Fig 1: CXCL12Col2-cre mice develop an ankylosis phenotype.(A and B) µCT images and quantitative analysis of pathological new bone formation in hind paw of DBA/1;CXCL12Col2 mice at the age of 20 weeks with or without EHT1864 administration. Scale bars, 500 µm. n = 6 per group. ANOVA (F2,15 = 34.40). (C) H&E staining, SOFG staining, immunohistochemical staining, and immunofluorescence staining of Prx1 in plantar surface of hind paw of DBA/1;CXCL12Col2 mice at the age of 20 weeks with or without EHT1864 administration. Scale bars, 200 µm. n = 6 per group. (D and E) Quantitative analysis of Prx1 in (C). ANOVA (F2,15 = 17.62; F2,15 = 71.39). (F) Photo of CXCL12Col2 mice at the age of 20 weeks with or without EHT1864 administration. Scale bar, 5 mm. (G and H) µCT images and quantitative analysis of pathological new bone formation in the spine of CXCL12Col2 PGIS mice at the age of 20 weeks with or without EHT1864 administration. Scale bars, 200 µm. n = 6 per group. ANOVA (F2,15 = 34.74). (I) H&E staining, TB staining, immunohistochemical staining, and immunofluorescence staining of Sca1 in spine of CXCL12Col2 PGIS mice at the age of 20 weeks with or without EHT1864 administration. Scale bars, 200 µm. n = 6 per group. (J and K) Quantitative analysis of Sca1 in (I). ANOVA (F2,15 = 29.55; F2,15 = 18.36). (L) Schematic diagram illustrating CXCL12-induced pathological new bone formation and recruitment of OPCs. Data are shown as means ± SEM. One-way ANOVA with Levene’s test, followed by the Tukey’s post hoc test was used.
Fig 2: CXCL12/CXCR4 induces migration of OPCs.(A and B) Immunofluorescence staining and quantitative analysis of PDGFRa in spinal tissues from patients with AS or non-AS. Scale bar, 100 µm. n = 6 tissues from patients with AS versus n = 8 tissues from non-AS patients. (C and D) H&E staining, TB staining, immunohistochemical staining, and quantitative analysis of Sca1 in spine of PGIS mice. Scale bar, 100 µm. n = 6 per group. (E and F) Immunofluorescence staining and quantitative analysis of Sca1 in spine of PGIS mice. Scale bar, 20 µm. n = 6 per group. (G and H) H&E staining, SOFG staining, immunohistochemical staining, and quantitative analysis of Prx1 in hind paws of male DBA/1 model at the age of 8 and 20 weeks. Scale bar, 200 µm. n = 6 per group. (I and J) Immunofluorescence staining and quantitative analysis of Prx1 in hind paws of male DBA/1 model. Scale bar, 20 µm. n = 6 per group. (K) Schematic diagram of the construction process of GFP-DBA/1 mice parabiosis. (L) H&E staining and immunofluorescence analysis of GFP in the skin tissues of GFP-DBA/1 mice. Scale bars, 500 µm. (M) H&E staining, immunofluorescence analysis of GFP and Sca1 in GFP-DBA/1 mice. Scale bars, 50 µm. n = 6 per group. (N and O) Immunofluorescence staining and quantitative analysis of GFP and CXCL12 in the plantar surface of hind paws of GFP-DBA/1 model at the age of 8 and 20 weeks. n = 6 per group. Scale bar, 100 µm. Data are shown as means ± SEM. Student’s t test with Shapiro-Wilk test was used. DAPI, 4',6-diamidino-2-phenylindole.
Fig 3: Locally delivered MSCs maintained their SCA-1 expression and increased SCA-1+ cell population during bone defect repair(A) Fluorescent microscopy showing cells with proliferative activity (KI67) and implanted cells (fNP); c, cortical bone; d, defect area. Scale bars, 80 µm; yellow dotted line, VOI.(B) XY (top) and XZ plane (middle) of zoomed-in regions enclosed by white dotted lines in (A), followed by 3D rendered surfaces (bottom). Scale bars, 5 µm; white dotted lines, fNP+ KI67+ cells.(C) Proportion of KI67+ cells in VOI from (A); *p < 0.05; n = 6 mice.(D) Distance distribution of fNP+ cells to KI67+ cells; region enclosed by red dotted line, fNP+ KI67+ cells (distance < 5 µm).(E and F) Proportion of fNP+ KI67+ cells over total fNP+ cells and total KI67+ cells, respectively, at each time point. n = 6 mice.(G) Fluorescent microscopy showing SCA-1+ cells and fNP+ implanted cells; c, cortical bone; d, defect area. Scale bars, 80 µm; yellow dotted lines, VOI; zoomed-in images of regions enclosed by white dotted line are shown to the right, followed by 3D rendered surfaces; white triangles, SCA-1+ fNP– cells; empty triangles, SCA-1+ fNP+ cells. Scale bars, 5 µm.(H) Co-expression analysis of SCA-1 and fNP. Scale bars, 80 µm.(I) Number of SCA-1+ cells in VOI from (G); *p < 0.05, ****p < 0.0001; n = 6.(J) Distance distribution of fNP+ cells to SCA-1+ cells; red dotted line, fNP+ SCA-1+ cells (distance < 5µm).(K) Proportion of fNP+ SCA-1+ cells over total fNP+ cells; ****p < 0.0001; n = 6.(L) Qualitative summary of implanted cells with stem cell marker (SCA-1) or osteogenic lineage cell marker (PRRX1, OSX) expression in defect area at each time point.
Fig 4: The CXCL12/CXCR4 axis mediates OPC migration through Rac1.(A and B) µCT images and quantitative analysis of pathological new bone formation in PGIS mice at the age of 32 weeks after EHT1864 administration. Scale bars, 500 µm. n = 6 per group. (C) H&E staining, TB staining, immunohistochemical staining, and immunofluorescence staining of Sca1 in PGIS mice. Scale bars, 100 µm. n = 6 per group. (D and E) Quantitative analysis of Sca1 in (C). (F) µCT images of pathological new bone formation in hind paw of male DBA/1 mice at the age of 32 weeks after EHT1864 administration. Scale bar, 50 µm. n = 6 per group. (G) H&E staining, SOFG staining, immunohistochemical staining, and immunofluorescence staining of Prx1 in plantar surface of hind paw of male DBA/1 mice at the age of 32 weeks after EHT1864 administration. Scale bars, 50 µm. n = 6 per group. (H) Quantitative analysis of pathological new bone formation in (F). (I and J) Quantitative analysis of Prx1 in (G). Data are shown as means ± SEM. Student’s t test with Shapiro-Wilk test was used. EHT1864, Rac1 inhibitor.
Fig 5: Inhibition of CXCL12/CXCR4 attenuates OPC migration and pathological new bone formation.(A and B) µCT images and quantitative analysis of pathological new bone formation in spine of PGIS mice. Scale bars, 500 µm. n = 6 per group. Veh., vehicle. (C) H&E staining, TB staining, immunohistochemical staining of Sca1 in spine of PGIS mice at the age of 32 weeks after AMD3100 administration. Scale bar, 200 µm. n = 6 per group. (D) Immunofluorescence staining of Sca1 and CXCL12 in PGIS mice. Scale bar, 20 µm. n = 6 per group. (E) Quantitative analysis of Sca1 in (C). (F) Quantitative analysis of Sca1 in (D). (G and H) µCT images and quantitative analysis of pathological new bone formation in hind paw of male DBA/1 model at the age of 32 weeks after AMD3100 administration. Scale bar, 500 µm. n = 6 per group. (I) H&E staining, SOFG staining, and immunohistochemical staining of Prx1 in hind paw of male DBA/1 model. Scale bar, 200 µm. n = 6 per group. (J) Immunofluorescence staining of Prx1 and CXCL12 in hind paw of male DBA/1 mice. Scale bar, 20 µm. n = 6 per group. (K) Quantitative analysis of Prx1 in (J). (L) Quantitative analysis of Prx1 in (I). (M and N) µCT images and quantitative analysis of pathological new bone formation in hind paw of DBA/1;CXCR4fl/fl mice and DBA/1;CXCR4prx1 mice. Scale bar, 500 µm. (O) H&E staining, SOFG staining, immunohistochemical staining of Prx1 in DBA/1;CXCR4fl/fl mice and DBA/1;CXCR4prx1 mice. Scale bar, 100 µm. n = 6 per group. (P) Immunofluorescence staining of Prx1 and CXCL12 in hind paw of DBA/1;CXCR4fl/fl mice and DBA/1;CXCR4prx1 mice. Scale bar, 20 µm. n = 6 per group. (Q) Quantitative analysis of Prx1 in (P). (R) Quantitative analysis of Prx1 in. (O) Data are shown as means ± SEM. Student’s t test with Shapiro-Wilk test was used. AMD3100, CXCR4 inhibitor.
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