gms | German Medical Science

German Congress of Orthopaedics and Traumatology (DKOU 2019)

22. - 25.10.2019, Berlin

Evaluation of viability and functionality of an ex vivo bone defect model for applications in bone tissue engineering and regenerative medicine

Meeting Abstract

  • presenting/speaker Tim Klüter - Universitätsklinikum Schleswig-Holstein, Campus Kiel, Klinik für Unfallchirurgie, Kiel, Germany
  • Rywan Hassan - Experimentelle Unfallchirurgie, Klinik für Unfallchirurgie, Kiel, Germany
  • Alexander Rasch - Universitätsklinikum Schleswig-Holstein, Campus Kiel, Klinik für Unfallchirurgie, Kiel, Germany
  • Hendrik Naujokat - Universitätsklinikum Schleswig-Holstein, Campus Kiel, Klinik für Mund-, Kiefer-, Gesicht-Chirurgie, Kiel, Germany
  • Fanlu Wang - Experimentelle Unfallchirurgie, Kiel, Germany
  • Andreas Seekamp - Universitätsklinikum Schleswig-Holstein, Campus Kiel, Klinik für Unfallchirurgie, Kiel, Germany
  • Sabine Fuchs - Experimentelle Unfallchirurgie, Klinik für Unfallchirurgie, Kiel, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2019). Berlin, 22.-25.10.2019. Düsseldorf: German Medical Science GMS Publishing House; 2019. DocAB41-1485

doi: 10.3205/19dkou335, urn:nbn:de:0183-19dkou3357

Published: October 22, 2019

© 2019 Klüter 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: Critical bone defects still remain a challenging issue for the treating surgeon. Currently, the grafting of autologous cancellous bone derived from the iliac crest remains the gold standard. The shortcomings of the biologically-derived bone grafts and the increasing demand for bone repair materials constitute the main driving forces for the continuous search of synthetic bone graft substitutes. Up to now these approaches are usually tested in vitro or in vivo for their regenerative performance. The aim of this study was to establish a novel ex vivo bone defect model with regeneration potential to investigate the potential of bone substitutes in order to support bone osteogenesis and vascularization.

Methods: Femoral heads derived from patients undergoing total hip replacement surgery were cut into cylinders of 20mm diameter and a bone defect was drilled centrally. These bone defect models were cultured for 28 days. Medium was changed every third day and determined for lactate dehydrogenase (LDH) and alkaline phosphatase (ALP). On day 28 cells in the bone slices were lysed in 1%Triton X-100 and tested for LDH. After staining with Calcein-AM, Hoechst and propidium iodide fluorescence microscopy and confocal microscopy (CLSM) images were taken after 7 and 28 days of culture. Finally, collagen-1 gel was injected into the bone defect and was investigated on day 21 using CLSM, DNA quantification and qPCR for the presence of SPP1, RUNX2, COLA1, BGLAP and ALPL in relation to isolated mesenchymal stem cells (MSC) after osteogenic differentiation.

Results and conclusion: LDH levels in the supernatants of the bone slices increased during the first 7 days and decreased significantly afterwards. After Triton-lysis on day 28, LDH levels raised significantly, demonstrating persisting viability. ALP assay showed high concentrations in the first 7 days, followed by a significant decrease up to day 28. Fluorescence and CLSM images of bone defect models demonstrated increasing number of vital cells over culture time, which was also confirmed by DNA quantification from bone slice samples. CLSM of collagen plugs in the defects documented a high number of vital and elongated cells migrated into the center of the gel. DNA quantification of harvested collagen gel show significant higher concentrations than the negative control consisting of collagen gel without cells. qPCR analysis of invaded cells indicates a typical osteogenic gene expression profile.

Conclusively the bone defect model showed vitality over the tested time period of 28 days. Probably, due to sawing procedure LDH and ALP results indicated higher cell death in the first week whereas fluorescence microscopy images showed higher cell density of elongated cells on day 28 than on day 7, showing a high regeneration process. Consequently, the bone defect model showed viability and self-regeneration potential enabling to evaluate different treatment strategies in bone replacement using hydrogels for instance.