Article
Identification and characterization of potential new biomarker for pseudarthrosis
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Authors
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Stefanie Kern
- Experimental Trauma Surgery, Justus Liebig University Gießen, Gießen, Germany
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Deeksha Malhan
- Experimental Trauma Surgery, Justus Liebig University Gießen, Gießen, Germany
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Felix Schulze
- Department of Pediatrics, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany
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Angela Rösen-Wolff
- Department of Pediatrics, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany
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Sabine Schulz
- Institute of Inorganic and Analytical Chemistry, Justus-Liebig University Gießen, Gießen, Germany
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Bernhard Spengler
- Institute of Inorganic and Analytical Chemistry, Justus-Liebig University Gießen, Gießen, Germany
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Thaqif El Khassawna
- Experimental Trauma Surgery, Justus Liebig University Gießen, Gießen, Germany
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Christian Heiß
- Department of Trauma, Hand and Reconstructive Surgery, University Hospital of Giessen-Marburg GmbH, Gießen, Germany
| Published: | November 6, 2018 |
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Outline
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Objectives: Bone regeneration after a fracture is an important subject amongst clinicians, researchers and scientists. In large defects, mineralized fibrocartiliganous callus leads to pseudarthrosis, a type of a non-union. Non-unions may occur in approximately 10% of all fractures and in up to 50% of open fractures of the tibia. Clinically a revision of non-unions is required and in many cases osteoblast growth factors like bone morphogenetic proteins (BMP) are applied. However, fibroblasts located abundantly in the site of non-union respond to the same growth factors as osteoblasts and differentiate to osteoblast-like cells. Decreasing of neurofibromin in pseudarthrosis encourages the examination of healing in a Nf1 knock-out mouse model.
Research Question: Protein-level differences between osteoblast-like fibroblasts and osteoblasts result pseudarthrosis. Analysis of mesenchymal stem cell (MSCs) and fibroblast response to BMP stimulation can assist in understanding pseudarthrosis. Moreover, a cellular marker for non-union prediction shall be identified and established in order to assist in treatment strategy.
Methods: MSCs and fibroblasts were derived from human reaming debris and divided to BMP stimulated and controls. LC-MS/MS based label free quantitative (LFQ) proteomics was applied to analyse these cells. Therefore, cells were homogenized; proteins were then enzymatically digested, purified and analyzed using nanoHPLC-MS/MS. In addition, relative gene expression was carried out using semi-quantitative real-time polymerase chain reaction (qPCR). Pseudarthrotic tissue sections were stained with Movat's pentachrome for histological analysis. Immunohistochemical (IHC) analysis was performed on patient pseudarthrotic tissue and on Nf1 knock-out mouse model samples with closed femoral fracture at day 10 to visualize potential biomarker. Statistical analysis was run in IBM SPSS software Version 24.
Results: 2470 proteins were found in total of which 193 proteins were found in all biological and technical replicates. Out of these 193 proteins, 10 were found to show relations with pseudarthrosis and NF1 related genes. Out of these 10, collagen alpha-2(I) chain, calmodulin and glycine-tRNA ligase (GARS) were found to be significantly down-regulated under osteogenic differentiation conditions. RT-PCR showed the same results. Immunostaining visualized calmodulin and GARS on patients' pseudarthrotic tissue and Nf1 KO-mice samples.
Discussion and Conclusion: Three proteins were found to be promising biomarkers for pseudarthrosis. Further histomorphometric analysis of IHC stained tissues are being carried out. Future studies will include analysis of those three proteins in human pseudarthrosis samples by mass spectrometric approach in order to validate their status as biomarker.
