Effect of polycaprolactone/collagen/hUCS microfiber nerve conduit on facial nerve regeneration

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Abstract

Nerve conduits have been used to bridge nerve gaps without the donor site morbidity associated with nerve grafting. To fabricate artificial nerve conduits, various biological, synthetic, and natural materials have been applied. In this study, we suggest a new fibrous nerve conduit consisting of PCL microfibers supplemented with collagen and human umbilical cord serum (hUCS). The proposed PCL-based nerve conduit exhibited significantly higher bioactivities in vitro and in vivo, compared to pure PCL and PCL/collagen fibrous conduits. Based on these results, we believe that the PCL/collagen/hUCS fibrous conduit provides more favorable micro-environmental conditions for facial nerve (FN) regeneration and has more therapeutic potential for the regeneration of FN transectional damage than the autograft technique.

Introduction

Facial nerve dysfunction causes noticeable disfigurement and emotional distress to the affected individuals. Facial nerve paralysis interrupts normal daily functions, such as eating and drinking. Direct reconnection of damaged nerves tumps and transplantation of autologous nerve graft are two techniques that have been commonly adopted for nerve regeneration. However,the limited application of direct reconnection to only cases of slight or small defects, the necessity of an additional surgical procedure to obtain a donor nerve using greater superficial auricular nerve or sural nerve, and permanent functional loss of the donor nerve following autologous nerve graft remain significant challenges in the clinical field [1]. Where a gap occurs between the cut ends of a nerve, proliferating Schwann cells develop from the ends, mostly the distal end, and form sequences of nucleated cellular strands,which fill the gap [2].

An artificial nerve conduit can provide a favorable microenvironment for nerve regeneration and properly guide the axonal sprouting from the proximal stump to the distal stump to reinnervate its original target. The use of artificial conduits has recently been suggested as an alternative strategy for treating damaged nerves [3], [4]. Nerve conduits can also be used to bridge nerve gaps without the donor site morbidity (paresthesia, neuroma formation, second incision) that is typically associated with nerve grafting. A variety of biological tissues and natural and synthetic polymers have been adapted for use as nerve conduits [5]. The transected nerve end sare placed into the conduit, which acts as a guide to direct sprouting axons toward the distal nerve stump and also asa barrier to prevent infiltration by fibrous tissue. There are several theoretical advantages of entubulation by conduit over suturere pair. For instance, damaged nerve endings are not further traumatized by placement of sutures. In addition, the tube facilitates a microenvironment, in which the nerve is bathed in its own secreted neurotropic factors and allows administration of exogenous growth factors to enhance regeneration and/or reduce inflammation and scarring. However, the relatively slow morphological and functional recovery, as well as the limited regeneration distance of peripheral nerves through the nerve conduit, remains critical drawbacks for clinical use [6].

The main goal of an artificial nerve conduit is to bridgethe nerve gap, joining the proximal and distal stumps of the severed nerve. Biodegradablepolymers, e.g. polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid(PLGA), and poly(ε-caprolactone) (PCL), enable nerve regeneration, showing comparable results to autografts [7]. The extracellular matrix (ECM) is the a cellular component of all tissues and organs, and is composed of two main categories of biomolecules: proteoglycans and fibrous proteins (i.e., collagen, elastin, fibronectin and laminin) [8]. The use of ECM-modified scaffolds can be a valid approach in peripheral nerve repair, working either to improve endogenous cell survival and migration at the injury site or to transplant exogenous cells by pre-loadingthem in a biomimetic conduit [8]

However, natural polymers are characterized by poor mechanical properties, causing the conduit to kink or collapse, and undergo fast degradation in vivo; henceco-polymers are thus required to improve scaffold stability [8]. PCL is an ideal biocompatible scaffold due to its slow rate of degradation. The use of nanotechnology, specifically nanofibers, to provide a more suitable format of artificial matrices for tissue engineering applications, has become an important direction to consider in the field, given the aforementioned structural features of the native ECM [8], [9]. Collagen is one of the ECM, and we have previously reported on the excellence of PCNF in aiding bone regeneration [10], [11].

Human umbilical cord serum (hUCS) contains various growth factors (GFs), such as trans-forming GF-beta, epidermoid GF, acidic and fibroblast GF, platelet-derived GF, hepatocyte GF, vitamin A, substance P, insulin-like growth factor (IGF)-1, nerve growth factor (NGF), fibronectin, and serumanti-proteases, such as α-2-macroglobulin [12], [13]. Previously, we have reported on the excellent effects of polycaprolactone/collagen nanofiber (PCL/CoNF) combined with hUCS for bone regeneration and tracheal regeneration [14], [15].

To date, there has been little data on the effect of the polycaprolactone/collagen microfiber (PCL/CoMF) nerve conduit after transection of the rat facial nerve. In this study, we evaluated facial nerve regeneration byPCL/CoMF and hUCS- coated PCL/CoMF(PCL/CoMF/UCS) nerve conduits using in vitro and in vivo approaches.

Section snippets

Materials and scaffold fabrication

PCL (Mn = 60,000) was obtained from Sigma–Aldrich (St. Louis, MO, USA) for electrospinning, and collagen type-I from porcine tendon was obtainedfrom Bioland Inc. (Matrixen-PSP; Bioland Inc., South Korea). For electrospinning, 10 wt% of PCL and solvent (a mixture of 4:1 ratio of n, n-dimethylformamide and methylene chloride) were used. The nerve conduit of PCL microfibers coated with collagen (PCL/CoMF) was obtained using an electrospinning and dipping/drying process (Fig. 1A). As shown in Fig. 1A,

Surface morphology of the fabricated conduit

Fig. 1B shows optical and SEM micrographs of pure PCL, PCL/CoMF, and PCL/CoMF/UCS conduits. As shown in the SEM images, the morphology of the inner and outer surface of the cylindrical conduits was similar, and the fibrous structure was randomly distributed through out the conduits. The diameter of the fibers consisting of the conduits was 2.19 ± 1.31 μm. The collagen and UCS stayed in the surface of the fabricated conduit in the concentration of 2.8 ± 0.1 μg/cm2 and 1.4 ± 0.1 μg/cm2, respectively.

In vitro activities (live/dead, cell proliferation, and DAPI/Phalloidin) of the conduits using PC12 cells

To

Discussion

After FN injury, the distal portions of axons separate from the trophic center (cell body) and degenerate in a series of steps called Wallerian degeneration [17]. Thereafter, the axons extend from the proximal to distal stump via the proliferation of Schwann cells, and finally reinnervate their distal targets, possibly restoring function. Thus, the nerve conduit may play the role of artificial Schwann cells and enable stable nerve growth in a controlled microenvironment. In the present study,

Conclusion

This study found that application of UCS could accelerate functional and histological recovery after transection of FN. From these results, PCL/CoMF/UCS provides a favorable environment for FN regeneration.

Acknowledgements

This study was partially supported by a grant from the National Research Foundation of Korea grant funded by the Ministry of Education, Science, and Technology (MEST) (Grant no. NRF-2015R1A2A1A15055305) and also supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (Grant no. HI15C3000).

References (20)

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