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 Distal Femur and Proximal Humerus with Fully Intact Articular Cartilage
<ol>
	<li>Euthanize the mouse according to institutional guidelines using a CO2 inhalation chamber followed by cervical dislocation. Follow aseptic technique throughout. 
	NOTE: Mice between 8 and 81 weeks old of any strain and sex may be used.</li>
	<li>Use the following surgical tools for dissections: micro-scissors (used for making incisions), standard dissecting scissors (used for cutting the bone), scalpel with #11 scalpel blade (used for cutting soft tissue), jeweler&#39;s forceps (used for removal of soft tissue) and standard forceps (used for peeling the soft tissue and the skin).</li>
	<li>For dissections of the distal femur follow the instructions below.
	<ol>
		<li>Position the mouse in a supine position.</li>
		<li>Make a small (~5 mm) incision in the skin on the anterior portion of the knee.</li>
		<li>Extend the incision all the way around the knee and pull back the skin to expose the knee joint and leg muscles.</li>
		<li>Starting from the proximal end of the femur, use a scalpel blade (#11) to cut along the distal direction. Position the blade between the quadriceps muscle-tendon unit and the anterior side of the femoral shaft. Extend the cut towards and past the patella and finish by cutting through the middle of the patellar tendon, thereby removing the quadriceps muscle-tendon unit.</li>
		<li>Starting from the proximal end of the femur, use a scalpel blade (#11) to cut along the distal direction. Position the blade between the hamstring muscle-tendon unit and the posterior side of the femoral shaft. Once the cut approaches the knee joint, begin cutting through the soft tissue, avoiding contact between the scalpel and the articular surface on the distal condyles of the femur. Finish the cut past the knee joint.</li>
		<li>Pull back the quadriceps and hamstring muscles to expose the femur. Cut away any excess muscle on the lateral and medial sides of the femur.</li>
		<li>Cut the calf muscle on the posterior side of the proximal tibia and flip the leg to visualize the posterior side of the femur.</li>
		<li>Expose the distal condyles of the femur and posterior surface of the proximal tibia by removing excess tissue around the knee joint.</li>
		<li>Cut the anterior and posterior cruciate ligaments using a scalpel, cutting away from the femoral condyles. Pull the tibia away from the femur and cut all the ligaments to separate the lower leg from the femur.</li>
		<li>Using standard dissecting scissors, cut through the femur at the proximal end of the bone (8 mm above the tibio-femoral joint) from the lateral side. After cutting the bone, wipe away any visible marrow on the outside of the bone to avoid possible contamination from bone marrow cells. 
		NOTE: Cutting from lateral side reduces the risk of crack propagation along the bone.</li>
		<li>Remove surrounding soft tissues (i.e., ligaments and excess muscle) from the femur using jeweler&#39;s forceps and expose the cartilage on both condyles at the distal end of the femur. Avoid contact between the cartilage and forceps.</li>
	</ol>
	</li>
	<li>For dissections of the humerus follow the instructions below.
	<ol>
		<li>Position the mouse in a supine position.</li>
		<li>Make an incision (~5 mm) in the skin on the posterior side of the elbow using micro-scissors, extend the incision around the elbow and pull back the skin to expose the muscles of the arm and shoulder.</li>
		<li>Starting from the proximal end of the humerus, use a scalpel blade (#11) to cut along the distal direction. Position the blade between the triceps muscle-tendon unit and the posterior side of the humerus. Extend the cut towards the distal end of the humerus and finish by cutting through the triceps tendon.</li>
		<li>Then pull back the triceps towards the proximal end of the humerus until the humeral head is exposed.</li>
		<li>Cut the connective tissue around the humeral head using a scalpel blade (#11) without touching the articular surface and remove the limb (arm and shoulder) from the body. Periodically hydrate the articular surface of the humeral head with HBSS.</li>
		<li>Disconnect the humerus from the arm by first breaking off the proximal end of the ulna on the posterior side of the arm using forceps and then cutting the connective tissue around the distal end of the humerus. Remove any excess tissue on the humerus.</li>
		<li>Cut off the deltoid tuberosity on the posterior side of the humerus using standard dissecting scissors.</li>
	</ol>
	</li>
	<li>Place the dissected specimen(s) into a 1.5 mL microcentrifuge tube containing HBSS buffer.</li>
</ol>
3. Live (Calcein AM) / Dead (Propidium Iodide) Staining Protocol
<ol>
	<li>Stain using calcein AM as follows.
	<ol>
		<li>Make a stock solution of calcein AM by adding 12.5 &#181;L of dimethyl sulfoxide (DMSO) into a vial of 50 &#181;g of calcein AM, resulting in a stock concentration of 4 mg/mL (4.02 mM).</li>
		<li>Dilute the stock calcein solution 1:400 in HBSS to reach a concentration of 10 &#181;g/mL (10.05 &#181;M) of calcium AM (for example, add 1.25 &#181;L of stock solution to 500 &#181;L of HBSS).</li>
		<li>Centrifuge the diluted staining solution for 5 s at 2,000 x g to ensure that all of the dye, which may occasionally stick to the walls of the tube, mixes with the buffer. Do not remove the supernatant. Vortex the solution to ensure proper mixing.</li>
		<li>Put the dissected specimen into a 1.5 mL microcentrifuge tube containing 500 &#181;L of the diluted staining solution. Incubate the specimen at 37 &#176;C for 30 min in a thermomixer while agitating at 800 rpm. Make sure the tubes are covered in aluminum foil to protect from exposure to light.</li>
		<li>Transfer the specimen into calcein AM-free HBSS buffer. At this point, the specimen is ready for mechanical testing.</li>
	</ol>
	</li>
	<li>Prepare propidium iodide (PI) staining solution as follows.
	<ol>
		<li>Prepare the PI staining solution by diluting the purchased stock solution (1 mg/mL, 1.5 mM) 1:25 in HBSS to reach 40 &#181;g/mL (60 &#181;M) of PI. For example, add 40 &#181;L of stock solution to 1000 &#181;L of HBSS.</li>
		<li>Keep the PI staining solution covered in aluminum foil to protect it from exposure to light. Use the staining solution immediate after the mechanical test to detect injured cells (see section 4). 
		NOTE: One can perform the PI staining prior to loading as well to more rigorously assess the quality of the dissections.</li>
	</ol>
	</li>
</ol>
4. Mechanical Testing Protocol
<ol>
	<li>Place the specimen (femur or humerus) onto the glass slide of a custom microscope-mounted mechanical testing device such that the articular surface on the posterior femoral condyles or humeral head is sitting on the glass (Figure 1). 
	NOTE: Fasten a 10 mm-long wooden applicator (diameter = 2 mm) to the distal side of the humerus using cyanoacrylate glue before placing it onto the device in order to stabilize the specimen on the flat surface. For a detailed description of the mechanical testing platform see Supplementary File 1: section 1.</li>
	<li>Hydrate the specimen with HBSS and place the device with the specimen onto a fluorescence microscope.</li>
	<li>Image the articular chondrocytes stained with calcein AM (excitation/emission wavelengths = 495/515 nm) before (baseline) application of the load under a fluorescence microscope with a 4X dry lens (NA = 0.13). Adjust the acquisition settings to optimize the image quality.</li>
	<li>Apply mechanical loading on top of the specimen such that articular cartilage is compressed against the cover glass.
	<ol>
		<li>For static loading, apply a prescribed static load (e.g., 0.5 N) on top of the specimen (femur or humerus). Hold the load for 5 min and then remove it.</li>
		<li>For impact, apply a prescribed impact energy (e.g., 1 mJ) onto the specimen by dropping a cylindrical impactor of known weight (e.g., 0.1 N) from a prescribed height (e.g., 1 cm). Release the load 5 s after the impact. 
		NOTE: The weight of the impactor (mg) and the prescribed height (h) can be converted into impact energy (E) using the following equation: E = mgh.</li>
	</ol>
	</li>
	<li>Incubate the specimen in PI staining solution for 5 min at room temperature.</li>
	<li>Image the articular chondrocytes stained with calcein AM and PI (excitation/emission wavelengths = 535/617 nm) under a fluorescence microscope (Figure 2).</li>
</ol>
5. Data Analysis
<ol>
	<li>Quantify the area of injured/dead cells due to the applied mechanical loading regimen.
	<ol>
		<li>Open the micrographs of the articular surface acquired before and after application of mechanical load in ImageJ18.</li>
		<li>Combine the images into a stack.</li>
		<li>Set the scale of the images based on the image resolution.</li>
		<li>Use the Polygon tool to define the area where the cells became calcein-negative (loss of green fluorescence) and PI-positive (gain of red fluorescence). These cells are considered to be injured or dead.</li>
		<li>Determine the area of injured/dead cells using the Measurement tool.</li>
	</ol>
	</li>
</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><tbody><tr><td>Calcein, AM&amp;nbsp;</td><td>Invitrogen by Thermo Fisher Scientific</td><td>C3100MP</td><td>20x50mg , Eugene, OR, USA</td></tr><tr><td>Propidium Iodide</td><td>Invitrogen by Thermo Fisher Scientific</td><td>P3566</td><td>1 mg/mL solution in water, 10mL, Eugene, OR, USA</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>276855</td><td>1L DMSO, anhydrous, &amp;ge;99.9%, St. Louis, MO, USA</td></tr><tr><td>HBSS (calcium, magnesium, no phenol red)&amp;nbsp;</td><td>Gibco by Thermo Fisher Scientific</td><td>14025-092</td><td>1X, 500mL, Grand Island, NY, USA</td></tr><tr><td>Feather surgical blade (#11)</td><td>VWR</td><td>102097-822</td><td>Hatfield, PA, USA</td></tr><tr><td>Vapor pressure osmometer, VAPRO</td><td>ELITechGroup</td><td>Model 5520</td><td>Puteaux, France</td></tr><tr><td>pH meter&amp;nbsp;</td><td>Beckman</td><td>Model Phi 32&amp;nbsp;</td><td>Brea, CA, USA</td></tr><tr><td>Eppendorf thermomixer&amp;nbsp;</td><td>Eppendorf AG&amp;nbsp;</td><td>Model 5350</td><td>Hamburg, Germany</td></tr><tr><td>Motorized inverted research microscope</td><td>Olypmus</td><td>Model IX-81</td><td>Center Valley, PA, USA</td></tr><tr><td>Wooden applicator</td><td>Puritan Medical Products Company, LLC</td><td>807</td><td>6&quot;x100, Guilford, ME, USA</td></tr><tr><td>1.5 Glass coverslips</td><td>Warner Instruments, LLC</td><td>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 ...

real-time-visualization-analysis-chondrocyte-injury-due-to-mechanical

Research

JoVE Journal

Bioengineering

6.8K Views.  University of Rochester. 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.

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

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Developmental Biology

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  ...

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 ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

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 ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Biology

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

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.

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 ...