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 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.
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 (<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.
All animal work was approved by the University of Rochester Committee on Animal Resources.
1. Solutions
2. Dissections of the Distal Femur and Proximal Humerus with Fully Intact Articular Cartilage
3. Live (Calcein AM) / Dead (Propidium Iodide) Staining Protocol
4. Mechanical Testing Protocol
5. Data Analysis
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 vulnerability of articular chondrocytes was affected by load magnitude and impact intensity. In particular, higher load magnitudes and higher impact intensities significantly exacerbated the spatial extent of cell injury in both femurs and humeri (Figure 3).
Figure 1: Schematic representation of the custom mechanical testing device. (a) Assembled device with the cylindrical impactor used to apply prescribed mechanical loads and/or impact energies to a specimen. The device is shown without a specimen. (b) Schematic representation of the experiment. Controlled static (e.g., 0.1 N) and/or impact (e.g., 1 mJ) loading can be applied on top of the dissected specimen such that articular cartilage is compressed against the cover glass. Please click here to view a larger version of this figure.
Figure 2: Representative micrograph of the articular surface after injurious mechanical loading. Representative micrograph of the articular surface on (a) the distal femoral condyles and (b) the humeral head after injurious mechanical loading (impact [1 mJ] and static loading [1 N], respectively). Green cells are vitally stained chondrocytes with intact cell membranes while red nuclei indicate injured cells with permeabilized cell membranes. (c) Zoomed-in view of the area of injured/dead cells (yellow contours) on the articular surface of the humeral head. Please click here to view a larger version of this figure.
Figure 3: Area of injured/dead cells. Area of injured/dead cells on the articular surface of the distal femoral condyles (a, b) and the humeral head (c, d) after (a, c) static (0.1 N, 0.5 N and 1 N; n = 6 per group) and (b, d) impact (1 mJ, 2 mJ and 4 mJ; n = 6 per group) loading. All cartilage-on-bone specimens were obtained from female BALB/c 8-10 weeks old mice. Data are mean + standard deviation; brackets denote statistical significance at α=0.05 determined by analysis of variance (ANOVA) test with Tukey post hoc comparisons. Please click here to view a larger version of this figure.
Supplementary File 1. Please click here to download this file.
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 on load magnitude and impact intensity (Figure 3). Importantly, the use of this method facilitates investigations of the cellular response to mechanical loading under physiologically relevant conditions. That is, it enables testing of articular cartilage on an intact joint under physiological and supraphysiological loads (see Supplementary File 1: section 2).
Given the steep learning curve in performing dissections of murine synovial joints and challenges in preserving viable in situ chondrocytes at baseline, some protocol modification and troubleshooting may be required. The greatest risk of damaging articular chondrocytes during dissection occurs during steps 2.3.5 through 2.3.10 and 2.4.5 through 2.4.7. To minimize cell injury/death during dissections, the researcher should avoid any contact between surgical tools (e.g., the scalpel when cutting the tissue or the jeweler's forceps when removing the soft tissue) and the articular surface of the specimen. However, touching the articular surface with a glove induces little cell injury/death. In order to improve baseline cell viability, it may also be necessary to reduce the amount of soft tissue removed from the joint. Additionally, using finer tools will generally reduce dissection-induced cell death. Ultimately, to rigorously confirm the absence of dissection-associated damage of articular chondrocytes at baseline, it is advisable to stain the specimens with both permeability dyes (calcein AM and PI, prior to loading) especially while the researcher is becoming familiar with the dissection procedure (see Supplemental File 1: section 3).
Given the small size of the mouse joint and the genetic manipulability of the mouse genome, murine models provide multiple advantages over large animal models to study vulnerability of articular chondrocytes to mechanical loading. However, to the authors' knowledge, no studies have previously been conducted to quantify injury/death of in situ articular chondrocytes due to mechanical loading of intact murine cartilage. Investigators typically use explants removed from joints of large animal models to investigate the extent of cell death due to mechanical injury3,4,5,6,7,8,9,10,11,12. In contrast, mouse models facilitate 1) visualization of nearly the entire articular surface on a given bone; and 2) analyses of post-loading or post-impact chondrocyte viability in intact joints without compromising native boundary conditions. Furthermore, while compressed, mouse specimens generate substantially smaller contact areas compared to large animal models; therefore, stresses are dramatically higher than in large animal models for a given load magnitude. Hence, cell injury can be induced by smaller loads. Additionally, murine models facilitate research on cartilage degeneration and, in particular, osteoarthritis, as this disease can be easily induced in mice through genetic19,20,21,22, dietary23,24 or surgical manipulations25,26,27,28. Moreover, spontaneous osteoarthritis occurs in several mouse strains including BALB/c and C57BL/629,30.
In our representative data, we used viability (live/dead) stains to quantify cell injury/death 5 min after the removal of mechanical loading. We acknowledge that these experiments may not discriminate injured cells (cells with temporarily ruptured membranes) from dead cells (cells with permanently ruptures membranes). That is, cells that are calcein negative and PI positive- indicating that the membrane was previously permeabilized-may repair their membranes over time scales ranging from seconds to several minutes31. In fact, in a separate set of experiments, we have determined that the fraction of "injured" cells that survive mechanical trauma is small (~5%) but significant (see Supplementary File 1: section 4). Therefore, live/dead staining is a direct measure of membrane integrity that is not always indicative of cell viability. In particular, PI-positive and calcein-negative cells are most appropriately defined as "injured", where injury is defined as a (potentially temporary) loss of plasma membrane integrity due to mechanical trauma. We also acknowledge that our representative data likely reflects only immediate (necrotic) cell death. Imaging specimens at later time points (e.g., 48 h after removal of load) should enable quantification of both necrotic and apoptotic cell death.
Several limitations must be considered when using these methods. In our trial experiments for this manuscript, we used 8-10-weeks-old female BALB/c mice to demonstrate the capabilities of the testing platform (Figure 3). However, due to noticeable alterations in cell density in femurs of older mice, the assessment of load-induced cell injury becomes more challenging (though feasible) in older femurs (success rate = 40%). In contrast, no noticeable alterations in cell density on humeri were observed in 61-81-week-old C57BL/6 mice (see Supplementary File 1: section 5), thereby making the testing platform useful for ages 8-81 weeks. Another limitation is that the method was used to analyze the spatial extent of cell injury only on the articular surface of femoral condyles and humeral head. However, the method could further be extended to analyze depth-dependent spatial extent of cell injury through use of laser scanning confocal microscopy. Note that the latter method would require usage of more expensive equipment, longer image acquisition time, and more involved and time-consuming analysis. Finally, although the contact location between the articular cartilage and the cover glass was physiologically relevant for mice32,33, this location was not varied. However, our testing platform allows for variation of the contact location if the femur or humerus is gripped with a rotating armature.
In conclusion, we have developed an in vitro murine injury model that enables application of controlled mechanical loads and/or impacts onto the articular surface of intact articular cartilage. This model enables visualization of fluorescently labeled articular chondrocytes in real-time and rapid single image-based analysis of cell injury/death. Importantly, the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on short- and/or long-term chondrocyte viability can be tested using this methodology. Thus, our platform provides a tool to interrogate basic science questions and screen therapeutic targets related to the mechanical vulnerability of chondrocytes.
The authors have nothing to disclose.
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.
Calcein, AM | Invitrogen by Thermo Fisher Scientific | C3100MP | 20x50mg , Eugene, OR, USA |
Propidium Iodide | Invitrogen by Thermo Fisher Scientific | P3566 | 1 mg/mL solution in water, 10mL, Eugene, OR, USA |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | 276855 | 1L DMSO, anhydrous, ≥99.9%, St. Louis, MO, USA |
HBSS (calcium, magnesium, no phenol red) | Gibco by Thermo Fisher Scientific | 14025-092 | 1X, 500mL, Grand Island, NY, USA |
Feather surgical blade (#11) | VWR | 102097-822 | Hatfield, PA, USA |
Vapor pressure osmometer, VAPRO | ELITechGroup | Model 5520 | Puteaux, France |
pH meter | Beckman | Model Phi 32 | Brea, CA, USA |
Eppendorf thermomixer | Eppendorf AG | Model 5350 | Hamburg, Germany |
Motorized inverted research microscope | Olypmus | Model IX-81 | Center Valley, PA, USA |
Wooden applicator | Puritan Medical Products Company, LLC | 807 | 6"x100, Guilford, ME, USA |
1.5 Glass coverslips | Warner Instruments, LLC | 64-1696 | #1.5, 0.17mm thick, 40mm diameter, Hamden, CT, USA |