A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells
© Jenkins et al. 2015
Received: 4 March 2015
Accepted: 3 June 2015
Published: 12 June 2015
Despite major advances in the pathophysiological understanding of peripheral nerve damage, the treatment of nerve injuries still remains an unmet medical need. Nerve guidance conduits present a promising treatment option by providing a growth-permissive environment that 1) promotes neuronal cell survival and axon growth and 2) directs axonal extension. To this end, we designed an electrospun nerve guidance conduit using a blend of polyurea and poly-caprolactone with both biochemical and topographical cues. Biochemical cues were integrated into the conduit by functionalizing the polyurea with RGD to improve cell attachment. Topographical cues that resemble natural nerve tissue were incorporated by introducing intraluminal microchannels aligned with nanofibers. We determined that electrospinning the polymer solution across a two electrode system with dissolvable sucrose fibers produced a polymer conduit with the appropriate biomimetic properties. Human neural stem cells were cultured on the conduit to evaluate its ability to promote neuronal growth and axonal extension. The nerve guidance conduit was shown to enhance cell survival, migration, and guide neurite extension.
Functional recovery following a severe peripheral nerve injury is a daunting challenge in the field of neuroscience. After nerve transection, collapse and sealing of the axonal membranes at each nerve stump lead to the formation of either a growth cone or a retraction bulb. As part of the initial response after injury, myelin breaks down and axonal degeneration occurs at both the proximal and distal nerve stumps further increasing the gap between the nerve ends. Peripheral nerves are able to effectively regenerate over short distances; however, when the space between nerve ends is too wide (greater than 2 cm), surgical intervention is required to reconnect the damaged nerve using nerve grafts [1–3]. Currently, the gold standard for this type of peripheral nerve injury repair is surgical implantation of autologous nerve grafts. However, autographs have many clinical limitations including donor site morbidity, mismatch of donor size, neuropathic pain, and limited donor nerve length [4, 5].
To overcome these obstacles, considerable attention has been given to synthetic nerve conduits as an alternative to autographs. Synthetic nerve conduits are easily fabricated and show good reproducibility in structure specifications . A variety of synthetic nerve conduits have been developed using different biomaterials such as polyamides , polyethylene terephthalate , poly(L-lactide) [8, 9], poly(ε-caprolactone) [10–12], poly(lactic glycolic acid) [13–15], and collagen [16, 17]. Blending of polymers has also been used to produce different types of conduits including simple hollow tubes , tubes with longitudinally aligned nanofibers along the interior lumen wall, and tubes with sheets of aligned nanofibers. Recently, more sophisticated conduits containing intraluminal microchannels of aligned nanofibers have shown promising results . These aligned nanofibers and microchannels increase the surface area for which cells may interact with the conduit. However, these polymers rely solely on topographical cues to guide axonal growth and cell migration. It is likely that incorporating biochemical cues into these nerve conduits will further induce cell attachment and spreading, accelerating axonal extension during nerve regeneration [20–25].
For optimal nerve regeneration, synthetic conduits should include both topographical and biochemical cues. One commonly employed biochemical cue is Arg-Gly-Asp (RGD), a short peptide motif derived from the active sites of extracellular matrix (ECM) proteins specific for cell binding. RGD has been shown to enhance cell proliferation, migration, and survival in most tissues . Previously, we reported that an RGD-functionalized, biomimetic poly(serinol hexamethylene urea) (PSHU) nanofiber conduit was shown to increase neurite guidance and axon extension in vitro . In this study, the PSHU-RGD was further modified into a conduit with microchannels of aligned nanofibers. While the incorporation of RGD was intended to mimic biochemical cues of natural connective tissue found in nerves, the inclusion of microchannels with aligned nanofibers were intended to mimic the topographical cues in natural nerve tissue. Human neural stem cells (hNSCs) were used to evaluate the ability of this conduit to promote neurite guidance and extension. hNSCs are multipotent cells that primarily differentiate into neurons, oligodendrocytes, and astrocytes, making them ideal for modeling neural regeneration.
All in vitro cell morphologies were examined on a Nikon DIAPHOT 300 equipped with CCD camera (SPOT RT 2.3.0, Diagnostic Instruments) using SPOT Advanced software for post hoc analysis and LSM 510 Laser Scanning Microscope. The microstructures of the electrospun nanofiber conduits were observed by field emission scanning electron microscopy (SEM) (JSM 7401F, JEOL).
Trifluoroacetic acid (TFA), 2,2,2-trifluoroethanol (TFE), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) were purchased from Alfa Aesar (Ward Hill, MA, USA). Anhydrous dichloromethane (DCM) was purchased from JT Baker (Phillipsburg, NJ, USA). The pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) was purchased from Biomatik (Wilmington, DE, USA). N,N-dimethylformamide (DMF), hexamethylene diisocyanate (HDI), N-BOC-serinol, urea, and poly(ε-caprolactone) (PCL, Mn: 70,000-90,000 g/mol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anhydrous diethyl ether was purchased from Fisher Scientific (Pittsburgh, PA, USA).
Synthesis of RGD conjugated PSHU (PSHU-RGD)
PSHU-RGD was synthesized as described previously . Briefly, N-BOC-Serinol, HDI, and urea were reacted for 7 days at 90 °C. The PSHU was recovered by precipitation in diethyl ether. Following synthesis, the BOC groups were removed from PSHU by reaction with TFA/DCM (1:1) for 1 h at room temperature. Finally, RGD (1:1 molar ratio of RGD: −NH2 groups in PSHU) was conjugated to deprotected PSHU using EDC/NHS chemistry to yield PSHU-RGD.
Scaffold synthesis by electrospinning
Fiber angle distribution
OrientationJ, a plug-in for ImageJ, was used to process SEM images and generate a table of pixels counted versus degree of orientation. The data was then exported to Microsoft Office Excel 2010 and normalized to center the maximum peak at 0°. After normalization, the data was exported to Origin 9.1 to plot a histogram and to determine the degree of fiber angle agreement.
All research met the requirements of the Helsinki Declaration. The method of induction of human neural stem cells from embryonic stem cells was modified from what was described previously . Stem cell induction began with hESCs UCSF-4 cultured in mTesR media (STEMCELL) on Cellstart (Invitrogen) coating plates. UCSF-4 cells were passaged by cutting colonies into small squares using StemPro EZPassage Disposable Stem Cell Passaging Tool (Gibco) at a dilution of 1:10. For induction, UCSF-4 cells were treated with 10 ng/ml hLIF (Millipore), 3 μM CHIR99021 (Cellagentech), and 2 μM SB431542 (Cellagentech) in neural induction media, N2B27, containing DMEM/F12:neurobasal (1:1), 0.5× N2, 1× B27, 1 % Glutmax, for 10 days. The culture was then split 1:3 for the next passages using Accutase and expanded in neural induction media supplemented with 10 ng/ml hLIF, 3 μM CHIR99021, and 2 μM SB431542 on Cellstart coating plate.
hNSC culture on nerve guidance channel
hNSCs (1 × 105 cells/100 μl) were injected into the very tip of one end of 3 PSHU-RGD/PCL conduits and 3 PCL conduits and cultured on 24-well plates. After 24 h, 200 μM ascorbic acid and 10 ng/ml BDNF and GDNF were added to N2B27 culture media and changed daily. After a 14-day culture, conduits were washed twice in PBS then fixed at room temperature for 4 h with 4 % paraformaldehyde. Once fixed, the conduits were prepared for cryosectioning by soaking overnight at 4 °C in a 30 % sucrose PBS solution. After cryosectioning (20-μm slices), each section was washed twice in PBS for 10 min. Then, the samples were treated with blocking buffer (3 % goat serum PBS with 0.01 % Triton X-100) for 1 h at room temperature before adding β-III tubulin goat anti-mouse primary antibody (1:200 dilution, Promega, Madison, WI, USA) overnight at 4 °C. Following three PBS washes, the samples were incubated with anti-mouse secondary antibody conjugated to Alexa 488 (1:300 dilutions, Invitrogen) for 1 h at room temperature in the dark. The samples were examined using confocal microscopy.
Results and discussion
The effect of RGD as a biochemical cue has been previously examined with PC12 cells and hNSCs. PC12 cells on surfaces coated with PSHU-RGD showed significantly higher levels of cell attachment, differentiation, and neurite outgrowth compared to surfaces coated without the presence of RGD . An increase in hNSC survival and differentiation was observed on PSHU-RGD surfaces compared to surfaces without RGD . In this study, a biochemically and topographically controlled synthetic nerve conduit was developed with emphasis on promoting neuronal cell survival, migration, and guided extension. After confirmation of the conduit structure and the presence of aligned microchannels, the conduits were assessed in vitro by hNSC cultures.
Scaffold synthesis by electrospinning
Dual two-electrode electrospinning has been shown to produce highly aligned nanofibers but utilizes a double nozzle system to produce a composite material. Our initial design was to fabricate a conduit with PSHU-RGD alone; however, PSHU-RGD nanofibers proved too brittle to be further processed into the desired conduit. Thus, to provide more flexibility, PSHU-RGD was blended with PCL.
Neurite extension and fiber alignment has been previously examined using PC12 cells. PC12 cells seeded on randomly aligned nanofibers had highly branched and randomly aligned neurite outgrowth. In contrast, PC12 cells seeded on aligned fibers were less branched, sprouted along the direction of the aligned fibers, and extended further from the cell bodies, similarly to native neurons .
hNSC response to nerve guidance conduit
Stem cell research has gained significant interest in the field of nerve regeneration. However, one obstacle to these cell-based therapies is the lack of a sufficient cell scaffold or conduit to enhance the survival and axon extension of the implanted cells . Through the presence of both topographical and biochemical cues, we expect that our conduit may overcome these obstacles and serve as a nerve guide for cell-based therapies. hNSCs were cultured on the nerve guidance conduits to determine if cell migration and neurite extension are enhanced.
A nerve guidance conduit was successfully fabricated for applications in neuronal tissue engineering. This conduit was designed to provide 1) biochemical cues through the conjugation of RGD and 2) topographical cues through microchannels with aligned nanofibers. The topographical and biochemical cues present on the PSHU-RGD/PCL conduit were shown to encourage hNSC growth and neurite extension when compared to the pure PCL conduit. These findings could have a significant impact on current nerve grafts and the treatment of peripheral nerve damage. Furthermore, the design concept of this conduit could provide a platform for improved nerve regeneration with enhanced cell survival, migration, and guided extension.
This work was supported by the University of Colorado Denver start-up funding.
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