Name: a protocol for bioinspired design: a ground sampler based on sea urchin jaws
Uploaded: 2016-04-24T14:00:00
Description: Watch this Scientific Journal Video about A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws at JoVE.com

This work is supported by Multi-University Research Initiative through the Air Force Office of Scientific Research of the United States (AFOSR-FA9550-15-1-0009) (M. B. F., S. E. N., J.-Y. J., J. M). Collection of pink sea urchins was supported by the University of California Ship Funds and the US National Marine Fisheries Service (K.N.S., J.R.A.T). The authors acknowledge the following people: Prof. Jerry Tustaniwskyj for helpful suggestions during development of the bioinspired Aristotle's lantern sampler, Prof. Marc A. Meyers (UCSD, Dept. of Mechanical and Aerospace Engineering, Materials Science and Engineering Program), Prof. Robert L. Sah and Esther Cory (UCSD, Dept. of Bioengineering), and Dr. Maya deVries (Marine Biology Research Division, Scripps Institution of Oceanography). We also thank undergraduate students Sze Hei Siu, Jerry Ng and Ivan Torres for polishing urchin teeth cross-sections.

1. Biological Materials Science
<ol>
	<li>Wear personal protective equipment (i.e., gloves, safety glasses and lab coat) and follow all applicable safety procedures for using dissecting tools.</li>
	<li>Rinse off the forceps and scalpel with distilled water to use for dissection.</li>
	<li>Thaw a frozen pink sea urchin at RT for 1 hr. Place a thawed specimen in a glass dish with sufficient space to be able to maneuver the urchin and cutting tools. Turn the urchin upside down so that the teeth tips face up.
	<o...

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Sea urchins use the Aristotle&#39;s lantern (Figure 1A) for a variety of functions (feeding, boring, pivoting, etc.). The fossil record indicates that the lantern has evolved in shape and function from the most primitive cidaroid type to the camarodont type of modern sea urchins14. Cidaroid lanterns have longitudinally grooved teeth (Figure 1B, top) and non-separated muscle attachment to its pyramid structure. This limits their up and down movement and robs them of th...

The fields of biology, biological materials science, biomaterials, bioengineering and biochemistry employ the premiere scientific techniques and minds in an attempt to provide a deeper understanding of the incredible natural world. This research has explained many of the most amazing biological structures and organisms; from the intrinsic toughness of human bone1,2 to the large beak of the toucan3. However, much of this knowledge is difficult to employ in a manner that can provide a benefit to society. As a result, the tangential field of bioinspiration employs the lessons learned from nature to modern materials in order to solve common problems. Examples include superhydrophobic surfaces inspired by lotus leaves4-6, adhesive surfaces inspired by the feet of geckos and insects7,8, tough ceramics inspired by the nacre of abalone9-11 and biopsy harvesters inspired by the mouthpiece of the sea urchin, also known as the Aristotle&#39;s lantern12,13.
Sea urchins are invertebrate animals covered with spines whose habitat most commonly consists of the rocky beds on the ocean floor. The body (called a test) in the largest urchin species can be more than 18 cm in diameter; test size in pink sea urchins (Strongylocentrotus fragilis) examined in this study can grow to 10 cm diameter. The Aristotle&#39;s lantern is composed of five predominately calcium carbonate teeth supported by pyramid structures composed of mineralized tissue and arranged into a dome-like formation that enclose all but the distal grinding tips of the teeth (Figure 1A).
The muscle structure of the jaws is capable of efficient chewing and scraping even against hard ocean rocks and corals. When the jaws open, the teeth protrude outwards and when the jaws close, the teeth retract inwards in a single smooth motion. Comparison between primitive (above) and modern (below) sea urchin tooth cross-sections (Figure 1B) indicates that a keeled tooth evolved to strengthen the tooth when grinding against hard substrates. Each individual tooth has a slightly convex curvature and a T-shaped morphology in the transverse plane (normal to the growth direction) due to the longitudinally attached keel (Figure 1C, D).
Bioinspiration begins with observation of interesting natural phenomena, such as the efficient chewing motion of the Aristotle&#39;s lantern in sea urchins. This natural structure initially captivated Aristotle because it reminded him of a horn lantern with the panes of horn left out. More than two millennia later, Scarpa was fascinated by the complexity of the Aristotle&#39;s lantern that he and later Trogu mimicked the natural chewing motion using only paper and rubber bands (Figure 2A)15,16. Similarly, Jelinek was bioinspired by the chewing motion of the Aristotle&#39;s lantern and developed a better biopsy harvester that could safely isolate tumorous tissue without spreading cancerous cells (Figure 2B, C)12,13. In this case, bioinspired design was utilized to make a biomedical device that fit a specific need for a desired application.
The design protocol described here applies to a sediment sampler bioinspired by sea urchins. Through biological materials science, the natural structure of the Aristotle&#39;s lantern is characterized. Bioinspired design identifies potential applications where the natural mechanisms can be enhanced through the use of modern materials and fabrication techniques. The final design is re-examined through the prism of bioexploration to understand how the natural tooth structure evolved (Figure 3). The last bioexploration step, proposed by Porter17,18, uses engineering analysis methods to explore and explain biological phenomena. All the important steps of the bioinspiration process are presented as an example for harnessing technology, pre-approved by nature, which can be used for solving modern problems. Our protocol, motivated by previous bioinspiration procedures presented for specific applications by Arzt7, is targeted for biologists, engineers and anyone else who is inspired by nature.

<table>
	<tbody>
		<tr>
			<td>BUEHLERMET II 8 PLN 600/P1200</td>
			<td>Buehler</td>
			<td>305308600102</td>
			<td>Abrasive paper for polishing</td>
		</tr>
		<tr>
			<td>TRIDENT POLISH CLOTH 8&#34; PSA</td>
			<td>Buehler</td>
			<td>407518</td>
			<td>Polish cloth for 3 um suspension</td>
		</tr>
		<tr>
			<td>METADI SUPREME POLY SUSP,3MIC&#160;</td>
			<td>Buehler</td>
			<td>406631</td>
			<td>Polish suspension (3 um)</td>
		</tr>
		<tr>
			<td>MICROCLOTH FOR 8 IN WHEEL PSA</td>
			<td>Buehler</td>
			<td>407218</td>
			<td>Polish cloth for 50 nm suspension</td>
		</tr>
		<tr>
			<td>MASTERPREP SUSPENSION, 6 OZ</td>
			<td>Buehler</td>
			<td>636377006</td>
			<td>Polish suspension (50 nm)</td>
		</tr>
		<tr>
			<td>Skyscan 1076 micro-CT Scanner</td>
			<td>Bruker</td>
			<td></td>
			<td>Micro-CT scanner equipment</td>
		</tr>
		<tr>
			<td>Amira software</td>
			<td>FEI Visualization Sciences Group</td>
			<td></td>
			<td>Software for 3D manipulation of Micro-CT scans</td>
		</tr>
		<tr>
			<td>FEI Philips XL30</td>
			<td>FEI Philips</td>
			<td></td>
			<td>ESEM equipment for characterization of polished tooth cross-sections</td>
		</tr>
		<tr>
			<td>SolidWorks Design software</td>
			<td>Dassault Systems</td>
			<td></td>
			<td>Design software for CAD drawing bioinspired device</td>
		</tr>
		<tr>
			<td>SolidWorks Simulation software</td>
			<td>Dassault Systems</td>
			<td></td>
			<td>Simulation software for stress test of CAD drawing bioinspired device</td>
		</tr>
		<tr>
			<td>Dimension 1200es</td>
			<td>Stratasys</td>
			<td></td>
			<td>3D printer for fabrication of bioinspired device from CAD drawing</td>
		</tr>
		<tr>
			<td>ABSplus</td>
			<td>Stratasys</td>
			<td></td>
			<td>3D printer plastic</td>
		</tr>
	</tbody>
</table>

a protocol for bioinspired design: a ground sampler based on sea urchin jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

Bioinspired design of the Aristotle&#39;s lantern sampling device depends heavily upon the quality of the characterization methods used. Non-invasive techniques like &#181;-CT are helpful for analyzing the whole lantern and individual teeth to apply application specific enhancements for the bioinspired design (Figure 4). Meanwhile, the tooth microstructure can be explored via secondary electron and back-scattered electron micrographs of the polished cross-section of an in...

Watch this Scientific Journal Video about A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws at JoVE.com

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

The aim of this protocol is to examine the natural structure of the sea urchin mouthpiece, Aristotle's lantern, to develop a sediment sampling prototype using engineering analysis to test the application of biological features in bio-inspired designs. This method can help answer key questions in the bio-inspired design field. Specifically why did a keeled tooth structure evolve in sea urchins and how might it be useful for sediment sampling.The main advantage of this technique is that it provides a general framework for bio-inspired design investigations of various natural structures. Other such structures include the seahorse tail, the scutes of the boxfish, and the spines of the porcupine fish. The implications of this technically makes enhancement a proprietary surface expoloration because mineral poles are fit with bio-spar sediment samplers can efficiently collect a little samples.Generally individuals new to this method will struggle because objectives for each bio-inspired protocol will vary depending on the type of natural structure examined. After thawing the sea urchin, set it up for dissection with the tips of the teeth pointed up. And start cutting away the connective tissue around the perimeter of the Aristotle's lantern.Then, carefully lift out the lantern and rinse it off with running distilled water. Use forceps to remove remnant tissue on the outside and carefully open the lantern to extract the teeth. Alternatively, a dried lantern that is rinsed off again with water will fall apart to allow for easier access to the teeth.Now, pick up a tooth and carefully submerge it in a tube of freshly prepared epoxy. Position the curved concave side facing up and do not let the tooth touch the tube wall. Then, let the epoxy cure at room temperature for 24 hours.The next day, use a sectioning saw to remove the epoxy around the tooth down to about one cubic centimeter. Then, with a piece of coarse sandpaper number 125 or higher and using light pressure, rub the sample in one direction for five minutes. Progressively change to finer and finer grit sandpapers, up to the number 2400.For each sandpaper grit, make sure to rotate the sample 90 degrees. Use a microscope as needed to verify that the rubbing is always in the same direction for each sandpaper grit. After the sanding, moisten a microcloth with a 50 percent by volume half micron aluminina polish in water.Then rub the sample in a back and forth motion for five minutes. After polishing use particle-free tissue paper to clean off the sample, and then wrap it up in the same paper for storage. Next, characterize the tooth's microstructure using scanning electron microscopy.After applying a sputter coat, take 250 to 500x micrograph images of the sample. Next take microcomputed tomography scans of the whole pink sea urchin and the freshly dissected Aristotle's lantern. Place the samples over a moistened tissue when in the imaging chamber.For the body, scan with an isotropic voxel size of 36 microns at 100 peak kilovoltage and 100 milliamps. For the lantern, scan with an isotropic voxel size of about nine microns at 70 peak kilovoltage and 141 milliamps. Use a one point zero millimeter aluminum filter for both.Using micro CT scans and CAD modeling a bio-inspired design was produced with five curved teeth with a height of six centimeters and a diameter of eight centimeters for the closed lantern, which is five times larger than the natural Aristotle's lantern. For each component of the bio-inspired jaw produce STL files for 3D printing. Prepare the 3D printer by first loading acrylonitrile butadiene styrene plastic, and support plastic material cartridges.Then insert the modeling base onto the z platform by first aligning the tabs on the base with the slots in the tray. Now open the STL file parts and follow the display screen steps to orient and print individual lantern parts at the same time. Shown here is an example for 3D printing a joint arm.After printing the files, release the modeling base from the tabs and slide the base out of the printer along the tray guides. Then, use a metal spatula to pry the printed parts off the base. Once detached, place the printed parts into a heated base bath until the support plastic material dissolves.Now, fasten each tooth to a joint arm with a link rod using e-retaining rings on either side and assemble the bio-inspired Aristotle's lantern. Using the CAD file for the bio-inspired tooth perform a finite element modeling stress analysis test. First, open the file.Start a new study. And choose to run a static test with a fixed geometry. Then, click on the interfaces to add fixtures to the mounting holes where the pins will go.Next, set the force of the external loads. Click on the tooth grinding tip faces to apply a 45 Newton force to the edges. Now, set the gravity.Indicate in the top plane setting that the gravity force should be applied normally to the plane. Next, create a mesh on the design surface, and set the mesh density to fine. Now, run the test to compare between a tooth with and without the keel.A colored scale will show the areas of highest stress. Non-invasive imaging of the sea urchin tooth and the Aristotle's lantern were instrumental in producing a bio-inspired design. Detailed analysis of the tooth's microstructure by SEM confirmed the structural importance of the magnesium-enriched stone part in the tooth grinding tip.Plate and fiber primary elements are connected together by a matrix of secondary elements in the hardest stone region of the tooth grinding tip. The harder stone portion shown in darker gray, is composed of up to 40 percent magnesium atoms which replace the calcium atoms. Thus, the bio-inspired lantern was designed with CAD software, 3D printed and assembled.The ultimate purpose of the design is to collect sand. The importance of the keel in the design was tested using computerized stress tests on the CAD design. For 45 Newton applied force, the maximum stress experienced by the keeled tooth design was about 16 percent less.However, the keel adds only four percent to the mass of the design. When attempting a bio-inspired design protocol, it's important to start with careful observation of the natural structure in its living form, or through usage of non-invasive characterization methods, like micro CT and SEM. Following the general procedure, bio-inspired designs from other natural structures can be implemented in useful applications in a wide range of engineering fields.The final bio-exploration step based on the work of Professor Michael Porter, is especially essential for using engineering analysis methods to quantify the mechanical advantage of the keel structure in the sea urchin tooth.

a-protocol-for-bioinspired-design-ground-sampler-based-on-sea-urchin

Research

JoVE Journal

Bioengineering

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

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  ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is desired. A novel neural-machine interface (NMI) based on neuromuscular-mechanical fusion developed in our previous study has demonstrated a great potential to accurately identify the intended movement of transfemoral amputees. However, this interface has not yet been integrated with a powered prosthetic leg for true neural control. This study aimed to report (1) a flexible platform to implement and optimize neural control of powered lower limb prosthesis and (2) an experimental setup and protocol to evaluate neural prosthesis control on patients with lower limb amputations. First a platform based on a PC and a visual programming environment were developed to implement the prosthesis control algorithms, including NMI training algorithm, NMI online testing algorithm, and intrinsic control algorithm. To demonstrate the function of this platform, in this study the NMI based on neuromuscular-mechanical fusion was hierarchically integrated with intrinsic control of a prototypical transfemoral prosthesis. One patient with a unilateral transfemoral amputation was recruited to evaluate our implemented neural controller when performing activities, such as standing, level-ground walking, ramp ascent, and ramp descent continuously in the laboratory. A novel experimental setup and protocol were developed in order to test the new prosthesis control safely and efficiently. The presented proof-of-concept platform and experimental setup and protocol could aid the future development and application of neurally-controlled powered artificial legs.

Neural-machine interfaces (NMI) have been developed to identify the user's locomotion mode. These NMIs are potentially useful for neural control of powered artificial legs, but have not been fully demonstrated. This paper presented (1) our designed engineering platform for easy implementation and development of neural control for powered lower limb prostheses and (2) an experimental setup and protocol in a laboratory environment to evaluate neurally-controlled artificial legs on patients with lower limb amputations safely and efficiently.

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...