Ciliotherapy Treatments to Enhance Biochemically- and Biophysically-Induced Mesenchymal Stem Cell Osteogenesis: A Comparison Study

Abstract

Introduction: New approaches to treat osteoporosis have focused on promoting bone formation through the targeting of osteoblasts and their progenitors, mesenchymal stem cells (MSCs). The primary cilium is a singular cellular extension known to play an important role in biochemical and biophysical osteogenic induction of MSCs. Defects in ciliary structure have been associated with a plethora of diseases. Therefore targeting the cilium therapeutically (ciliotherapies) has emerged as a potential new treatment modality. Therefore, this study performed a comparison analysis on known ciliotherapies and their potential effects in mediating MSC osteogenic differentiation.

Methods: MSCs were treated with forskolin, lithium chloride (LiCl) or fenoldopam to investigate the effect on ciliogenesis and cilia-associated signalling. Moreover, both early and long term biochemical and biophysical (fluid shear) induced osteogenic differentiation was examined in terms of osteogenic gene expression and bone matrix deposition following each treatment.

Results: LiCl and fenoldopam were found to enhance MSC ciliogenesis to a similar degree. LiCl significantly altered hedgehog (HH) and Wnt signalling which was associated with inhibited osteogenic gene expression, while fenoldopam demonstrated enhanced early osteogenesis. Long term treatment with both ciliotherapies did not enhance osteogenesis, however LiCl had detrimental effects on cell viability. Intriguingly both ciliotherapies enhanced MSC mechanosensitivity as demonstrated by augmented osteogenic gene expression in response to fluid shear, which over longer durations resulted in enhanced matrix deposition per cell.

Conclusions: Therefore, ciliotherapies can be utilised to enhance MSC ciliogenesis resulting in enhanced mechanosensitivity, however, only fenoldopam is a viable ciliotherapeutic option to enhance MSC osteogenesis.

Keywords: Bone; Hedgehog; Mechanobiology; Oscillatory fluid shear; Primary cilium; Wnt.

Figure 1

Representative images of MSCs following treatment with (a) forskolin or vehicle, DMSO, (b) lithium chloride (LiCl) or vehicle, H₂O and (c) fenoldopam or vehicle, DMSO, for 1 and 24 h at 100 × fluorescence (red channel—acetylated alpha tubulin, green channel—pericentrin and DAPI) (scale bar 10 µm), inset shows zoom on cilium (scale bar 1 µm). (d, f, h) Percentage of cells in each group with a primary cilium N = 3, n = 194–275. Bars illustrate % ciliated cells, mean ± SEM, statistical analysis, two-way ANOVA, ⁺p < 0.05, ⁺⁺p < 0.01 effect of time in culture, ^&p < 0.05 different effect of treatment at different time points, *p < 0.05 significant difference between treatments in the 1-h time point group. (e, g, i) Length of cilium following each treatment, N = 3, n = 194–275. Box plot illustrates 25th and 75th percentile values, whiskers represent 5th and 95th percentile values, line at median, black dots represent points outside of the 5th and 95th percentile, statistical analysis, one-way ANOVA with Bonferroni post-test, *p < 0.05, *p < 0.01, ***p < 0.001, significant difference between labelled groups.

Figure 2

Effect of biochemical primary cilium elongation on hedgehog and Wnt pathway activity. (a, b) Ptch1 and Gli1 expression following 24 h of (a) 100 mM LiCl or (b) 50 µM fenoldopam treatment, N = 3, n = 7–10. Bars illustrate mean ± SEM, NS, not significant, statistical analysis, unpaired two tailed t-test. (c, f) Representative images of immunofluorescent staining of β-catenin in MSCs at 100 × fluorescence (red channel—β-catenin, green channel—phalloidin and DAPI) (scale bar 10 µm) (d, g) fold change intensity of β- catenin in the nucleus of MSCs (N = 3, n = 36–37), (e, h) Axin2 expression (N = 2, n = 3–6) following 24 h of (c–e) 100 mM LiCl and (f–h) 50 µM fenoldopam treatment, bars illustrate mean ± SEM, NS, not significant, statistical analysis, unpaired two tailed t-test.

Figure 3

Effect of biochemical primary cilium elongation on osteogenic gene expression. Cox2, Opn, Runx2 and Dlx5 expression following 24 h of (a) 100 mM LiCl or (b) 50 µM fenoldopam treatment, N = 3, n = 4–6. Bars illustrate mean ± SEM, NS, not significant, *p < 0.05, statistical analysis, unpaired two tailed t-test.

Figure 4

Osteogenic marker expression following 2 h of 2 Pa 2 Hz OFF in each group. (a–d) Lithium chloride or vehicle, H₂O and (e–h) fenoldopam or vehicle, DMSO represented as Mean ± SEM, N = 3, n = 5–12. No flow data is reproduced from Fig. 3 as control. Statistical analysis, 2 way ANOVA with Bonferroni post hoc test; ^$$p < 0.01, ^$$$p < 0.001 effect of flow on gene expression, ^#p < 0.05, ^###p < 0.001 effect of treatment on gene expression, *p < 0.05, **p < 0.01 effect of flow within individual treatment groups.

Figure 5

Quantification of DNA content following treatment with (a) 100 mM LiCl or vehicle, H₂O and (b) 50 µM fenoldopam or vehicle, DMSO for 21 days with full osteogenic supplements under static conditions. N = 1, n = 5–6, Mean ± SEM, *p < 0.05, Student’s t-test with Welch’s correction. (c, d, g, h) Representative images of extracellular matrix staining (c, d) picrosirius and (g, h) alizarin red, following treatment with (c, g) LiCl or vehicle, H₂O and (d, h) fenoldopam or vehicle, DMSO after 21 days culture in full osteogenic supplements, scale = 500 and 100 µm in the inset. (e, f, i, j) Quantification of matrix staining (e, i) total collagen and collagen normalized to DNA content and (f, j) total calcium and calcium normalized to DNA content from each group, Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

(a) Quantification of DNA content following treatment with LiCl or vehicle, H₂O for 21 days cultured in either static, no flow, or mechanically stimulated, flow. (b, c) Representative images of extracellular matrix staining (b) picrosirius, (c) alizarin red staining, following treatment, scale = 500 µm and 100 µm in the inset. (d–g) Quantification of matrix staining (d, e) total collagen and calcium concentration extracted from each group. (f, g) Extracellular matrix normalized to DNA quantification (f) collagen and (g) calcium, two-way ANOVA, ^$p < 0.05, effect of flow on collagen and calcium concentration. ^###p < 0.001 effect of treatment, *p < 0.05, ***p < 0.001 Bonferroni post test, effect of flow on individual treatment groups, ^&p < 0.05, ^&&p < 0.01 difference between the effect of flow in each treatment.

Figure 7

(a) Quantification of DNA content following treatment with fenoldopam or vehicle, DMSO for 21 days cultured in either static, no flow, or mechanically stimulated, flow. (b, c) Representative images of extracellular matrix staining (b) picrosirius, (c) alizarin red staining, following treatment, scale = 500 µm and 100 µm in the inset. (d–g) Quantification of matrix staining (d, e) total collagen and calcium concentration extracted from each group. (f, g) Extracellular matrix normalized to DNA quantification (f) collagen and (g) calcium, two-way ANOVA, ^$p < 0.05, effect of flow on collagen and calcium concentration. ^###p < 0.001 effect of treatment, *p < 0.05, ***p < 0.001 Bonferroni post test, effect of flow on individual treatment groups, ^&p < 0.05, ^&&p < 0.01 difference between the effect of flow in each treatment.