This work was supported by the EC within the framework of the STIFF-FLOP FP7-ICT-2011.2.1 European Project (#287728).

Note: This protocol describes the fabrication phases of a single module, which includes the fluidic chambers, stiffening channel, actuation pipelines and external sheath. The following procedure has to be executed under a fume hood and wearing lab coat and gloves for safety reasons. As previously mentioned, the fabrication process of the elastomeric unit is based on the sequential use of molds designed with CAD software. They are composed of the 13 pieces shown in Figure 2 and listed in Table 1.
1. Preparation of the Silicone
<ol>
	<li>Weigh 12 g of part A and 12 g of part B in the same plastic glass or Petri dish and mix them together, stirring. 
	Note: Material proportions can vary depending on the specific silicone used, in this case it consists of two parts: part A (the base) and part B (the catalyst). They are used in proportion 1A:1B in weight.</li>
	<li>Place the glass containing the mixed silicone materials in a degasser machine at 1 bar vacuum pressure. Keep the glass under vacuum until all the bubbles are removed from the silicone material. For the employed silicone the degassing process takes about 10 min. Once the materials are completely free from the presence of bubbles, restore the atmospheric pressure into the machine and use the silicone.</li>
</ol>
2. Fabrication of the Siliconic Module
<ol>
	<li>Assembly of the mold.
	<ol>
		<li>Insert the stiffening cylinder and the top of the chambers into cap_A (Figure 3a).</li>
		<li>Close the shells around the second layer of cap_A.</li>
	</ol>
	</li>
	<li>First silicone casting.
	<ol>
		<li>Pour the silicone inside the assembled mold up to the edge of the shells (Figure 3b).</li>
		<li>Place the mold in an oven at 60 &#176;C for about 30 min.</li>
	</ol>
	</li>
	<li>Rearrangement of the mold.
	<ol>
		<li>Remove the external shells and cap_A (Figure 3c).</li>
		<li>Insert the cylinders from the bases of the chambers and the stiffening cylinder inside cap_B (Figure 3d).</li>
		<li>Close the shells again around the module, sliding them of 10 mm upward in order to have a gap of 10 mm between the top surface of the module and the edges of the shells (Figure 3e).</li>
	</ol>
	</li>
	<li>Second silicone casting.
	<ol>
		<li>Pour the silicone inside the rearranged mold up to the edge of the shells on the top side (i.e. also up to the stiffening cylinder) (Figure 3f).</li>
		<li>Put the mold into an oven at 60 &#176;C for about 30 min.</li>
		<li>Remove the external shells, cap_B and the chambers (except the stiffening cylinder) (Figure 3g).</li>
	</ol>
	</li>
</ol>
3. Insertion of the Tubes
<ol>
	<li>Cut 3 tubes to the same desired length (300 mm for example).</li>
	<li>Put siliconic glue around one end of each tube for 10 mm, without obstructing the tubes.</li>
	<li>Insert the tubes inside the 2 mm dedicated channels in the siliconic unit (Figure 3h).</li>
	<li>Allow a curing time of 12 min at room temperature or put the module inside an oven at a higher temperature (50&#176; - 60&#176;) to speed up the drying process.</li>
</ol>
4. Fabrication of the Crimped Braided Sheath
<ol>
	<li>Cut 700 mm of an expandable braided sheath (about 15 times the height of the module).</li>
	<li>Insert a metallic cylinder of 30 mm in diameter and 250 mm in length inside the sheath.</li>
	<li>Push down and force the sheath by sliding over the cylinder, in order to create crimps.</li>
	<li>Mechanically fix the sheath in place with a clamp and heat with a heating gun at 350 &#176;C for 2-3 min until a permanent deformation is obtained.</li>
	<li>Let the sheath cool down and remove the internal cylinder.</li>
</ol>
5. Integration of the External Sheath
<ol>
	<li>Pass the tubes through the holes of cap_C.</li>
	<li>Pour 3 g of silicone into cap_C.</li>
	<li>Clamp cap_C to a support that is higher than the work plane.</li>
	<li>Insert the bottom side of the module previously fabricated into cap_C.</li>
	<li>Slide the crimped sheath around the module.</li>
	<li>Push the first crimps of the sheath inside cap_C and dip them into the freshly poured silicone (Figure 3i).</li>
	<li>Put the mold into an oven at 60 &#176;C for about 20 min.</li>
	<li>Repeat the same procedure from point 5.1-5.6 to fix the sheath at the top side, using cap_D (Figure 3j).</li>
	<li>Remove cap_C and cap_D.</li>
	<li>Remove the central cylinder (Figure 3k).</li>
</ol>
6. Fabrication of the Granular Jamming Membrane
<ol>
	<li>Pour 5 g of liquid latex into a plastic glass.</li>
	<li>Immerse the cylinder for the membrane (last piece shown in Figure 2) inside the liquid latex until the surface is completely covered.</li>
	<li>Let it dry under a hood for 20 min.</li>
	<li>Repeat points 6.2 and 6.3.</li>
	<li>Remove the membrane from the mold.</li>
</ol>
7. Insertion of the Granular Jamming Membrane
<ol>
	<li>Cut a tube (2 mm in diameter) to the desired length (300 mm for example).</li>
	<li>Cut a squared piece of about 100 mm2 of nylon tissue and close one end of the tube with this tissue using a plastic paraffin film or superglue.</li>
	<li>Weigh 4 g of coffee powder and fill the membrane.</li>
	<li>Insert the tube (the end with the filter) inside the filled membrane and fix it around the tube using a plastic paraffin film.</li>
	<li>Apply a vacuum on the other side of the tube (the membrane becomes stiffer).</li>
	<li>Insert the membrane inside the empty central channel of the siliconic module (Figure 3l).</li>
	<li>Glue the ends of the stiffening membrane to the silicone module.</li>
	<li>Close the rings around the top side of the module (Figure 3m).</li>
	<li>Pour 2 g of silicone into the rings in order to level the surface.</li>
	<li>Let the silicone dry under hood or in an oven at 60&#176;.</li>
	<li>Remove the rings.</li>
	<li>Repeat from points 7.8 to 7.11 for the bottom side (Figure 3n).</li>
</ol>

<ol>
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S., Dasgupta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+Robotics.">Flexible Robotics.</a> BJU International. 107, 187-189 (2011).</li><li>Degani, A., Choset, H., Wolf, A., Zenati, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+articulated+robotic+probe+for+minimally+invasive+surgery.">Highly articulated robotic probe for minimally invasive surgery.</a> Proceedings of IEEE International Conference on Robotics and Automation. , 4167-4172 (2006).</li><li>Bajo, A., Dharamsi, L. M., Netterville, J. L., Garrett, C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trunks,+tongues,+and+tentacles:+Moving+with+skeletons+of+muscle.">Trunks, tongues, and tentacles: Moving with skeletons of muscle.</a> American Scientist. 77, 28-35 (1989).</li><li>Walker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+issues+in+creating+&#8220;invertebrate&#8221;+robots.">Some issues in creating &#8220;invertebrate&#8221; robots.</a> , (2000).</li><li>McMahan, W., Jones, B., Walker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+a+multi-section+continuum+robot:+Air-octor.">Design and implementation of a multi-section continuum robot: Air-octor.</a> IEEE/RSJ International Conference on Intelligent Robots and Systems. , 2578-2585 (2005).</li><li>Laschi, C., Mazzolai, B., Cianchetti, M., Margheri, L., Follador, M., Dario, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Soft+Robot+Arm+Inspired+by+the+Octopus.">A Soft Robot Arm Inspired by the Octopus.</a> Advanced Robotics (Special Issue on Soft Robotics). 26, 709-727 (2012).</li><li>Chianchetti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+robotics+technologies+to+address+shortcomings+in+today&#8217;s+minimally+invasive+surgery:+the+STIFF-FLOP+approach.">Soft robotics technologies to address shortcomings in today&#8217;s minimally invasive surgery: the STIFF-FLOP approach.</a> Soft Robotics. 1, 122-131 (2014).</li><li>Cheng, N. G., 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=Design+and+Analysis+of+a+Robust,+Low-cost,+Highly+Articulated+manipulator+enabled+by+jamming+of+granular+media.">Design and Analysis of a Robust, Low-cost, Highly Articulated manipulator enabled by jamming of granular media.</a> , 4328-4333 (2012).</li><li>Cianchetti, M., Ranzani, T., Gerboni, G., De Falco, I., Laschi, C., Menciassi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIFF-FLOP+Surgical+Manipulator:+mechanical+design+and+experimental+characterization+of+the+single+module.">STIFF-FLOP Surgical Manipulator: mechanical design and experimental characterization of the single module.</a> Proceedings of IEEE/RSJ International Conference on Intelligent Robots and Systems (IROS. , 3576-3581 (2013).</li><li>De Falco, I., Cianchetti, M., Menciassi, 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+soft+and+controllable+stiffness+manipulator+for+minimally+invasive+surgery:+preliminary+characterization+of+the+modular+design).">A soft and controllable stiffness manipulator for minimally invasive surgery: preliminary characterization of the modular design).</a> Proceedings of 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC). , (2014).</li><li>De Falco, I., Cianchetti, M., Menciassi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIFF-FLOP+surgical+manipulator:+design+and+preliminary+motion+evaluation).">STIFF-FLOP surgical manipulator: design and preliminary motion evaluation).</a> Proceedings of 4th WorkShop on Computer/Robot Assisted Surgery (CRAS). , 131-134 (2014).</li></ol>

The authors have nothing to disclose.

The technique described in this protocol enables the fabrication of a pneumatically actuated soft unit usable for modular compliant structures. Thanks to the design of the molds and their simple assembly, it is possible to fabricate one complete module in about 4 hours with 7 main steps. The process of fabrication involves specific materials, which are easily available, and work should be carried out under a fume hood. An external set up including air valves, air compressor and vacuum pump is necessary to activate the mo...

Recent trends in the medical field are pushing for a reduction in the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully improved in the last few years for abdominal operations. MIS procedures are based on the use of tools introduced through four or five access points (trocars) placed on the abdominal wall. In order to reduce the number of trocars, the instruments can be inserted by Single Port Laparoscopy (SPL) or Natural Orifice Translumenal Endoscopic surgery (NOTES)1. These procedures prevent external visible scars, but increase the difficulty for the clinicians in executing the surgery. This limitation is mainly due to the reduced points of access and to the rigid and semi-rigid nature of the instruments, which are not able to avoid or pass around organs2, 3. Dexterity and motility can be improved using articulated and hyper-redundant robots which can cover a wider and more complex workspace, thus enabling a specific target in the body to be reached more easily4, 5, 6 and to work as retraction systems when necessary7. A flexible manipulator can improve tissue compliance, thus making contact safer than by traditional tools.
However, these manipulators often lack stability when the target is reached and generally they cannot control the contact with the surrounding tissues8, 9. Studies on biological structures, such as the octopus arm10 and the elephant trunk11, have recently inspired the design of flexible, deformable and compliant manipulators with a redundant number of Degrees of Freedom (DoFs) and controllable stiffness12. These kinds of devices utilize passive springs, smart materials, pneumatic elements, or tendons13, 14, 15. Generally, manipulators fabricated with soft and flexible materials do not guarantee the generation of high forces.
The STIFF-FLOP (STIFFness controllable Flexible and Learnable manipulator for surgical OPerations) manipulator has been recently presented as a novel surgical device for NOTES and SPL inspired by the octopus&#8217;s capabilities. In order to overcome the limitations of previous soft manipulators, it has a soft body as well as high dexterity, high force and controllable stiffness16.
The architecture of the manipulator is based on a modular approach: multiple units, with the same structure and functionalities, are integrated together. The single unit is shown in Figure 1. It is based on an elastomeric cylinder obtained by a multiphase fabrication. The assembly steps of the mold components and the casting processes enable three empty chambers (for fluidic actuation) and one hollow central channel17 (for housing a granular jamming-based mechanism18) to be embedded. The chambers are placed at 120&#176;, so that their combined inflation produces omnidirectional motion and elongation. In addition an external braided sheath is placed externally to limit the outward radial expansion of the fluidic chambers when pressurized, thus optimizing the effect of the chamber actuation in the module motion (bending and elongation).
The central channel houses a cylindrical device composed of an external membrane filled with granular material. When a vacuum pressure is applied, it changes its elastic properties causing a stiffening which affects the entire module&#8217;s properties.
Motion and stiffness performances are controlled by an external setup including an air compressor and three pressure valves for actuating the chambers and one vacuum pump for activating the vacuum in the stiffening channel. An intuitive user interface allows control of actuation and vacuum pressures inside the module.
This paper details the fabrication process of the single module of this manipulator and reports the most significant results on basic motion capabilities. Considering the modular nature of the device, the assessment of the fabrication and performance of just one single module also enables the results to be extended and to predict the basic behavior of a multi-module manipulator integrating two or more modules.

<table border="0">
	<tbody>
		<tr>
			<td>Ecoflex 00-50 Trial Kit</td>
			<td>SmoothOn</td>
			<td></td>
			<td>Used for the fabrication of the soft unit, combining equal amounts of liquid parts A (the base) and B (the catalyst)</td>
		</tr>
		<tr>
			<td>Latex</td>
			<td>Antichit&#224; Belsito</td>
			<td></td>
			<td>Used for the fabrication of the granular jamming membrane</td>
		</tr>
		<tr>
			<td>Peroxide-Cured Silicone Tubing</td>
			<td>Cole Parmer</td>
			<td>T-06411-59</td>
			<td>Used for actuating the chambers and applying vacuum</td>
		</tr>
		<tr>
			<td>PET expandable braided sleeving</td>
			<td>RS</td>
			<td>408-249</td>
			<td>Used for the fabrication of the external braided sheath</td>
		</tr>
		<tr>
			<td>Silicone Rubber</td>
			<td>Momentive</td>
			<td>127374</td>
			<td>Used to fix the actuation tubes to the module</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Cole Parmer</td>
			<td>EW-06720-40</td>
			<td>Used to fix the latex membrane to the vacuum tube</td>
		</tr>
		<tr>
			<td>Fume hood Secuflow</td>
			<td>Groupe Waldner</td>
			<td></td>
			<td>Working space</td>
		</tr>
		<tr>
			<td>Precision scale</td>
			<td>KERN EW</td>
			<td></td>
			<td>Used to weight silicone, latex and coffee powder</td>
		</tr>
		<tr>
			<td>Oven/degasser</td>
			<td>Heraeus</td>
			<td></td>
			<td>Used to degass the silicone and reduce its cure time</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>DVP Vacuum Technology</td>
			<td></td>
			<td>Used to apply vacuum to the latex membrane</td>
		</tr>
	</tbody>
</table>

design and fabrication of an elastomeric unit for soft modular robots in minimally invasive surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

The various phases of the fabrication, described in the Protocol, are illustrated in Figure 3.
In order to evaluate the effectiveness of the technique and the outcomes of the final prototype, the module was tested in different working conditions. An external setup allows control of both the actuation and stiffness of the module. It includes an air compressor that activates three valves. They are connected to the siliconic tubes integrated in the chambers and allow their pressu...

Watch this Scientific Journal Video about Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery at JoVE.com

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

design-fabrication-an-elastomeric-unit-for-soft-modular-robots

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Bioengineering

8.9K Views.  Scuola Superiore Sant'Anna. This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported. 

Video: Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

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

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

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

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

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

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 ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of complex organs due to the fundamental limitations in accuracy, quality, and efficiency of the current modeling paradigm. This has largely ignored the soft tissue critical to most surgical specialties where the interior of an object matters and anatomical boundaries transition gradually. Therefore, the needs of the biomedical industry to replicate human tissue, which displays multiple scales of organization and varying material distributions, necessitate new forms of representation. 
Presented here is a novel technique to create 3D models directly from medical images, which are superior in spatial and contrast resolution to current 3D modeling methods and contain previously unachievable spatial fidelity and soft tissue differentiation. Also presented are empirical measurements of novel, additively manufactured composites that span the gamut of material stiffnesses seen in soft biological tissues from MRI and CT. These unique volumetric design and printing methods allow for deterministic and continuous adjustment of material stiffness and color. This capability enables an entirely new application of additive manufacturing to presurgical planning: mechanical realism. As a natural complement to existing models that provide appearance matching, these new models also allow medical professionals to "feel" the spatially varying material properties of a tissue simulant-a critical addition to a field in which tactile sensation plays a key role.

This method demonstrates a voxel-based 3D printing workflow, which prints directly from medical images with exact spatial fidelity and spatial/contrast resolution. This enables the precise, graduated control of material distributions through morphologically complex, graduated materials correlated to radiodensity without loss or alteration of data.

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of ...