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

Date Published: January 26, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Yun Qian, Jialin Song, Xiaotian Zhao, Wei Chen, Yuanming Ouyang, Weien Yuan, Cunyi Fan.


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.

Partial Text

Although peripheral nerves exhibit some self‐healing potential after mild and moderate trauma as they spontaneously start new axons sprouting after injury,1 successful reinnervation cannot be achieved, especially for long‐range nerve defects and it calls for implantation of a nerve graft to bridge the gap.2 Conventional surgical treatment has plateaued because the gold‐standard protocol‐autologous nerve transplantation causes unavoidable secondary damage to the donor site.3 Tissue engineering and bioactive materials are ideal alternatives for use in the circulatory, digestive, respiratory and nervous systems.4, 5, 6, 7 Many studies have reported that nerve guidance conduits (NGCs) exhibit extensive capabilities for repairing large nerve defects in the peripheral nervous system.8, 9, 10 NGCs are expected to direct cell migration in a targeted manner with their physical and chemical characteristics and to facilitate cell proliferation and differentiation. Synthetic materials such as polycaprolactone (PCL) possess several advantages such as biodegradability, nontoxicity, and structural stability. PCL has been tested in various applications, and has been shown to positively influence cardiovascular, nervous and soft tissues.11, 12, 13

In this study, an integration molding method was used to fabricate GO/PCL nerve nanoscaffolds (Figure1). A tubular mold consisting of four tubes of concentric circles had been previously prepared. Two complex tubes were located between the inner tube and outer‐most tubes to form a concentric circle structure. A mixed solution of GO and PCL was injected into the space between the outer‐most and second outer‐most layers. After solidifying, the second outer‐most layer was removed, and the GO/PCL solution was injected into the space between the second and third outer‐most layers. The procedure was repeated a third time 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. The multi‐layered structure strengthened the mechanical properties of the nerve conduit. In addition, the several porous layers facilitated biodegradation and optimized the long‐term in vivo performance of the NGC because the macropores between the different layers increased the possibility of internal contact with body fluid. Another major challenge in nerve tissue engineering is how to treat long‐range nerve defect over a long‐term regeneration. A strategy to do this is a controlled release system and a slow but steady biodegradation substrate material. In this study, the 3D printing enabled us to fabricate the conduit with a bottom‐up style, which indicates integrated multi‐layered fabrication. In addition, the printer also permitted digital control of mixed solution injection, and this resulted in an even distribution of different biomaterials in the conduit. Furthermore, the 3D printing enabled us to create a conduit with certain volume of different elements and to fabricate it from any angle, position, or plane. With high resolution, 3D printing could better improve RSC proliferation, attachment and neural expression.

In this study, the effects of GO/PCL nanoscaffolds in nerve repair were evaluated both in vitro and in vivo, and the results showed excellent functional and morphological recovery equivalent to those of autografts. We focused on the pro‐angiogenic characteristic of GO and the potential mechanism behind this key phenomenon. The nanoscaffolds directly contributed to successful functional restoration in a long nerve defect model. We plan to continue studying the complex interplay between GO nanomaterials and nerve regeneration.

Integration Molding Method for Producing GO‐Coated PCL Nanofiber Scaffolds and Conduit: GO and PCL were purchased from Sigma Aldrich. The GO nanoparticles were mixed with PCL in a uniform solution and sonicated for 5 min. The injectable suspension was then contained in a previously designed mold and further prepared by a jet spraying process. Using a nozzle and compressed air, the solution was sprayed from a collection container. The dichloromethane evaporated with the formation of the GO/PCL membrane. The integration molding method was used to create a multi‐layered GO/PCL conduit. Finally, a 3D printer was used to create various evenly distributed pores in the surface of the GO/PCL conduit.

The authors declare no conflict of interest.




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