Roles of reinforced nerve conduits and low-level laser phototherapy for long gap peripheral nerve repair

Roles of reinforced nerve conduits and low-level laser phototherapy for long gap peripheral nerve repair

Peripheral nerve injuries are common in clinical practice because of traumas such as crushing and sectioning. Lesions of the nerve structure result in lost or diminished sensitivity and/or motor activity in the innervated territory. The degree of lesion depends on the speci fi c nerve involved, the magnitude and type of pressure exerted, and the duration of the compression. The results of such injuries commonly include axonal degeneration and retrograde degeneration of the corresponding neurons in the spinal medulla, followed by very slow regeneration (Rochkind et al., 2001). The adverse effects on the daily activities of patients with a peripheral nerve injury are a determining factor in establishing the goals of early recovery (Rodriguez et al., 2004). The most severe form of nerve damage involves complete transection of the nerve, which results in the loss of sensory and motor function at the site of injury. Although a degree of recovery can be expected in most untreated nerve injuries, the process is slow and often incomplete. Moreover, despite considerable advances in microsurgical techniques, the functional results of peripheral nerve repair remain largely unsatisfactory. The regrowth of nerves across large gaps is particularly challenging, usually requiring a nerve graft to correctly bridge the proximal and distal nerve stumps. At present, nerve autografting is the most common treatment used to repair peripheral nerve defects. However, this recognized “gold standard” technique has a number of inherent disadvantages, such as limited availability of donor tissue (IJkema-Paassen et al., 2004), secondary deformities, potential differences in tissue structure and size (Nichols et al., 2004), and numbness at donor sites (Bini et al., 2004). Although xenografts and allografts have been proposed as alternatives to autografts, the success rate of these techniques remains poor, often resulting in immune rejection. Thus, researchers have invested considerable effort in developing synthetic nerve conduits for the repair of peripheral nerve defects.

Nerve guide conduit

Scientists have developed various non-degradable (Chen et al., 2000) and biodegradable (Wang et al., 2001; Bini et al., 2004; Liu, 2008; Hsu et al., 2011) materials as synthetic nerve conduits, for example, PLGA (Bini et al., 2004) and PLA (Hsu et al., 2011). Of these materials, doctors have widely used non-degradable materials such as silicone rubber in general clinical cases because of its inert and mechanical properties. However, the main disadvantages of non-degradable conduits are that they remain in situ as foreign bodies following nerve regeneration and may require removal via a second surgery, which could possibly cause damage to the nerve. In contrast, biodegradable materials potentially avoid these problems. Therefore, biodegradable conduits seem a more promising alternative for reconstructing nerve gaps. Nerve guidance channels fabricated out of collagen have already shown rather favorable results in nerve repair (Itoh et al., 2002). However, clinical experience with collagen products has demonstrated that cracks and tears can occur when the suture needle penetrates the conduits. In addition, biodegradable conduits that degrade with time may lose their functionality as a structural cuff. Accordingly, an ideal biodegradable conduit should maintain its structural integrity during the regenerative processes (Yannas and Hill, 2004).

Gelatin is less expensive and much easier to obtain in concentrated solutions than collagen. Moreover, gelatin is a biodegradable polymer with excellent biocompatibility, plasticity, and adhesiveness. However, swelling of the degradable tube walls caused by absorption of body fl uids may occur during the nerve regenerative processes. This swelling could occlude the lumen and therefore impair axonal regeneration. In addition, the handling characteristics are unsatisfactory for suturing, and the lumen of gelatin channels may collapse or be obliterated following implantation. Therefore, the use of proper cross-linking agents to modulate the mechanico-chemical characteristics of gelatin is desirable in order to prevent toxicity and generate stable materials for biomedical applications. Various cross-linkers, such as formaldehyde, glutaraldehyde (Chen et al., 2005), genipin (Yang et al., 2010, 2011), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Chang et al., 2007) have been used to compensate for the disadvantages inherent in gelatin, and to make gelatin nerve substitutes resistant to natural biodegradation following transplantation. Since crosslinked gelatin may have low mechanical strength under physiological conditions, its applications may prove limited.

Previous studies (Yang et al., 2010, 2011) developed a biodegradable composite (GGT conduit) consisting of genipin crosslinked gelatin annexed with β-tricalcium phosphate (β-TCP) ceramic particles to enhance the mechanical strength as a nerve guidance channel-material for axon regeneration (Figure 1). The results of that study revealed that the TCP ceramic particles provided structural reinforcement to the genipin-cross-linked gelatin (GG) structure. Macroscopic observations show that this study does not observe any unsatisfactory swelling or deformation of the GGT nerve guide conduits. The improvement in the water uptake and swelling ratios may have been attributable to the presence of TCP ceramic particles in the GG matrix. Consequently, the GGT conduits swelled slowly and maintained a lower water uptake and swelling ratios than the GG conduits. Therefore, the hydrated GGT conduits (when grafted in vivo to repair nerve defects) did not stenose and collapse to compress the regenerating nerve fi ber of the lumen. Mechanical measurements showed that these good mechanical properties, which bene fi ted from the addition of TCP ceramic particles, rendered it possible for the GGT conduit to resist the muscular contraction and keep its cylindrical shape unchanged within a considerable period after implantation into the body. Since the collapse of an un fi lled circular conduit is a major block to nerve regeneration in tubulization, the properties of a gelatin tube that can be molded into various con fi gurations and compounded with TCP, can effectively enhance nerve regeneration. Besides, as tricalcium phosphate dissolves during the degradation of GGT, calcium ions could be released from the conduits, and a previous study (Kulbatski et al., 2004) has shown that a post-neuritotomy rise in calcium in fl ux through calcium channels is a necessity for neurite regeneration. In addition, the GGT conduit had the strength necessary to withstand the muscular forces that surrounded it, meaning that a stable support structure for the extended regeneration processes was maintained. These results demonstrate the feasibility of designed GGT conduits in the applications of peripheral nerve repair.

Laser therapy

Clinicians have focused on developing more effective methods to promote nerve regeneration, target organ reinnervation, and restore function at the site of injury. Many physical and neurotrophic factors, as well as pharmaceutical drugs, in fl uence nerve regeneration. Physiotherapy commonly involves the use of thera-peutic instruments for regenerative purposes (Gigo-Benato et al., 2005). Various forms of external stimulation have been employed to accelerate the process of regeneration, which in turn accelerates functional recovery. Such techniques include electrical (Mendon?a et al., 2003), ultrasound (Raso et al., 2005), and low-level laser (LLL) stimuli. Clinical and experimental studies have provided evidence that lasers can increase nerve function, reduce the formation of wounds, increase the metabolic activity of neurons, and enhance myelin production (Bagis et al., 2002). The non-invasive nature of laser phototherapy enables treatment without surgical intervention. LLL therapy began to be used in the regeneration and functional recuperation process of peripheral nerves in the 1970s, and the results obtained so far have been inconsistent. Many animal experiments and clinical studies have indicated that LLL irradiation can attenuate injury, promote repair, and stimulate axonal sprouting and propagation, but its mechanism of action is not well understood (Amat et al., 2006). A review of the literature on phototherapy for peripheral nerve repair found that the use of laser was based on several wavelengths (632-904 nm) (Masoumipoor et al., 2014), lesion types (crushing, neurorrhaphy, and tubulation), sample types, the duration and manner of the emission (Marcolino et al., 2013; Akgul et al., 2014), and the assessment types (such as functional, electrophysiological, and morphometric) (Gigo-Benato et al., 2005).

In many studies, descriptions of the irradiation parameters, such as dose, average power, time, and application methods, have expressly varied, hampering the methodological comprehension required for the reproduction of results and hindering comparisons between studies. Barbosa et al. (2010) sought to analyze the effects of two different GaAlAs laser wavelengths (660 nm and 830 nm) on sciatic nerve regeneration by using the same crushing injuries for a novel comparison of studies reported in the literature. They observed that the 660 nm wavelength treatment group had the best SFI scores on average, indicating that the use of these parameters was more ef fi cient. The possibility that neural tissue is located in more super fi cial layers may have favored a better response to the shorter wavelength. Data also suggested that 660 nm LLL therapy with low (10 J/cm2) or moderate (60 J/ cm2) energy densities is able to accelerate neuromuscular recovery after nerve crush injury in rats (Gigo-Benato et al., 2010). Our own previous studies investigated the in fl uence of large-area irradiation using an aluminum-gallium-indium phosphide (Al-GaInP) diode laser (660 nm) (Shen et al., 2011) and trigger point therapy using gallium-aluminum-arsenide phosphide (GaAlAsP) laser diodes (660 nm) (Shen et al., 2013a, 2013b) on the neurorehabilitation of transected sciatic nerves in rats after bridging them with the GGT nerve conduit (Figure 2). The results for these studies indicated that the GGT/laser system may be very helpful for long-gap nerve regeneration as well as for acceleration of the reinnervation rate of regenerated nerves, which may lead to suf fi cient morphologic and functional recovery of the peripheral nerve.

It has also previously been shown that LLL enhances Schwann cell proliferation in vitro. Schwann cells myelinate axons of the peripheral nervous system and play a crucial role in post-injury nerve regeneration. They promote neuronal survival, guide axons to their proper targets, and secrete neurotrophic factors that aid axonal elongation (Bhatheja and Field, 2006). Morphological changes in the mitochondria of lymphocytes, as well as in the proliferation of mononuclear cells, have also been observed after radiation with a red laser, and these responses might be bene fi cial in the process of tissue repair (Gulsoy et al., 2006). The underlying mechanism of phototherapy in nerve regeneration has been proposed in previous in vitro studies which showed that phototherapy induced Schwann cell proliferation, as well as massive neurite sprouting and outgrowth in cultured neuronal cells. It has also been suggested that phototherapy may enhance the recovery of neurons by altering the oxidative metabolism of mitochondria (Elles et al., 2003). The same mechanism may guide neuronal growth cones in vitro, perhaps through interaction with cytoplasmic proteins and, in particular, by enhancing actin polymerization at the leading edge of the axon (Ehrlicher et al., 2002). One possible molecular explanation is the increase in growth-associated protein-43 (GAP-43) immunoreactivity during the early stages of nerve regeneration proceeding phototherapy (Shin et al., 2003). In summary, all of the aforementioned effects may play a role in accelerating axonal regeneration and preventing the loss of neurons.

Although the preliminary results support the mechanical strength and biocompatibility of the GGT conduit and are encouraging in regards to peripheral nerve regeneration, further studies should attempt to improve the design of GGT nerve guide conduits. Examples of such studies could include an introduction of neurotrophic factors or seeding cells to establish the possibility of using GGT grafts as a suitable alternative to nerve autografts for peripheral nerve regeneration. With regard to clinical applicability, LLL phototherapy makes an important contribution towards the development of a safe and effective strategy for rehabilitating peripheral nerve injuries. Further studies on the use of LLL therapy as a noninvasive treatment modality for various nerve diseases and injuries could pave the way for mainstream acceptance and standardization of this innovative therapy.

Bai-Shuan Liu1, Tsung-Bin Huang2, Shiuh-Chuan Chan3

1 Department of Medical Imaging and Radiological Sciences, Central Taiwan University of Science and Technology, Taichung, Taiwan, China

2 Department of Bioscience Technology, Chang Jung Christian University, Tainan, Taiwan, China

3 Graduate Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan, China

Akgul T, Gulsoy M, Gulcur HO (2014) Effects of early and delayed laser application on nerve regeneration. Lasers Med Sci 29:351-357.

Amat A, Rigau J, Waynant RW, Ilev IK, Anders JJ (2006). The electric fi eld induced by light can explain cellular responses to electromagnetic energy: a hypothesis of mechanism. Photochem Photobiol 82:152-160.

Bagis S, Comelekoglu U, Sahin G, Buyukakilli B, Erdogan C, Kanik A (2002) Acute electrophysiologic effect of pulsed galliumarsenide low energy laser irradiation on configuration of compound nerve action potential and nerve excitability. Lasers Surg Med 30:376-380.

Barbosa RI, Marcolino AM, Guirro RRD, Mazzer N, Barbieri CH, Fonseca MDR (2010) Comparative effects of wavelengths of low-power laser in regeneration of sciatic nerve in rats following crushing lesion. Lasers Med Sci 25:423-430.

Bhatheja K, Field J (2006) Schwann cells: Origins and role in axonal maintenance and regeneration. Int J Biochem Cell Biol 38:1995-1999.

Figure 1 Smacrograph and scanning electron micrograph (SEM) of the genipin-crosslinked gelatin annexed with tricalcium phosphate (GGT) conduit.

Bini TB, Gao S, Xu X, Wang S, Ramakrishna S, Leong KW (2004) Peripheral nerve regeneration by microbraided poly(L-lactide-co-glycolide) biodegradable polymer fi bers. J Biomed Mater Res A 68:286-295.

Chang JY, Lin JH, Yao CH, Chen JH, Lai TY, Chen YS (2007) In vivo evaluation of a biodegradable EDC/NHS-cross-linked gelatin peripheral nerve guide conduit material. Macromol Biosci 7:500-507.

Chen PR, Chen MH, Lin FH, Su WY (2005) Release characteristics and bioactivity of gelatin-tricalcium phosphate membranes covalently immobilized with nerve growth factors. Biomaterials 26:6579-6587.

Chen YS, Hsieh CL, Tsai CC, Chen TH, Cheng WC, Hu CL, Yao CH (2000) Peripheral nerve regeneration using silicone rubber chambers fi lled with collagen, laminin and fi bronectin. Biomaterials 21:1541-1547.

Ehrlicher A, Betz T, Stuhrmann B, Koch D, Milner V, Raizen MG, Kas J (2002) Guiding neuronal growth with light. Proc Natl Acad Sci U S A 99:16024-16028.

Elles JT, Henry MM, Summerfelt P, Wong-Riley MT, Buchmann EV, Kane M, Whelan NT, Whelan HT (2003) Therapeutic photobiomodulation for methanol-induced retinal toxicity. Proc Natl Acad Sci U S A 100:3439-3444.

Gigo-Benato D, Geuna S, Rochkind S (2005) Phototherapy for enhancing peripheral nerve repair: a review of the literature. Muscle Nerve 31:694-701.

Gigo-Benato D, Russo TL, Tanaka EH, Assis L, Salvini TF, Parizotto NA (2010) Effects of 660 and 780 nm low-level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve. Lasers Surg Med 42: 673-682.

Gulsoy M, Ozer GH, Bozkulak O, Tabakoglu HO, Aktas E, Deniz G, Ertan C (2006) The biological effects of 632.8-nm low energy He-Ne laser on peripheral blood mononuclear cells in vitro. J Photochem Photobiol B 82: 199-202.

Hsu SH, Chan SH, Chiang CM, Chen CC, Jiang CF (2011) Peripheral nerve regeneration using a microporous polylactic acid asymmetric conduit in a rabbit long-gap sciatic nerve transection model. Biomaterials 32:3764-3775.

IJkema-Paassen J, Jansen K, Gramsbergen A, Meek MF (2004) Transection of peripheral nerves, bridging strategies and effect evaluation. Biomaterials 25:1583-1592.

Itoh S, Takakuda K, Kawabata S, Aso Y, Kasai K, Itoh H, Shinomiya K (2002) Evaluation of cross-linking procedures of collagen tubes used in peripheral nerve repair. Biomaterials 23:4475-4481.

Kulbatski I, Cook DJ, Tator CH (2004) Calcium entry through L-type calcium channels is essential for neurite regeneration in cultured sympathetic neurons. J Neurotrauma 21:357-374.

Liu BS (2008) Fabrication and evaluation of a biodegradable proanthocyanidin-crosslinked gelatin conduit in peripheral nerve repair. J Biomed Mater Res Part A 87:1092-1102.

Marcolino AM, Barbosa RI, das Neves LM, Mazzer N, de Jesus Guirro RR, de Cássia Registro Fonseca M (2013) Assessment of functional recovery of sciatic nerve in rats submitted to low-level laser therapy with different fluences. An experimental study: laser in functional recovery in rats. J Hand Microsurg 5:49-53.

Masoumipoor M, Jameie SB, Janzadeh A, Nasirinezhad F, Soleimani M, Kerdary M (2014) Effects of 660- and 980-nm low-level laser therapy on neuropathic pain relief following chronic constriction injury in rat sciatic nerve. Lasers Med Sci [Epub ahead of print].

Figure 2 Transected nerve was subjected to a large-area irradiated therapy with the 660-nm aluminum-gallium-indium phosphide (AlGaInP) low-level laser (A) or a transcutaneous trigger point therapy with the 660-nm gallium aluminum arsenide phosphide (GaAlAsP) low-level laser (B).

Mendon?a AC, Barbieri CH, Mazzer N (2003) Directly applied low intensity direct electric current enhances peripheral nerve regeneration in rats. J Neurosci Methods 129:183-190.

Nichols CM, Brenner MJ, Fox IK, Tung TH, Hunter DA, Rickman, SR, Mackinnon SE (2004) Effects of motor versus sensory nerve grafts on peripheral nerve regeneration. Exp Neurol 190:347-355.

Raso VVM, Barbieri CH, Mazzer N, Fasan VS (2005) Can therapeutic ultrasound in fl uence the regeneration of the peripheral nerves? J Neurosci Methods 142:185-192.

Rochkind S, Nissan M, Alon M, Shamir M, Salame K (2001) Effects of laser irradiation on the spinal cord for the regeneration of crushed peripheral nerve in rats. Lasers Surg Med 28:216-219.

Rodriguez FJ, Valero-Cabré A, Navarro X (2004) Regeneration and functional recovery following peripheral nerve injury. Drug Discov Today Dis Models 1:177-185.

Shen CC, Yang YC, Huang TB, Chan SC, Liu BS (2013a) Low-level laser-accelerated peripheral nerve regeneration within a reinforced nerve conduit across a large gap of the transected sciatic nerve in rats. Evid Based Complement Alternat Med 2013:175629.

Shen CC, Yang YC, Huang TB, Chan SC, Liu BS (2013b) Neural regeneration in a novel nerve conduit across a large gap of the transected sciatic nerve in rats with low-level laser phototherapy. J Biomed Mater Res A 101:2763-2777.

Shen CC, Yang YC, Liu BS (2011) Large-area irradiated low-level laser effect in a biodegradable nerve guide conduit on neural regeneration of peripheral nerve injury in rats. Injury 42:803-813.

Shin DH, Lee E, Hyun JK, Lee SJ, Chang YP, Kim JW, Choi YS, Kwon BS (2003) Growth-associated protein-43 is elevated in the injured rat sciatic nerve after low power laser irradiation. Neurosci Lett 344:71-74.

Wang S, Wan ACA, Xu X, Gao S, Mao HQ, Leong KW, Yu H (2001) A new nerve guide conduit material composed of a biodegradable poly(-phosphoester). Biomaterials 22:1157-1169.

Yang YC, Shen CC, Cheng HC, Liu BS (2011) Sciatic nerve repair by reinforced nerve conduits made of gelatin-tricalcium phosphate composites. J Biomed Mater Res A 96:288-300.

Yang YC, Shen CC, Huang TB, Chang SH, Cheng HC, Liu BS (2010) Characteristics and biocompatibility of a biodegradable genipin-crosslinked gelatin/β-tricalcium phosphate reinforced nerve guide conduit. J Biomed Mater Res B Appl Biomater 95:207-217.

Yannas IV, Hill BJ (2004) Selection of biomaterials for peripheral nerve regeneration using data from the nerve chamber model. Biomaterials 25:1593-1600.

Bai-Shuan Liu, Ph.D., Department of Medical Imaging and Radiological Sciences, Central Taiwan University of Science and Technology, No. 666, Buzih Road, Beitun District, Taichung 406,

10.4103/1673-5374.135323 http://www.nrronline.org/

Taiwan, China, bsliu@ctust.edu.tw

Accepted: 2014-05-28

Liu BS, Huang TB, Chan SC. Roles of reinforced nerve conduits and low-level laser phototherapy for long gap peripheral nerve repair. Neural Regen Res. 2014;9(12)：1180-1182.