Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Summary

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.

Abstract

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.

Introduction

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 population^1,2,3. 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....

Protocol

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. The enzymatic treatment steps must be performed in the described chronological order.

1. Harvesting of Articular Cartilage f....

Results

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 settings^15,16,17. For this specific decellularization method, DNA was below the detection range, which started at 13.6.......

Discussion

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.......

Materials

List of materials used in this article

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
Superlite S UV	Lumatec	2001AV
Incubator
Microtome
Sieve (mesh size 0.71 mm)	VWR	34111229
Scalpel
Scalpel holder
Small laddle