Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat.

Bone marrow stromal cells (BMSCs) have been studied as effective transplants for the treatment of spinal cord injury (SCI). Our previous study showed that BMSCs infused into the cerebrospinal fluid (CSF) exhibited distinct effects on the recovery of acute SCI. The present study examined the effects of BMSCs in sub-acute SCI (2weeks post-injury) by transplanting them directly into the lesion. The spinal cord was crush-injured at the Th8-9 level in rats, and 2weeks later, cultured BMSCs (5x10(5)) derived from GFP-transgenic rats of the same strain were transplanted into the lesion. Tissue repair and nerve regeneration were examined by immunohistochemistry and electron microscopy. GFP-labeled BMSCs survived as cell assemblies in the spinal cord for 1-2weeks after transplantation. The dorsal side of BMSC assemblies in the spinal cord usually showed an expanded GFAP-negative, astrocyte-devoid area, in which extracellular matrices including collagen fibrils were deposited. Numerous regenerating axons associated with Schwann cells grew out through such astrocyte-devoid extracellular matrices. Ascending (CGRP-containing) and descending (5HT- and TH-containing) axons were included in these regenerating axons. Regenerated axons were myelinated by Schwann cells beyond 2weeks post-transplantation. Cavity formation was reduced in the cell transplantation group. Locomotory behavior assessed by the BBB scale improved to 9.8 points in the cell transplantation group, while it was to 5.5-5.7 in the control. BMSC transplantation into lesions of advanced SCI has markedly beneficial effects on tissue repair and axonal outgrowth, leading to improved locomotion in rats.

In situ tissue engineering for tracheal reconstruction using a luminar remodeling type of artificial trachea.

BACKGROUND: After successful trials of tracheal reconstruction using mesh-type prostheses in canine models, the technique has been applied clinically to human patients since 2002. To enhance tissue regeneration, we have applied a new tissue engineering approach to this mesh-type prosthesis. METHODS: The prosthesis consists of a polypropylene mesh tube reinforced with a polypropylene spiral and atelocollagen layer. The cervical tracheas of 18 beagle dogs were replaced with the prosthesis. The collagen layer was soaked with peripheral blood in 6 of the dogs, with bone marrow aspirate in another 6, and with autologous multipotential bone marrow-derived cells (mesenchymal stem cells) in another 6. The dogs were humanely killed at 1 to 12 months after the operation. RESULTS: All 18 dogs survived the postoperative period. Bronchoscopically, 3 of 4 dogs in the peripheral blood group showed stenosis, whereas no stenosis was evident in all 8 of the dogs in the bone marrow and mesenchymal stem cell groups 6 months after the operation. Faster epithelialization and fewer complications, such as mesh exposure and luminal stenosis, were observed in these two groups than in the peripheral blood group. Histologically, the cells from autologous bone marrow were found to proliferate into the tracheal tissue during the first month. Cilial movement in these two groups was faster than that in the peripheral blood group and recovered to 80% to 90% of the normal level. CONCLUSIONS: Bone marrow aspirate and mesenchymal stem cells enhance the regeneration of the tracheal mucosa on this prosthesis. This in situ tissue engineering approach may facilitate tracheal reconstruction in the clinical setting.

Regeneration of central nervous tissue using a collagen scaffold and adipose-derived stromal cells.

Adipose-derived stromal cells (ASCs) include stem cells, which have the potential to differentiate into a variety of cell lineages. The regeneration of central nerves was examined using ASCs and a collagen scaffold. A cerebral cortex defect (3 x 4 x 3 mm(3)) was created in the left frontal lobe of 16 male rats. In one group (n = 8), collagen (3 x 4 x 3 mm(3)) seeded with DiI-labeled ASCs was implanted in the defect. In order to seed the ASCs, a combination of the rotary cell culture system and pressing the collagen scaffold gently several times with a glass rod was applied. In the control group (n = 8), collagen was implanted without ASCs. The rats were sacrificed at 1 month after the scaffold implantation. Histologically, 0.2% of the implanted ASCs were positive for anti-human/rat microtubule-associated protein 2 (MAP2) antibody and microvessels were present at a density of 4.6 +/- 1.2/mm(2) within the collagen scaffold-implanted area in each coronal section. In the control group, no MAP2-positive cells were detected and the microvessel density was 0.6 +/- 0.4/mm(2). These data suggest that ASCs seeded into a collagen scaffold may have the potential to promote regeneration of nervous tissue after cerebral cortex injury.

Development of new nerve guide tube for repair of long nerve defects.

A novel nerve guide tube (poly (L-lactic) acid (PLLA)/ polyglycolic acid (PGA)-c-tube) capable of repairing long peripheral nerve injuries in a canine model has been developed. The tube was created by braiding together PLLA and PGA and then coating it with collagen. PLLA was newly added to the formulation to achieve higher sustainability. The tube was compared with a PGA-collagen tube in clinical use since 2002 having the same structure with a collagen coating but composed of PGA alone (PGA-c-tube). When tested for repair of a 40-mm gap in the left peroneal nerve, using PLLA/PGA-c-tube (n = 15), PGA-c-tube (n = 15), and a negative control group where the cut stump was capped using a silicone cap (n = 15), the lumen structure essential for securing the space for nerve regeneration was maintained in PLLA/PGA-c-tube for over 12 months with a higher number of axons both within the tube and at the distal nerve end. Electrophysiological evaluation revealed that the amplitude of compound muscle action potentials and sensory nerve action potentials after nerve regeneration with PLLA/PGA-c-tube were significantly higher. When assessed using magnetic resonance imaging (MRI), the volume of the tibialis anterior (TA) muscle in dogs that had undergone nerve repair using PLLA/PGA-c-tube was approximately 80% that of the positive control at 12 months. Functional analysis conducted by assessing the ankle angle revealed faster recovery in the PLLA/PGA-c-tube group. Better regeneration was achieved using a PLLA/PGA-c-tube that contains the slowly decomposing fiber material, PLLA. This indicates potential for repair of even longer nerve gaps or defects located near joints, and also clinical application.

Experimental repair of phrenic nerve using a polyglycolic acid and collagen tube.

OBJECTIVE: The feasibility of a nerve guide tube for regeneration of the phrenic nerve with the aim of restoring diaphragmatic function was evaluated in a canine model. METHODS: The nerve tube, made of woven polyglycolic acid mesh, had a diameter of 3 mm and was filled with collagen sponge. This polyglycolic acid-collagen tube was implanted into a 10-mm gap created by transection of the right phrenic nerve in 9 beagle dogs. The tubes were implanted without a tissue covering in 5 of the 9 dogs (group I), and the tubes were covered with a pedicled pericardial fat pad in 4 dogs (group II). Chest x-ray films, muscle action potentials, and histologic samples were examined 4 to 12 months after implantation. RESULTS: All of the dogs survived without any complications. x-ray film examination showed that the right diaphragm was paralyzed and elevated in all dogs until 3 months after implantation. At 4 months, movement of the diaphragm in the implanted side was observed during spontaneous breathing in 1 dog of group I and in 3 dogs of group II. In the dogs showing diaphragm movement, muscle action potentials were evoked in the diaphragm muscle, indicating restoration of nerve function. Regeneration of the phrenic nerve structure was also examined on the reconstructed site using electron microscopy. CONCLUSION: The polyglycolic acid-collagen tube induced functional recovery of the injured phrenic nerve and was aided by coverage with a pedicled pericardial fat pad.

Surgical relief of causalgia with an artificial nerve guide tube: Successful surgical treatment of causalgia (Complex Regional Pain Syndrome Type II) by in situ tissue engineering with a polyglycolic acid-collagen tube.

Two patients with causalgia associated with allodynia and finger contracture were treated surgically with a bioresorbable nerve guide tube made from polygycolic acid and collagen: the injured segment of the digital nerve was resected and the resulting gap (25 and 36mm) was bridged with the tube. In both cases, a neuroma was found on the injured nerve and many sprouting branches were. After reconstruction, the causalgia and allodynia disappeared and movement of the fingers recovered during the following 6 months. Functional recovery was objectively identified for 1 year and 9 months. Both patients regained full use of their finger and were free of discomfort for up to 24 and 18 months, respectively. Since the first description of causalgia in 1864, there has been no definitive treatment for this intractable burning pain. Our experience shows that at least some types of causalgia can be resolved successfully by surgery.

New canine spinal cord injury model free from laminectomy.

The present report details the successful development of a model for spinal cord injury (SCI). This model is simple, reproducible, and requires no laminectomy. Development of the model was carried out using fourteen dogs. A balloon catheter was inserted into the extradural space via the intervertebral foramen of each dog, then the balloon was inflated at the L1 level by injection of saline. Six dogs underwent compression with a balloon volume of 1.5 ml, three dogs with a volume of 1.0 ml, and the remaining five dogs were used as uninjured controls. We applied the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale to the dogs. Compression of the spinal cord for 10 min at 1.5 ml produced severe paraplegia (BBB remained zero or one for 6 months following surgery), while compression for the same time interval at 1.0 ml produced moderate paraplegia. Electrophysiological tests showed no hindlimb movement upon stimulation cranial to the site of injury in the 1.5-ml group. The volume of abnormal-intensity lesions in the 1.0-ml group calculated using MR imaging showed no marked changes in either high- or low-intensity lesions after 3 months, whereas in the 1.5-ml group, the low-intensity lesions alone showed a marked increase. Pathological examination of the damaged spinal cord showed the formation of cavities surrounded by scar tissue containing high levels of collagen. These findings closely resembled those of clinical cases. It was concluded that 10 min of balloon compression with a volume of 1.5 ml caused irreversible paraplegia in dogs.

Experimental study on the regeneration of peripheral nerve gaps through a polyglycolic acid-collagen (PGA-collagen) tube.

We have developed a bioabsorbable polyglycolic acid (PGA) tube filled with collagen sponge (PGA-collagen tube) as a nerve connective guide, and compared its effectiveness with that of autograft in terms of nerve regeneration across a gap. The PGA-collagen tube was implanted into 24 beagle dogs across a 15-mm gap in the left peroneal nerve. The right peroneal nerve was reconstructed with the autograft harvested from the left side, as a control. After the surgery, the connective tissue extended from both cut ends in the PGA-collagen tube and connected again at the center. Pathologically, the collagen sponge in the tube provided adequate scaffolding for nerve tissue extension, and the nerve tissue reconnected within 3 weeks. Electrophysiologically, muscle-evoked potentials (MEPs) and compound nerve action potentials (CNAPs) were detected 18 days after the surgery. For up to 6 months postsurgery, CNAPs and somatosensory-evoked potentials (SEPs) on the PGA-collagen side had a shorter latency and larger peak voltage than those on the autograft side. The myelinated axons on the PGA side were larger in diameter than those on the autograft side. It is suggested that the PGA-collagen tube has the potential to be an effective alternative to conventional autografting for the repair of some peripheral nerve defects.

A novel morphological technique to investigate a single climbing fibre synaptogenesis with a Purkinje cell in the developing mouse cerebellum: DiI injection into the inferior cerebellar peduncle.

To study morphologically the relationship between climbing fibre and Purkinje cell in the developing mouse cerebellum, we established a novel tract tracing using injection of 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the inferior cerebellar peduncle, the half point of olivocerebellar projection. In this tracing method, only a certain number of climbing fibres were labelled with DiI and they revealed the single-fibre resolution, individually. These technical advantages enabled us to follow the projection of a climbing fibre to a Purkinje cell at the light microscopic level. To further investigate how a single labelled olivocerebellar axon interacts with a Purkinje cell, we introduced a photoconversion method into this tracing method and successfully observed the photo-oxidized climbing fibre terminals at the electron microscopic level. At postnatal days 7 and 9, a single DiI-labelled climbing fibre arborized around some adjacent Purkinje cell bodies in a distinguishable nest. At this pericellular nest stage, we first demonstrated that the terminal arborization stemmed from a single climbing fibre formed synapses simultaneously on both a soma and dendrites of a Purkinje cell. This finding suggests that the pericellular nest may be such an efficient form that a single climbing fibre innervates a Purkinje cell at both perisomatic and peridendritic sites. Thus, we succeeded in establishing an effective tracing method to investigate a single climbing fibre synaptogenesis with a Purkinje cell both at the light and electron microscopic levels.

Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration.

The human amnion consists of the epithelial cell layer and underlying connective tissue. After removing the epithelial cells, the resulting acellular connective tissue matrix was manufactured into thin dry sheets called amnion matrix sheets. The sheets were further processed into tubes, amnion matrix tubes (AMTs), of varying diameters, with the walls of varying numbers of amnion matrix sheets with or without a gelatin coating. The AMTs were implanted into rat sciatic nerves. Regenerating nerves extended in bundles through tubes of 1-2 mm in diameter and further elongated into host distal nerves 1-3 weeks after implantation. Morphometrical analysis of the regenerated nerve cable at the middle of each amnion matrix tube 3 weeks after implantation was performed. The average numbers of myelinated axons were almost the same (ca. 80-112/10(4) microm(2)) in AMTs of 1-2 mm in diameter, as in the normal sciatic nerve (ca. 95/10(4) microm(2)). No myelinated fibers were found in AMTs composed of multiple thin tubes of 0.2 mm in diameter. The myelinated axons were thinner in implanted tubes than those in the normal sciatic nerve. The rate of occurrences of myelinated axons less than 4 microm in diameter was significantly higher in the AMTs, whereas axons in the normal sciatic nerve were diverse in distribution, with the highest population at 8-12 microm in diameter. Reinnervation to the gastrocnemius muscle was demonstrated electrophysiologically 9 months after implantation. It was concluded that the extracellular matrix sheet from the human amnion is an effective conduit material for peripheral nerve regeneration.

Repair of facial nerve with alginate sponge without suturing: an experimental study in cats.

A bioabsorbable material, alginate, was used to repair a defect in the facial nerve. In five cats a 5-mm gap was created in the dorsal ramus of the facial nerve on one side selected at random, which was repaired by implantation of alginate sponge without suturing. In the control group, two cats had a similar nerve injury but without implantation of alginate. Behavioural, electrophysiological, and histological examinations were made over a period of 16 weeks postoperatively. Movement of the upper eyelids and electrophysiological function were restored 12 weeks postoperatively, and many myelinated axons were observed both in the gap of facial nerve and its branches 16 weeks after operation, whereas no alginate residue was detected remaining within gap. In the control group, no movement or electrophysiological restoration was recorded, and there were few regenerated axons accompanied by a large amount of scar tissue. The nerve repaired with alginate showed remarkable regeneration. These results suggest that alginate is a promising material for facial nerve repair, and sutureless repair with alginate is a possible option for treating defects in the facial nerve.

Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors.

In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: alpha2betagammadelta (gamma-AChR) or alpha2betaepsilondelta (epsilon-AChR). After birth, gamma-AChRs are replaced by epsilon-AChRs (gamma/epsilon-switch). The two receptors have different conductances and open times. During perinatal period, the long open time gamma-AChRs generate random myofiber action potentials from uniquantal miniature end-plate potentials (mEPPs). epsilon-AChRs are suitable for strong adult muscle activities. Since the effect of the gamma/epsilon-switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate gamma-subunit-deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included epsilon-subunits, and muscle fibers had multiple neuromuscular junctions. Both pre- and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for gamma-AChRs in mediating synaptically-induced action potential activity critical for neuromuscular development.

Electrophysiological and horseradish peroxidase-tracing studies of nerve regeneration through alginate-filled gap in adult rat spinal cord.

The spinal cord segments at T(9-10) were totally excised and the resulting gap was filled by implantation of alginate sponge in adult rats. A horseradish peroxidase-tracing study at 21 weeks after operation showed that numerous ascending and many but less numerous descending regenerating fibres traversed the alginate-filled gap, and that after re-entering the distal stump of the transected spinal cord, they extended randomly over a long distance away from the gap. Intracellular electrophysiological recording at the same postoperative time showed that both ascending and descending regenerating axons formed functional synapses with host neurons located beyond the gap. These findings suggest that alginate could be a promising material for the support of regenerating axons in the spinal cord.