3D Printing for Peripheral Nerve Regeneration

3D Printed PU-based conduits with 3D engineered Schwann cell blocks for enhancing the peripheral nerve regeneration

The nervous system is part of our body and plays a key role in coordinating action and sensory information as well as communicating between different body parts through the electrical signal transmission. The system works by coordinating inputs from different sources and transforming them into actions and autonomous outputs that respond to environmental changes. Therefore, severe neurological dysfunction or injury can lead to disability and have a significant negative impact on the quality of life of patients [1]. The human nervous system can be identified as two subsystems, one is the central nervous system (CNS) and the other is the peripheral nervous system (PNS). The CNS includes the brain and spinal cord, while the PNS contains other nerve tissues besides the brain and spinal cord [2]. Patients involved in major trauma often suffer from peripheral nerve damage due to the extent of the impact, and such injuries typically include nerve compression, nerve damage or tearing or even ischemic injury. Other causes of peripheral nerve injury include surgery and other complications. These damages can then lead to a variety of neurological dysfunctions and diseases. In addition, studies have shown that approximately 2.8% of patients still claim peripheral neuropathy after treatment, some have neuropathic pain and permanent dysfunction, and some patients have a permanent disability even after years of recovery [3]. With the advancement of medical technology and knowledge, the success rate of peripheral nerve therapy has increased significantly. However, due to the limited regenerative capacity of the nerves, there is still a huge gap between the upon solutions and the complete recovery of neurological function. To date, the inclusion of regenerative medicine in peripheral nerve therapy remains challenges [4] due to major obstacles including lengthy duration of regeneration, limited neuronal sources of autografting, and autologous transplant rejection. Therefore, scientists are always looking for new solutions to overcome these challenges. Tissue engineering has long been regarded as a potential replacement for nerve transplantation since the past decade, and it has become an important research topic in the field of nerve regeneration [5].

Figure 1. (a) Schematic drawing of the conduit
Figure 1. (a) Schematic drawing of the conduit
Figure 1. (b) The top-view photograph of 3D-printed PU-based conduits.
Figure 1. (b) The top-view photograph of 3D-printed PU-based conduits.

Attempting to regenerate or replace severe nerve damage is a major challenge nowadays. Temporary loss of neurological function after any nerve injury is unavoidable due to the disruption of communication between nerves. Previous cases have shown that patients who underwent >15 mm neurological repair often claimed loss of function even after recovery. In addition, nerve damage involving > 15 mm distance may significantly reduce nerve regeneration due to the disruption of internal nerve contact between the proximal and distal ends [3]. Clinically, autologous nerve grafting is currently the mainstream treatment for nerve damage. Neural grafts from autologous sources are optimal because they are not only proven to protect damaged nerve tissue, but also help guide axonal regeneration and connect to one nerve ending to another. In recent years, it has been suggested that 3d porous scaffolds only provide temporary support for cell growth in cells [6]. However, modern stents have evolved into a key factor in tissue engineering applications, and these stents have proven to be more than just containers or supports [7]. Therefore, they are also rich in growth factors, which can significantly promote nerve regeneration [8]. Among them, Zhang conducted a 20-year study involving peripheral nerve defects, and the data collected were used to establish research guidelines for peripheral nerve conduits [9]. Studies have shown that neuropathy with a size of 9.7 ± 1.8 mm can be successfully regenerated by the use of a nerve conduit. However, for the repair of rat sciatic nerves greater than 10 mm, several different proteins such as laminin [10], fibronectin [11] and collagen [12] need to be incorporated into the nerve conduit to achieve optimal nerves regeneration.

Figure 2. The process of S manufacturing cell-containing blocks and a method of assembling procedures: (a) Degradable mold; (b) cell block adhesion; (c) cell block collection.
Figure 2. The process of S manufacturing cell-containing blocks and a method of assembling procedures: (a) Degradable mold; (b) cell block adhesion; (c) cell block collection.

China Medical University (CMU) adapted the advancement of additive manufacturing technology to fabricate innovative nerve conduits with complex pore patterns and dimensions with high precision. We used Digital light processing (DLP), a photo-polymerization technique to cross-link photo-curable materials, to fabricate nerve conduits. This printed nerve conduit will present as a scaffold for two reasons in this research, one is to hold/contain the decellularized extracellular matrix (dECM) of nerves and the other is to exist as a scaffold for nerve regeneration from the damaged nerve ends. The dECM was usually obtained by exposing harvested nerves with chemical or physical methods to remove unnecessary cellular components, thus only maintaining the structural proteins and several growth factors that were supposed to mimic the microenvironment of normal tissues. In the CMU 3D Printing group, we fabricate water-based light-cured polyurethane (PU) nerve conduits with DLP technology. To improve the resolution and biocompatibility of PU, homogenous polydopamine (PDA) and dECM were mixed into the raw material. Mechanical properties and chemical composition were analyzed using the EZ test and electron spectroscopy for chemical analysis (ESCA). In general, PU/PDA/dECM conduits (Figure 1) were able to influence and enhance stem cell adhesion, proliferation and neural differentiation of stem cells.

Figure 3 (a) 3D schwann cell bocks;(b) Live/Death of 3D schwann cell after 3 days culture; (c) schematic of schwann cell blocks into 3D printed PU-based conduits and the animal implantation.
Figure 3 (a) 3D Schwann cell bocks;(b) Live/Death of 3D Schwann cell after 3 days culture; (c) schematic of Schwann cell blocks into 3D printed PU-based conduits and the animal implantation.

Furthermore, our group also adopted an updated method to manufacture the three-dimensional cell-containing blocks, as Figure 2. This innovative 3D cell-containing blocks achieved several advantages including first, being able to be efficiently expanded to achieve rapid production; secondly, having standardized size in order to prevent them from blocking or destroying the nozzle during their processing or passing through the nozzle of the bioprinter; thirdly, manufacturing methods thereof not able to induce significant cell damage and/or gene (DNA) damage; and fourthly, manufacturing methods thereof not able to impair its ability to integrate into tissues. In nerve regeneration, we used the microRNA-transfected Schwann cells (SC) to fabricate the SC blocks and loaded SC blocks into the nerve conduit to enhance nerve regeneration rate. Signal-channel neural conduits with SC blocks were implanted into Sprague-Dawley rats [Figure 3 and 4] and observe the SC blocks will secrete more nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). In an animal in vivo study, there was no foreign body membrane formed around the nerve conduit and could be incorporated with natural growth factors for optimal biocompatibility in regenerative nerve tissue engineering.

Figure 4. (a) 3D schwann cell bocks;(b) Live/Death of 3D schwann cell after 3 days culture; (c) schematic of schwann cell blocks into 3D printed PU-based conduits and the animal implantation
Figure 4 Animal Implantation results, with and without SC cell block

References:

1. Cheney, F. W.; Domino, K.; Caplan, R. A.; Posner, K. L. Nerve injury associated with anesthesia. Anesthesiology 1990, 90, 1062–1069.

2. Kouyoumdjian, J. A. Peripheral nerve injuries: A retrospective survey of 456 cases. Muscle & Nerve 2006, 34, 785–788.

3. Tajdaran, K.; Gordon, T.; Wood, M. D.; Shoichet, M. S.; Borschel, G. H. A glial cell line-derived neurotrophic factor delivery system enhances nerve regeneration across acellular nerve allografts. Acta Biomater 2016, 29, 62–70.

4. Johnson, B. N.; Lancaster, K. Z.; Zhen, G.; He, J.; Gupta, M. K.; Kong, Y. L.; Engel, E. A.; Krick, K. D.; Ju, A.; Meng, F.; Enquist, L. W.; Jia, X.; McAlpine, M. C. 3D printed anatomical nerve regeneration pathways. Adv Funct Mater 2015, 25, 6205–6217.

5. Xia, B.; Lv, Y. Dual-delivery of VEGF and NGF by emulsion electrospun nanofibrous scaffold for peripheral nerve regeneration. Mater Sci Eng C Mater Biol Appl 2018, 82, 253–264.

6. Maiti, B.; Díaz Díaz, D. 3D printed polymeric hydrogels for nerve regeneration. Polymers 2018, 10, 1041

7. Kankala, R. K.; Xu, X. M.; Liu, C. G.; Chen, A. Z.; Wang, S. B. 3D-printing of microfibrous porous scaffolds based on hybrid approaches for bone tissue engineering. Polymers 2018, 10, 807.

8. Jubran, M.; Widenfalk, J. Repair of peripheral nerve transections with fibrin sealant containing neurotrophic factors. Exp Neurol 2003, 181, 204–212.

9. Ko, C. H.; Shie, M. Y.; Lin, J. H.; Chen, Y. W.; Yao, C. H.; Chen, Y. S. Biodegradable bisvinyl sulfonemethyl-crosslinked gelatin conduit promotes regeneration after peripheral nerve injury in adult rats. Sci Rep 2017, 7, 1062.

10. Wu, T.; Li, D.; Wang, Y.; Sun, B.; Li, D.; Li, D.; Morsi, Y.; El-Hamshary, H.; Al-Deyab, S. S.; Mo, X. Laminin-coated nerve guidance conduits based on poly(l-lactide-co-glycolide) fibers and yarns for promoting Schwann cells’ proliferation and migration. J Mater Chem B 2017, 5, 3186–3194.

11. Toll, E. C.; Seifalian, A. M.; Birchall, M. A. The role of immunophilin ligands in nerve regeneration. Regen Med 2011, 6, 635–652.

12. Yao, L.; Daly, W.; Newland, B.; Yao, S.; Wang, W. C.; Chen, B. K. K.; Madigan, N.; Windebank, A.; Pandit, A. Improved axonal regeneration of transected spinal cord mediated by multichannel collagen conduits functionalized with neurotrophin-3 gene. Gene Ther. 2013, 20, 1149–1157.

About the Author:

Dr. YiWen Chen
Dr. YiWen Chen

Dr. YiWen Chen received her M.S. and Ph.D. degrees in Industrial and Manufacturing Engineering (Nanomaterial Group) Florida State University, USA. She joined China Medical University Hospital and funded the 3D Printing Medical Research Center in 2014. She is responsible for leading the team and developing and implementing the 3D printed medical research and clinical application integration. She is also the Associate Professor of Graduate Institute of Biomedical Science at China Medical University since then. Dr. Chen’s research interests focus on to develop and deliver advanced and affordable 3D printed medical care including biomedical devices, implants, and therapeutics for medical applications. Several of her patents and technologies have to tech-transfer to industries. She was awarded the 2017 and 2018 Innovation in Taiwan. She is also a member of the Board of Supervisors of the Additive Manufacturing Association in Taiwan(AMAT) and a board of directors of China Medical Derivatives Corporation – Everyoung Biomedical International. She is responsible for providing relevant technical advice and is often invited to serve as lecturers at many international academic conferences. Many achievements of her research team are also recognized by high-impact journals, media, conference scientific publications, and published works. She has published 35 peer-reviewed articles and holds 15 issued/pending patents.

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