Biomarkers of inflammation and innate immunity in atrophic nonunion fracture.
Nonunion 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.



Thirty-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.



From the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF-MS using CM10 and IMAC-Cu(2+) 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.



Two 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.
Laboratory of Rheumatology, Department of Rheumatology, GIGA Research, University of Liège, Tour GIGA, +2, CHU, 4000, Liège, Belgium. ddeseny@chu.ulg.ac.be.
Dominique
D
de Seny
Laboratory of Rheumatology, Department of Rheumatology, GIGA Research, University of Liège, Tour GIGA, +2, CHU, 4000, Liège, Belgium.|Laboratory for the Analysis of Medicines, Department of Pharmacy, CIRM, University of Liège, 4000, Liège, Belgium.
Gaël
G
Cobraiville
GIGA-Neurosciences, University of Liège, 4000, Liège, Belgium.
Pierre
P
Leprince
Laboratory for the Analysis of Medicines, Department of Pharmacy, CIRM, University of Liège, 4000, Liège, Belgium.
Marianne
M
Fillet
Laboratory of Rheumatology, Department of Rheumatology, GIGA Research, University of Liège, Tour GIGA, +2, CHU, 4000, Liège, Belgium.
Charlotte
C
Collin
Laboratory of Bone and Metabolic Biochemistry, Department of Rheumatology, Université Libre de Bruxelles (ULB), 1000, Brussels, Belgium.
Myrielle
M
Mathieu
Laboratory of Rheumatology, Department of Rheumatology, GIGA Research, University of Liège, Tour GIGA, +2, CHU, 4000, Liège, Belgium.
Jean-Philippe
JP
Hauzeur
Laboratory of Bone and Metabolic Biochemistry, Department of Rheumatology, Université Libre de Bruxelles (ULB), 1000, Brussels, Belgium.|Department of Rheumatology and Physical Medicine, Hôpital Erasme, Université Libre de Bruxelles (ULB), 1000, Brussels, Belgium.
Valérie
V
Gangji
Laboratory of Rheumatology, Department of Rheumatology, GIGA Research, University of Liège, Tour GIGA, +2, CHU, 4000, Liège, Belgium.
Michel G
MG
Malaise
D000293:Adolescent; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D015415:Biomarkers / Q000378:metabolism*; D016022:Case-Control Studies; D003710:Demography; D005260:Female; D005599:Fractures, Ununited / Q000276:immunology* / Q000473:pathology*; D064451:Hepcidins / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D007249:Inflammation / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D040901:Proteomics; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D057825:Two-Dimensional Difference Gel Electrophoresis; D055815:Young Adult
D015415:Biomarkers; D064451:Hepcidins
2D-DIGE; Apolipoprotein; Biomarkers; Complement; Hepcidin; Innate immunity; Nonunion; Proteomics; S100A8; SELDI