Zebrafish caudal fin as a model to investigate the role of probiotics in bone regeneration

Abstract

Probiotics are live microorganisms that confer several beneficial effects to the host, including enhancement of bone mineralization. However, probiotic action on bone regeneration is not well studied and therefore we analysed various effects of probiotic treatment on the caudal fin regeneration of zebrafish. Morphological analysis revealed an increased regenerated area with shorter and thicker lepidotrichia segments after probiotic treatment. Fourier transform infrared spectroscopy imaging analysis highlighted the distribution of phosphate groups in the regenerated fins and probiotic group showed higher amounts of well-crystallized hydroxyapatite. At the midpoint (5 days post amputation) of regeneration, probiotics were able to modulate various stages of osteoblast differentiation as confirmed by the upregulation of some key marker genes such as runx2b, sp7, col10a1a, spp1 and bglap, besides suppressing osteoclast activity as evidenced from the downregulation of ctsk. Probiotics also caused an enhanced cell cycle by regulating the expression of genes involved in Retinoic acid (rarga, cyp26b1) and Wnt/β-catenin (ctnnb1, ccnd1, axin2, sost) signaling pathways, and also modulated phosphate homeostasis by increasing the entpd5a levels. These findings provide new outlooks for the use of probiotics as a prophylactic treatment in accelerating bone regeneration and improving skeletal health in both aquaculture and biomedical fields.

Figure 1

(a) Representative photographs showing the fins before amputation, post amputation and at 1, 5 and 10 DPA in control (C) (n = 7) and probiotic-treated (P) (n = 7) groups (Scale bar = 2,000 µm); (b) Picture of a representative amputated fin, showing the regenerated area (REG, green dotted tracing), the stump width or width of the amputation plane (STU, red dotted line), the fin ray width (RAY, pink line), the segment length (SEG, yellow line) and the peduncle width (PED, blue dotted line); (c) Statistical analysis of regeneration rate (expressed as %) between C and P groups calculated at 10 DPA with respect to the fin before amputation of the same fish. (d–g) Various morphometric parameters used to analyze the regenerated areas in C and P fins (n = 7 per group per time point) (d) Regenerated area (REG)/Peduncle width (PED) ratio; (e) Regenerated area (REG)/Stump width (STU) ratio; (f) Mean ray width (RAY) and (g) Mean segment length (SEG). T-test was used to analyze the differences among the groups. % Values were converted to respective decimal values before applying T-test and statistical significance was set at p < 0.05.

Figure 2

(a) Representative picture showing the areas analyzed by FTIRI in the fourth bifurcated fin ray of the dorsal lobe of the regenerating fin. The two regions assayed after amputation were: proximal-corresponding to the first formed bifurcation after amputation and distal-corresponding to the last segment joint preceding the actinotrichia; (b and c) Representative microphotograph (left) and false color images (right) denoting the topographical distribution of phosphate groups in the proximal and distal areas of the regenerated fin from C and P fins (n = 3) at (b) 5 DPA and (c) 10 DPA time points. The same color scale (0–3) was used for all false color images: white/light pink colors indicate the areas with the highest amount of phosphates, red/orange/yellow indicate the areas with an intermediate amount and black/dark blue shows the areas with the lowest amount of phosphates. The black dotted squares indicate the region where spectra were extracted for the curve fitting analysis; (d–f) Biochemical composition and bone mineralization assessed by FTIRI analysis.  Histograms representing band area ratios (d) A₁₆₅₅/A₁₀₂₄, (e) A₁₂₄₀/A₁₀₂₄ and (f) A₁₀₂₄/A₁₀₉₀ are calculated on proximal and distal regions of the regenerating fin from C (n = 3) and P (n = 3) fins at 5 DPA and 10 DPA. Data are presented as mean ± S.D. Different letters over the histograms indicate statistically significant difference among groups. Two-way ANOVA and Tukey’s multiple comparison test are used, and statistical significance was set at p < 0.05.

Figure 3

runx2b, sp7, col10a1a, bglap and spp1 mRNA values normalized against rplp0 and rpl13 in fins collected from C (n = 3) and P (n = 3) groups at 0, 5 and 10 DPA. Data are presented as mean ± S.D. Different letters over the histograms indicate statistically significant difference among groups. Two-way ANOVA and Tukey’s multiple comparison test are used, and statistical significance was set at p < 0.05.

Figure 4

rarga, cyp26b1 and entpd5a mRNA values normalized against rplp0 and rpl13 in fins collected from C (n = 3) and P (n = 3) groups at 0, 5 and 10 DPA. Data are presented as mean ± S.D. Different letters over histograms indicate statistically significant difference among groups. Two-way ANOVA and Tukey’s multiple comparison test are used, and statistical significance was set at p < 0.05.

Figure 5

ctnnb1, axin2, sparc, sost, ctsk and ccnd1 mRNA values normalized against rplp0 and rpl13 in fins collected from C (n = 3) and P (n = 3) groups at 0, 5 and 10 DPA. Data are presented as mean ± S.D. Different letters over histograms indicate statistically significant difference among groups. Two-way ANOVA and Tukey’s multiple comparison test are used, and statistical significance was set at p < 0.05.