Figure 4. Mechanical stimuli regularly and reversibly compromise the integrity of osteoblast cell membrane.

(A–F) Membrane permeability in micropipette-stimulated Fura2-loaded osteoblasts. (A) Top: Schematic of dye-leakage assay. Bottom: 340 ex/510 em before (a) and after (b) CB-OB stimulation (white outline). (B) Representative Fura2 traces in C2-OB with minor (mIn), intermediate (iIn) or severe (sIn) cell injury and relative occurrence frequency (right, n = 35–40 stimulated cells). (C) C2-OB [Ca²⁺]_i responses and differences (inset) for mIn (green), iIn (orange) and sIn (red), n = 8–14 stimulated cells. (D–F) Primary [Ca²⁺]_i response amplitudes (D) and decay constants (E), and secondary responsiveness (F) in CB-OB (black; n = 40 stimulated cells) or C2-OB (white; n = 35 stimulated cells), grouped by cell injury status. (G–K) Membrane permeability in tFSS-stimulated osteoblasts. (G) Top: schematic of dye-uptake assay. Bottom: CB-OB stained with TB (top) or R-dextran (bottom) prior to (0 s, left) or after (300 s, right) tFSS application (10x). Uptake of TB (H) or R-dextran (I) added prior to (0 s) or after (300 s) tFSS stimulation of CB-OB (n = 4 independent cultures). (J) Leakage of LDH from CB-OB 5 min after tFSS (n = 8 independent cultures, normalized to total LDH). (K) Viability of C2-OB 1 hr after tFSS assessed by alamarBlue (n = 8–16 independent cultures). (L) Uptake of TB added at indicated times after micropipette-stimulation of C2-OB (L-a, n = 4–23 stimulated cells) compared to relative frequencies of mIn, iIn and sIn assessed by Fura2-leakage assay (L-b). (M) Calculated minimum membrane lesion radius R_min required for permeability to LDH, R-dextran, TB, Fura2 and ATP. For Figure 4, means ± SEM, dashed lines: linear regression, *significance by ANOVA. Source data for Figure 4 is provided in Figure 4—source data 1.

Figure 4—figure supplement 1. Involvement of hemichannels in ATP release and membrane resealing in murine osteoblasts.

(A–B) To demonstrate the presence of functional GAP-junctions and validate GAP-junction/hemi channel blockers, C2-OB were pretreated (10 min) with vehicle (n = 22), Cbx (n = 11), FFA (n = 6) and Oct (n = 10) and scraped and stained with LY. Sample sizes are number of regions of interest imaged from three independent cultures. Fluorescence (A, top) and bright field (A, bottom) images show dye transfer and cell-cell coupling, respectively. Mean ± SEM percentage of cells that were not initially scraped but were coupled and LY-positive following LY staining, normalized to vehicle (B). (C–D) Fura2-loaded CB-OB pretreated with GAP-junction/hemi-channel blockers were mechanically stimulated with glass micropipette and [Ca²⁺]_i elevation amplitude (C, mean ± SEM amplitude, normalized to vehicle) and percentage of sIn cells (D, mean ± SEM percentage) was determined (n = 6–9 stimulated cells). (E–F) Membrane injury of CB-OB following tFSS (10x resuspensions) was assessed by T.B. uptake at 0 and 300 s (E, n = 5–8 separate cultures, means ± SEM), and ATP release ([ATP]_e) was measured using bioluminescence assay (F, n = 6 separate cultures, means ±SEM attomoles released per cell over 60 s after simulation) following pre-treatment with GAP-junction/hemichannel blockers. Comparisons to vehicle; *p<0.05, **p<0.01 and ***p<0.001, or as specified; ^#p<0.05, ^##p<0.01 and ^###p<0.001, indicate significance assessed by ANOVA followed by post-hoc Bonferroni test.