Degradation of Unsintered Hydroxyapatite and Poly-L-Lactide Composite Sheets In Vivo and In Vitro.

Bioabsorbable sheet-shaped implants made of forged composites of unsintered hydroxyapatite and poly-L-lactide (F-u-HA/PLLA) have been used for orbital fracture repair with good results. This is the first report using multiple specimens implanted in the human orbit to demonstrate the biodegradation and loss of strength of F-u-HA/PLLA sheets. Among the patients who underwent various facial fracture repairs with F-u-HA/PLLA sheets implanted in their orbits, those whose sheets were subsequently extracted were included in the study. Viscosity-average molecular weight, crystallinity, and bending strength of the extracted implants were measured. An in vitro degradation test was also performed for comparison. Among the 111 patients who underwent F-u-HA/PLLA sheet implantation, 13 subsequently underwent surgical extraction of implants; the majority were due to secondary correction of complex fractures. One patient developed an infection; none developed foreign body reactions. Overall, 11 specimens from 10 patients with consent were examined. The time from implantation to extraction ranged from 43 to 632 days (median: 210 d). Compared with the results of the in vitro degradation test, the viscosity-average molecular weight and bending strength had a slower decrease. The F-u-HA/PLLA sheets retained more than 50% of their initial bending strength after 12 months. Crystallinity varied widely. F-u-HA/PLLA sheets implanted in human orbits did not degrade faster than those of in vitro testing. Sheet-shaped implants made of forged composites of unsintered hydroxyapatite and poly-L-lactide can be considered appropriate reconstructive materials for orbital fractures as they retained sufficient strength to support the orbital contents at 12 months postoperatively, and no case of delayed foreign body reactions was observed.

The combined use of hyoid bone flap and radial forearm free flap for reconstruction following partial laryngopharyngectomy.

In hypopharyngeal carcinoma, even partial laryngopharyngectomy, results in functional disorders involving swallowing and speaking. We reconstructed partial defects following partial laryngopharyngectomy using a combined flap of a hyoid bone flap and radial forearm free flap. Before ablative surgery, we prepared an ipsilateral hyoid bone as a bone flap with sternohyoidal muscle. Then a radial forearm free flap was prepared simultaneously with tumor surgery. Thereafter, we reconstructed the epiglottis and pyriform recess using the combined flap. We successfully reconstructed 7 patients suffering from squamous cell carcinoma of the hypopharynx. The tracheostoma in all patients could be closed. Six patients could swallow without dysphagia within 48 days. This is the first report of the successful combined use of a hyoid bone flap and radial forearm free flap for hypopharyngeal carcinoma.

Two cases of Hailey-Hailey disease effectively treated with apremilast and a review of reported cases.

Hailey-Hailey disease (HHD) is an autosomal dominant genetic disease caused by a mutation of the ATP2C1 gene. Corticosteroids, antibiotics or cyclosporine have been administered to reduce inflammation and prevent flare-ups, but the efficacy is not always sufficient. We herein report two cases of HHD effectively treated with apremilast and review the previous literature. Patient 1 was a 28-year-old male and patient 2 was a 35-year-old female. Both patients were diagnosed with HHD based on histological and genetic analyses. Both patients were treated with oral antibiotics or topical corticosteroids, but their symptoms were refractory, therefore apremilast was administered to both patients. Two weeks later, the skin lesion of both patients was improved. No adverse reaction was observed except for mild headache in patient 2. There have been 13 reported cases of HHD treated with apremilast, including our cases. Eight cases showed a good response to apremilast, whereas five cases showed no response. There seems to be no association between the disease severity and efficacy of apremilast, although the reason remains unknown. Interestingly, an early improvement of the HHD lesion was observed in all good response cases. Although digestive symptoms, headache, and myalgia were observed as adverse events, the treatment was well-tolerated. The accumulation of a greater number of similar cases and further research will be required. We hypothesize that apremilast may be a useful therapeutic option for skin lesions of HHD.

Induction of activating transcription factor 3 after different sciatic nerve injuries in adult rats.

Staining by activating transcription factor 3 (ATF3), a neuronal marker of nerve injury, was examined by immunocytochemistry in neurons and Schwann cells after crush or transection (regeneration inhibited) of rat sciatic nerve. ATF3 immunoreactivity peaked in neurons after three days and then gradually subsided to normal within 12 weeks after the crush. The response lasted somewhat longer and declined over time in spinal cord neurons but not in those of dorsal root ganglia (DRG) after transection, indicating a differential regulation of sensory and motor neurons. ATF3 expression was more pronounced in Schwann cells, and remained longer after transection, implying that to some extent regenerating axons produce signals that reduce ATF3 expression in Schwann cells. However, even after transection without repair (no contact with regenerating axons), ATF3 expression in Schwann cells in the distal segment decreased over time suggesting that regenerating axons are not entirely responsible for the down-regulation. These findings have clinical implications on when it is worthwhile to reconstruct nerve injuries.

End-to-side nerve repair induces nuclear translocation of activating transcription factor 3.

We wanted to find out if any of three different types of manipulations: a piece of muscle or nerve put parallel to a nerve; an epineurial window made, or sutures inserted into a nerve, or both; or pieces of nerve sutured to an epineurial window end-to-side to the musculocutaneous or sciatic nerve, resulted in activation of activating transcription factor 3 (ATF3) in neurons and in non-neuronal cells. ATF3, a marker of cell activation, was investigated by immunocytochemistry one week after manipulation. A piece of nerve or muscle parallel to a nerve did not induce ATF3 locally in the nerve and induced ATF3 only rarely in neurons. In contrast, an epineurial window or insertion of sutures, or both, with or without attachment of a piece of nerve placed end-to-side, induced robust ATF3 expression. We conclude that an injury to a peripheral nerve trunk associated with end-to-side nerve repair, activates neurons and non-neuronal cells and may contribute to sprouting of axons into the nerve attached end-to-side.

Alginate enhances elongation of early regenerating axons in spinal cord of young rats.

Freeze-dried alginate sponge cross-linked with covalent bonds has been demonstrated to enhance nerve regeneration in peripheral nerves and spinal cords. The present study examined, at early stages after surgery, the outgrowth of regenerating axons and reactions of astrocytes at the stump of transected spinal cord in young rats. Two segments (Th7-8) were resected, and alginate was implanted in the lesion. As controls, collagen gel was implanted in place of alginate or the lesion was left without implantation. Two and 4 weeks after surgery, nerve outgrowth and astrocyte reactions were examined. Many regenerating axons, some of which were accompanied by astrocytic processes, were found to extend from the stump into the alginate-implanted lesion. In the all nonimplanted animals, large cystic cavities were formed at both interfaces with no definite axonal outgrowth into the lesion. In collagen-implanted animals, cavity formation was found in some rats, and regenerating axons once formed at the stumps did not extend further into the lesion. Astrocytic processes extending into alginate-implanted lesion had no basal laminae, whereas those found in control experiments were covered by basal laminae. These findings suggest that alginate contributed to reducing the barrier composed of connective tissues and reactive astrocytic processes, and served as a scaffold for the outgrowth of regenerating axons and elongation of astrocytic processes.

Implantation of neural stem cells via cerebrospinal fluid into the injured root.

In avulsion injury of the dorsal root, regenerating axons cannot extend through the entry zone, i.e. the transition zone between peripheral and central nervous systems, due to the discontinuity between Schwann cells and astrocytes. We infused neural stem cells through the 4th ventricle in an attempt to enhance axonal growth in injured dorsal roots. Infused stem cells were attached to, and integrated into, the lesion of the root and became associated with axons in the same manner as Schwann cells or perineurial sheath cells in the peripheral nerve, and as astrocytes in the central nerve area. These findings suggest that neural stem cells integrated by infusion through CSF might have a beneficial effect on nerve regeneration by inducing a continuity of Schwann cells and astrocytes at the transition zone.

Dissemination and proliferation of neural stem cells on the spinal cord by injection into the fourth ventricle of the rat: a method for cell transplantation.

We examined the distribution of hippocampus-derived neural stem cells on the spinal cord surface for up to 3 weeks following injection through the fourth ventricle. The injected cells were disseminated as tiny spots on the pia mater of the spinal cord and proliferated into large cell-clusters. On both the dorsal and ventral side, cell clusters increased in number rapidly up to 5 days after injection and thereafter decreased gradually due to the coalition of neighbouring clusters. Concomitantly, individual cell clusters continuously increased in size, occupying almost 50% of the spinal cord surface. Cell attachment was usually found around blood vessels, along which cells invaded into the spinal cord. In the injured site, cells migrated into the lesion and were integrated into the spinal cord tissue, some of which had differentiated into astrocytes 1-2 weeks after injection. BrdU-uptake experiments demonstrated that the transplanted cells proliferated within the host cerebrospinal fluid. These results indicate that application of neural stem cells through the ventricle is an effective method to disseminate cells all over the spinal cord and that they can migrate and be integrated into the injured spinal cord.

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.

New method for transplantation of neurosphere cells into injured spinal cord through cerebrospinal fluid in rat.

Here we report a novel method of supplying cultured neurosphere cells to the injured spinal cord, by injection of cells into the cerebrospinal fluid (CSF) through the fourth ventricle or cisterna magna. Hippocampus-derived neurosphere cells, isolated from a transgenic rat fetus expressing green fluorescent protein, were transplanted into the CSF of a rat with spinal cord injury. It was found that injected cells were extensively transported by CSF within the subarachnoidal space, and survived as clusters on the pial surface of the spinal cord. The most notable finding was that a large number of injected cells migrated into the lesion site and integrated into the injured spinal cord tissues.

Peripheral nerve regeneration by transplantation of BMSC-derived Schwann cells as chitosan gel sponge scaffolds.

It is said that bone marrow stromal cells (BMSCs) are able to differentiate into different kinds of cells, including Schwann cells, under relevant conditions (Dezawa et al., Eur J Neurosci 2001;14:1771-1776). In the previous paper, we demonstrated that chitosan gel sponge is one of the effective scaffolds for regenerating axons of the rat sciatic nerve (Ishikawa et al., J Biomed Mater Res A 2007;83:33-40). In the present study, we examined whether BMSC-derived Schwann cells with chitosan gel sponges were one of the effective scaffolds for peripheral nerve regeneration in rats. BMSC-derived cells with Schwann cell characteristics were labeled by green fluorescent protein using a retrovirus. An 8-mm gap was made by removing a nerve segment from the rat peripheral nerve, and chitosan gel sponges containing BMSC-derived Schwann cells were grafted, sandwiching the proximal and distal stumps of the transected nerve. Rats were sacrificed at 7, 14, and 28 days, and 2 and 4 months after transplantation. Immunohistochemistry demonstrated that regenerating axons were found near transplanted Schwann cells 7 days after surgery and extended into the host distal nerve segment at 14 days after surgery. Electron microscopy showed that transplanted Schwann cells formed myelin sheaths on regenerating axons 1 month after transplantation. The mean diameter of myelinated fibers was increased from 2.58 mum at 2 months to 2.84 mum at 4 months postsurgery. This study indicates that chitosan gel sponges containing BMSC-derived Schwann cells have strong potentiality as a graft that can be used for peripheral nerve regeneration.

Transmaxillary and transmandibular approach to a C1 chordoma.

STUDY DESIGN: Case report. OBJECTIVES: To demonstrate the efficacy of a transmaxillary and transmandibular approach in achieving a wide view and the aggressive resection of a retropharyngeal chordoma originating from C1. SUMMARY OF BACKGROUND DATA: Although aggressive surgical resection has been recommended for the treatment of chordomas, wide exposure of the tumors in the upper cervical region is a challenge. METHODS: A 19-year-old man presented with a large ossified retropharyngeal chordoma (6 cm in diameter) originating from the right side of the anterior arch of C1, and extending from the clivus to the C2/3 intervertebral disc level in the sagittal plane. A posterior occipitocervical (O-C3) fusion with an iliac bone graft was first performed. Ten days after the fusion, the tumor was resected using a mandible and tongue midsplitting approach combined with a Le Fort I (transmaxillary) osteotomy, which allowed us to expose the entire tumor. The tumor was hard and immovable because of ossification. The main part of the tumor was resected from the anterior arch of C1, and then the C1 anterior arch was resected en bloc. The retropharyngeal wall was reconstructed using a vascularized radial forearm flap. Radiation therapy (60 Gy) was performed after surgery. RESULTS: No local recurrence or metastasis was observed 3 years after the operation. The patient had no complaints and has returned to his previous job as a manual laborer. CONCLUSIONS: A transmaxillary and transmandibular approach allowed us to obtain a complete view of a large immovable chordoma located ventral to the upper cervical spine. This enabled us to resect totally the tumor into 2 pieces without major complications or sequelae. This approach is useful for the resection of large tumors located in the median upper cervical spine.

Immunohistochemical and electron microscopic study of invasion and differentiation in spinal cord lesion of neural stem cells grafted through cerebrospinal fluid in rat.

Neurospheres were obtained by culturing hippocampal cells from transgenic rat fetuses (E16) expressing green fluorescent protein (GFP). The neurosphere cells were injected into the cerebrospinal fluid (CSF) through the 4th ventricle of young rats (4 weeks old) that had been given a contusion injury at T8-9 of the spinal cord. The injected neural stem cells were transported through the CSF to the spinal cord, attached to the pial surface at the lesion, and invaded extensively into the spinal cord tissue as well as into the nerve roots. The grafted stem cells survived well in the host spinal cord for as long as 8 months after transplantation. Immunohistochemical study showed that many grafted stem cells had differentiated into astrocytes at 1-4 months, and some into oligodendrocytes at 8 months postoperatively. Immunoelectron microscopy showed that the grafted stem cells were well integrated into the host tissue, extending their processes around nerve fibers in the same manner as astrocytes. In addition, grafted stem cells within nerve roots closely surrounded myelinated fibers or were integrated into unmyelinated fiber bundles; those associated with myelinated fibers formed basal laminae on their free surface, whereas those associated with unmyelinated fibers were directly attached to axons and Schwann cells, indicating that grafted stem cells behaved like Schwann cells in the nerve roots.

Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord.

Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord-derived neurosphere cells by either cell mixing or making monolayered-feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered-feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting.