Novel protein biomarkers for advanced pseudarthrosis treatment revealed by mass spectrometry

Abstract

Fracture healing is a complex physiological process, requiring long and complicated treatment. Delayed healing and pseudarthrosis occur in 5-10% of all fractures. In surgically challenging pseudarthrosis revisions, osteoinductive growth factors are often applied locally to stimulate the differentiation of mesenchymal stromal cells (MSC) into bone-forming osteoblasts, thereby accelerating fracture healing. However, fibroblasts are also stimulated by those growth factors. Particularly in the case of large-area defects, fibrous tissue can lead to pronounced pseudarthroses. Aim of the present study was therefore to find biomarkers that help to identify when the application of growth factors leads to increased fibroblast differentiation and thus worsens the course of bone healing. Biomarkers would allow early detection pseudoarthrosis development and thus a more targeted treatment. In addition to identifying such biomarkers, this work also used retrospective studies to identify patient-related risk factors that influence the type of pseudoarthrosis. Differences in the proteomes of MSCs and fibroblasts before and after osteogenic stimulation were analyzed with mass spectrometry. Liquid chromatography-nano-electrospray ionization-mass spectrometry (LC-nano-ESI-MS) based quantitative proteomics, integrated with network analysis, was applied. Proteome analysis revealed three potential biomarkers, significantly down-regulated (p<0.05) in osteogenic stimulated fibroblasts compared to osteogenic stimulated MSCs. The promising proteins are alpha-2 type I collagen (COL1A2), calmodulin-1 (CALM1), and glycine tRNA synthetase (GARS). Corresponding gene network analysis showed connection of CALM1 and GARS with genes related to bone diseases and associated with pseudarthrosis formation. In the next steps, the successfully identified biomarkers must be tested further for their diagnostic potential and their role in fracture healing. Thus, future studies should include human serum analysis. A correlation between biomarkers discrepancies in the tissue and their serum level concentration could provide additional information about healing status of surgically revised pseudarthrosis. In the second part of this study, two retrospective clinical studies were conducted to gain insight into systemic risk factors for pseudarthrosis. The results of this work show the importance of retrospective clinical studies to assess effectiveness of current surgical and pharmacological treatment approaches. Evaluation of individual patient characteristics can enable tailored and better pseudarthrosis treatment.