The Selective Serotonin Reuptake Inhibitor Fluoxetine Directly Inhibits Osteoblast Differentiation and Mineralization During Fracture Healing in Mice

Abstract

Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to osteoporosis. In this study, we investigated the effect of chronic SSRI use on fracture healing in two murine models of bone regeneration. First, we performed a comprehensive analysis of endochondral bone healing in a femur fracture model. C57/BL6 mice treated with fluoxetine, the most commonly prescribed SSRI, developed a normal cartilaginous soft-callus at 14 days after fracture and demonstrated a significantly smaller and biomechanically weaker bony hard-callus at 28 days. In order to further dissect the mechanism that resulted in a smaller bony regenerate, we used an intramembranous model of bone healing and revealed that fluoxetine treatment resulted in a significantly smaller bony callus at 7 and 14 days postinjury. In order to test whether the smaller bony regenerate following fluoxetine treatment was caused by an inhibition of osteogenic differentiation and/or mineralization, we employed in vitro experiments, which established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. However, histologic and μCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus. In conclusion, fluoxetine exerts a direct, inhibitory effect on osteoblast differentiation and mineralization, shown in two disparate murine models of bone repair. Discontinuation of the drug did not result in restoration of the healing potential, but rather led to complete arrest of the repair process. Besides the well-established effect of SSRIs on bone homeostasis, our study provides strong evidence that fluoxetine use negatively impacts fracture healing. © 2017 American Society for Bone and Mineral Research.

Keywords: BONE; DEPRESSION; OSTEOBLAST; OSTEOPOROSIS; OSTEOPROGENITOR CELL.

Fig. 1

Fluoxetine inhibits osteogenic phase of endochondral ossification. (A–D) Pentachrome staining of representative longitudinal sections through fracture callus. Insets in C and D show high magnification of aniline blue staining of the callus region demonstrating difference in trabecular architecture. (E) Histomorphometry of cartilage volume at day 14 and (F) bony callus at day 14 and 28 (n = 4, p < 0.05). (G–J) 3D reconstruction of µCT analysis of the fracture callus at 14 and 28 days (n = 4). (K–N) Analysis of µCT data showing BV/TV, Tb.N, Tb.Th, and Tb.Sp. (O) Maximum and minimum bending moment of inertia (I_max and I_min, respectively), as well as the polar moment of inertia, J, of at POD 28 (n = 4, p < 0.01). (P) Four-point bending analysis of uninjured and injured femurs (POD 28) (n = 4). Asterisk denotes statistical significance. Scale bar = 2 mm, 100 µm in insets. d = day; POD = postoperative day; BV/TV = bone volume/total volume; Tb.N = trabecular number; Tb.Th = trabecular thickness; Tb.Sp = trabecular spacing.

Fig. 2

Decreased callus volume of femur fractures in fluoxetine treated mice is not a result of increased osteoclast number. (A–D) TRAP staining of the fracture callus at 14 and 28 days after fracture. (E) Histomorphometric quantification of activated, bone lining osteoclasts (n = 4). Scale bar = 2 mm. c = cortical bone; TRAP = tartrate-resistant acid phosphatase.

Fig. 3

Fluoxetine treatment disrupts intramembranous bone formation and results in smaller bony regenerate. (A–D) Aniline blue staining of representative longitudinal sections through monocortical defect sites. (E) Histomorphometric analysis of bony regenerate size (n = 4, p = 0.04 [POD7], p < 0.001 [POD14]). (F–I) 3D reconstruction of µCT analysis of the monocortical defect site at 7 and 14 days. (J–L) Analysis of µCT data showing BV/TV, Tb.N, Tb.Th, and Tb.Sp (n = 4, p < 0.05). Asterisk denotes statistical significance. Scale bar = 500 µm. d = day; POD = postoperative day; BV/TV = bone volume/total volume; Tb.N = trabecular number; Tb.Th = trabecular thickness; Tb.Sp = trabecular spacing.

Fig. 4

Proliferation, osteogenic differentiation, and mineralization is decreased in fluoxetine treated mice, while osteoclast activity remains unchanged. (A, B) Alizarin red (red) and calcein (green) labeling of fracture callus (outer dotted line) at POD 28. Inserts show representative high-magnification images used for quantification (arrow depicts ossification front). (C) Dynamic histomorphometry quantifying the mineral apposition rate of control and fluoxetine treated mice (n = 3, p < 0.05). (D, E) mRNA and protein expression of 5HTR2B is upregulated in response to injury at d7, while fluoxetine treatment resulted in downregulation (n = 3). (F, G) Immunohistochemistry for PCNA revealing decreased proliferation in fluoxetine-treated animals. (H) Quantification of PCNA-positive cells within the injury site (n = 4, p < 0.001). (I, J) Runx2 immunolabeling at POD 7 revealed an abundance of runx2-positive cells within the periosteum and the injury site of control animals, whereas the fluoxetine-treated animals exhibited fewer runx2-positive cells. (K) This finding was confirmed by Western blot analysis, which showed a significant decrease in Runx2 protein levels within the injury site of fluoxetine-treated animals compared to control animals. Osteocalcin levels were unchanged among groups. (L–O) Representative longitudinal section stained for TRAP in fluoxetine-treated and control animals at 7 and 14 days postsurgery. Quantification of activated osteoclasts reveals no significant differences between the two groups. Asterisk denotes statistical significance (p ≤ 0.05). Scale bar = 300 µm (A, B), 20 µm (A, B, insets), 100 µm (F, G, I, J), 500 µm (L–O). bm = bone marrow; c = callus; cb = cortical bone; d = day; oc = osteocalcin; PCNA = proliferating cell nuclear antigen; POD = postoperative day; 5HTR2B = serotonin receptor 2B.

Fig. 5

Fluoxetine inhibits osteogenic differentiation of bone marrow stromal cell in vitro. (A) TUNEL assay of BMSCs in GM with varying concentrations of fluoxetine. (B) Proliferation assay using BrdU labeling of BMSC in growth media with varying concentrations of fluoxetine. (C) Alizarin red staining was quantified using absorbance, representative images of the mineralization assay are shown below. (D) ALP activity of BMSCs in OM and varying concentrations of fluoxetine. (E) Gene expression of col 1, oc, and ALP in BMSC cultured in OM and fluoxetine. Asterisk denotes statistical significance (p < 0.05), statistical comparison against growth/osteogenic media control. alp = alkaline phosphatase; BrdU = Bromodeoxyuridine; col 1 = collagen type 1; f = fluoxetine; GM = growth media; oc = osteocalcin; OM = osteogenic differentiation media; BMSC = bone marrow stromal cell.

Fig. 6

Inhibitory effect of fluoxetine on osteogenic differentiation in vitro is serotonin-independent. (A) Proliferation assay, (B) alkaline phosphatase assay, and (C) gene expression of collagen type 1 of BMSCs in serotonin-free media with supplementation of serotonin and varying doses of fluoxetine as outlined in the tables below the figures. Asterisk denotes statistical significance (p < 0.05), statistical comparison against media control. F = fluoxetine; ser = serotonin; BMSC = bone marrow stromal cell.

Fig. 7

Discontinuation of fluoxetine treatment results in bony non-union in femoral fracture model. (A) Schematic representing the three experimental groups. (B–H) µCT analyses of femur fractures in control, fluoxetine treated, and fluoxetine-discontinued animals showing prolonged effect of SSRI on bone healing and incomplete fracture healing with callus gap (n = 4, p < 0.05). (I–K) Pentachrome staining of representative section showing fibrous non-union formation (dotted line in K, O, S, W) in the drug cessation group. (L) Histomorphometric evaluation of the callus reveals persistent cartilage at POD 28 in the fluoxetine-discontinued group (n = 4, p < 0.05). (M–P) Aniline blue staining demonstrating bony regenerate size in the three experimental groups. Histomorphometry shows mild increase in bone formation, but still significantly below the control (n = 4, p < 0.05). (Q–T) TRAP staining and analysis confirmed that the phenotype is not caused by an increase in osteoclastic resorption (n = 4, p < 0.04). (U, V) Alkaline phosphatase staining indicating ongoing osteogenic differentiation, with increased staining at the ossification front within the persistent fracture gap (W, dotted line). Scale bar = 2 mm. Asterisk denotes statistical significance (p < 0.05). bm = bone marrow; BV/TV = bone volume/total volume; c = cortical bone; ca = callus; d = day; mu = muscle; POD = postoperative day; Tb.N = trabecular number; Tb.Sp = trabecular spacing; Tb.Th = trabecular thickness.