The influence of calcitonin gene-related peptide on markers of bone metabolism in MG-63 osteoblast-like cells co-cultured with THP-1 macrophage-like cells under virtually osteolytic conditions.

BACKGROUND: The neuropeptide calcitonin gene-related peptide (CGRP) has been described to have an inhibitory effect on endotoxin- and wear particle-induced inflammation in the early stages of periprosthetic osteolysis. In the present study, the crosstalk between immune cells and osteoblasts in osteolytic conditions treated with CGRP has been analyzed to evaluate whether the anti-inflammatory properties of the peptide also have a beneficial, i.e. an anti-resorptive and osteo-anabolic impact on bone metabolism. METHODS: MG-63 osteoblast-like cells were co-cultured with THP-1 macrophage-like cells stimulated with either ultra-high molecular weight polyethylene (UHMWPE) particles or different concentrations of bacterial lipopolysaccharides (LPS) and simultaneously treated with CGRP. Inflammation was monitored in terms of measuring the levels of tumor necrosis factor (TNF)-α secretion. Furthermore, the production of the osteoblast markers osteoprotegerin (OPG), receptor activator of nuclear factor κB ligand (RANKL), alkaline phosphatase (ALP) and osteopontin (OPN) was quantified. Also, ALP enzymatic activity was measured. RESULTS: Stimulation of co-cultured THP-1 macrophages with either high levels of LPS or UHMWPE induced the secretion of TNF-α which could be inhibited by CGRP to a great extent. However, no remarkable changes in the OPG/RANKL ratio or bone ALP activity were observed. Interestingly, OPN was exclusively produced by THP-1 cells, thus acting as a marker of inflammation. In addition, TNF-α production in THP-1 single cell cultures was found to be considerably higher than in co-cultured cells. CONCLUSIONS: In the co-culture system used in the present study, no obvious relation between inflammation, its mitigation by CGRP, and the modulation of bone metabolism became evident. Nonetheless, the results suggest that during the onset of periprosthetic osteolysis the focus might lie on the modulation of inflammatory reactions. Possibly, implant-related inflammation might merely have an impact on osteoclast differentiation rather than on the regulation of osteoblast activity.

Calcitonin gene-related peptide modulates the production of pro-inflammatory cytokines associated with periprosthetic osteolysis by THP-1 macrophage-like cells.

OBJECTIVE: An anti-resorptive impact of the neuropeptide calcitonin gene-related peptide (CGRP) on periprosthetic osteolysis, the leading cause of early prosthesis loosening, has been shown previously. In this study, the impact of CGRP on pro-inflammatory cytokine production associated with periprosthetic osteolysis was analysed using THP-1 macrophage-like cells. METHODS: Cells were stimulated with ultra-high-molecular-weight polyethylene (UHMWPE) particles (cell-to-particle ratios of 1:100 and 1:500) and lipopolysaccharides (LPS; 1 µg/ml) to establish osteolytic conditions, and simultaneously treated with CGRP (10(-8)M). Receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and tumour necrosis factor (TNF)-α mRNA expression were measured by quantitative RT-PCR. RANK protein was detected by Western blot. Secreted protein levels of TNF-α as well as interleukin (IL)-1ß and IL-6 were quantified in cell culture supernatants by ELISA and Bio-Plex cytokine assay, respectively. RESULTS: Activation of macrophage-like cells failed to enhance the production of RANK but led to a dose- and time-dependent increase of TNF-α mRNA and secreted protein levels of TNF-α, IL-1ß and IL-6. Application of CGRP time-dependently suppressed TNF-α mRNA expression induced by low-particle concentrations and LPS, while both particle- and LPS-induced secretion of TNF-α was inhibited. A pronounced inhibitory effect of CGRP on LPS-induced cytokine production at 24 h of incubation was also observed with IL-1ß and IL-6. CONCLUSIONS: CGRP shows a time-dependent inhibitory effect on the secretion of osteolysis-associated pro-inflammatory cytokines, indicating an indirect anti-resorptive influence of the neuropeptide on both aseptic prosthesis loosening and bacterially induced bone resorption which might enhance the life time of total joint replacements.

A single intraperitoneal injection of bovine fetuin-A attenuates bone resorption in a murine calvarial model of particle-induced osteolysis.

Particle-induced osteolysis, which by definition is an aseptic inflammatory reaction to implant-derived wear debris eventually leading to local bone destruction, remains the major reason for long-term failure of orthopedic endoprostheses. Fetuin-A, a 66kDa glycoprotein with diverse functions, is found to be enriched in bone. Besides being an important inhibitor of ectopic calcification, it has been described to influence the production of mediators of inflammation. Furthermore, a regulatory role in bone metabolism has been assigned. In the present study, the influence of a single dose of bovine fetuin-A, intraperitoneally injected in mice subjected to particle-induced osteolysis of the calvaria, was analyzed. Twenty-eight male C57BL/6 mice, twelve weeks of age, were randomly divided into four groups. Groups 2 and 4 were subjected to ultra-high molecular weight polyethylene (UHMWPE) particles placed on their calvariae while groups 1 and 3 were sham-operated. Furthermore, groups 3 and 4 received a single intraperitoneal injection of 20mg bovine fetuin-A while groups 1 and 2 were treated with physiologic saline. After 14days calvarial bone was qualitatively and quantitatively assessed using microcomputed tomography (µCT) and histomorphometrical approaches. Application of fetuin-A led to a reduction of particle-induced osteolysis in terms of visible osteolytic lesions and eroded bone surface. The reduction of bone thickness and bone volume, as elicited by UHMWPE, was alleviated by fetuin-A. In conclusion, fetuin-A was found to exert an anti-resorptive effect on particle-induced osteolysis in-vivo. Thus, fetuin-A could play a potentially osteoprotective role in the treatment of bone metabolic disorders.