Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

PLoS One. 2016 Jul 21;11(7):e0159580. doi: 10.1371/journal.pone.0159580. eCollection 2016.

Abstract

Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis.

MeSH terms

  • Adsorption
  • Animals
  • Biomarkers / metabolism
  • Body Fluids / metabolism*
  • Bone and Bones / drug effects
  • Bone and Bones / pathology*
  • Chemokines / metabolism
  • Cytokines / metabolism*
  • Enzyme-Linked Immunosorbent Assay
  • Extracellular Matrix Proteins / metabolism
  • Fracture Healing / drug effects
  • Fractures, Bone / metabolism
  • Fractures, Bone / pathology
  • Hematoma / metabolism
  • Hematoma / pathology
  • Immunomodulation / drug effects
  • Intercellular Signaling Peptides and Proteins / metabolism
  • Male
  • Metabolome* / drug effects
  • Metabolomics
  • Microdialysis*
  • Principal Component Analysis
  • Protease Inhibitors / pharmacology
  • Proteomics
  • Rats, Wistar
  • Reproducibility of Results
  • Signal Transduction / drug effects
  • Soft Tissue Injuries / metabolism
  • Soft Tissue Injuries / pathology
  • Wounds and Injuries / metabolism*
  • Wounds and Injuries / pathology*

Substances

  • Biomarkers
  • Chemokines
  • Cytokines
  • Extracellular Matrix Proteins
  • Intercellular Signaling Peptides and Proteins
  • Protease Inhibitors

Grant support

YF, JS, and SK are thankful to the Deutsche Forschungsgemeinschaft (DFG), Transregio 67 for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.