Takao Kohgo

Early bone formation around calcium‐ion‐implanted titanium inserted into rat tibia

Rat tibia tissue into which calcium ion (Ca2+)-implanted titanium was surgically placed was histologically analyzed to investigate the performance of the Ca(2+)-implanted titanium as a biomaterial. Calcium ions were implanted into only one side of titanium plates at 10(17) ions/cm2 and the Ca(2+)-treated titanium was surgically implanted into rat tibia for 2, 8, and 18 days. Tetracycline and calcein were used as hard-tissue labels. After excision of the tibia, the tissues were fixed, stained, embedded in polymethyl methacrylate, and sliced. The specimens were observed using a fluorescence microscope. A larger amount of new bone was formed on the Ca(2+)-treated side than on the untreated side, even at 2 days after surgery. In addition, part of the bone made contact with the Ca2(+)-treated surface. On the other hand, bone formation on the untreated side was delayed and the bone did not make contact with the surface. Mature bone with bone marrow formed in 8 days. Neither macrophage nor inflammatory cell infiltration was observed. The results indicated that Ca(2+)-implanted titanium is superior to titanium alone for bone conduction.

Development of calcium phosphate cement using chitosan and citric acid for bone substitute materials.

We developed a calcium phosphate cement that could be molded into any desired shape due to its chewing-gum-like consistency after mixing. The powder component of the cement consists of alpha-tricalcium phosphate and tetracalcium phosphate, which were made by decomposition of hydroxyapatite ceramic blocks. The liquid component consists of citric acid, chitosan and glucose solution. In this study, we used 20% citric acid (group 20) and 45% citric acid (group 45). The mechanical properties and biocompatibility of this new cement were investigated. The setting times of cements were 5.5 min, in group 20 and 6.4 min, in group 45. When incubated in physiological saline, the cements were transformed to hydroxyapatite at 3, and 6 weeks, the compressive strengths were 15.6 and 20.7 MPa, in group 45 and group 20, respectively. The inflammatory response around the cement implanted on the bone and in the subcutaneous tissue in rats was more prominent in group 45 than in group 20 at 1 week after surgery. After 4 weeks, the inflammation disappeared and the cement had bound to bone in both groups. These results indicate that this new calcium phosphate cement is a suitable bone substitute material and that the concentration of citric acid in the liquid component affects its mechanical properties and biocompatibility.

Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation

Three different types of porous hydroxyapatite with pore sizes of 100-200 micrometer in diameter-porous particles of hydroxyapatite (PPHAP), porous blocks of hydroxyapatite (PBHAP), and honeycomb-shaped hydroxyapatite (HCHAP)-were compared in terms of their abilities to induce osteogenesis when implanted subcutaneously with recombinant human BMP-2 into rats and extracted at 1, 2, 3, and 4 weeks. Histologically, direct bone formation occurred in PPHAP and PBHAP while only endochondral ossification took place in HCHAP. Interestingly, cartilage in the central zones and bone in the orifice zones of the tunnels of the HCHAP were observed at 2 weeks. After 3 weeks, the cartilage disappeared and bone formation occurred throughout the inner surface of the tunnels of the HCHAP, always leaving space for capillaries within the tunnels. Alkaline phosphatase activity and osteocalcin content were the highest in HCHAP among the three hydroxyapatite implants. These results clearly indicate that BMP-induced bone formation is highly dependent on the geometry of the carrier, which provides feasible structural factors for vascularization.

Detection of Human Papillomavirus DNA Sequences in Oral Squamous Cell Carcinomas and Their Relation to p53 and Proliferating Cell Nuclear Antigen Expression

Background. The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site.

Bone augmentation osteogenesis using hydroxyapatite and β-tricalcium phosphate blocks

PURPOSE In this study, we investigated the differences in osteogenesis and resorption between hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) implanted on the parietal bone of rats. MATERIALS AND METHODS HA and beta-TCP were used in blocks with macropores and micropores. They were implanted between the parietal bone and the cranial periosteum in rats. Osteogenesis around the implanted materials was investigated histopathologically and histomorphometrically at 1, 2, 4, 8, and 24 weeks after surgery. RESULTS At 2 weeks, osteogenesis from the parietal bone was observed around both materials, and new bone had attached directly to the surfaces of both materials. New bone grew into the pores of the upper regions of both materials with time. The beta-TCP block had a characteristic basophilic reticular structure in which the dissolution of the materials was observed close to the new bone. The HA blocks were stable for 24 weeks, whereas parts of the beta-TCP blocks were fractured and resorbed at 24 weeks. Histomorphometrically, the volume of new bone around HA was larger than that around beta-TCP. There was no remarkable change in the amount of remaining HA, but that of beta-TCP was decreased. CONCLUSION HA blocks in this model are suitable for onlay grafts because of its stability and osteogenesis, beta-TCP is not stable. Therefore, when beta-TCP blocks are used for onlay grafts, the mechanical stress on the recipient site should be taken into consideration because of resorption and fracture.

Ultrastructure of ceramic-bone interface using hydroxyapatite and β-tricalcium phosphate ceramics and replacement mechanism of β-tricalcium phosphate in bone

Abstract Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) are useful for grafting and augmentation of bone tissue. Observation by transmission electron microscopy (TEM) was done to investigate the ultrastructures at the interfaces between the biomaterials and the adjacent tissue, and osteogenesis around the biomaterials in the present study. HA and β-TCP ceramics were used in disk forms which had macropores and micropores, and were implanted between the parietal bone and the cranial periosteum of rats. Specimens were prepared for observation at 4 and 8 weeks postoperatively. The microscopic results indicated that an intervening layer was present on the surface of HA, whereas it was not present on the surface of β-TCP. A characteristic fibrillar structure was observed in the intervening layer between HA and bone under decalcification by HCl. In β-TCP, in reticular structures observed close to the bone tissue by optical microscopy, calcification and sparse collagen fibers were interspersed among the granules of β-TCP. In addition, close to the interface between β-TCP and bone, many osteocytes with numerous processes were present. Some processes were elongated towards the interface. These results revealed the difference in the ultrastructures of the interfaces between HA and β-TCP, and the dissolution mechanism of β-TCP in bone.

nm23-H1 Suppresses Invasion of Oral Squamous Cell Carcinoma-Derived Cell Lines without Modifying Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 Expression

nm23-H1 is a candidate gene for the suppression of cancer metastasis. Several studies on human breast, hepatocellular, gastric, ovarian, and colon carcinomas and melanomas have shown that reduced nm23-H1 expression was closely related to metastatic progression with poor prognosis. However, the biochemical mechanism by which nm23-H1 suppresses the metastasis has yet to be elucidated. In this study, we analyzed the correlation between nm23 expression, cell motility, and the invasive abilities of six different oral squamous cell carcinoma cell lines (HSC2, HSC3, HSC4, KB, OSC19, and OSC20). Reduced mRNA/protein expression of the nm23-H1 was observed in three cell lines (HSC2, HSC3, and HSC4). These cell lines exhibited increased cell motility and an invasive character on organotypic raft culture. On the other hand, the cell lines (KB, OSC19, and OSC20) that showed a higher expression of nm23-H1 exhibited a threefold to fivefold reduced motility and also reflected fewer invasions compared to the former three cell lines. Because the HSC3 cells demonstrated the lowest nm23-H1 expression with the highest cell motility and invasive character, we established nm23-H1-transfected HSC3 cell lines to investigate whether exogenous nm23-H1 protein could inhibit cell migration and invasive activity. These transfectants showed a significant reduction in cell motility with exogenous nm23-H1 in a dose-dependent manner, and exhibited a noninvasive character. An immunofluorescence study demonstrated a distinct stress-fiber distribution at peripheral region of these transfectants. However, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 expression was observed between mock transfectant and nm23-H1-transfected cells. These findings suggest that nm23-H1 inhibits the invasive activity of oral squamous cell carcinoma by suppression of cell motility without altering the MMP-2 and MMP-9 status.

Expression of E1AF, an ets-family transcription factor, is correlated with the invasive phenotype of oral squamous cell carcinoma

E1AF is a newly identified ets-oncogene family transcription factor. Previous reports have noted that E1AF can upregulate promoter activities of several matrix metalloproteinase (MMP) genes and showed that invasive potentials of oral squamous cell carcinoma-derived cell lines are correlated with expression of E1AF and MMPs. The invasive phenotype is restrained by transfection with an antisense E1AF expression vector. Thus, E1AF is thought to be highly correlated with malignant potentials of cancer cells. However, little is known about E1AF expression and cancer cell malignancies in in vivo tumours. In the present study, 27 oral squamous cell carcinoma (SCC) specimens were examined using RT-PCR, Southern blot hybridisation and in situ hybridisation (ISH) and compared to the clinicopathological parameters. Among the 27 patients, E1AF was detected in 15 cases. E1AF mRNA was detected in 13 of 17 invasive SCCs, whereas the majority of SCCs not expressing E1AF showed an expansive growth pattern. Increased prevalence of E1AF-positive oral SCC was observed in cases with nodal metastasis. These results indicate that E1AF may be involved in cancer cell malignancies through its ability to promote invasive potential.

Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism

E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

BAG-1 expression correlates highly with the malignant potential in early lesions (T1 and T2) of oral squamous cell carcinoma

BAG-1 is a Bcl-2-binding protein that functions as an anti-apoptotic molecule. In this report we show a possible correlation between BAG-1 expression levels and the probability of oral squamous cell carcinoma (SCC) progression. We investigated BAG-1 expression levels in 22 patients diagnosed with early lesions (T1 and T2) of oral SCCs using immunohistochemistry and western blotting. High steady-state levels of BAG-1 were detected in 13 out of 22 cases (59%). High BAG-1 expression was observed more frequently in cases with nodal metastasis (89%) than in those without nodal metastasis (38%) (P<0. 03), suggesting that BAG-1 expression levels may correlate with the pathological stage of oral SCCs. Furthermore, BAG-1 expression levels correlated with the WHO grade, i.e. 45% in grade-I cases as opposed to 72% in grade-II cases (P<0.02). These data suggest that an analysis of BAG-1 expression may be useful in establishing a prognosis for patients with oral SCCs, and especially in predicting the metastatic potential of SSCs.