Ibudilast Mitigates Delayed Bone Healing Caused by Lipopolysaccharide by Altering Osteoblast and Osteoclast Activity

Abstract

Bacterial infection in orthopedic surgery is challenging because cell wall components released after bactericidal treatment can alter osteoblast and osteoclast activity and impair fracture stability. However, the precise effects and mechanisms whereby cell wall components impair bone healing are unclear. In this study, we characterized the effects of lipopolysaccharide (LPS) on bone healing and osteoclast and osteoblast activity in vitro and in vivo and evaluated the effects of ibudilast, an antagonist of toll-like receptor 4 (TLR4), on LPS-induced changes. In particular, micro-computed tomography was used to reconstruct femoral morphology and analyze callus bone content in a femoral defect mouse model. In the sham-treated group, significant bone bridge and cancellous bone formation were observed after surgery, however, LPS treatment delayed bone bridge and cancellous bone formation. LPS inhibited osteogenic factor-induced MC3T3-E1 cell differentiation, alkaline phosphatase (ALP) levels, calcium deposition, and osteopontin secretion and increased the activity of osteoclast-associated molecules, including cathepsin K and tartrate-resistant acid phosphatase in vitro. Finally, ibudilast blocked the LPS-induced inhibition of osteoblast activation and activation of osteoclast in vitro and attenuated LPS-induced delayed callus bone formation in vivo. Our results provide a basis for the development of a novel strategy for the treatment of bone infection.

Keywords: bone bridge; bone healing; callus bone; femoral defect; ibudilast; lipopolysaccharide; osteoblast; osteoclast.

Figure 1

Lipopolysaccharides (LPS) delayed new bone formation and bone healing in mice with femoral bone defects. (A) Schematic representation of the experimental setup. (B) Micro-computed tomography (microCT) three-dimensional image of the bone defect site show that low-dose LPS delays bone healing and high-dose LPS promotes bone fracture in the bone defect area. (C) Representative transverse images of the fractured femur by microCT. (D) LPS decreases the trabecular number and thickness. (E) MT stained histological sections demonstrate LPS inhibits bone bridge formation during bone healing. (F) LPS impairs body weight recovery. (G) LPS increases the serum levels of CTX1 and osteocalcin, an indicator of the bone turnover rate. Data are presented as the mean ± standard error of the mean. Analyses were conducted with two-way analysis of variance followed by Bonferroni’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LPS, lipopolysaccharide; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; MT, Masson’s trichrome stain; CTX1, C-terminal telopeptides of type I collagen; #, full fracture. Sham, n = 5; Low-LPS, n = 7; and High-LPS, n = 5.

Figure 2

LPS inhibits osteoblastic cell differentiation in vitro. (A,B) LPS inhibits both ALP (alkaline phosphatase) staining and ALP concentrations during osteoblast differentiation. (C,D) LPS inhibits calcium deposits during osteoblast differentiation. (E) LPS decreased OPN production during osteoblast differentiation. Data are presented as the mean ± standard error of the mean. Analyses were conducted with two-way analysis of variance followed by Bonferroni’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: OS, osteogenic factor; ALP, alkaline phosphatase; OPN, osteopontin.

Figure 3

LPS enhances osteoclastic cell differentiation in vitro. (A–C) LPS not only increases cathepsin K expression but also increases the number of cathepsin K-positive cells during osteoclast differentiation. (D,E) LPS increases both the expression intensity of TRAP and the percentage of TRAP-positive cells during osteoclast differentiation. Data are presented as the mean ± standard error of the mean. Analyses were conducted with two-way analysis of variance followed by Bonferroni’s post-hoc test. ** p < 0.01. Abbreviations: TRAP, tartrate-resistant acid phosphatase; LPS, lipopolysaccharides; OC, osteoclasts; C, vehicle control.

Figure 4

Ibudilast not only reverses the LPS-induced decrease in ALP production but also prevents the LPS-induced increase in osteoclast activation. (A–C) Ibudilast reversed the LPS-induced inhibition of alkaline phosphatase and bone calcium parameters in vitro. (D,E) Ibudilast prevented the LPS-induced increase in TRAP-positive osteoclast percentage in vitro. Data are presented as the mean ± standard error of the mean. Analyses were conducted with two-way analysis of variance followed by Bonferroni’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: OS, osteogenic factor; ALP, alkaline phosphatase.

Figure 5

Ibudilast prevents LPS-induced decreases in trabecular number in vivo. (A) Schematic representation of the experimental setup. (B,C) MicroCT three-dimensional image and transverse images of the bone defect site show that Ibudilast reversed the LPS-induced decrease in trabecular number in vivo. (D) Ibudilast not only promoted bone bridge formation but also promoted mature osteocyte formation. Data are presented as the mean ± standard error of the mean. Analyses were conducted with two-way analysis of variance followed by Bonferroni’s post-hoc test. * p < 0.05, ** p < 0.01. Abbreviations: LPS, lipopolysaccharide; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation. Sham, n = 5; LPS, n = 7; and LPS + Ibudilast, n = 5.