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Bioengineering

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Published: January 7th, 2019

DOI:

10.3791/58656

1Department of Orthopedics, University Medical Center Utrecht, The Netherlands, 2Department of Veterinary Medicine, Equine Surgery, University of Utrecht, The Netherlands

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Articular cartilage defects in the knee caused by traumatic events can lead to discomfort, and above all can have a large impact on the lives of the young and active population1,2,3. Moreover, cartilage damage at a young age may lead to a more rapid onset of osteoarthritis later in life4. Currently, the only salvage therapy for generalized osteoarthritis of the knee is joint replacement surgery. As cartilage is a hypocellular, aneural, and avascular tissue, its regenerative capacity is severely limited. Therefore, regenerative medicine approaches are s....

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For this protocol, equine stifle cartilage was obtained from horses that had died from other causes than osteoarthritis. Tissue was obtained with permission of the owners, in line with the institutional ethical regulations.

NOTE: This protocol describes the fabrication of scaffolds from decellularized equine cartilage, which can be used for applications such as in vitro tissue culture platforms or for in vivo implantation in regenerative medicine strategies. .......

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Decellularization of CDM scaffolds must always be confirmed using histological stainings as well as using DNA quantification to measure the amount of DNA remnants. Insufficient decellularization might lead to undesired immunological responses that influence the results in in vivo settings15,16,17. For this specific decellularization method, DNA was below the detection range, which started at 13.6.......

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The ECM of articular cartilage is very dense and quite resilient to different enzymatic treatments. The multi-step decellularization protocol described in this article addresses such resistance and successfully generates decellularized matrices. To achieve that, the process spans over several days. Many decellularization processes have been proposed for different types of tissues18, and this article describes a protocol suitable for the decellularization of cartilage. In this protocol, it is, howe.......

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The authors would like to acknowledge W. Boot for assistance in the production of the scaffolds. K.E.M. Benders is supported by the Alexandre Suerman Stipendium from the University Medical Center. R. Levato and J. Malda are supported by the Dutch Arthritis Foundation (grant agreements CO-14-1-001 and LLP-12, respectively).

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Name Company Catalog Number Comments
Cadaveric joint This can be obtained as rest material from the local butcher or veterinary center.
Sterile phosphate-buffered saline (PBS)
Penicillin-Streptomycin Gibco 15140
Amphotericin B Thermo Fischer Scientific 15290026
Liquid nitrogen
Trypsin-EDTA (0.25%), phenol red Thermo Fischer Scientific 25200072
Tris-HCl pH 7.5
Deoxyribonuclease I from bovine pancreas Sigma-Aldrich DN25
Ribonuclease A from bovine pancreas Sigma-Aldrich R6513
Triton X-100 (octoxynol-1) Sigma-Aldrich X100
Papain Sigma-Aldrich P3125
Trisodium citrate dihydrate Sigma-Aldrich S4641
Alginate Sigma-Aldrich 180947
Formalin
CaCl2
Ethanol
Xylene
Paraffin
Ethylene oxide sterilization Synergy Health, Venlo, the Netherlands
Multipotent Stromal cells/chondrocytes from equine donors MSCs and chondrocytes can be isolated from donor joints that are rest material, coming from the local butcher or veterinary center.
MEM alpha Thermo Fischer Scientific 22561
L-ascorbic acid 2-phosphate Sigma-Aldrich A8960
DMEM Thermo Fischer Scientific 41965
Heat inactivated bovine serum albumin Sigma-Aldrich 10735086001
Fibroblast growth factor-2 (FGF-2) R & D Systems 233-FB
DNA quantification kit (Quant-iT PicoGreen dsDNA Reagent) Thermo Fischer Scientific P7581
1,9-Dimethyl-Methylene Blue zinc chloride double salt Sigma-Aldrich 341088
Freeze-dryer SALMENKIPP ALPHA 1-2 LD plus
Analytical mill IKA A 11 basic
mortar/pestle Haldenwanger 55/0A
Roller plate CAT RM5
Centrifuge (for 50 mL tubes) Eppendorf 5810R
Capsule (cylindric mold) TAAB 8 mm flat
Superlight S UV Lumatec 2001AV
Incubator
Microtome
Sieve (mesh size 0.71 mm) VWR 34111229
Scalpel
Scalpel holder
Small laddle

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