (BMSCs-OVX) as well as the expression of osteoclastogenic factors was evaluated. The results showed that akermanite could enhance cell proliferation, ALP activity, expression of Runx2, BMP-2, BSP, OPN, OCN, OPG and angiogenic factors including VEGF and ANG-1. Meanwhile, akermanite could repress expression of osteoclastogenic factors including RANKL and TNF-α. Moreover, akermanite could activate ERK, P38, AKT and STAT3 signaling pathways, while crosstalk among these signaling pathways was evident. More importantly, the effect of akermanite extract on RANKL-induced osteoclastogenesis was evaluated by TRAP staining and real-time PCR assay. The results showed that akermanite could suppress osteoclast formation and expression of TRAP, cathepsin K and NFATc1. The in vivo experiments revealed that akermanite bioceramics dramatically stimulated osteogenesis and angiogenesis in an OVX rat critical-sized calvarial defect model. All these results suggest that akermanite bioceramics with the effects of Mg and Si ions on osteogenesis, angiogenesis and osteoclastogenesis are promising biomaterials for osteoporotic bone regeneration.

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Figures

Figure 1
Figure 1. Material characterization.
(A) SEM images of the fractured surface of the prepared β-TCP and akermanite bioceramic scaffolds; (B) XRD patterns of the prepared β-TCP and akermanite bioceramic scaffolds; (C) The concentrations of Ca, Mg and Si ions in β-TCP and akermanite extracts. ★indicates significant differences as compared with DMEM; ▲indicates significant differences as compared with β-TCP, p < 0.05. Scale bar = 500 μm.
Figure 2
Figure 2. Cell proliferation assay.
(A) MTT assay for the effect of various concentrations of β-TCP and akermanite extracts on cell proliferation of BMSCs-OVX; (B) The optimal concentration of 1/16 dilution of β-TCP and akermanite extracts on cell proliferation of BMSCs-OVX. The cells cultured without β-TCP and akermanite extracts was treated as control group (Control). ★indicates significant differences as compared with control group; ▲indicates significant differences as compared with β-TCP group, p < 0.05.
Figure 3
Figure 3. ALP activity assay.
(A) ALP staining of BMSCs-OVX treated with β-TCP and akermanite extracts for 10 days; (B) ALP quantitative assay of BMSCs-OVX treated with β-TCP and akermanite extracts at days 4, 7 and 10. The cells cultured without β-TCP and akermanite extracts was treated as control group (Control). ★indicates significant differences as compared with control group; ▲indicates significant differences as compared with β-TCP group, p < 0.05. Scale bar = 10 mm.
Figure 4
Figure 4. The effect of akermanite extract on the expression of osteogenic, angiogenic and osteoclastogenic genes and the activation of signaling pathways.
(A) Rea-time PCR analysis of Runx2 (a), BMP-2 (b), BSP (c), OPN (d), OCN (e), OPG (f), RANKL (g), TNF-α (h), VEGF (i) and ANG-1 (j) in BMSCs-OVX treated with β-TCP and akermanite extracts at days 4 and 7. The cells cultured without β-TCP and akermanite extracts was treated as control group (Control). ★indicates significant differences as compared with control group; ▲indicates significant differences as compared with β-TCP group, p < 0.05; (B) Western blot assay for key protein expression of MAPK, AKT and STAT3 signaling pathways for BMSCs-OVX treated with β-TCP and akermanite extracts at 30, 60 and 90 min; (C) The quantitative assay for the ratios of p-P38/P38 (a), p-ERK/ERK (b), p-JNK/JNK (c), p-AKT/AKT (d) and p-STAT3/STAT3 (e). ★indicates significant differences as compared with β-TCP group, p < 0.05.
Figure 5
Figure 5. The effect of inhibitors SB202190, PD98059, LY294002 and AG490 on BMSCs-OVX treated with akermanite extract.
(A) Western blot assay for the expression of p-P38, p-ERK, p-AKT and p-STAT3 for BMSCs-OVX cultured in akermanite extract with inhibitors SB202190, PD98059, LY294002 and AG490 for 90 min, respectively; (B) The quantitative assay for the ratios of p-P38/P38 (a), p-ERK/ERK (b), p-AKT/AKT (c) and p-STAT3/STAT3 (d) for BMSCs-OVX cultured in akermanite extract with inhibitors SB202190, PD98059, LY294002 and AG490 for 90 min, respectively; (C) ALP staining for BMSCs-OVX cultured in akermanite extract with inhibitors SB202190, PD98059, LY294002 and AG490 for 10 days, respectively; (D) Rea-time PCR analysis of Runx2 (a), BMP-2 (b), BSP (c), OPN (d), OCN (e), OPG (f), RANKL (g), TNF-α (h), VEGF (i) and ANG-1 (j) for BMSCs-OVX cultured in akermanite extract with inhibitors SB202190, PD98059, LY294002 and AG490 for 7 days, respectively. BMSCs-OVX cultured in akermanite extract without any inhibitors was treated as akermanite group. ★indicates significant differences as compared with akermanite group, p < 0.05. Scale bar = 10 mm.
Figure 6
Figure 6. In vitro osteoclastogenesis assay.
(A) TRAP staining for BMMs cultured in M-CSF and RANKL supplement with β-TCP and akermanite extracts, respectively; (B,C) The quantitative assay for number (B) and area (C) of osteoclasts according to TRAP staining; (D–F) Rea-time PCR analysis of TRAP (D), cathepsin K (E) and NFATc1 (F) for BMMs cultured in M-CSF and RANKL supplement with β-TCP and akermanite extracts, respectively. The cells cultured in M-CSF and RANKL without β-TCP and akermanite extracts was treated as control group (Control). ★indicates significant differences as compared with control group; ▲indicates significant differences as compared with β-TCP group, p < 0.05. Scale bar = 100 μm.
Figure 7
Figure 7. Micro-CT assay and sequential fluorescent labeling assay for in vivo bone regeneration.
(A) Representative micro-CT 3D superficial images of new bone formation (a: β-TCP; b: akermanite) and morphometric analysis of BV/TV (c) and Tb.Th (d) for each group at 8 weeks postoperation; (B) The images in yellow (TE; a1, b1), red (AL; a2, b2) and green (CA; a3, b3) represented bone formation and mineralization at 2, 4 and 6 weeks after operation, while the images of a4–a5 and b4–b5 represented the merged images of the three fluorochromes, or together with a brightfield confocal laser microscope image for β-TCP (a1–a5) and akermanite (b1–b5) groups, respectively; The percentages of TE, AL and CA staining (c) and mineral apposition rate (d) of 2–4 weeks and 4–6 weeks for β-TCP and akermanite groups assessed at week 8 after implantation by histomorphometric analysis. ▲indicates significant differences between β-TCP and akermanite groups, p < 0.05. A: Scale bar = 1 mm; B: Scale bar = 100 μm.
Figure 8
Figure 8. Histological and histomorphometric assay.
(A,B) Histological images of newly formed bone in β-TCP (A1–A2) and akermanite (B1–B2) groups at week 8 after operation; (C–E) The percentage of new bone area (C), vessel area (D) and residual material area (E) assessed for β-TCP and akermanite groups by histomorphometric analysis; (F) Real-time PCR for the expression of Runx2, OCN, OPG, RANKL, TRAP and CD31 in calvarial defect area. ★indicates significant differences between β-TCP and akermanite groups, p < 0.05. A1, B1: Scale bar = 1 mm; A2, B2: Scale bar = 100 μm.
Figure 9
Figure 9. The crosstalk among P38, ERK, AKT and STAT3 signaling pathways for the effect of akermanite bioceramics on BMSCs-OVX.
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