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Published Online: 1 December 2016

Systematic Comparison of Protocols for the Preparation of Human Articular Cartilage for Use as Scaffold Material in Cartilage Tissue Engineering

Publication: Tissue Engineering Part C: Methods
Volume 22, Issue Number 12

Abstract

Natural extracellular matrix-derived biomaterials from decellularized allogenic tissues are of increasing interest for tissue engineering because their structure and composition provide a complexity that is not achievable with current manufacturing techniques. The prerequisite to bring allogenic tissue from bench to bedside as a functional biomaterial is the full removal of cells while preserving most of its native characteristics such as structure and composition. The exceptionally dense structure of articular cartilage, however, poses a special challenge for decellularization, scaffold preparation, and reseeding. Therefore, we tested 24 different protocols aiming to remove cells and glycosaminoglycans (GAG) while preserving the collagen backbone and ultrastructure. The resulting matrices were analyzed for cell removal (DNA quantification, haematoxylin and eosin staining), GAG content (dimethyl methylene blue assay, Alcian blue staining and micro-computed tomography), collagen integrity (immunohistochemistry and ultrastructure), and biomechanics (compression test). Furthermore, seeding tests were conducted to evaluate cell viability and attachment to the scaffolds. Sodium dodecyl sulfate-based protocols yielded satisfactory reduction of DNA content, yet had negative effects on cell viability and attachment. Hydrochloric acid efficiently decellularized the scaffold and pepsin emerged as best option for GAG depletion. Combining these two reagents led to our final protocol, most efficient in DNA and GAG depletion while preserving the collagen architecture. The compressive modulus decreased in the absence of GAG to ∼1/3 of native cartilage, which is significantly higher than that by commercially available scaffolds tested as a reference (ranging from 1/25 to 1/100 of native cartilage). Cytocompatibility tests showed that human adipose-derived stromal cells readily adhered to the scaffold. In this study, we established a protocol combining freeze–thaw cycles, osmotic shock, and treatment with hydrochloric acid followed by a pepsin digestion step, achieving successful decellularization and GAG depletion within 1 week, resulting in a cytocompatible material with intact collagen structure. The protocol provides a basis for the generation of allogeneic scaffolds, potentially substituting manufactured scaffolds currently used in clinical articular cartilage treatment.

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Information & Authors

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Published In

cover image Tissue Engineering Part C: Methods
Tissue Engineering Part C: Methods
Volume 22Issue Number 12December 2016
Pages: 1095 - 1107
PubMed: 27846786

History

Published in print: December 2016
Published online: 1 December 2016
Published ahead of production: 15 November 2016
Accepted: 28 October 2016
Received: 16 September 2016

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Cornelia Schneider
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Johannes Lehmann
Department of Otorhinolaryngology and Cell Biology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands.
Gerjo J.V.M. van Osch
Department of Otorhinolaryngology and Orthopaedics, Erasmus MC, University Medical Center, Rotterdam, The Netherlands.
Florian Hildner
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Red Cross Blood Transfusion Center of Upper Austria, Linz, Austria.
Andreas Teuschl
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Department of Biochemical Engineering, University of Applied Sciences Technikum Wien, Vienna, Austria.
Xavier Monforte
Department of Biochemical Engineering, University of Applied Sciences Technikum Wien, Vienna, Austria.
David Miosga
Department of Trauma Surgery, Medical University, Vienna, Austria.
Patrick Heimel
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Karl Donath Laboratory for Hard Tissue and Biomaterial Research, School of Dentistry Medical University of Vienna, Austria.
Eleni Priglinger
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Heinz Redl
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Suanne Wolbank
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Sylvia Nürnberger
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Department of Trauma Surgery, Medical University, Vienna, Austria.
Bernhard Gottlieb University Clinic of Dentistry, Vienna, Austria.
School of Dentistry Medical University of Vienna, Competence Center for Morphology, Vienna, Austria.

Notes

Address correspondence to:Sylvia Nürnberger, PhDDepartment of Trauma SurgeryAustrian Cluster for Tissue RegenerationMedical University of ViennaWähringer Gürtel 18-20Vienna 1090Austria
E-mail: [email protected]

Disclosure Statement

No competing financial interests exist.

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