Nobuyuki Shimohata

Trehalose treatment suppresses inflammation, oxidative stress, and vasospasm induced by experimental subarachnoid hemorrhage

BackgroundSubarachnoid hemorrhage (SAH) frequently results in several complications, including cerebral vasospasm, associated with high mortality. Although cerebral vasospasm is a major cause of brain damages after SAH, other factors such as inflammatory responses and oxidative stress also contribute to high mortality after SAH. Trehalose is a non-reducing disaccharide in which two glucose units are linked by α,α-1,1-glycosidic bond, and has been shown to induce tolerance to a variety of stressors in numerous organisms. In the present study, we investigated the effect of trehalose on cerebral vasospasm, inflammatory responses, and oxidative stress induced by blood in vitro and in vivo.MethodsEnzyme immunoassay for eicosanoids, pro-inflammatory cytokines, and endothelin-1, and western blotting analysis for cyclooxygenase-2, inducible nitric oxide synthase, and inhibitor of NF-κB were examined in macrophage-like cells treated with hemolysate. After treatment with hemolysate and hydrogen peroxide, the levels of lipid peroxide and amounts of arachidonic acid release were also analyzed. Three hours after the onset of experimental SAH, 18 Japanese White rabbits received an injection of saline, trehalose, or maltose into the cisterna magna. Angiographic and histological analyses of the basilar arteries were performed. In a separate study, the femoral arteries from 60 rats were exposed to fresh autologous blood. At 1, 3, 5, 7, 10, and 20 days after treatment, cryosections prepared from the femoral arteries were histologically analyzed.ResultsWhen cells were treated with hemolysate, trehalose inhibited the production of several inflammatory mediators and degradation of the inhibitor of NF-κB and also suppressed the lipid peroxidation, the reactive oxygen species-induced arachidonic acid release in vitro. In the rabbit model, trehalose produced an inhibitory effect on vasospasm after the onset of experimental SAH, while maltose had only a moderate effect. When the rat femoral arteries exposed to blood were investigated for 20 days, histological analysis revealed that trehalose suppressed vasospasm, inflammatory response, and lipid peroxidation.ConclusionsThese data suggest that trehalose has suppressive effects on several pathological events after SAH, including vasospasm, inflammatory responses, and lipid peroxidation. Trehalose may be a new therapeutic approach for treatment of complications after SAH.

Tenomodulin expression in the periodontal ligament enhances cellular adhesion.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.

Bone healing by sterilizable calcium phosphate tetrapods eluting osteogenic molecules

Although bone grafts and prosthetic implants have shown some clinical success in the treatment of bone defects, the graft availability, biocompatibility, function, and longevity still remain to be improved. One possible solution to these problems is to develop bone implants acting on host cells to induce rapid bone regeneration. Here, we demonstrate bone healing by means of a sterilizable and osteogenic molecule-eluting implant system in which two small molecules, a smoothened agonist (SAG) and a helioxanthin derivative (TH), are loaded onto tetrapod-shaped calcium phosphate granules (Tetrabone). We succeeded in directing progenitor cells toward mature osteoblasts with the combined application of the two small molecules acting on different stages of osteogenesis. Tetrabone released SAG and TH for prolonged periods when loaded with these molecules. EOG sterilization did not affect the osteogenic activity of the SAG- and TH-loaded Tetrabones. The combinatorial use of SAG- and TH-loaded Tetrabones achieved bone healing without cell transplantation in a rat femur bone defect model within two weeks. This system will allow us to vary the combination rate of implants loaded with different osteogenic factors depending on the types and sizes of defects, potentially allowing full temporal and spatial control of the bone regeneration.

Trehalose inhibits oral dryness by protecting the cell membrane

This study assessed the clinical efficacy and acceptability of trehalose solution for oral dryness caused by dental treatment. The efficacy of trehalose on oral dryness under drying conditions was assessed by measuring the surface area of the fungiform papillae and the moisture content of the tongue in seven healthy volunteers. Based on the data from this pilot study, a clinical study was performed, in which the efficacy of oral trehalose spray was evaluated on oral dryness in 10 patients undergoing root canal treatment. The effects of trehalose on cell viability were also assessed under drying conditions in vitro. Trehalose suppressed oral dryness and associated pain caused by dental treatment and protected cells from dryness-related damage. These results indicate that pretreatment application of trehalose solution on the oral mucosa is effective in preventing oral dryness caused by dental treatment.

Repair of rabbit segmental femoral defects by using a combination of tetrapod-shaped calcium phosphate granules and basic fibroblast growth factor-binding ion complex gel.

The effect of tetrapod-shaped alpha tricalcium phosphate granules (TB) as a scaffold combined with basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) on neovascularization and bone regeneration was evaluated in segmental femoral defects of rabbits. The defects were stabilized using a plate with a polypropylene mesh cage (PMC) containing one of the following: PMC alone (PMC group), TB (TB group), TB and bFGF (TB/f group), TB and IC gel (TB/IC group), or TB and f-IC gel (TB/f-IC group). Four rabbits from each group were euthanized at 2 and 4 weeks after surgery. Histomorphometry showed that the number of vessels and the volume of new bone in the TB/f-IC group were significantly higher than those in the other groups at all time points. There were no differences in the extent of neovascularization and new bone formation between the TB and TB/f groups. These findings suggest that the combination of TB and f-IC gel facilitated both neovascularization and new bone formation in segmental femoral defects of rabbits. This combination may be of considerable use for treating segmental long bone defects.

Bone Regeneration by the Combined Use of Tetrapod-Shaped Calcium Phosphate Granules with Basic Fibroblast Growth Factor-Binding Ion Complex Gel in Canine Segmental Radial Defects

ABSTRACT The effect of tetrapod-shaped alpha tricalcium phosphate granules (Tetrabones® [TB]) in combination with basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) on bone defect repair was examined. Bilateral segmental defects 20-mm long were created in the radius of 5 dogs, stabilized with a plate and screws and implanted with 1 of the following: TB (TB group), TB and bFGF solution (TB/f group), and TB and f-IC gel (TB/f-IC group). Dogs were euthanized 4 weeks after surgery. Radiographs showed well-placed TB granules in the defects and equal osseous callus formation in all the groups. Histomorphometry revealed that the number of vessels and volume of new bone in the TB/f-IC group were significantly higher than those in the other groups. However, no significant differences in neovascularization and new bone formation were observed between the TB/f and TB groups. Furthermore, no significant difference in the lamellar bone volume or rate of mineral apposition was observed among groups. These results suggest that increased bone formation might have been because of the promotion of neovascularization by the f-IC gel. Therefore, the combinatorial method may provide a suitable scaffold for bone regeneration in large segmental long bone defects.

Dermal cell damage induced by topical application of non-steroidal anti-inflammatory drugs is suppressed by trehalose co-lyophilization in ex vivo analysis.

ABSTRACT Topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) is generally considered safer than oral administration, although the former can occasionally induce cutaneous irritation. We hypothesized that the cutaneous irritation by topical NSAIDs might be suppressed by trehalose, which has protective effects on biological membranes. Using the three-dimensional cultured human skin model, Living Skin Equivalent-high, we found that cutaneous damage due to NSAIDs was reduced by concomitant use of trehalose and that this effect of trehalose was reinforced by co-lyophilization of NSAIDs with trehalose. The anti-inflammatory effect of co-lyophilized NSAIDs with trehalose was comparable to that seen with NSAIDs alone in a rat model. Our results suggest that co-lyophilization of NSAIDs with trehalose might be a novel procedure that can help prevent NSAIDs-induced skin irritation.

Prevention of intubation-induced mucosal damage using a tube coated with 2-methacryloyloxyethyl phosphorylcholine polymer.

Background and objective Tracheal intubation is associated with various complications that include epithelial injury. Abrasion of the fragile tracheal epithelium can occur at the points of contact between the tube and mucosa subject to respiratory movement. In this original experiment, we examined the mucosal protective effect of coating endotracheal tubes with poly[2-methacryloyloxyethl phosphorylcholine (MPC)-co-n-butyl methacrylate] (PMB). Methods We prepared four types of tubes: tube A (control, no coating), tube B (two coats, 0.5% PMB), tube C (10 coats, 0.5% PMB) or tube D (one coat, 5% PMB). Twenty-nine beagle dogs were divided into four groups and orally intubated with tube A, B, C or D for 4 ± 0.5 h. The cuffs of extubated tubes were stained with haematoxylin. Paraffin sections from tracheal walls in contact with the inflated cuff were stained with haematoxylin/eosin and periodic acid-Schiff. Results Cuffs of tubes A and B were strongly stained with haematoxylin because of attached epithelial cells. Stained areas in those of tubes C and D were significantly reduced. Histological analysis showed that a single coat of 5% PMB prevented epithelial abrasion and proliferation of goblet cells. Excess tracheal mucus was observed in the tube A group, but not in the tube D group. Conclusion Tracheal epithelial damage caused by intubation was greatly reduced or eliminated by PMB coating on the surface of the tracheal tube.