Biomimetic Nerve Guidance Conduit Containing Intraluminal

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Biomimetic nerve guidance conduit containing intraluminal microchannels with aligned nanofibers markedly facilitates in nerve regeneration David Jay Lee, Arjun Fontaine, Xianzhong Meng, and Daewon Park ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00344 • Publication Date (Web): 28 Jun 2016 Downloaded from http://pubs.acs.org on July 2, 2016

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ACS Biomaterials Science & Engineering

Biomimetic nerve guidance conduit containing intraluminal microchannels with aligned nanofibers markedly facilitates in nerve regeneration David J. Lee†, Arjun Fontaine†, Xianzhong Meng‡, Daewon Park*,† †

Department of Bioengineering, University of Colorado Denver Anschutz Medical Campus,

12800 E. 19th Avenue, Aurora, CO 80045 USA ‡

Department of Surgery, University of Colorado Denver Anschutz Medical Campus, 12700 E.

19th Avenue, Aurora, CO 80045 USA * Corresponding author Email: [email protected]

KEYWORDS: biomimetic, peripheral nerve regeneration, nerve guidance conduit, rat sciatic nerve injury model

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ABSTRACT

Although the gold standard for the surgical treatment of peripheral nerve injury, the autograft is associated with many drawbacks, including a second surgical procedure, donor site morbidity, mismatch of donor nerve size, and limited donor nerve length. As an alternative to the autograft, nerve guidance conduits may be used to promote neuronal growth and guide axonal extension after nerve injury. Using a blend of RGD-conjugated polyurea and polycaprolactone, a nerve guidance conduit was designed consisting of intraluminal microchannels with aligned nanofibers. A 10 mm sciatic nerve transection rat model was used to evaluate the efficacy of the conduit up to 8 weeks after nerve transection and conduit implantation. Restoration of electrophysiological activity from the nerve guidance conduit was significantly improved compared to the autograft. Functional and histological assessments indicated that the nerve guidance conduit is comparable to autograft in functional recovery and target muscle reinnervation, respectively.


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INTRODUCTION Peripheral nerve injury (PNI) often occurs from physical distress or compression from traumatic injury. Common causes of PNI include vehicular accidents, penetrating trauma after stabbing incidents, falls inducing stretching or crushing of the nerve, gun-shot wounds, and sports.1 PNI is reported in 3 % of all trauma patients.2 Over 200,000 peripheral nerve repair procedures are performed and $150 billion is spent yearly in the United States.3,4 Despite advances in the reconstruction of segmented nerves following PNI, functional recovery remains inadequate and all surgical treatment options are associated with major drawbacks, either from surgical implications or lack of efficacy in PNI repair. Neurorrhaphy is one surgical repair technique involving the direct suturing of discontinued nerve stumps. Although relatively successful at recovering nerve function, neurorrhaphy is limited to gaps no greater than 5 mm in length as excessive tension on the nerve disrupts connective tissue matrices and reduces blood flow, inducing necrosis and chronic ischemia.5,6 The gold standard for longer nerve gaps are autografts.7 Although autografts offer the best nerve regenerative characteristics, they are associated with many drawbacks including a second surgical procedure, donor site morbidity, mismatch of donor nerve size, and limited donor nerve length. As an alternative, allografts avoid several of the limitations of autografts, but the complexity and cost of producing allografts remains a challenge.8 Allografts are not as effective in restoring nerve function due to their tendency to elicit an immune response and acellular allografts can only support regenerating axons across short nerve gaps.9 Another alternative for PNI repair is the hollow tube that can be fabricated using natural (e.g. collagen, chitosan, fibrin) or synthetic (e.g. polyglycolic acid (PGA), poly-lactic-co-glycolic acid (PLGA), polycaprolactone (PCL)) materials. The use of hollow tubes avoid many of the drawbacks associated with autografts and allografts, but neurite


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outgrowth has been limited to short nerve gaps and functional recovery is often not achieved as the random dispersion of regenerating axons leads to inappropriate target reinneraction.10 An alternative to the autograft for PNI repair remains an unmet medical need. A synthetic nerve guidance conduit (NGC) has previously been developed to promote and facilitate nerve regeneration.11 Surface functionalization of the NGC was used to enhance the interactions between the conduit and the cells involved in nerve regeneration. A biochemical cue to promote cell attachment was incorporated using Arg-Gly-Asp (RGD). As a cell-binding peptide motif, RGD promotes cell adhesion, spreading, focal contact formation, and cytoskeletal organization.12,13 Topographical signals for directing cellular function in regenerating axons were integrated using intraluminal channels with longitudinally aligned fibers. By introducing intraluminal microchannels, both cell attachment and migration are improved as surface area is increased and axonal extension is confined to a restricted area once a regenerating axon enters a single channel.14,15 As axonal growth generally occurs more randomly in direction, longitudinally aligned fibers were used to guide axon growth linearly, improving neurite extension across large distances.16 Longitudinally aligned fibers contribute to high surface area to volume which is favorable for cell attachment and growth,17,18 and aligned fibers facilitate in sensory and motor nerve regeneration, improving functional recovery.19 The potential use of a NGC composed of an RGD-conjugated poly(serinol hexamethylene urea) (PSHU-RGD) and PCL blended nanofiber scaffold has been examined for nerve regeneration. The efficacy of RGD modification and cytotoxicity, cell viability, cell proliferation, cell differentiation, neurite outgrowth, and guided neurite sprouting in PC12 cell culture has been investigated for PSHURGD.20,21 A method of introducing intraluminal microchannels with aligned nanofibers was developed, confirmed with scanning electron microscope (SEM) imaging, and was found to


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promote neuronal growth and guide axonal extension, in addition to enhancing cell attachment, survival, and migration in human neural stem cell (hNSC) culture.11 The focus of this study was to evaluate the NGC for peripheral nerve regeneration in a rat sciatic nerve injury (SNI) model. MATERIALS AND METHODS Materials. Serinol, urea, hexamethylene diisocyanate (HDI), anhydrous N,Ndimethylformamide (DMF), PCL (Mn 80,000 g/mol), and Masson’s trichrome stain kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Di-tert-butyl dicarbonate, ethyl acetate, trifluoroacetic acid (TFA), 2,2,2-trifluoroethanol (TFE), N-(3-dimethylamino- propyl)-N′ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 1,1,1,3,3,3hexafluoro-2-propanol (HFP) were purchased from Alfa Aesar (Ward Hill, MA, USA). Hexane and anhydrous diethyl ether were purchased from Fisher Scientific (Pittsburgh, PA, USA). Anhydrous methylene chloride (DCM) was purchased from JT Baker (Phillipsburg, NJ, USA). Gly–Arg–Gly–Asp–Ser (GRGDS) was purchased from Biomatik (Wilmington, DE, USA). Neurofilament-medium (NF-M) (rabbit IgG), Alexa Fluor 594 (goat anti-rabbit IgG), S100b (mouse IgG1), Alexa Fluor 488 (goat anti-mouse IgG), and SlowFade Diamond antifade mountant with DAPI were purchased from Life Technologies (Carlsbad, CA, USA). Equipment. SEM images were obtained using a JEOL JSM-6010LA. Electrophysiological assessment involving compound action potential (CAP) measurements was performed using Axon CNS MultiClamp 700B, Axon Digidata 1440A, and Grass SD9 Stimulator. Confocal images were taken using an Olympus FV1000. Brightfield images were taken using a Nikon Eclipse 80i. ImageJ was used to quantify variables for image analysis.


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Synthesis of PSHU-RGD. Serinol (1.96 g, 21.5 mmol) was dissolved in ethanol (20.0 ml) and di-tert-butyl dicarbonate (5.97 ml, 26.0 mmol) dissolved in ethanol (10.0 ml) was added dropwise cooled at 4 °C. The solution was heated at 37 °C for 1 hour and subsequently rotoevaporated at 50 °C and 20 mbar to remove ethanol, yielding a white powder. The white powder was dissolved in a 1:1 volume mixture of ethyl acetate and hexane at 60 °C. Hexane was added dropwise until crystalline structures were formed and excess hexane was added to ensure complete precipitation. The precipitate was allowed to settle at 4 °C and filtered to remove hexane, yielding N-BOC serinol as a crystalline white powder. PSHU-RGD was synthesized as described previously.20 Briefly, N-BOC serinol, urea, and HDI were dissolved in DMF and heated at 90 °C for 7 days. PSHU was recovered by precipitation in diethyl ether and rotoevaporation, yielding the polyurea as a white powder. PSHU was dissolved in DCM and TFA. Deprotection occurred by hydrogenation at room temperature for 45 min. Deprotected PSHU was recovered by precipitation in diethyl ether and rotoevaporation. GRGDS was conjugated to all free amine groups of deprotected PSHU (2.05 mmol NH2 / g PSHU) using EDC/NHS chemistry. PSHU-RGD was recovered by precipitation in diethyl ether and rotoevaporation, yielding a white powder. Nerve guidance conduit fabrication. Prior to electrospinning, sucrose fibers with diameters between 200-250 µm were formed using a fiber drawing method. Sucrose was heated to 75 °C until melted with a thick consistency and fibers were drawn. The collector was constructed using two copper wire electrodes spaced 3.5 cm apart and the sucrose fibers were fit to span the gap between the copper wire electrodes. 8 w/w % polymer solutions in HFP were prepared for PSHU-RGD/PCL (30/70) blend, PSHU/PCL (30/70) blend, and pure PCL. A two electrode electrospinning setup was used to fabricate the NGC. The collector was placed 10 cm from the


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needle of the syringe filled with polymer solution. The polymer solution was ejected at 1.0 ml/h through a 21 gauge stainless steel flat-tip needle at room temperature and relative humidity at 30 %. A positive 7.5 kV electrostatic potential was applied to the needle for the two blended solutions, and a 9 kV potential for the pure PCL solution. For PSHU-RGD/PCL conduits, the PSHU-RGD/PCL blend was electrospun for the initial 10-15 min, and then the PSHU/PCL blend was used to deposit the remainder of the nanofibers for an additional 10-15 min. The pure PCL conduits were electrospun for 20-30 min using its respective solution during the entire electrospinning process. The flat sheet was then removed from the collector and rolled into a tube. Using the same electrospinning setup, the rolled tube was held in front of a flat collector and manually rotated for 30-60 s until a thin layer of polymer was coated onto the surface, sealing the outer layer of the tube and preventing it from unraveling. The tubes were then cut into 10 mm long conduits using a razor blade and submerged in ultrapure water for 48 hours to dissolve out the sucrose fibers. The conduit structure including intraluminal microchannels with aligned nanofibers was confirmed with SEM imaging (Figure 1).


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Figure 1. SEM images of PSHU-RGD/PCL conduit cross-sections: (A-B) transverse sections, (C-D) longitudinal sections revealing aligned nanofibers inside intraluminal channels. Scale bars represent 200 µm. Surgical procedures. The sciatic nerve transection and NGC implantation procedure was approved by the Institutional Animal Care and Use Committee (IACUC). A total of 24 Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were used for the study, 4 rats per each graft (autograft, PCL, PSHU-RGD/PCL) for 2 time points (4, 8 weeks). The rats weighing 250-275 g were maintained on a 14/10-hour light/dark cycle with access to food and water ad libitum. The rats were anaesthetized using isoflurane. Preoperative doses of ketoprofen (5 mg/kg) and bupivacaine (0.5 % Marcaine) (2 mg/kg) were administered via subcutaneous injection. The sciatic nerve that underwent transection and implantation was determined randomly, either the left or right sciatic nerve. The sciatic nerve that was not selected for transection did not undergo any experimental manipulation and was used as a control for normal nerve function. The rats were placed on their side and the skin around the gluteal region on the randomly selected side was shaved and disinfected with chlorhexidine and isopropyl alcohol. A longitudinal skin incision from the knee to the hip was made to expose the underlying muscles that were retracted to isolate the sciatic nerve. An incision of the sciatic nerve was made 5 mm in each direction from mid-thigh for a total of a 10 mm gap. In the case of the autograft, the ends of the 10 mm transected nerve were reversed and sutured to the proximal and distal nerve stumps using Prolene polypropylene 7-0 sutures. For PCL and PSHU-RGD/PCL conduits, the ends of the conduit were sutured to the proximal and distal nerve stumps using Prolene polypropylene 7-0. After graft implantation, the muscle layer was closed with coated Vicryl 4-0 sutures using a continuous


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suturing pattern. The skin incision was closed using coated Vicryl 4-0 sutures using a continuous subcuticular suture technique. Postoperative doses of ketoprofen (5 mg/kg) were administered daily for 3 days. The rats were maintained for 4 or 8 weeks after implantation. Walking track analysis. A walking alley with a darkened goal box at one end was used to assess functional recovery after implantation. The floor of the walking alley with dimensions 45 cm × 8 cm × 5 cm was covered with white paper. The hind feet of the rats were smeared with finger paint and the rats were allowed to walk down the track leaving footprints on the paper. Several measured variables were used to calculate sciatic function index (SFI), including print length (PL), toe spread (TS), and intermediate toe spread (ITS) of both the experimental or grafted side (EPL, ETS, EITS) and the normal contralateral side (NPL, NTS, NITS) for three sets of prints that were averaged. PL was measured as the distance from the heel to the third toe, TS from the first to the fifth toe, and ITS from the second to the forth toe. SFI was calculated using an equation as described previously, SFI = -38.3((EPL − NPL)/NPL) + 109.5((ETS − NTS)/NTS) + 13.3((EITS − NITS)/NITS) − 8.8.22 The rats were trained once on the walking track before the implantation surgery and once before taking actual measurements at a specific time point. Successful training involved three consecutive trials across the walking track that yielded clear hind foot prints with measurable PL, TS, and ITS. The SFI was measured once for each time point and reported as the average of SFI values measured from three trials. Euthanasia and tissue harvest. Following the walking track analysis, the rats were euthanized by carbon dioxide and bilateral thoracotomy. Both of the sciatic nerves for the grafted and healthy contralateral sides were exposed similarly to the initial implantation protocol, and the sciatic nerves from the spinal cord to the terminal branching site of the tibial, sural, and common peroneal nerves were harvested for a total length of 15 mm. The gastrocnemius muscle samples


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were harvested by making an incision of the skin just above the heel of the hind foot and the skin surrounding the gastrocnemius muscle was removed. Both the gastrocnemius muscles from the grafted and healthy contralateral sides were harvested. CAP recordings. Immediately after nerve tissue harvest, the CAPs of both the sciatic nerves of the grafted and healthy contralateral sides were recorded. When taking measurements, a nerve was placed on a platform with parallel conducting wires where the nerve perpendicularly spanned across the parallel conducting wires (Figure S1). The stimulating electrode was connected to the conducting wire in contact with the proximal region of the nerve and the recording electrode connected to the conducting wire in contact with the distal region of the nerve. The proximal end of the graft or healthy nerve was stimulated for 0.15 ms at 10 V and the CAP was recorded at the distal end for 50 ms. Three CAP measurements were taken for each nerve for each time point and averaged and the CAP amplitude ratio of the grafted nerve to the healthy contralateral nerve was calculated for comparison between the grafts. Immunohistochemistry of nerve grafts. After recording CAP measurements of the sciatic nerves, the grafted nerves were fixed using 4 % PFA in PBS for 1 hour, cryoprotected with 30 % sucrose in PBS for 2 days, embedded in optimal cutting temperature (OCT) compound, and frozen at -80 °C. The nerves were sectioned longitudinally with a thickness of 18 µm and placed on glass slides. The sections were fixed in acetone for 10 min and washed 2 times in PBS for 3 min each. Clear nail polish was used at the very ends of each nerve and allowed to dry to secure the nerves onto the slides. The sections were blocked in 3 % hydrogen peroxide in PBS for 10 min to block endogenous peroxidase activity and washed 3 times in PBS for 3 min each. Blocking Buffer (5% goat serum, 0.4% Triton X-100, PBS) was used to block the sections for 30 min. All antibodies were diluted in Dilution Buffer (1% goat serum, 0.4% Triton X-100, PBS).


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The sections were stained with NF-M (1:500) for 60 min, washed 3 times in PBS for 3 min each, stained with Alexa Fluor 594 (1:500) for 30 min, and washed 3 times in PBS for 3 min each. The sections were double immunostained with S100b (1:1000) for 60 min, washed 3 times in PBS for 3 min each, stained with Alexa Fluor 488 (1:500) for 30 min, and washed 3 times in PBS for 3 min each. SlowFade Diamond antifade mountant with DAPI was used to mount the slides. Axon growth was evaluated by calculating the pixel intensity per unit area of the images stained with NF-M and Alexa Fluor 594 at the proximal end, middle, and distal end of each conduit. Masson’s trichrome staining of gastrocnemius muscle. The gastrocnemius muscle of both the sides with the graft implantation and the healthy contralateral side were fixed in 10 % formalin for 24 hour, cryoprotected with 30 % sucrose in PBS for 24 hours, embedded in OCT compound, and frozen at -80 °C. The muscles were sectioned with a thickness of 5 µm and placed on glass slides. Sections were stained using Masson’s trichrome staining kit. Gastrocnemius muscle fiber area was quantified using the cross-sectional area of muscle fibers. The amount of collagen was quantified by calculating the area of collagen to muscle fiber area ratio for the grafted side to the healthy contralateral side. Statistical analysis. All results are expressed as means ± standard error of the mean. Analysis of variance (ANOVA) was used to determine significant differences between groups and followed by Tukey’s post hoc test when applicable. Statistical significance was considered when p < 0.05. RESULTS PNI was modeled with sciatic nerve transection and implanted nerve grafts were used to facilitate in nerve regeneration. An autograft, a PCL conduit, or a PSHU-RGD/PCL conduit was implanted into each rat. The autografts were expected to provide the greatest amount of


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functional recovery and was used as a positive control. As seen previously, the PCL conduit is associated with limited neuronal activity due to the absence of RGD in vitro, while PSHURGD/PCL demonstrated the promotion of neuronal growth and guided axonal extension.11 After making a longitudinal skin incision and retracting the underlying muscles surrounding the sciatic nerve, a 10 mm long transection was made and a graft was implanted at the transection site (Figure 2). Nerve regeneration was assessed at 4 and 8 weeks. No complications arose during the suturing of the grafts to the proximal and distal nerve stumps with the exception for one rat (autograft, 8 weeks), which was not included for statistical analysis.

Figure 2. Sciatic nerve transection and graft implantation: (A) isolation of sciatic nerve, (B) sutured autograft, (C) sutured PSHU-RGD/PCL conduit. An implanted PCL conduit was similar to the PSHU-RGD/PCL conduit. Walking track analysis showed that functional motor recovery was similar to the autograft. Clinically, the objective in the use of a graft or conduit after a nerve transection is to restore function to denervated tissues. Although functional tests are highly variable, functional assessment is the most direct method of evaluating restored nerve function and can be assessed using a walking track analysis. Through the walking track analysis, motor function is quantified through several variables that are measured after recording footprints of the hind feet (Figure 3). The quantified value, the SFI, ranges from 0 to -100, where 0 indicates normal nerve function and -100 indicates total impairment. No significant difference in motor function was observed


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between each graft at either 4 or 8 weeks, but a significant improvement was observed in sciatic nerve function between 4 and 8 weeks for the PSHU-RGD/PCL conduit. This suggests that the PSHU-RGD/PCL conduit may allow for more rapid functional recovery compared to the autograft and the PCL conduit. Overall, the PSHU-RGD/PCL conduit seemed to show similar, if not enhanced, axon regeneration and selective target reinnervation characteristics compared to the autograft.

Figure 3. Functional assessment using walking track analysis. SFI ranges from 0 to -100, where 0 indicates normal sciatic nerve function and -100 indicates total sciatic nerve impairment. Error bars represent standard error of the mean. * indicates p < 0.05. CAPs revealed improved restoration of electrophysiological activity to the autograft. As nerves regenerate, it is imperative that the regenerated axons are capable of propagating action potentials. The recovery of this electrophysiological activity is an important step towards achieving functional recovery. As axons and myelin sheath regenerate, CAP recording can be used to assess the ability to propagate electrical impulses across the transection gap. Electrical


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stimulation at the proximal end was used to evoke a CAP that was recorded at the distal end (Figure 4A). The electrophysiological measurements consisted of two peaks. The first peak was the stimulus artifact that is produced from stimulation, while the second peak is the CAP. The amplitude was determined as the maximum peak value of the CAP, and the CAP amplitude ratio of the grafted nerve to the healthy contralateral nerve was calculated to compare the different grafts due to the variability of CAPs of the healthy contralateral sciatic nerves between each rat (Figure 4B). The CAP amplitude ratio was significantly higher for the PSHU-RGD/PCL conduit compared to the autograft and PCL conduit after 4 weeks. However, no statistical difference was observed at 8 weeks, presumably due to the high variability associated with taking recordings and limited sample size. Even so, it appears that electrophysiological activity was increased with the use of PSHU-RGD/PCL over the other grafts. The CAP recordings seem to imply that PSHU-RGD/PCL may provide an improved environment for promoting axonal extension and myelination to the autograft.


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Figure 4. Electrophysiological assessment by stimulating and recording CAPs. (A) representative electrophysiological measurements for all grafts and the healthy contralateral sciatic nerve after 8 weeks. The first peak is a stimulus artifact, while the second peak is the desired CAP. (B) ratio of CAP amplitude of the grafted to healthy contralateral nerve. Error bars represent standard error of the mean. * indicates p < 0.05. Immunohistochemistry was used to observe substantial axonal extension and Schwann cell presence in the conduits. Although functional and electrophysiological assessments can be used to evaluate nerve regeneration, histological examination is essential to complement these assessments. Axons were stained with NF-M and Alexa Fluor 594, while Schwann cells were stained with S100b and Alexa Fluor 488 (Figure 5). For both the PCL and PSHU-RGD/PCL conduits, axons and Schwann cells were observed to span the entire length of the transection site. Axons and Schwann cells are much more prevalent in the autograft compared to the two conduits. However, a direct comparison of regenerating axon and Schwann cell density between the conduits and the autograft cannot be made based on these images, as the autograft initially contained axons and Schwann cells with implantation and both of the stains are not specific for only regenerating axons and Schwann cells involved in regeneration. Also, the physical characteristics of the conduits prevent direct comparison. As the wall thickness of the intraluminal channels is larger than the section thickness that were cut for staining, axons and Schwann cells within a specific microchannel may leave the field of view if sections were not cut perfectly parallel to the microchannels. Although axonal extension was not always continuous through each section imaged, the presence of axons at the distal end was used to infer that axons have spanned the entire length of the transection gap. As for the PCL conduit, substantial axonal extension was observed along the outer surface of the conduit with minimal axonal extension


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through the intraluminal channels (Figure 5C-D). However, the PSHU-RGD/PCL conduit allowed for axonal extension along the entire length of an intraluminal channel in a continuous linear path. Axon regeneration was also observed in multiple microchannels and throughout the diameter of the conduits (Figure S2). One of the limitations of the immunohistochemical analysis is the background signal associated with the S100b antibody and the associated secondary antibody. S100b or the associated Alexa Fluor 488 antibody appears to have high affinity to the conduits and the signal observed with the stain is not representative of specific Schwann cell binding. However, closer examination of areas with overlapping neurofilament and Schwann cell signals indicates myelinated axons (Figure 5F-G). Higher magnification images of areas with high axon density reduced signal from the conduit and revealed the close association of axons and Schwann cells (Figure 5E, Figure 5H).


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Figure 5. Immunohistochemical assessment with double immunostaining for axonal regeneration and Schwann cell activity. Representative images after 8 weeks of (A-B) autograft (C-E) PCL conduit, and (F-H) PSHU-RGD/PCL conduit; (A-D, F-G) longitudinal sections from left to right represent proximal to distal ends, (E,H) higher magnification images of areas with high axon density. The outer surface of the (C-D) PCL conduit annotated with a dashed line showed substantial axonal extension along the outer surface of the conduit. Closer examination of areas with overlapping neurofilament and Schwann cell signals annotated with the region of interest (F-G) indicates myelinated axons. Higher magnification images (E, H) of areas with high axon density reduced signal from the conduit and revealed the close association of axons and Schwann cells. Axons were stained with NF-M and Alexa Fluor 594 and appear in red. Schwann cells were stained with S100b and Alexa Fluor 488 and appear in green. Scale bar represents: (AD, F-G) 500 µm, (E,H) 100 µm. Axon regeneration was quantified by calculating pixel intensity per unit area at (Figure 6). PSHU-RGD/PCL consistently showed statistically superior axonal growth compared to the PCL conduit at the proximal end, middle, and distal end of the conduits. Although a direct comparison between the conduits and autograft cannot be made due to the reasons mentioned previously, axon density of the autografts was still quantified and compared to conduit values to observe for any regenerating trends. As expected, the autograft showed the highest axon density. The autograft had a significant increase in axon density compared to the PCL conduit across the entire length of the graft, but showed statistically significant increase only in more distal ends of the nerves. This may indicate regenerating nerves more readily span a nerve gap with an autograft, rather than the conduits.


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Figure 6. Quantification of axon regeneration using pixel intensity per unit area: (A) proximal end, (B) middle, and (C) distal end of conduits. Error bars represent standard error of the mean. * indicates p < 0.05. Masson’s trichrome stain indicated similar muscle atrophy and fibrotic tissue formation compared to the autograft. As a target tissue of the sciatic nerve, the gastrocnemius muscle undergoes morphological changes and collagenous tissue formation with denervation. By comparing muscle atrophy and fibrotic tissue formation of the muscle from the grafted side and healthy contralateral side, sciatic nerve function can be assessed. Masson’s trichrome stain was used to examine the morphology of the gastrocnemius muscle fibers (Figure 7A-C). Crosssectional muscle fiber area and the amount of collagen were quantified to assess muscle atrophy and fibrotic tissue formation (Figure 7D-E). At 4 weeks after implantation, no statistical difference was observed between any of the grafts, but after 8 weeks, the autograft and PSHURGD/PCL conduit had a statistically significant higher muscle fiber area and lower presence of collagen compared to the PCL conduit. The results suggest that the PSHU-RGD/PCL conduit


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had comparable motor neuron regeneration and muscle reinnervation characteristics to the autograft.

Figure 7. Masson’s trichrome stain of gastrocnemius muscle after 8 w: (A) autograft, (B) PCL conduit, (C) PSHU-RGD/PCL conduit. Muscle fibers are stained in red, while collagen is stained in blue. Scale bars represent 100 µm. (D) ratio of cross-sectional muscle fiber area of the grafted side to the healthy contralateral side. (E) area of collagen to muscle fiber area ratio for the grafted side to the healthy contralateral side. Error bars represent standard error of the mean. * indicates p < 0.05.


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DISCUSSION Functional, electrophysiological, and histological assessments were used to evaluate nerve regeneration 4 and 8 weeks post nerve transection and graft implantation. The results suggest that the biomimetic nerve guidance conduit containing intraluminal microchannels with aligned nanofibers markedly facilitates in nerve regeneration. The walking track analysis utilized decreased PL, increased TS, and increased ITS characteristics from the initial intrinsic loss of muscle function to measure functional recovery of the selective target tissue of the sciatic nerve.23 The calculated SFI values showed that both the PSHU-RGD/PCL and PCL conduits seemed to attribute to equal, if not superior, functional recovery compared to the autograft. This encouraging level of functional regeneration of the PSHU-RGD/PCL conduit can be attributed to RGD and intraluminal microchannels with aligned nanofibers. Although the PCL conduit was expected to induce a very limited amount of functional recovery, the positive effect of intraluminal microchannels with aligned nanofibers may have been underestimated. Axons in the peripheral nervous system are known to be able to regenerate on their own, but the topical cues associated with the microchannels and aligned nanofibers may have accelerated this regeneration process. These results suggest that intraluminal microchannels with aligned nanofibers may positively contribute topographical signals for regenerating axons, and the presence of RGD for cell binding may further improve the overall functional recovery. The CAP recordings obtained from the electrophysiological assessment for all the grafts had low latency, characteristic of the short delay of the peak after the stimulus artifact. This suggests that the majority of the axons that are contributing to the CAP have high degrees of


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myelination. Within 4 weeks after implantation, a CAP was observed for all grafts. This implies that a sufficient number of myelinated axons have regenerated across the entire length of the nerve gap. Immediately after sciatic nerve transection, CAPs are expected to be unmeasurable. Due to the disconnection of axons, the nerve is incapable of propagating electrical impulses across the nerve gap. As the number of regenerating axons across the transection site increase during the regeneration process, the CAP amplitude is expected to increase. A statistically significant increase in CAP amplitude ratio was observed for the PSHU-RGD/PCL graft after 4 weeks compared to other grafts. Immunohistochemical assessment was used to directly observe axonal extension and growth. Axons were observed with a stain specific for neurofilament, while Schwann cells were present in areas of high axon density, detected with a stain specific for calcium-binding proteins. Axonal extension from the proximal end of the graft across the entire length towards the distal end was observed for the PSHU-RGD/PCL conduit. As measurable CAPs were recorded for all the grafts at both 4 and 8 week time points, it was expected to see axonal extension across the entire length. Compared to the PCL conduit, the PSHU-RGD/PCL conduit provided a statistically significant improvement in axonal extension and growth. The PCL conduit showed considerable axonal extension, but extensively along the outer surfaces of the conduit. The high density of axons along the circumferential surface and the low density of axons through the intraluminal channels are undesirable as axonal extension through the intraluminal channels are more likely to contribute to selective target reinnervation. Without RGD, regenerating axons may have been unable to attach to the surfaces of the intraluminal microchannels. It is speculated that the regeneration of axons along the outer surface of the conduit was due to the inflammatory response following the invasive surgical procedure, creating an extracellular environment with


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abundant connective tissue that promoted cell attachment. PCL conduits have been shown to be surrounded by fibrous connective tissue formation after implantation,24 and axons have been associated to extend along fibrous tissue.25 This formation of connective tissue may have allowed for favorable cell attachment along the outer surface of the conduits compared to the intraluminal channels without RGD. The morphology associated with the reinnervation of the gastrocnemius muscle was observed using Masson’s trichrome stain. Both cross-sectional muscle fiber area and the quantity of collagen present were used to assess the disease state of the muscle. Muscle atrophy occurs with the denervation of the gastrocnemius muscle following sciatic nerve transection and fibrotic tissue is formed. With the regeneration of motor neurons and reinnervation of the muscles, the degree of muscle atrophy and fibrotic tissue will gradually decrease. Muscle fiber area was used to assess muscle atrophy and the quantity of collagen present in muscle was used to assess the disease state of the muscle. The PSHU-RGD/PCL conduit had comparable motor neuron regeneration and muscle reinnervation characteristics to the autograft and statistically reduced muscle atrophy and fibrotic tissue formation compared to the PCL conduit. CONCLUSIONS Although nerve regeneration using the PSHU-RGD/PCL conduit did not have statistically significant improvements for all assessments considered for evaluation, the NGC consistently showed similar or improved nerve regeneration characteristics compared to the autograft. Functional motor recovery and reductions in muscle atrophy and fibrotic tissue formation were similar to the autograft, while restoration of electrophysiological activity was improved over the autograft. These results are encouraging as autografts are associated with many drawbacks and it


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was determined that the PSHU-RGD/PCL conduit is a functionally comparable alternative for surgical treatment of PNI. ASSOCIATED CONTENT Supporting Information. Supplemental Figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. DL performed all aspects of the study including polymer synthesis, conduit fabrication, animal surgeries, data collection, and data analysis. AF assisted in preparing electrophysiology setup. XM provided access to cryostat. DP conceived the study and provided the means and direction for the synthesis and characterization of this polymer conduit. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was partially supported by NIH 1R21EY023711-01A1. Dr. John H Caldwell helped arrange all the equipment needed for electrophysiological assessment. Melissa Card dedicated


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much of her time on animal care training and surgical techniques. Dr. Chris Manuel provided training for the sciatic nerve transection surgical procedure. Yufeng Zhai provided instruction for sectioning both nerves and muscle. REFERENCES (1)

Kouyoumdjian, J. A. Peripheral nerve injuries: A retrospective survey of 456 cases. Muscle Nerve 2006, 34 (6), 785–788 DOI: 10.1002/mus.20624.

(2)

Robinson, L. R. Traumatic injury to peripheral nerves. Muscle Nerve 2000, 23 (6), 863– 873 DOI: 10.1002/(SICI)1097-4598(200006)23:63.0.CO;2-0 [pii].

(3)

Taylor, C. A.; Braza, D.; Rice, J. B.; Dillingham, T. The incidence of peripheral nerve injury in extremity trauma. Am. J. Phys. Med. Rehabil. 2008, 87 (May), 381–385 DOI: 10.1097/PHM.0b013e31815e6370.

(4)

Ichihara, S.; Inada, Y.; Nakamura, T. Artificial nerve tubes and their application for repair of peripheral nerve injury: an update of current concepts. Injury 2008, 39, 29–39 DOI: 10.1016/j.injury.2008.08.029.

(5)

Daly, W. T.; Knight, A. M.; Wang, H.; de Boer, R.; Giusti, G.; Dadsetan, M.; Spinner, R. J.; Yaszemski, M. J.; Windebank, A. J. Comparison and characterization of multiple biomaterial conduits for peripheral nerve repair. Biomaterials 2013, 34 (34), 8630–8639 DOI: 10.1016/j.biomaterials.2013.07.086.

(6)

Siemionow, M.; Brzezicki, G. Chapter 8: Current techniques and concepts in peripheral nerve repair. Int Rev Neurobiol 2009, 87, 141–172 DOI: 10.1016/s0074-7742(09)87008-6.

(7)

Kang, S. B.; Ju, Y. M.; Lee, S. J.; Atala, A.; Yoo, J. J. Functional recovery of denervated muscle by neurotization using nerve guidance channels. Journal of Tissue Engineering and Regenerative Medicine. 2013.

(8)

Moore, A. M.; Ray, W. Z.; Chenard, K. E.; Tung, T.; Mackinnon, S. E. Nerve allotransplantation as it pertains to composite tissue transplantation. Hand 2009, 4, 239–


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 29

244 DOI: 10.1007/s11552-009-9183-x. (9)

Tajima, K.; Tohyama, K.; Ide, C.; Abe, M. Regeneration through nerve allografts in the cynomolgus monkey (Macaca fascicularis). J. Bone Joint Surg. Am. 1991, 73, 172–185.

(10)

Kehoe, S.; Zhang, X. F.; Boyd, D. FDA approved guidance conduits and wraps for peripheral nerve injury: A review of materials and efficacy. Injury 2012, 43 (5), 553–572 DOI: 10.1016/j.injury.2010.12.030.

(11)

Jenkins, P. M.; Laughter, M. R.; Lee, D. J.; Lee, Y. M.; Freed, C. R.; Park, D. A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells. Nanoscale Res. Lett. 2015, 10 (1), 972 DOI: 10.1186/s11671015-0972-6.

(12)

Van der Flier, A.; Sonnenberg, A. Function and interactions of integrins. Cell and Tissue Research. 2001, pp 285–298.

(13)

Lagunas, A.; Comelles, J.; Martínez, E.; Prats-Alfonso, E.; Acosta, G. A.; Albericio, F.; Samitier, J. Cell adhesion and focal contact formation on linear RGD molecular gradients: Study of non-linear concentration dependence effects. Nanomedicine Nanotechnology, Biol. Med. 2012, 8, 432–439 DOI: 10.1016/j.nano.2011.08.001.

(14)

Ma, P. X.; Zhang, R. Microtubular architecture of biodegradable polymer scaffolds. J. Biomed.

Mater.

Res.

2001,

56

(4),

469–477

DOI:

10.1002/1097-

4636(20010915)56:43.0.CO;2-H. (15)

De Ruiter, G. C.; Spinner, R. J.; Malessy, M. J. A.; Moore, M. J.; Sorenson, E. J.; Currier, B. L.; Yaszemski, M. J.; Windebank, A. J. Accuracy of motor axon regeneration across autograft, single-lumen, and multichannel poly(lactic-co-glycolic acid) nerve tubes. Neurosurgery 2008, 63, 144–153 DOI: 10.1227/01.NEU.0000335081.47352.78.

(16)

Rangappa, N.; Romero, A.; Nelson, K. D.; Eberhart, R. C.; Smith, G. M. Laminin-coated poly(L-lactide) filaments induce robust neurite growth while providing directional orientation. J. Biomed. Mater. Res. 2000, 51 (4), 625–634 DOI: 10.1002/10974636(20000915)51:43.0.CO;2-U.


26

Page 27 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(17)

Jeffries, E. M.; Wang, Y. Incorporation of parallel electrospun fibers for improved topographical guidance in 3D nerve guides. Biofabrication 2013, 5, 035015 DOI: 10.1088/1758-5082/5/3/035015.

(18)

Chew, S. Y.; Mi, R.; Hoke, A.; Leong, K. W. Aligned protein-polymer composite fibers enhance nerve regeneration: A potential tissue-engineering platform. Adv. Funct. Mater. 2007, 17, 1288–1296 DOI: 10.1002/adfm.200600441.

(19)

Kim, Y. T.; Haftel, V. K.; Kumar, S.; Bellamkonda, R. V. The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps. Biomaterials 2008, 29, 3117–3127 DOI: 10.1016/j.biomaterials.2008.03.042.

(20)

Yun, D.; Lee, Y. M.; Laughter, M. R.; Freed, C. R.; Park, D. Substantial Differentiation of Human Neural Stem Cells Into Motor Neurons on a Biomimetic Polyurea. Macromol. Biosci. 2015 DOI: 10.1002/mabi.201500066.

(21)

Yun, D.; Famili, A.; Lee, Y. M.; Jenkins, P. M.; Freed, C. R.; Park, D. Biomimetic poly(serinol hexamethylene urea) for promotion of neurite outgrowth and guidance. J. Biomater.

Sci.

Polym.

Ed.

2014,

25

(March),

354–369

DOI:

10.1080/09205063.2013.861170. (22)

Bain, J. R.; Mackinnon, S. E.; Hunter, D. A. Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast. Reconstr. Surg. 1989, 83 (1), 129–138.

(23)

Sarikcioglu, L.; Demirel, B. M.; Utuk, a. Walking track analysis: An assessment method for functional recovery after sciatic nerve injury in the rat. Folia Morphol. (Warsz). 2009, 68 (1), 1–7.

(24)

Niu, Y.; Chen, K. C.; He, T.; Yu, W.; Huang, S.; Xu, K. Scaffolds from block polyurethanes based on poly(ɛ-caprolactone) (PCL) and poly(ethylene glycol) (PEG) for peripheral nerve regeneration. Biomaterials 2014, 35 (14), 4266–4277 DOI: 10.1016/j.biomaterials.2014.02.013.

(25)

Yamamoto, T.; Osako, Y.; Ito, M.; Murakami, M.; Hayashi, Y.; Horibe, H.; Iohara, K.;


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Takeuchi, N.; Okui, N.; Hirata, H.; et al. Trophic Effects of Dental Pulp Stem Cells on Schwann Cells in Peripheral Nerve Regeneration. Cell Transplant. 2016, 25 (1), 183–193 DOI: 10.3727/096368915X688074.


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Biomimetic Nerve Guidance Conduit Containing Intraluminal

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