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 is essential to complement other preclinical models, both in vitro, such as chondrocyte monolayer cultures, and in vivo, such as surgery-induced OA models or the autoimmune collagen-induced arthritis model (CIA). Numerous studies have highlighted the differences between how cells behave in 2D monolayer cultures and 3D structures or in their native tissue^3,4. Many cells in 2D layers adopt unnatural morphologies, including differences in cell polarity and tissue attachment, resulting in both visual and functional differences in cells within native tissues⁵. The differences are also apparent in the functionality of the cells, which may shift protein expression, leading to profoundly altered differentiation patterns, regulatory machinery, and cell functionality^5,6,7,8.

The cartilage ECM consists mainly of type II collagen providing a matrix framework, and aggrecan, a proteoglycan that helps retain fluid within the tissue. Other matrix molecules such as collagen type IV, VI, IX, X, XI, XII, fibronectin, cartilage oligomeric protein (COMP), biglycan, decorin, and perlecan are also present⁹.

While the aetiology of OA remains unclear^10,11, the onset of the disease is believed to be caused by imbalances in tissue turnover and repair processes^12,13. The degradation of the articular cartilage is a hallmark of OA. Cartilage-resident chondrocytes or cells in the surrounding tissues increase their release of cytokines, stimulating elevated production of proteinases such as matrix metalloproteinases (MMPs) and aggrecanases, which increase degradation of cartilage ECM¹⁴. This degradation results in the release of small unique protein fragments called neo-epitopes, which can be quantified in serum, urine, or culture medium¹⁵. Upon formation and maturation of collagen, so-called profragments are also released; these can be quantified as a measure of matrix production¹⁶.

The aim of this protocol is to establish an ex vivo cartilage model to compare the effect of stimulation and/or drug treatment on ECM tissue turnover. Cartilage turnover is profiled by measuring matrix-derived neo-epitope biomarkers directly in the conditioned culture medium using ELISA: AGNx1 (reflecting aggrecanase activity), C2M (reflecting matrix MMP activity), and ProC2 (reflecting type II collagen formation). The findings can be verified by histological staining of the ECM, which also visualizes the organization of chondrocytes in the individual explants. The described protocol can be used to test the effect of novel treatments on chondrocyte function and cartilage ECM turnover. A number of studies have used cartilage explants to describe biological processes or the effect of intervention on cytokine-challenged explants using quantitative histological or immunohistochemical approaches, mRNA, protein expression, or proteomics^2,17,18. However, these protocols are outside the scope of the current manuscript.

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 release them from the articular surface by cutting with a scalpel parallel to and as close to the subchondral bone as possible. The hard structure of the subchondral bone should ensure that explants do not contain calcified matrix. Strive for explants with uniform height.
   5. Immediately store and mix the explants in DMEM/F12- GlutaMAX + 1% P/S culture medium in a 50 mL tube or Petri dish. Do not mix explants from different cow knees but keep separate for each study.
2. Tissue culturing
   1. Transfer the explants to a sterile 96-well plate in a laminar flow hood.
   2. Wash the explants 3 times in culture medium or PBS and culture them in 200 μL of culture medium per well until the start of the experiment. Use a washout period of 1 day to synchronize biopsy cellular activity and passive biomarker release.
   3. Culture the explants up to 10 weeks in a 37 °C incubator with 5% CO₂. Place all replicates within each group diagonally in the culture plate to minimize the variation induced by evaporation. To further avoid evaporation of the supernatant, add PBS to the outer wells of the culture plate.

2. Bovine cartilage explant treatment and assessment of metabolic activity

1. Culture medium change and treatment
   1. Change the culture medium every 2-3 days in a laminar flow hood.
   2. If applying any treatments, prepare these prior to changing the medium. Prepare the treatments to the wanted concentration in the explant wells by dilution in the culture medium.
   3. Gently remove the supernatant from each well and transfer it to a new 96 well plate. Store the supernatant with sealing tape at −20 °C for biomarker analysis of tissue turnover and protein expression.
   4. Immediately add 200 µL of fresh culture medium or treatment per well. Do not let the explants dry out during the medium change and ensure that all the explants are completely submerged in the new medium.
2. Resazurin staining
   1. Measure metabolic activity once weekly as an indirect measurement of cell viability. The resazurin test is an easy way to assess if the metabolic activity of the explants deteriorates for an individual explant due to cell death or cellular changes. Explants in culture medium alone have a relatively stable resazurin reading throughout the experiment period.
   2. Make a solution of culture medium with 10% resazurin.
   3. Harvest the supernatant as described in step 2.1.3.
   4. Immerse the explants in 10% resazurin solution for 3 h at 37 °C or until the supernatants turn purple. Include 4 wells without explants as background controls.
   5. Transfer the conditioned and background control resazurin solution to a black microtiter plate and measure fluorescence at 540 nm excitation/590 nm emission.
   6. Wash thoroughly 3 times in culture medium or PBS and submerge the explants in wash medium for 5-10 min to allow the resazurin to completely diffuse out. Add new culture medium or treatments if used.

3. Termination, fixation, and sample storage

1. Termination of culturing period
   1. Measure the metabolic activity as described in step 2.2. Add 200 µL of PBS per well.
2. Fixation and storage
   1. Remove the PBS, add 200 µL of formaldehyde per well, and leave for 2 h at room temperature.
   2. Dispose of the formaldehyde and add 200 µL of PBS per well. Cover the plate with sealing tape, and store at 4 °C for histochemical analysis. We recommend performing histochemical analysis within 3 months.

4. Tissue turnover biomarkers (ELISA)

1. Indirect competitive ELISAs
   1. Coat a streptavidin-plate with the specific biotinylated assay target-peptide diluted 1:100 in assay buffer (100 µL per well) for 30 min at 20 °C.
   2. Wash 5 times with standard washing buffer and add sample-supernatant (20 µL per well) together with primary monoclonal antibody against the assay target-peptide diluted 1:93.3 for ProC2 and 1:100 in assay buffer for AGNx1 (100 µL per well) and incubate for 2 h at 20 °C with shaking for ProC2 and 3 h at 20 °C for AGNx1.
      NOTE: The sample volume is directly taken from the stored supernatant plates. If the measured concentration is out of the assay measuring range, dilute the supernatant in a v-bottomed dilution plate in PBS or assay buffer.
   3. Wash 5 times with standard washing buffer and incubate with peroxidase-labeled secondary antibody diluted 1:100 in assay buffer (100 µL per well) for 1 h at 20 °C.
   4. Wash 5 times with standard washing buffer and incubate with shaking for 15 min in the dark at 20 °C with tetramethylbenzidine (TMB) as a peroxidase substrate (100 µL per well).
   5. End the reaction with standard stop solution, 0.1 M H₂SO₄ (100 µL per well).
   6. Read the colorimetric reaction at 450 nm absorbance using a reference absorbance at 650 nm on a standard laboratory plate reader.
2. Direct Competitive ELISAs for measurement of the cartilage tissue turnover in the supernatant
   NOTE: This quantifies C2M.
   1. Coat a streptavidin-plate with specific biotinylated assay target-peptide diluted 1:100 in assay buffer (100 µL per well) for 30 min at 20 °C.
   2. Wash 5 times with washing buffer and add sample-supernatant together with 100 µL of peroxidase-labeled monoclonal antibody against the assay target-peptide diluted 1:100 in assay buffer (20 µL per well). Incubate for 20 h at 2–8 °C with shaking.
      NOTE: The sample volume is directly taken from the stored supernatant plates. If the measured concentration is out of the assay measuring range, dilute the supernatant in a v-bottomed dilution plate in PBS or assay buffer.
   3. Wash 5 times with standard washing buffer and incubate with shaking for 15 min in the dark at 20 °C with TMB as a peroxidase substrate (100 µL per well).
   4. End the reaction with standard stop solution, 0.1 M H₂SO₄ (100 µL per well).
   5. Read the colorimetric reaction at 450 nm absorbance with a reference absorbance at 650 nm on a standard laboratory plate reader.
3. AGNx1
   1. Quantify aggrecan degradation by measuring the release of the AGNx1 neo-epitope. This indirect competitive ELISA assay targets the aggrecan C-terminal peptide (NITEGE³⁷³) generated by ADAMTS-4 and 5 cleavage. The monoclonal antibody recognizes all fragments with an exposed NITEGE epitope. The experimental details of the assay have been published elsewhere¹⁹.
4. ProC2
   1. Quantify type II collagen formation by measuring the release of the profragment of type II collagen. This indirect competitive ELISA assay targets the epitope of the PIIBNP propeptide (QDVRQPG) generated by N-propeptidases during trimming of newly synthesized type II collagen. The experimental details of the assay have been published elsewhere¹⁶.
5. C2M
   1. Quantify type II collagen degradation by measuring the release of the C2M neo-epitope fragment. This direct competitive ELISA recognizes the MMP-cleaved C-terminal peptide (KPPGRDGAAG¹⁰⁵³). This assay differs from AGNx1 and ProC2 as it is the primary antibody that is peroxidase-labeled and thus, used as detector. The experimental details of the assay have been published elsewhere²⁰.

5. Histological analysis

1. Infiltration, embedding, and cutting
   1. Place the fixated explants (see step 3.2) into individually labeled cassettes. Include both a label within the cassette and label cassettes to ensure identification.
   2. Transfer the cassettes containing explants to a tissue processor machine. Then infiltrate the explants with paraffin in a series of dehydration and paraffin infiltration steps.
      1. Dehydrate with 96% ethanol for 90 min with no temperature adjustment. Repeat this step 3 times.
      2. Clear the ethanol with toluene for 90 min with no temperature adjustment. Repeat this step 2 times.
      3. Clear the ethanol with toluene for 90 min at 60 °C.
      4. Infiltrate with paraffin wax for 30 min at 60 °C.
      5. Infiltrate with paraffin wax for 60 min at 60 °C.
      6. Infiltrate with paraffin wax for 90 min at 60 °C.
      7. For each step, add the solutions into the sample chamber with slow pump-out and pump-in flows under 33–34 kPa. Run the infiltration process in a pressure/vacuum cycle with a maximum vacuum of −65 to −70 kPa.
   3. Following infiltration, place the cassettes on a heating block to allow careful removal of the explants from the cassette. Gently embed the infiltrated explants into individual paraffin blocks. With heated forceps, place the explants with the superficial articular cartilage and subchondral bone sides perpendicular to the cutting surface, ensuring visualization of the different cartilage layers within each specimen section.
   4. Cut 5 µm sections of cooled paraffin-blocks with embedded explants on a microtome. Transfer the cut sections to a cold-water bath. If necessary, sections can be separated using either a scalpel or a cover glass.
   5. Using an uncoated glass slide carefully, transfer the sections to a warm water bath (50 °C), where the sections unfold. Lift each section onto a labeled cover slide and place on a hot plate for 30 min.
   6. Place the slides in a basket and incubate at 60 °C for 1 h and then keep them overnight at 37 °C. Hereafter, store slides in closed containers at 4 °C until staining.
2. Safranin O/Fast Green staining and visualization
   1. Place the slides to be stained in a basket and incubate the slides at 60 °C for 1 h.
   2. Prepare and filter all reagents with a 0.45 mm filter.
   3. In preparation for staining, pour the filtered reagents in beakers to a volume that allows the solutions to completely cover the slides when submerging the basket. The beakers used required a volume of 250 mL to cover the slides.
   4. Deparaffinize the melted slides by submerging the basket in toluene for 10 min twice, 99% ethanol for 2 min twice, 96% ethanol for 2 min twice, and 70% ethanol for 2 min twice. Then, hydrate the slides in water for 2 min.
   5. Stain the deparaffinized and hydrated slides by submerging the basket in Weigert’s Iron Hematoxylin solution (pH 1.5) for 10 min, dip in 1% HCl once, and rinse with running tap water for approximately 5 min or until excess color has washed away.
   6. Next, stain in 0.05% Fast Green solution (pH: 5.75) for 5 min, dip in 1% CH₃COOH once, and stain in 0.1% Safranin O (pH: 6.5) for 20 min.
   7. Dehydrate and clear the slides by dipping twice in 70% ethanol, 96% ethanol for 2 min twice, 99% ethanol for 2 min twice, and toluene 2 min twice.
   8. Mount the uncoated glass slides with resinous medium covering the histology slides.

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 (I...

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

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