Glucocorticoid‐induced osteoporosis development and scatter plots showing the de novo mineralization vs. the overall regeneration. Lepidotrichia fluorescence signal before (A) and after (B) the treatment with PN. Representation of fluorescence intensities for all the fish analyzed (C). Healthy fish regenerating in fish tank water only (D); fish with osteoporosis phenotype regenerating in fish tank water only (E); fish with osteoporosis phenotype regenerating in fish tank water containing ALN (F); all treatments plotted in the same graphic with linear regression analysis showing the strong relationship between the variables (G). The P1 and P2 (linear increase) regions are displayed in “D” separated by the dashed line. R² showed the strong and positive interaction between variables. Different capital letters indicate statistically significant difference (p < .01).

Scheme and SEM images of proximal and distal crushed bones extracted from zebrafish caudal fins before and after the proposed treatments, in ×20 k and ×100 k magnifications. (A) Processes performed prior to SEM analysis. Proximal crushed parts showed minerals with bigger size and well‐defined shapes, which was not commonly found in distal counterparts (white arrowheads), while the distal sites showed minerals resembling amorphous phases (yellow arrowheads). (B) The distal region of ALN_REGEN showed grouped small spherical‐like (light blue arrowheads) and plate‐like morphologies (red arrowheads).

Montage containing a representative zebrafish caudal fin bony ray and the Raman spectra obtained. (A) Representation of the 17th lepidotrichia (dorsal region of the fin) of the ALN_REGEN group isolated from the fluorescence images obtained in confocal microscope; five different spots of interest along the growth direction were measured (A1–5) and the respective Raman peaks were obtained for each region. (B) Heat color plots representing the total area below the curve for the phosphate peak of ~962 cm⁻¹; the black arrow indicates the bony ray represented in the Raman spectra shifts above. The legend box shows the total values of the areas divided by 10³. The darker the spots, the higher the mineral intensity of the signal.

Surface topography profile of zebrafish bony rays and roughness calculation. (A) Procedure steps from the resection of the zebrafish caudal fins, to the analysis with AFM in tapping‐mode. (B) 3D representation of the cross‐section slices (obtained by cryosection process) analyzed by AFM. (C) The distal part of CTRL_REGEN group showing collagen fibers bundles (white single arrows) in flattened representations. (D) Approximation of the collagen fiber bundle from the white square in “B”; the double arrows shows the diameter of a periodic unit from a fibril (d ~ 75 nm). (E) Box‐plots with means (red horizontal line) and median (black horizontal line) of the roughness obtained from proximal and distal parts of fins by AFM. Capital letters indicate significant statistically difference between the different regions (proximal and distal) within the same treatment (p < .01). Lowercase letters indicate significant statistically difference between the same region (proximal or distal) among different treatments (p < .01).

Mechanical performance of zebrafish caudal fin bony rays. (A) Procedure steps to obtain the caudal fin's slices for mechanical evaluation. Box‐plots with means (red horizontal line) and median (black horizontal line) of the E_r (B) and H (C) obtained from proximal and distal parts of fins with a triboindenter. Capital letters indicate significant statistically difference between the different regions (proximal and distal) within the same treatment (p < .01). Lowercase letters indicate significant statistically difference between the same region (proximal or distal) among different treatments (p < .01). The average values of the proximal parts of both E_r (D) and H (E) plotted against the Ca/P ratio. The regression lines show a weak but positive correlation between the variables.