Name: fabrication and characterization of a conformal skin-like electronic system for quantitative, cutaneous wound management
Uploaded: 2015-09-02T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management at JoVE.com

This work was supported by the startup funding from the School of Engineering, Virginia Commonwealth University and some of electronic devices were prepared at the microfabrication facilities in the Wright Virginia Microelectronics Center. We acknowledge researchers who made contributions for the device and clinical data (Figures 3 and 5 in this paper), acquired from the published article10. W.-H.Y thanks Yoshiaki Hattori for the custom-made, data recording software.

The experiments for device fabrication, skin lamination, and characterization shown in Figures 1, 2, and 4 involved two volunteers, all performed in the Bio-interfaced NanoEngineering Laboratory at Virginia Commonwealth University (VCU), Richmond, VA, USA. This study was approved by the VCU Institutional Review Board (protocol number: HM20001454) and followed the research guidelines from the VCU Human Research. The device and clinical data shown in Figures 3 and 5 were acquired from the published article10 where the experiments on patients were conducted under the protocol (number: STU69718) approved by the Institutional Review Board, Northwestern University, Chicago, IL, USA.
1. Device Fabrication &#160;
NOTE: Figure 2 presents schematic illustrations for the overall fabrication process.
<ol>
	<li>Prepare a carrier substrate
	<ol>
		<li>Cut a bare 3 in&#160;silicon (Si) wafer into the desired size of the electronics by using a diamond blade. 
		NOTE: About half Si wafer gives an ideal size for the wound device.</li>
		<li>Degrease Si wafer with acetone and isopropyl alcohol (IPA). Rinse the wafer with deionized (DI) water and then dry with nitrogen and dehydrate on a hotplate at 110 &#176;C for 3 min.</li>
		<li>Prepare 11 g of polydimethylsiloxane (PDMS) mixture with 10:1 volume ratio of base and curing agent and degas the mixture in a vacuum chamber for an hour. 
		NOTE: PDMS is used for a dry pattern retrieval and transfer printing after the microfabrication, which is preferable to the wet chemical (acetone)-based approach from the previous study7.</li>
		<li>Spin coat 5 g of mixed PDMS solution on the wafer at 3,000 rpm for 1 min&#160;and fully cure on a hotplate at 150 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Deposit materials and pattern electronics
	<ol>
		<li>Treat the PDMS-coated wafer with ultraviolet (UV)/ozone by using a UV lamp (8.9 mW/cm2) for 3 min&#160;to make the surface hydrophilic. 
		NOTE: The hydrophilic surface offers uniform coating of additional layers on the PDMS.</li>
		<li>Spincoat polyimide (PI; 2 ml) on the PDMS coated wafer, by pipetting, at 4,000 rpm for 1 min&#160;to form a 1.2-&#181;m-thick layer, pre-bake on a hotplate at 150 &#176;C for 5 min, and post-bake at 250 &#176;C for 2 hr.</li>
		<li>Deposit chromium (Cr) to form a 20 nm-thick layer and then deposit copper (Cu) to form a 3 &#181;m-thick layer by using electron-beam (e-beam) evaporation (base pressure: ~1&#215;10-7 Torr, deposition pressure: ~1&#215;10-6 Torr, deposition rate: 1 &#8211; 5 &#197;/s). Monitor the film thickness by the deposition controller interface embedded in the evaporator. 
		NOTE: The thick layer of Cu provides enough levels of electrical conductivity on the microscale resistors of the device and thin Cr layer is used to promote adhesion between PI and Cu.</li>
		<li>Spincoat a photoresist (2 ml) with three steps at 900 rpm for 10 sec, 1,100 rpm for 60 sec, and 4,000 rpm for 20 sec&#160;and then cure it on a hotplate at 75 &#176;C for 30 min. 
		NOTE: The sequential steps described above were used to deposit a thick (&#62; 10 &#181;m) photoresist.</li>
		<li>Align Cu electronic patterns (sensors; fractal &#8216;Peano&#8217; design with 35 &#181;m in width and interconnects; serpentine open mesh design with 50 &#181;m in width) at the center of the Si wafer by using a UV aligner (power: 10 mW/sec) with exposure time 25 s. 
		NOTE: The fractal structures are used to provide superior mechanical stretchability, compared to merely meandering features14.</li>
		<li>Develop the photoresist in a diluted base developer (1:2 ratio of developer and DI water) for a minute, rinse with DI water, and dry with nitrogen. Inspect the patterns (Cu fractals and interconnects) using a microscope to confirm the feature size and find any defects from particulates. 
		NOTE: If there are any unwanted defects, then remove photoresist by rinsing with acetone/IPA/DI water. After drying with nitrogen, repeat the steps from 1.2.4 to 1.2.6.</li>
		<li>Etch the Cu layer on the Si wafer by immersing in a wet chemical etchant for ~6 min&#160;(10 ml; mixture of ammonium persulfate and water in ratio of 1:4; etch rate of 8 nm/sec&#160;in 40 &#176;C), rinse with DI water, and dry with nitrogen. Inspect the patterns using a microscope for any over-etched patterns. 
		NOTE: If the patterns are over-etched, it may cause unwanted sharp edges of the features, which could lead to mechanical fracture during device handling and washing process. The prior testing results showed that more than ~20% over-etching of the original patterns caused the aforementioned issues.</li>
		<li>Etch the Cr layer with reactive ion etching (RIE; pressure: 300 mTorr, power: 200 W, CF4 gas: 5 sccm, O2 gas: 10 sccm) for 5 min. Inspect the patterns. 
		NOTE: For etching of the Cr layer, the RIE process is preferable to wet chemical etching that causes unfavorable reaction with the Cu layer.</li>
		<li>Remove the remained photoresist on the metal layers by immersing the wafer in acetone (10 ml), IPA (10 ml), and DI water (20 ml), respectively. Then, dry it with nitrogen.</li>
		<li>Spincoat PI (2 ml) on the metal deposited wafer, by pipetting, at 4000 rpm for 1 min&#160;to form a 1.2-&#181;m-thick layer, pre-bake on a hotplate at 150 &#176;C for 5 min, and post-bake at 250 &#176;C for 2 hr.</li>
		<li>Spincoat a photoresist (2 ml) with three steps at 900 rpm for 10 sec, 1,100 rpm for 60 sec, and 4,000 rpm for 20 sec&#160;and then cure it on a hotplate at 75 &#176;C for 30 min.</li>
		<li>Align PI patterns to encapsulate the Cu electronics (sensors; fractal &#8216;Peano&#8217; design with 35 &#181;m in width and interconnects; serpentine open mesh design with 250 &#181;m in width) with the pre-defined Cu fractals and interconnects by using a UV aligner (power: 10 mW/sec) with exposure time 25 sec.</li>
		<li>Develop the photoresist with a diluted developer (1:2 ratio of developer and DI water) for a minute, rinse with DI water, and dry with nitrogen. Inspect the patterns using a microscope to confirm the feature size and find any defects from particulates. 
		NOTE: If there are any unwanted defects, then remove photoresist by rinsing with acetone/IPA/DI water. After drying with nitrogen, repeat the steps from 1.2.10 to 1.2.13.</li>
		<li>Etch the PI layer with RIE (pressure: 170 mTorr, power: 150 W, O2 gas: 20 sccm) for 25 min. Inspect the patterns.</li>
		<li>Remove the remained photoresist by immersing the wafer in acetone (10 ml), IPA (10 ml), and DI water (20 ml), respectively. Then, dry it with nitrogen.</li>
	</ol>
	</li>
	<li>Prepare an elastomeric membrane
	<ol>
		<li>Prepare a 10 g of encapsulating silicone mixture (1:1 volume ratio of base and curing agent) and add a black ink15 with one to one volume ratio, which is to facilitate control measurements of temperature variation on the skin using an infrared camera. 
		NOTE: The utilized silicone (clear encapsulating rubber) provides unique characteristics of low viscosity, optical clarity, and electrical isolation/protection to the device16.</li>
		<li>Spincoat 8 g of the mixture in a petri dish at 150 rpm for 1 min&#160;to form a 500 &#181;m thick elastomeric membrane and cure at RT for O/N. 
		NOTE: The material needs to be placed on a flat surface for uniform thickness.</li>
		<li>Cut the membrane into the desired size of 70 mm x 30 mm by using a sharp razor blade and gently detach it from the petri dish.</li>
	</ol>
	</li>
	<li>Retrieve and transfer electronics
	<ol>
		<li>Cut a water soluble tape (25 mm x 80 mm) and gently laminate onto the fabricated electronic patterns and place them on a hotplate at 130 &#176;C for 3 min. 
		NOTE: Temperature elevation expands the PDMS layer on the Si wafer to help dissociation of the electronic patterns from the surface.</li>
		<li>Detach the tape rapidly from the PDMS/Si wafer to retrieve the electronic patterns.</li>
		<li>Deposit a 20 nm thick Cr (for adhesion) followed by a 50 nm thick silicon dioxide (SiO2) on retrieved patterns by e-beam evaporation.</li>
		<li>Treat UV/ozone by using UV lamp (365 nm, 8.9 mW/cm2) on the targeted silicone membrane for 2 min&#160;to activate the surface.</li>
		<li>Transfer the patterns to the silicone membrane by placing the retrieved patterns on the tape to the desired location and evenly adding pressure from top side of the patterns down to the substrate. Apply water to dissolve the tape for 5 min. 
		NOTE: The described process of materials transfer is facilitated by covalent bonding (Si-O-Si) between the deposited silicon dioxide&#160;and the UV-activated silicone substrate17.</li>
		<li>Peel off the tape, rinse with DI water, and dry on a hotplate at 90 &#176;C for 1 min.</li>
	</ol>
	</li>
	<li>Encapsulate the device using a silicone membrane
	<ol>
		<li>Prepare an 10 g of encapsulating silicone mixture (1:1 volume ratio of base and curing agent).</li>
		<li>Cover the cable contact pads with a rectangular PDMS piece (22 &#215; 6 &#215; 1 mm3) by van der Waals bonding with the bottom silicone membrane, to avoid silicone coating the pads.</li>
		<li>Spincoat the 5 g of silicone mixture at 4,000 rpm for 1 min&#160;to form a 5 &#181;m thick layer on the transferred electronics and then cure at RT for O/N.</li>
	</ol>
	</li>
	<li>Connect a flexible ribbon cable for data acquisition
	<ol>
		<li>Apply liquid steel flux (0.5 ml), by pipetting, on the connector pads for 3 sec&#160;to make clean surface.</li>
		<li>Bond a thin, flexible ribbon cable on the contact points with pressure at high temperature (&#62; 60 &#176;C). A typical hair straightener offers easy handling and bonding. 
		NOTE: The micro-film cable is preferable to the conventional hard-wire soldering to avoid any mechanical fracture of the transferred metal membranes on a silicone.</li>
		<li>Check the electrical connection using a digital multimeter. The resistance value is expected less than 1 Ohm between the sensor pad with one end and the other end of the film cable (distance: ~1 cm apart).</li>
		<li>Bond the other end of the ribbon cable to a customized printed circuit board with the same strategy described in the step 1.6.2.</li>
		<li>Check the electrical connection using a digital multimeter.</li>
		<li>Connect the device with the data acquisition hardware by soldering conventional wires on PCB.</li>
	</ol>
	</li>
</ol>
2. Clinical Testing &#160;
<ol>
	<li>Clean the device using a disinfectant solution
	<ol>
		<li>Prepare 205 g of a diluted disinfectant solution (40:1 volume ratio of water and solution).</li>
		<li>Spray the solution (10 g) on the device and soak it for 10 min. 
		NOTE: The diluted disinfectant cleaner can be stored at RT.</li>
		<li>Rinse with water three times and dry it with clean tissues.</li>
	</ol>
	</li>
	<li>Set up a series of equipment for device testing
	<ol>
		<li>Prepare and connect a lock-in amplifier with a current source, a multiplexer, and the custom software installed on a laptop computer for data recording.</li>
		<li>Place an infrared camera on a tripod and focus on a target object for thermography as a reference.</li>
		<li>Set up system parameters of a lock-in amplifier to measure thermal conductivity (frequency: 1 &#38; 3 Hz; time constant: 3 and 1 sec; sensitivity: 1 mV; dynamic reserve: high reserve) and temperature (frequency: 997 Hz; time constant: 300 msec; sensitivity: 2 mV; dynamic reserve: low noise) with the applied constant current (2 mA).</li>
		<li>Connect two wound devices, prepared by microfabrication and transfer printing and mounted on wound and contralateral sites, to the multiplexer right before recording data from a patient.</li>
	</ol>
	</li>
	<li>Record temperature and thermal conductivity 
	NOTE: The data acquisition software is custom-made, which can remote control the lock-in amplifier for real-time data monitoring and saving. In the temperature measurement, each data point is measured every 300 msec for 20 sec. The set of data for the first 10 sec&#160;and next 10 sec&#160;are used to calculate the average temperature value and standard deviation, respectively. The recorded data are then saved as a comma-separated value file, which is used to plot a graph for comparison with data from infrared thermography. In the measurement of thermal conductivity, the 3&#937; signals are directly read from the hardware screen (amplifier), which is then utilized to calculate the thermal conductivity analytically.
	<ol>
		<li>Gently rub the device application sites on the skin using antiseptic alcohol wipes10.</li>
		<li>2.3.2) Laminate two devices on the desired skin locations by gently pressing the device to the skin with fingers to facilitate the soft bonding: one on the surgical wound site and the other on the contralateral location as a reference.</li>
		<li>Measure the electrical voltage (3&#937;), related to thermal conductivity, of the device by starting the data acquisition.</li>
		<li>Evaluate the obtained data to verify the conformal contact of the device to the skin; abnormal value (&#60; 0.1 W/mK) shows bad contact of the device.</li>
		<li>Measure the electrical resistance to determine temperature distribution and record data through the custom software.</li>
		<li>Take optical and IR images of two devices on the skin.</li>
		<li>Compare temperature values from the IR images with the data recorded from the wound device (2.3.5). Add both values to separate columns in a customized spreadsheet.</li>
	</ol>
	</li>
	<li>Analyze the recorded data
	<ol>
		<li>Export the recorded data to the customized template to automatically calculate temperature and thermal conductivity from an array of sensors in the device.</li>
		<li>Plot the data (temperature and thermal conductivity according to the sensor location on a different time scale) for comparison over the course of one month (four sets of data on day 1, 3, 15, and 30).</li>
		<li>Analyze the data by comparing a series of temperature and thermal conductivity data according to time; Values with sudden elevation or drop tell the change of wound healing phase and/or unexpected abnormality on wound sites.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

This article highlights the methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination near the wound tissues, which offers quantitative measurement of skin temperature and thermal conductivity mapping on the skin. 
The key features include the utilization of novel techniques of materials transfer printing and hard-soft materials integration to design and develop the flexible/stretchable, soft electronic device. The use of biocompatible, elec...

In clinical study and biomedical research, monitoring of wound healing has focused on an invasive method that is based on the histological evaluation of tissue morphologic change in wounds1,2. Recently, rapid advancements in electronic technologies enable the development of high-precision imaging and analysis tools that can visually inspect the wound healing process via digital imaging3,4 or confocal scanning microscopy and spectroscopy4,5. However, those imaging approaches require high cost, complicated optical tools and operations, and more importantly, patients need to be immobilized during testing. Therefore, there exists a need for new devices and systems that are quantitative, non-invasive, easy-to-use, inexpensive, and multifunctional to offer more accurate wound management.
Here, we introduce a skin-like electronic system that provides precise, real-time mapping of temperature and thermal conductivity and delivers a precise level of heating at wound sites via conformal lamination of the device non-invasively. This device presents a class of technology, skin-mounted epidermal electronic systems that are designed to match to mechanical and material properties (total thickness, bending stiffness, effective moduli, and mass density) of the epidermis6-9.
The device is designed in a biocompatible, skin-friendly, water-proof, and reusable form that can be washed and disinfected for clinical applications on patients10. The conformal electronic device mounted near the wound tissues captures the inflammation phase (one of wound healing process), caused by increased blood flow and enzymatic reactions to the wound11,12, through quantitative recording of temperature8 and thermal conductivity13, correlated to hydration. Experimental and computational studies determine an optimal mechanics design to accommodate natural motions and applied strains without mechanical fracture and capture the underlying physics of stretching mechanics of the skin-like electronics that laminates conformally on the skin surface, which offers acquisition of high fidelity signals.
The protocols described in this article present the methods of microfabrication for skin-like electronic systems, testing preparation including device cleaning, equipment setup in a clinical setting, and clinical applications for quantitative monitoring of temperature and thermal conductivity on cutaneous wounds.

<table><tbody><tr><td>3&quot; Silicon wafer</td><td>University Wafer, USA</td><td></td><td>Use as carrier to fabricate the device</td></tr><tr><td>Acetone</td><td>Fisher Scientific, USA</td><td>A18-1</td><td>Use to clean a wafer and to remove photoresist</td></tr><tr><td>Isopropanol (IPA)</td><td>Fisher Scientific, USA</td><td>A459-1</td><td>Use to clean a wafer</td></tr><tr><td>AZ4620 photoresist</td><td>AZ Electrionic Materials, USA</td><td></td><td>Use to make patterns on metals and polymers</td></tr><tr><td>AZ400K developer</td><td>AZ Electrionic Materials, USA</td><td></td><td>Use to develop AZ4620 photoresist</td></tr><tr><td>Chromium etchant</td><td>Transene, USA</td><td>1020AC</td><td>Use to etch Cr layer of device</td></tr><tr><td>Copper etchant</td><td>Transene, USA</td><td>ASP-100</td><td>Use to etch Cu layer of device</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit (PDMS)</td><td>Dow Corning, USA</td><td>39100000</td><td>Use as a substrate for &#039;dry&#039; retrieval</td></tr><tr><td>PI2545 polyimide</td><td>HD MicroSystem, USA</td><td></td><td>Use to encapsulate metal layer</td></tr><tr><td>Solaris</td><td>Smooth-On, USA</td><td></td><td>Use as substrate and to encapsulate device</td></tr><tr><td>Petridish</td><td>Carolina, USA</td><td>741255</td><td>Use as mold to make substrate</td></tr><tr><td>Water-Soluble Wave Solder Tape 5414</td><td>3M, USA</td><td>AM000000217</td><td>Use to retrive device from PDMS layer</td></tr><tr><td>High Activity Liquid Stainless Steel Flux</td><td>Worthington, USA</td><td>331929</td><td>Use to remove oxidation layer on Cu</td></tr><tr><td>Flexible, micro-film cable</td><td>Elform, USA</td><td></td><td>Use to make the electrical connection between the electronic device and the data acquisition system</td></tr><tr><td>pH Neutral Cleaner</td><td>Australian Gold, USA</td><td></td><td>Use as disinfectant solution to clean device in clinical testing</td></tr><tr><td>Solder</td><td>Kester, USA</td><td>24-6337-9703</td><td>Use as material to solder hard wires</td></tr><tr><td>Ultraviolet lamp</td><td>Cole-Parmer, USA</td><td>97600-00</td><td>Use to activate PDMS layer as hydrophilic surface</td></tr><tr><td>Multiplexer</td><td>FixYourBoard, USA</td><td>U802</td><td>Use to acquire measurements from six sensing components&amp;nbsp;</td></tr><tr><td>DC/AC current source</td><td>Keithley, USA</td><td>6221</td><td>Use to supply current</td></tr><tr><td>SMD Digital Hot Air Rework Station</td><td>Aoyue, China</td><td>968A+</td><td>Use to solder hard wires, to electrically connect between the device and external instruments</td></tr><tr><td>Infrared camera</td><td>FLIR, USA</td><td>435-0001-01-00</td><td>Use to take infrared images in experiment</td></tr><tr><td>Digital multimeter</td><td>Fluke, USA</td><td>117</td><td>Use to check electrical connection</td></tr><tr><td>Lock-in amplifier</td><td>Stanford Research System, USA</td><td>SR830</td><td>Use to perform four-point-probe-measurement</td></tr><tr><td>Electron beam evaporator</td><td>9 scale Vacuum Products, USA</td><td></td><td>Use to deposit thin films (Cu and SiO&lt;sub&gt;2&lt;/sub&gt;)</td></tr></tbody></table>

fabrication and characterization of a conformal skin-like electronic system for quantitative, cutaneous wound management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

Figure 1 presents an overview of the characteristics of the conformal, skin-like electronic system, designed for quantitative, cutaneous wound management on patients. The multifunctional electronic device consists of microscale fractal structures3,14 and filamentary serpentine traces9,17 on a thin elastomeric membrane that offers exceptional mechanical stretchability and bendability. The compliant device that is completely enclosed by silicone layers enables gentle, reversible lamin...

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Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

fabrication-characterization-conformal-skin-like-electronic-system

Research

JoVE Journal

Bioengineering

8.8K Views.  Virginia Commonwealth University. This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management. 

Video: Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Medicine

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Developmental Biology

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Conformable Wearable Electrodes: From Fabrication to Electrophysiological Assessment

Wearable electronic devices are becoming key players in monitoring the body signals predominantly altered during physical activity tracking. Considering the growing interest in telemedicine and personalized care driven by the rise of the Internet of Things era, wearable sensors have expanded their field of application into healthcare. To ensure the collection of clinically relevant data, these devices need to establish conformable interfaces with the human body to provide high-signal-quality recordings and long-term operation. To this end, this paper presents a method to easily fabricate conformable thin tattoo- and soft textile-based sensors for their application as wearable organic electronic devices in a broad spectrum of surface electrophysiological recordings. 
The sensors are developed through a cost-effective and scalable process of cutaneous electrode patterning using poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS), the most popular conductive polymer in bioelectronics, on off-the-shelf, wearable substrates. This paper presents key steps in electrode characterization through impedance spectroscopy to investigate their performance in signal transduction when coupled with the skin. Comparative studies are required to position the performance of novel sensors with respect to the clinical gold standard. To validate the fabricated sensors' performance, this protocol shows how to perform various biosignal recordings from different configurations through a user-friendly and portable electronic setup in a laboratory environment. This methods paper will allow multiple experimental initiatives to advance the current state of the art in wearable sensors for human body health monitoring.

Two recent technologies-tattoo and textiles-have demonstrated promising results in cutaneous sensing. Here, we present the fabrication and evaluation methods of tattoo and textile electrodes for cutaneous electrophysiological sensing. These electronic interfaces made of conductive polymers outperform the existing standards in terms of comfort and sensitivity.

Wearable electronic devices are becoming key players in monitoring the body signals predominantly altered during physical activity tracking. Considering ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Immunology and Infection

Developing a Long-Term Swine Model for Biofilm-Infected Burn Wound Studies

Swine Model of Biofilm Infection and Invisible Wounds

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the involvement of the host immune system. Iterative changes in the host and pathogen during the formation of such clinically relevant biofilm can only occur in vivo. The swine wound model is recognized for its advantages as a powerful pre-clinical model. There are several reported approaches for studying wound biofilms. In vitro and ex vivo systems are deficient in terms of the host immune response. Short-term in vivo studies involve acute responses and, thus, do not allow for biofilm maturation, as is known to occur clinically. The first long-term swine wound biofilm study was reported in 2014. The study recognized that biofilm-infected wounds may close as determined by planimetry, but the skin barrier function of the affected site may fail to be restored. Later, this observation was validated clinically. The concept of functional wound closure was thus born. Wounds closed but deficient in skin barrier function may be viewed as invisible wounds. In this work, we seek to report the methodological details necessary to reproduce the long-term swine model of biofilm-infected severe burn injury, which is clinically relevant and has translational value. This protocol provides detailed guidance on establishing an 8 week wound biofilm infection using P. aeruginosa (PA01). Eight full-thickness burn wounds were created symmetrically on the dorsum of domestic white pigs, which were inoculated with (PA01) at day 3 post-burn; subsequently, noninvasive assessments of the wound healing were conducted at different time points using laser speckle imaging (LSI), high-resolution ultrasound (HUSD), and transepidermal water loss (TEWL). The inoculated burn wounds were covered with a four-layer dressing. Biofilms, as established and confirmed structurally by SEM at day 7 post-inoculation, compromised the functional wound closure. Such an adverse outcome is subject to reversal in response to appropriate interventions.

Author Spotlight: Studying Host-Microbe Interactions in Wound Biofilm Formation 

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the ...