The authors thank Jana Moon for her expert lab management and technical assistance and Charles Hwang for his imaging expertise. Experiments in this paper were in part funded through Plastic Surgery Foundation grants to SS (3135146.4) as well as the National Institute of Child Health and Human Development under Award Number 1F32HD100286-01 to SS, and the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number P30 AR069620.

All animal procedures and experiments were carried out with the approval of the University of Michigan&#39;s Institutional Care and Use of Animals Committee (IACUC). Male and female Fischer F344 and Lewis rats (~200-300 g) at 3-6 months of age are most frequently utilized in experiments, but any strain can theoretically be utilized. If utilizing donor rats instead of autologous muscle grafts, donor rats must be isogenic to the experimental strain. Rats are allowed free access to food and water both pre- and post-operatively. Following terminal endpoint evaluations, euthanasia is performed under deep anesthesia with intra-cardiac potassium chloride injection followed by a secondary method of bilateral pneumothorax.
1. Experimental preparation of the rat
<ol>
	<li>Anesthetize the experimental rat utilizing a solution of 5% isoflurane in oxygen at 0.8-1.0 L/min in an induction chamber. Once adequate anesthesia is achieved and confirmed with the absence of corneal reflex, place the rat on a rebreather nose cone with isoflurane lowered to 1.75%-2.25% for maintenance of anesthesia.</li>
	<li>Inject a solution of 0.02-0.03 mL Carprofen (50 mg/mL) in 0.2 mL of sterile saline with 27 G needle in the subcutaneous plane between the shoulder blades for peri- and post-operative analgesia.</li>
	<li>Apply sterile eye ointment to both eyes to prevent corneal ulcers while anesthetized.</li>
	<li>Using an electric razor, shave the lateral portion of the bilateral lower limbs, extending from the hip joint, over the thigh, and to the dorsal surface of the paw.</li>
	<li>Sterilize the surgical site&#160;by first wiping with an alcohol prep pad, followed by povidone-iodine solution application, ending with a final cleanse with a new alcohol prep pad to remove the residual povidone-iodine solution.&#160;Repeat this alternating cleansing process three times to maintain sterility. 
	NOTE: This can be a dermatological irritant; ensure the majority of the solution is removed.</li>
</ol>
2. Preparation of the muscle graft
<ol>
	<li>Place the rat on a heating pad underneath a surgical microscope with an intraoral body temperature probe of choice for body temperature monitoring. Maintain isoflurane at 1.75%-2.25% and oxygen at 0.8-1.0 L/min.</li>
	<li>Make a longitudinal incision along the anterior aspect of the desired donor hindlimb extending from just above the ankle to just below the knee with a #15 scalpel.</li>
	<li>Dissect through the underlying subcutaneous tissue using sharp iris scissors to expose the underlying musculature and distal tendons just proximal to the ankle joint. Tibialis anterior (TA) is the largest and most anterior of the muscles; the extensor digitorum longus (EDL) muscle can be found just deep and posterior to this muscle. Isolate the EDL muscle and its distal tendon from the surrounding musculature.</li>
	<li>Ensure isolation of the correct tendon by inserting both tines of a forceps or iris scissor underneath the distal tendon just proximal to the ankle joint. Exert upward pressure on the tendon by opening either the forceps or iris scissors. This motion should produce a simultaneous extension of all of the toes simultaneously. If isolated ankle dorsiflexion, ankle eversion, or single toe dorsiflexion occurs, the wrong tendon has been isolated.</li>
	<li>Perform a distal tenotomy of the EDL muscle at the level of the ankle with sharp iris scissors and dissect the muscle free from surrounding tissues working proximally towards its tendinous origin.</li>
	<li>Once the proximal tendon is visualized, perform a proximal tenotomy utilizing sharp iris scissors to free the graft.</li>
	<li>Trim both tendinous ends of the muscle graft and cut to the desired length with sharp iris scissors. 
	NOTE: Grafts measuring 8-13 mm have been utilized with success; however, the most common length utilized is 10 mm.</li>
	<li>On one side of the muscle graft, make a longitudinal incision along the entire trimmed length to facilitate placement of the nerve within the muscle graft and provide contact of the nerve with endomysium.</li>
	<li>Place the prepared muscle graft in a saline-moistened gauze to prevent tissue desiccation.</li>
	<li>Close the skin overlying the donor site with 4-0 chromic suture in a running fashion.</li>
</ol>
3. Common peroneal nerve isolation and preparation
<ol>
	<li>Mark the surgical incision, which will extend from a line ~5 mm from the sciatic notch, extending to just inferior to the knee joint. Ensure that this marking is inferior to, and angled away from, the femur that can be palpated below.</li>
	<li>Incise through the skin and subcutaneous tissues along the marked incision line with a #15 blade. Carefully incise through the underlying biceps femoris fascia, taking care not to extend through the entire depth of the muscle as the sciatic nerve lies just below.</li>
	<li>Utilizing blunt-tipped small scissors or a hemostat, dissect carefully through the biceps femoris muscle. 
	NOTE: The sciatic nerve travels in this space underlying the biceps, oriented in approximately the same direction as the incision marked on the skin. There are three notable sciatic nerve branches: sural (most posterior and smallest of the nerves), tibial (typically most anterior, but this nerve always dives deep to the knee joint), and common peroneal (typically located between tibial and sural, always travels above the knee joint).</li>
	<li>Identify the common peroneal (CP) nerve and carefully isolate it from the surrounding nerves using a pair of micro-forceps and micro-scissors. Remove any surrounding connective tissue from the middle 2 cm of the nerve. Take care not to crush the CP nerve with forceps in this process, as crush injury can alter endpoint results.</li>
	<li>Over the central-most portion of the freed CP nerve, perform an epineurial window by removing 25% of the epineurium along the length of the nerve that matches the desired length of the muscle graft.</li>
	<li>To perform this hold the proximal epineurium with micro-forceps, cut into the epineurium immediately underlying with micro-dissection scissors, and remove ~25% of the epineurium traveling distally along the nerve. Take care to remove this segment in one piece, as multiple attempts can cause irregular epineurial removal, increasing the risk of nerve injury.&#160; 
	NOTE: The nerve tissue underlying the epineurium will have a goo-like texture; noting this quality of nerve ensures the correct tissue plane has been removed.</li>
</ol>
4. MC-RPNI construct fabrication
<ol>
	<li>Remove the muscle graft from the saline-moistened gauze and place it under the central portion of the CP nerve where the epineurial window was created. Rotate the nerve 180&#176; so the epineurial window section contacts intact muscle and does not underlie the eventual suture line.</li>
	<li>Using an 8-0 nylon suture, suture the epineurium of the CP nerve both proximally and distally to the muscle graft within the groove created in step 2.8 using simple interrupted sutures to secure epineurium to endomysium. 
	NOTE: Place these stitches, ensuring the muscle is at normal resting length. Stretching or compressing the muscle too much can impact regeneration and signaling capabilities later on.</li>
	<li>Circumferentially wrap the edges of the muscle graft surrounding the now-secured nerve and suture in place utilizing simple interrupted 8-0 nylon stitches (~4-6 depending on length).</li>
	<li>Once hemostasis is achieved, close the biceps femoris fascia over the construct with 5-0 chromic suture in running fashion.</li>
	<li>Close the overlying skin in running fashion with a 4-0 chromic suture.</li>
	<li>Cleanse the surgical area with an alcohol prep pad and apply antibiotic ointment.</li>
	<li>Terminate inhalational anesthetic and place the rat in a clean cage isolated from cage mates and allow to recover with food and water ad lib.</li>
	<li>Once the rat has suitably recovered, place it back with cage mates in a clean cage. 
	NOTE: These constructs require maturation of three months at the minimum to produce adequate nerve signal amplification.</li>
</ol>

The authors have no disclosures.

The MC-RPNI is a novel construct that allows for amplification of an intact, peripheral motor nerve&#39;s efferent action potentials in order to accurately control an exoskeleton device. Specifically, the MC-RPNI confers a particular benefit to those individuals with extremity weakness caused by significant muscle disease and/or absence of muscle where EMG signals cannot be recorded. Reducing already compromised muscle function would be devastating in this population; however, the MC-RPNI has the ability to provide this ...

In the United States alone, approximately 130 million people are affected by neuromuscular and musculoskeletal disorders, resulting in over $800 billion in annual economic impact1,2. This group of disorders is typically secondary to pathology within the nervous systems, at the neuromuscular junction, or within the muscle itself3. Despite the variety of pathologic origins, the majority share some degree of extremity weakness1,3. Unfortunately, this weakness is often permanent given the limitations in neural and muscle tissue regeneration, especially in the setting of severe trauma4,5,6.
Extremity weakness treatment algorithms have classically focused on rehabilitative and supportive measures, often relying on harnessing the capabilities of the remaining intact limbs (canes, wheelchairs, etc.)7. This strategy falls short, however, for those whose weakness is not limited to a single extremity. With recent innovations in robotic technologies, advanced exoskeleton devices have been developed that restore extremity functionality to those living with extremity weakness8,9,10,11,12,13. These robotic exoskeletons are often powered, wearable devices that can assist with initiation and termination of movement or maintenance of limb position, providing a varying amount of force that can be individually tailored for the user8,9,10,11,12,13. These devices are classified as either passive or active depending on how they provide motor assistance to the user: active devices contain electrical actuators that augment power to the user, whereas passive devices store energy from the user&#39;s motions in order to release it back to the user when necessary14. As active devices have the ability to increase a user&#39;s power capabilities, these devices are utilized far more frequently in the setting of extremity weakness[14].
In order to determine motor intent in this population, modern exoskeletons commonly rely on pattern recognition algorithms generated from either electromyography (EMG) of distal limb muscles8,15,16,17 or surface electroencephalography (sEEG) of the brain18,19,20. Despite the promise of these detection modalities, both options have significant limitations that preclude widespread utilization of these devices. As sEEG detects microvolt-level signals transcranially18,19,20, criticisms frequently focus on the inability to differentiate these signals from background noise21. When background noise is similar to the desired recording signal, this produces low signal-to-noise ratios (SNRs), resulting in inaccurate motor detection and classification22,23. Accurate signal detection additionally relies on stable, low-impedance scalp contact21, which can be significantly affected by the presence of coarse/thick hair, user activity, and even sweating22,24. In contrast, EMG signals are several magnitudes larger in amplitude, facilitating greater motor signal detection accuracy15,18,25. This comes at a cost, however, as nearby muscles can contaminate the signal, decreasing the degrees of freedom able to be controlled by the device16,17,25 and an inability to detect deep muscle motion25,26,27,28. Most importantly, EMG cannot be used as a control method when there is significant muscle compromise and complete absence of tissue29.
In order to advance the development of robotic exoskeletons, consistent and accurate detection of motor intent of the intended user is required. Interfaces that utilize the peripheral nervous system have arisen as a promising interface technique, given their relatively simple access and functional selectivity. Current peripheral nerve interfacing methods can be invasive or non-invasive and typically fall within one of three categories: extraneural electrodes30,31,32,33, intrafascicular electrodes34,35,36 and penetrating electrodes37,38,39,40. As peripheral nerve signals are generally on the level of microvolts, it can be difficult to differentiate these signals from similar amplitude background noise41,42, which reduces the overall motor detection accuracy capabilities of the interface. These low signal-to-noise (SNR) ratios often worsen over time secondary to worsening electrode impedance43 produced from either degradation of the device39,43, or local foreign body reaction producing scar tissue around the device and/or local axonal degeneration37,44. Although these shortcomings can generally be resolved with reoperation and implantation of a new peripheral nerve interface, this is not a viable long-term solution as foreign-body-associated reactions would continue to occur.
To avoid these local tissue reactions generated from peripheral nerves&#39; interaction with abiotic interfaces, an interface incorporating a biologic component is necessary. To address this shortcoming, the Regenerative Peripheral Nerve Interface (RPNI) was developed to integrate transected peripheral nerves in the residual limbs of those with amputations with prosthetic devices45,46,47,48. Fabrication of the RPNI involves surgical implantation of a transected peripheral nerve into a segment of autologous free muscle graft, with revascularization, regeneration, and reinnervation occurring over time. Through the generation of milli-volt level compound muscle action potentials (CMAPs), the RPNI is able to amplify its contained nerve&#39;s micro-volt level signal by several magnitudes, facilitating accurate detection of motor intent45,48,49. There has been considerable development of the RPNI over the past decade, with notable success in amplifying and transmitting efferent motor nerve signals in both animal50,51 and human47 trials, facilitating high accuracy prosthetic device control with multiple degrees of freedom.
Individuals with extremity weakness but intact peripheral nerves would similarly benefit from high accuracy detection of motor intent through peripheral nerve interfaces in order to control exoskeleton devices. As the RPNI was developed for integration with transected peripheral nerves, such as in persons with amputations, surgical modifications were necessary. Building from experience with the RPNI, the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) was developed. Consisting of a similar segment of free muscle graft as in the RPNI, it is instead secured circumferentially to an intact peripheral nerve (Figure 1). Over time, it regenerates and becomes reinnervated through collateral axonal sprouting, amplifying and translating these efferent motor nerve signals to EMG signals that are several orders of magnitude larger52. As the MC-RPNI is biologic in origin, it avoids the inevitable foreign body reaction that occurs with peripheral nerve interfaces currently in use52. Furthermore, the MC-RPNI confers the ability to control multiple degrees of freedom simultaneously as they can be placed on distally dissected nerves to individual muscles without significant cross-talk, as has been previously demonstrated in RPNIs49. Finally, the MC-RPNI can operate independent of distal muscle function as it is placed on the proximal nerve. Given its advantages over current peripheral nerve interfaces, the MC-RPNI holds substantial promise for providing a safe, accurate, and reliable method of exoskeleton control.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#15 Scalpel</td>
			<td>Aspen Surgical, Inc</td>
			<td>Ref 371115</td>
			<td>Rib-Back Carbon Steel Surgical Blades (#15)</td>
		</tr>
		<tr>
			<td>2-N-thin film load cell (S100)</td>
			<td>Strain Measurement Devices, Inc</td>
			<td>SMD100-0002</td>
			<td>Measures force generated by the attached muscle</td>
		</tr>
		<tr>
			<td>4-0 Chromic Suture</td>
			<td>Ethicon</td>
			<td>SKU# 1654G</td>
			<td>P-3 Reverse Cutting Needle</td>
		</tr>
		<tr>
			<td>5-0 Chromic Suture</td>
			<td>Ethicon</td>
			<td>SKU# 687G</td>
			<td>P-3 Reverse Cutting Needle</td>
		</tr>
		<tr>
			<td>8-0 Monofilament Suture</td>
			<td>AROSurgical</td>
			<td>T06A08N14-13</td>
			<td>Black polyamide monofilament suture on a threaded tapered needle</td>
		</tr>
		<tr>
			<td>Experimental Rats</td>
			<td>Envigo</td>
			<td>F344-NH-sd</td>
			<td>Rats are Fischer F344 Strain</td>
		</tr>
		<tr>
			<td>Fine Forceps - mirror finish</td>
			<td>Fine Science Tools</td>
			<td>11413-11</td>
			<td>Fine tipped forceps with mirror finish ideal for handling delicate structures like nerves</td>
		</tr>
		<tr>
			<td>Fluriso (Isofluorane)</td>
			<td>VetOne</td>
			<td>13985-528-40</td>
			<td>Inhalational Anesthetic</td>
		</tr>
		<tr>
			<td>Force Measurement Jig</td>
			<td>Red Rock</td>
			<td>n/a</td>
			<td>Custom designed force measurement jig that allows for immobilization of hindlimb to allow for accurate muscle force recording</td>
		</tr>
		<tr>
			<td>MATLAB software</td>
			<td>Mathworks, Inc</td>
			<td>PR-MATLAB-MU-MW-707-NNU</td>
			<td>Calculates active force for each recorded force trace from passive and total force measurements</td>
		</tr>
		<tr>
			<td>Nicolet Viasys EMG EP System</td>
			<td>Nicolet</td>
			<td>MFI-NCL-VIKING-SELECT-2CH-EMG</td>
			<td>Portable EMG and nerve signal recording system capable of simultaneous 2 channel recordings from nerve and/or muscle</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Cryogenic Gases</td>
			<td>UN1072</td>
			<td>Standard medical grade oxygen canisters</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>APP Pharmaceuticals</td>
			<td>63323-965-20</td>
			<td>Injectable form, 2 mEq/mL</td>
		</tr>
		<tr>
			<td>Povidone Iodine USP</td>
			<td>MediChoice</td>
			<td>65517-0009-1</td>
			<td>10% Topical Solution, can use one bottle for multiple surgical preps</td>
		</tr>
		<tr>
			<td>Puralube Vet Opthalmic Ointment</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td>Corneal protective ointment for use during procedure</td>
		</tr>
		<tr>
			<td>Rimadyl (Caprofen)</td>
			<td>Zoetis, Inc.</td>
			<td>NADA# 141-199</td>
			<td>Injectable form, 50 mg/mL</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Leica</td>
			<td>Model M60</td>
			<td>User can adjust magnification to their preference</td>
		</tr>
		<tr>
			<td>Surgical Instruments</td>
			<td>Fine Science Tools</td>
			<td>Various</td>
			<td>User can choose instruments according to personal preference or from what is currently available in their lab</td>
		</tr>
		<tr>
			<td>Triple Antibiotic Ointment</td>
			<td>MediChoice</td>
			<td>39892-0830-2</td>
			<td>Ointment comes in sterile, disposable packets</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 2mm cutting edge</td>
			<td>Fine Science Tools</td>
			<td>15000-04</td>
			<td>Curved micro-dissection scissors used to perform the epineurial window</td>
		</tr>
		<tr>
			<td>VaporStick 3</td>
			<td>Surgivet</td>
			<td>V7015</td>
			<td>Anesthesia tower with space for isofluorane and oxygen canister</td>
		</tr>
		<tr>
			<td>Webcol Alcohol Prep</td>
			<td>Coviden</td>
			<td>Ref 6818</td>
			<td>Alcohol prep wipes; use a new wipe for each prep</td>
		</tr>
	</tbody>
</table>

the muscle cuff regenerative peripheral nerve interface for the amplification of intact peripheral nerve signals

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those individuals with extremity weakness. However, their use remains largely confined to research institutions, frequently operating as a means of static extremity support as motor detection methods remain unreliable. Peripheral nerve interfaces have arisen as a potential solution to this shortcoming; however, due to their inherently small amplitudes, these signals can be difficult to differentiate from background noise, lowering their overall motor detection accuracy. As current interfaces rely on abiotic materials, inherent material breakdown can occur alongside foreign body tissue reaction over time, further impacting their accuracy. The Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) was designed to overcome these noted complications. Consisting of a segment of free muscle graft secured circumferentially to an intact peripheral nerve, the construct regenerates and becomes reinnervated by the contained nerve over time. In rats, this construct has demonstrated the ability to amplify a peripheral nerve's motor efferent action potentials up to 100 times the normal value through the generation of compound muscle action potentials (CMAPs). This signal amplification facilitates high accuracy detection of motor intent, potentially enabling reliable utilization of exoskeleton devices.

MC-RPNI surgical fabrication is considered a peri-operative failure if rats do not survive emergence from surgical anesthesia or develop an infection within a week of the operation. Previous research has indicated a 3 month maturation period will result in reliable signal amplification from this constructs42,45,48,49. At that time or thereafter, surgical exposure of the constructs and evaluatio...

Watch this Scientific Journal Video about The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals at JoVE.com

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those ...

the-muscle-cuff-regenerative-peripheral-nerve-interface-for

Research

JoVE Journal

Bioengineering

1.9K Views.  The University of Michigan Health System. This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices. 

Video: The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

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  ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This paper presents a cleanroom "light" fabrication process of carbon fiber neural electrode arrays that can be learned quickly by an inexperienced cleanroom user. This carbon fiber electrode array fabrication process requires just one cleanroom tool, a Parylene C deposition machine, that can be learned quickly or outsourced to a commercial processing facility at marginal cost. This fabrication process also includes hand-populating printed circuit boards, insulation, and tip optimization. 
The three different tip optimizations explored here (Nd:YAG laser, blowtorch, and UV laser) result in a range of tip geometries and 1 kHz impedances, with blowtorched fibers resulting in the lowest impedance. While previous experiments have proven laser and blowtorch electrode efficacy, this paper also shows that UV laser-cut fibers can record neural signals in vivo. Existing carbon fiber arrays either do not have individuated electrodes in favor of bundles or require cleanroom fabricated guides for population and insulation. The proposed arrays use only tools that can be used at a benchtop for fiber population. This carbon fiber electrode array fabrication process allows for quick customization of bulk array fabrication at a reduced price compared to commercially available probes.

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...