Name: real-time visualization and analysis of chondrocyte injury due to mechanical loading in fully intact murine cartilage explants
Uploaded: 2019-01-07T14:00:00
Description: 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

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 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.

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		<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>
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			<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>
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			<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>
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		<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>
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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.

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

Research

JoVE Journal

Bioengineering

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  ...

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 ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during an impact are important factors in protecting the helmet wearer from impact-induced injury. This manuscript aims to investigate impact-induced injury mechanisms in different helmet fit scenarios through analysis of simulated helmeted impacts with an anthropometric test device (ATD), an array of headform acceleration transducers and neck force/moment transducers, a dual high speed camera system, and helmet-fit force sensors developed in our research group based on Bragg gratings in optical fiber. To simulate impacts, an instrumented headform and flexible neck fall along a linear guide rail onto an anvil. The test bed allows simulation of head impact at speeds up to 8.3 m/s, onto impact surfaces that are both flat and angled. The headform is fit with a crash helmet and several fit scenarios can be simulated by making context specific adjustments to the helmet position index and/or helmet size. To quantify helmet retention, the movement of the helmet on the head is quantified using post-hoc image analysis. To quantify head and neck injury potential, biomechanical measures based on headform acceleration and neck force/moment are measured. These biomechanical measures, through comparison with established human tolerance curves, can estimate the risk of severe life threatening and/or mild diffuse brain injury and osteoligamentous neck injury. To our knowledge, the presented test-bed is the first developed specifically to assess biomechanical effects on head and neck injury relative to helmet fit and retention.

Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact.

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.

This protocol determines equilibrium uptake, depth of penetration and non-equilibrium diffusion rate for cationic peptide carriers in cartilage. Characterization of transport properties is critical for ensuring an effective biological response. These methods can be applied for designing an optimally charged drug carriers for targeting negatively charged tissues.

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.