3D Fabrication with Integration Molding of a Graphene Oxide/Polycaprolactone Nanoscaffold for Neurite Regeneration and Angiogenesis

Abstract

Treating peripheral nerve injury faces major challenges and may benefit from bioactive scaffolds due to the limited autograft resources. Graphene oxide (GO) has emerged as a promising nanomaterial with excellent physical and chemical properties. GO has functional groups that confer biocompatibility that is better than that of graphene. Here, GO/polycaprolactone (PCL) nanoscaffolds are fabricated using an integration molding method. The nanoscaffolds exhibit many merits, including even GO nanoparticle distribution, macroporous structure, and strong mechanical support. Additionally, the process enables excellent quality control. In vitro studies confirm the advantages of the GO/PCL nanoscaffolds in terms of Schwann cell proliferation, viability, and attachment, as well as neural characteristics maintenance. This is the first study to evaluate the in vivo performance of GO-based nanoscaffolds in this context. GO release and PCL biodegradation is analyzed after long-term in vivo study. It is also found that the GO/PCL nerve guidance conduit could successfully repair a 15 mm sciatic nerve defect. The pro-angiogenic characteristic of GO is evaluated in vivo using immunohistochemistry. In addition, the AKT-endothelial nitric oxide synthase (eNOS)-vascular endothelial growth factor (VEGF) signaling pathway might play a major role in the angiogenic process. These findings demonstrate that the GO/PCL nanoscaffold efficiently promotes functional and morphological recovery in peripheral nerve regeneration, indicating its promise for tissue engineering applications.

Keywords: graphene oxide; nerve conduits; peripheral nerve injuries; signaling pathways; vascular endothelial growth factor.

Figure 1

Schematic illustration of GO/PCL nanoscaffold fabrication by the integration molding method A) and NGC implantation in the rat model B). We prepared a tubular mold that included four tubes of concentric circles. Two complex tubes are located inside the inner‐most tube and the outer‐most tube, forming a concentric circle structure. A GO and PCL mixture was injected into the space between the outer‐most layer and the second outer‐most layer. After solidifying, the second outer‐most layer was removed, and the GO/PCL mixture was injected into the space between the second outer‐most layer and the third outer‐most layer. The same procedure was repeated again between the third outer‐most layer and the inner‐most layer. Finally, a 3D printer was used to create multiple aligned pores in the surface of the GO/PCL conduit.

Figure 2

Characterization of the GO/PCL NGC. Optical images of the GO/PCL NGC A). SEM images showing the nanoporous structure of the GO/PCL NGC B,C) and the multilayered structure of an ultra‐thin section D). TEM images showing the uniform distribution of GO nanoparticles in the PCL scaffolds E,F). Mechanical and electrical properties, i.e., scaffold thickness, elongation at break, elastic modulus, and electrical conductivity of the GO/PCL and PCL scaffolds G).

Figure 3

Cell viability as assayed by LIVE/DEAD cell staining on GO/PCL scaffolds A–C), PCL scaffolds D–F) and TCP G–I). Live cells (green fluorescence, A, D, and G). Dead cells (red fluorescence, B, E, and H). Merged images (C, F, and I). The scale bar is 50 µm. CCK‐8 assay for RSCs cultured on GO/PCL scaffolds with different concentrations of GO, PCL scaffolds and TCP at 24, 72, 120, and 168 h J). *P < 0.05 compared with 0.5% GO/PCL. ^# P < 0.05compared with 2% GO/PCL. ΔP < 0.05 compared with 4% GO/PCL. Relative cell viability was evaluated by the LIVE/DEAD cell staining for 1% GO/PCL scaffolds, PCL scaffolds and TCP K).

Figure 4

SEM images showing RSC morphology on GO/PCL and PCL scaffolds. RSCs were cultured on GO/PCL, and PCL nanoscaffolds for 4 d before observation. GO/PCL scaffold A–C). PCL scaffold D–F). The scale bars are 100 µm A,D), 50 µm B,E), and 20 µm C,F), respectively.

Figure 5

Immunofluorescence staining for Tuj1 A,B), Ki67 C,D), and phalloidin E,F). All samples were washed three times, fixed with 4% paraformaldehyde for 20 min at 25 °C and blocked with BSA overnight. DAPI staining appears blue. GO/PCL scaffolds A1–A3, C1–C3, and E1–E3). PCL scaffolds B1–B3, D1–D3, and F1–F3). The scale bar is 50 µm.

Figure 6

Western blot and qPCR results for S100 A), GFAP B), nestin C), Tuj1 D), Ki67 E), GAP‐43 F), N‐cadherin G), vinculin H), and integrin I) expression of RSCs on the GO/PCL and PCL scaffolds. Three independent replicates were included for each group. Relative mRNA expression is shown compared with GAPDH. *P < 0.05 compared with PCL.

Figure 7

Immunofluorescence staining for S100 A,B), GFAP C,D), and nestin E,F). All samples were washed three times, fixed with 4% paraformaldehyde for 20 min at 25 °C and were blocked with BSA overnight. DAPI staining appears blue. GO/PCL scaffolds (A1–A3, C1–C3, and E1–E3). PCL scaffolds (B1–B3, D1–D3, and F1–F3). The scale bar is 50 µm.

Figure 8

Morphological evaluation of sciatic nerve and muscle regeneration at 18 weeks postoperatively. Optical images of the GO/PCL NGC at implantation A) and at 18 weeks after surgery B), as well as a dissected regenerated nerve section from the in vivo study C). Toluidine blue staining of a GO/PCL conduit D), a PCL conduit E), and an autograft F) at 18 weeks postoperatively. All samples were dissected from 15 mm sections of regenerated nerves. Ultra‐thin 5 µm thick sections were created using a cryostat. The gastrocnemius muscle from the injured side was also collected at 18 weeks postoperatively. The results for the GO/PCL conduits G), PCL conduits H), and autografts I) are displayed. The scale bar is 100 µm.

Figure 9

TEM images for transverse sections of regenerated nerves from a GO/PCL conduit A–C), a PCL conduit D–F), and an autograft G–I) at 18 weeks postoperatively. We evaluated cross‐sections from different samples and used uranyl acetate and lead citrate for staining. All specimens were observed by TEM. The scale bars are 10 µm A, D, and G), 2 µm B, E, and H) and 1 µm C, F, and I), respectively.

Figure 10

Assessment of angiogenesis in sciatic nerve regeneration at 18 weeks postoperatively. Immunohistochemistry staining for CD31 in regenerated nerve samples from a GO/PCL conduit A), a PCL conduit B), and an autograft C) at 18 weeks after surgery. CD31 is important for endothelial cell intercellular junctions and is extensively involved in angiogenesis. CD31⁺ cells are indicated by arrows in each picture. CD34 is a transmembrane protein that is associated with vascular tissues. Immunofluorescence staining for CD34 in regenerated nerve samples is also shown for a GO/PCL conduit D), a PCL conduit E), and an autograft F) at 18 weeks after surgery. The scale bars are 100 µm A–C) and 50 µm D–F), respectively.

Figure 11

Quantification of CD31⁺‐ and CD34⁺ cells based on various measurements of sciatic nerve samples from the GO/PCL conduit, PCL conduit and autograft groups at 6, 12, and 18 weeks postoperatively. CD31 area (mm²)/region of interest (ROI) (mm²) A). VLS area (mm²)/ROI (mm²) B). VLS number/ROI (mm²) C). Average VLS area (mm²) D). Quantification of CD34⁺ region via MVD (per mm²) E). Quantification of muscle fiber area (%) (F). *P < 0.05 compared with autograft; ^# P < 0.05 compared with PCL conduit.

Figure 12

Western blot results for AKT, p‐AKT, eNOS, p‐eNOS, VEGFR, and p‐VEGFR expression in regenerated nerves from the GO/PCL conduit, PCL conduit and autograft groups at 18 weeks after surgery. The relative expression shown was normalized to that of β‐actin. From left to right: GO/PCL conduit, autograft and PCL conduit. *P < 0.05 compared with autograft; ^# P < 0.05 compared with PCL conduit.