Myrielle Mathieu

Decreased pool of mesenchymal stem cells is associated with altered chemokines serum levels in atrophic nonunion fractures

Nonunion fractures can cause severe dysfunction and are often difficult to treat mainly due to a poor understanding of their physiopathology. Although many aspects of impaired fracture healing have been extensively studied, little is known about the cellular and molecular mechanisms leading to atrophic nonunion. Therefore, the aim of the present study was to assess the pools and biological functions of bone marrow-derived mesenchymal stem cells (hMSCs) and circulating endothelial progenitor cells (EPCs) in atrophic nonunion patients compared to healthy subjects, and the systemic levels of growth factors involved in the recruitment, proliferation and differentiation of these cells. In nonunions, the pool of hMSCs was decreased and their proliferation delayed. However, once committed, hMSCs from nonunions were able to proliferate, differentiate into osteoblastic cells and mineralize in vitro as efficiently as hMSCs from healthy subjects. In parallel, we found altered serum levels of chemokines and growth factors involved in the chemotaxis and proliferation of hMSCs such as leptin, interleukin-6 (IL-6) and its soluble receptor, platelet-derived growth factor-BB (PDGF-BB), stem cell factor (SCF) and insulin-like growth factor-1 (IGF-1). Moreover, we showed that the number of EPCs and their regulating growth factors were not affected in nonunion patients. If nonunion is generally attributed to a vascular defect, our results also support a role for a systemic mesenchymal and osteogenic cell pool defect that might be related to alterations in systemic levels of factors implicated in their chemotaxis and proliferation.

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

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.