Materials Today
Volume 21, Issue 9, November 2018, Pages 951-959
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Research
Rapid continuous 3D printing of customizable peripheral nerve guidance conduits

https://doi.org/10.1016/j.mattod.2018.04.001Get rights and content

Abstract

Engineered nerve guidance conduits (NGCs) have been demonstrated for repairing peripheral nerve injuries. However, there remains a need for an advanced biofabrication system to build NGCs with complex architectures, tunable material properties, and customizable geometrical control. Here, a rapid continuous 3D-printing platform was developed to print customizable NGCs with unprecedented resolution, speed, flexibility, and scalability. A variety of NGC designs varying in complexity and size were created including a life-size biomimetic branched human facial NGC. In vivo implantation of NGCs with microchannels into complete sciatic nerve transections of mouse models demonstrated the effective directional guidance of regenerating sciatic nerves via branching into the microchannels and extending toward the distal end of the injury site. Histological staining and immunostaining further confirmed the progressive directional nerve regeneration and branching behavior across the entire NGC length. Observational and functional tests, including the von Frey threshold test and thermal test, showed promising recovery of motor function and sensation in the ipsilateral limbs grafted with the 3D-printed NGCs.

Introduction

Peripheral nervous system (PNS) injuries require approximately 200,000 surgeries in the United States and 300,000 in Europe annually [1], [2], [3]. Injuries result from trauma, tumors, and other illnesses, and may cause complete or partial paralysis. Current repair strategies for PNS injuries after a complete nerve transection involve suturing the distal and proximal nerve ends without introducing tension, which is only suitable for repairing short nerve-gaps (<5 mm), or placing an cadaveric or autologous nerve graft (autograft) harvested from another anatomical location to treat larger defects [4], [5], [6]. The autograft is the current “gold standard” but requires additional surgical procedures to harvest the graft, leading to loss of function at the donor site and potentially neuroma formation [1]. Furthermore, the size and geometry of the graft cannot always match the injury site (e.g., diameter mismatch, branched nerves), and the total length of the autologous donor grafts is limited, especially in children, thereby limiting reconstructive options [6], [7]. Consequently, it is highly desirable to develop engineered alternatives to the autografts with design flexibility and improved performance.

Nerve guidance conduits (NGCs) have been engineered as a promising alternative to repair large-gap nerve injuries [7], [8], [9], [10], [11]. NGCs are tubular structures that are used to bridge the gap of a severed nerve, thereby acting as a guide for the regenerating axons and as a barrier against the in-growth of scar-forming tissue. A number of natural (e.g., vein, collagen, chitosan, agarose, and silk) and synthetic materials (e.g., silicone, polyglycolide, gelatin methacryloyl, hyaluronic acid, poly(ethylene glycol) diacrylate, and polyhydroxybutyrate) have been researched to develop NGCs with various fabrication techniques, including electrospinning, microdrilling, molding, and microstereolithography [3], [9], [12], [13], [14], [15]. Nonetheless, these fabrication techniques generally can only offer NGCs with simple architectures (e.g., straight hollow conduits) and limited choices in materials, as well as dimensions. On the other hand, the effects of complex geometrical features and mechanical cues on axonal growth and neuronal behavior have been of great interest to the neural engineering field with the potential of being employed to modulate nerve development and regeneration [16], [17], [18]. Recently, the fabrication of a custom-bifurcated NGC based on patient anatomy was done by an extrusion-based 3D printer, demonstrating the potential of using 3D-printing technology to build NGCs with complex customized designs [19]. However, there are also inherent limitations associated with the extrusion-based 3D-printing approach, such as low printing resolution limited by the physical confinement of the nozzles, compromised structural integrity caused by the interfacial artifacts between the extruded lines, and limited printing speed due to the serial writing fashion [20], [21]. Thus, there remains an unmet need for a manufacturing technique that can fabricate designer scaffolds with superior resolution, speed, flexibility, and scalability so that customized NGCs with specified diameters and complex architectures (such as branches) can be created for regeneration of more complicated nerve gaps (e.g., nerve gaps joining the proximal stump of the common digital nerve with two distal stumps of the proper distal nerve or the complex branching pattern of the extratemporal facial nerve).

In this work, we present the use of a digital light processing (DLP)-based rapid continuous 3D-printing platform for the fabrication of NGCs with customizable architectures and material properties for guided peripheral nerve regeneration. NGCs featuring a wide range of different designs were printed to demonstrate the flexibility of our 3D-printing technique, and the fabrication parameters were varied to fine-tune the material property of the printed NGCs to match the injury site. 3D-printed NGCs with microchannels and sleeves were implanted in vivo to guide mouse sciatic nerve regeneration. The progression of the nerve regeneration from the proximal end of the injury to the distal end within the 3D-printed NGCs was evaluated by both histological staining and immunohistochemistry. The functional recovery of the experimental animals was also assessed by the von Frey threshold testing and thermal testing.

Section snippets

Rapid continuous 3D printing

The schematic setup of our rapid continuous 3D printer is shown in Figure 1. A 405-nm visible light LED is used as the light source for photopolymerization, eliminating the concern of potential cell damage by UV light sources. A digital micromirror device (DMD) chip modulates the optical pattern projected onto the prepolymer solution for selective photopolymerization. The DMD chip is composed of approximately four million micromirrors (2560 × 1600), which can be controlled by user-defined

Discussion

In the past five years, a considerable number of efforts have been made to engineer NGCs as a potential alternative to the autograft for PNS injuries [7], [8], [9], [10], [11]. However, most of these engineered NGCs feature simple architectures that may not be sufficient to promote structural and functional recovery, especially in the case of longer nerve gaps (i.e., 15 mm or more in rodents or over 100 mm in humans). For instance, it has been demonstrated that conduits with a luminal filler or

Conclusion

In summary, we have developed a rapid continuous 3D-printing platform to create customizable NGCs featuring intricate architectures, as well as tunable material properties. Using the concepts of projection printing (instead of point-by-point writing) and continuous fabrication, our printing technique is simple, fast, and capable of 3D scaffold fabrication from any digital images (e.g., CAD design, MRI, and CT scans). Various NGC designs have been demonstrated with unprecedented speed and

3D printing of NGCs

PEGDA (Mn = 700) was purchased from Sigma–Aldrich (USA). GelMA and LAP were synthesized as described in the previous work [38], [43]. The prepolymer solutions were prepared by dissolving GelMA, PEGDA, and LAP in Dulbecco’s phosphate-buffered saline (DPBS) following the recipes (wt.%) listed in the table of Figure 3. The 3D printer setup shown in Figure 1 is used to print all NGCs as well as the mechanical test samples. The NGCs were first designed in CAD software (Autodesk 3ds Max) and the 3D

Acknowledgments

The work was supported in part by grants from the National Institutes of Health (R21HD090662, R01EB021857) and National Science Foundation (CMMI-1547005 and CMMI-1644967) to S.C. and a grant from National Institutes of Health (1TL1TR001443) to K.R.T. The UCSD Neuroscience Microscopy Shared Facility was supported by Grant P30 (NS047101). We thank Laarni Gapuz for the help with cryosectioning and H&E staining.

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    W.Z. and K.R.T. contribute equally to this work.

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