Fabrication and characterization of graphene nanoscaffolds

In this study, an integrated LBLC method was used to fabricate the 3D porous graphene conduit (Fig. 1). A 3D printer was composed of a rolling tube and a sprayer. A tubular mode with evenly distributed microneedles was placed on a roller. The nozzle above this rolling tube directly sprayed different solutions on the rolling tube. The tube was rolling at a constant speed and added different layers to form a tubular structure. The first and also the inner-most part was RGD and PDA mixed layer. And then graphene/PCL dichloromethane solution was sprayed on the rolling tube and crosslinked with PDA/RGD layer. Another single-layered or multi-layered graphene and PCL mixed solution was sprayed again after previous layers were solidified. Finally, RGD and PDA mixed solution was sprayed again to form the outer-most layer. The inner-most and outer-most layers of RGD and PDA were beneficial to cell adhesion and proliferation. The graphene/PCL double layers could intensify the tubular structure and allow certain stiffness for long-term in vivo study.

Fig. 1 Schematic illustration of graphene nerve conduit fabrication with LBLC method. a The inner-most and outer-most green layers are PDA/RGD mixed layers. The purple layer is single-layered or multi-layered graphene and PCL mixed layer. The blue layer is a repetition of the graphene and PCL mixed layer. b An illustration of the single-layered or multi-layered graphene/PCL nerve conduit in a sciatic nerve defect model in the SD rats Full size image

A reasonable pore size is vital to successful nerve conduit fabrication because it allows free entrance of water and oxygen into the lumen. Either too big or too small pore size interferes with ideal peripheral nerve restoration23. In this study, we used microneedles to create multiple aligned macropores with 50 μm in diameter in the conduit. We removed the microneedles and the rolling tube mold after the conduit was solidified.

We characterized the morphology of the porous 3D graphene conduit by optical imaging and scanning electron microscopy (SEM) (VEGA3 TESCAN, Fig. 2). The nanoporous and multi-layered 3D structure was shown at different magnifications. From Raman spectra results, the 2D peak is a single and sharp one in single-layered graphene sheet. However, it was relatively low in multi-layered graphene sheet. The rescaled image showed a higher intensity for 2D peak in single-layered graphene. The 2D peak was relatively 1/2 the height of G peak in multi-layered graphene, while the 2D peak was two times as high as G peak in single-layered graphene. The position of 2D band from single-layered graphene sheet was around 2676 cm−1, however it was 2682 cm−1 for multi-layered graphene sheet (Supplementary Fig. 1). The Raman spectrum helped us distinguish the different carbon structures of single-layered and multi-layered graphene because the reduction in layers resulted in different electronic dispersions. To test the mechanical property of the 3D conduit, we measured the elastic modulus and found that the average value for single-layered graphene/PCL conduit was 68.74 MPa, in contrast with 58.63 MPa for multi-layered graphene/PCL conduit. The mechanical test indicated that the porous 3D graphene conduit could hold the structure and allow nerve regrowth by offering ideal flexibility and rigidity. We also evaluated the electric conductivity for different scaffolds. The single-layered graphene/PCL conduit displayed a high conductivity of 8.92 × 10−3 S cm−1. The electric conductivity of multi-layered graphene/PCL conduit was 6.37 × 10−3 S cm−1. This was consistent with previous research that the electric conductivity decreased with more layers of graphene24. In addition, it displayed relatively good electric conductivity like some conductive materials. For instance, Song and colleagues focused on excellent conductive material polypyrrole (PPY). The electric conductivity of PPY based conduit was 6.72 × 10−5 S cm−125. Our multi-layered macroporous nerve conduit could allow exchanges of nutrients and oxygen via excellent permeability, strong mechanical support, appropriate biodegradation rate, and flexibility for complete nerve regrowth.

Fig. 2 Characterization of graphene nerve conduit. a–d SEM images for evaluation of the nanoporous and multi-layered 3D structure in graphene-based nanomaterials. e Thickness, elastic modulus, and electric conductivity of PDA/RGD-SG/PCL and PDA/RGD-MG/PCL scaffolds (evaluation of both materials was repeated for five times) Full size image

After nerve conduit fabrication, we seeded Schwann cells on the nanoscaffolds and evaluated the neural expression. Furthermore, this 3D conduit was also implanted in a long-range sciatic nerve defect model in Sprague Dawley (SD) rats.

Cell proliferation and attachment on graphene nanoscaffolds

To verify rat Schwann cell (RSC) viability on the nanoscaffolds, different graphene/PCL was designed to determine the concentration dependent cytotoxicity on Schwann cells by cell counting kit 8 (CCK8) assay, including 0.1%, 0.5%, 1%, 2%, and 4% single-layered and multi-layered graphene in PCL. CCK8 assay showed that 1% single-layered and multi-layered graphene displayed lower cytotoxicity than 2% and 4% single-layered and multi-layered graphene. In addition, cells were more proliferative in 1% than 0.5% and 0.1% single-layered and multi-layered graphene. Therefore, we chose 1% graphene and further evaluated its effects in peripheral nerve regeneration. This was consistent with previous research. Park and colleagues evaluated the toxicity of graphene nanoparticle both in vitro and in vivo. They found 0.5% graphene was the maximal concentration for a relatively high cell viability and low systematic organ damages. In contrast, 1% and 2% graphene nanoplatelets exerted a negative influence on normal cell and tissue function26. In this study, we modified single-layered and multi-layered graphene with PDA and RGD in a controlled release way and confirmed 1% graphene nanoparticle was suitable for optimal cell biocompatibility.

To verify that graphene scaffolds could support cell proliferation and attachment, we seeded Schwann cells on the different scaffolds for 1, 3, 5, and 7 days respectively and examined their proliferative state by CCK8 assay. After 1, 3, and 5 days respectively, SCs on PDA/RGD-SG/PCL, PDA/RGD-MG/PCL, PDA/RGD-PCL, and PCL nanoscaffolds were similarly proliferative as tissue culture plate (TCP) (analysis of variance (ANOVA), p > 0.05, Fig. 3). At day 7, the outcomes of PDA/RGD-SG/PCL and PDA/RGD-MG/PCL were significantly better than other groups, indicating the positive role of PDA and RGD-modified graphene in cell proliferation. In addition, PDA/RGD-SG/PCL nanoscaffolds could improve the greatest extent of cell proliferation among three PDA/RGD coated scaffolds.

Fig. 3 Cell viability assay of LIVE/DEAD cell staining and CCK8. a–c Live/dead/merge pictures for PDA/RGD-SG/PCL. d–f Live/dead/merge pictures for PDA/RGD-MG/PCL. g–i Live/dead/merge pictures for PDA/RGD-PCL. j–l Live/dead/merge pictures for PCL. The scale bar is 50 μm. m Cytotoxicity assay for 0.1%, 0.5%, 1%, 2%, and 4% SG/PCL at different time points. n Cytotoxicity assay for 0.1%, 0.5%, 1%, 2%, and 4% SG/PCL at different time points. *p < 0.05 compared with 0.1% SG(MG)/PCL; #p < 0.05 compared with 0.5% SG(MG)/PCL; Δp < 0.05 compared with 2% SG(MG)/PCL. фp < 0.05 compared with 4% SG(MG)/PCL. o CCK8 assay for five groups. p Relative cell viability by live and dead staining. All data are displayed as mean ± standard deviation. *p < 0.05 compared with PDA/RGD-PCL; #p < 0.05 compared with PCL; Δp < 0.05 compared with TCP (the statistical test is ANOVA) Full size image

LIVE/DEAD cell kit was used for cell viability analysis. Figure 3 exhibited live and dead cells which were stained with Calcein AM and Ethidium homodimer-1. The results of various nanoscaffolds did not display a notable difference and all of them showed optimal cell viability after one-day co-culture.

We performed immunofluorescence and western blotting (WB) to further evaluate cell proliferation and attachment on the different scaffolds. N-cadherin and vinculin are adhesion-associated proteins and can activate chemical signaling via extracellular matrix (ECM). The expression of vinculin and N-cadherin on PDA/RGD-SG/PCL and PDA/RGD-MG/PCL nanoscaffolds was significantly increased compared with that on PDA/RGD-PCL and PCL nanoscaffolds (Fig. 4). This indicated that graphene could also prominently contribute to SCs adhesion just like PDA and RGD. In addition, we evaluated the proliferative ability of SCs on the different nanoscaffolds by Ki67 and Brdu. The expression of Brdu and Ki67 on PDA/RGD-PCL and PCL nanoscaffolds was relatively lower than that on PDA/RGD-SG/PCL and PDA/RGD-MG/PCL nanoscaffolds. Ki67 showed a higher expression on PDA/RGD-SG/PCL nanoscaffolds (Fig. 4). Immunofluorescent staining of Ki67 was shown in Fig. 5.

Fig. 4 Gene expression compared between nanoscaffolds. a WB assay of Ki67, Brdu, GFAP, Tuj1, N-cadherin, and vinculin. b–g Their relative expression from SC seeded PDA/RGD-SG/PCL, PDA/RGD-MG/PCL, PDA/RGD-PCL, and PCL nanoscaffolds. All data are displayed as mean ± standard deviation. *p < 0.05 compared with PDA/RGD-MG/PCL; #p < 0.05 compared with PDA/RGD-PCL; Δp < 0.05 compared with PCL (the statistical test is ANOVA) Full size image

Fig. 5 Immunofluorescent staining for Ki67 and F-actin. a, b Ki67 expression of SC on PDA/RGD-SG/PCL. e, f Ki67 expression of SC on PDA/RGD-MG/PCL. i, j Ki67 expression of SC on PDA/RGD-PCL. m, n Ki67 expression of SC on PCL. c, d Phalloidin staining on PDA/RGD-SG/PCL. g, h Phalloidin staining on PDA/RGD-MG/PCL. k, l Phalloidin staining on PDA/RGD-PCL. o, p Phalloidin staining on PCL. q Relative expression of Ki67. r Cell density evaluation from phalloidin staining. The scale bar is 50 μm. All data are displayed as mean ± standard deviation. *p < 0.05 compared with PDA/RGD-MG/PCL; #p < 0.05 compared with PDA/RGD-PCL; Δp < 0.05 compared with PCL (the statistical test is ANOVA) Full size image

SEM was performed to evaluate cell morphology after SCs were seeded on the different nanoscaffolds for 3 days. Cells were evenly distributed on the entire nanoscaffolds and almost covered all the fields. Protuberances of most cells were extended on PDA/RGD-SG/PCL and PDA/RGD-MG/PCL nanoscaffolds. By phalloidin staining, the cell density was also increased in PDA/RGD-SG/PCL and PDA/RGD-MG/PCL nanoscaffolds (Fig. 5). These results indicated that the graphene nanoscaffolds were able to improve cell attachment.

Neural expression on graphene nanoscaffolds

The glial fibrillary acidic protein (GFAP), Class III β-tubulin (Tuj1), and S100 were involved in the immunofluorescence assay to validate single-layered graphene/PCL and multi-layered graphene/PCL scaffolds could promote neural expression (Fig. 6). Tuj1 can distinguish neurons from glial cells. GFAP is expressed in many nerve cell types in the central and peripheral nerve system. The relative expression level of GFAP on PDA/RGD-SG/PCL was respectively 3.8-fold, 5.4-fold, and 7.5-fold greater than that of cells cultured on PDA/RGD-MG/PCL, PDA/RGD-PCL, and PCL nanoscaffolds. Furthermore, the relative expression level of Tuj1 on PDA/RGD-SG/PCL nanoscaffold was respectively 1.2-fold, 2.3-fold, and 2.8-fold greater than that of cells cultured on PDA/RGD-MG/PCL, PDA/RGD-PCL, and PCL nanoscaffolds. For further validation, WB was also shown in Fig. 4. Neurotrophic factors have profound implications in peripheral nerve regeneration, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF). The expression of NGF, BDNF, GDNF, and CNTF on PDA/RGD-SG/PCL nanoscaffolds was apparently higher than that on other nanoscaffolds (Supplementary Fig. 2). Conclusively speaking, PDA/RGD-MG/PCL and PDA/RGD-SG/PCL had huge potentials in promoting neural expression and differentiation.

Fig. 6 Immunofluorescent staining for GFAP, Tuj1, and S100. a, d GFAP expression of SC on PDA/RGD-SG/PCL. g, j GFAP expression of SC on PDA/RGD-MG/PCL. m, p GFAP expression of SC on PDA/RGD-PCL. s, v GFAP expression of SC on PCL. b, e Tuj1 expression of SC on PDA/RGD-SG/PCL. h, k Tuj1 expression of SC on PDA/RGD-MG/PCL. n, q Tuj1 expression of SC on PDA/RGD-PCL. t, w Tuj1 expression of SC on PCL. c, f S100 expression of SC on PDA/RGD-SG/PCL. i, l S100 expression of SC on PDA/RGD-MG/PCL. o, r S100 expression of SC on PDA/RGD-PCL. u, x S100 expression of SC on PCL. y Relative GFAP expression. z Relative Tuj1 expression. aa Relative S100 expression. The scale bar is 50 μm. All data are displayed as mean ± standard deviation. *p < 0.05 compared with PDA/RGD-MG/PCL; #p < 0.05 compared with PDA/RGD-PCL; Δp < 0.05 compared with PCL (the statistical test is ANOVA) Full size image

Functional improvement of graphene conduits

The in vitro study confirmed the potential effects of graphene-based nanoscaffolds in promoting cell growth and neural expression. Further in vivo evaluation would help us identify the long-term performance of graphene-based conduit in peripheral nerve restoration. Ninety SD rats were allocated into six groups randomly and equally, including Schwann cell-loaded PDA/RGD-SG/PCL, Schwann cell-loaded PDA/RGD-MG/PCL, PDA/RGD-SG/PCL, PDA/RGD-MG/PCL, PDA/RGD-PCL, and autograft groups. Each group was evaluated at 6 weeks, 12 weeks, and 18 weeks after surgery.

We did not observe severe complications like delay of wound healing, ulcer, infection at 6, 12, and 18 weeks after surgery. No nerve conduits degraded at 18 weeks after surgery. All regenerated nerves were observed by optical imaging (Supplementary Fig. 3).

To evaluate functional recovery in all experimental rats, walking track analysis was performed according to a previous research27. The sciatic function index (SFI) results were displayed (Supplementary Fig. 4). At 6 and 12 weeks after surgery, the recovery of sciatic nerves was significantly faster in Schwann cell-loaded PDA/RGD-SG/PCL and Schwann cell-loaded PDA/RGD-MG/PCL nerve conduits than the remaining scaffolds (ANOVA, p < 0.05), but it was not as good as the autograft group (ANOVA, p < 0.05). At 18 weeks after surgery, the Schwann cell-loaded PDA/RGD-SG/PCL and Schwann cell-loaded PDA/RGD-MG/PCL nerve conduits showed similar results compared with the autograft group (ANOVA, p > 0.05). In addition, we also evaluated extensor postural thrust, which was also an important indicator for motor performance. The Schwann cell-loaded single-layered and multi-layered graphene/PCL conduit could improve the motor functions better than non-cell loading conduit groups and PCL conduit group at 6, 12, and 18 weeks post operatively (ANOVA, p < 0.05). Meanwhile, they were as excellent as the autograft group at 18 weeks after surgery (ANOVA, p > 0.05, Supplementary Fig. 3). It also showed the beneficial effects of graphene-based nerve conduit and cell loading in the sciatic nerve functional recovery.

Gastrocnemius muscle recovery can also indicate nerve function because it is dominated by sciatic nerves. We weighed the gastrocnemius muscle and calculated the average weight. At 6 and 12 weeks, there was a statistical difference among Schwann cell-loaded PDA/RGD-SG/PCL, Schwann cell-loaded PDA/RGD-MG/PCL nerve conduits, and other conduits (ANOVA, p < 0.05). Without cell loading, muscles from PDA/RGD-SG/PCL and PDA/RGD-MG/PCL groups also showed significant higher weight than PDA/RGD-PCL group (ANOVA, p < 0.05). Cell-loaded conduit groups showed similar results in comparison with the autograft group at 18 weeks after surgery (ANOVA, p > 0.05, Supplementary Fig. 4). It indicated that single- and multi-layered graphene could reverse muscle atrophy and help nerve recovery to a certain extent, which was further improved by Schwann cell loading.

Apart from locomotor function recovery, we also evaluated sensory functional recovery in all groups. Significant increase in response time was observed in Schwann cell-loaded PDA/RGD-SG/PCL, Schwann cell-loaded PDA/RGD-MG/PCL, PDA/RGD-SG/PCL, PDA/RGD-MG/PCL, and PDA/RGD PCL groups compared with the autograft group at 6 and 12 weeks. In contrast, the values of cell-loaded conduit groups were close to the autograft group at 18 weeks, and were significantly better than those of the other groups (ANOVA, p < 0.05, Supplementary Fig. 3). This indicated a successful sensory recovery after graphene conduit implantation and cell loading therapy.

Electrophysiological improvement of graphene conduits

SD rats were subjected to electrophysiological analysis to evaluate electrophysiological performance. We performed electrophysiological analysis at 6 and 12 weeks post operatively. The nerve conducting velocity (NCV) of the Schwann cell-loaded PDA/RGD-SG/PCL (14.8 m s−1, 21.2 m s−1) and Schwann cell-loaded PDA/RGD-MG/PCL nerve conduits (13.4 m s−1, 20.7 m s−1) was notably higher than that of the remaining conduit groups (PDA/RGD-SG/PCL: 11.1 m s−1, 17.1 m s−1; PDA/RGD-MG/PCL: 10.9 m s−1, 16.2 m s−1; PDA/RGD-PCL: 9.4 m s−1, 13.7 m s−1, ANOVA, p < 0.05). However, it was significantly lower than the autograft group (17.2 m s−1, 25.2 m s−1, ANOVA, p < 0.05). We achieved similar results at 18 weeks post operatively. The NCV of Schwann cell-loaded PDA/RGD-SG/PCL and Schwann cell-loaded PDA/RGD-MG/PCL groups was significantly higher than that of PDA/RGD-SG/PCL, PDA/RGD-MG/PCL, and PDA/RGD-PCL groups. However, it showed no significant differences compared with the autograft group (ANOVA, p > 0.05). We evaluated the distal compound motor action potential (DCMAP) and achieved similar results at 6, 12, and 18 weeks after surgery (Supplementary Fig. 4).

Nerve regeneration improvement of graphene conduits

To evaluate morphological nerve regeneration and nerve expression, regenerated nerves were dissected immediately after electrophysiological analysis. Samples were processed by Hematoxylin & Eosin (HE) staining, 1% toluidine blue (TB) staining, and transmission electron microscopy (TEM). We displayed representative images of SC-loaded PDA/RGD-SG/PCL, SC-loaded PDA/RGD-MG/PCL, PDA/RGD-SG/PCL, PDA/RGD-MG/PCL, PDA/RGD-PCL, and autograft groups (Figs. 7 and 8, Supplementary Figs. 5–8). The appearance of regenerated axon fibers was exhibited and calculated via TB staining and TEM observation. Most of the regenerated nerves were well organized and lacked scar tissues. Four parameters were included for evaluation: number of myelinated axons, thickness of myelin sheath, regenerated axon area, and average myelinated axon diameter. The number of myelinated axons was significantly higher in the autograft group, followed by Schwann cell-loaded PDA/RGD-SG/PCL, SC-loaded PDA/RGD-MG/PCL, PDA/RGD-SG/PCL, PDA/RGD-MG/PCL, and PDA/RGD-PCL groups. At 6 and 12 weeks, the results of SC-loaded PDA/RGD-SG/PCL and PDA/RGD-MG/PCL groups were significantly better than those of other groups (ANOVA, p < 0.05). But they were lower than that of the autograft group (ANOVA, p < 0.05). However, at 18 weeks, the value from SC-loaded PDA/RGD-SG/PCL and PDA/RGD-MG/PCL groups showed no significant differences from the autograft group (ANOVA, p > 0.05). So axon area was regenerated (Supplementary Fig. 9).

Fig. 7 Nerve regeneration at 18 weeks postoperatively. HE (a–f) and TB (g–l) staining for regenerated nerves at 18 weeks post operatively. a, g SC-loaded PDA/RGD-SG/PCL. b, h SC-loaded PDA/RGD-MG/PCL. c, i PDA/RGD-SG/PCL. d, j PDA/RGD-MG/PCL. e, k PDA/RGD-PCL. f, l Autograft. The scale bar is 100 μm Full size image

Fig. 8 TEM for regenerated myelinated axons at 18 weeks post operatively. a–c SC-loaded PDA/RGD-SG/PCL. d–f SC-loaded PDA/RGD-MG/PCL. g–i PDA/RGD-SG/PCL. j–l PDA/RGD-MG/PCL. m–o PDA/RGD-PCL. p–r Autograft. The scale bar in a, d, g, j, m, and p is 10 μm. The scale bar in b, e, h, k, n, and q is 2 μm. The scale bar in c, f, i, l, o, and r is 1 μm Full size image

For axonal regrowth and nerve remyelination evaluation, Tuj1, NF200, S100, and myelin basic protein (MBP) were involved in immunofluorescence assay. We performed Tuj1 and NF200 triple staining as well as S100 and MBP triple staining. NF200 and Tuj1 represented regenerated neurofilaments and axons. S100 represented migration of Schwann cells and MBP indicated myelinated fibers. At 6 and 12 weeks after surgery, the autograft group showed better results in Tuj1, NF200, and S100 expression than all other groups (ANOVA, p < 0.05). At 18 weeks, the expression of Tuj1, NF200, and S100 was notably increased in SC-loaded PDA/RGD-SG/PCL and PDA/RGD-MG/PCL groups. It was significantly higher than that of PDA/RGD-SG/PCL, PDA/RGD-MG/PCL, and PDA/RGD-PCL groups (ANOVA, p < 0.05) and was slightly lower than that of the autograft group (ANOVA, p > 0.05). The MBP expression was generally low in each group at 6, 12, and 18 weeks (Figs. 9 and 10, Supplementary Figs. 10–13). This indicated that PDA/RGD-SG/PCL and PDA/RGD-MG/PCL nerve conduits could promote nerve regeneration after long-term restoration in nerve conduit area and the effect was further enhanced by Schwann cell loading.

Fig. 9 Triple immunofluorescent staining of Tuj1 and NF200 at 18 weeks post operatively. Tuj1 (green), NF200 (red), and nuclei (blue) were exhibited from different groups respectively. a–d SC-loaded PDA/RGD-SG/PCL. e–h SC-loaded PDA/RGD-MG/PCL. i–l PDA/RGD-SG/PCL. m–p PDA/RGD-MG/PCL. q–t PDA/RGD-PCL. u–x Autograft. The scale bar is 100 μm Full size image