Promoting Neurite Growth and Schwann Cell Migration by the

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Biological and Medical Applications of Materials and Interfaces

Promoting Neurite Growth and Schwann Cells Migration by Harnessing Decellularized Nerve Matrix onto Nanofibrous Guidance Shihao Chen, Zhaoyi Du, Jianlong Zou, Shuai Qiu, Zilong Rao, Sheng Liu, Xiumin Sun, Yiwei Xu, Qingtang Zhu, Xiaolin Liu, Hai-Quan Mao, Ying Bai, and Daping Quan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01066 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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ACS Applied Materials & Interfaces

Promoting Neurite Growth and Schwann Cell Migration by Harnessing Decellularized Nerve Matrix onto Nanofibrous Guidance Shihao Chena,b, Zhaoyi Dua, Jianlong Zouc, Shuai Qiuc, Zilong Raoa, Sheng Liua, Xiumin Suna, Yiwei Xua, Qingtang Zhuc, Xiaolin Liuc, Hai-Quan Maod,e, Ying Baib,*, Daping Quana,b,*

aPCFM

Lab, GD HPPC Lab, School of Chemistry, Sun Yat-Sen University, Guangzhou 510275,

China bGuangdong

Functional Biomaterials Engineering Technology Research Center, School of Materials Science and Engineering, Sun Yat-Sen University, Guangzhou 510275, China cGuangdong

Peripheral Nerve Tissue Engineering and Technology Research Center, Department of Orthopedic and Microsurgery, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China dTranslational

Tissue Engineering Center, and Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA eInstitute

for NanoBioTechnology, and Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA

KEYWORDS Neurite, Schwann cell, Decellularized nerve matrix, Electrospun nanofiber, Remyelination, Fasciculation, Topographical guidance, Cell migration

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ABSTRACT Synergistic intercellular interactions have been widely acknowledged in tuning functional cell behaviors in vivo, and these interactions have inspired the development of a variety of scaffolds for regenerative medicine. In this paper, the promotion of Schwann cell-neurite interactions through the use of a nerve extracellular matrix-coated nanofiber composite in vitro was demonstrated using a cell culturing platform consisting of either random or aligned electrospun poly(L-lactic acid) nanofibers and decellularized peripheral nerve matrix gel (pDNM gel) from porcine peripheral nervous tissue. The pDNM-coated nanofiber platform served as a superior substrate for dorsal root ganglion culturing. Furthermore, Schwann cell migration was facilitated by the pDNM gel coating on the nanofibers, accompanied with a much faster axonal extension, in comparison with effect of topographical guidance from the aligned electrospun fibers only. Finally, the decellularized nerve matrix promoted the ability of Schwann cells to wrap around bundled neurites, triggering axonal remyelination towards nerve fiber functionalization.

1. INTRODUCTION Intercellular communications are integral to tissue development processes, including cell proliferation, growth and migration.1,2 Different cell line origins, homogeneously or heterogeneously, often result in varied stem cell differentiation and functionalization. For example, enhanced chondrogenesis was reported when mesenchymal stem cells (MSCs) were co-cultured with chondrocytes in vitro,3 however, the cellular interactions between the MSCs and degenerate nucleus pulposus (NP) cells stimulated MSC differentiation into the NP-like phenotype.4 Meanwhile, experiments in vivo revealed that the growth and maturation of each tissue type are highly dependent on the intercellular interactions of heterogeneous cells and their compatibility when co-existing in the same microenvironment. Schwann cells (SCs), a major type of neuroglia in the peripheral nervous system, were reported to regulate the direction of neurite and axonal outgrowth due to the secretion of many nerve growth factors.5 However, such cell-cell interaction has hardly been studied in vivo, due to the complexity of the living environment and the technical difficulties of characterization. Surgical repair of transected peripheral nerve after injury requires a precise reconnection between the proximal and distal ends, with the goal of complete functional restoration.6 A fast regeneration rate and directed growth of neurites are the key factors in nerve tissue regeneration and functional recovery.7 Without clinical treatment such as implantation or injection, there are very few spontaneous responses in situ for nerve fiber regeneration, especially for long distance defects.8 Many nerve grafts have been developed by synthetic biodegradable polymers, such as poly(lactic acid) and poly(ε-caprolactone).9-11 However, these inert materials have very limited capability in


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promoting axonal extension and nerve fiber functionalization. It has been widely known that the biochemical interactions between SCs and neurites are crucial in nerve fiber outgrowth, their myelination and functional maintenance.5,12 In order to enhance interactions between SCs and neurites, it is desirable to develop a biomaterial composite that facilitates neurite outgrowth while recapitulating the in vivo nerve regenerative microenvironment. We have reported that the decellularized peripheral nerve matrix hydrogel (pDNM gel) derived from porcine peripheral nerve effectively promoted neurite remyelination and inhibited synaptogenesis.13,14 However, the pDNM gel’s ability to support neurite extension was inferior to that of Matrigel and similar to that of Collagen I. Full characterization of protein composition has indicated that many non-extracellular matrix (ECM) proteins and growth factors were present in the pDNM gel. Axonal inhibitory factors such as chondroitin sulfate proteoglycans (CSPGs) were also identified. It was articulated that the pDNM gel could promote functionalization of the newborn nerve fibers, but the mechanism for neurite extension and guidance was not fully demonstrated, since the topological cues were not taken into consideration. Many reports confirmed that ordered patterns in scaffolds guide axonal elongation and SC migration.15 Electrospinning has been widely used in fabricating nanofibers with ordered or unordered architectures.16,17 Electrospun nanofibers have been found to guide nerve fiber growth due to their spatial and physical confinement, mimicking the ECM’s intrinsic nanostructures.18-23 It was also reported that the aligned nanofibers accelerated neurite extension compared to the nanofibers with random alignment. Additionally, the SCs arranged and migrated along the aligned nanofibers, facilitating maturation of the SCs.23-25 Introducing the extracellular matrix (ECM) contents onto the nanofibrous surfaces would further promote axonal extension by providing nerve nutrients and biological cues, including collagen, fibronectin, laminin, glycosaminoglycans, hyaluronic acid, and a small amount of growth factors such as nerve growth factor (NGF). In this work, biocompatible and biodegradable poly(L-lactic acid) (PLLA) was electrospun into aligned nanofibers for directing axonal extension and pre-coated with pDNM gel to further enhance SC migration. This novel scaffold provided both physical and biological functionalities; the composite materials served as the substrate for dorsal root ganglion (DRG) culturing, which contains neurons and SCs. The intercellular behavior between neurites and SCs was examined in terms of the rate and direction of both axonal extension and SC migration, towards the functionalization of regenerative nerve fibers. 2. MATERIALS AND METHODS 2.1. Materials PLLA was purchased from PURAC (Groningen, Holland); 2,2,2-trifluoroethanol (TFE) was purchased from Aladdin (Shanghai, China); Triton X-100, sodium deoxycholate and



trypsin were purchased from Sigma-Aldrich (Missouri, the United States). Neurobasal medium, B27, L-glutamine, penicillin–streptomycin and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Massachusetts, the United States). NGF was purchased from PeproTech (New Jersey, the United States). Ascorbic acid and mitomycin (MMC) was purchased from Sigma-Aldrich (Missouri, the United States). 4% Paraformaldehyde was purchased from Biosharp (Anhui, China). Donkey serum was purchased from Millipore (Massachusetts, the United States). Anti-NF200 antibody produced in rabbit, Anti-S100 antibody produced in mouse and Anti-MBP antibody produced in mouse were purchased from Sigma-Aldrich (Missouri, the United States). Alexa Fluor-488 and Fluor-594 were purchased from Thermo Fisher Scientific (Massachusetts, the United States). DAPI was purchased from Sigma-Aldrich (Missouri, the United States). Other chemical reagents were purchased from Guangzhou Chemical Reagent Corporation (Guangzhou, China). 2.2. Electrospun Nanofibrous Scaffolds 7.5% (w/v) solution which was prepared by dissolving PLLA in TFE, then transferred into a syringe with a 20G stainless-steel needle. The polymer solution was ejected at 1 mL/h, driven by a syringe pump (Tonli; Shenzhen, China). While the PLLA solution was shooting out and formed the nanofibrous film, a high voltage supply (Tonli; Shenzhen, China) was connected to the needle at 15 kV. For random nanofibrous films, a steel plate was used as a collector. The distance between the needle and the plate was 100 mm. When producing the aligned nanofibers, a roller wheel was used as the collector. The rotational speed was 3000 rpm. Glass slides were placed on the top of the both collectors. After 20 min of nanofiber collection, the glass slides with PLLA films were cut off, followed by vacuum drying for 24 h to remove residual solvent. 2.3. Porcine Decellularized Nerve Matrix (pDNM) The production of pDNM followed the process reported in our previous work.8,9 Briefly, fresh sciatic nerve obtained from miniature pigs (supplied by Experimental Animal Center of the First Affiliated Hospital of Sun Yat-sen University) was extracted by 3.0% Triton X-100, 4.0% sodium deoxycholate and rinsed by sterile water to complete decellularization. The resulting scaffold was then treated with ethanol/dichloromethane (1/2) to remove lipids, followed by rinsed with sterile water and lyophilization. Finally, the pDNM scaffold was smashed into powder using a Thomas Wiley mini-Mill (Thomas Scientific; Swedesboro, USA) and stored at -40 ℃. All solutions were sterile and all procedures were carried out in a sterilized environment. 2.4. pDNM gel Coatings pDNM was solubilized in pepsin containing HCl (0.01 M) to 1% (w/v) pDNM solution (pDNM/pepsin=10/1 (w/w)), after digestion at room temperature for 24 hours. The digested solution was centrifuged at 20,000 rpm (Beckman Coulter, Brea, USA) for 30 min to remove all undissolved particulates. Then the solution was lyophilized and smashed to powder. The resulting powder was re-dissolved in 0.01 M HCl for 2% (w/v) solution, the pH


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value was adjusted to 7.5 by addition of 0.1 M NaOH and 10 PBS (1/9 to final neutralized volume) was added to form pDNM-sol. The sol was diluted to lower concentration with 0.25% (w/v) and 1% (w/v) by adding 1 PBS and used respectively. To prepare pDNM gel-coated PLLA nanofibrous films or on glass slides (pre-sterilization with 75% ethanol) were immersed in the 0.25% (w/v) and 1% (w/v) pDNM-sol for 10 min at 4 ℃, followed by gelation at 37 ℃ for 20 min. All the films were then lyophilized and stored at -40 ℃ before cell culturing. All the solution was sterile, and all the procedure proceeded in a sterilized environment. The fabrication process for pDNM-coated nanofibrous films is illustrated in Figure S1, supplementary information. 2.5. Dorsal Root Ganglion (DRG) Culturing All the films were immersed in 75% ethanol for 30 min for sterilization, then washed by PBS 3 times (5 min for each wash) and kept wet before cell culturing. The DRGs were isolated from newborn SD rats (postnatal day 1, P1), supplied by Laboratory Animal Center of Sun Yat-sen University, China). The residual nerve roots were cut off under a stereomicroscope. Then DRGs were plated on pDNM-coated PLLA films/glass slides, which were put in a 48-well plate with neurobasal medium containing 2% B27, 0.3% L-glutamine and 1% penicillin–streptomycin in a 37 ℃ incubator with 5% CO2 and 92% humidity. The medium was changed every 2 days and the experiment was sustained for 7 days.

2.6. DRG Dissociation and Nerve Fiber Remyelination DRGs were taken from newborn SD rats and then treated with 0.25% trypsin at 37 ℃ for 15 min and mechanically dissociated by pipetting up and down until there was few fragments. Then the cells were washed with 10% FBS to inactivate trypsin and then centrifuged at 800 rpm for 2 min to separate the cells. The cells were resuspended in neurobasal medium containing 2% B27, 0.3% L-glutamine, 50 ng/ml NGF and 1% penicillin–streptomycin and plated on pDNM coated PLLA films. 7 days after plating, 50 μg/mL ascorbic acid was added to the medium to promote myelination of SCs for another 14 days. The medium was changed every 2 days.

2.7. Immunofluorescence Staining Cells were fixed by 4% paraformaldehyde in PBS for 20 min, rinsed by PBS, permeabilized and blocked with 0.1% Triton X-100 and 10% donkey serum in PBS for 30 min. Cells were incubated with primary antibodies against NF200 (dilution 1/150) and S100 (dilution 1/1000) with DRG culture samples and NF200 (dilution 1/150) and MBP (dilution 1/500) with neuron-Schwann cell co-culture samples for 2 h at room temperature, followed by secondary antibodies conjugated to Fluro 488 (dilution 1/1000) and Fluro 594 (dilution 1/1000) incubation for 1h. The cells were then rinsed with PBS for 10 min (3 times) and stained with DAPI (dilution 1/2500) for 20 min. The



fluorescence was observed and imaged under the laser scanning confocal microscope. The parameters and cells were measured by image analysis software (Image J® ) for at least 3 samples.

2.8. Scanning Electron Microscopy All the films were attached on conducting resin and coated with platinum. The micromorphology was observed and imaged by Hitachi S4800 (Japan) at 15kv. The diameters of nanofibers were measured by image analysis software (Image J® ).

2.9. X-Ray Photoelectron Spectroscopy The surface of all films was analyzed using X-ray photoelectron spectroscopy (XPS) (ESCALAB 250; Thermo-VG Scientific, England) with a focused monochromatic. Al-Ka source (1486.7 eV) for excitation to determine the chemical compositions. The electron take-off angle was 151° and the hemispherical analyzer was operated in the constant analyzer energy mode for all measurements. XPS survey spectra over a binding energy (BE) range of 0–1400 eV were acquired. Data analysis was carried out with Multipak software provided by the manufacturer. The BE scale was set by carbon only bound to carbon and hydrogen at 284.8 eV. XPS spectra were analyzed using the XPSPEAK41 free software.

2.10. Statistical Analysis Data are presented as the mean ± standard deviation (SD). Differences between groups were evaluated by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) posthoc tests. A p-value < 0.05 indicates statistically significant difference. For characterization of SC migration, we focused only on the most significantly migrated SCs. The maximum distances of SC migration were measured by averaging the distance of six most migrated SCs within every 15°from the long axis of the electrospun nanofibers (illustrated in Figure S2, supplementary information). Additional to the maximum SC migration, the frequency of SC appearance 350 μm radially away from the tangential surface of the DRG explants was also recorded.

3. RESULTS 3.1. Preparation and Characterization of pDNM Gel-coated PLLA Nanofibers Electrospun PLLA nanofibrous films was first fabricated by controlling the alignment of the nanofibers. The conventional electrospinning approach resulted in randomly aligned nanofibers (denoted as “random”, Fig. 1), with an average diameter of 820 ± 90 nm (Fig. 1A4). The direction of the electrospun nanofibers was regulated by a


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high-speed rotating wheel (3000 rpm), which resulted in thin films of well-oriented nanofibrous structures with an average diameter of 650 ± 90 nm (denoted as “aligned”, Fig. 1). Small and short nanofibrous structures were clearly evident in the 0.25% pDNM gel-coated films, bridging the neighboring PLLA nanofibers (as highlighted in the red boxes in Figure A2 and B2). The diameters of these nanostructures ranged from 30 to 100 nm (Fig. 1A4 and B4), which is close to the size of nanostructures in the peripheral nerve-derived decellularized matrix26 and pDNM gel13. The appearance of a significant N1s peak in the X-ray photoelectron spectra (XPS) also revealed the successful incorporation of the protein-rich decellularized matrix content (Fig. 1C1, C2 and C3). Distinct morphological contrast was observed by increasing the concentration of pDNM gel. The 1% pDNM gel coating underwent clotting on the electrospun films during gelation, and the PLLA nanofibers were almost fully embedded inside the pDNM gel. Therefore, no significant patterning or protrusion was observed on the surfaces of 1% pDNM gel-coated films (Figure 1, A3 and B3).

Figure 1. Characterization of electrospun PLLA nanofibrous films coated with pDNM gel. The SEM images of (A1) PLLA-random, (A2) PLLA-random/0.25% pDNM gel, (A3) PLLA-random/1% pDNM gel, (B1) PLLA-aligned, (B2) PLLA-aligned/0.25% pDNM gel and (B3) PLLA-aligned/1% pDNM gel. Scale bars = 1 μm. The size distribution of the nanofibrous structures is shown in (A4) and (B4), representing the PLLA nanofibers alone and with pDNM-coatings (nanofibers from the decellularized matrix), respectively. XPS characterization of the (C 1) PLLA, (C2) PLLA/0.25% pDNM gel and (C3) PLLA/0.25% pDNM gel films.

3.2. Neuron and SC Co-culturing Improves SC Migration DRG contains both neurons and SCs, serving as a classic model for nerve cell



co-culturing. Fresh DRGs isolated from newborn rats with residual nerve roots removed, were placed on the aligned PLLA nanofibrous film, pDNM gel-coated glass slide, and pDNM gel-coated aligned electrospun film. After 7 days of co-culturing, the growing neurites were characterized by immunofluorescence staining (NF200, green). After proliferation and migration, the SCs were stained by S100 (red) while the nuclei were stained by DAPI (blue), showing the location of each cell (Fig. 2, merged channels, and Fig. S3, individual channels). On the aligned electrospun PLLA nanofibrous film, neurite outgrowth followed the fiber orientation and SC migration followed the associated neurites, but very few moved further than the axonal length (Fig. 2A). Without such topographical cues, neurite growth from the DRG was radial but not well-controlled, as seen on the pDNM gel-coated glass slide (Fig. 2B). This suggests that the alignment of the axonal extension was highly regulated by the underlying nanofibrous structures. Interestingly, when the aligned PLLA nanofibers were coated with pDNM gel, the SCs migrated ahead of the axons (Fig. 2C1) with the growing neurites (Fig. 2 C1-C3). The zoom-in images (Fig. 2C2-C3) showed that the oval-shaped SCs stretched out along the nanofiber direction, and further beyond the length of neurites, thus facilitating axonal extension.

Consistent with previous reports,20,27 both the length of neurites and the distance of SC migration were significantly augmented due to cues introduced by the topographical and biological environments, which were in turn created by aligned nanofibers and pDNM gel. To determine which factor is more important to nerve regeneration, we calculated the average length of extended neurites and the distance of SC migration by measuring the longest axons and the furthest SCs away from their original DRG in all directions (at least 30 neurites and SCs per DRG), respectively. As shown in Figure 2D, the aligned nanofibers without coating enabled axonal extension to 1.6 ± 0.35 mm, which is about 1.5-fold higher than that of the pDNM gel-coated glass slide. Within the same time period (7 days), the length of the newly grown neurites reached 2.1 ± 0.32 mm on the pDNM-coated aligned nanofibers, likely due to the combined effect of the physical and biological effects of the nanofibers and the pDNM gel. Consistent with our previous observation (Fig. 2C), the distance of SCs migration was remarkably enlarged by the complex co-culturing on the aligned PLLA nanofibers that was coated with pDNM gel, which is almost three fold that of the migration distances seen in the single cued groups, on either aligned nanofibers film or pDNM gel-coated glass slide. There was no significant difference observed in the SC migration distances between the latter two groups.

Notably, neurite extension and SC migration are not mutually independent cell behaviors. As illustrated in Figure S2 (supplementary information), the most significant neurite growth and the associated five farthest SCs from the DRG explant were selected to measure the extension length and SC migration distance within the same region on


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the pDNM gel-coated aligned nanofibrous film. Figure 2E shows that when DRG and SCs were co-cultured on pDNM gel-coated directional nanofibrous films, both axonal extension and SC migration were significantly accelerated, shown as the triangular data points within the purple region, compared to the either the nanofibers or pDNM gel (on glass slides) alone (the blue and incarnadine regions). This clearly demonstrates that the combination of topographical and biological cues enables intercellular interaction.

Figure 2. Confocal micrographs of DRGs cultured on (A) aligned PLLA electrospun films, (B) pDNM gel-coated glass slide and (C) aligned nanofibrous film coated with pDNM gel after 7 days, scale bars =200 μm. (D) Statistics of the neurite length and distance of SC migration on the substrates. (E) Statistical significance of aligned PLLA nanofibers and pDNM gel coating in terms of neurites growth and SCs migration, compared to the aligned PLLA nanofibers and pDNM gel-coated glass slide.

3.3. Neurite Outgrowth is Assisted by Nanofibers and Decellularized Nerve Matrix In order to understand how axonal extension and SC migration are regulated with respect to the topological nanofiber alignment and integration of pDNM gel coating, further studies were carried out by focusing on neurite outgrowth and SC migration



separately. The promotion of neurite growth was studied by conducting DRG culturing on both random and aligned nanofibrous films, with or without pDNM gel coating. In order to mitigate the experimental variation in different groups, the data from Figures S4 and S5 in supplementary materials was normalized by the neurite length resulting from the random or aligned electrospun PLLA nanofibers without pDNM gel coating, respectively.

On randomly oriented nanofibrous films, the neurites underwent radial outgrowth from the DRG explants (Figs. 3A and S4), similar to neurite outgrowth on pDNM gel-coated glass slide (Fig. 2B). Once the pDNM gel was integrated on the surfaces of the random electrospun films, the distribution of neurites was unordered, but the axonal extension was significantly enhanced by providing a mimicry of in vivo microenvironment for nerve cell growth. Moreover, neurite growth was further extended by increasing the pDNM gel concentration of the surface coating from 0.25% to 1% (Fig. 3B). On 1% pDNM gel coatings, the axons were much longer than both the uncoated PLLA electrospun films and the 0.25% pDNM gel-coated surfaces. Interestingly, axonal bundling was also observed, as several nerve fibers tended to merge together away from the DRG explants (Fig. S4C), most likely due to the higher contents of nerve ECM molecules, triggering nerve fiber fasciculation.

On the aligned nanofibrous film (Figs. 3C and S5), almost all the neurites distributed in the region within 0-15° from the long axis of aligned PLLA-nanofibers, with or without 0.25% pDNM gel coating. However, when 1% pDNM gel was coated on the aligned nanofibrous films, the nanostructures were fully covered with diminished topographical cues, which led to a randomly oriented neurite growth, which was close to the random nanofibrous films (Fig. 3C). Neurite length on the 0.25% pDNM gel-coated aligned group was about two-fold greater than that of the aligned nanofibers group, but the extension of neurites on the 1% pDNM gel coating film was not enhanced due to the lack of topographical alignment (Fig. 3D).

Thus, the integration of pDNM gel resulted in significant neurite extension (Fig. 3B). However, topographical guidance compensated for the difference in pDNM gel concentration on aligned nanofibrous substrates (Fig. 3D). Additionally, the highly enhanced SC migration may play an important role in biological guidance of the neurite extension, which may also explain why the decellularized nerve matrix and its concentration contribute in the increment of neurite length without topological alignment.


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Figure 3. Distribution and length of neurite outgrowth. Neurites were seeded on the random (A, B) and aligned (C, D) nanofibrous films. (A) and (C): Analysis on the angle of neurites deviation DRG explant. (B) and (D): Statistical graph of normalized neurites length of DRG culturing on PLLA, PLLA/0.25% pDNM gel, PLLA/1% pDNM gel in the random and aligned groups. (L= neurites length on each film; L0, random= average neurites length on random nanofibrous films without coating; L0,aligned= average neurites length on aligned nanofibrous films without coating).

3.4. SC Migration is Promoted by pDNM Gel Integration The integration of pDNM gel contents was found to trigger the promotion in the SCs migration (Fig. 2C). Since the majority of the SCs remained close to the DRG explants (within ~1.0 mm), the total amount of the SCs was easily distinguished within certain regions nearby. By focusing only on the most significantly migrated SCs, the maximum distances of SCs migration were measured by averaging the distance of six most migrated SCs within 15°from the long axis of the electrospun nanofibers (Fig. 4A). Each experimental group was performed at least three times with six independent DRG samples. Similar to neurite growth, SC relocation and distribution followed the guidance of the nanofibers, and the 15° angular distribution of SC migration again maintained non-directional and directed characteristics based on the random and aligned nanofibrous films, respectively (Fig. 4B, C). In addition, the frequency of SC appearance



every 350 μm radially away from the tangential surface of the DRG explants was also recorded (Fig. 4A). Figures 4D and E, show that the majority of SCs with minor migration stayed within 350 μm region closest to the surface of the explant. This was particularly true on the PLLA-nanofibrous films alone, without pDNM gel coating, where more than 95% of the SCs maintained within the periphery regions of the DRG. Generally, the number of SCs decreased further from the DRG surface. However, with integration of the pDNM gel, especially with increasing concentration of pDNM gel (1% w/v), SC migration was highly enhanced, so much so that the migrated SCs were identified 2-2.5 mm away from the DRG surface (Fig. 4E). This suggests the decellularized nerve matrix contents from the surface coating effectively facilitated SC migration. Furthermore, such enhancement of SC migration was augmented by the directional confinement produced by tuning the electrospun nanofibers from random to controlled alignment.

For each fabrication method, random or aligned, the distance of SC migration (D) was normalized by the corresponding PLLA electrospun film without pDNM gel (D0, Figs. 4F, G). The integration of pDNM gel was significantly effective for SC migration, with 3-6 fold greater maximum migration distance compared to the average. For random nanofibrous film groups, the pDNM gel with a higher concentration (1% pDNM gel) exhibited much stronger capability in promoting SC migration. Compared to the 1% pDNM gel coating, the biological deficiency of the 0.25% pDNM gel coating was again compensated for by topographical alignment (Fig. 4). Both factors contributed cooperatively in facilitating faster and further SC migration along the nanofibers. The highly migrated SCs resulted consequentially in promoting axonal extension.


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Figure 4. Distribution and distance of SC migration. Cells were seeded on the random (B, D, F) and aligned (C, E, G) nanofibrous films. (A) Schematic diagram of the measurements of SCs distribution regarding angular spacing (15 degrees) or migration distance from the tangential surface of the DRG (every 350 μm), respectively; maximum distance of SC migration (averaged by six SCs) from the explants in every 15-degree angular spacing from the long axis, random (B) and aligned (C); frequency of the SCs appearance in different regions every 350 μm from the tangential surface of the DRG, random (D) and aligned (E), the red lines denote the SCs migration distribution as function of radial displacement from



the surface of the DRG explants; and the distance of SCs migration on random (F) and aligned (G) PLLA, PLLA/0.25% pDNM, PLLA/1% pDNM normalized by the uncoated electrospun nanofibrous films, respectively. Where, D = distance of SCs migration; D0,random = averaged SCs migration on random nanofibrous films without pDNM gel coating, D0,aligned = averaged SCs migration on aligned nanofibrous films with absence of pDNM gel coating.

3.5. SCs Perform Guidance of Neurite Outgrowth The synergistic interaction between SCs and neurites is inevitable and critical for understanding the key mechanism of nerve tissue regeneration both in vivo and in vitro. Neurite elongation and SC migration were both elevated by introducing either directionality or integration of decellularized ECM contents to the nanofibers, and further enhanced by combining both conditions. Meanwhile, the SCs also served as an effective biological cue, as their proliferation and migration often lead to promoted axonal extension. This can be attributed to the secretion of neurotrophic factors (e.g. NGF, neurotrophin-3, glial cell line-derived neurotrophic factor, and extracellular matrix proteins such as laminin, fibronectin, collagen).28 When SC proliferation was inhibited by addition of mytomycin (MMC) in the DRG cell culture (Fig. S6). Very few SCs (S100+ cells) were observed on the pDNM gel-coated glass slide and little to no neurite outgrowth was identified. However, by integrating the topological cues, i.e. DRG cultured on the aligned electrospun nanofibers, about 5-10 neurites were found extended from the explant. Meanwhile, there was no significant difference between the average lengths of such neurites with or without the pDNM gel coating. The decellularized extracellular matrix was a greater and more effective contributor to SC migration. The migrated SCs communicated with the growth cone, guiding the pathway for axonal extension. Using the same data analysis approach in Figure 2E, the most significant SC migration distances was collected along with the length of their closest neurites. On the random and aligned nanofibrous films, the neurite length and distance of SCs migration (D) was both normalized by the averaged neurite length cultured on the PLLA electrospun films without pDNM gel (L0, random and L0,aligned). In Figure 5A and B, the red dashed lines were drawn as the function y = x, representing an ideal circumstance when the normalized length of neurites was equal to the normalized SCs migration distance. Notably with absence of the pDNM gel coating on both random and aligned electrospun nanofibers, the blue regions with square dots are mostly located above the red dashed lines, implying that the neurites often grew faster than the migration of the SCs, in which case the topographical guidance dominated the axonal extension and the guidance effect from the SCs were minimized since they were all migrating behind or along with the neurite growth cone (Figs. 5C, D). By introducing the pDNM gel, it was noted that the SCs migrated much faster and further along the nanofibers with 0.25% pDNM gel coating, compared to the neurite outgrowth (Figs. 5E, F). The promotion in neurite growth was therefore attributed more to the directional surface topology and the SCs guidance. When the topological cues were fully covered by 1% pDNM gel, the neurite extension was facilitated with highly promoted SCs migration, but non-directional (Figs. 5G, H). Almost all the corresponding data points (circular and triangular dots) were found lying on or underneath the red dashed lines (random and aligned) in Figure 5A and B, due to the “reconstruction” of cell


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growth microenvironment. While SC migration was more significant than neurite outgrowth with pDNM gel integration, the length of neurites and the SC migration distance were inherently correlated and promoted (the corresponding data points, both circular and triangular, were all moving further away from the origin but closely along the red dashed lines in Figure 5A and B), which resulted in a simultaneous nerve cells behavior, in other words, a synergistic intercellular interaction. When the pDNM concentration was large enough to cover the topographical nanofibers, i.e. the randomly aligned nanofibers were enshrouded by the 1% pDNM gel coating, as seen in the green region with triangular dots in Figure 5A, and the pDNM gel content effectively facilitated SC migration. The purple region (0.25% pDNM gel-coated on aligned PLLA nanofibers) in Figure 5B exhibited nerve cell co-culturing, resulting from both pDNM gel mimicking the neurite growth microenvironment and topographical guidance. The aligned nanofibrous surface topography compensated for the concentration difference in pDNM gel coatings from 0.25% to 1%. Owing to the pDNM gel, the SCs migrated ahead of the neurite growth cone on the same nanofibers, providing the biological guidance for axonal extension. The significance in promoting the axonal extension may rely on the fact that the leading edge of the growth cone kept sensing the SCs secretion, thus the directional motility of the neurites was greatly enlarged by the regenerative microenvironment and synergistic nerve cells interaction. Though we have not yet reached enough quantification for showing exactly how the SCs facilitate the neurite growth, conclusively from the existing experimental demonstration, the total promotion in neurite extension exhibited positive correlation with (1) the effective decellularized nerve matrix gel concentration in the cell culture, as well as (2) the regulation in nanofiber alignment, both of which also resulted in augment of SCs migration, which served as the third factor for facilitating the neurite growth.



Figure 5. Normalized neurite length-distance and Schwann cell migration after 7 days of co-culturing. (A) Random PLLA fibrous film; (B) aligned PLLA fibrous film. (L= neurites length on every film, D= distance of SCs migration, L0= average neurites length on PLLA films in the same group corresponding to L (random or aligned). The red dashed line represents a specific situation when the neurite length is equal to its corresponding distance of SCs migration. The violet dashed line was drawn from the origin of coordinate and tangential to the edge of the purple regions which represents the PLLA/0.25% pDNM gel films. The green dashed line was drawn from the origin of coordinate and tangential to the edge of the green regions which represents the PLLA/1% pDNM gel films. The SCs (S100+) migrated with the neurite extension (NF 200+) on (C) random and (D) aligned PLLA electrospun films. SCs migrated ahead of the neurite extension on 0.25% pDNM gel-coated (E) random and (F) aligned


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electrospun films. And the SCs migration and neurite extension are also shown on 1% pDNM gel-coated (G) random and (H) aligned electrospun films. Scale bars in (C-H) =10 μm.

3.6. Fasciculation and Remyelination towards SC Maturation Besides the enhanced SC migration and highly increased neurite lengths, the neurites were also relatively thicker than a single newborn axon on the pDNM gel-coated nanofibrous films, which were conjectured to precede neurite bundling or fasciculation (Fig. S7). The average diameter of the bundled neurites on the 0.25% pDNM gel coatings (Figs. S7B, F) was much higher than the nanofibrous films alone (Figs. S6A, E). Though 1% pDNM gel-coated nanofibers exhibited clear bundled neurites (Figs. S6C, G) but led to slightly thinner nerve fibers compared to the 0.25% pDNM gel-coated films. The initiation of fasciculation, as many have reported,29 represents a clear phenotype as another navigational cue towards nerve fiber functionalization. The pDNM gel contents, such as laminin, fibronectin, strengthened the adhesion between nerve fibers. Meanwhile, the alignment of neurites magnified the function of bundling due to higher density of neurofilaments in the local region along the direction of neurite growth. Yet the mechanism of axons fasciculation has not been fully understood, it is reasonable that the guidance from extracellular matrix, as well as the intracellular interaction between the neighboring neurites, triggered the bundling of axons and further consolidated the long-distance nerve fiber regeneration into their distal targets.

The remyelination of the nerve fibers is another landmark in their functionalization in peripheral nerve regeneration. The effect of myelination highly depends on the synergistic interaction between neurites and their surrounding SCs. The decellularized nerve molecules derived from peripheral nerve tissues have shown their capability in SCs migration. Previously, we reported that the pDNM gel-enabled SCs wrapping around neurites, thereafter, promoting remyelination.13 For DRG cultured on PLLA nanofibers, very few neurites and SCs were observed (Fig. S8A) due to the lack of pDNM gel into limited nerve cells growth and inhibitory for remyelination. Therefore, ascorbic acid was employed for triggering the myelination effect in the following parallel experiments (Figs. 6 and S9).

Neurons and SCs were co-cultured on the electrospun nanofibrous films (with and without pDNM gel coating) for 7 days, followed by addition of ascorbic acid (50 μg/mL) for another 14 days to trigger remyelination by the SCs. It was noted that the remyelination effect was minimal without the pDNM gel coating, since very few SCs immunofluorescence stained by myelin basic protein (MBP) were observed associating with the neurites (Fig. 6A). The integration of pDNM gel significantly facilitated the neurite myelination (Fig. 6B), the SCs were found wrapping around the growing neurites with formation of a myelin sheath-like shape. Simply by the percentage of SCs attached



to the neurites, the myelination effect was effectively elevated by introduction of pDNM gel. Additionally, from the merged immunofluorescence micrographs, the MBP+ signal was found associating with the bundled axons, implying the initiation of nerve fiber remyelination (Fig. 6B). It was noticed that, on the 1% pDNM gel-coated nanofibrous films (Figs. 6C, S9C), the neurite bundling was commonly observed, as well as the SCs wrapping around the bundled axons. The addition of ascorbic acid only triggered the remyelination in absence of ECM contents on the PLLA nanofibrous films, leading to an increased probability for SCs attaching to neurites. The overall SC-neurite attachment was highly elevated on both pDNM gel-coated films even without ascorbic acid, compared to the nanofibers alone (Fig. 6D). Therefore, the integration of peripheral nerve-derived extracellular matrix hydrogel accelerates the SCs maturation and was confirmed to be critical in the functionalization of the regenerated nerve fibers.

Figure 6. Confocal micrographs of isolated DRG culturing. Cultured for 7 days, followed by addition of ascorbic acid for another 14 days on (A) aligned PLLA nanofibers; (B) PLLA-aligned nanofibrous film coated with 0.25% pDNM gel and (C) PLLA-aligned nanofibrous film coated with 1% pDNM gel. The nuclei of the cells, neurites and SCs were immunofluorescence stained by antibodies against DAPI, NF200 and MBP, respectively. The merged-channel images are also shown. (D) The percentage of Schwann cells attached to neurites with and without addition of ascorbic acid. Scale bars = 200 μm.


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4. DISCUSSION AND CONCLUSIONS In vitro cell-culturing provides an effective and powerful approach for understanding fundamental biological and physical processes by mimicking the cell microenvironments. For peripheral nerve regeneration, it has been widely realized that the directional fiber-like or channel-like architectures ensured the guidance of neurite outgrowth.18-23,30 However, the interfacial topography alone, even with additional growth factors, cannot fully recapitulate or mimic the complex microenvironment of the nerve cells in vitro. Our recent studies have shown that decellularized matrix hydrogels not only maintain most of the bioactive contents and the nanofibrous structures for restoration in cell living microenvironment, but also exhibit high tissue-specificity.13,14 For instance, the peripheral nerve-derived pDNM gel was proven to be helpful in nerve fiber remyelination and inhibitory to synapse formation.13 In this study, the neuron and the SC co-culturing system had both topographical and biological guidance by the aligned nanofibrous films coated with pDNM gel. The surface nano-topography was critical to axonal extension, not only for directional guidance, but also for triggering neurite formation. Meanwhile, the pDNM gel contributed more to promoting SC migration. The SCs have been reported to secrete nerve growth factors which stimulate the growth of nerve fibers. Meanwhile, the SCs wrap around axons for remyelination and nerve fiber functionalization.5,12 Such synergistic intercellular behaviors trigger nerve regeneration following peripheral nerve lesion or trauma. First, the intercellular interaction between the neurites and SCs remodeled on the PLLA nanofibrous films resulted in enhanced guidance of axonal extension. When the pDNM gel was introduced to the nanofibers, it effectively accelerated SC migration. On 0.25% pDNM gel coated aligned-nanofibers, SCs were found ahead of neurite growth, spontaneously serving as cellular guidance for promoting directed axonal extension. Second, such peripheral nerve-derived decellularized ECM hydrogel also triggered axonal fasciculation and remyelination with high density SC attachment on and wrapping around the neurites. Overall, the SCs enhanced the topographical guidance of the nanofibrous structure and promoted neurite outgrowth. Additionally, the highly extended axons and their surrounding SCs facilitated remyelination towards nerve cell maturation and functionalization of regenerative nerve fibers. Furthermore, we noticed that when 1% pDNM gel was employed, the topological cues in electrospun nanofibers were fully concealed. In comparing the 0.25% pDNM gel-coated groups with the 1% groups, the biological contents were found to compete against the surface topography. The directional nanofibrous structures compensated the reduction in pDNM gel concentration (from 1% to 0.25%) in terms of neurite outgrowth and SC migration. However, the higher concentration of the pDNM gel played a dominant role in fasciculation and remyelination rather than the nanofibrous surface topography,



owing to more concentrated protein complexes and peripheral nerve-derived growth factors. Finally, the compositional biomaterial design of combining a naturally-sourced bioactive material with a synthetic scaffold leads to a clear solution for three goals in repairing peripheral nerve tissue with long-distance defects: (1) fast and controllable rate of neurite growth, (2) accurate connection, and (3) complete functionalization of the regenerative nerve fibers. The development of such an in vitro evaluation system not only provides further evidence of nerve tissue regeneration in scaffold design, but also offers a springboard in experimental investigation regarding intercellular interactions of many living organisms. Thus, the integration of tissue-specific decellularized matrix hydrogels with other biomaterials possesses great translational potential in regenerative tissue engineering, drug screening and minimally invasive treatment.

ASSOCIATED CONTENT Supporting Information Figure S1. Schematic illustration for preparation of PLLA nanofibers coated with pDNM gel. Figure S2. Schematic diagram of statistics measurement of neurites length and distance of Schwann cells (SCs) migration. Figure S3. Individual channel micrographs in Figure 2. Figure S4. Confocal micrographs on PLLA-random nanofibers alone or coated with two concentrations of pDNM gel after 7 days of DRG culturing. Figure S5. Confocal micrographs on PLLA-aligned nanofibers alone or coated with two concentrations of pDNM gel after 7 days of DRG culturing. Figure S6. DRG cultured in medium with mitomycin (MMC) on substrates. Figure S7. Neurites bundling on pDNM gel-coated random and aligned electrospun nanofibrous films. Figure S8. Myelination effect by isolated DRG culturing without ascorbic acid. Figure S9. Myelination effect by isolated DRG culturing with addition of ascorbic acid on random nanofibrous groups. Supplementary methods

AUTHOR INFORMATION Corresponding Authors


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* E-mail: [email protected]. Tel: 86-20-84114030 (D.Q.). * E-mail: [email protected]. (Y.B.)

Author contributions S.C., Y.B. and D.Q. conceived the study. D.Q., Q.Z., X.L. and H.-Q.M. designed the experiments. S.C. performed the materials and experiments. S.C. and Y.B. analyzed the data and edited the figures. S.Q., Z.R. and S.L. helped the preparation of pDNM gel. Z.D. assisted in electrospinning. J.Z., X.S. and Y.X. assisted in cell culture and data analysis. S.C., Y.B. and D.Q. wrote the manuscript with contributions from all authors.

Notes The authors declare that they have no competing interests.

ACKNOWLEDGMENTS The authors thank Weizhen Tang for experimental assistance. This work was supported by National Key R&D Program of China (No. 2016YFC1100103, 2016YFC1101603), the National Natural Science Foundation of China (No. 51673220, 5107378 and U1134007), Science and Technology Planning Project of Guangdong Province, China (2015B010125001, 2015B020233012, 2017A050501017), the Major Project of Health and Medical Collaborative Innovation of Guangzhou (201508020251) and foreign cooperation project of Guangzhou (201807010082), Guangdong Innovative and Entrepreneurial Research Team Program (NO.2013S086).

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Table of Contents (TOC)


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Figure 1. Characterization of electrospun PLLA nanofibrous films coated with pDNM gel. The SEM images of (A1) PLLA-random, (A2) PLLA-random/0.25% pDNM gel, (A3) PLLA-random/1% pDNM gel, (B1) PLLAaligned, (B2) PLLA-aligned/0.25% pDNM gel and (B3) PLLA-aligned/1% pDNM gel. Scale bars = 1 μm. The size distribution of the nanofibrous structures is shown in (A4) and (B4), representing the PLLA nanofibers alone and with pDNM-coatings (nanofibers from the decellularized matrix), respectively. XPS characterization of the (C1) PLLA, (C2) PLLA/0.25% pDNM gel and (C3) PLLA/0.25% pDNM gel films.



Figure 2. Confocal micrographs of DRGs cultured on (A) aligned PLLA electrospun films, (B) pDNM gel-coated glass slide and (C) aligned nanofibrous film coated with pDNM gel after 7 days, scale bars =200 μm. (D) Statistics of the neurite length and distance of SC migration on the substrates. (E) Statistical significance of aligned PLLA nanofibers and pDNM gel coating in terms of neurites growth and SCs migration, compared to the aligned PLLA nanofibers and pDNM gel-coated glass slide.


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Figure 3. Distribution and length of neurite outgrowth. Neurites were seeded on the random (A, B) and aligned (C, D) nanofibrous films. (A) and (C): Analysis on the angle of neurites deviation DRG explant. (B) and (D): Statistical graph of normalized neurites length of DRG culturing on PLLA, PLLA/0.25% pDNM gel, PLLA/1% pDNM gel in the random and aligned groups. (L= neurites length on each film; L0, random= average neurites length on random nanofibrous films without coating; L0,aligned= average neurites length on aligned nanofibrous films without coating).



Figure 4. Distribution and distance of SC migration. Cells were seeded on the random (B, D, F) and aligned (C, E, G) nanofibrous films. (A) Schematic diagram of the measurements of SCs distribution regarding angular spacing (15 degrees) or migration distance from the tangential surface of the DRG (every 350 μm), respectively; maximum distance of SC migration (averaged by six SCs) from the explants in every 15degree angular spacing from the long axis, random (B) and aligned (C); frequency of the SCs appearance in different regions every 350 μm from the tangential surface of the DRG, random (D) and aligned (E), the red lines denote the SCs migration distribution as function of radial displacement from the surface of the DRG explants; and the distance of SCs migration on random (F) and aligned (G) PLLA, PLLA/0.25% pDNM, PLLA/1% pDNM normalized by the uncoated electrospun nanofibrous films, respectively. Where, D = distance of SCs migration; D0,random = averaged SCs migration on random nanofibrous films without pDNM gel coating, D0,aligned = averaged SCs migration on aligned nanofibrous films with absence of pDNM gel coating.


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Figure 5. Normalized neurite length-distance and Schwann cell migration after 7 days of co-culturing. (A) Random PLLA fibrous film; (B) aligned PLLA fibrous film. (L= neurites length on every film, D= distance of SCs migration, L0= average neurites length on PLLA films in the same group corresponding to L (random or aligned). The red dashed line represents a specific situation when the neurite length is equal to its corresponding distance of SCs migration. The violet dashed line was drawn from the origin of coordinate and tangential to the edge of the purple regions which represents the PLLA/0.25% pDNM gel films. The green dashed line was drawn from the origin of coordinate and tangential to the edge of the green regions which represents the PLLA/1% pDNM gel films. The SCs (S100+) migrated with the neurite extension (NF 200+) on (C) random and (D) aligned PLLA electrospun films. SCs migrated ahead of the neurite extension on 0.25% pDNM gel-coated (E) random and (F) aligned electrospun films. And the SCs migration and neurite extension are also shown on 1% pDNM gel-coated (G) random and (H) aligned electrospun films. Scale bars in (C-H) =10 μm.



Figure 6. Confocal micrographs of isolated DRG culturing. Cultured for 7 days, followed by addition of ascorbic acid for another 14 days on (A) aligned PLLA nanofibers; (B) PLLA-aligned nanofibrous film coated with 0.25% pDNM gel and (C) PLLA-aligned nanofibrous film coated with 1% pDNM gel. The nuclei of the cells, neurites and SCs were immunofluorescence stained by antibodies against DAPI, NF200 and MBP, respectively. The merged-channel images are also shown. (D) The percentage of Schwann cells attached to neurites with and without addition of ascorbic acid. Scale bars = 200 μm.


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Promoting Neurite Growth and Schwann Cell Migration by the

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