Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

Abstract

BackgroundNonunion is a failure of healing following a bone fracture. Its physiopathology remains partially unclear and the discovery of new mediators could promote the understanding of bone healing.MethodsThirty-three atrophic nonunion (NU) patients that failed to demonstrate any radiographic improvement for 6 consecutive months were recruited for providing serum samples. Thirty-five healthy volunteers (HV) served as the control group. Proteomics studies were performed using SELDI-TOF–MS and 2D-DIGE approaches, associated or not with Proteominer® preprocessing, to highlight biomarkers specific to atrophic nonunion pathology. Peak intensities were analyzed by two statistical approaches, a nonparametric Mann–Whitney U tests (univariate approach) and a machine-learning algorithm called extra-trees (multivariate approach). Validation of highlighted biomarkers was performed by alternative approaches such as microfluidic LC–MS/MS, nephelometry, western blotting or ELISA assays.ResultsFrom the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF–MS using CM10 and IMAC-Cu2+ ProteinChip arrays, and 665 peaks were integrated for extra-trees multivariate analysis. Accordingly, seven biomarkers and several variants were identified as potential NU biomarkers. Their levels of expression were found to be down- or up-regulated in serum of HV vs NU. These biomarkers are inter-α-trypsin inhibitor H4, hepcidin, S100A8, S100A9, glycated hemoglobin β subunit, PACAP related peptide, complement C3 α-chain. 2D-DIGE experiment allowed to detect 14 biomarkers as being down- or up-regulated in serum of HV vs NU including a cleaved fragment of apolipoprotein A-IV, apolipoprotein E, complement C3 and C6. Several biomarkers such as hepcidin, complement C6, S100A9, apolipoprotein E, complement C3 and C4 were confirmed by an alternative approach as being up-regulated in serum of NU patients compared to HV controls.ConclusionTwo proteomics approaches were used to identify new biomarkers up- or down-regulated in the nonunion pathology, which are involved in bone turn-over, inflammation, innate immunity, glycation and lipid metabolisms. High expression of hepcidin or S100A8/S100A9 by myeloid cells and the presence of advanced glycation end products and complement factors could be the result of a longstanding inflammatory process. Blocking macrophage activation and/or TLR4 receptor could accelerate healing of fractured bone in at-risk patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1019-1) contains supplementary material, which is available to authorized users.

Knockout of TLR4 promotes fracture healing by activating Wnt/β-catenin signaling pathway

“…Inflammation is an important cause of delayed union or nonunion of fracture 21 . Previous studies have confirmed that TLR4 can mediate inflammatory response in bone 22 .…”

Maillard Proteomics: Opening New Pages

“…A surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS), relying on selective affinity-based retention of specific molecules on an affinity chip, integrated with MALDI target, represents a promising alternative to gel-based techniques [ 89 ], and is mostly used for analysis of low molecular weight proteins [ 90 ]. Recently, Nedic et al reported an application of SELDI to the analysis of serum fractions obtained by boronic acid- and lectin-affinity chromatography [ 91 ].…”

“…Recently, Nedic et al reported an application of SELDI to the analysis of serum fractions obtained by boronic acid- and lectin-affinity chromatography [ 91 ]. SELDI was also successfully used for characterization of glyoxal-derived modifications of bovine erythrocyte superoxide dismutase [ 92 ], for identification of inflammatory biomarkers, and for characterization of innate immunity in atrophic nonunion fracture [ 90 ]. The main advantage of this technique is high sensitivity, i.e., the ability to detect analytes, present in rather low concentrations [ 93 ].…”

Antimicrobial Peptide Combined with BMP2-Modified Mesenchymal Stem Cells Promotes Calvarial Repair in an Osteolytic Model

“…Thus, multiple regulatory mechanisms modulate the effectors of innate immunity to balance between host defense and inflammation. 11 Neutrophils are one of the most prominent effector cells of the innate immune system, 12 which are recruited to the site of infection and exert various antimicrobial effector mechanisms. 13 Antimicrobial peptides (AMPs) are effector molecules of the innate immune system with various activities.…”