This work was supported by the&#160;United Soybean Board grant&#160;1940-352-0701-C and the U.S.&#160;Department of Agriculture\Forest&#160;Service. We appreciate the support and detailed information&#160;from Phil Humphrey of AES.

1. Preparation of substrates
<ol>
	<li>Use a substrate surface that is suitable for the application. For wood, use a sliced veneer of about 0.6 to 0.8 mm thick from a reliable producer because these veneers are used for plywood and laminated veneer lumber (LVL) manufacturing. These are obtained from a veneer supplier, as sheets of 0.6 to 0.8 mm thickness and cut into 305 mm on a side. A consistent substrate is a hard maple (Acer saccharum) face veneer because of its surface smoothness and consistent thickness, and it is a diffuse porous and high modulus hardwood. Maple face veneers are commonly used in cabinetry construction and are usually free from defects.</li>
	<li>Condition the wood, unstacked, at 21 &#176;C and 50% relative humidity (RH) for at least a day prior to use. Avoid veneers that are excessively wavy, have an uneven surface, and contain defects including discoloration. 
	NOTE: Other wood species can be used to understand the bond performance of the adhesive with these species. However, diffuse-porous hardwoods and softwoods with a gradual earlywood-to-latewood transition are recommended for their uniformity. Take care because wood can be acidic or basic or have extractives on the surface that can alter the adhesive curing process. In addition, the processing of the tree from time of cutting to the veneer production can alter the bond strength12,13. Because the ABES uses a small amount of wood, it is less affected by wood variations that occur with other tests, such as wood moisture content and veneer check depth.</li>
	<li>Ensure that the sides of the veneer are free of any loose fibers along the edge and the bonded product does not have any significant adhesive squeeze out as these will tend to overestimate the bond strength since there is no post bonding modification of the samples.</li>
</ol>
2. Preparation of specimens
<ol>
	<li>Condition the wood specimens at 21 &#176;C and 50% RH for at least a day. Check the veneer for any cracks, discoloration, or grain irregularities to be avoided when cutting the specimens.</li>
	<li>Make sure that the pneumatically driven specimen cutting device is operational.</li>
	<li>Use a special die cutter that cuts the required specimen size of 20 mm by 117 mm from 0.6 to 0.8 mm thick maple veneer (Figure 1, Table of Materials).
	<ol>
		<li>Place a piece of veneer, at least 150 mm by 300 mm, under the cutting blades so that the veneer grain is parallel with the long direction and depress the air pressure button to cut each piece of wood of 20 mm by 117 mm.</li>
		<li>Move the piece of veneer under the cutting blades to an uncut area and depress the button again to cut another piece of wood. Continue until the piece of veneer is completely cut into pieces. 
		NOTE: If the long direction of the specimen is not parallel with the grain direction, during a test early fracture can occur in the wood away from the bonded portion.</li>
	</ol>
	</li>
	<li>For materials other than wood, cut the specimens using the appropriate techniques. If the material cannot be cut with the specimen cutter, use whatever will cut the material to cut it to the required size. Due to the small bonding area, it is important that cutting be accurate and the specimens free of debris along the edges and on the bonding surfaces.</li>
</ol>
3. Operability of the equipment
<ol>
	<li>For the bonding process, make sure that the ABES equipment is operating properly according to a standard operating procedure11. The settings on the front of the ABES unit for bonding and breaking samples are: LP Press 0.2 MPa, HP Press 0.2 MPa, Pull 0.65 MPa, and Cool Air 0.2 MPa.</li>
	<li>Use air supply pressure of at least 0.62 MPa (90 psig) because pressure that is too low will cause the gripping clamps and platens to close too slowly or unevenly on the sample resulting in incorrect bond strengths (Figure 2, top).</li>
	<li>Clean the platens of any adhesive resulting from squeeze out from the prior sample. Adjust the temperature of the platens to the desired temperature and equilibrate before bonding samples.</li>
	<li>To bond wood, operate the equipment in a room that is at 21 &#176;C and 50% RH. If this is not possible, keep the conditioned specimens in a plastic bag until bonding because of the rapid change in wood moisture due to the small size of the specimens.</li>
	<li>For obtaining kinetic cure data, design the method such that the mechanical and electronic speeds are sufficient to collect data accurately as outlined in ASTM D7998-1911.</li>
</ol>
4. Bonding of specimens with the adhesive
NOTE: The application of the adhesive is a critical issue for wood adhesives because of the wide variation in viscosity and percent solids going from a lamination adhesive as in plywood to a spray able adhesive for binder applications. Wood adhesives are generally water-borne so evaporation is only a minor problem. However, water soaking into the porous wood is important.
<ol>
	<li>Spread 5 mg of the adhesive being studied over the terminal 0.5 cm sufficiently to cover the bonding area and transfer to the other specimen but without excessive squeeze out. To obtain a relatively constant adhesive spread rate, tare the wood specimen on a balance and re-weigh after adhesive application.</li>
	<li>Exercise great care in distributing the adhesive, overlapping the specimens and making sure the two specimens are aligned, since a small bonding area is used and strengths are determined as the pull force over the bonded area (Figure 2 bottom). Different bonding areas can be used, but the strength is not necessarily comparable due to variation in the mechanics of lap shear tests. 
	NOTE: The literature recommends several ways to apply the adhesive to the wood depending on the adhesive consistency. The originally recommended adhesive application method used a purpose-designed microspraying device10, but this was found to be messy, slow, and very dependent on the adhesive rheology. Although this method applied the adhesive as discrete dots as used in binder applications for particleboard and oriented strandboard, a printing method seems more reliable14. The micro-pipette application method can supply a reproducible volume of adhesive10, but it is somewhat difficult to distribute evenly. The spatula method has worked the best for obtaining an even distribution of the adhesive on the bonding area, and a microbalance for obtaining a measured amount is recommended11.</li>
	<li>Final strength data
	<ol>
		<li>Bond the specimens at 120 &#176;C for 2 min and condition them overnight at 21 &#176;C and 50% RH since the hot pressing during bonding dries out the wood. To bond the wood, lock a sample in place by closing the grips on the ABES tester, making sure that the sample is aligned with the tester. Then press the start button on the machine to have the 120 &#176;C platens press on the overlapped section for 2 min, before retracting the platens and loosening the grips so that the samples can be removed. 
		NOTE: The time and temperature for curing are dictated by the application and adhesive chemistry. The bonding temperature and time should be optimized so that the strength reaches the highest plateau by using different bonding temperatures and times to determine conditions for maximum strength. For wood bonds, testing dry shear strength is valuable, but wet testing is generally more critical to determine adhesive durability and requires a 4 hour room temperature soak of the sample in water.</li>
		<li>For testing, lock a sample in place by closing the grips on the ABES tester making sure that the sample is aligned with the tester. Then by pressing the start button, the instrument pulls on one end through a servodrive while the other end of the sample pulls on a load cell attached to the grips. This pulling continues until the bond breaks. The computer records the maximum force the sample can withstand, which is recorded as bond strength.
		<ol>
			<li>Use the same procedure for the dry and water-soaked samples. In measuring the breaking force, take care to ensure that the grips hold the wood tightly because if the adhesive is very strong, the wood might slip. If the sample breaks outside the bonded area, discard the value since this is measuring the wood strength, not the adhesive.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Kinetic strength development
	<ol>
		<li>Determine the rate of strength development of an adhesive to estimate the press time required for large scale products. Follow the same procedure as in step 4.3, except vary the temperature and time. Begin the testing of strength at 100 &#176;C platen temperature, using bonding times of 10, 30, 60, 90, 120, 150, 180, and 210 seconds. Subsequently, raise the temperature by 10 &#176;C, and repeat the bonding times until there is no longer any linear section of strength versus time at the low bonding times.</li>
		<li>After bonding, retract the platens and use the air cooling feature of the ABES to cool the sample to near room temperature and then measure the sample strength. By starting at a low pressing time and increasing first the time for subsequent samples, collect the strength versus time data until increasing time results in little or no increasing strength. Then doing the same sequence at higher temperatures will yield the resulting plot of strength versus time and the cure rate as the slope (Figure 3). 
		NOTE: The phenolic adhesive data in Figure 3a10 shows the effect of temperature on the strength development at different times. Figure 3b shows the regressed isothermal strength development rate versus temperature. To obtain the isothermal strength development, the sample was cooled before testing. A few adhesives, such as urea formaldehyde15, have an optimum bonding time and temperature before degradation starts to take place. This method can detect this problem and determine optimum conditions.</li>
	</ol>
	</li>
	<li>Heat resistance
	<ol>
		<li>If the product needs to meet a certain temperature resistance, clamp the bonded sample into the ABES unit. After the platens are heated to that temperature, for example 220 &#176;C, above which wood starts to degrade, close them onto the pre-bonded sample for 2 min and then open to measure the bond strength as in 4.3.2 to determine any thermal softening of the adhesive compared to the bonding temperature of 120&#176;C.</li>
		<li>Repeat this test except that the platens are closed on the sample for 30 min and then tested for strength to determine strength if the adhesive is thermally degraded. Release of the platens and testing strength will determine the heat resistance of the sample compared to the value before heating. This type of procedure was used to test wood adhesives16. Since the ABES uses rapid heating and can measure strength while hot without moving the sample to another machine, it can be used to differentiate between the two modes of failure (i.e., thermal softening or degradation). Thermal softening produces strength loss immediately upon heating, and is typically recoverable. Chemical degradation occurs gradually over time at high temperature and does not recover mechanical strength on cooling. 
		NOTE: Adhesive manufacturers need to differentiate whether strength loss is from thermal softening or chemical degradation, because these problems require different solutions. There are many methods which can measure softening transitions including other thermal analyses, but they do not distinguish between a change in mechanical properties and chemical structure.</li>
	</ol>
	</li>
</ol>
5. Image analysis of failed bonding surface
<ol>
	<li>Because the main objective is to determine the adhesive strength or rate of cohesive strength development, make sure that failure is within the adhesive and not with adhesion to the substrate (Figure 4) or substrate failure. If substrate failure occurs, then the adhesive has sufficient strength. Alternatively, cohesive failure in the bulk adhesive indicates adhesive weakness. However, deciding between adhesion and adhesive interphase failure can be difficult17. A variety of methods have been developed for wood analysis18.</li>
</ol>

<ol>
	<li>Lambuth, A., Pizzi, A., Mittal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+adhesives+for+wood.">Protein adhesives for wood.</a> Handbook of Adhesive Technology. , 457-477 (2003).</li><li>Keimel, F. A., Pizzi, A., Mittal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+development+of+adhesives+and+adhesive+bonding.">Historical development of adhesives and adhesive bonding.</a> Handbook of Adhesive Technology. , 1-12 (2003).</li><li>Marra, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Technology of Wood Bonding: Principles in Practice. , 454 (1992).</li><li>Dunky, M., Pizzi, A., Mittal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesives+in+the+Wood+Industry.">Adhesives in the Wood Industry.</a> Handbook of Adhesive Technology. , 511-574 (2017).</li><li>River, B. H., Vick, C. B., Gillespie, R. H., Minford, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wood+as+an+adherend.">Wood as an adherend.</a> Treatise on Adhesion and Adhesives. , (1991).</li><li>Liswell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+Wood+DCB+Specimens+Using+Southern+Yellow+Pine+for+Monotonic+and+Cyclic+Loading.">Exploration of Wood DCB Specimens Using Southern Yellow Pine for Monotonic and Cyclic Loading.</a> Engineering Mechanics. , (2004).</li><li>Frihart, C. R., Rowell, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wood+Adhesion+and+Adhesives.">Wood Adhesion and Adhesives.</a> Handbook of Wood Chemistry and Wood Composites. , 255-313 (2013).</li><li>Humphrey, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+device+to+test+adhesive+bonds.">A device to test adhesive bonds.</a> U.S. Patent. , (2003).</li><li>Humphrey, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+reactant+injection+effects+on+the+bonding+kinetics+of+thermosetting+adhesives.">Temperature and reactant injection effects on the bonding kinetics of thermosetting adhesives.</a> Wood adhesives. , (2005).</li><li>Humphrey, P. E., Frihart, C. R., Hunt, C., Moon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outline+Standard+for+Adhesion+Dynamics+Evaluation+Employing+the+ABES+(Automated+Bonding+Evaluation+System)+Technique.">Outline Standard for Adhesion Dynamics Evaluation Employing the ABES (Automated Bonding Evaluation System) Technique.</a> International Conference on Wood Adhesives 2009. , 213-223 (2010).</li><li>ASTM International. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> D 7998-19 Standard Test Method for Measuring the Effect of Temperature on the Cohesive Strength Development of Adhesives using Lap Shear Bonds under Tensile Loading, in Vol. 15.06. , (2019).</li><li>Rohumaa, A., 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=The+influence+of+felling+season+and+log-soaking+temperature+on+the+wetting+and+phenol+formaldehyde+adhesive+bonding+characteristics+of+birch+veneer.">The influence of felling season and log-soaking temperature on the wetting and phenol formaldehyde adhesive bonding characteristics of birch veneer.</a> Holzforschung. 68 (8), 965-970 (2014).</li><li>Rohumaa, A., 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=Effect+of+Log+Soaking+and+the+Temperature+of+Peeling+on+the+Properties+of+Rotary-Cut+Birch+(Betula+pendula+Roth)+Veneer+Bonded+with+Phenol-Formaldehyde+Adhesive.">Effect of Log Soaking and the Temperature of Peeling on the Properties of Rotary-Cut Birch (Betula pendula Roth) Veneer Bonded with Phenol-Formaldehyde Adhesive.</a> Bioresources. 11 (3), 5829-5838 (2016).</li><li>Smith, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+some+process+variables+on+the+lap-shear+strength+of+aspen+strands+uniformly+coated+with+pmdi-resin.">The effect of some process variables on the lap-shear strength of aspen strands uniformly coated with pmdi-resin.</a> Wood and Fiber Science. 36 (2), 228-238 (2004).</li><li>Pizzi, A., Pizzi, A., Mittal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urea-formaldehyde+adhesives.">Urea-formaldehyde adhesives.</a> Handbook of Adhesive Technology. , 635-652 (2003).</li><li>O'Dell, J. L., Hunt, C. G., Frihart, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+temperature+performance+of+soy-based+adhesives.">High temperature performance of soy-based adhesives.</a> Journal of Adhesion Science and Technology. 27 (18-19), 2027-2042 (2013).</li><li>Frihart, C. R., Beecher, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+that+lead+to+failure+with+wood+adhesive+bonds.">Factors that lead to failure with wood adhesive bonds.</a> World Conference on Timber Engineering 2016. , (2016).</li><li>Hunt, C. G., Frihart, C. R., Dunky, M., Rohumaa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+wood+bonds:+going+beyond+what+meets+the+eye.">Understanding wood bonds: going beyond what meets the eye.</a> Reviews of Adhesives and Adhesion. 6 (4), 369-440 (2018).</li><li>Frihart, C. R., Dally, B. N., Wescott, J. M., Birkeland, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-Based+Adhesives+and+Reliable+Rapid+Small+Scale+Bond+Strength+Testing.">Bio-Based Adhesives and Reliable Rapid Small Scale Bond Strength Testing.</a> International Symposium on Advanced Biomass Science and Technology for Bio-based Products. , (2009).</li></ol>

The authors have nothing to disclose.

Critical steps in the procedure are as follows: selection of substrates, preparation of specimens, operability of the equipment, and bonding of samples.
The substrate must be strong, have minimal defects (smooth, flat, no cracks and no discoloration. Unsanded, rotary cut cabinetry face veneer of a diffuse porous hardwood with sugar maple (Acer saccharum) preferred. Sanding creates a less even and more fragmented surface7. After conditioning the veneer at 21 &#1...

Wood bonding is the largest single adhesive market and has led to efficient use of forest resources. For many centuries, solid wood was used for most applications, except for furniture construction, with no test criteria except product in-use durability. However, bonded wood products became more common, starting with plywood and glulam beams, using bio-based adhesives1,2. Although these products were satisfactory at the time, the replacement of soy, casein, and blood glues by synthetic adhesives containing formaldehyde led to improved properties. The higher performance of these new adhesives led to defined testing standards with higher performance expectations than achievable with most bio-based adhesives. The synthetic adhesives also made possible the bonding of particles including sawdust to form particleboards, fibers to form fiberboards with varying densities, chips to provide oriented strandboard and parallel strand lumber, veneers to yield plywood and laminated veneer lumber, as well as finger jointed lumber, glulam, cross laminated lumber, and wood I-joists3. Each of these products have their own testing criteria4. Thus, the development of a new adhesive can require a lot of formulation work and extensive testing to determine if there is any potential for developing sufficient strength. This time-consuming testing and the complexity of wood properties and wood bonding5 has limited the development of new adhesives. In addition, the mechanical properties of wood adhesives can be different when cured between wood surfaces as opposed to neat6. Curing in contact with wood allows water and low molecular weight components from the adhesive to escape, in addition to complex interphase and chemical interactions of the adhesive with the wood3,7.
The development of the Automated Bonding Evaluation System (ABES) has been very helpful for understanding the strength development of wood adhesives because it is rapid and easy to use8,9,10. The system is an integral unit that bonds lap-shear samples and then measures the force under tension needed to break the bond. Its utility has led to development of ASTM method D7998-19 that uses this system11. Although this system was originally designed to measure adhesive strength development as a function of temperature and time, it can also measure the heat resistance of cured adhesives, as well as routine bond strength evaluation. Although the ABES test is a very useful preliminary screening tool, like any test, it has its limitations and does not replace all specific product strength and durability testing.
While there are many means of gauging the curing characteristics of adhesives, ranging from gel-time rheometry to differential scanning calorimetry, dynamic mechanical analysis, and spectroscopy of many types, only the ABES method measures the development of mechanical strength. This requires an instrument that is tightly controlled for heating, cooling, and in-place tensile testing11.

<table>
	<tbody>
		<tr>
			<td>Adhesive</td>
			<td>Supplied by user</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Normal supply house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mark II Automated Bonding Evaluation System (ABES-II)</td>
			<td>Adhesive Evaluation Systems Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatically driven sample cutting device</td>
			<td>Adhesive Evaluation Systems Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Regular spatula</td>
			<td>Normal supply house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wood supply &#8211; Hard maple</td>
			<td>Besse Forest Products Group</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

standard test method astm d 7998-19 for the cohesive strength development of wood adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

The procedure has been used extensively for the study of protein adhesives at the Forest Products Laboratory. It has been found that less than 2 MPa wet bond strength was insufficient to warrant further wood adhesive testing, while greater than 3 MPa was a promising result for further testing19. It has been shown to be useful in demonstrating sensitivity of wood processing conditions12,13. Further examples can be found in Frihart publicati...

Watch this Scientific Journal Video about Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives at JoVE.com

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

standard-test-method-astm-d-7998-19-for-cohesive-strength-development

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JoVE Journal

Bioengineering

2.8K Views.  USDA, Forest Service. We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 °C.

Video: Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary Service Support Detachments, Combat Support Hospitals, and Area Medical Laboratories to sterilize surgical instruments and to sterilize pathological specimens prior to disposal in operating rooms, emergency treatment areas, and intensive care units. The following ensemble of novel, &#8216;clean and green&#8217; chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination6,15. Specifically, the Portable Chemical Sterilizer (PCS) was invented to meet urgent battlefield needs and close critical capability gaps for energy-independence, lightweight portability, rapid mobility, and rugged durability in high intensity forward deployments3. As a revolutionary technological breakthrough in surgical sterilization technology, the PCS is a Modern Field Autoclave that relies on on-site, point-of-use, at-will generation of chlorine dioxide instead of steam. Two (2) PCS units sterilize 4 surgical trays in 1 hr, which is the equivalent throughput of one large steam autoclave (nicknamed &#8220;Bertha&#8221; in deployments because of its cumbersome size, bulky dimensions, and weight). However, the PCS operates using 100% less electricity (0 vs. 9 kW) and 98% less water (10 vs. 640 oz.), significantly reduces weight by 95% (20 vs. 450 lbs, a 4-man lift) and cube by 96% (2.1 vs. 60.2 ft3), and virtually eliminates the difficult challenges in forward deployments of repairs and maintaining reliable operation, lifting and transporting, and electrical power required for steam autoclaves.

The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally-friendly Decontamination, All-purpose (D-FEND ALL) produce aqueous chlorine dioxide in a collapsible spray bottle and other potential embodiments. These versatile decontamination technologies kill microbes in myriad diverse Dual-use applications for military and civilian consumers.

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.