Hideaki Kinoshita

Distribution of elastic fibers in the head and neck: a histological study using late-stage human fetuses

There is little or no information about the distribution of elastic fibers in the human fetal head. We examined this issue in 15 late-stage fetuses (crown-rump length, 220-320 mm) using aldehyde-fuchsin and elastica-Masson staining, and we used the arterial wall elastic laminae and external ear cartilages as positive staining controls. The posterior pharyngeal wall, as well as the ligaments connecting the laryngeal cartilages, contained abundant elastic fibers. In contrast with the sphenomandibular ligament and the temporomandibular joint disk, in which elastic fibers were partly present, the discomalleolar ligament and the fascial structures around the pterygoid muscles did not have any elastic fibers. In addition, the posterior marginal fascia of the prestyloid space did contain such fibers. Notably, in the middle ear, elastic fibers accumulated along the tendons of the tensor tympani and stapedius muscles and in the joint capsules of the ear ossicle articulations. Elastic fibers were not seen in any other muscle tendons or vertebral facet capsules in the head and neck. Despite being composed of smooth muscle, the orbitalis muscle did not contain any elastic fibers. The elastic fibers in the sphenomandibular ligament seemed to correspond to an intermediate step of development between Meckels cartilage and the final ligament. Overall, there seemed to be a mini-version of elastic fiber distribution compared to that in adults and a different specific developmental pattern of connective tissues. The latter morphology might be a result of an adaptation to hypoxic conditions during development.

Influence of sulfide concentration on the corrosion behavior of titanium in a simulated oral environment

This study assessed the corrosion behavior of titanium in response to sulfide by determining the effects of sulfide concentration and pH over immersion period. Corrosion was evaluated through changes in color, glossiness, surface characterization, and titanium release. Sulfide solutions were prepared in 3 different concentrations with Na2S, each in pH unadjusted (sulfide-alkaline) and pH adjusted to 7.5 (sulfide-neutral). Titanium discoloration increased and glossiness decreased as sulfide concentration and immersion period increased in sulfide-alkaline solutions. Coral-like complexes were observed on the surface of these specimens, which became more pronounced as concentration increased. Small amounts of titanium release were detected in sulfide-alkaline solutions; however, this was not affected by immersion periods. Corrosion was indicated through considerable surface oxidation suggesting the formation of a thick oxide layer. No significant changes in color and glossiness, or titanium release were indicated for titanium specimens immersed in sulfide-neutral solutions indicating that pH had a significant effect on corrosion. Our findings suggest that a thick oxide layer on the titanium surface was formed in sulfide-alkaline solutions due to excessive oxidation.

Prevention of Lethal Murine Hypophosphatasia by Neonatal Ex Vivo Gene Therapy Using Lentivirally Transduced Bone Marrow Cells

Hypophosphatasia (HPP) is an inherited skeletal and dental disease caused by loss-of-function mutations in the gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). The major symptoms of severe forms of the disease are bone defects, respiratory insufficiency, and epileptic seizures. In 2015, enzyme replacement therapy (ERT) using recombinant bone-targeted TNALP with deca-aspartate (D10) motif was approved to treat pediatric HPP patients in Japan, Canada, and Europe. However, the ERT requires repeated subcutaneous administration of the enzyme because of the short half-life in serum. In the present study, we evaluated the feasibility of neonatal ex vivo gene therapy in TNALP knockout (Akp2(-/-)) HPP mice using lentivirally transduced bone marrow cells (BMC) expressing bone-targeted TNALP in which a D10 sequence was linked to the C-terminus of soluble TNALP (TNALP-D10). The Akp2(-/-) mice usually die within 20 days because of growth failure, epileptic seizures, and hypomineralization. However, an intravenous transplantation of BMC expressing TNALP-D10 (ALP-BMC) into neonatal Akp2(-/-) mice prolonged survival of the mice with improved bone mineralization compared with untransduced BMC-transplanted Akp2(-/-) mice. The treated Akp2(-/-) mice were normal in appearance and experienced no seizures during the experimental period. The lentivirally transduced BMC were efficiently engrafted in the recipient mice and supplied TNALP-D10 continuously at a therapeutic level for at least 3 months. Moreover, TNALP-D10 overexpression did not affect multilineage reconstitution in the recipient mice. The plasma ALP activity was sustained at high levels in the treated mice, and tissue ALP activity was selectively detected on bone surfaces, not in the kidneys or other organs. No ectopic calcification was observed in the ALP-BMC-treated mice. These results indicate that lentivirally transduced BMC can serve as a reservoir for stem cell-based ERT to rescue the Akp2(-/-) phenotype. Neonatal ex vivo gene therapy thus appears to be a possible treatment option for treating severe HPP.

Treatment of hypophosphatasia by muscle-directed expression of bone-targeted alkaline phosphatase via self-complementary AAV8 vector

Hypophosphatasia (HPP) is an inherited disease caused by genetic mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). This results in defects in bone and tooth mineralization. We recently demonstrated that TNALP-deficient (Akp2−/−) mice, which mimic the phenotype of the severe infantile form of HPP, can be treated by intravenous injection of a recombinant adeno-associated virus (rAAV) expressing bone-targeted TNALP with deca-aspartates at the C-terminus (TNALP-D10) driven by the tissue-nonspecific CAG promoter. To develop a safer and more clinically applicable transduction strategy for HPP gene therapy, we constructed a self-complementary type 8 AAV (scAAV8) vector that expresses TNALP-D10 via the muscle creatine kinase (MCK) promoter (scAAV8-MCK-TNALP-D10) and examined the efficacy of muscle-directed gene therapy. When scAAV8-MCK-TNALP-D10 was injected into the bilateral quadriceps of neonatal Akp2−/− mice, the treated mice grew well and survived for more than 3 months, with a healthy appearance and normal locomotion. Improved bone architecture, but limited elongation of the long bone, was demonstrated on X-ray images. Micro-CT analysis showed hypomineralization and abnormal architecture of the trabecular bone in the epiphysis. These results suggest that rAAV-mediated, muscle-specific expression of TNALP-D10 represents a safe and practical option to treat the severe infantile form of HPP.

The cricothyroid joint in elderly Japanese individuals

Using 15 cricothyroid joint (CT joint) specimens obtained from donated cadavers of elderly individuals, we examined the morphologies of the ceratocricoid ligaments as well as the synovial tissue. The ligaments consistently contained abundant elastic fibers: the fibers tended to be straight on the anterior side in contrast to a mesh-like arrangement on the posterior side. Thick and/or long synovial folds were often evident in the CT joint. The synovial tissue usually contained CD68-positive macrophages, but the positive cells were often restricted to certain parts of the tissue. Factor VIII-positive capillaries were present but few in number, and CD3- or IgM-positive lymphocytes were absent in the synovial tissue. Degenerative changes in the joint cartilage, such as roughness or thinning, were often present, but no cartilage defects were evident. Therefore, in contrast to the small, non-weight-bearing joints of the musculoskeletal system, we considered the degeneration of the CT joint to be a specific, silent form of osteoarthritis. For high-pitched phonation and ossification of the laryngeal cartilage, the CT joint in elderly individuals seemed to maintain its anterior gliding and rotation with the aid of elastic fiber-rich tissues compensating for the loss of congruity between the joint cartilage surfaces.

Stochastic Multi-Scale Finite Element Analysis of the Drilling Force of Trabecular Bone During Oral Implant Surgery

To avoid procedural accidents during/after oral implant surgery in a jawbone (e.g., perforation of the lingual side due to an inadequate drilling angle or scratching the mandibular canal), it is im...

Fetal development of the minor lung segment.

The mediobasal segment (S7) of the right lung has been considered to correspond to the cardiac lobe generally seen in mammals. To investigate fetal development of the right mediobasal segmental bronchus (B7), we examined paraffin-embedded serial sections of 15 embrynic and fetal lungs at 7-8 weeks (serial sections) as well as semiserial sections of 8 fetuses at 15-18 weeks (semiserial sections). All of the smaller specimens did not contain B7, but 2 of the 8 larger specimens carried B7: one was found in the immediately anterior side of the inferior pulmonary vein, while in the other, the subdivisions (B7a, B7b) were overriding the vein. Although the incidence might be underestimated because of observations using semiserial sections, the B7 was most likely to develop secondarily during a period from 8 to 15 weeks. Fetal topographical changes (mainly, the descent) of the middle lobe and the inferior pulmonary vein might relate with the secondarily budding of B7. The present result does not reduce a clinical relevance of B7 as a segmental bronchus of the lung segment system.

Effects of calcium hydroxide reagent on the bond strength of resin cements to root dentin and the retention force of FRC posts

The aim of this study was to determine the effects of calcium hydroxide (Ca(OH)2) treatment on bond strength of resin cements to root dentin and retention force of fiber-reinforced composite (FRC) posts. Bovine root dentin was endodontically prepared and treated with Ca(OH)2 for 7 days. Root dentin for bond strength test was adhered to resin-composite with resin cements. For pull-out test, posts consisting of FRC posts and resin-composites were fabricated and cemented to root. Shear bond and pull-out tests were performed using a universal testing machine. No significant differences in bond strength and post retention force were found between Ca(OH)2 treated and untreated groups. Significant differences were found among the cements. A positive correlation was indicated between bond strength of cements and retention force of FRC posts. In conclusion, Ca(OH)2 treatment on root dentin did not affect bond strength of resin cements and retention force of FRC posts.

707. Prolonged Survival and Improved Phenotypes of Lethal Hypophosphatasia Model Mice By Adeno-Associated Virus-Mediated Muscle Transduction of Bone-Targeted Alkaline Phosphatase

Muscle directed transduction was demonstrated after the injection of scAAV8-MCK-EGFP. The plasma ALP activity was elevated and sustained at therapeutic level, (>1.0 unit/ml) for at least 3 months of scAAV8-MCK-TNALP-D10 treated Akp2–/– mice. The treated Akp2–/– mice grew well and survived over 3 months with healthy appearance (9/10), while untreated Akp2 −/- mice died within 3 weeks (n=10; P<0.001). Improved mineralization of the knee joints was demonstrated by X-ray images and micro CT analysis. Ectopic calcification under this condition was not detected in the treated mice. These results suggest that rAAV-mediated muscle TNALP-D10 transduction strategy would be promising to treat the severe infantile form of HPP.