Kazuya Masuno

Detection of HPV in Japanese and Chinese oral carcinomas by in situ PCR

Human papillomavirus (HPV) is established as the cause of almost 100% of cervical carcinomas. However, the association of HPV with oral squamous cell carcinomas (SCCs) is less well understood. We examined the prevalence of HPV in oral SCCs in samples of Japanese and Chinese populations. Using in situ polymerase chain reaction (PCR) analysis (MY09 and MY11 consensus primers), HPV was detected in the nucleus of epithelia and tumor cells in oral lesions. Analysis revealed the specific presence of HPV DNA in all cases of SCC in our Japanese (10/10) and Chinese (10/10) population samples. These results suggest that HPV infection could be one of several risk factors contributing to oral SCC in Japanese and Chinese.

Bone repair analysis in a novel biodegradable hydroxyapatite/collagen composite implanted in bone.

The purpose of this study was to evaluate a biodegradable hydroxyapatite/collagen composite and to examine the use of the calcium ion contained for bone formation and growth. Surgical holes were prepared in the femora and tibiae of beagle dogs, and were filled with the hydroxyapatite/collagen composite labeled with alizarin red. After 4 weeks, calcein was administered to the experimental dogs. After 1 additional week, the femora and tibiae were removed surgically and fixed in formalin. Light microscopy and confocal laser scanning microscopy were used to examine the surgical holes with their implanted materials and the surrounding bone. There were only a few inflammatory cells adjacent to the hydroxyapatite/collagen composite. The newly formed bone in the cortical bone was stained with calcein, which binds to serum calcium, and new bone near the hydroxyapatite/collagen composite in the holes was stained positive for alizarin red, which binds tothe calcium in the hydroxyapatite/collagen composite. In addition, osteoblasts near the hydroxyapatite/collagen composite as well as newly formed bone adjacent to the osteoblasts showed alizarin red staining, but the new bone at a distance from the hydroxyapatite/collagen implant reacted only to calcein staining. These results, using the tissue labeling method with calcein and alizarin red, suggested that the calcium bound to the alizarin red released from the hydroxyapatite/collagen composite materials might have been translocated to sites of new bone formation. The present experiment showed that the novel hydroxyapatite/collagen composite is a useful implant material for bone augmentation and that the calcium in the newly formed bone might have been released from the implant.

Calcification at the Interface Between Titanium Implants and Bone: Observation With Confocal Laser Scanning Microscopy

It has not been previously possible to observe bone formation in undecalcified sections with titanium implants at high magnification because of the difficulty in sectioning bone together with implants. A method for examining the bone-implant interface in undecalcified sections is described in which implants are left in situ and confocal laser scanning microscopy (CLSM) is used to examine both the implant surface and adjacent bone. Pulsing of animals at different times with the fluorescent dyes calcein and alizarin red permitted assessment of temporal patterns of bone formation by CLSM. Reflectivity of the polished implant surface permitted accurate assessment of the position of the implant relative to labeled bone. The analysis showed that bone first formed as thin processes towards and across the implant surface, followed by further bone formation behind these processes. The interface between calcified bone tissue and the implant surface was characterized by a 10-microm space. The CLSM technique enabled detailed observations of new bone formation at the titanium implant interface.

Salivary protein histatin 3 regulates cell proliferation by enhancing p27(Kip1) and heat shock cognate protein 70 ubiquitination.

Histatins are salivary proteins with antimicrobial activities. We previously reported that histatin 3 binds to heat shock cognate protein 70 (HSC70), which is constitutively expressed, and induces DNA synthesis stimulation and promotes human gingival fibroblast (HGF) survival. However, the underlying mechanisms of histatin 3 remain largely unknown. Here, we found that the KRHH sequence of histatin 3 at the amino acid positions 5-8 was essential for enhancing p27(Kip1) (a cyclin-dependent kinase inhibitor) binding to HSC70 that occurred in a dose-dependent manner; histatin 3 enhanced the binding between p27(Kip1) and HSC70 during the G1/S transition of HGFs as opposed to histatin 3-M(5-8) (substitution of KRHH for EEDD in histatin 3). Histatin 3, but not histatin 3-M(5-8), stimulated DNA synthesis and promoted HGF survival. Histatin 3 dose-dependently enhanced both p27(Kip1) and HSC70 ubiquitination, whereas histatin 3-M(5-8) did not. These findings provide further evidence that histatin 3 may be involved in the regulation of cell proliferation, particularly during G1/S transition, via the ubiquitin-proteasome system of p27(Kip1) and HSC70.

The Effect of Ozone on Collagen Type-1 and Inflammatory Cytokine Production in Human Gingival Fibroblasts

Ozone is currently being considered as a possible oral antiseptic agent because it is strongly antimicrobial and does not induce microbial resistance. In the article, we examined the effects of ozone exposure on the production of collagen type-1 and inflammatory cytokines in primary human gingival fibroblasts (HGFs) in vitro using enzyme-linked immunosorbent assays. The addition of 0.5 ppm ozone significantly enhanced collagen type-1 production by HGFs within 24 h. Secretion of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 by HGFs treated with lipopolysaccharide decreased when ozone was present in the medium. Together, these results suggest that clinical use of ozone would facilitate the positive balance between HGF-mediated periodontal tissue maintenance and repair and the stimulation of inflammation and tissue degeneration following exposure to microbial pathogens.