The authors wish to thank Jana Moon for expert technical assistance. Studies presented in this paper were funded through an R21 (R21NS104584) grant to SK.

All animal experiments are performed under the approval of the University of Michigan&#39;s Committee on the Use and Care of Animals.
NOTE: Donor rats are allowed free access to food and water prior to skin and muscle donation procedures. Euthanasia is performed under deep anesthesia followed by intra-cardiac potassium chloride injection with a secondary method of bilateral pneumothorax. Any strain of rat can theoretically be utilized with this experiment; however, our laboratory has achieved consistent results in both male and female Fischer F344 rats (~200-250 g) at two to four months of age. Donor rats must be isogenic to the experimental rats.
1. Preparation of the dermal graft
<ol>
	<li>Anesthetize donor rat in an induction chamber utilizing a solution of 5% isoflurane in oxygen at 0.8-1 L/min. Once the rat has been anesthetized, remove from induction chamber and place on a rebreathing nose cone, lowering the isoflurane to 2-2.5% for maintenance of anesthesia.</li>
	<li>Administer a solution of 0.02-0.03 mL Carprofen (50 mg/mL) in 0.2 mL of sterile saline subcutaneously between the shoulder blades for analgesia.</li>
	<li>Apply artificial tears ointment to both eyes to prevent corneal ulcers.</li>
	<li>Using clippers, shave the entire lower hindlimb(s), ankle region, and sides of paw(s).</li>
	<li>Cleanse chosen hindlimb and plantar surface of paw with alcohol, followed by iodopovidone solution, ending with a final cleanse with alcohol to remove residual iodopovidone.</li>
	<li>Using a hand-held micro motor high speed drill with a removable round fine grit polishing stone (4000 rpm), burr the plantar surface of the paw to remove the epidermis. While burring, apply drips of saline as to not burn the skin. The underlying dermis will have a shiny appearance with pinpoint bleeding.</li>
	<li>Apply a tourniquet to the lower extremity to slow blood flow.</li>
	<li>Remove the plantar skin sharply with a #15 scalpel and place in saline-moistened gauze to prevent desiccation. Some tendinous and connective tissue will inherently be removed with the skin in this step and will be removed later.</li>
	<li>Apply gauze wrap to the bleeding foot to slow hemorrhage. Repeat steps 1.5-1.9 if doing two constructs.</li>
	<li>Under a microscope (20x magnification), remove the tendinous and connective tissue from the deep layer of the skin graft using micro-scissors. Take care not to make holes in the graft. The thinned dermal graft should be slightly opaque containing only dermis, measuring approximately 0.5 cm x 1.0cm in size.</li>
	<li>Place in saline-moistened gauze until ready for C-RPNI construct fabrication. Grafts should be utilized within 2 hours of harvest.</li>
</ol>
2. Preparation of the muscle graft
<ol>
	<li>Make a longitudinal incision along the anterior aspect of the lower hindlimb from just above the ankle to just below the knee with a #15 scalpel. Dissect through subcutaneous tissue to expose the underlying musculature.</li>
	<li>At the distal aspect of the incision, expose the tendinous insertions of the lower limb musculature. Tibialis anterior (TA) is typically the largest and most anterior of the muscles, and just underneath and posterior to this muscle lies the extensor digitorum longus (EDL). Isolate the distal EDL tendon from the other tendons in the area, taking care not to incise its insertion at this point.</li>
	<li>Ensure isolation of the correct tendon by inserting both tines of a forceps underneath the tendon and exerting upward pressure by opening the forceps to cause tendon excursion. Manipulation of this tendon should cause all of the toes to extend simultaneously.</li>
	<li>Perform a distal tenotomy with sharp iris scissors and separate the muscle from the surrounding tissues bluntly with tenotomies (or other blunt-tipped scissor) working proximally to find the tendinous origin.</li>
	<li>Once the proximal tendon is visualized, again perform a tenotomy utilizing sharp iris scissors. Place the muscle graft in a saline-moistened gauze to prevent desiccation.</li>
	<li>Once all desired grafts have been removed from a donor rat, euthanize primarily by intra-cardiac KCl injection (1-2 mEq K+/kg) followed by secondary euthanasia with bilateral puncture pneumothorax with a #15 blade.</li>
</ol>
3. Common peroneal nerve isolation and preparation
<ol>
	<li>Anesthetize and provide analgesia to the experimental rat according to protocol outlined in steps 1.1-1.3.</li>
	<li>Shave the desired thigh and cleanse with alcohol, betadine, ending with alcohol to remove traces of betadine.</li>
	<li>Move animal from surgical prep table to surgical microscope table and place on heating pad with temperature probe for body temperature maintenance. Maintain isoflurane at 2-2.5% and oxygen at 0.8-1 L/min.</li>
	<li>Mark the incision, extending from just distal to sciatic notch to the inferior portion of the knee. This marking should be inferior to, and angled away from, the femur. Make the incision with a #15 blade incising through the underlying biceps femoris fascia.</li>
	<li>Carefully dissect through the biceps femoris muscle with either a hemostat or blunt-tipped micro-scissors to the space underlying biceps femoris. 
	NOTE: The sciatic nerve travels approximately in the same direction as the initial incision that was made. There are three branches, typically with sural nerve posterior and common peroneal and tibial nerve traveling superficial and deep to the knee, respectively.</li>
	<li>Following identification of the common peroneal (CP) nerve, using a pair of micro-, fine-tipped forceps and micro-scissors, carefully isolate the CP nerve from the other sciatic branches and remove any lingering connective tissue distally.</li>
	<li>At the point where the nerve crosses the surface of the knee, sharply transect the nerve with a pair of micro-scissors. 
	NOTE: Using sharp scissors is extremely important in this step as causing significant trauma to the nerve could increase the risk of neuroma formation.</li>
	<li>Carefully free any remaining connective tissue from the CP nerve and work proximally to free the nerve to a length of approximately 2 cm.</li>
</ol>
4. C-RPNI construct fabrication
<ol>
	<li>Remove the muscle graft from saline-moistened gauze and remove all central tendinous tissue as well as a small central segment of epimysium. Leave the tendinous ends intact.</li>
	<li>Using an 8-0 nylon suture, secure the epineurium of the transected end of the CP nerve to the area of the muscle graft devoid of epimysium with two interrupted stitches on either side of the nerve.</li>
	<li>Secure the muscle graft to the femur periosteum with a single 6-0 nylon interrupted stitch both proximally and distally with the nerve-muscle junction facing away from the femur. 
	NOTE: Secure the muscle so that it is at normal relaxed length. Try not to stretch the muscle significantly or leave too much laxity when securing.</li>
	<li>Place an 8-0 nylon stitch at the inferior, central margin of the muscle graft epimysium, securing it to the CP nerve epineurium in a way as to create laxity in the nerve within the muscle graft and help to relieve any future tension it may be exposed to with later ambulation.</li>
	<li>Remove the skin graft from the saline-moistened gauze and arrange it on the muscle graft in such a way to completely cover the nerve and the majority of the muscle. Ensure that the deep margin of the dermis is resting on the muscle. Trim any dermis that extends beyond the border of the muscle.</li>
	<li>Secure the skin graft to the muscle graft circumferentially using 8-0 nylon interrupted sutures. Typically, 4-8 total sutures are used depending on the size of the construct.</li>
	<li>Close the biceps femoris fascia over the construct in a running fashion with 5-0 chromic suture.</li>
	<li>Close the overlying skin with 4-0 chromic suture in running fashion.</li>
	<li>Swab the surgical area with an alcohol pad and apply antibiotic ointment.</li>
	<li>Cease inhalational anesthetic and allow rat to recover with food and water sources separate from cage mates.</li>
</ol>

<ol>
	<li>Biddiss, E. A., Chau, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upper+limb+prosthesis+use+and+abandonment:+A+survey+of+the+last+25+years.">Upper limb prosthesis use and abandonment: A survey of the last 25 years.</a> Prosthetics and Orthotics International. 31 (3), 236-257 (2007).</li><li>Kung, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+peripheralnerve+interface+viability+and+signal+transduction+with+an+implanted+electrode.">Regenerative peripheralnerve interface viability and signal transduction with an implanted electrode.</a> Plastic and Reconstructive Surgery. 133 (6), 1380-1394 (2014).</li><li>Larson, J. V., 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=Prototype+Sensory+Regenerative+Peripheral+Nerve+Interface+for+Artificial+Limb+Somatosensory+Feedback.">Prototype Sensory Regenerative Peripheral Nerve Interface for Artificial Limb Somatosensory Feedback.</a> Plastic and Reconstructive Surgery. 133 (3 Suppl), 26-27 (2014).</li><li>Hijjawi, J. B., 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=Improved+myoelectric+prosthesis+control+accomplished+using+multiple+nerve+transfers.">Improved myoelectric prosthesis control accomplished using multiple nerve transfers.</a> Plastic and Reconstructive Surgery. 118 (7), 1573-1578 (2006).</li><li>Pylatiuk, C., Schulz, S., D&#246;derlein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+an+Internet+survey+of+myoelectric+prosthetic+hand+users.">Results of an Internet survey of myoelectric prosthetic hand users.</a> Prosthetics and Orthotics International. 31 (4), 362-370 (2007).</li><li>Baghmanli, Z., 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=Biological+and+electrophysiologic+effects+of+poly(3,4-ethylenedioxythiophene)+on+regenerating+peripheral+nerve+fibers.">Biological and electrophysiologic effects of poly(3,4-ethylenedioxythiophene) on regenerating peripheral nerve fibers.</a> Plastic and Reconstructive Surgery. 132 (2), 374-385 (2013).</li><li>Dhillon, G. S., Horch, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+neural+sensory+feedback+and+control+of+a+prosthetic+arm.">Direct neural sensory feedback and control of a prosthetic arm.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 13 (4), 468-472 (2005).</li><li>Romo, R., Hern&#225;ndez, A., Zainos, A., Salinos, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatosensory+discrimination+based+on+cortical+microstimulation.">Somatosensory discrimination based on cortical microstimulation.</a> Nature. 392, 387-390 (1998).</li><li>O'Doherty, J., 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=Active+tactile+exploration+using+a+brain-machine-brain+interface.">Active tactile exploration using a brain-machine-brain interface.</a> Nature. 479, 228-231 (2011).</li><li>Stein, R. B., Walley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+comparison+of+upper+extremity+amputees+using+myoelectric+and+conventional+prostheses.">Functional comparison of upper extremity amputees using myoelectric and conventional prostheses.</a> Archives of Physical Medicine and Rehabilitation. 64 (6), 243-248 (1983).</li><li>Millstein, S. G., Heger, H., Hunter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prosthetic+Use+in+Adult+Upper+Limb+Amputees:+A+Comparison+of+the+Body+Powered+and+Electrically+Powered+Prostheses.">Prosthetic Use in Adult Upper Limb Amputees: A Comparison of the Body Powered and Electrically Powered Prostheses.</a> Prosthetics and Orthotics International. 10 (1), 27-34 (1986).</li><li>Zollo, L., 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=Restoring+tactile+sensations+via+neural+interfaces+for+real-time+force-and-slippage+closed-loop+control+of+bionic+hands.">Restoring tactile sensations via neural interfaces for real-time force-and-slippage closed-loop control of bionic hands.</a> Science Robotics. 4 (27), eaau9924 (2019).</li><li>Tan, D. W., 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=A+neural+interface+provides+long-term+stable+natural+touch+perception.">A neural interface provides long-term stable natural touch perception.</a> Science Translational Medicine. 6 (257), 257ra138 (2014).</li><li>Stieglitz, T., 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=On+Biocompatibility+and+Stability+of+Transversal+Intrafascicular+Multichannel+Electrodes-TIME.">On Biocompatibility and Stability of Transversal Intrafascicular Multichannel Electrodes-TIME.</a> Converging Clinical and Engineering Research on Neurorehabilitation II. 15, 731-735 (2017).</li><li>Petrini, F. 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=Six-months+assessment+of+a+hand+prosthesis+with+intraneural+tactile+feedback.">Six-months assessment of a hand prosthesis with intraneural tactile feedback.</a> Annals of Neurology. 85 (1), 137-154 (2019).</li><li>Jung, R., Abbas, J., Kuntaegowdanahalli, S., Thota, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bionic+intrafascicular+interfaces+for+recording+and+stimulating+peripheral+nerve+fibers.">Bionic intrafascicular interfaces for recording and stimulating peripheral nerve fibers.</a> Bioelectronics in Medicine. 1 (1), 55-69 (2018).</li><li>Micera, S., Navarro, X., Yoshida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacing+With+the+Peripheral+Nervous+System+to+Develop+Innovative+Neuroprostheses.">Interfacing With the Peripheral Nervous System to Develop Innovative Neuroprostheses.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 17 (5), 417-419 (2009).</li><li>Stieglitz, T., Schuettler, M., Schneider, A., Valderrama, E., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+measurement+of+torque+development+in+the+rat+foot:+measurement+setup+and+results+from+stimulation+of+the+sciatic+nerve+with+polyimide-based+cuff+electrodes.">Noninvasive measurement of torque development in the rat foot: measurement setup and results from stimulation of the sciatic nerve with polyimide-based cuff electrodes.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 11 (4), 427-437 (2003).</li><li>Polasek, K. H., Hoyen, H. A., Keith, M. W., Tyler, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+nerve+stimulation+thresholds+and+selectivity+using+a+multi-contact+nerve+cuff+electrode.">Human nerve stimulation thresholds and selectivity using a multi-contact nerve cuff electrode.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 15 (1), 76-82 (2007).</li><li>Nielson, K. D., Watts, C., Clark, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+injury+from+implantation+of+chronic+stimulating+electrodes+for+pain+control.">Peripheral nerve injury from implantation of chronic stimulating electrodes for pain control.</a> Surgical Neurology. 5 (1), 51-53 (1976).</li><li>Waters, R. L., McNeal, D. R., Faloon, W., Clifford, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+electrical+stimulation+of+the+peroneal+nerve+for+hemiplegia.+Long-term+clinical+follow-up.">Functional electrical stimulation of the peroneal nerve for hemiplegia. Long-term clinical follow-up.</a> Journal of Bone and Joint Surgery. 67 (5), 792-793 (1985).</li><li>Larsen, J. O., Thomsen, M., Haugland, M., Sinkjaer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+and+regeneration+in+rabbit+peripheral+nerve+with+long-term+nerve+cuff+electrode+implant:+a+stereological+study+of+myelinated+and+unmyelinated+axons.">Degeneration and regeneration in rabbit peripheral nerve with long-term nerve cuff electrode implant: a stereological study of myelinated and unmyelinated axons.</a> Acta Neuropathologica. 96 (4), 365-378 (1998).</li><li>Krarup, C., Loeb, G. E., Pezeshkpour, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conduction+studies+in+peripheral+cat+nerve+using+implanted+electrodes:+III.+The+effects+of+prolonged+constriction+on+the+distal+nerve+segment.">Conduction studies in peripheral cat nerve using implanted electrodes: III. The effects of prolonged constriction on the distal nerve segment.</a> Muscle Nerve. 12 (11), 915-928 (1989).</li><li>Micera, S., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+interfaces+with+the+peripheral+nervous+system.">Bidirectional interfaces with the peripheral nervous system.</a> International Review of Neurobiology. 86, 23-38 (2009).</li><li>Urbanchek, M. 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=Microscale+Electrode+Implantation+during+Nerve+Repair:+Effects+on+Nerve+Morphology,+Electromyography,+and+Recovery+of+Muscle+Contractile+Function.">Microscale Electrode Implantation during Nerve Repair: Effects on Nerve Morphology, Electromyography, and Recovery of Muscle Contractile Function.</a> Plastic and Reconstructive Surgery. 128 (4), 270e-278e (2011).</li><li>Yoshida, K., Horch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+stimulation+of+peripheral+nerve+fibers+using+dual+intrafascicular+electrodes.">Selective stimulation of peripheral nerve fibers using dual intrafascicular electrodes.</a> IEEE Transactions on Biomedical Engineering. 40 (5), 492-494 (1993).</li><li>Branner, A., Stein, R. B., Normann, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+stimulation+of+cat+sciatic+nerve+using+an+array+of+varying+length+microelectrodes.">Selective stimulation of cat sciatic nerve using an array of varying length microelectrodes.</a> Journal of Neurophysiology. 85 (4), 1585-1594 (2001).</li><li>Zheng, X. J., Zhang, J., Chen, T., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinally+implanted+intrascicular+electrodes+for+stimulating+and+recording+fascicular+physioelectrical+signals+in+the+sciatic+nerve+of+rabbits.">Longitudinally implanted intrascicular electrodes for stimulating and recording fascicular physioelectrical signals in the sciatic nerve of rabbits.</a> Microsurgery. 23, 268-273 (2003).</li><li>del Valle, J., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfaces+with+the+peripheral+nerve+for+the+control+of+neuroprostheses.">Interfaces with the peripheral nerve for the control of neuroprostheses.</a> International Review of Neurobiology. 109, 63-83 (2013).</li><li>Stiller, A. 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=A+Meta-Analysis+of+Intracortical+Device+Stiffness+and+Its+Correlation+with+Histological+Outcomes.">A Meta-Analysis of Intracortical Device Stiffness and Its Correlation with Histological Outcomes.</a> Micromachines. 9 (9), 443 (2018).</li><li>Hanson, T., Diaz-Botia, C., Kharazia, V., Maharbiz, M., Sabes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"sewing+machine"+for+minimally+invasive+neural+recording.">The "sewing machine" for minimally invasive neural recording.</a> bioRxiv. , (2019).</li><li>Yang, X., 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=Bioinspired+neuron-like+electronics.">Bioinspired neuron-like electronics.</a> Nature Materials. 18, 510-517 (2019).</li><li>Irwin, Z. T., 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=Chronic+recording+of+hand+prosthesis+control+signals+via+a+regenerative+peripheral+nerve+interface+in+a+rhesus+macaque.">Chronic recording of hand prosthesis control signals via a regenerative peripheral nerve interface in a rhesus macaque.</a> Journal of Neural Engineering. 13 (4), 046007 (2016).</li><li>Kubiak, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abstract+24:+Successful+Control+of+Virtual+and+Robotic+Hands+using+Neuroprosthetic+Signals+from+Regenerative+Peripheral+Nerve+Interfaces+in+a+Human+Subject.">Abstract 24: Successful Control of Virtual and Robotic Hands using Neuroprosthetic Signals from Regenerative Peripheral Nerve Interfaces in a Human Subject.</a> Plastic and Reconstructive Surgery Global Open. 6 (4), 19-20 (2018).</li><li>Sando, I. C., 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=Dermal-Based+Peripheral+Nerve+Interface+for+Transduction+of+Sensory+Feedback.">Dermal-Based Peripheral Nerve Interface for Transduction of Sensory Feedback.</a> Plastic and Reconstructive Surgery. 136 (4 Suppl), 19-20 (2015).</li><li>Kubiak, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abstract+36:+Viability+and+Signal+Transduction+with+the+Composite+Regenerative+Peripheral+Nerve+Interface+(C-RPNI).">Abstract 36: Viability and Signal Transduction with the Composite Regenerative Peripheral Nerve Interface (C-RPNI).</a> Plastic and Reconstructive Surgery Global Open. 7 (4), 26-27 (2019).</li><li>Kubiak, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abstract+QS18:+Neural+Signal+Transduction+with+the+Muscle+Cuff+Regenerative+Peripheral+Nerve+Interface.">Abstract QS18: Neural Signal Transduction with the Muscle Cuff Regenerative Peripheral Nerve Interface.</a> Plastic and Reconstructive Surgery Global Open. 7 (4 Suppl), 114 (2019).</li><li>Woo, S. L., 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=Utilizing+nonvascularized+partial+skeletal+muscle+grafts+in+peripheral+nerve+interfaces+for+prosthetic+control.">Utilizing nonvascularized partial skeletal muscle grafts in peripheral nerve interfaces for prosthetic control.</a> Journal of the American College of Surgeons. 219 (4), e136-e137 (2014).</li><li>Sporel-&#214;zakat, R. E., Edwards, P. M., Hepgul, K. T., Savas, A., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+reducing+autotomy+in+rats+after+peripheral+nerve+lesions.">A simple method for reducing autotomy in rats after peripheral nerve lesions.</a> Journal of Neuroscience Methods. 36 (2-3), 263-265 (1991).</li><li>Carr, M. M., Best, T. J., Mackinnon, S. E., Evans, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+differences+in+autotomy+in+rats+undergoing+sciatic+nerve+transection+or+repair.">Strain differences in autotomy in rats undergoing sciatic nerve transection or repair.</a> Annals of Plastic Surgery. 28 (6), 538-544 (1992).</li></ol>

The authors have no disclosures.

The C-RPNI is a novel construct that provides simultaneous amplification of a target nerve&#39;s motor efferent signals with provision of afferent sensory feedback. In particular, the C-RPNI has unique utility for those living with proximal amputations as their motor and sensory fascicles cannot easily be mechanically separated during surgery. Instead, the C-RPNI utilizes the inherent preferential reinnervation properties of the nerve itself to encourage sensory fiber reinnervation to dermal sensory end organs and motor ...

Extremity amputations affect nearly 1 in 190 Americans1, and their prevalence is projected to increase from 1.6 million today to over 3.6 million by 20502. Despite documented use for over a millennium, the ideal prosthetic has yet to be realized3. Currently, there exist complex prosthetics capable of multiple joint manipulations with the potential to reproduce many motor functions of the native extremity4,5. However, these devices are not considered intuitive as the desired prosthetic motion is typically functionally separate from the input control signal. Users typically consider these &#34;advanced prosthetics&#34; difficult to learn and therefore not suitable for everyday use1,6. Additionally, complex prosthetics currently on the market do not provide any appreciable degree of subtle sensory feedback for adequate control. The sense of touch and proprioception are vital to carrying out daily tasks, and without these, simple acts such as picking up a cup of coffee become burdensome as it relies entirely on visual cues7,8,9. For these reasons, advanced prostheses are associated with a significant degree of mental fatigue and are often described as burdensome and unsatisfactory5,10,11. To address this, some research laboratories have developed prosthetics capable of providing a limited degree of sensory feedback via direct neural interaction12,13,14,15, but feedback is often limited to small, scattered areas on the hands and fingers12,13, and sensations were noted to be painful and unnatural at times15. Many of these studies unfortunately lack any appreciable long-term follow-up and nerve histology to delineate local tissue effects, while noting interface failure on the scale of weeks to months16.
For this population, the ideal prosthetic device would provide high fidelity motor control alongside meaningful somatosensory feedback from the individual&#39;s environment throughout their lifetime. Critical to the design of said ideal prosthetic is the development of a stable, reliable interface that would allow for simultaneous transmission of afferent somatosensory information with efferent motor signals. The most promising of current human-machine interfaces are those that interact with the peripheral nervous system directly, and recent developments in the field of neuro-integrated prosthetics have worked towards bridging the gap between bioelectric and mechanical signals17. Current interfaces utilized include: flexible nerve plates14,15,18, extra-neural cuff electrodes13,19,20,21,22,23, tissue penetrating electrodes24,25,31,32, and intrafascicular electrodes26,27,28. However, each of these methods has demonstrated limitations with regards to nerve specificity, tissue injury, axonal degeneration, myelin depletion, and/or scar tissue formation associated with chronic indwelling foreign body response16,17,18,19. More recently, it has been postulated that a driver behind eventual implanted electrode failure is the significant difference in Young&#39;s moduli between electronic material and native neural tissue. Brain tissue is subject to significant micromotion on a daily basis, and it has been theorized that the shear stress induced by differences in Young&#39;s moduli causes inflammation and eventual permanent scarring30,31,32. This effect is often compounded in the extremities, where peripheral nerves are subject to both physiologic micromotion and intentional extremity macromotion. Due to this constant motion, it is reasonable to conclude that utilization of a completely abiotic peripheral nerve interface is not ideal, and an interface with a biologic component would be more suitable.
To address this need for a biologic component, our laboratory developed a biotic nerve interface termed the Regenerative Peripheral Nerve Interface (RPNI) to integrate transected peripheral nerves in a residual limb with a prosthetic device. RPNI fabrication involves surgically implanting a peripheral nerve into an autologous free muscle graft, which subsequently revascularizes and reinnervates. Our lab has developed this biologic nerve interface over the past decade, with success in amplifying and transmitting motor signals when combined with implanted electrodes in both animal and human trials, allowing for suitable prosthetic control with multiple degrees of freedom2,34. In addition, we have separately demonstrated sensory feedback through the use of peripheral nerves embedded in dermal grafts, termed the Dermal Sensory Interface (DSI)3,35. In more distal amputations, using these constructs simultaneously is feasible as motor and sensory fascicles within the target peripheral nerve can be surgically separated. However, for more proximal level amputations, this is not feasible due to intermingling of motor and sensory fibers. The Composite Regenerative Peripheral Nerve Interface (C-RPNI) was developed for more proximal amputations, and it involves implanting a mixed sensorimotor nerve into a construct consisting of free muscle graft secured to a segment of dermal graft (Figure 1). Peripheral nerves demonstrate preferential targeted reinnervation, thus sensory fibers will re-innervate the dermal graft and motor fibers, the muscle graft. This construct thus has the ability to simultaneously amplify motor signals while providing somatosensory feedback36 (Figure 2), allowing for the realization of the ideal, intuitive, complex prosthetic.

<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>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>6-0 Ethilon Suture</td>
			<td>Ethicon</td>
			<td>SKU# 697G</td>
			<td>P-1 Reverse Cutting Needle (Nylon suture)</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>Fluriso (Isofluorane)</td>
			<td>VetOne</td>
			<td>13985-528-40</td>
			<td>Inhalational Anesthetic</td>
		</tr>
		<tr>
			<td>Micro Motor High Speed Drill with Stone</td>
			<td>Master Mechanic</td>
			<td>Model 151369</td>
			<td>Handheld rotary tool; kit comes with multiple fine grit stones</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>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>

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.

Construct fabrication is considered unsuccessful if rats develop an infection or do not survive surgical anesthesia. Previous research has indicated these constructs require approximately three months to revascularize and reinnervate2,3,17,36. Following the three-month recovery period, construct testing can be pursued to examine viability. Surgical exposure of t...

Watch this Scientific Journal Video about Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat at JoVE.com

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.

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 ...

fabrication-composite-regenerative-peripheral-nerve-interface-c-rpni

Research

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Bioengineering

8.1K Views.  University of Michigan, Ann Arbor. 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.

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

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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%.

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 ...

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 ...

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 ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

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.

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 ...

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...