Name: tape: a biodegradable hemostatic glue inspired by a ubiquitous compound in plants for surgical application
Uploaded: 2016-06-08T13:00:00
Description: Watch this Scientific Journal Video about TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application at JoVE.com

This study was supported by National Research Foundation of South Korea: Mid-career scientist grant (2014002855), and Ministry of Industry, Trade, and Natural Resources: World Premier Material Development Program. This work is also supported by in part by Center for Nature-inspired Technology (CNiT) in KAIST Institute for NanoCentury (KINC).

All animal care and experiments are performed in accordance with the ethical protocol provided by the KAIST (Korea Advanced Institute of Science and Technology) IRB (Institutional Review Board).
1. TAPE Formation
<ol>
	<li>For preparing a TA solution, place a 4 ml-sized glass vial on a magnetic stirrer, and add 1 ml of distilled water with a stirring bar. Add 1 g of tannic acid to the vial, and dissolve it in the water by gentle stirring at 200 rpm for more than 1 hr. When the TA is completely dissolved, the mix...

<ol>
	<li>Leggat, P. A., Smith, D. R., Kedjarune, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+applications+of+cyanoacrylate+adhesives:+a+review+of+toxicity.">Surgical applications of cyanoacrylate adhesives: a review of toxicity.</a> ANZ J Surg. 77 (4), 209-213 (2007).</li><li>MacGillivray, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+Sealants+and+Glues.">Fibrin Sealants and Glues.</a> J Cardiac Surg. 18 (6), 480-485 (2003).</li><li>Radosevich, M., Goubran, H. A., Burnouf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+sealant:+scientific+rationale,+production+methods,+properties+and+current+clinical+use.">Fibrin sealant: scientific rationale, production methods, properties and current clinical use.</a> Vox. Sang. 72 (3), 133-143 (1997).</li><li>Nomori, H., Horio, H., Suemasu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+and+side+effects+of+gelatin-resorcinol+formaldehyde-glutaraldehyde+(GRFG)+glue+for+preventing+and+sealing+pulmonary.">The efficacy and side effects of gelatin-resorcinol formaldehyde-glutaraldehyde (GRFG) glue for preventing and sealing pulmonary.</a> Surg. Today. 30 (3), 244-248 (2000).</li><li>Duarte, A. P., Coelho, J. F., Bordado, J. C., Cidade, M. T., Gil, M. 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D., Burks, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemostats,+sealants,+and+adhesives:+components+of+the+surgical+toolbox.">Hemostats, sealants, and adhesives: components of the surgical toolbox.</a> Transfusion (Paris). 48 (7), 1502-1516 (2008).</li><li>Erel, I., Schlaad, H., Demirel, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+structural+isomerism+and+polymer+end+group+on+the+pH-stability+of+hydrogen-bonded+multilayers.">Effect of structural isomerism and polymer end group on the pH-stability of hydrogen-bonded multilayers.</a> J Colloid Interface Sci. 361 (2), 477-482 (2011).</li><li>Shutava, T. G., Prouty, M. D., Agabekov, V. E., Lvov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+Properties+of+Layer-by-Layer+films+on+the+Basis+of+Tannic+Acid.">Antioxidant Properties of Layer-by-Layer films on the Basis of Tannic Acid.</a> Chem Lett. 35 (10), 1144-1145 (2006).</li><li>Schmidt, D. J., Hammond, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemically+erasable+hydrogen-bonded+thin+films.">Electrochemically erasable hydrogen-bonded thin films.</a> Chem Commun. 46 (39), 7358-7360 (2010).</li><li>Shutava, T., Prouty, M., Kommireddy, D., Lvov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+Responsive+Decomposable+Layer-by-Layer+Nanofilms+and+Capsules+on+the+Basis+of+Tannic+Acid.">pH Responsive Decomposable Layer-by-Layer Nanofilms and Capsules on the Basis of Tannic Acid.</a> Macromolecules. 38 (7), 2850-2858 (2005).</li><li>Erel, I., Zhu, Z., Zhuk, A., Sukhishvili, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen-bonded+layer-by-layer+films+of+block+copolymer+micelles+with+pH-responsive+cores.">Hydrogen-bonded layer-by-layer films of block copolymer micelles with pH-responsive cores.</a> J Colloid Interface Sci. 355 (1), 61-69 (2011).</li><li>Kim, B. -. S., Lee, H. -. I., Min, Y., Poon, Z., Hammond, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen-bonded+multilayer+of+pH-responsive+polymeric+micelles+with+tannic+acid+for+surface+drug+delivery.">Hydrogen-bonded multilayer of pH-responsive polymeric micelles with tannic acid for surface drug delivery.</a> Chem Commun. 45 (28), 4194-4196 (2009).</li><li>Murakami, Y., Yokoyama, M., Nishida, H., Tomizawa, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hemostasis+model+for+the+quantitative+evaluation+of+hydrogel-based+local+hemostatic+biomaterials+on+tissue+surface.">A simple hemostasis model for the quantitative evaluation of hydrogel-based local hemostatic biomaterials on tissue surface.</a> Colloids Surf B Biointerfaces. 65 (2), 186-189 (2008).</li><li>Kim, 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=TAPE:+A+Medical+Adhesive+Inspired+by+a+Ubiquitous+Compound+in+Plants.">TAPE: A Medical Adhesive Inspired by a Ubiquitous Compound in Plants.</a> Adv Funct Mater. 25 (16), 2402-2410 (2015).</li><li>Suzuki, S., Ikada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+cells+and+tissues+to+bioabsorbable+polymeric+materials:+scaffolds,+surgical+tissue+adhesives+and+anti-adhesive+materials.">Adhesion of cells and tissues to bioabsorbable polymeric materials: scaffolds, surgical tissue adhesives and anti-adhesive materials.</a> J Adhes. 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We developed an entirely new class of hemostatic adhesive named TAPE inspired by the water-resistant molecular interaction of a plant-derived polyphenolic compound, TA. TA is a representative hydrolysable tannin that has significantly attracted attention due to its anti-oxidant, anti-bacterial, anti-mutagenic, and anti-carcinogenic properties.
The process of making TAPE is extremely simple, scalable, and environmentally friendly, as it is just the one-step mixing of two aqueous solutions follo...

In a past decade, efforts have been made to replace current surgical sutures and staples to close wounds with biodegradable/bioabsorbable adhesives due to their convenience in usage and low tissue invasiveness during surgical treatments. Commercially available tissue-adhesives are classified into four types: (1) cyanoacrylate derivatives1, (2) fibrin glues formed by enzymatic conversion from fibrinogen to fibrin polymers by thrombin2,3, (3) protein-based materials such as chemically or physically cross-linked albumin and/or gelatin4,5, and (4) synthetic polymer-based ones6. Although they have been used in many clinical applications, all adhesives have their own intrinsic disadvantages and drawbacks that can be obstacles to their widespread usage. Cyanoacrylate-based glues show high adhesion strength to tissues, but their toxic by-products such as cyanoacetate and formaldehyde formed during degradation, often cause significant degrees of inflammatory responses7. Fibrin glues and albumin or gelatin-based materials have safety issues regarding the transmission of infectious components, such as viruses from animal sources: human blood plasma for fibrin glues and animals including cattle, chicken, pigs, and fish for gelatin-based glues8. Although a few synthetic polymer-based adhesives have been approved by the Federal Drug Administration (FDA), most adhesives made of synthetic polymers continue to have difficulties in minimizing the manufacturing process steps and achieving biocompatibility9. Most importantly, all glues suffer from poor mechanical and adhesion strength to wet tissues10. Recently, biomimetic tissue adhesives inspired by marine mussels11-13, geckos14, gecko with mussel15, and endoparasitic worms16 have been emerging as promising alternatives to current medical glues due to their tunable mechanical and adhesive properties with biocompatibility. However, to this day, there are still issues to be addressed before they become commercial products17.
Here, we report an entirely new type of medical glue called TAPE that is prepared by the intermolecular hydrogen bonding between a plant-derived adhesive molecule, Tannic acid (TA), and a bio-inert polymer Poly(ethylene glycol) (PEG), as its name indicates. TA is a representative hydrolysable tannin ubiquitously found during the secondary metabolism of plants. It has attracted much attention due to its anti-oxidant, anti-mutagenic, and anti-carcinogenic properties and has been shown to participate in supramolecular interactions with many polymers, such as poly(N-isopropylacrylamide) (PNIPAM) and poly(N-vinylpyrrolidone) (PVPON), to form layer-by-layer (LbL) films18-20 and drug-releasing microcapsules21-23. In this study, we discover that TA can act as an efficient water-resistant adhesive functional moiety to form a medical adhesive, TAPE. By simple mixing with TA, a non-fouling polymer PEG becomes a supramolecular glue with 2.5-fold increased adhesion strength compared with commercial fibrin glue, and this adhesion was maintained throughout up to 20 cycles of attachment and detachment, even in the presence of water. Its hemostatic ability was tested on a liver bleeding model in vivo and showed good hemostatic ability to stop bleeding within a few seconds. TAPE has its significant meaning in a related field as the first plant-derived adhesive that can reveal new insight into solving the drawbacks of current problems with bio-inspired approaches. We also expect the widespread use of TAPE in a variety of medical and pharmaceutical applications such as muco-adhesives, drug-releasing patches, wound-care dressings, and others due to its simple preparation method, scalability, tunable biodegradation rate, as well as highly wet-resistant adhesion properties.

<table>
	<tbody>
		<tr>
			<td>Tannic acid</td>
			<td>Sigma-aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene oxide), 4-arm, hydroxy terminated</td>
			<td>Aldrich</td>
			<td>565709</td>
			<td>Averge Mn ~10,000</td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>Aldrich</td>
			<td>373001</td>
			<td>Average Mn 4,600</td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Miltex</td>
			<td>33-36</td>
			<td>Diameter = 6 mm</td>
		</tr>
		<tr>
			<td>Aron Alpha&#174;</td>
			<td>Toagosei Co., Ltd.</td>
			<td></td>
			<td>Instant glue</td>
		</tr>
		<tr>
			<td>Universal testing machine (UTM)</td>
			<td>Instron</td>
			<td>5583</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>SPL life science</td>
			<td>60015</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SPL life science</td>
			<td>10090</td>
			<td>90 x 15 mm</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S5136</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Duchefa biochemie</td>
			<td>S0520.5000</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Incubating shaker</td>
			<td>Lab companion</td>
			<td>SIF6000R</td>
			<td></td>
		</tr>
		<tr>
			<td>ICR mice</td>
			<td>Orient bio</td>
			<td>Normal ICR mouse</td>
			<td>6 weeks, 30-35 g, male</td>
		</tr>
		<tr>
			<td>Tiletamine-zolazepam (Zoletil 50)</td>
			<td>Virbac</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zylazine (Rompun)</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlideTM needle (18 G)</td>
			<td>BD</td>
			<td>302032</td>
			<td>18 G</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1001 125</td>
			<td>Diameter = 125 mm</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis Flexible Pakaging</td>
			<td>PM996</td>
			<td></td>
		</tr>
	</tbody>
</table>

tape: a biodegradable hemostatic glue inspired by a ubiquitous compound in plants for surgical application

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant adhesion strength than commercial tissue adhesives. Medical adhesives have attracted great attention as potential alternative tools to sutures and staples due to their convenience in usage with minimal invasiveness. Although there are several protocols for developing tissue adhesives including those commercially available such as fibrin glues and cyanoacrylate-based materials, mostly they require a series of chemical syntheses of organic molecules, or complicated protein-purification methods, in the case of bio-driven materials (i.e., fibrin glue). Also, the development of surgical glues exhibiting high adhesive properties while maintaining biodegradability is still a challenge due to difficulties in achieving good performance in the wet environment of the body. We illustrate a new method to prepare a medical glue, known as TAPE, by the weight-based separation of a water-immiscible supramolecular aggregate formed after a physical mixing of a plant-derived, wet-resistant adhesive molecule,&#160;Tannic Acid (TA), and a well-known biopolymer, Poly(Ethylene) glycol (PEG). With our approach, TAPE shows high adhesion strength, which is 2.5-fold more than commercial fibrin glue in the presence of water. Furthermore, TAPE is biodegradable in physiological conditions and can be used as a potent hemostatic glue against tissue bleeding. We expect the widespread use of TAPE in a variety of medical settings and drug delivery applications, such as polymers for muco-adhesion, drug depots, and others.

TAPE is a supramolecular aggregate that settles down after centrifuging the mixture of two aqueous solutions containing TA (1 g/ml in distilled water) and PEG (1 g/ml in distilled water) with 2:1 volume ratio (Figure 1A). The mixing ratio is the key factor in achieving high adhesion strength; when TAPE is formed by a 2:1 mixing ratio, 20 units of the hydroxyl group (-OH) in 25 units of TA interact with each ether group (-O-) in PEG, resulting in the highest intermolecular...

Watch this Scientific Journal Video about TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application at JoVE.com

TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

We describe the simplest protocol to prepare biodegradable medical glue that has an effective hemostatic ability. TAPE is a water-immiscible supramolecular aggregate prepared by mixing of tannic acid, a ubiquitous compound found in plants, and poly(ethylene) glycol, yielding a 2.5 times greater water-resistant adhesion compared with commercial fibrin glue.

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant ...

The overall goal of this protocol is to develop a biodegradable hemostatic glue for surgical application inspired by a ubiquitous compound in plants. This method can help us understand the water resistant adhesion mechanism of biometrics, such as tissue adhesives and sealant in biomedical application. The main advantage of this technique is that the process of making TAPE is extremely simple, scalable, and environmentally friendly.First, prepare a Tannic Acid solution in a four milliliter glass vial on a magnetic stirrer. Add one milliliter of distilled water and one gram of Tannic Acid with stirring at 200 rpm. After an hour, the Tannic Acid will be completely dissolved in a brown solution.Next, add one milliliter of distilled water to one gram of PEG powder and vortex them into a white slurry. Keep the slurry at 60 degrees for 10 minutes. It should become completely clear.Then, return it to room temperature. Next, in a microcentrifuge tube, mix 329 microliters of PEG solution with 671 microliters of the Tannic Acid solution. Take the aliquat slowly as the solutions are both quite viscous.Gently blend the solutions into a honey-like mixture with a narrow spatula. Spin the mixture at 12, 300 gs for three minutes in a fixed angle rotor. Then, carefully pipette off as much of the supernatant as possible, leaving the fully formed TAPE.Store the TAPE at four to eight degrees Celsius for several weeks. To measure the adhesive strength of the TAPE, prepare a test tissue from a porcine biopsy skin punch. Remove all the fat and then prepare two six millimeter diameter test tissues.Apply commercial cyanoacrylate glue to the outer side of each tissue and attach the rods or a similar object to be gripped by the test machine. Next, apply a drop of TAPE to one side of the tissue and spread it uniformly between the inner sides of the two tissues so they are attached. Then, attach and detach the tissues several times to make a homogeneous mix that fully covers the interface.Next, attach the rods to the testing apparatus. Begin with applying a force of 20 Newtons for one minute. Next, have the rods pull apart at a rate of one millimeter per minute until the tissues are completely detached.The data is given as a force-distance curve. To test adhesion strength in the presence of water, add 20 microliters of water to the detached area between the two tissues and immediately attach the wet surfaces with the TAPE. Repeat the test as before with no other changes.First, cut an eight millimeter diameter cap off a microcentrifuge tube and weigh it. Next, fill the cap with 150 milligrams of TAPE and measure the total weight. Do not fill the cap beyond the lip, which will be needed as a barrier.Add 50 milliliters of PBS buffer to the 75 square centimeter cell culture flask. Put the loaded cap into the flask and submerge the cap. Incubate the flask at 37 degrees Celsius on an orbital shaker set to 50 rpm, but not higher.At the desired time, remove the cap and dry it with nitrogen gas. Then, weigh the cap. Next, replace the PBS in the flask and continue the incubation with gentle shaking until the next time point.From the measurements, calculate the relative remaining weight. Begin with a fully anesthetized six-week old male mouse tested with a toe pinch. Be sure to apply vet ointment to the eyes prior to beginning surgery.Now, expose the liver using a midline abdominal incision, and place filter paper of known mass under the liver to immobilize it. Next, prick the liver with an 18-gauge needle to induce bleeding. While absorbing the blood with sterile gauze, immediately put 100 microliters of TAPE on the site of the incision.For positive controls, use cyanoacrylate or a similar glue and do nothing for negative controls. No further suturing is needed. After placing the adhesive, collect the blood from the damage site onto the paper.Replace the paper every 30 second for two minutes. Measure the mass of the absorbed blood on the four filter papers. The animal should be euthanized.Several ratios of TA and PEG were tested and the ideal combination to make TAPE was a two-to-one volume ratio. Such TAPE was applied between two porcine skins with a diameter of six millimeters then tested on a tensile machine as described. The force needed to detach two porcine skins was initially measured at 200 kilopascals and increased to 250 kilopascals with repeated cycles.When the skin samples were moistened after each cycle of attachment/detachment, the tensile strength of the bond dropped to 90 kilopascals initially and decreased to 50 kilopascals after 20 cycles. The durability of TAPE was tested in small volumes diluted in PBS with shaking at physiological light conditions. Mass was retained for 21 days using the two-to-one ratio formulation.A one-to-one ratio formulation of TAPE only lasted 13 days under these conditions. Next, a hemostatic test performed in vivo was conducted. The TAPE sealed liver was compared with commercial fibrin glue.The TAPE sealed liver bled significantly less than the fibrin glue sealed liver. Once mastered, the preparation of TAPE can be done in five minutes if it is performed properly. While attempting this procedure, it is important to remember to keep the optimized ratio between Tannic Acid and PEG because it can dramatically affect the adhesion force of TAPE.After its development, this technique paved the way for researchers in the field of biometrics to explore the nature inspired way to solve current problems in developing water resistant adhesive materials. Though this method can provide insight into hemostatic materials, it can also be applied to other systems such as tissue sealant for wound healing and implantable patch for drug delivery.

tape-biodegradable-hemostatic-glue-inspired-ubiquitous-compound

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Sustained Administration of &amp;#946;-cell Mitogens to Intact Mouse Islets &lt;em&gt;Ex Vivo&lt;/em&gt; Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...