title: Systematic Comparison of Protocols for the Preparation of Human Articular Cartilage for Use as Scaffold Material in Cartilage Tissue Engineering.
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.
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration, Vienna, Austria .
authors.forename: Cornelia
authors.initials: C
authors.lastname: Schneider
authors.affiliation: 3 Department of Otorhinolaryngology and Cell Biology, Erasmus MC, University Medical Center , Rotterdam, The Netherlands .
authors.forename: Johannes
authors.initials: J
authors.lastname: Lehmann
authors.affiliation: 4 Department of Otorhinolaryngology and Orthopaedics, Erasmus MC, University Medical Center , Rotterdam, The Netherlands .
authors.forename: Gerjo J V M
authors.initials: GJ
authors.lastname: van Osch
authors.affiliation: 2 Austrian Cluster for Tissue Regeneration, Vienna, Austria .|5 Red Cross Blood Transfusion Center of Upper Austria , Linz, Austria .
authors.forename: Florian
authors.initials: F
authors.lastname: Hildner
authors.affiliation: 2 Austrian Cluster for Tissue Regeneration, Vienna, Austria .|6 Department of Biochemical Engineering, University of Applied Sciences Technikum Wien , Vienna, Austria .
authors.forename: Andreas
authors.initials: A
authors.lastname: Teuschl
authors.affiliation: 6 Department of Biochemical Engineering, University of Applied Sciences Technikum Wien , Vienna, Austria .
authors.forename: Xavier
authors.initials: X
authors.lastname: Monforte
authors.affiliation: 7 Department of Trauma Surgery, Medical University, Vienna, Austria .
authors.forename: David
authors.initials: D
authors.lastname: Miosga
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration, Vienna, Austria .|8 Karl Donath Laboratory for Hard Tissue and Biomaterial Research, School of Dentistry Medical University of Vienna , Austria .
authors.forename: Patrick
authors.initials: P
authors.lastname: Heimel
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration, Vienna, Austria .
authors.forename: Eleni
authors.initials: E
authors.lastname: Priglinger
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration, Vienna, Austria .
authors.forename: Heinz
authors.initials: H
authors.lastname: Redl
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration, Vienna, Austria .
authors.forename: Suanne
authors.initials: S
authors.lastname: Wolbank
authors.affiliation: 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .|2 Austrian Cluster for Tissue Regeneration, Vienna, Austria .|7 Department of Trauma Surgery, Medical University, Vienna, Austria .|9 Bernhard Gottlieb University Clinic of Dentistry , Vienna, Austria .|10 School of Dentistry Medical University of Vienna , Competence Center for Morphology, Vienna, Austria .
authors.forename: Sylvia
authors.initials: S
authors.lastname: Nürnberger
mesh_terms: D000273:Adipose Tissue / Q000166:cytology* / Q000502:physiology; D002358:Cartilage, Articular / Q000166:cytology* / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000502:physiology; D005109:Extracellular Matrix; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds; D055114:X-Ray Microtomography
keywords: allogenic scaffold; articular cartilage; cartilage regeneration; decellularization; fine structure; matrix depletion