Tranexamic Acid Promotes Murine Bone Marrow-Derived Osteoblast Proliferation and Inhibits Osteoclast Formation In Vitro

Abstract

Despite modern surgical trauma care, bleeding contributes to one-third of trauma-related death. A significant improvement was obtained through the introduction of tranexamic acid (TXA), which today is widely used in emergency and elective orthopedic surgery to control bleeding. However, concerns remain regarding potential adverse effects on bone turnover and regeneration. Therefore, we employed standardized cell culture systems including primary osteoblasts, osteoclasts, and macrophages to evaluate potential effects of TXA on murine bone cells. While osteoblasts derived from calvarial digestion were not affected, TXA increased cell proliferation and matrix mineralization in bone marrow-derived osteoblasts. Short-term TXA treatment (6 h) failed to alter the expression of osteoblast markers; however, long-term TXA stimulation (10 days) was associated with the increased expression of genes involved in osteoblast differentiation and extracellular matrix synthesis. Similarly, whereas short-term TXA treatment did not affect gene expression in terminally differentiated osteoclasts, long-term TXA stimulation resulted in the potent inhibition of osteoclastogenesis. Finally, in bone marrow-derived macrophages activated with LPS, simultaneous TXA treatment led to a reduced expression of inflammatory cytokines and chemokines. Collectively, our study demonstrates a differential action of TXA on bone cells including osteoanabolic, anti-resorptive, and anti-inflammatory effects in vitro which suggests novel treatment applications.

Keywords: bone regeneration; macrophages; osteoblast; osteoclast; tranexamic acid.

Figure 1

TXA does not affect the differentiation or function of calvaria-derived osteoblasts. (a) Representative alizarin red stainings of calvaria-derived osteoblasts from WT mice differentiated in the presence of indicated concentrations of TXA for 10 days in osteogenic medium. Scale bar 10 mm. The quantification of extracellular matrix mineralization is indicated below. (b) qRT-PCR expression analysis for the indicated genes in calvaria-derived osteoblasts at day 10 of osteogenic differentiation, stimulated with TXA (1 mg/mL) during the entire course of cell differentiation. (c) qRT-PCR expression analysis for the indicated genes in the same samples. (d) MTT proliferation assay of calvaria-derived osteoblasts stimulated with 1 mg/mL TXA for 6 h at the indicated days of differentiation. For (a–d), n = 4–6 independent cultures per group were used, as indicated by individual data points. Data presented are means ± SD. Gene abbreviations: runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), alpha-1 type I collagen (Col1a1), osteocalcin (Bglap), sclerostin (Sost), Rankl (Tnfsf11), osteoprotegerin (Tnfrsf11b).

Figure 2

TXA promotes extracellular matrix mineralization. (a) Representative alizarin red stainings of bone marrow-derived osteoblasts from WT mice differentiated in the presence of indicated concentrations of TXA for 10 days in osteogenic medium. Scale bar 10 mm. The quantification of extracellular matrix mineralization is indicated below. (b) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoblasts at day 10 of osteogenic differentiation, stimulated with TXA (1 mg/mL) during the entire course of cell differentiation. (c,d) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoblasts at day 2 (c) and day 10 (d) of osteogenic differentiation, stimulated with TXA for 6 h at the indicated concentrations after serum starvation overnight. (e) qRT-PCR expression analysis for the indicated genes in the same cells at day 2 or (f) day 10 of differentiation. For (a–f), n = 3–4 independent cultures per group were used. Data presented are means ± SD. Gene abbreviations: runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), alpha-1 type I collagen (Col1a1), osteocalcin (Bglap), sclerostin (Sost), Rankl (Tnfsf11), osteoprotegerin (Tnfrsf11b).

Figure 3

TXA enhances cell proliferation of bone marrow-derived osteoblasts. MTT proliferation assay of bone marrow-derived osteoblasts stimulated with 1 mg/mL TXA for 6 h at the indicated time points; n = 3–5 independent cultures per group were used. Data presented are means ± SD.

Figure 4

Inhibition of early osteoclastogenesis through TXA. (a) TRAP activity staining of WT bone marrow cells at day 7 of differentiation, cultured in the presence of M-CSF and RANKL and continuous exposure to vehicle or TXA at the indicated concentrations. Scale bars = 50 μm. The quantification of osteoclast numbers per viewing field is depicted on the left (Ocl.N./VF). (b) TRAP activity staining of WT bone marrow cells at day 7 of differentiation, cultured in the presence of M-CSF and RANKL, and exposure to vehicle or TXA at the indicated concentrations only at day 1 of osteoclast differentiation. The quantification of osteoclast numbers per viewing field is depicted on the left (Ocl.N./VF). (c) TRAP activity staining of WT osteoclasts at day 7 of differentiation, cultured in the presence of M-CSF and RANKL and exposed to vehicle or TXA at the indicated concentrations for 24 h. (d) MTT proliferation assay of WT osteoclasts at day 7 of differentiation stimulated with the indicated concentrations of TXA for 6 h. (e) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoclasts at day 7, stimulated with TXA (1 mg/mL) during the entire course of cell differentiation. (f) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoclasts at the indicated stages of cell differentiation, stimulated with TXA (1 mg/mL) for 6 h. (g) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoclasts at day 5 of differentiation, stimulated with TXA (1 mg/mL) during the entire course of cell differentiation. For (a–g), n = 3–4 independent cultures per group were used. Data presented are means ± SD. Gene abbreviations: chloride channel 7 (Clcn7), nuclear factor kappa B subunit 1 (Nfkb1), receptor activator of nf-κb (Tnfrsf11a), tartrate-resistant acid phosphatase (Acp5), cathepsin K (Ctsk).

Figure 5

Modulation of cytokine responses through TXA in resting and activated macrophages. (a) qRT-PCR expression analysis for the indicated genes in bone marrow-derived macrophages at day 7 of differentiation, stimulated with TXA (1 mg/mL) for 6 h. (b) qRT-PCR expression analysis for the indicated genes in bone marrow-derived macrophages at day 7 of differentiation, activated with 1 μg/mL LPS and simultaneously co-stimulated with TXA (1 mg/mL) for 6 h. (c) MTT proliferation assay of bone marrow-derived macrophages stimulated with the indicated concentrations of TXA. (d) Representative images of macrophage migration assays using Ibidi chambers of the indicated groups and time points. Scale bars = 50 μm. Blue lines indicate spreading cell fronts. The quantification of cell migration is shown on the right. For (a–d), n = 3–5 independent macrophage cultures per group were used as indicated with individual data points. Data presented are means ± SD. Gene abbreviations: interleukin-1 alpha (Il1a), interleukin-1 beta (Il1b), interleukin-4 (Il4), interleukin-6 (Il6), interleukin-10 (Il10), tumor necrosis factor alpha (Tnfa), CC-chemokine ligand 5 (Ccl5), cluster of differentiation 14 (Cd14), interleukin-1 receptor antagonist (Il1ra), inducible NO synthase (iNos), toll-like receptor 4 (Tlr4), transforming growth factor beta (Tgfb).