Microdialysis in critical size bone defects � quantitative assessment of cytokines and proteins in the early stage of fracture repair

Abstract

Event Abstract Back to Event Microdialysis in critical size bone defects – quantitative assessment of cytokines and proteins in the early stage of fracture repair Yvonne Förster1, Johannes Schmidt2, Dirk K. Wissenbach3, Susanne E. Pfeiffer3, Martin Von Bergen2, 3, Stefan Kalkhof2 and Stefan Rammelt1 1 University Hospital "Carl Gustav Carus" at TU Dresden, UniversityCenter of Orthopedics and Trauma Surgery, Germany 2 Helmholtz-Centre for Environmental Research-UFZ, Department of Proteomics, Germany 3 Helmholtz-Centre for Environmental Research-UFZ, Department of Metabolomics, Germany Fracture repair is a highly complex process influenced by growth factors, cytokines and other mediators. The early stages of the bone healing are of specific interest as they are essential for later consolidation but still poorly understood. From model systems we know about the significance of single proteins, but knowledge about the processes directly within the bone defect is limited. This is also caused by a lack of methods that allow the sampling of growth factors, cytokines and metabolites in situ. Microdialysis is a promising approach to obtain time-resolved data of specific growth factors and cytokines directly from the fracture site in vivo. When combined with a comprehensive analysis of the proteins adsorbed at the catheter tip, complementary data can be gathered from the wound site of the same defect. A 5 mm defect was created in the femur of 12 adult male Wistar rats and stabilized with an internal fixator. The microdialysis catheter was placed within the defect and the wound fluid was collected continuously under anesthesia for 24 h. Twelve rats with only a soft tissue wounds without bone defects were included as controls. Samples were collected in 3 h intervals and used to quantify cytokines and growth factors by ELISA. At the end of experiment the microdialysis catheters were explanted, the proteins adsorbed to the membrane of the microdialysis catheter were extracted and were analyzed after separation by HPLC-MS/MS. The functional annotation clustering was performed using the software packages PANTHER and DAVID.[1],[2] Analysis of the wound fluid by ELISA resulted in concentration profiles for 9 different cytokines and growth factors. Detailed analysis of Interleukin-6 (IL-6) showed that the concentration rapidly increased after surgery and declined to baseline after 24 h (Fig. 1A). In contrast, the concentration of the potentially relevant chemokine CXCL7 ((C-X-C) motif ligand 7) showed a rapid decrease immediately after surgery suggesting the release of the protein from activated platelets (Fig. 1B). More than 500 proteins were identified at the membrane of the catheter. Among them 222 proteins were detected in at least 3 of 4 replicates in both groups indicating a good reliability of the method. We could detect 78 proteins that occurred either exclusively (31) or with significantly increased abundance (47) within bone defect. For the control group, only 11 proteins were exclusive or significant increased compared to the group with a bone defect (Fig. 2). The combination of microdialysis with proteomics and metabolomics is a promising approach to obtain a detailed and direct insight into the local extracellular microenvironment of a bone defect. With these methods it is possible to obtain comprehensive and time-resolved information from the early stages of fracture repair. These data are of potential use for the development of new biomaterials that are designed to enhance bone repair and regeneration of critical size defects. Fig 1: A) Protein concentration of IL-6 in the wound fluid from bone defect (n=6) and soft tissue wound (n=8). B) Protein concentration of CXCL7 in the wound fluid from bone defect (n= 4) and soft tissue wound (n=4). Fig. 2: Distribution of the identified proteins at the membrane of the catheter. For each defect scenario 4 catheters were analyzed. This study was supported by grants from Deutsche Forschungsgemeinschaft Transregio 67 (subprojects B1, B5, and Z4). The authors thank Ute Hempel, Carolin Preissler, Sven Baumann, and Jacqueline Kober for technical assistance.