A new nerve coaptation technique using a biodegradable honeycomb-patterned film.

We developed a biodegradable poly-lactide (PLA) film with a honeycomb-patterned porous structure (honeycomb film). This study investigated the use of this film in neurorrhaphy. Three types of PLA film were tested following bilateral sciatic nerve transection and neurorrhaphy in 35 rats: 7- and 10-µm thick honeycomb films, and cast film with no porous structures. Initially, following two-stitch neurorrhaphy, 40 limbs (20 rats) underwent wrapping in 7- or 10-µm honeycomb film, cast film, no wrapping, or extra two-stitch neurorrhaphy (8 limbs each). Breaking strength was tested 2 days postoperatively. Another 30 limbs (15 rats) then underwent wrapping in 7- or 10-µm honeycomb film, cast film, no wrapping, or sham operation (six limbs each). Histological and functional analyses were performed 6 weeks postoperatively. Breaking strength was significantly higher for the 10-µm honeycomb film than for no wrapping (P = 0.013), although no significant difference was observed between the 7-µm honeycomb and no wrapping (P = 0.085). Breaking strength for the cast film was almost equal to that for no wrapping (P = 0.994). Extra two-stitch (four-stitch) neurorrhaphy was significantly stronger than all groups, except the 10-µm honeycomb group. No significant difference was observed between the 10-µm honeycomb and the four-stitch (P = 0.497). No negative effects on functional recovery were identified. No adhesions or inflammation were observed between the film and surrounding tissues in the honeycomb groups. Honeycomb film may offer a suitable reinforcing material for adhesion-free neurorrhaphy.

Artificial perineurium to enhance nerve recovery from damage after neurolysis.

We have developed a novel biodegradable poly-lactide (PLA) film (honeycomb film) with a micropatterned porous structure on one side. We hypothesized that this film could be used as a substitute for perineurium. We used two types of thin PLA film: honeycomb film and cast film with smooth surfaces on both sides. In a rat extensive internal neurolysis model, the nerve was wrapped with honeycomb film (group H) or cast film (group CA), or left unwrapped (group C). Histological and functional analyses were performed. The honeycomb film closely attached to the nerve surface but did not adhere to surrounding tissues. In contrast, nerves in group C displayed severe adhesion to the neural bed. Mean percent wet muscle weight and motor nerve conduction velocity were significantly higher in group H than in group C. The honeycomb film prevents nerve adhesion and enhances functional recovery after extensive neurolysis.

A Novel Treatment with Stem Cells from Human Exfoliated Deciduous Teeth for Hypoxic-Ischemic Encephalopathy in Neonatal Rats.

Recently, cell therapy has been developed as a novel treatment for perinatal hypoxic-ischemic encephalopathy (HIE), which is an important cause of neurological disorder and death, and stem cells from human exfoliated deciduous teeth (SHED) express early markers for mesenchymal and neuroectodermal stem cells. We investigated the treatment effect of SHED for HIE in neonatal rats. Seven-day-old rats underwent ligation of the left carotid artery and were exposed to 8% hypoxic treatment. SHED (1 × 105 cells) were injected via the right external jugular vein 24 h after the insult. The effect of intravenous administration of SHED cells was evaluated neurologically and pathophysiologically. In the evaluation of engraftment using quantum dots 655, only a few SHED were detected in the injured cortex. In the immunohistological evaluation 24 h after injection, the numbers of positive cells of active caspase-3 and anti-4 hydroxynonenal antiserum were lower in the SHED group than in the vehicle group. The number of Iba-1+ cells in the cortex was higher in the SHED group. However, the proportion of M1 microglia (Iba-1+/ED-1+) was significantly decreased, whereas M2 microglia (Iba-1+/CD206+) tended to increase in the SHED group. In the behavioral tests performed 5 months after hypoxic treatment, compared to the vehicle group, the SHED group showed significant elongation of the endurance time in the rotarod treadmill test, significantly ameliorated proportion of using the impaired hand in the cylinder test, significantly lower ratio of right/left front paw area in gait analysis, and significantly higher avoidance rate in the active avoidance test. In the in vitro experiment with cultured neurons exposed to oxygen-glucose deprivation, we confirmed the neuroprotective effect of the condition medium of SHED. These results suggested that intravenous administration of SHED exerted a treatment effect both histologically and functionally, possibly via a paracrine effect.

Chondrotinase ABC enhances axonal regeneration across nerve gaps.

We evaluated the effects of chondroitinase ABC on axonal regeneration across peripheral nerve gaps. We compared axonal regeneration after 15-mm tibial nerve resection and repair with a silicone tube filled with type I collagen gel (negative control group), with a silicone tube filled with type I collagen gel containing chondroitinase ABC at three different concentrations (2.5 units/mL, 5 units/mL, 10 units/mL) (chondroitinase ABC groups), and with an autologous nerve segment (nerve autograft group). Electrophysiological and histological assessments were carried out 12 weeks after surgery. In the electrophysiological study, compound muscle action potentials (CMAPs) and nerve conduction velocities (NCVs) were recorded in all groups except the negative control group. Although both CMAPs and NCVs were highest in the nerve autograft group, there were no significant differences among the three chondroitinase ABC groups in either parameter. Histological findings were consistent with electrophysiological results. Based on these findings, we conclude that topical injection of chondroitinase ABC can significantly increase the critical length of nerve gap repair by tubulization or artificial nerve placement.

Efficacy and safety of novel collagen conduits filled with collagen filaments to treat patients with peripheral nerve injury: A multicenter, controlled, open-label clinical trial.

INTRODUCTION: The safety and efficacy of using artificial collagen nerve conduits filled with collagen filaments to treat nerve defects has not been fully studied in humans. We conducted a multicenter, controlled, open-label study to compare the safety and efficacy of artificial nerve conduit grafts with those of autologous nerve grafts. METHODS: We included patients with a sensory nerve defect of ≤30 mm, at the level of the wrist or a more distal location, with the first-line surgical methods selected according to a patient's preference. We compared sensory recovery using static two-point discrimination and adverse events between the artificial collagen nerve conduit and autologous nerve grafting. RESULTS: The artificial nerve conduit group included 49 patients, with a mean age of 42 years and nerve defect of 12.6 mm. The autologous nerve graft group included 7 patients, with historical data of an additional 31 patients, with a mean age of 36 years and nerve defect of 18.7 mm. The rate of recovery of sensory function at 12 months was 75% (36/49) for the artificial nerve conduit group and 73.7% (28/38) in the autologous nerve group. No serious adverse events directly associated with use of the artificial nerve conduit were identified. CONCLUSIONS: The treatment of nerve defects ≤30 mm using artificial collagen nerve conduits was not inferior to treatment using autologous nerve grafts. Based on our data, the new artificial collagen nerve conduit can provide an alternative to autologous nerve for the treatment of peripheral nerve defects.

Development of a new method for endovascular aortic repair: combination therapy of cell transplantation and stent grafts with a drug delivery system.

BACKGROUND: Endovascular aortic repair by stent grafts (S/Gs) has been developed as a less invasive treatment for aortic aneurysms. However, some aneurysmal cavities can remain without organization, causing re-expansion. We demonstrated previously that transplantation of a cell combination (myoblasts and fibroblasts) promoted thrombus organization in a rat model. We also developed basic fibroblast growth factor (bFGF) slow-delivery S/Gs coated with elastin and impregnated with bFGF. Here, we evaluated the effects of cell transplantation combined with bFGF slow release on canine thoracic aortic aneurysmal sacs after S/Gs repair. METHODS AND RESULTS: Thoracic aortic aneurysms were surgically created with jugular vein patches in 15 beagles. Myoblasts and fibroblasts of autologous skeletal muscle were isolated and cultured for cell transplantation. The S/Gs had 6 holes and produced endoleaks in the excluded cavities. Collagen gel (gel group, n=5) or a mixture of skeletal myoblasts and fibroblasts with collagen gel (cell group, n=5) were injected into the aneurysmal sacs excluded by the S/Gs. We also studied the effects of combined therapy of bFGF slow-release S/Gs and cell transplantation (hybrid group, n=5). After 14 days, histological analyses revealed that the excluded aneurysmal cavities of the gel group were filled with fresh thrombus, whereas the excluded cavities in the cell-transplanted groups were occupied by organized tissue. The percentages of the organized areas relative to the excluded cavities, evaluated by Masson's trichrome staining, were 18.1+/-4.0%, 52.6+/-4.0%, and 77.1+/-6.9% in the gel, cell, and hybrid groups, respectively. Collagen fibers had already appeared, and increased numbers of alpha-smooth muscle actin-positive cells were observed in the hybrid group. CONCLUSIONS: Cell transplantation accelerated thrombus organization. Moreover, slow release of bFGF enhanced the effects of cell transplantation. Cell transplantation into unorganized spaces may improve the outcomes of endovascular treatments of aortic aneurysms.

Novel Anti-Adhesive CMC-PE Hydrogel Significantly Enhanced Morphological and Physiological Recovery after Surgical Decompression in an Animal Model of Entrapment Neuropathy.

We developed a novel hydrogel derived from sodium carboxymethylcellulose (CMC) in which phosphatidylethanolamine (PE) was introduced into the carboxyl groups of CMC to prevent perineural adhesions. This hydrogel has previously shown excellent anti-adhesive effects even after aggressive internal neurolysis in a rat model. Here, we confirmed the effects of the hydrogel on morphological and physiological recovery after nerve decompression. We prepared a rat model of chronic sciatic nerve compression using silicone tubing. Morphological and physiological recovery was confirmed at one, two, and three months after nerve decompression by assessing motor conduction velocity (MCV), the wet weight of the tibialis anterior muscle and morphometric evaluations of nerves. Electrophysiology showed significantly quicker recovery in the CMC-PE group than in the control group (24.0 ± 3.1 vs. 21.0± 2.1 m/s (p < 0.05) at one months and MCV continued to be significantly faster thereafter. Wet muscle weight at one month significantly differed between the CMC-PE (BW) and control groups (0.148 ± 0.020 vs. 0.108 ± 0.019%BW). The mean wet muscle weight was constantly higher in the CMC-PE group than in the control group throughout the experimental period. The axon area at one month was twice as large in the CMC-PE group compared with the control group (24.1 ± 17.3 vs. 12.3 ± 9 µm2) due to the higher ratio of axons with a larger diameter. Although the trend continued throughout the experimental period, the difference decreased after two months and was not statistically significant at three months. Although anti-adhesives can reduce adhesion after nerve injury, their effects on morphological and physiological recovery after surgical decompression of chronic entrapment neuropathy have not been investigated in detail. The present study showed that the new anti-adhesive CMC-PE gel can accelerate morphological and physiological recovery of nerves after decompression surgery.

Trophic Effects of Dental Pulp Stem Cells on Schwann Cells in Peripheral Nerve Regeneration.

Recently, mesenchymal stem cells have demonstrated a potential for neurotrophy and neurodifferentiation. We have recently isolated mobilized dental pulp stem cells (MDPSCs) using granulocyte-colony stimulating factor (G-CSF) gradient, which has high neurotrophic/angiogenic potential. The aim of this study is to investigate the effects of MDPSC transplantation on peripheral nerve regeneration. Effects of MDPSC transplantation were examined in a rat sciatic nerve defect model and compared with autografts and control conduits containing collagen scaffold. Effects of conditioned medium of MDPSCs were also evaluated in vitro. Transplantation of MDPSCs in the defect demonstrated regeneration of myelinated fibers, whose axons were significantly higher in density compared with those in autografts and control conduits only. Enhanced revascularization was also observed in the MDPSC transplants. The MDPSCs did not directly differentiate into Schwann cell phenotype; localization of these cells near Schwann cells induced several neurotrophic factors. Immunofluorescence labeling demonstrated reduced apoptosis and increased proliferation in resident Schwann cells in the MDPSC transplant compared with control conduits. These trophic effects of MDPSCs on proliferation, migration, and antiapoptosis in Schwann cells were further elucidated in vitro. The results demonstrate that MDPSCs promote axon regeneration through trophic functions, acting on Schwann cells, and promoting angiogenesis.

Enhanced repair of large osteochondral defects using a combination of artificial cartilage and basic fibroblast growth factor.

The purpose of this study was to examine the efficacy of a combination of artificial cartilage and basic fibroblast growth factor (bFGF) for the repair of large osteochondral defects. The artificial cartilage was a three-dimensional fabric (3-DF) composed of an ultra-high molecular weight polyethylene fiber with a triaxial three-dimensional structure. We implanted 3-DF impregnated with type I collagen gel containing 500 ng of bFGF (bFGF-treated group) or 3-DF impregnated with type I collagen gel alone (non-treated group) into a large full-thickness osteochondral defect (6 x 6 x 3 mm) of the patellar groove of rabbits. The defect area was examined grossly, histologically and biomechanically 4-48 weeks after surgery. Bone ingrowth into and around the 3-DF was evaluated with micro-computed tomography (micro-CT). Addition of bFGF to the 3-DF greatly accelerated cartilage formation on the articular surface and subchondral bone formation into and around the 3-DF, and improved biomechanical properties. These findings suggest that a combination of artificial cartilage and bFGF is clinically useful in cases involving large osteochondral defects.

Novel polysaccharide-derived hydrogel prevents perineural adhesions in a rat model of sciatic nerve adhesion.

We investigated the effects of a novel carboxymethylcellulose (CMC)-derived hydrogel, in which phosphatidylethanolamine (PE) was introduced into the carboxyl groups of CMC, for preventing perineural adhesion after extensive internal neurolysis of rat sciatic nerve. Sciatic nerves were randomly assigned to one of the following groups: the Control group, operated but no treatment; the HA group, operated and treated with 1% hyaluronan; the CMC-PE(L) group, operated and treated with low-viscosity CMC-PE hydrogel; and the CMC-PE(H) group, operated and treated with high-viscosity CMC-PE hydrogel. Perineural adhesions were evaluated at 6 weeks. Nerves were also subjected to biomechanical testing to assess ultimate breaking strength. Electrophysiological and wet muscle weight measurements were performed. Breaking strengths were significantly lower for the CMC-PE(L) group than for the Control and HA groups. Latency was significantly longer for the Control group than for the CMC-PE(L) group at 20 days. The mean percentage of wet muscle weight to body weight was significantly lower for the Control group than for the CMC-PE(L) group at 6 weeks. Low-viscosity CMC-PE hydrogel appears to prevent perineural adhesions and allow early restoration of nerve function.

Enhancement of perineurial repair and inhibition of nerve adhesion by viscous injectable pure alginate sol.

BACKGROUND: Clinical treatment of recurrent symptoms following peripheral nerve decompression is problematic. Removal of scar tissue can produce an inappropriately pronounced healing response and thereby lead to more serious tethering or compression of the nerve. The goal of the present study was to test the ability of viscous injectable pure alginate sol to prevent perineurial adhesion using a rat neurolysis model. METHODS: The antiadhesive effect of viscous injectable pure alginate sol after neurolysis was evaluated histologically and biomechanically. A total of 40 rats were used in this study. The viscous injectable pure alginate sol was applied topically on the right side after external (six rats) or extensive internal (six rats) neurolysis, whereas the left side received no treatment. The nerves were harvested over 6 weeks, and histologic analysis was performed with hematoxylin and eosin staining and Masson trichrome staining. Functional analysis of the blood and perineurial barriers was also evaluated with Evans blue albumin (four rats). Sixteen rats were used for biomechanical analysis, and eight rats were used as a control group. The results were analyzed statistically using a post hoc test. RESULTS: Histologic examination showed that the use of viscous injectable pure alginate sol was associated with excellent biocompatibility and strong inhibition of perineurial granulation. Viscous injectable pure alginate sol was absorbed completely within 6 weeks without inducing inflammation. In addition, viscous injectable pure alginate sol enhanced repair of the perineurium associated with regeneration of epithelial-like cell layers, a key structure necessary for barrier function of the inner layer of the perineurium. Functional analysis with Evans blue albumin clearly demonstrated that, although blood nerve barrier function recovered by 6 weeks postoperatively in all nerves, the perineurial barrier function recovered only in the sol-treated nerves. Biomechanical study showed a significant antiadhesive effect of viscous injectable pure alginate sol. CONCLUSIONS: Viscous injectable pure alginate sol inhibited adhesion formation around nerves and enhanced regeneration of the perineurium with barrier function. Because excessive perineurial fibrosis and tethering at the neurolysis or neurorrhaphy site is a common postoperative problem in peripheral nerve surgery, viscous injectable pure alginate sol appears to have potentially broad clinical applications.