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Fibrin glue as a stabilization strategy in peripheral nerve repair when using porous nerve guidance conduits.

ABSTRACT: Porous conduits provide a protected pathway for nerve regeneration, while still allowing exchange of nutrients and wastes. However, pore sizes >30 µm may permit fibrous tissue infiltration into the conduit, which may impede axonal regeneration. Coating the conduit with Fibrin Glue (FG) is one option for controlling the conduit's porosity. FG is extensively used in clinical peripheral nerve repair, as a tissue sealant, filler and drug-delivery matrix. Here, we compared the performance of FG to an alternative, hyaluronic acid (HA) as a coating for porous conduits, using uncoated porous conduits and reverse autografts as control groups. The uncoated conduit walls had pores with a diameter of 60 to 70 µm that were uniformly covered by either FG or HA coatings. In vitro, FG coatings degraded twice as fast as HA coatings. In vivo studies in a 1 cm rat sciatic nerve model showed FG coating resulted in poor axonal density (993 ± 854 #/mm²), negligible fascicular area (0.03 ± 0.04 mm²), minimal percent wet muscle mass recovery (16 ± 1 in gastrocnemius and 15 ± 5 in tibialis anterior) and G-ratio (0.73 ± 0.01). Histology of FG-coated conduits showed excessive fibrous tissue infiltration inside the lumen, and fibrin capsule formation around the conduit. Although FG has been shown to promote nerve regeneration in non-porous conduits, we found that as a coating for porous conduits in vivo, FG encourages scar tissue infiltration that impedes nerve regeneration. This is a significant finding considering the widespread use of FG in peripheral nerve repair.

SUBMITTER: Bhatnagar D

PROVIDER: S-EPMC5384961 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rationale: Peripheral nerve injury (PNI) is a great challenge for regenerative medicine. Nerve autograft is the gold standard for clinical PNI repair. Due to its significant drawbacks, artificial nerve guidance conduits (NGCs) have drawn much attention as replacement therapies. We developed a combinatorial NGC consisting of longitudinally aligned electrospun nanofibers and porcine decellularized nerve matrix hydrogel (pDNM gel). The in vivo capacity for facilitating nerve tissue regeneration and functional recovery was evaluated in a rat sciatic nerve defect model. Methods: Poly (L-lactic acid) (PLLA) was electrospun into randomly oriented (PLLA-random) and longitudinally aligned (PLLA-aligned) nanofibers. PLLA-aligned were further coated with pDNM gel at concentrations of 0.25% (PLLA-aligned/0.25% pDNM gel) and 1% (PLLA-aligned/1% pDNM gel). Axonal extension and Schwann cells migration were evaluated by immunofluorescence staining of dorsal root ganglia cultured on the scaffolds. To fabricate implantable NGCs, the nanofibrous scaffolds were rolled and covered with an electrospun protection tube. The fabricated NGCs were then implanted into a 5 mm sciatic nerve defect model in adult male Sprague-Dawley rats. Nerves treated with NGCs were compared to contralateral uninjured nerves (control group), injured but untreated nerves (unstitched group), and autografted nerves. Nerve regeneration was monitored by an established set of assays, including T2 values and diffusion tensor imaging (DTI) derived from multiparametric magnetic resonance imaging (MRI), histological assessments, and immunostaining. Nerve functional recovery was evaluated by walking track analysis. Results: PLLA-aligned/0.25% pDNM gel scaffold exhibited the best performance in facilitating directed axonal extension and Schwann cells migration in vitro due to the combined effects of the topological cues provided by the aligned nanofibers and the biochemical cues retained in the pDNM gel. Consistent results were obtained in animal experiments with the fabricated NGCs. Both the T2 and fractional anisotropy values of the PLLA-aligned/0.25% pDNM gel group were the closest to those of the autografted group, and returned to normal much faster than those of the other NGCs groups. Histological assessment indicated that the implanted PLLA-aligned/0.25% pDNM gel NGC resulted in the largest number of axons and the most extensive myelination among all fabricated NGCs. Further, the PLLA-aligned/0.25% pDNM gel group exhibited the highest sciatic nerve function index, which was comparable to that of the autografted group, at 8 weeks post-surgery. Conclusions: NGCs composed of aligned PLLA nanofibers decorated with 0.25% pDNM gel provided both topological and biochemical guidance for directing and promoting axonal extension, nerve fiber myelination, and functional recovery. Moreover, T2-mapping and DTI metrics were found to be useful non-invasive monitoring techniques for PNI treatment.

Project description:Conductivity is a desirable property of an ideal nerve guide conduit (NGC) that is being considered for peripheral nerve regeneration. Most of the conductive polymers reported in use for fabrication of tissue engineering scaffolds such as polypyrrole (PPy), polyaniline, polythiophene, and poly(3,4-ethylenedioxythiophene) are non-biodegradable and possess weak mechanical properties to be fabricated into 3D structures. In this study, a biodegradable and conductive block copolymer of PPy and Polycaprolactone (PPy-b-PCL) was used to fabricate 3D porous NGCs using a novel electrohydrodynamic jet 3D printing process which offers superior control over fiber diameter, pore size, porosity, and fiber alignment. PCL/PPy scaffolds with three different concentrations of PPy-b-PCL (0.5, 1, and 2% v/v) were fabricated as a mesh (pore size 125 ± 15 μm) and the effect of incorporation of PPy-b-PCL on mechanical properties, biodegradability, and conductivity of the NGCs were studied. The mechanical properties of the scaffolds decreased with the addition of PPy-b-PCL which aided the ability to fabricate softer scaffolds that are closer to the properties of the native human peripheral nerve. With increasing concentrations of PPy-b-PCL, the scaffolds displayed a marked increase in conductivity (ranging from 0.28 to 1.15 mS/cm depending on concentration of PPy). Human embryonic stem cell-derived neural crest stem cells (hESC-NCSCs) were used to investigate the impact of PPy-b-PCL based conductive scaffolds on the growth and differentiation to peripheral neuronal cells. The hESC-NCSCs were able to attach and differentiate to peripheral neurons on PCL and PCL/PPy scaffolds, in particular the PCL/PPy (1% v/v) scaffolds supported higher growth of neural cells and a stronger maturation of hESC-NCSCs to peripheral neuronal cells. Overall, these results suggest that PPy-based conductive scaffolds have potential clinical value as cell-free or cell-laden NGCs for peripheral neuronal regeneration.

Project description:Peripheral nerve regeneration requires stepwise and well-organized establishment of microenvironment. Since local delivery of VEGF-A in peripheral nerve repair is expected to promote angiogenesis in the microenvironment and Schwann cells (SCs) play critical role in nerve repair, combination of VEGF and Schwann cells may lead to efficient peripheral nerve regeneration. VEGF-A overexpressing Schwann cells were established and loaded into the inner wall of hydroxyethyl cellulose/soy protein isolate/polyaniline sponge (HSPS) conduits. When HSPS is mechanically distorted, it still has high durability of strain strength, thus, can accommodate unexpected strain of nerve tissues in motion. A 10 mm nerve defect rat model was used to test the repair performance of the HSPS-SC (VEGF) conduits, meanwhile the HSPS, HSPS-SC, HSPS-VEGF conduits and autografts were worked as controls. The immunofluorescent co-staining of GFP/VEGF-A, Ki67 and MBP showed that the VEGF-A overexpressing Schwann cells could promote the proliferation, migration and differentiation of Schwann cells as the VEGF-A was secreted from the VEGF-A overexpressing Schwann cells. The nerve repair performance of the multifunctional and flexible conduits was examined though rat behavioristics, electrophysiology, nerve innervation to gastrocnemius muscle (GM), toluidine blue (TB) staining, transmission electron microscopy (TEM) and NF200/S100 double staining in the regenerated nerve. The results displayed that the effects on the repair of peripheral nerves in HSPS-SC (VEGF) group was the best among the conduits groups and closed to autografts. HSPS-SC (VEGF) group exhibited notably increased CD31+ endothelial cells and activation of VEGFR2/ERK signaling pathway in the regenerated nerve tissues, which probably contributed to the improved nerve regeneration. Altogether, the comprehensive strategy including VEGF overexpressing Schwann cells-mediated and HSPS conduit-guided peripheral nerve repair provides a new avenue for nerve tissue engineering.

Project description:Nerve conduits enhance nerve regeneration in the repair of long-distance peripheral nerve defects. To help optimize the effectiveness of nerve conduits for nerve repair, we developed a multi-step electrospinning process for constructing nerve guide conduits with aligned nanofibers. The alignment of the nerve guide conduits was characterized by scanning electron microscopy and fast Fourier transform. The mechanical performance of the nerve guide conduits was assessed by testing for tensile strength and compression resistance. The biological performance of the aligned fibers was examined using Schwann cells, PC12 cells and dorsal root ganglia in vitro. Immunohistochemistry was performed for the Schwann cell marker S100 and for the neurofilament protein NF200 in PC12 cells and dorsal root ganglia. In the in vivo experiment, a 1.5-cm defect model of the right sciatic nerve in adult female Sprague-Dawley rats was produced and bridged with an aligned nerve guide conduit. Hematoxylin-eosin staining and immunohistochemistry were used to observe the expression of ATF3 and cleaved caspase-3 in the regenerating matrix. The recovery of motor function was evaluated using the static sciatic nerve index. The number of myelinated fibers, axon diameter, fiber diameter, and myelin thickness in the distal nerve were observed by electron microscopy. Gastrocnemius muscle mass ratio was also determined. The analyses revealed that aligned nanofiber nerve guide conduits have good mechanical properties and can induce Schwann cells, PC12 cells and dorsal root ganglia to aggregate along the length of the nanofibers, and promote the growth of longer axons in the latter two (neuronal) cell types. The aligned fiber nerve conduits increased the expression of ATF3 and cleaved caspase-3 at the middle of the regenerative matrix and at the distal nerve segment, improved sciatic nerve function, increased muscle mass of the gastrocnemius muscle, and enhanced recovery of distal nerve ultrastructure. Collectively, the results show that highly aligned nanofibers improve the performance of the nerve conduit bridge, and enhance its effectiveness in repairing peripheral nerve defects.

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Fibrin glue as a stabilization strategy in peripheral nerve repair when using porous nerve guidance conduits.

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