In Vitro Impact Model to Generate Sublethal Chondrocyte Injury in Bovine Cartilage Explants

Summary

To better understand the chondrocyte injury response to a mechanical cartilage overload, the protocol describes the development of an ex vivo cartilage impact model that was sublethal for 24 h.

Abstract

Post-Traumatic Osteoarthritis (PTOA) is a prevalent, degenerative, and painful progressive joint disease that typically arises after significant joint trauma, affecting over 6 million Americans. Impact-induced cartilage trauma results in a chondrocyte injury response that culminates in PTOA. Understanding the molecular events associated with chondrocyte injury is necessary to develop therapies to prevent PTOA or slow its progression. To facilitate this, an ex vivo cartilage impact model that was sublethal for 24 h was developed. Osteochondral explants were harvested from bovine metacarpophalangeal joints, and a drop tower was used to deliver impact loads to the explants. To ensure that the stress and impact were applied relatively uniformly to the articular cartilage, explants were positioned with the bone surface facing upward, and the bone was impacted from above. Different loads were applied to the explants from carriage heights of 4.0 cm, 4.5 cm, and 5.0 cm, resulting in average peak stresses of 10.87 ± 1.84 MPa, 11.69 ± 0.67 MPa, and 12.98 ± 0.75 MPa, respectively. The study analyzed the role of subchondral bone presence in cartilage during post-impact culture and the effect of using either a fitted or loose holder during impact, assessing cell viability and apoptosis. Articular cartilage impacted from 4.5 cm, combined with a fitted holder and immediate bone removal, provided the optimal model to induce cartilage injury with minimal cell death at 24 h post-impact.

Introduction

Post-traumatic osteoarthritis (PTOA) is a debilitating disease that affects over 5 million adults in the United States¹. A single traumatic impact to cartilage can trigger a chondrocyte injury response, leading to early-onset symptoms and a lifelong struggle with joint pain and dysfunction². Cartilage has little to no intrinsic repair capability, and thus, there exists an unfilled need to develop therapies to modify the progression of the disease³. Because PTOA is correlated with a single, clearly defined traumatic event, there is an opportunity to intervene before the disease progresses. However, the development of these treatments is hindered by the gap in knowledge about the molecular signaling that regulates the chondrocyte injury response and leads to post-traumatic osteoarthritis^2,3,4. A better understanding of the molecular events following chondrocyte injury could help design new therapeutic treatments that rescue the chondrocyte injury response and prevent or delay the development of PTOA^2,5,6.

To better understand the molecular signaling of chondrocyte injury, previous research has utilized drop towers or similar devices that mechanically impact cartilage explants so that an injury response is initiated. These studies have sought to characterize many aspects of this response, including changes in mechanical properties and structure^7,8, matrix damage, and synthesis^6,9,10,11, mitochondrial dysfunction^12,13,14,15, ion transport pathways^16,17,18,19, and gene expression^{9,11,20,21,22,23}. However, in many of these studies, cell viability was either not reported or was compromised, with 50% or lower cell viability in some studies^24,25,26. Confounding effects of low cell viability may make it difficult to study the injury response. Chondrocyte death that immediately follows joint trauma is irreversible, but therapies that treat chondrocyte injury mitigate the subsequent PTOA²⁷. In order to identify molecular changes that are associated with chondrocyte injury and not a loss of viable cells, a cartilage impact model that maintains chondrocyte viability for a period of time is necessary. Additionally, a cartilage impact that generates a chondrocyte injury response that is relatively uniform throughout the cartilage tissue would provide the most consistent results in analyzing cell behavior.

The objective of the study was to develop and characterize a cost-effective ex vivo impact injury model for articular cartilage that conserves the ability to study signaling pathways inherent to the acute cartilage injury response. Additionally, such a model could be used to evaluate potential treatments to rescue the injury response. To accomplish this, we first developed a sterile technique to harvest cartilage explants from bovine metacarpophalangeal joints from skeletally mature animals using a diamond-tipped coring bit mounted on a drill press. Metacarpophalangeal joints are discarded by the abattoir when the animals are processed for food consumption, making this joint an easily accessible and inexpensive source of tissue. Next, we established an impact injury model where the osteochondral specimen was placed in a holder under a drop tower device. In this model, the osteochondral specimens were placed with the articular surface facing downward on a #8 mirror-polished stainless-steel surface, and the bone was subjected to an impact from above, delivering a relatively uniform load to the cartilage tissue. Impact parameters that maintain chondrocyte viability for 24 h post-impact while still triggering an apoptotic molecular injury response were determined. Holders that allowed for different amounts of lateral motion of the specimen during impact were assessed for their effect on cell viability and apoptosis; finally, the effect of removing cartilage from the subchondral bone after impact was evaluated.

Protocol

The use of bovine limbs obtained from a local abattoir did not require ethical approval, as the tissues were collected post-mortem from animals processed for food consumption.

1. Osteochondral explant harvest

1. Sterilize the following materials using an autoclave prior to explant harvest: Phosphate buffered saline (PBS; approximately 1 L per joint), Coring drill bit, Water swivel adapter, one autoclavable wash bottle, one scalpel handle, two forceps, and one autopsy saw blade.
2. Set up tissue culture hood with drill press and vise inside a basin to contain PBS. Assemble coring drill bit and swivel adapter in the drill press and connect the swivel adapter nozzle with a hose to a carboy filled with sterilized PBS. Assemble the autopsy saw and place inside the hood (Figure 1A-B).
3. Spread disposable under-pads in a stack in the tissue culture hood, one for each joint.
   NOTE: Under-pads absorb PBS and synovial fluid that may drip while opening the joint. Stacking several under-pads allows for easy removal of the top layer when it becomes too wet, providing a clean and dry workspace throughout the procedure.
4. Prepare one 50 mL test tube containing 70% ethanol and another 50 mL test tube containing 0.13% v/v betadine in PBS, and place them in the hood. Place forceps and scalpel handle into the ethanol test tube to maintain sterility during tissue handling and place a sterile 6-well cell culture plate at the back of the hood.
5. Prepare 3 mL of cell culture media per anticipated specimen. Culture media consists of Modified Eagle's Medium (DMEM) with 0.9 mM sodium pyruvate, insulin, transferrin, and selenium (ITS), 50 mg/mL L-ascorbic acid, minimal essential medium (MEM) nonessential amino acids, penicillin, streptomycin, and amphotericin B. Add L-ascorbic acid and ITS on the day of use²⁸.
6. Obtain bovine forelegs from skeletally mature animals from a local abattoir within 6 h of sacrifice. Keep tissue on ice during transportation.
7. Isolate the metacarpophalangeal joint by removing the hoof using a meat band saw without opening the joint capsule. Remove skin using #22 scalpel blades without puncturing the joint capsule.
8. Rinse each leg thoroughly outside the biosafety cabinet with cold water to remove dirt and coat with betadine to disinfect.
9. Inside the tissue culture hood, open the joint capsule using a new #22 scalpel blade. Isolate the distal joint and keep articular cartilage hydrated with PBS from a wash bottle (Figure 1C). Cover the articular cartilage with sterile gauze soaked with PBS to maintain hydration.
10. Further isolate the distal metacarpophalangeal joint by cutting through the metatarsus with the autopsy saw (Figure 1D). Place the distal joint into the vise with the covered cartilage facing upwards and tighten until secure. During the entire process, moisten the gauze on the cartilage regularly with PBS.
11. Fold gauze away from one condyle and adjust the position of the vise so that the coring bit is above the flattest region of the articular surface (Figure 1E).
12. Open the carboy valve to allow PBS to flow through the coring bit. Use a wash bottle to continually hydrate the tissue with PBS and cool the samples during drilling.
    NOTE: PBS from the carboy and the wash bottle has a cooling effect during the coring process.
13. Core into the condyle slowly, by pressing the drill bit into the surface of the condyle. Release the pressure frequently and allow heat to dissipate. Continue until the coring depth is approximately 2 mm into the subchondral bone. Repeat for each side (Figure 1E).
    NOTE: Signs of overheating include rapid fluid evaporation or a burning smell.
14. Use the autopsy saw to release osteochondral cores by cutting the bone parallel to the articular cartilage surface, continuing to squeeze PBS from the wash bottle to the interface of the autopsy saw and osteochondral bone while cutting. During this step, maintain the moistened gauze on the surface of the articular cartilage to avoid dehydration.
15. Store osteochondral cores (Figure 1F) in dilute betadine solution (see step 1.3) until all joints have been processed.
16. Rinse osteochondral cores in PBS and transfer the samples into individual wells of a sterile 6-well plate containing 3 mL of pre-warmed culture media (DMEM with 0.9 mM sodium pyruvate, insulin-transferrin-selenium (ITS), 50 mg/mL L-ascorbic acid, nonessential amino acids, penicillin, streptomycin, and amphotericin B)²⁸. Maintain samples in a humidified incubator at 37 °C with 5% CO₂ until the impact procedure on the following day.

2. Osteochondral explant impact

1. Fabricate stainless-steel impact holder with a mirror-polished impact surface (Supplementary Figure 1 and Supplementary Figure 2) and drill corresponding holes in drop tower base plate (Supplementary Figure 3).
2. Sterilize the following materials using an autoclave prior to the impact procedure: 15 mm diameter stainless steel spherical impactor head, stainless-steel holder with mirror-polished impact surface, 4 6/32 screws, 4 washers, forceps.
3. Place a drop tower that had previously been designed and fabricated²⁹ into a biosafety cabinet. Use a drop tower with a 15 mm diameter spherical impactor head mounted on a carriage equipped with a load cell and accelerometer (Figure 2A), with a total carriage mass of 358.4 g for this study.
4. Fix holder to the base plate of drop tower with four autoclaved screws (Figure 2B). In this study, holders with circular openings of 8.7 mm and 9.1 mm in diameter were tested.
5. Place an osteochondral core into the holder (Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3) under the impactor head of the drop tower with the bone facing upwards.
   NOTE: Placing the osteochondral core with the bone-facing upward and the articular surface facing downward³⁰ was chosen for more homogeneous loading.
6. Adjust the drop tower carriage to the desired height relative to the top of the bone. Determine the approximate drop tower height from pilot studies. Heights of 4.0, 4.5, and 5.0 cm were evaluated in this study.
7. Click on the Start button on the data acquisition software (Supplementary Coding File 1) just before releasing the drop tower carriage by freeing the spindle stop (Figure 2A) and allowing the impactor head to drop under the weight of gravity onto the sample. Immediately lift the carriage and rest it on the spindle stop.
   NOTE: During the impact, data will be collected at 100 kHz using data acquisition software connected to a data acquisition module, which records signals from a load cell positioned between the impactor head and carriage, as well as from an accelerometer. While we lift the carriage as soon as possible after the impact, it does not have an anti-rebound feature or other mechanism to prevent a second impact.
8. Place the .txt file generated by the data acquisition software in the same folder as the Matlab data analysis code (Supplementary Coding File 2, also available at https://github.com/sogol-younesi/cartilage-impact-analysis) and run the data analysis code to filter raw data and calculate impact parameters.
   NOTE: The data analysis code uses a cartilage thickness of 0.563 mm to calculate the impact energy. This was determined as the average cartilage thickness taken from > 50 locations on each of five specimens in a separate study using the needle method³¹ and a Biomomentum Mach-1 mechanical tester. Impact parameters are calculated as previously described³².
9. Remove cartilage from bone with a #11 scalpel blade, as appropriate. Return the sample to the media and humidified incubator at 37 °C with 5% CO₂ until imaging on the following day.
   NOTE: The effect of removing cartilage from the bone immediately after impact or 24 h after impact was assessed in this study.

3. Characterization of cell viability in cartilage specimens

1. Remove cartilage from bone if necessary. Cut the cartilage specimen in half with a sharp scalpel. Avoid making multiple cuts; aim for a single incision to ensure a smooth edge.
2. Submerge each half-specimen of interest in a solution consisting of 5 µM ethidium homodimer and 2.5 µM calcien AM in PBS. Protect from light and incubate at room temperature for 30 min to complete live-dead staining.
3. Rinse samples with PBS 2x and keep samples in PBS until imaging. Image as soon as possible after staining is complete.
4. Stand cartilage sample upright on a few drops of PBS in a microscopy dish so that the full semicircle is visible from the front of the microscope. Capture images with an inverted confocal microscope. Bend the cartilage slightly with forceps if it fails to stand upright on its own.
5. Using FIJI, a distribution of ImageJ with bundled plugins³³, first convert the image to 8-bit by clicking on Image > Type > 8-bit. Then, set the threshold under Image > Adjust > Threshold, and click Apply. Next, perform the Watershed function under Process > Binary, and run Analyze Particles… under Analyze for both the green and red channels. Analyze images captured at a consistent z-height for each specimen for accurate comparisons between groups.
6. Calculate cell viability by dividing the number of live cells by the total number of cells in the sample.

4. Characterization of apoptosis in cartilage specimens

1. Submerge each half-specimen of interest in 8 µM Caspase-3/7 Green Detection Reagent in PBS. Protect from light and incubate at 37 °C for 30 min.
2. Wash samples with PBS 3x. Fix the stained samples by incubating in 4% paraformaldehyde (PFA) for 15 min. Wash with PBS again and store in PBS until imaging. Image within 24 h of staining.
3. Image with confocal microscope (see step 3.4 above). For valid comparisons of fluorescence intensity between groups, maintain consistent imaging parameters for all samples, including objective lens, excitation laser power, photomultiplier tube detector gain, pinhole size, optical slice thickness, scan speed, line averaging, and image resolution.
4. Using the FIJI extension of ImageJ, select Mean gray value under the Analyze > Set Measurements… menu.
5. Use the Polygon Section Tool to outline the entire area of the sample shown in an image. Perform the Measure function under the Analyze menu and record the mean gray value, using the same processing parameters across groups.
6. Use the Polygon Section tool to select an area of the image that does not contain the sample and perform the Measure function again. Record this as the background mean gray value.
7. Calculate the fluorescence intensity by subtracting the background mean gray value from the sample mean gray value.

Results

The mechanical impact load to osteochondral explants was applied using a drop tower with carriage heights of 4.0 cm, 4.5 cm, and 5.0 cm above the tissue (n = 3 or 4). Increasing the height of the carriage before it was released resulted in higher impact forces, average peak stresses, loading rates, and impact energies (Table 1).

We sought to develop an ex vivo cartilage injury model that was sublethal for 24 h after impact. Characterization of Live/Dead stain 24 h pos...

Discussion

The objective of this study was to characterize and optimize a cartilage impact model that does not compromise cell viability for studying molecular events following chondrocyte injury, with the eventual goal of developing a treatment that rescues the injured cells to prevent PTOA progression. In pursuit of this goal, a mechanical load was delivered to the bone portion of osteochondral cores, transferring the load to the cartilage tissue. Furthermore, we examined different impact heights, the effects of different holder ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#11 sterile scalpel blade	Thermo Fisher Scientific	10-001-823	Remove cartilage from the bone
#22 sterile scalpel blade	Thermo Fisher Scientific	10-001-833	Remove skin from bovine tissues and open the joint by cutting the ligaments
10-1000 ul pipette tips	Thermo Fisher Scientific	02-707-411	Measure and transfer  PBS, culture media and other solutions
200 proof ethanol	Thermo Fisher Scientific	03-007-647	Used to prepare 70% ethanol as sterilizing agent to disinfect surgical tools and maintain a sterile environment during tissue preparation and processing.
50 mL centrifuge tube (sterile)	Thermo Fisher Scientific	339652	Used to store media and samples
5-gallon carboy	Thermo Fisher Scientific	04-355-233	Sterile PBS container which is connected to swivel adapter by hose.
6-well plate (sterile)	Thermo Fisher Scientific	140685	Used to temporarily store osteochondral cores in a sterile environment with pre-warmed media during collection, incubation before impact studies, and imaging
Accelerometer	Kistler	8743A5	Used to measure the acceleration produced during an impact
Apoptosis staining kit (CellEvent)	Thermo Fisher Scientific	C10423	Used to detect and visualize apoptotic cells
Autoclavable wash bottles	Nalgene	2405-0500	Fill with sterile PBS to hydrate the cartilage during tissue harvesting.
Autopsy saw	Mopec	BD 810	Remove any excess bone either after opening the joint or extracting the osteochondral core
Autopsy saw blade	Mopec	BD 101	Used for cutting through bone to isolate and release osteochondral explants.
Betadine	Avrio Health L.P.	n/a	Used for disinfection of the exterior of bovine samples before moving them to the hood and diluted for disinfecting the final osteochondral samples.
Clear 4 ft hose	n/a	n/a	Used to connect the 5-liter carboy containing PBS to the swivel adapter of the drill press  to transport PBS.
Coring drill bit	UKAM Industrial Superhard Tools	3EDCD7160	Used for extracting osteochondral explants from bone by drilling into the articular cartilage and subchondral bone.-  diameter of the drill bit: 7/16" (Electroplated Diamond Core Drills)
Crescent wrench	McMaster Carr	5362A1	Used to tighten clamp (that holds joint in place) and tighten the coring drill bit
Disposable serological pipets	Thermo Fisher Scientific	13-678-11	Measure and transfer PBS and culture media
Disposable underpad	Thermo Fisher Scientific	23-666-062	Used to cover counterspace to absorb water and liquids during harvesting for more convenience and hygiene
Disposable vacuum filter units	Thermo Fisher Scientific	SCGP00525	Filter PBS and other solutions
Drill press	WEN	433TV 3.25-Inch	Apply force to drill osteochondral samples- WEN 433TV 3.25-Inch Industrial Strength Benchtop
Drill press tilting angle vise	WEN	TV434	Secures osteochondral samples in a fixed position at the desired angle during the coring process.
Drop tower	custom made	n/a	Used to impact the samples
FIJI (ImageJ Extension)	n/a	n/a	Software used for processing and analyzing microscopy images, including cell viability and apoptosis quantification
Forceps	Fisherbrand	12-000-163	Used for cutting tissue in hood, opening the joint and transfering the samples
Gauze (sterile)	Dukal	6412	Used to hydrate the articular cartilage after opening the joint capsule
Glass 1 liter media bottle	Sigma Aldrich	CLS13951LHTC	Used to store sterile PBS
Gloves	Thermo Fisher Scientific	19-130-1597D	Used to protect collection team
Insulin-Transferrin-Selenium (ITS)	Gibco	41400-045	Media supplement
LabVIEW data acquisition software	National Instruments	n/a	Captures data from the load cell and accelerometer during impact testing for subsequent analysis.
L-ascorbic acid	Merck	A4403	Media supplement
Laser confocal scanning microscope	Olympus	FV1000D	Used to capture high-resolution, three-dimensional images of stained tissue samples.
Live and dead assay staining kit	Thermo Fisher Scientific	L3224	Used to differentiate between live and dead cells in tissue samples to asses cell viability
Load cell	Kistler	9712B5000	Used to measure the force applied during an impact.
MATLAB	The MathWorks Inc.	n/a
Meat band saw	Skymsen	MSKLE	Remove carpal hoof from bovine samples
Micropipette 10-100 uL	Thermo Fisher Scientific	FBE00100	Measuring and transferring  PBS and culture media
Minimal essential medium (MEM) nonessential amino acid solution	Gibco	11140-050	Media supplement
Modified Eagle’s Medium (DMEM)	Gibco	10566-016	Osteochondral explant culture.
Penicillin-Streptomycin	Thermo Fisher Scientific	15-140-148	Media supplement
Phosphate buffered saline	Thermo Fischer Scientific	BP399-20	Used for rinsing, hydrating, and cooling tissues to maintain physiological conditions, prevent dehydration and preparing staining solutions
Sample holder on drop tower	custom made	n/a	Secures the osteochondral core during impact.
Scalpel handle	Thermo Fisher Scientific	16-100-107	Used for precise cutting, tissue dissection, and opening the joint.
Screws (add to cad)			6-32 thread Size-18-8 Stainless Steel Socket Head Screws
Sodium pyruvate	Gibco	11360-070	Media supplement
Spherical impactor head	McMaster Carr	9292K52	Autoclave before impact procedure
Surgical masks	Filtermask	375101	Used to protect collection team
Water swivel adapter	UKAM Industrial Superhard Tools	7035812	Connects to the coring drill bit to provide a continuous flow of PBS for cooling  during the drilling process.