Probiotics protect mice from CoCrMo particles-induced osteolysis

Abstract

Wear particle-induced inflammatory osteolysis is the primary cause of aseptic loosening, which is the most common reason for total hip arthroplasty (THA) failure in the med- and long term. Recent studies have suggested an important role of gut microbiota (GM) in modulating the host metabolism and immune system, leading to alterations in bone mass. Probiotic bacteria administered in adequate amounts can alter the composition of GM and confer health benefits to the host. Given the inflammatory osteolysis that occurs in wear debris-induced prosthesis loosening, we examined whether the probiotic Lactobacillus casei could reduce osteolysis in a mouse calvarial resorption model. In this study, L. casei markedly protected mice from CoCrMo particles (CoPs)-induced osteolysis. Osteoclast gene markers and the number of osteoclasts were significantly decreased in L. casei-treated mice. Probiotic treatment decreased the M1-like macrophage phenotype indicated by downregulation of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and inducible nitric oxide synthase (iNOS) and increased the M2-like macrophage phenotype indicated by upregulation of IL-4, IL-10 and arginase. Collectively, these results indicated that the L. casei treatment modulated the immune status and suppressed wear particle-induced osteolysis in vivo. Thus, probiotic treatment may represent a potential preventive and therapeutic approach to reduced wear debris-induced osteolysis.

Keywords: aseptic loosening; gut microbiota; inflammatory cytokines; macrophage polarization; nanotoxicity; wear particles.

Figure 1

Characterizations of CoPs. Notes: (A) Representative TEM images of CoPs. (B) CoPs size distribution. (A) Scale bar 100 nm. (B) Particles with sizes of 52.2±27.5 (mean ± SD). Abbreviations: CoPs, CoCrMo particles; TEM, transmission electron microscopy; SD, standard deviation.

Figure 2

Lactobacillus casei ameliorated CoPs-induced mouse calvarial osteolysis. Notes: (A) The 8-week-old mice were treated with either L. casei or vehicle preoperatively for 8 weeks (3 times/week) and postoperatively for 2 weeks (3 times/week). The animals were then sacrificed, and tissues were collected for later analysis. (B) Representative micro-CT with three-dimensional reconstructed images from each group. (C) BV/TV and the percentage of total porosity of each sample were measured. (D) Representative HE-stained images of calvaria from each group. (E) The erosion area (%) of each group was measured in (D). The ROI in (B) was indicated by the yellow square box. The data are presented as the mean ± SEM (n=5–7). *P<0.05. (D) Scale bar: 250 µm. Abbreviations: CoPs, CoCrMo particles; CT, computed tomography; BV/TV, bone volume/total volume; HE, hematoxylin and eosin; ROI, region of interest; SEM, standard error of the mean; L. casei, Lactobacillus casei.

Figure 3

Lactobacillus casei inhibited CoPs-induced osteoclast formation. Notes: (A) The levels of ALP and OCN mRNAs from each group were examined using real-time PCR. (B) The levels of TRAP, CTR, c-fos and cathepsin K mRNAs from each group were examined using real-time PCR. (C) Representative TRAP-stained images of calvaria from each group. Osteoclasts are indicated by arrows. (D) The number of TRAP-positive cells in each group was measured in (C). The data are presented as the mean ± SEM (n=5–7). *P<0.05. (C) Scale bar: 60 µm. Abbreviations: CoPs, CoCrMo particles; ALP, alkaline phosphatase; OCN, osteocalcin; mRNA, messenger RNA; PCR, polymerase chain reaction; TRAP, tartrate-resistant acidic phosphatase; CTR, calcitonin receptor; SEM, standard error of the mean; ns, not significant; L. casei, Lactobacillus casei.

Figure 4

Lactobacillus casei did not affect RANKL and OPG expression in calvaria. Notes: The levels of (A) RANKL, (B) OPG and (C) RANKL/OPG from each group were examined by ELISA assays. The data are presented as the mean ± SEM (n=5–7). Abbreviations: RANKL, receptor activator of nuclear factor (NF)-κB ligand; OPG, osteoprotegerin; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean; CoPs, CoCrMo particles; ns, not significant; L. casei, Lactobacillus casei.

Figure 5

Lactobacillus casei decreased the levels of M1 markers and enhanced the levels of M2 markers. Notes: (A) M1 polarization was inhibited in the L. casei-treated group. Expression levels of the M1 signature gene (iNOS) were measured by real-time PCR, and cytokine concentrations (TNF-α and IL-6) in the culture supernatants were measured by ELISA. (B) M2 polarization was enhanced in the L. casei-treated group. Expression levels of the M2 signature gene (arginase) were measured by real-time PCR, and cytokine concentrations (IL-4 and IL-10) in the culture supernatants were measured by ELISA. The data are presented as the mean ± SEM (n=5–7). *P<0.05. Abbreviations: iNOS, inducible nitric oxide synthase; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor; IL, interleukin; SEM, standard error of the mean; CoPs, CoCrMo particles; L. casei, Lactobacillus casei.