The authors would like to thank Dr. Richard Waugh and Luis Delgadillo for the generous use of their pH meter and osmometer. Additionally, the authors would like to thank Andrea Lee for contributing to the initial development of the mechanical testing system. This study was funded by NIH P30 AR069655.

All animal work was approved by the University of Rochester Committee on Animal Resources.
1. Solutions
<ol>
	<li>Prepare Hank&#39;s Balanced Salt Solution (1X HBSS) containing calcium, magnesium and no phenol red. Adjust the pH to 7.4 by adding small amounts of HCl or NaOH.</li>
	<li>Adjust the osmolarity to 303 mOsm by adding NaCl or deionized water. Use the buffer during the dissections, specimen preparation and mechanical testing.</li>
</ol>
2. Dissections of the Di...

<ol>
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The authors have nothing to disclose.

The methods described above were successfully employed to visualize viable and injured/dead in situ articular chondrocytes from mouse joints after prescribed mechanical loads or impacts. In particular, we were able to analyze the mechanical vulnerability of chondrocytes within fully intact articular cartilage from two different synovial joints: the knee joint (distal femurs) and shoulder (humeri). Our representative results show that the spatial extent of cell injury on the articular surface depends sensitively ...

Articular cartilage (AC) is a load bearing tissue that covers and protects bones in synovial joints, providing smooth joint articulation. Tissue homeostasis is dependent on the viability of chondrocytes, the sole cell type residing in AC. However, exposure of cartilage to extreme forces due to trauma (e.g., falls, vehicle accident or sports injuries) or due to post-traumatic joint instability can induce chondrocyte death, leading to irreversible breakdown of the joint (osteoarthritis)1. Furthermore, in osteochondral grafting procedures that aim to repair local defects in damaged cartilage, graft insertion-associated mechanical trauma reduces chondrocyte viability and has detrimental effects on surgical outcomes2.
Cartilage explant models are commonly used to study the susceptibility of articular chondrocytes to mechanically-induced cell death. These models typically use explants from large animals to study the effects of loading conditions, environmental conditions and other factors on cell vulnerability3,4,5,6,7,8,9,10,11,12,13,14,15. However, due to the large size of the native joints, these models generally require removal of a plug from the articular surface of an intact joint, thereby compromising native boundary conditions. Moreover, they generally require application of large mechanical loads to induce cell injury. Alternatively, murine cartilage explant models provide several advantages over larger animal models in studying the mechanical vulnerability of in situ chondrocytes. In particular, due to their smaller dimensions, these models facilitate testing of fully intact articular cartilage without altering native tissue integrity. In addition, loading of murine cartilage occurs over small contact areas such that chondrocyte death/injury can be induced with small loads (&#60;1 N). Finally, the mouse genome is easily manipulated, enabling testing of how specific genes impact the susceptibility of in situ chondrocytes to mechanical injury.
The overall goal of the method introduced in this manuscript is to quantify and visualize-in real-time-the spatial extent of in situ cell death/injury due to applied mechanical loads on fully intact mouse cartilage-on-bone explants in vitro. This method requires careful dissection of mouse synovial joints without compromising chondrocyte viability, followed by mechanical testing of vitally stained explants using a microscope-mounted device similar to a testing platform that we recently developed to quantify murine cartilage mechanical properties16. During mechanical testing, a large portion of the (intact) articular surface of the dissected bone is visible on a single fluorescence micrograph, enabling rapid analysis of cell viability after a load is applied. A similar analysis of surface cell viability in murine cartilage explants has been performed previously, but without simultaneous application of load17. Potential applications of our method include comparative studies to investigate the vulnerability of articular chondrocytes to different controlled environmental and mechanical conditions, as well as screening of treatments aimed at reducing the sensitivity of chondrocytes to mechanical loading.

<table border="0" cellpadding="0" cellspacing="0" style="width:1027px;" width="1026">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Calcein, AM&#160;</td>
			<td style="width:205px;">Invitrogen by Thermo Fisher Scientific</td>
			<td style="width:220px;">C3100MP</td>
			<td style="width:379px;">20x50mg , Eugene, OR, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Propidium Iodide</td>
			<td style="width:205px;">Invitrogen by Thermo Fisher Scientific</td>
			<td style="width:220px;">P3566</td>
			<td>1 mg/mL solution in water, 10mL, Eugene, OR, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:205px;">Sigma-Aldrich</td>
			<td style="width:220px;">276855</td>
			<td>1L DMSO, anhydrous, &#8805;99.9%, St. Louis, MO, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">HBSS (calcium, magnesium, no phenol red)&#160;</td>
			<td style="width:205px;">Gibco by Thermo Fisher Scientific</td>
			<td style="width:220px;">14025-092</td>
			<td>1X, 500mL, Grand Island, NY, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Feather surgical blade (#11)</td>
			<td style="width:205px;">VWR</td>
			<td style="width:220px;">102097-822</td>
			<td>Hatfield, PA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Vapor pressure osmometer, VAPRO</td>
			<td style="width:205px;">ELITechGroup</td>
			<td style="width:220px;">Model 5520</td>
			<td>Puteaux, France</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">pH meter&#160;</td>
			<td style="width:205px;">Beckman</td>
			<td style="width:220px;">Model Phi 32&#160;</td>
			<td>Brea, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Eppendorf thermomixer&#160;</td>
			<td style="width:205px;">Eppendorf AG&#160;</td>
			<td style="width:220px;">Model 5350</td>
			<td>Hamburg, Germany</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Motorized inverted research microscope</td>
			<td style="width:205px;">Olypmus</td>
			<td style="width:220px;">Model IX-81</td>
			<td>Center Valley, PA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Wooden applicator</td>
			<td style="width:205px;">Puritan Medical Products Company, LLC</td>
			<td style="width:220px;">807</td>
			<td>6&#34;x100, Guilford, ME, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">1.5 Glass coverslips</td>
			<td style="width:205px;">Warner Instruments, LLC</td>
			<td style="width:220px;">64-1696</td>
			<td>#1.5, 0.17mm thick, 40mm diameter, Hamden, CT, USA</td>
		</tr>
	</tbody>
</table>

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.

Six different applied loading protocols (static loading: 0.1 N, 0.5 N and 1 N for 5 min; and impact loading: 1 mJ, 2 mJ and 4 mJ) reproducibly induced quantifiable localized areas of cell injury in femoral and humeral cartilage obtained from 8-10-week-old BALB/c mice (Figure 2). Importantly, the spatial extent of chondrocyte injury on the articular surface was measured quickly and easily in ImageJ. Representative results demonstrate that the mechanical vulner...

Watch this Scientific Journal Video about Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants at JoVE.com

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

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 ...

This method enables investigation of the effects of different mechanical loading regimens, environmental conditions, or stages of cartilage degeneration on the vulnerability of in situ articular chondrocytes. The physical forces. Unlike other techniques, these measurements are performed in real time and in fully intact cartilage without compromising native boundary conditions.Another advantage of this technique is it uses a mouse model, allowing testing of how specific genes affect the susceptibility of in situ chondrocytes in mechanical injury. For dissection of the distal femur, place the mouse in the supine position and make a five millimeter skin incision on the anterior portion of the knee. Extend the incision all the way around the knee.And pull back the skin to expose the knee joint and leg muscles. Starting from the proximal end of the femur, use a number eleven scalpel blade to cut along the bone in the distal direction. Positioning the blade between the quadriceps muscle tendon unit and the anterior side of the femoral shaft.Extend the incision past the patella. Cutting through the middle of the patellar tendon to remove the quadriceps muscle tendon unit. Next, starting from the proximal end of the femur, position the scalpel blade between the hamstring muscle tendon unit and the posterior side of the femoral shaft and cut along the bone in the distal direction.When the incision approaches the knee joint, cut through the soft tissue, and finish the cut past the knee joint. Pull back the quadriceps and hamstring muscles to expose the femur. And cut away any excess muscle on the lateral and medial sides of the bone.Cut the calf muscle on the posterior side of the proximal tibia. And flip the leg to visualize the posterior side of the femur. Remove the excess tissue around the knee joint to expose the distal condyles of the femur and the posterior surface of the proximal tibia.Cut the anterior and posterior cruciate ligaments away from the femoral condyles. And pull the tibia away from the femur, cutting all the ligaments to separate the lower leg from the femur. Using standard dissection scissors, cut through the femur at the proximal end of the bone about eight millimeters above the tibiofemoral joint from the lateral side.And use jeweler's forceps to remove the surrounding soft tissues from the femur. Then expose the cartilage on both condyles at the distal end of the femur. Periodically hydrate the articular surface with HBSS.For dissection of the humerus, place the mouse in the supine position. And use micro scissors to make a five millimeter incision on the posterior side of the elbow. Extending the incision around the elbow and pulling back the skin to expose the muscles on the arm and shoulder.Starting from the proximal end of the humerus, position the blade of a number eleven scalpel between the triceps muscle tendon unit and the posterior side of the humerus. And cut along the bone in the distal direction. Extend the incision toward the distal end of the humerus, through the triceps tendon and pull back the triceps toward the proximal end of the humerus until the humeral head is exposed.Cut the connective tissue around the humeral head without touching the articular surface. And remove the limb from the body. Hydrate the articular surface of the humeral head with HBSS.To disconnect the humerus from the arm, use forceps to break off the proximal end of the ulna on the posterior side of the arm. And cut the connective tissue around the distal end of the humerus, removing any excess tissue on the bone. Then use standard dissecting scissors to remove the deltoid tuberosity on the posterior side of the humerus.And place the dissected specimen into a 1.5 milliliter microcentrifuge tube containing HBSS. For Calcein AM staining of the samples, transfer the bones into a 1.5 milliliter microcentrifuge tube containing 500 microliters of 10.05 micro molar Calcein in HBSS. And place the tube in a thermo mixer at 37 degrees Celsius at 800 rpm, protected from light.After thirty minutes, wash the specimen in fresh HBSS for ten minutes. And transfer the specimen onto the glass slide of a custom microscope mounted mechanical testing device, such that the articular surface of the posterior femoral condyles or the humeral head is sitting on the glass. Hydrate the specimen with HBSS and place the device with the specimen onto a fluorescence microscope stage.Image the articular chondrocytes stained with Calcein AM before the load application under a 4x magnification. Adjust the acquisition settings to optimize the image quality. To apply static mechanical loading, hold a prescribed static load on top of the specimen such that the articular cartilage is compressed against the cover glass for five minutes before removing the load.To apply an impact mechanical load, drop a cylindrical impactor of known weight onto the specimen from a prescribed height, releasing the load five seconds after the impact. Immediately after either load has been removed, incubate the specimen in freshly prepared 60 micro molar propridium iodide in one milliliter of HBSS. For five minutes at room temperature, then re-image the articular chondrocytes by fluorescence microscopy as just demonstrated.To quantify the area of injured and dead cells due to the applied mechanical loading regimen, first open the micrographs of the articular surface acquired before and after application of mechanical load in ImageJ. Combine the images into a stack. Set the scale of the images based on the image resolution.Next, define the area where the cells became Calcein negative and PI positive. These cells are considered to be injured or dead. Then determine the area of injured dead cells using the measurement tool.At least six tested applied the loading protocols reproducibly induced quantifiable localized areas of cell injury in femoral and humeral cartilages obtained from eight to ten week old valve C mice. In all of the loading protocols tested, higher load magnitudes and higher impact intensities significantly exacerbated the spacial extent of cell injury in both femurs and humeri. While performing the dissections it is important to remember not to contact the articular cartilage with the surgical tools, as this may compromise the baseline by ability of the specimens.The use of this mirroring model may facilitate research in osteoarthritis, as the disease is easily induced in mice through genetic, dietary, or surgical manipulations. Our platform also provides a tool for interrogating basic science questions and for screening therapeutic interventions targeting the mechanical vulnerability of chondrocytes.

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Research

JoVE Journal

Bioengineering

6.7K visualizações.  University of Rochester. Este método permite a investigação dos efeitos de diferentes regimes mecânicos de carga, condições ambientais ou estágios de degeneração da cartilagem sobre a vulnerabilidade dos condrócitos in situ articular. As forças físicas. Ao contrário de outras técnicas, essas medidas são realizadas em tempo real e em cartilagem totalmente intacta sem comprometer as condições de fronteira nativa.Outra vantagem dessa técnica é que ele usa um modelo de mouse, permitindo testes d...