Elsevier

Biomedicine & Pharmacotherapy

Volume 103, July 2018, Pages 588-597
Biomedicine & Pharmacotherapy

Bacitracin promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by stimulating the bone morphogenetic protein-2/Smad axis

https://doi.org/10.1016/j.biopha.2018.04.084Get rights and content

Abstract

Bacitracin, a widely used metallopeptide antibiotic, has been reported to be locally used in treating wounds without systemic adverse reactions. Our preliminary study showed that bacitracin might enhance the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). The present study investigated whether bacitracin affects the osteogenic differentiation of HBMSCs and the molecular mechanisms involved. The proliferation of HBMSCs in the presence of bacitracin was examined using a cell counting kit-8 (CCK-8) assay. The effects of bacitracin on the cell cycle and apoptosis of HBMSCs were observed using flow cytometry assay. Staining and quantitative assays for alkaline phosphatase (ALP) staining, collagen deposition (Sirius Red), and mineralization (Alizarin Red) were used to study osteogenic differentiation of HBMSCs. The expression of osteogenic differentiation markers was detected using quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses. The activation of related signaling pathways was examined using a luciferase reporter assay and western blotting. Bacitracin treatment increased osteogenic differentiation of HBMSCs without cytotoxicity and did not adversely affect cell cycle progression or apoptosis. The luciferase reporter assay showed that bacitracin activated the transcription of bone morphogenetic protein-2 (BMP2) gene, a key gene in the BMP2/Smad signaling axis. Western blotting indicated that this axis was markedly activated by bacitracin stimulation of osteogenesis. Moreover, the activation of Smad phosphorylation and osteogenic differentiation by bacitracin was inhibited by a transforming growth factor (TGF)-β/Smad inhibitor (LDN-193189 HCl) and small interfering RNA (siRNA) gene silencing (si-BMP2). In conclusion, our results suggest that bacitracin can promote osteogenesis of HBMSCs by activating the BMP2/Smad signaling axis.

Introduction

Infection is a common complication in orthopedics and often occurs in numerous types of orthopedic surgery. Bone infection can lead to a sequence of serious consequences such as protracted infected lesions, necrotic bone formation, loosened implants, and failed surgery, which require further treatments [1]. In addition to surgical removal of the infected tissue, use of antibiotics in bone-related infections is of vital importance [2]. Antibiotics are used intravenously to treat soft tissue or deep infections and may be impregnated into cement preparations or loaded into beads that are placed directly into the wound. As the infection is gradually controlled and the necrotic bone tissue is absorbed, the resulting osteolysis from the infection needs to be resolved [3]. However, common antibiotics currently used in the clinical treatment of bone-related infections, including β-lactams, cephalosporins, aminoglycosides, macrolides, and quinolones, do not effectively promote bone regeneration and differentiation. Gentamicin, an antibiotic commonly used for the clinical treatment of bone-related infections, inhibits the proliferation and viability of osteoblasts [4], while vancomycin inhibits osteoblast proliferation at certain concentrations [5]. It is important to find a drug that exhibits both antibacterial and osteogenic effects.

Human bone marrow mesenchymal stem cells (HBMSCs) are bone marrow-derived cells that play a key role in the renewal and regeneration of osteoblasts. HBMSCs can differentiate into bone-forming osteoblasts and have been shown to be a primary source of osteoprogenitor cells [[6], [7], [8]]. Moreover, HBMSCs can be used as bone graft materials to treat bone defects [9]. HBMSCs have been shown to differentiate into osteoblasts by treatment with dexamethasone, ascorbic acid, and β-glycerophosphate [10,11]. After a local infection is cleared, HBMSCs are activated and differentiate into osteoblasts to complete the repair of local bone dissolution. Failure of HBMSCs to completely repair the local bone defects caused by infection may lead to local osteoporosis and even pathological fractures [3]. Currently, drugs that function as bone absorption inhibitors by suppressing osteoclast activity are used for osteoporosis treatment [12], including vitamin D analogs and calcitonin [13]. However, these drugs cannot promote osteogenic differentiation and do not possess the necessary antibacterial properties to be used to treat orthopedic infection.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β superfamily that play a significant regulatory role in bone formation and are potent osteoblastic differentiation factors [14,15]. Among the members of the BMP subfamily, BMP-2 induces bone formation and differentiation in vivo and in vitro [16]. BMP-2 activates Smad1/5/9 proteins and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinases (JNKs) [[17], [18], [19], [20]]. Following activation, these signal transduction molecules increase the expression of Runt-related transcription factor 2 (Runx2) and core-binding factor alpha 1 (Cbfa1) [[21], [22], [23]].

Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and B. licheniformis. It strongly inhibits gram-positive bacteria and has a significant antagonistic effect on the development of Staphylococcus aureus resistance [24]. Bacitracin possesses a thiazoline ring formed by condensation of the Ile-1 carboxylate and contains a unique cyclic heptapeptide structure formed via an amide linkage between the side chain of Lys-6 and the C-terminus of Asn-12. This particular structure might be associated with the ability of bacitracin to resist protease degradation [25]. Partial use of bacitracin in wound treatments could reduce adverse systemic reactions such as renal dysfunction, which is the most common complication of bacitracin use [26]. In our preliminary study, we found that bacitracin also enhanced the osteogenic differentiation of HBMSCs. Thus, its excellent bioactivity and multi-functional properties suggest that bacitracin might be a suitable agent for the treatment of local bone infection and osteolysis.

Considering the important role of the BMP2/Smad signaling axis in osteoblast differentiation and bone formation, we hypothesized that this signaling axis mediates the characteristic effects of bacitracin in promoting the osteogenic differentiation of HBMSCs. To verify this hypothesis in the present study, we investigated the effects of different concentrations of bacitracin on the osteogenic differentiation of HBMSCs and the degree of activation of BMP2/Smad signaling.

Section snippets

Reagents

Bacitracin, dexamethasone, ascorbic acid, β-glycerophosphate, 16-alkyl pyridine, and sodium phosphate were purchased from Sigma-Aldrich (MO, USA). Dexamethasone was dissolved in dimethyl sulfoxide (DMSO), while ascorbic acid and β-glycerophosphate were dissolved in phosphate-buffered saline (PBS). Fetal bovine serum (FBS) and trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA, 0.5%) were purchased from Gibco (USA). Alpha-modified Eagle medium (α-MEM) and penicillin/streptomycin solution were

Effects of bacitracin on HBMSCs proliferation, cell cycle, and apoptosis

To investigate the potential cytotoxicity of bacitracin, its effects on the proliferation of HBMSCs were tested using the CCK-8 assay. As shown in Fig. 1B, bacitracin was not cytotoxic at concentrations up to 100 μM after 1, 3, and 5 days of incubation. However, 500 μM bacitracin inhibited cell proliferation. The effects of bacitracin on the cell cycle were determined using PI staining and assessed with a flow cytometer. Compared with that of the control group, bacitracin at concentrations

Discussion

Bone infection and osteolysis caused by osteomyelitis are common clinical orthopedic complications that require the use of systemic and topical antibiotics [1,3]. However, antibiotics commonly used to control infections clinically do not effectively promote new bone formation and even inhibit the local osteogenic microenvironment. Several studies have shown that antibiotics, including gentamicin and vancomycin, inhibit osteoblast proliferation and osteogenic differentiation [4,5]. As a commonly

Conclusion

In conclusion, in HBMSCs, bacitracin at concentrations up to 10 μM, promoted osteogenic differentiation in a concentration-dependent manner. Bacitracin at concentrations of 100 μM also promoted osteogenic differentiation, although the effect was lower than that at 10 μM. Furthermore, the BMP2/Smad signaling axis might play a crucial role in this process. This research study indicates that bacitracin may be a useful agent for the prevention and treatment of local bone infection and related

Competing interests

The authors declare no competing interests linked to this study.

Authors’ contributions

Hui Li, Bin’en Nie, Zhe Du, Shutao Zhang and Teng Long carried out the experiments. Hui Li wrote the manuscript. Bing Yue and Teng Long designed the experiments. Bing Yue revised the manuscript. All authors reviewed the manuscript.

Acknowledgements

This research was supported by National Natural Science Foundation of China (81472119 and 81672196) and Shanghai municipal education commission-Gaofeng clinical medicine grant support (20161423).

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