gms | German Medical Science

German Congress of Orthopedic and Trauma Surgery (DKOU 2018)

23.10. - 26.10.2018, Berlin

Tolloid proteinases and their regulation in a blunt force trauma model

Meeting Abstract

  • presenting/speaker Daniel Kronenberg - Department of Regenerative Musculoskeletal Medicine, Institute of Musculoskeletal Medicine (IMM), Westfaelische Wilhelms-University, Muenster, Germany, Münster, Germany
  • Jens Everding - Department of Trauma, Hand and Reconstructive Surgery, University Hospital Muenster, Germany, Münster, Germany
  • Catherine Moail - Tissue Biology and Therapeutic Engineering Unit, UMR 5305 CNRS - University of Lyon 1, Lyon, France, Lyon, France
  • Sandrine Vadon-Le Goff - Tissue Biology and Therapeutic Engineering Unit, UMR 5305 CNRS - University of Lyon 1, Lyon, France, Lyon, France
  • Melanie Timmen - Department of Regenerative Musculoskeletal Medicine, Institute of Musculoskeletal Medicine (IMM), Westfaelische Wilhelms-University, Muenster, Germany, Münster, Germany
  • Richard Stange - Department of Regenerative Musculoskeletal Medicine, Institute of Musculoskeletal Medicine (IMM), Westfaelische Wilhelms-University, Muenster, Germany, Münster, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2018). Berlin, 23.-26.10.2018. Düsseldorf: German Medical Science GMS Publishing House; 2018. DocPT20-189

doi: 10.3205/18dkou708, urn:nbn:de:0183-18dkou7088

Published: November 6, 2018

© 2018 Kronenberg et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.


Outline

Text

Objectives: Tolloid proteinases and its best known member BMP-1 are enzymes responsible for activating many components of the extracellular matrix. Fibrillar Collagens are secreted as a proprotein, of whom the C-terminal propeptide is cut off by BMP-1, thereby leading to a reduction of solubility and the spontaneous formation of collagen fibrils. During fracture repair, the synthesis of fibrillary collagens in great quantities occurs at two occasions. Firstly during the formation of a cartilaginous callus, in which collagen type II is mostly involved and later on when remodelling the callus to a mineralized matrix which organic part consists mostly out of collagen type I. In this study, we have investigated the role of tolloid proteinases in the early stage of fracture healing. For this purpose, we applied recombinant BMP-1 and several of its regulators in a closed fracture model. As inhibitor of Tolloid proteinases we used the sizzled protein expressed recombindant from the clawed frog xenopus. We also analyzed the effect of procollagen C-proteinase enhancer (PCPE-1) which can increase the procollagen processing activity of tolloid proteinases.

Methods: After anesthesia the left femur was fractured with a 3-point bending machine and stabilized with an intramedullary screw (RIS systems, Davos, Switzerland). Tolloid proteinase, sizzled or PCPE-1 were injected directly into the fracture gap during the operation, whereas saline was used in the negative control group. The mice were sacrificed after 7-28 days and the femurs were dissected. The fractured femurs were scanned at a resolution of 9 μm with the μCT. A segmentation of the newly formed mineralized tissue was performed and quantified. Paraffin thin slices were prepared and evaluated by staining with either Azan staining for collagenous areas or alcian blue for cartilage. Overall, mineralized and cartilaginous callus area were determined by histomorphometry.

Results and conclusion: Histological quantification revealed more mineralized callus after injection of BMP-1 although not significant. However, after 28 days, some of the legs where we injected BMP-1 still showed some cartilaginous zones in the fracture area, pointing to a delayed remodeling. The injection of PCPE-1 led to no differences. However, when the tolloid proteinase inhibitor was injected, a significant increase of cartilaginous callus at the expense of soft tissue occurred at 7 days. This difference is mitigated during the healing time, as remodelling took place. After 28 days of healing, no significant differences between the injection of the tolloid inhibitor and the control were observed. The collagen maturation aspect of BMP-1 appears to play a neglectable role when injecting it at the early stage of fracture healing. This leads to the conclusion that the activity of Tolloid protease although beneficial for the generation of extracellular matrix, has a time critical window of application potentially leading to rather adverse effects when applying directly after fracture.