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.
Highlights
Nonunion is a failure of healing following a bone fracture
Four experimental conditions were used in this study: (1) crude serum on CM10 arrays; (2) crude serum on IMAC-Cu2+ arrays; (3) Proteominer® eluate on CM10 and (4) Proteominer® eluate on IMAC-Cu2+ arrays
For the 35 healthy volunteers (HV) and 33 NU, crude serum samples and Proteominer® eluates were run in duplicate. 136 spectra were collected on CM10 and IMAC-Cu2+ ProteinChip arrays for each of the four experimental conditions: (1) crude serum on CM10 arrays; (2) crude serum on IMAC-Cu2+ arrays; (3) Proteominer® eluate on CM10 and (4) Proteominer® eluate on IMAC-Cu2+ arrays (Additional file 2: Appendix Figure 2)
Summary
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. Two main forms of radiographic nonunion exist: atrophic nonunion for which there is no evidence of callus formation after 6 months of fracture and hypertrophic de Seny et al J Transl Med (2016) 14:258 nonunion for which there is abnormal callus formation. Atrophic nonunion result from a failure of normal cellular response necessary for bone reconstruction [1]. Several studies have demonstrated that the pool of bone marrow-derived mesenchymal stem cells (hMSCs) in nonunions was decreased and their proliferation delayed [1,2,3]. Hypertrophic nonunion would rather appear following instability at the site of fracture. Angiogenesis is necessary to promote revascularization of the injured site. New vessels enhance mobilization of osteoprogenitor cells and of growth factors involved in osteogenesis
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