홍합 접착 단백질 바이오엔지니어링 기반의 기능적 신경 재생 플랫폼 개발

Abstract

Tissue engineering is a field of study which pursues development of practical medical applications by searching harmonious combination of principles, scaffolds, and cellular responses. Among many fields of tissue engineering studies, one of big parts to be resolved is the neuronal regeneration that has become the motivation for this research. After fetal development, the formation of central nervous system (CNS) and peripheral nervous system (PNS) are strongly regulated by growth inhibition mechanism. This growth inhibition mechanism keeps neurons from straying outside their path, which is an advantageous function in healthy state. However, when the nervous system suddenly needs active growth or reconstruction, this inhibition mechanism hinders the process. Thus, a traumatic nerve injury often leaves sequelae or aftereffect in a patient even if a proper treatment was performed. Luckily, PNS has relatively larger capacity of regeneration than the CNS has owing to the plasticity of Schwann cell that PNS can be a good starting target to understand the nerve regeneration and to develop a treatment. This thesis presents bioengineering and biomedical application of mussel adhesive protein (MAP) for functional nerve regeneration in various perspectives. Specifically, a potential of bioengineered MAP, which is a biomimicry of adhesive proteins from mussel byssus, was exploited as a promising biomaterial for the neural tissue regeneration. In addition, virtues and vices of various strategies for neuronal regeneration were explored to overcome current limitations of today’s methodologies. To achieve breakthroughs for current limitations, MAP was engineered with various types of biofunctional peptides to provide biofunctional cues; and MAPs were fabricated into aligned nanofiber conduit and glue hydrogel to develop application for treating peripheral nerve injury (PNI). Firstly, a biomaterial for nerve regeneration should be able to strongly influence the nerve cells, Schwann cells, and precursor cells that the effects of MAPs on various cell types were explored. One way of powerful strategies for PNI treatment is to direct the stemness and fate of mesenchymal stem cells (MSCs) into nerve cells; however, proliferation and differentiation of MSCs are complex processes to control. Subsequently, an application of MSCs demands biochemical and biophysical cues that bio-mimic niches to support proliferation and differentiation. Herein, we established a MAP-based platform for MSC neural differentiation by taking advantage of its intrinsic adhesiveness, incorporating extracellular matrix (ECM)-derived biofunctional peptides, and constructing aligned nanofiber. MAP flexibly and maneuverably bio-mimic niches in terms of both biochemical and biophysical cues that enhanced proliferation and neural differentiation of MSCs. Secondly, the limited regenerative capacity of the nervous system makes treating traumatic nerve injuries with conventional polymer-based nerve grafting a challenging task. Consequently, utilizing natural polymers and biomimetic topologies became obvious strategies for nerve conduit designs. As a bioinspired natural polymer from a marine organism, MAPs fused with biofunctional peptides from ECM were engineered for accelerated nerve regeneration by enhancing cell adhesion, proliferation, neural differentiation, and neurite formation. To physically promote contact guidance of neural and Schwann cells and to achieve guided nerve regeneration, MAP was fabricated into an electrospun aligned nanofiber conduit by introducing synthetic polymer poly(lactic-co-glycolic acid) (PLGA) to control solubility and mechanical property. In vitro and in vivo experiments demonstrated that the multi-dimensional tactics of combining adhesiveness from MAP, integrin-mediated interaction from ECM peptides (in particular, IKVAV derived from laminin α1 chain), and contact guidance from aligned nanofibers synergistically accelerated functional nerve regeneration. Thus, MAP-based multi-dimensional approach provides new opportunities for neural regenerative applications including nerve grafting. Thirdly, the treatments for traumatic PNIs have been mainly focused on developing nerve conduits. However, replacing suture has been hardly considered because secure anastomosis of severed nerves is the priority in neurorrhaphy. In a tissue engineering perspective, suture method causes additional traumatic injuries to already injured tissues. Thus, developing a sutureless neurorrhaphy system can save the limited regeneration capacity of nervous system and improve functional nerve regeneration. Herein, a practical bio-inspired application of sutureless neurorrhaphy was proposed using MAP-based glue hydrogel. MAP-based glue hydrogel provided both suitable adhesiveness and biocompatibility as a neurorrhaphy adhesive. In addition, the fusion protein of MAP and Substance P, which is a neurotransmitter biofunctional peptide, successfully induced M2 macrophage polarization for the tissue remodeling process that significantly improved functional nerve regeneration. The efficacy of MAP-based macrophage polarizing glue hydrogel as sutureless neurorrhaphy system was intensely evaluated in vitro and in vivo Overall, the several potential strategies for developing biofunctional MAPs as a nerve regeneration platform were intensively investigated in this research. Hence, MAP-based aligned nerve conduit and sutureless glue hydrogel systems successfully demonstrated advances in volumetric regeneration and functional recovery of a critically damaged nerve tissue.