The authors thank the technical staff at Nordic Bioscience for laboratory support, as well as the Danish Research Foundation for general support of our research.

1. Tissue isolation
<ol>
	<li>Tissue sourcing
	<ol>
		<li>Perform the entire tissue sourcing section outside a laminar flow hood in an aseptic environment.</li>
		<li>From the local slaughterhouse, obtain an entire fresh bovine tibiofemoral knee joint from calves between 1.5 and 2 years of age.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Tissue culturing
	<ol>
		<li>Transfer the explants to a sterile 96-well plate in a laminar flow hood.</li>
		<li>Wash the explants 3 times in culture medium or PBS and culture them in 200 &#956;L 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.</li>
		<li>Culture the explants up to 10 weeks in a 37 &#176;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.</li>
	</ol>
	</li>
</ol>
2. Bovine cartilage explant treatment and assessment of metabolic activity
<ol>
	<li>Culture medium change and treatment
	<ol>
		<li>Change the culture medium every 2-3 days in a laminar flow hood.</li>
		<li>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.</li>
		<li>Gently remove the supernatant from each well and transfer it to a new 96 well plate. Store the supernatant with sealing tape at &#8722;20 &#176;C for biomarker analysis of tissue turnover and protein expression.</li>
		<li>Immediately add 200 &#181;L 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.</li>
	</ol>
	</li>
	<li>Resazurin staining
	<ol>
		<li>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.</li>
		<li>Make a solution of culture medium with 10% resazurin.</li>
		<li>Harvest the supernatant as described in step 2.1.3.</li>
		<li>Immerse the explants in 10% resazurin solution for 3 h at 37 &#176;C or until the supernatants turn purple. Include 4 wells without explants as background controls.</li>
		<li>Transfer the conditioned and background control resazurin solution to a black microtiter plate and measure fluorescence at 540 nm excitation/590 nm emission.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Termination, fixation, and sample storage
<ol>
	<li>Termination of culturing period
	<ol>
		<li>Measure the metabolic activity as described in step 2.2. Add 200 &#181;L of PBS per well.</li>
	</ol>
	</li>
	<li>Fixation and storage
	<ol>
		<li>Remove the PBS, add 200 &#181;L of formaldehyde per well, and leave for 2 h at room temperature.</li>
		<li>Dispose of the formaldehyde and add 200 &#181;L of PBS per well. Cover the plate with sealing tape, and store at 4 &#176;C for histochemical analysis. We recommend performing histochemical analysis within 3 months.</li>
	</ol>
	</li>
</ol>
4. Tissue turnover biomarkers (ELISA)
<ol>
	<li>Indirect competitive ELISAs
	<ol>
		<li>Coat a streptavidin-plate with the specific biotinylated assay target-peptide diluted 1:100 in assay buffer (100 &#181;L per well) for 30 min at 20 &#176;C.</li>
		<li>Wash 5 times with standard washing buffer and add sample-supernatant (20 &#181;L 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 &#181;L per well) and incubate for 2 h at 20 &#176;C with shaking for ProC2 and 3 h at 20 &#176;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.</li>
		<li>Wash 5 times with standard washing buffer and incubate with peroxidase-labeled secondary antibody diluted 1:100 in assay buffer (100 &#181;L per well) for 1 h at 20 &#176;C.</li>
		<li>Wash 5 times with standard washing buffer and incubate with shaking for 15 min in the dark at 20 &#176;C with tetramethylbenzidine (TMB) as a peroxidase substrate (100 &#181;L per well).</li>
		<li>End the reaction with standard stop solution, 0.1 M H2SO4 (100 &#181;L per well).</li>
		<li>Read the colorimetric reaction at 450 nm absorbance using a reference absorbance at 650 nm on a standard laboratory plate reader.</li>
	</ol>
	</li>
	<li>Direct Competitive ELISAs for measurement of the cartilage tissue turnover in the supernatant 
	NOTE: This quantifies C2M.
	<ol>
		<li>Coat a streptavidin-plate with specific biotinylated assay target-peptide diluted 1:100 in assay buffer (100 &#181;L per well) for 30 min at 20 &#176;C.</li>
		<li>Wash 5 times with washing buffer and add sample-supernatant together with 100 &#181;L of peroxidase-labeled monoclonal antibody against the assay target-peptide diluted 1:100 in assay buffer (20 &#181;L per well). Incubate for 20 h at 2&#8211;8 &#176;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.</li>
		<li>Wash 5 times with standard washing buffer and incubate with shaking for 15 min in the dark at 20 &#176;C with TMB as a peroxidase substrate (100 &#181;L per well).</li>
		<li>End the reaction with standard stop solution, 0.1 M H2SO4 (100 &#181;L per well).</li>
		<li>Read the colorimetric reaction at 450 nm absorbance with a reference absorbance at 650 nm on a standard laboratory plate reader.</li>
	</ol>
	</li>
	<li>AGNx1
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>ProC2
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>C2M
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Histological analysis
<ol>
	<li>Infiltration, embedding, and cutting
	<ol>
		<li>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.</li>
		<li>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.
		<ol>
			<li>Dehydrate with 96% ethanol for 90 min with no temperature adjustment. Repeat this step 3 times.</li>
			<li>Clear the ethanol with toluene for 90 min with no temperature adjustment. Repeat this step 2 times.</li>
			<li>Clear the ethanol with toluene for 90 min at 60 &#176;C.</li>
			<li>Infiltrate with paraffin wax for 30 min at 60 &#176;C.</li>
			<li>Infiltrate with paraffin wax for 60 min at 60 &#176;C.</li>
			<li>Infiltrate with paraffin wax for 90 min at 60 &#176;C.</li>
			<li>For each step, add the solutions into the sample chamber with slow pump-out and pump-in flows under 33&#8211;34 kPa. Run the infiltration process in a pressure/vacuum cycle with a maximum vacuum of &#8722;65 to &#8722;70 kPa.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>Cut 5 &#181;m 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.</li>
		<li>Using an uncoated glass slide carefully, transfer the sections to a warm water bath (50 &#176;C), where the sections unfold. Lift each section onto a labeled cover slide and place on a hot plate for 30 min.</li>
		<li>Place the slides in a basket and incubate at 60 &#176;C for 1 h and then keep them overnight at 37 &#176;C. Hereafter, store slides in closed containers at 4 &#176;C until staining.</li>
	</ol>
	</li>
	<li>Safranin O/Fast Green staining and visualization
	<ol>
		<li>Place the slides to be stained in a basket and incubate the slides at 60 &#176;C for 1 h.</li>
		<li>Prepare and filter all reagents with a 0.45 mm filter.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Stain the deparaffinized and hydrated slides by submerging the basket in Weigert&#8217;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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Mount the uncoated glass slides with resinous medium covering the histology slides.</li>
	</ol>
	</li>
</ol>

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CST, ACBJ and MK are employees of Nordic Bioscience. ACBJ and MK holds shares in Nordic Bioscience. The remaining authors have nothing to disclose.

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...

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 environment1,2. 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 tissue3,4. 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 functionality5,6,7,8.
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 unclear10,11, the onset of the disease is believed to be caused by imbalances in tissue turnover and repair processes12,13. 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 proteomics2,17,18. However, these protocols are outside the scope of the current manuscript.

<table><tbody><tr><td>45% Iron(III) chloride solution</td><td>Sigma-Aldrich</td><td>12322</td><td></td></tr><tr><td>Acetic acid</td><td>Merck</td><td>1.00056.2500</td><td></td></tr><tr><td>Alamar Blue</td><td>Life tech Invitrogen</td><td>DAL1100</td><td></td></tr><tr><td>Biopsy processing cassettes &amp;ndash; green</td><td>IHCWORLD</td><td>BC-0109G</td><td></td></tr><tr><td>Biopsy punch W/Plunger (3 mm)</td><td>Scandidat</td><td>MTP-33-32</td><td></td></tr><tr><td>Bovine cartilage (Bovine knees)</td><td>Local slaughterhouse</td><td></td><td></td></tr><tr><td>C2M</td><td>Nordic Bioscience</td><td></td><td>Fee for service</td></tr><tr><td>Corning 96-well plate</td><td>Sigma-Aldrich</td><td>CLS7007</td><td></td></tr><tr><td>Cover Glass &amp;Oslash; 13 mm</td><td>VWR</td><td>631-0150P</td><td></td></tr><tr><td>DMEM/F12-GlutaMAX Dulbecco&#039;s Modified Eagle Medium/Nutrient Mixture F-12) without HEPES</td><td>Gibco</td><td>31331-028</td><td></td></tr><tr><td>Ethanol &amp;ge;96%</td><td>VWR</td><td>83804.36</td><td></td></tr><tr><td>Ethanol absolute &amp;ge;99.5%</td><td>VWR</td><td>83813.36</td><td></td></tr><tr><td>exAGNx1</td><td>Nordic Bioscience</td><td></td><td>Fee for service</td></tr><tr><td>exPRO-C2</td><td>Nordic Bioscience</td><td></td><td>Fee for service</td></tr><tr><td>Fast green</td><td>Sigma-Aldrich</td><td>F7252</td><td></td></tr><tr><td>Formaldehyde solution 4%</td><td>Merck</td><td>1004965000</td><td></td></tr><tr><td>GM6001</td><td>Sigma-Aldrich</td><td>M5939-5MG</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>H3136</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>30721-M</td><td></td></tr><tr><td>IGF-1</td><td>Sigma-Aldrich</td><td>I3769-50UG</td><td></td></tr><tr><td>Oncostatin M</td><td>Sigma-Aldrich</td><td>O9635-10UG</td><td></td></tr><tr><td>Penicillin-streptomycin (P/S)</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Pertex (mounting medium for light microscopy)</td><td>HistoLab</td><td>811</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Safranin O</td><td>Sigma-Aldrich</td><td>S2255</td><td></td></tr><tr><td>Sterile Standard Scalpels</td><td>Integra Miltex</td><td>12-460-451</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>30743</td><td></td></tr><tr><td>SUPERFROST PLUS Adhesion Microscope Slides</td><td>Thermo scientific</td><td>J1800AMNT</td><td></td></tr><tr><td>TNF-alpha</td><td>R&amp;amp;D Systems</td><td>210-TA-100</td><td></td></tr><tr><td>Toluene</td><td>Merck</td><td>1.08327.2500</td><td></td></tr><tr><td>Vacuum Filtration &quot;rapid&quot;-Filtermax</td><td>TPP</td><td>99955</td><td></td></tr></tbody></table>

an ex vivo tissue culture model of cartilage remodeling in bovine knee explants

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.

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 TNF&#945; (O+T); 2) O+T + GM6001 (GM6001); 3) Insulin like Growth Factor-1 (I...

Watch this Scientific Journal Video about An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants at JoVE.com

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

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.

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 ...

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Research

JoVE Journal

Biology

9.3K Views.  Nordic Bioscience. 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.

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

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Bioengineering

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Medicine

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

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.

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 ...

In Vitro Preparation of Actively Maturing Bovine Articular Cartilage Explants for X-Ray Phase Contrast Imaging

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of tissue engineering strategies and potentially repairing diseased or damaged cartilage. In general, postnatal maturation of the articular cartilage, which is a wholesale change in collagen structure and function of the tissue to accommodate growth of the organism, occurs over a timescale ranging from months to years. Conversely dissolution of the structural organization of the cartilage that also occurs over long timescales is the hallmark of tissue degeneration. Our ability to study these biological processes in detail have been enhanced by the findings that growth factors can induce precocious in vitro maturation of immature articular cartilage. The developmental and disease related changes that occur in the joint involve bone and cartilage and an ability to co-image these tissues would significantly increase our understanding of their intertwined roles.
The simultaneous visualization of soft tissue, cartilage and bone changes is nowadays a challenge to overcome for conventional preclinical imaging modalities used for the joint disease follow-up. Three-dimensional X-ray Phase-Contrast Imaging methods (PCI) have been under perpetual developments for 20 years due to high performance for imaging low density objects and their ability to provide additional information compared to conventional X-ray imaging.
In this protocol we detail the procedure used in our experiments from biopsy of the cartilage, generation of in vitro matured cartilage to data analysis of image collected using X-ray phase contrast imaging.

This protocol describes the preparation of in vitro bovine articular cartilage for imaging in high resolution with X-rays. These explants actively undergo postnatal maturation. We describe here the necessary steps from the biopsy to data analysis of 3D X-ray phase contrast imaging, passing through explant culture, tissue fixation and synchrotron preparation.

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...