Blood permeability of a novel ceramic scaffold for bone morphogenetic protein-2.

A functionally graded apatite (fg-HAp) with body fluid permeability was developed from bovine bone. The tissue reaction of fg-HAp and its efficacy as a scaffold for recombinant human bone morphogenetic protein-2 (BMP-2) were evaluated histomorphometrically, and a component of permeable fluid into the fg-HAp was analyzed by immunoblotting assay. The fg-HAp block (27 mm(3)) combined with and without BMP-2 (5 microg) was implanted subcutaneously in 4-week-old Wistar rats. Histological examination showed that the surface and bulk degradations of the fg-HAp proceeded extensively and giant cells appeared on the fg-HAp at 2 weeks. Body fluid permeation was found inside the fg-HAp, and the fluid component was immunopositive for albumin. In addition, albumin was detected as a main component among proteins collected from the in vivo implanted fg-HAp. The bioabsorption of the fg-HAp was accelerated as BMP-2-induced bone matured. Histomorphometrical analysis at 4 weeks in the BMP-2/fg-HAp implant showed 59.0% in the total volume of bone and marrow. These results indicate that fg-HAp is an innovative, bioabsorbable bioceramic with fluid permeability characteristic, and may become a biointegrated scaffold for bone engineering.

Treatment of habitual temporomandibular joint dislocation with miniplate eminoplasty: a report of nine cases.

Eminoplasty using T-shaped titanium miniplate was performed on 15 joints in nine patients suffering from recurrent dislocation of the temporomandibular joint with several general complications. After the conventional pre-auricular approach to the zygomatic arch and eminence, the bent over miniplate was inserted anteriorly against the articular eminence and fixed to the zygomatic arch with miniscrews to limit the over-movement of the condyle. In one case, the miniplate fractured, but no recurrence of dislocation was observed. In another case, it was possible to evaluate the mandibular movement by Sirognathograph analysis, which proved satisfactory function of the joints.

Bone induction in subcutaneous tissue in rats by a newly developed DNA-coated atelocollagen and bone morphogenetic protein.

A unique biomaterial, a mixture of DNA and collagen (DNA/collagen), was developed and its efficacy as a carrier matrix for bone morphogenetic protein (BMP) was evaluated histologically. The material was prepared as a composite of DNA from salmon milt and pepsin-digested type I collagen (atelocollagen) from bovine dermis. Phase-contrast and fluorescence microscopy showed atelocollagen fibres with DNA coating. The dose-response and time-course of bone induction by BMP in DNA/collagen (5 x 10 x 1 mm) in the subcutaneous tissue was investigated in 20 male Wistar rats. The BMP/DNA/collagen induced new bone in a dose-dependent manner (0, 25, 50 or 100 micrograms of BMP). Histological examination in the time-course study showed that the BMP (100 micrograms)/DNA/collagen induced bone formation, while the DNA/collagen alone resulted in the accumulation of fibroblasts. These results indicate that the DNA/collagen is effective as a carrier matrix for BMP. It provides a cell anchorage for differentiation of osteoblasts and is absorbed as bone matures.

Immunohistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin.

In the present study, we investigated the localization of amelogenin in odontogenic tumors, using an anti-amelogenin polyclonal antibody. In order to make the antibody, antisera against an amelogenin fraction obtained from the enamel matrix of unerupted bovine tooth was raised in rabbits. By Western blot analysis, a main band of 25 kDa and six minor bands (6.8, 12, 18, 20, 23, and 27 kDa) were detected under nonreducing conditions. Immunoreactivity for the amelogenin was observed in ameloblasts and in the immature enamel matrix of 4-day-old rats. In odontogenic tumors, positive reactions for amelogenin were localized in limited areas in adenomatoid odontogenic tumor, calcifying odontogenic cyst, primary intraosseous carcinoma and odontoma. The strongest immunoreactions were shown in enamel matrices in odontomas. Small mineralized foci in epithelial nests showed positive reactions, and a few reactions were observed in epithelium adjacent to the mineralized foci. In calcifying odontogenic cysts, some ghost cells in the lining epithelium were strongly stained. The results indicate that the present antibody for amelogenin is useful for the determination of odontogenic tumors, especially in those in which small mineralized foci are present in the epithelial nests.

Inhibitory effects of malotilate on in vitro invasion of lung endothelial cell monolayer by human oral squamous cell carcinoma cells.

We have previously reported that malotilate (MT) inhibited the invasion and metastasis of rat mammary carcinoma cells through the modification of host endothelial cells. In this study, we examined the inhibitory effects of MT on invasion of human cancer, using five oral squamous cell carcinoma cells (SAS, Ca9-22 and HSC-2, -3 and -4). MT did not affect the growth of these tumor cells and the invasion of reconstituted basement membrane, Matrigel. In an in vitro invasion assay using rat lung endothelial (RLE) cells, invasion of tumor cells which had been treated with MT (10 ng/ml, 24 h) was not affected; however, when RLE cells had been treated with MT, invasion was significantly inhibited in three cell lines (SAS, Ca9-22 and HSC-4) and a tendency to inhibition was also observed in other cell lines. Electron-microscopical examination of the RLE monolayer treated with MT (MT-RLE) showed the development of gap and tight junction-like structures. Subsequently, junction-associated proteins, connexin 43, zonula occludin and desmoglein, were examined by Western blotting. Protein levels of connexin 43 and zonula occludin were elevated dose dependently, and connexin 43 was chronologically enhanced by MT whereas desmoglein was not. The enhanced gap junctional communication of MT-RLE cells was observed in the scrape-loading assay using lucifer yellow CH. These results suggest that MT promotes the development of cell-to-cell adhesion, e. g. gap junction and tight junction in endothelial cells, resulting in the inhibition of invasion by the tumor cells.

Bone augmentation by onlay implant using recombinant human BMP-2 and collagen on adult rat skull without periosteum.

The purpose of this study was to determine whether bone augmentation could be obtained by the composite of recombinant human bone morphogenetic protein-2 (rhBMP-2) and bioabsorbable atelocollagen when the periosteum was resected, and to compare the efficacy of the rhBMP-2/collagen implant and the collagen alone implant. The onlay implant was inserted into the space between the elevated galea aponeurotica and the skull without the periosteum of 10-month-old rats. The rhBMP-2/collagen implant resulted in osteoblasts differentiation under the galea at 1 week and active bone formation without a prior formation of cartilage. At 4 weeks, the bony trabeculae were interconnected and connected directly with the compact bone of the skull. Histomorphometric analysis at 4 weeks demonstrated that the rhBMP-2/collagen implant showed 92.5% in the volume of bone tissue, whereas the collagen alone showed 0%. The implanted collagen was gradually replaced by bone tissue in the presence of rhBMP-2. Our present results indicate that rhBMP-2 stimulates undifferentiated mesenchymal cells in the galea overlying the implant to proliferate and differentiate directly into osteoblasts on the carrier collagen fibers. The collagen matrix was stably placed on the skull and suitable as a substitute for rhBMP-2. The rhBMP-2/collagen onlay implant might be clinically applicable for bone augmentation even under the condition without the periosteum.

Bone augmentation by recombinant human BMP-2 and collagen on adult rat parietal bone.

A composite of recombinant human bone morphogenetic protein-2 (rhBMP-2) and collagen was implanted beneath the cranial periosteum of 10-month-old rats to observe bone development and absorbent change of carrier collagen. The rhBMP-2/collagen onlay implant resulted in active bone formation and the augmented bone was connected directly with the original bone, whereas the collagen alone resulted in neither bone nor cartilage. The ossification process in the rhBMP-2/collagen occurred directly through bone formation, similar to intramembranous ossification. The carrier collagen fibers were found in the woven bone and were completely absorbed at 8 weeks in the presence of rhBMP-2, while the collagen alone implant remained encapsulated by a thin, fibrous connective tissue. Our results indicate that rhBMP-2/collagen is an effective material as a biological onlay implant, showing osteoinductive properties and being completely replaced by new bone. Carrier collagen not only plays a role in rhBMP-2 delivery, but also provides a cell anchorage for cell differentiation and remains as an artificial matrix in woven bone.

Bilirubin pigmentation of human teeth caused by hyperbilirubinemia.

This study was conducted to identify bilirubin in deciduous teeth obtained from two patients with a history of severe liver dysfunction. Teeth were histologically analyzed and bilirubin was extracted and quantified spectrophotometrically. Histological analysis revealed a green line in the dentine running parallel to the incremental lines. A chloroform/methanol/acetic acid (30:10:0.5, v/v) extract of the teeth was evaporated and the residue dissolved in chloroform. Absorption spectra were prepared before and after the diazo reaction. The absorption maximum shifted from 450 nm before to 540 nm after the diazo reaction and was higher than that of normal deciduous teeth. These results indicate that the discolouration of teeth in patients with severe liver dysfunction is due to bilirubin deposition.

Unsaturated fatty acid feeding prevents the development of acute hepatitis in Long-Evans cinnamon (LEC) rats.

To investigate the effects of dietary alpha-linolenic acid (18:3, n-3; alpha-LNA) and linoleic acid (18:2, n-6; LA) on the development of hereditary hepatitis, we compared incidences and grades of acute hepatitis between the Long-Evans cinnamon (LEC) rats fed with safflower oil-supplemented diet and perilla oil-supplemented diet. Both safflower and perilla oil supplemented diets reduced the incidence of hepatitis and significantly prolonged its onset as compared to the non-supplemented conventional diet. No significant difference was observed between safflower and perilla oil diets in the rats of incidence of hepatitis. At the age of 16 weeks, just before the onset of hepatitis, serum levels of transaminase (AST, ALT) and concentration of copper in rats fed with both test diets were significantly reduced as compared with that of rats fed alpha-linolenate and linoleate have an inhibitory effect on the development of hepatitis in LEC rats due to the prevention of serum copper elevation.

Carrier-dependency of cellular differentiation induced by bone morphogenetic protein in ectopic sites.

Partially purified bone morphogenetic protein (BMP) was delivered into two different types of carriers, porous particles of hydroxyapatite (PPHAP) and particles of insoluble bone matrix (IBM), and the ossification process was examined after subcutaneous implantation of the BMP/PPHAP and BMP/IBM in rats. The ossification in the BMP/PPHAP system was predominantly direct through bone formation similar to intramembranous ossification, whereas in the BMP/IBM system it was predominantly endochondral. The differences observed between the BMP/PPHAP and the BMP/IBM indicate the importance of the structure and nature of the carrier in the process of bone induction. The findings suggest that bone and cartilage differentiation is controlled not only by the regulation factor (BMP), but also by its interaction with the carrier, and that the BMP-induced cell differentiation is dependent upon the microenvironment derived from the carrier.

Basaloid-squamous cell carcinoma of the oral mucosa: report of two cases and study of the proliferative activity.

Two cases of basaloid-squamous cell carcinoma (BSC) of the oral mucosa are described. The first case occurred at the floor of the mouth in a 58-year-old man, and the second case occurred at the mandibular gingiva in a 79-year-old woman. The laboratory data of the first case showed a positive response to hepatitis C virus antibody. In the first case, the tumor mass measured 4 x 4 cm in size, and was located at the lingual side of the median mandible beside the sublingual gland. In the second case, the tumor mass measured 25 x 15 mm in size, and was located in the alveolar mucosa of the right mandible. Histologically, both tumors showed a neoplastic epithelium arranged in a solid pattern with evidence of peripheral palisading, central necrosis, and some squamous differentiation. The proliferative activities of the BSC were compared with conventional squamous cell carcinomas (SCC) in the oral floor and gingiva, respectively, by employing a sensitive argyrophilic nuclear organizer region (AgNOR) staining method. The number of AgNOR per nucleus of the BSC was higher than that of any other SCC cases. The results support the opinion that BSC of the oral mucosa has a worse prognosis than conventional SCC.

Immunological analysis of enhanced spontaneous metastasis in WKA rats following cryosurgery.

We have previously reported that inhibition of anti-tumor immune responses and enhancement of metastatic tumor growth occurred in rats following cryosurgery of the transplantable 3-methlcholanthrene-induced rat fibrosarcoma KMT-17. In this study, to elucidate the immunological responses in rats following cryosurgery, we examined whether rat serum obtained from rats which underwent cryosurgery (c-serum) might affect the in vivo neutralizing activity of the Winn assay. In this assay, c-serum did not reduce the anti-tumor immunity, though spleen cells obtained from rats undergoing surgical excision indicated strong anti-tumor immunity as compared with cryosurgery. Thus, we examined the anti-tumor responses of spleen cells. Macrophages were obtained from the glass adherent fraction of rat spleen cells following cryosurgery and these macrophages were used for cytostatic activity against KMT-17 cells. Cytostatic activity was not reduced by cryosurgery. The spleen cells obtained from rats receiving cryosurgery were intravenously transferred into other rats that were previously immunized with 80 Gy-irradiated KMT-17 cells, and an alteration of tumor growth modulated by this adoptive cell transfusion was observed. The anti-tumor resistance of rats was diminished by the adoptive transfusion of spleen cells treated with cryosurgery, though this diminution disappeared following anti-T serum and immune complement treatment of spleen cells. These results suggest that immuno-suppression following cryosurgery may be mainly caused by suppressor T cells.

Inhibitory effects of malotilate on invasion and metastasis of rat mammary carcinoma cells by modifying the functions of vascular endothelial cells.

Malotilate (diisopropyl,1,3-dithiol-2-ylidenemalonate, MT) is clinically used as a hepatoprotective agent. Because we noticed that MT induced the differentiation of cultured vascular endothelial cells, we have examined its effects on lung metastasis of the highly metastatic rat mammary carcinoma c-SST-2. MT was orally administered to syngeneic SHR rats from 7 days before or after s.c. inoculation of c-SST-2 cells to the end of the experiments. In the MT-treated rats, pulmonary metastasis was markedly suppressed compared with the non-treated rats. In the rats treated with MT for 19 days after i.v. inoculation of c-SST-2 cells, lung metastasis was also significantly suppressed. An in vitro invasion assay using a rat lung endothelial (RLE) cell monolayer revealed that pretreatment of the RLE cells with MT, but not c-SST-2 cells, significantly reduced the invasion of the RLE monolayer by c-SST-2 cells. An in vitro vascular permeability assay demonstrated that MT prevented the increase in permeability of the RLE monolayer by serum starvation. On the other hand, in vivo and in vitro growth, gelatinase production and adhesion to the RLE cell monolayer of c-SST-2 cells were not affected by MT treatment. These findings suggest that MT suppressed tumour metastasis by intensifying the cell-to-cell contact of endothelial cells, thus preventing tumour cells from invading vascular endothelium.

Enhancement of experimental pulmonary metastasis and inhibition of subcutaneously transplanted tumor growth following cryosurgery.

We have previously reported that inhibition of anti-tumor immune responses and a corresponding enhancement of metastatic tumor growth occurred in rats following cryosurgery of 3-methylcholanthrene-induced WKA rat fibrosarcoma (KMT-17). In this study, to evaluate the enhancement of metastasis arising from the inhibition of anti-tumor immune responses following cryosurgery, we examined how cryosurgery affected experimental pulmonary metastasis and the growth of subcutaneously transplanted tumor. To reveal the effect of cryosurgery on pulmonary metastasis, rats received a subcutaneous inoculation of KMT-17 tumor in the right flank (1 x 10(6)) and i.v. injection (1 x 10(5)) on the same day or 4 days later. The right flank tumors were treated with cryosurgery 5 days after subcutaneous transplantation. The pulmonary metastasis of the rats, which were injected i.v. one day before treatment, was enhanced by cryosurgery as compared with surgical excision, though the pulmonary metastasis of rats, which were injected i.v. 5 days before treatment, was un-affected by cryosurgery. These observations suggest that cryosurgery may enhance the pulmonary metastasis in its early steps but has no effects in its later stages. To reveal the effect of cryosurgery on the growth of distant tumors, rats received subcutaneous inoculations of KMT-17 tumor in the right (1 x 10(6)) and left (1 x 10(4) approximately 10(5)) flanks. Tumors in the right flank were treated with cryosurgery 5 days after inoculation and the growth of untreated left flank tumors was observed. In this double grafted tumor system, however, cryosurgery significantly inhibited the growth of the untreated left flank tumors. Spleen cells obtained from rats which had undergone cryosurgery 4 or 10 days previously (cryo-spleen cells) were used for in vivo neutralizing Winn assay. Antitumor activity of cryo-spleen cells was decreased as compared with that of rats after surgical excision in both spleen cells from 4 and 10 days after treatment. These findings suggest that effector cells in the spleen may not participate in subcutaneous tumor regression and that the evaluation of antitumor effect using the double grafted tumor system needs caution.

Comparison between submucosal (extra-nodal) and nodal non-Hodgkin's lymphoma (NHL) in the oral and maxillofacial region.

Fifty-two cases of non-Hodgkin's lymphoma (NHL) in the oral and maxillofacial region, comprising 31 submucosal (extra-nodal) and 21 cervical node NHLs, were investigated. The patients' ages ranged from 5 to 86 years, with a bimodal age distribution among young people below 12 years of age (average 8 years) and in those aged 30 years or older (average 60.3 years). The male-to-female gender difference ratio was 1.3:1. Patients presented with swelling as the major symptom. Histologically, diffuse, large cell malignant lymphoma was the most frequent type and 67.9% of lymphomas were of intermediate malignancy as defined by the Working Formulation for Clinical Usage. All submucosal lymphomas showed diffuse proliferation patterns, although follicular proliferation was identified in 5 of the 21 nodal lymphomas. Immunohistochemistry showed that the B-cell type was predominant, especially in nodal lymphomas.

Enhancement of tumor associated antigen expression during the regression phase of xenogenized tumor cell growth in vivo.

Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression was caused by immunological mechanisms, because the tumor cells were renogenized. In this study, we have tried to find out whether tumor-associated antigen (TAA) expression in these xenogenized tumor cells can be modulated by xenogenization. FV-KMT-17 cells (1 x 10(7)), which were subcutaneously transplanted into ten rats, spontaneously regressed after temporary growth. All rats which rejected FV-KMT-17 cells showed strong resistance to rechallenge with KMT-17 (1 x 10(6)) cells. To reveal the chronological modulation of TAA and virus-associated antigen (VAA), a single-cell suspension was obtained from the subcutaneous tumors and expression of these antigens was chronologically measured. TAA, termed CE7 antigen, was examined by anti-CE7 monoclonal antibody (MoAb) and VAA was examined by anti-FK1 MoAb which recognizes the FV env gene product (gp 70). Expression of VAA was not modulated through either the progression or the regression phase, but expression of TAA was strongly enhanced in the regression phase. These results show that enhancement of TAA expression occurs during the regression phase of FV-KMT-17 growth in vivo and that TAA-expressing cells may stimulate anti-tumor immunity, resulting in acquisition of resistance against parental KMT-17 cells.

The LEC (Long-Evans Cinnamon) rat as an animal model for bilirubin-induced tooth pigmentation.

The LEC (Long-Evans Cinnamon) rat is well known as a useful animal model for hepatic disease. We noticed the green pigmentation in incisors 2-3 weeks after acute hepatitis accompanied by severe jaundice. This study was undertaken to elucidate the cause of this phenomenon. Half of the pigmented teeth were examined by histopathological analysis and microradiographic analysis. Pigmentation was observed as a green stripe that ran parallel to the incremental line in the dentine. The microradiographic analysis disclosed enhanced permeability of the pigmented area as compared with other areas. The rest of pigmented teeth were dried, powdered and bilirubin was extracted with chloroform /methanol/acetic acid, 30:10:0.5; v/v under sonication. After centrifugation, the supernatant was collected and evaporated. The residue was dissolved in chloroform and its absorption spectrum measured after diazo reaction to reveal the presence of bilirubin. The spectral characteristics indicated the presence of bilirubin in the pigmented teeth. Thus, the LEC rat may be useful animal model for bilirubin-induced tooth pigmentation.

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) in 23 cases of ameloblastoma.

Ameloblastoma is the most frequent odontogenic tumour. It occurs mainly in the mandible and grows expansively. The treatment of ameloblastoma, which influences the prognosis, is decided in consideration of many factors, especially the age and size of the tumour. Conservative treatment sometimes leads to the recurrence of tumours and poor prognosis, but the relationships between the prognosis and the cytological features of tumour cells are still unclear. In the present study, we examined the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) in 23 cases of ameloblastoma and evaluated the correlation between the positive index of PCNA and the clinical and histological character. Our results revealed the higher the age of the patient the greater was the incidence of a positive index of PCNA. It was also shown that the mean positive PCNA index in the follicular type (34.56 +/- 14.00 S.D.) was higher than that of the plexiform type (24.436 +/- 15.74 S.D., P < 0.10). The cystic type showed a low positive PCNA index (14.75 +/- 8.41 S.D.). In the follicular type, the localisation of PCNA-positive cells was different according to the histological patterns of tumours. Additionally, the positive indices of the same patient differed at different periods of treatment.

Experimental bilirubin pigmentation of rat dentine and its detection by a qualitative analytical method.

An animal model of bilirubinemia was used to determine whether bilirubin present in pigmented teeth can be extracted and qualitatively analysed. The bile ducts of 10 Long-Evans Agouti rats were ligated and bilirubin (14 mg/kg per day) was injected intraperitoneally for 4 days. When the animals were killed 2 weeks later, pigmented lower incisors were observed in three animals. These teeth were dried, powdered and bilirubin was extracted with chloroform/methanol/acetic acid, 30:10:0.5, v/v for 10 min under sonication. After centrifugation, the supernatant was collected and evaporated. The residue was dissolved in chloroform and its absorption spectrum measured before and after diazo reaction. This resulted in a shift of the absorption maximum from 450 to 540 nm and indicated the presence of bilirubin in pigmented teeth. No bilirubin was found in the lower incisors of untreated control rats. This technique may be useful in distinguishing bilirubin staning from other intrinsic discolorations of teeth.

Modulation of the rat tumor-associated shedding antigen (CE7) and augmentation of immunogenicity by irradiation.

We have previously reported that rat fibrosarcoma KMT-17 cells and their in vitro counterparts, cloned A3 cells, shed a tumor-associated antigen (TAA), termed CE7, from the cell surface on vesicular membranes, under growth-enhancing conditions. This study shows that irradiation (1 approximately Gy) from a 60Co source, inhibited A3 cell growth dose-dependently and correspondingly increased CE7 expression by A3 cells as determined by anti-CE7 monoclonal antibody using flow cytometry. CE7 expression gradually increased with increasing doses of irradiation and reached a peak level at 30Gy. After 30Gy irradiation, CE7 expressing A3 cells were fixed with 1% paraformaldehyde and were used to intradermally immunize syngenic rats. Immunized rats developed transplantation resistance to the parent KMT-17 cells as compared to rats immunized with unirradiated A3 cells. Rat MHC class 1 antigen expression was slightly decreased by irradiation and therefore, resistance to tumor transplantation appeared to arise solely due to the enhancing effects of irradiation on TAA expression which increases the antigenicity of the tumor cells coverting them to an effective stimulator of antitumor effector cells. This phenomenon may offer a possibility of the resistance to the re-emergence and metastasis of the tumor like a KMT-17 through the induction of antitumor memory cells.