This work was supported by the National Science Foundation CAREER award no. CBET-1254767 and National Institute of Allergy and Infectious Diseases grant no. R01 AI104960. We gratefully acknowledge Patrick Griffin and Dan Tran for their assistance with characterization measurements and the Texas A&amp;M Cardiovascular Pathology Laboratory for micro-CT imaging.

1. Selection and Verification of Matrix Material Properties
<ol>
	<li>Before starting the phantom fabrication process (Figure 1), find the absorption and reduced scattering coefficients for the biological tissue of interest at the imaging wavelength(s). Preliminary estimates may be found in the references35,36. However, validation of the optical coefficients might be necessary.</li>
	<li>Using the look-up tables for absorption coefficient, &#181;a, and reduced scattering coefficient, &#181;s&#39;, at 488, 535, 632, and 775 nm wavelengths (Tables 1&#8211;4 and Figures 2&#8211;3), select the concentrations of India ink and titanium dioxide (TiO2) that approximate the desired optical properties. These recipes are specific to phantoms fabricated with PDMS. As these tables provide experimental data at discrete wavelengths, optimization of the recipe may be required for the specific application.</li>
	<li>Fabricate a polydimethylsiloxane (PDMS) slab of the selected recipe for confirmation of optical properties.
	<ol>
		<li>Using a 10:1 ratio in weight of PDMS resin to curing agent, pour ingredients into the mixing cup in the following order: PDMS resin, TiO2, India ink, PDMS curing agent. 
		NOTE: Here, we test two recipes: 1) 2 mg TiO2 + 3.5 &#181;L India ink per g PDMS and 2) 1 mg TiO2 + 10 &#181;L India ink per g PDMS. For each recipe, 4.5 g PDMS resin and 0.45 g PDMS curing agent are used with the corresponding amounts of optical particles.</li>
		<li>Mix in a speed mixer (see Table of Materials) for 60 s. If TiO2 particles stick to the mixing cup (probable with high concentrations of TiO2), mix by hand to remove the particles from the base of the cup, and mix in the mixer for another 30 s.</li>
		<li>Pour the mixture into wells or Petri dishes to make thin (0.1-1 mm) slabs of the mixture.</li>
		<li>Degas the slabs for 10 min by placing them in an air-tight negative pressure chamber, then place in a pre-heated oven at 80 &#176;C for 30-60 min. Remove from oven and let cool.</li>
		<li>Remove the cooled polymer slab from its container. Trim off the edges to leave a flat, uniform slab. Measure the thickness of the slab using calipers.</li>
	</ol>
	</li>
	<li>Measure transmittance (T) and reflectance (R) of slab(s) using an integrating sphere. Additional details and instructions can be found in the Inverse-Adding Doubling (IAD) manual37.
	<ol>
		<li>Turn on the light source and spectrometer of the integrating sphere setup. Check the alignment of the system to ensure a small, collimated beam is centered on the entry and exit ports of the integrating sphere.</li>
		<li>Calibrate the integrating sphere system.
		<ol>
			<li>Turn off the source, cap the exit port of the integrating sphere, and record three dark spectra.</li>
			<li>Turn the source back on to obtain the transmission reference with the exit port capped and the entrance port empty. Record three spectra.</li>
			<li>Obtain reflectance reference measurements using reflectance standard(s). Place each standard at the exit port of the sphere. Record three spectra for each reflectance standard.</li>
		</ol>
		</li>
		<li>Measure the transmittance of the slab. With the cap on the exit port, place the slab on the entry port of the integrating sphere for the transmission measurement. Record three spectra.</li>
		<li>Measure the reflectance of the slab. Remove the exit port cap and place the slab on the exit port for the reflectance measurement. Record three spectra.</li>
	</ol>
	</li>
	<li>Determine optical properties using IAD software. A full tutorial on the software can be found in the IAD manual with the software download37,38.
	<ol>
		<li>Average the three spectra acquired for each measurement.</li>
		<li>Using the equations in the IAD manual37, convert these measurements to R and T values. If necessary, condense the files by reducing the sampling rate along the spectrum.</li>
		<li>Prepare the input .rxt file (Supplemental Material 1) for IAD with the wavelengths, reflectance, transmittance, and sample thickness as described in the IAD manual37. Using the command prompt (Windows OS) or terminal (Mac OS), navigate to the correct path. Type &#34;iad &#39;input file name&#39;&#34; to run IAD. The software will produce an output text file with the estimated optical properties.</li>
	</ol>
	</li>
	<li>If the optical properties are not within an acceptable range (~15%) of the desired values, modify the recipe accordingly and repeat steps 1.3&#8211;1.5.</li>
</ol>
2. Preparation of Dissolvable 3D Printed Internal Structure
<ol>
	<li>Design internal structure using computer aided design (CAD) software. Convert structure solid model to a stereolithography file for fabrication on a 3D printer. If available, a segmented CT scan can also be converted into a stereolithography file rather than drawing a solid model of the internal structure. 
	NOTE: The CAD file for the fractal tree structure used here is provided in Supplemental Material 2. The printer used in this paper is an extruding printer, so the part was designed to have break-away support material.</li>
	<li>Select a dissolvable material for printing, such as poly-vinyl alcohol (PVA) or high-impact polystyrene (HIPS) (see Table of Materials). Print the solid model in this dissolvable material.</li>
	<li>When printed parts are sufficiently cooled, break, dissolve, or machine the support material off of the printed part. File or sand off any large imperfections.</li>
	<li>Vapor polish the printed part to reduce surface roughness.
	<ol>
		<li>With the printed part secured in a vice, drill a through hole with clearance for a thin steel or nitinol wire in the base of the printed part.</li>
		<li>Thread a stainless steel or nitinol wire through the hole. Bend ends of wire and hook together. This will allow for the part to be fully immersed in acetone vapor within the beaker. Set wire and part aside.</li>
		<li>Fill a large beaker roughly 10% full of acetone. Place beaker on a hot plate while heating to 100 &#176;C. CAUTION: Perform this step in a fume hood to prevent inhalation of acetone vapor.</li>
		<li>When acetone vapor condensation reaches about halfway up the wall of the beaker, hang the looped wire with the mock airway on a second wire and suspend in acetone vapor for 15&#8211;30 s. Ensure printed parts do not touch the beaker walls or each other (if vapor polishing multiple parts at once).</li>
		<li>Remove printed part and suspend over empty beaker or container. Let part dry for at least 4 h.</li>
	</ol>
	</li>
	<li>Verify the dimensions of the internal structure are within tolerance to the CAD design, as needed. Depending on accuracy requirements, calipers or a 3D laser scanner can be used to measure the structure.</li>
</ol>
3. Construction of Heat Resistant Mold
NOTE: Prepare a leak-proof, heat-resistant mold to form the PDMS phantom. Select a mold geometry to best fit the final phantom design. Here, a reusable rectangular mold is described.
<ol>
	<li>Design a solid model base of the mold to 3D print. This mold is designed for a phantom with a base of 1.17 cm x 1.79 cm. The base of the mold has a 1 mm thick and 5 mm deep recess with inner dimensions matching the base of the phantom. This allows the mold to sidings to be removed and the mold to be disassembled and re-used.</li>
	<li>Print a base for the mold with an inset of sufficient width to secure the sidings of the mold.</li>
	<li>Place sidings in the recesses of the mold base. Here, 1 mm thick polycarbonate sheets are used as mold sidings.</li>
	<li>Using heat resistant tape, seal the edges of the mold. It is imperative that all corners and edges are sufficiently sealed with no bubbles in the tape to prevent any leakage during the molding process.</li>
	<li>Place a polycarbonate base plate inside the mold prepared in step 3.4. This base plate is the same 1 mm thick polycarbonate sheet as the mold siding and gives the phantom base a smooth surface without the roughness of the 3D printed surface of the mold base. Glue the fully-dried vapor polished part to the base plate. Allow sufficient time for glue to dry.</li>
</ol>
4. Fabrication of Polymer Phantom
NOTE: Use the verified recipe for the bulk matrix material determined in step 1 for the specific application. The protocol here provides the steps for a healthy murine lung tissue phantom at 535 nm with &#181;s&#39; of 40 cm-1 and &#181;a of 2 cm-1. It may be useful to fabricate a second phantom with no optical particles to use as a reference in the fabrication process.
<ol>
	<li>Pour 9.1 g of PDMS resin into a plastic mixing cup. Add 20 mg of rutile TiO2, followed by 35 &#181;l of India ink. Finally add 0.91 g of curing agent to the top of the mixture. Follow the mixing protocol in step 1.3.2.</li>
	<li>Pour final polymer mixture into the heat-resistant mold.</li>
	<li>Pour a small amount of the mixture into a separate container to create a polymer slab for confirmation of material optical properties. Ensure enough polymer is poured to have a slab of at least 100 &#181;m thickness.</li>
	<li>Place both the mock airway mold and the separate slab into a bell jar for degassing. Begin vacuum process. If the polymer in the mock airway mold starts to rise, let the air back into the bell jar to burst the surface bubbles, then begin to pull air again. Repeat this process until the polymer does not rise significantly. This will take between 5-10 min depending on how much air was trapped during step 4.2. Once the PDMS no longer rises, continue to degas for another 15 min.</li>
	<li>After degassing, slowly let the air back into the chamber. Remove both the mock airway phantom and the polymer slab and place in level oven at 80 &#176;C for 2 h.</li>
	<li>Remove the phantom and slab from the oven and let cool for 20 min. Disassemble the polymer mold with a scalpel without cutting the cured polymer. Snap the base plate off of the mock-airway base.</li>
	<li>Place phantom in a heated (60 &#176;C) ~0.5 M sodium hydroxide (NaOH) base bath until the internal part is fully dissolved. An optically clear reference phantom may help to determine the dissolving time for the internal component. Once internal structure is dissolved, take phantom out of the bath and let fully dry (~24 h) before taking any optical measurements.</li>
</ol>
5. Verification of Phantom Fabrication
<ol>
	<li>Verify phantom geometry using high resolution magnetic resonance imaging (MRI) or micro-computed tomography (CT) imaging, if desired. These methods provide a 3D verification of internal structures within turbid material with axial resolutions of &#60;400 &#181;m39,40. Alternatively, an optically clear reference phantom can be optically imaged for verification that the printed part is fully dissolved and the remaining void is the correct geometry. 
	NOTE: We have verified the internal geometry of an optically opaque phantom (2 mg TiO2 + 3.5 &#181;l India ink) with micro-CT on a North Star Imaging (NSI) X50. The phantom was imaged with 20 &#181;m resolution in all dimensions (Supplemental Materials 3, 4).</li>
	<li>Verify optical properties of the phantom using the polymer slab and the integrating sphere (described in steps 1.5&#8211;1.6).</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparative+Study+of+Trabecular+Bone+Properties+in+the+Spine+and+Femur+Using+High+Resolution+MRI+and+CT.">A Comparative Study of Trabecular Bone Properties in the Spine and Femur Using High Resolution MRI and CT.</a> J Bone Miner Res. 13 (1), 122-132 (1998).</li><li>Batiste, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+MRI+and+micro-CT+in+an+ex+vivo+rabbit+anterior+cruciate+ligament+transection+model+of+osteoarthritis.">High-resolution MRI and micro-CT in an ex vivo rabbit anterior cruciate ligament transection model of osteoarthritis.</a> Osteoarthr cartil. 12 (8), 614-626 (2004).</li><li>Greening, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+thin+poly(dimethylsiloxane)-based+tissue-simulating+phantoms+with+tunable+reduced+scattering+and+absorption+coefficients+at+visible+and+near-infrared+wavelengths.">Characterization of thin poly(dimethylsiloxane)-based tissue-simulating phantoms with tunable reduced scattering and absorption coefficients at visible and near-infrared wavelengths.</a> J Biomed Opt. 19 (11), 115002 (2014).</li><li>Meissner, S., Knels, L., Krueger, A., Koch, T., Koch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+three-dimensional+optical+coherence+tomography+and+intravital+microscopy+for+imaging+subpleural+pulmonary+alveoli+in+isolated+rabbit+lungs.">Simultaneous three-dimensional optical coherence tomography and intravital microscopy for imaging subpleural pulmonary alveoli in isolated rabbit lungs.</a> J Biomed Opt. 14 (5), 054020 (2009).</li><li>Morris, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+nuclear+magnetic+resonance+property+of+lung.">A new nuclear magnetic resonance property of lung.</a> J Appl Phys. 58 (3), 759-762 (1985).</li><li>Hearon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+Shape+Memory+Polymers.">Porous Shape Memory Polymers.</a> Polym Rev (Phila Pa). 53 (1), 41-75 (2013).</li></ol>

The authors have nothing to disclose.

We have demonstrated a method for creating optical phantoms to represent a murine lung with an internal branching structure to simulate the internal air-tissue interface. The optical properties of murine lung tissue are achieved by incorporating unique concentrations of optically scattering and absorbing particles distributed homogenously within the bulk matrix polymer. These optical properties can be tuned to mimic the physiological values within different spectral ranges of tissues in different states (i.e. he...

Tissue phantoms are used widely for system characterization and calibration of optical imaging and spectroscopy instruments, including multimodality systems incorporating ultrasound or nuclear modalities1,2,3,4. Phantoms provide a controlled optical environment for system characterization and quality control of multiple biological imaging techniques. Tissue-mimicking phantoms are useful tools in predicting system performance and optimizing system design for the physiological task at hand; for example, to predict the probing depth of spectroscopic probes for assessing tumor margins5. Optical properties and structural design of the phantoms can be tuned to mimic the specific physiological environment in which the instrument will be used, therefore allowing for both feasibility studies and verification of system performance3,6,7. Verification of imaging system performance with realistic optical phantoms prior to entering pre-clinical or clinical trials reduces the risk of malfunction or acquisition of unusable data during in vivo studies. The reproducibility and stability of optical phantoms make them customizable calibration standards for optical techniques to monitor intra- and inter-instrument variability, particularly in multicenter clinical trials with different instruments, operators, and environmental conditions8,9.
Tissue-mimicking phantoms also serve as tunable and reproducible physical models for validation of theoretical optical models. Simulations aid in the design and optimization of in vivo optical instruments, while reducing the need for animal experiments10,11. The development and validation of optical simulations to accurately represent the in vivo environment can be encumbered by the complexity of the tissue structure, the biochemical content, and the location of the target or tissue within the body. Variability between subjects makes validation of theoretical models challenging using animal or human measurements. Polymer optical tissue phantoms allow for validation of theoretical models by supplying a known and reproducible optical environment in which to study photon migration12,13,14,15.
For the purpose of system calibration, solid optical phantoms may consist of a single homogeneous slab of cured polymer with the optical scattering, absorption, or fluorescence tuned for the wavelengths of interest. Layered polymer phantoms are frequently used to mimic the depth variance of the tissue optical properties in epithelial tissue models16,17. These phantom structures are sufficient for epithelial imaging and modeling, because the tissue structure is fairly homogeneous through each layer. However, larger scale and more complex structures affect radiative transport in other organs. Methods to create more complex phantoms have been developed to simulate the optical environment of subcutaneous blood vessels18,19 and even whole organs, such as the bladder20. Modeling light transport in the lungs provides a unique problem due to the branching structure of the air-tissue interface; a solid phantom would not likely replicate radiative transport in the organ accurately21. To describe a method for incorporating complex structure into an optical phantom, we describe a method to create an internal, reproducible fractal tree void that represents the three-dimensional (3D) macroscopic structure of the airway (Figure 1).
In the past few decades, 3D printing has become a predominant method for rapid prototyping of medical devices and models22, and optical tissue phantoms are no exception. 3D printing has been used as an additive manufacturing tool for fabricating optical phantoms with channels23, blood vessel networks24, and whole-body small animal models25. These methods use one or two printing materials with unique optical properties. Methods have also been developed to tune the optical properties of the printing material to mimic general, turbid biological tissue25,26. However, the range of achievable optical properties are limited by the printing material, usually a polymer such as acrylonitrile butadiene styrene (ABS)26, so this method is not suitable for all biological tissues. Polydimethylsiloxane (PDMS) is an optically clear polymer that can be readily mixed with scattering and absorbing particles with a higher level of tunability27,28. PDMS has also been used to mold phantoms with aneurysm models for deployment of embolic devices29,30. These phantoms also utilize a dissolvable 3D printed part, but remain optically clear for visualizing device deployment. Here, we combine this method with tunability of the optical properties of PDMS with scattering and absorbing particles to fabricate a preliminary model of the tissue and airways of the murine lung.
While the phantom presented here is specific to the lungs, the process can be applied to a variety of other organs. 3D printing of the internal structure of the phantom allows the design to be customizable for any purpose and printable scale, whether it be a blood or lymph vessel network, bone marrow, or even the four chambered structure of the heart31. Because we are interested in optical imaging and modeling of the lung32,33,34, we have opted to use a four-generation fractal tree as the internal structure to replicate within the polymer phantom. This structure was designed to approximate the branching structure of the airway and have break-away support material for the 3D printing process. A more anatomically correct airway could be printed if break-away support material is not necessary. Although this particular model represents an airway, the internal structure of the phantom does not have to remain a material void. Once the surrounding polymer is cured and the 3D printed part is dissolved, the internal structure can be used as a flow pathway or as a secondary mold for a material with its own unique absorption and scattering characteristics. For example, if the internal structure from this protocol was designed as a digital bone rather than an airway, the bone structure could be 3D printed, molded with PDMS with optical properties of the finger, and then dissolved out of the phantom. The void could then be filled with a PDMS mixture with different optical properties. Additionally, each mold is not limited to a single dissolvable part. A phantom of the finger could be created to include bone, veins, arteries, and a general soft tissue layer, each with its own unique optical properties.

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			<td height="42" style="height:42px;width:228px;">Dow Corning Sylgard 184 Silicone Encapsulant Clear 0.5 kg Kit</td>
			<td style="width:195px;">Ellsworth Adhesives</td>
			<td style="width:187px;">184 SIL ELAST KIT 0.5KG</td>
			<td style="width:259px;">Polydimethylsiloxane: polymer base for optical phantoms</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:228px;">White Rutile Titanium Dioxide powder</td>
			<td style="width:195px;">Atlantic Equipment Engineers</td>
			<td style="width:187px;">TI-602</td>
			<td style="width:259px;">Scattering particles for optical phantoms</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Higgins Fountain Pen India Ink</td>
			<td style="width:195px;">Michaels Craft Stores</td>
			<td style="width:187px;">&#160;10015483</td>
			<td style="width:259px;">Absorbing particles for optical phantom</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Heat Resistant tape</td>
			<td style="width:195px;">Uline</td>
			<td style="width:187px;">S-7595</td>
			<td style="width:259px;">Heat resistant tape for polymer molds</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Fortus 360mc 3D printer</td>
			<td style="width:195px;">Stratasys</td>
			<td style="width:187px;">N/A</td>
			<td style="width:259px;">Able to switch build and support material with this model printer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:228px;">ABS Ivory Model Material</td>
			<td style="width:195px;">Stratasys</td>
			<td style="width:187px;">SDS-000001</td>
			<td style="width:259px;">Material for printing mold parts and/or using as support for printing internal structure&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">SR-30 Soluble Support</td>
			<td style="width:195px;">Stratasys</td>
			<td style="width:187px;">400638-0001</td>
			<td style="width:259px;">Base soluble support material for printing internal structure</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Flacktek Speedmixer</td>
			<td style="width:195px;">Flacktek Inc.</td>
			<td style="width:187px;">DAC 150.1 FV</td>
			<td style="width:259px;">For efficient mixing of polymer and particles&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Integrating sphere</td>
			<td style="width:195px;">Edmund Optics</td>
			<td style="width:187px;">58-585</td>
			<td style="width:259px;">For measuring optical properties</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Polycarbonate build plates (1 mm)</td>
			<td style="width:195px;">Stratasys</td>
			<td style="width:187px;">N/A</td>
			<td style="width:259px;">Used polycarbonate build plates from Stratasys printer can also be used</td>
		</tr>
	</tbody>
</table>

fabrication and characterization of optical tissue phantoms containing macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

To demonstrate the phantom fabrication technique, mouse lung tissue phantoms were fabricated to simulate measured optical properties of excised healthy and inflamed murine lung tissue at 535 nm (Table 5). This wavelength of interest is the excitation wavelength for tdTomato fluorescent protein used in recombinant reporter strains of mycobacteria in previous studies33. Optical measurements of mouse lung tissue were obtained with the same methods des...

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Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

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10.4K Views.  Texas A&M University. Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Video: Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges1,2. These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production3. In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (&lambda;=532 nm, Fluence~110 J/cm2, spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 &plusmn; 6 kPa, (n=30). The adhesion strength drops to 2 &plusmn; 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.

Sutures are usually needed to repair tissue during surgical procedures. However, their application can be problematic as they are invasive and may damage tissue. The fabrication and application methods of a novel tissue adhesive are here reported. This adhesive film is laser-activated and does not require the use of sutures.

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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.

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 ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the tissue motion and infer or quantify the underlying biomechanical characteristics. For abdominal aortic aneurysms (AAA), biomechanical properties such as changes in the tissue's elastic modulus and estimates of the tissue stress may be essential for assessing the need for the surgical intervention. Abdominal aortic aneurysms US elastography could be a useful tool to monitor AAA progression and identify changes in biomechanical properties characteristic of high-risk patients.
A preliminary goal in the development of an AAA US elastography technique is the validation of the method using a physically relevant model with known material properties. Here we present a process for the production of AAA tissue-mimicking phantoms with physically relevant geometries and spatially modulated material properties. These tissue phantoms aim to mimic the US properties, material modulus, and geometry of the abdominal aortic aneurysms. Tissue phantoms are made using a polyvinyl alcohol cryogel (PVA-c) and molded using 3D printed parts created using computer aided design (CAD) software. The modulus of the phantoms is controlled by altering the concentration of PVA-c and by changing the number of freeze-thaw cycles used to polymerize the cryogel. The AAA phantoms are connected to a hemodynamic pump, designed to deform the phantoms with the physiologic cyclic pressure and flows. Ultra sound image sequences of the deforming phantoms allowed for the spatial calculation of the pressure normalized strain and the identification of mechanical properties of the vessel wall. Representative results of the pressure normalized strain are presented.

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the ...