The work was funded by NSF CAREER award grant #1751050.

The protocol provided in this article presents a method for determining the mechanical properties of the murine vagina and cervix. The mechanical properties analyzed in this protocol include both the passive and basal tone conditions of the organs. Passive and basal tone conditions are induced by altering the biochemical environment in which the organ is submerged. For this protocol, the media involved in basal testing contains calcium. Testing the basal tone condition permits isolation of the smooth muscle cell mechanic...

The vaginal wall is composed of four layers, the epithelium, lamina propria, muscularis, and adventitia. The epithelium is primarily composed of epithelial cells. The lamina propria has a large amount of elastic and fibrillar collagen fibers. The muscularis is also composed of elastin and collagen fibers but has an increased amount of smooth muscle cells. The adventitia is comprised of elastin, collagen, and fibroblasts, albeit in reduced concentrations compared to the previous layers. The smooth muscle cells are of interest to biomechanically motivated research groups as they play a role in the contractile nature of the organs. As such, quantifying the smooth muscle cell area fraction and organization is key to understanding the mechanical function. Previous investigations suggest that the smooth muscle content within the vaginal wall is primarily organized in the circumferential and longitudinal axis. Histological analysis suggests that the smooth muscle area fraction is approximately 35% for both the proximal and distal sections of the wall1.
The cervix is a highly collagenous structure, that until recently, was thought to have minimal smooth muscle cell content2,3. Recent studies, however, have suggested that smooth muscle cells may have a greater abundance and role in the cervix4,5. The cervix exhibits a gradient of smooth muscle cells. The internal os contains 50-60% smooth muscle cells where the external os only contains 10%. Mouse studies, however, report the cervix to be composed of 10-15% smooth muscle cells and 85-90% fibrous connective tissue with no mention of regional differences6,7,8. Given that the mouse model differs from the frequently reported human model, further investigations concerning the mouse cervix are needed.
The purpose of this protocol was to elucidate the mechanical properties of the murine vagina and cervix. This was accomplished by using a pressure myograph device that enables assessment of mechanical properties in the circumferential and axial directions simultaneously while maintaining native cell-matrix interactions and organ geometry. The organs were mounted on two custom cannulas and secured with silk 6-0 sutures. Pressure-diameter tests were performed around the estimated physiological axial stretch to determine the compliance and tangent moduli9. Force-length tests were conducted to confirm the estimated axial stretch and to ensure that mechanical properties were quantified in the physiological range. The experimental protocol was performed on the nonpregnant murine vagina and cervix at 4-6 months of age in estrus.
The protocol is divided into two main mechanical testing sections: basal tone and passive testing. A basal tone is defined as the baseline partial constriction of smooth muscle cells, even in the absences of external local, hormonal, and neural stimulation10. This baseline contractile nature of the vagina and cervix yields characteristic mechanical behaviors which are then measured by the pressure myograph system. The passive properties are assessed by removing the intercellular calcium that maintains the baseline state of contraction, resulting in relaxation of the smooth muscle cells. In the passive state, collagen and elastin fibers provide the dominant contributions for the mechanical characteristics of the organs.
The murine model is used extensively to study pathologies in women&#8217;s reproductive health. The mouse offers several advantages for quantifying the evolving relationships between ECM and mechanical properties within the reproductive system11,12,13,14. These advantages include short and well-characterized estrous cycles, relatively low cost, ease of handling, and a relatively short gestational time15. Additionally, the genome of laboratory mice is well-mapped and genetically-modified mice are valuable tools to test mechanistic hypotheses16,17,18.
Commercially available pressure myograph systems are used extensively to quantify the mechanical responses of various tissues and organs. Some notable structures analyzed on the pressure myograph system include elastic arteries19,20,21,22, veins and tissue engineered vascular grafts23,24, the esophagus25, and the large intestines26. The pressure myograph technology permits simultaneous assessment of properties in the axial and circumferential directions while maintaining the native cell-ECM interactions and in vivo geometry. Despite the extensive use of myograph systems in soft tissue and organ mechanics, a protocol utilizing the pressure myograph technology had not previously been developed for the vagina and cervix. Prior investigations into the mechanical properties of the vagina and cervix were assessed uniaxially27,28. These organs, however, experience multiaxial loading within the body29,30, thus quantifying their biaxial mechanical response is important.
Moreover, recent work suggests smooth muscle cells may play a potential role in soft tissue pathologies5,28,31,32. This provides another attraction of utilizing the pressure myograph technology, as it preserves the native cell-matrix interactions, thus permitting delineation of the contribution that smooth muscle cells play in physiological and pathophysiological conditions. Herein, we propose a protocol to quantify the multiaxial mechanical properties of the vagina and cervix under both basal tone and passive conditions.

<table>
	<tbody>
		<tr>
			<td>2F catheter</td>
			<td>Millar</td>
			<td>SPR-320</td>
			<td>catheter to measure cervical pressure</td>
		</tr>
		<tr>
			<td>6-0 Suture</td>
			<td>Fine Science Tools</td>
			<td>18020-60</td>
			<td>larger suture ties</td>
		</tr>
		<tr>
			<td>CaCl2 (anhydrous)</td>
			<td>VWR</td>
			<td>97062-590</td>
			<td>HBSS concentration: 140 mg/ mL</td>
		</tr>
		<tr>
			<td>CaCl2-2H20</td>
			<td>Fischer chemical</td>
			<td>BDH9224-1KG</td>
			<td> 
			KRB concentration: 3.68 g/L</td>
		</tr>
		<tr>
			<td>Dextrose (D-glucose)</td>
			<td>VWR</td>
			<td>101172-434</td>
			<td>HBSS concentration: 1000 mg/mL 
			KRB concentration: 19.8 g/L</td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>curved forceps</td>
		</tr>
		<tr>
			<td>Dumont SS Forceps</td>
			<td>Fine Science Tools</td>
			<td>11203-25</td>
			<td>straight forceps</td>
		</tr>
		<tr>
			<td>Eclipse</td>
			<td>Nikon</td>
			<td>E200</td>
			<td>microscope used for imaging</td>
		</tr>
		<tr>
			<td>Flow meter</td>
			<td>Danish MyoTechnologies</td>
			<td>161FM</td>
			<td>flow meter within the testing apparatus</td>
		</tr>
		<tr>
			<td>Force Transducer - 110P</td>
			<td>Danish MyoTechnologies</td>
			<td>100079</td>
			<td>force transducer</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>SciJava</td>
			<td>ImageJ1</td>
			<td>used to measure volume</td>
		</tr>
		<tr>
			<td>Instrument Cases</td>
			<td>Fine Science Tools</td>
			<td>20830-00</td>
			<td>casing to hold dissection tools</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Chemical</td>
			<td>97061-566</td>
			<td>HBSS concentration: 400 mg/ mL 
			KRB concentration: 3.5 g/L</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>G-Biosciences</td>
			<td>71003-454</td>
			<td>HBSS concentration: 60 mg/ mL</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>VWR</td>
			<td>97064-150</td>
			<td> 
			KRB concentration: 1.14 g/L</td>
		</tr>
		<tr>
			<td>MgCl2-6H2O</td>
			<td>VWR</td>
			<td>BDH9244-500G</td>
			<td>HBSS concentration: 100 mg/ mL</td>
		</tr>
		<tr>
			<td>MgSO4-7H20</td>
			<td>VWR</td>
			<td>97062-134</td>
			<td>HBSS concentration: 48 mg/ mL</td>
		</tr>
		<tr>
			<td>Mircosoft excel</td>
			<td>Microsoft</td>
			<td>6278402</td>
			<td>program used for spreadsheet</td>
		</tr>
		<tr>
			<td>Na2HPO4 (dibasic anhydrous)</td>
			<td>VWR</td>
			<td>97061-588</td>
			<td>HBSS concentration: 48 mg/mL 
			KRB concentration: 1.44 g/L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>97061-274</td>
			<td>HBSS concentration: 8000 mg/mL 
			KRB concentration: 70.1 g/L</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>VWR</td>
			<td>97062-460</td>
			<td>HBSS concentration: 350 mg/ mL 
			KRB concentration: 21.0 g/L</td>
		</tr>
		<tr>
			<td>Pressure myograph systems</td>
			<td>Danish MyoTechnologies</td>
			<td>110P and 120CP</td>
			<td>Pressure myograph system: 
			prorgram, cannulation device, 
			and controller unit</td>
		</tr>
		<tr>
			<td>Pressure Transducer</td>
			<td>Danish MyoTechnologies</td>
			<td>100106</td>
			<td>pressure transducer</td>
		</tr>
		<tr>
			<td>Student Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td>straight forceps</td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>91500-09</td>
			<td>micro-scissors</td>
		</tr>
		<tr>
			<td>Tissue dye</td>
			<td>Bradley Products</td>
			<td>1101-3</td>
			<td>ink to measure in vivo stretch</td>
		</tr>
		<tr>
			<td>Ultrasound transducer</td>
			<td>FujiFilm Visual Sonics</td>
			<td>LZ-550</td>
			<td>ultrasound transducer used; 256 elements, 40 MHz center frequency</td>
		</tr>
		<tr>
			<td>VEVO2100</td>
			<td>FujiFilm Visual Sonics</td>
			<td>VS-20035</td>
			<td>ultrasound used for imaging</td>
		</tr>
		<tr>
			<td>Wagner Scissors</td>
			<td>Fine Science Tools</td>
			<td>14069-12</td>
			<td>larger scissors</td>
		</tr>
	</tbody>
</table>

biaxial basal tone and passive testing of the murine reproductive system using a pressure myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

Successful analysis of the mechanical properties of the female reproductive organs is contingent on appropriate organ dissection, cannulation, and testing. It is imperative to explant the uterine horns to the vagina without any defects (Figure 1). Depending on the organ type, the cannula size will vary (Figure 2). Cannulation must be done so that the organ cannot move during the experiment but also not damage the wall of the organ during the procedure (Figure 3</...

Watch this Scientific Journal Video about Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph at JoVE.com

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

biaxial-basal-tone-passive-testing-murine-reproductive-system-using

Research

JoVE Journal

Bioengineering

9.4K Views.  Tulane University. This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

Video: Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

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 ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...