gms | German Medical Science

German Congress of Orthopaedics and Traumatology (DKOU 2016)

25.10. - 28.10.2016, Berlin

Decellularization of the porcine tendon-bone interface for tissue-engineering

Meeting Abstract

  • presenting/speaker Kai Xu - TU München, Klinikum rechts der Isar, Klinik für Orthopädie und Sportorthopädie, München, Germany
  • Lara Kuntz - TU München, Klinikum rechts der Isar, Klinik für Orthopädie und Sportorthopädie, München, Germany
  • Katharina Kümpel - TU München, Klinikum rechts der Isar, Klinik für Orthopädie und Sportorthopädie, München, Germany
  • Peter Föhr - TU München, Klinikum rechts der Isar, Klinik für Orthopädie und Sportorthopädie, München, Germany
  • Alexandra Wagner - TU München, Klinikum rechts der Isar, Klinik für Orthopädie und Sportorthopädie, München, Germany
  • Jutta Tübel - TU München, Klinikum rechts der Isar, Klinik für Orthopädie und Sportorthopädie, München, Germany
  • Rüdiger von Eisenhart-Rothe - TU München, Klinikum rechts der Isar, Klinik für Orthopädie und Sportorthopädie, München, Germany
  • Rainer Burgkart - TU München, Klinikum rechts der Isar, Klinik für Orthopädie und Sportorthopädie, München, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2016). Berlin, 25.-28.10.2016. Düsseldorf: German Medical Science GMS Publishing House; 2016. DocPO29-1169

doi: 10.3205/16dkou776, urn:nbn:de:0183-16dkou7766

Published: October 10, 2016

© 2016 Xu 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: High stress levels occur at interfaces between mechanically dissimilar materials. Therefore, fixation of soft tissue to bone, e.g. after bone tumor removal, has a high incidence of failure after surgical repair. Regenerating tendon/ligament-to-bone insertions, "entheses", by tissue engineering offers a promising solution to this challenge. Decellularization of porcine Achilles tendon entheses might elicit physiologically relevant scaffolds for tissue engineering. In this study, we establish a protocol to decellularize porcine entheses as potential scaffold for interface tissue engineering.

Methods: Achilles tendons with attached calcaneus were harvested from 6 months old pigs obtained from a local abattoir. Entheses were cut into 2x6x10mm3 samples using a diamond saw and frozen at -20°C. Samples underwent different treatment methods; group 1 was chemically treated with a solution of ddH2O containing 0.5% sodium dodecyl sulfate (SDS) and 1% triton x-100 for 48h, group 2 for 72h. In both groups, incubation was performed on a shaker at room temperature with exchange of detergents every 24h. Furthermore, we investigated the effect of perfusion on decellularization using a costum-made decellularization perfusion device for group 3. Untreated/ PBS-treated samples were used as controls. Decellularization was assessed histologically with HE staining and Masson staining. DAPI fluorescent dye was used to detect double-stranded DNA. Cell counts were performed on randomized regions of interest (ROI sized 200x200µm²) in tendon, bone, and interface and subsequently averaged. Statistical analysis was conducted using GraphPad Prism 6 software. Different treatment groups were compared to evaluate decellularization efficiency.

Results and Conclusion: Average cell counts (cells/mm²) were 106±39, 13±5 and 340±16 for treated groups 1, 2, and 3, respectively. Ratio of remaining cells was 16.24%±6.68%, 1.84%±0.48% and 52.14%±8.62%, respectively, compared to the negative control (663±93) (p < 0.001). Group 2 showed very efficient decellularization. HE and Masson staining indicated that the structure of the composite scaffold as well as the remaining matrix were rich in intact collagen fibers in all treated groups. No cell nuclei or remaining DNA could be detected in group 2 with DAPI staining.

Treatment of porcine Achilles tendon entheses with 0.5% SDS + 1% triton x-100 under washing for 72h resulted in efficient decellularization.