Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: In vitro validation of D-Bmp2@M osteogenic efficacy and inhibition of ectopic ossification. a. Schematic diagram of the osteoblast-bone Transwell model. Bmp2/D-Bmp2@M microspheres or free Bmp2/D-Bmp2 were loaded in the upper chambers, MC3T3-E1 cells were cultured on two coverslips (one of which was precoated with HA) in the lower compartments, and the medium was refreshed every day for 7 or 14 days. Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed at days 7 and 14, respectively. b. Osteogenic differentiation staining: ALP (early-stage, day 7) and ARS (late-stage, day 14) staining. Scale bar: 200 μm. c. ALP activity was quantitatively analyzed using an ALP kit (n = 3 per group). d. Relative quantitative analysis of ARS staining was performed at an OD of 562 nm (n = 3 per group). e. qPCR analysis of Bmp2 signaling-related mRNA in MC3T3-E1 cells (n = 3 per group). f. Schematic diagram of the muscle-bone Transwell model. Bovine bone slices were co-incubated with Bmp2/D-Bmp2@M or free Bmp2/D-Bmp2 in the upper chambers, and C2C12 cells were cultured in the lower chambers and the medium was refreshed every day for 7 days. D-Bmp2 and Bmp2 retention on bone slices and ALP staining of C2C12 cells were analyzed on day 7. g. Representative fluorescence imaging of bone slices incubated with AF647-conjugated anti-Flag antibodies (above) (yellow arrows: bone slice) and C2C12 ALP staining images (below), scale bar: 200 μm. h. AF647-conjugated anti-Flag antibody fluorescence intensity quantification in bone slices (n = 3 per group). i. Quantification of ALP activity in C2C12 cells (n = 3 per group). j. qPCR analysis of Bmp2 signaling-related mRNA in C2C12 cells (n = 3 per group). The data are presented as the means ± SDs. One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Calcium nodules were stained with Alizarin Red S staining kit (Beyotime, China) at room temperature for 15 min, and staining conditions were monitored.

Techniques: In Vitro, Biomarker Discovery, Inhibition, Cell Culture, Staining, Activity Assay, Incubation, Fluorescence, Imaging

Journal: JBMR Plus

Article Title: Follistatin-like protein 1 (FSTL1) modulates bone remodeling and attenuates bone loss in a mouse model of postmenopausal osteoporosis

doi: 10.1093/jbmrpl/ziag037

Figure Lengend Snippet: Fstl1 enhances the osteogenic differentiation potential of MC3T3-E1 cells. (A) Representative alkaline-phosphatase (ALP) staining at 7 d. (B) Quantitative ALP activity. (C) Representative Alizarin Red S (ARS) staining at 14 d. (D) Quantification of ARS absorbance (OD 562 ). (E-H) Relative mRNA expression of Runx2, Sp7, Ocn, and Opn at 14 d determined by RT-qPCR. (I) Western-blot bands for RUNX2 and SP7. (J and K) Densitometric ratios of RUNX2/GAPDH and SP7/GAPDH. Sample sizes: con ( n = 3), OE-Fstl1 ( n = 3), and sh-Fstl1 ( n = 3). Data are presented as mean ± SD; inter-group comparisons were performed using 1-way ANOVA. * p < .05, ** p < .01, *** p < .001, **** p < .0001; ns, not significant.

Article Snippet: On day 21, ARS staining was carried out with an Alizarin Red S calcium-staining kit (Beyotime Biotechnology): after fixation, working solution was applied, and the cells were incubated for 30 min at room temperature in the dark, and the reaction was terminated once red mineralized nodules became visible.

Techniques: Staining, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot