An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Summary

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

Abstract

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.

Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.

Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Introduction

Chondrocytes produce and maintain the cartilage matrix. In order to study the biology of chondrocytes and how they and the surrounding ECM react to external stimuli, it is crucial to interrogate them in their native environment^1,2. Studying cartilage tissue turnover is important to augment the understanding of the underlying mechanisms in joint diseases such as OA, a disease for which there is currently no disease modifying treatment. Consequently, there is a significant need for better translation models².

Ex vivo characterization of cell and tissue effects ....

Protocol

1. Tissue isolation

1. Tissue sourcing
   1. Perform the entire tissue sourcing section outside a laminar flow hood in an aseptic environment.
   2. From the local slaughterhouse, obtain an entire fresh bovine tibiofemoral knee joint from calves between 1.5 and 2 years of age.
   3. Gently dissect the calf knee by first removing the excess flesh, uncovering the condyles, meniscus, tendons, and synovial membrane. Cut the tendons and synovial membrane, allowing the joint to dismember. Remove the meniscus to expose the femoral condyles.
   4. Isolate explants from the load-bearing area of the femoral condyles using a 3 mm biopsy puncher and ....

Results

Bovine full-depth explants were isolated, cultured, and treated for 3 weeks (Figure 1). The culture medium was changed with the addition of treatment 3 times per week. Once weekly, metabolic activity was measured by the resazurin assay. Biomarkers of ECM turnover were measured in the supernatant harvested from the culture plate 3 times per week. Explants were divided into 4 groups for treatment: 1) Oncostatin M and TNFα (O+T); 2) O+T + GM6001 (GM6001); 3) Insulin like Growth Factor-1 (IGF-1).......

Discussion

The protocol presented here for the profiling of cartilage tissue turnover in bovine cartilage explants can be used for characterizing treatment effects of many types of drugs, including inhibitors of inflammatory intracellular pathways, inhibitors of proteolytic enzymes, or anabolic growth factors.

Two different setups were described in this protocol: an anabolic setup where explants were stimulated with insulin-like growth factor 1 (IGF-1), and a catabolic setup comprising stimulation with T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
45% Iron(III) chloride solution	Sigma-Aldrich	12322
Acetic acid	Merck	1.00056.2500
Alamar Blue	Life tech Invitrogen	DAL1100
Biopsy processing cassettes – green	IHCWORLD	BC-0109G
Biopsy punch W/Plunger (3 mm)	Scandidat	MTP-33-32
Bovine cartilage (Bovine knees)	Local slaughterhouse
C2M	Nordic Bioscience		Fee for service
Corning 96-well plate	Sigma-Aldrich	CLS7007
Cover Glass Ø 13 mm	VWR	631-0150P
DMEM/F12-GlutaMAX Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) without HEPES	Gibco	31331-028
Ethanol ≥96%	VWR	83804.36
Ethanol absolute ≥99.5%	VWR	83813.36
exAGNx1	Nordic Bioscience		Fee for service
exPRO-C2	Nordic Bioscience		Fee for service
Fast green	Sigma-Aldrich	F7252
Formaldehyde solution 4%	Merck	1004965000
GM6001	Sigma-Aldrich	M5939-5MG
Hematoxylin	Sigma-Aldrich	H3136
Hydrochloric acid	Merck	30721-M
IGF-1	Sigma-Aldrich	I3769-50UG
Oncostatin M	Sigma-Aldrich	O9635-10UG
Penicillin-streptomycin (P/S)	Sigma-Aldrich	P4333
Pertex (mounting medium for light microscopy)	HistoLab	811
Phosphate Buffered Saline (PBS)	Sigma-Aldrich	D8537
Safranin O	Sigma-Aldrich	S2255
Sterile Standard Scalpels	Integra Miltex	12-460-451
Sulfuric acid	Sigma-Aldrich	30743
SUPERFROST PLUS Adhesion Microscope Slides	Thermo scientific	J1800AMNT
TNF-alpha	R&D Systems	210-TA-100
Toluene	Merck	1.08327.2500
Vacuum Filtration "rapid"-Filtermax	TPP	99955