Oral-specific ablation of Klf4 disrupts epithelial terminal differentiation and increases premalignant lesions and carcinomas upon chemical carcinogenesis.

BACKGROUND: Squamous cell carcinoma (SSC) of the head and neck is the sixth most common cancer and is rarely diagnosed in early stages. The transcription factor Krϋppel-like factor 4 (Klf4) suppresses cell proliferation and promotes differentiation. Inducible mice carrying an oral-specific ablation of Klf4 (K14-CreER(tam) /Klf4(flox/flox) ) develop mild dysplastic lesions and abnormal differentiation in the tongue. Aiming to analyze whether Klf4 cooperate in oral chemical carcinogenesis,we applied 4-nitroquinoline 1-oxide (4NQO), a tobacco surrogate, to this conditional Klf4 knockout mice. METHODS: K14-CreER(tam) /Klf4(flox/flox) and control mice were treated with 4NQO for 16 weeks and monitored until week 30. Histopathological samples were used for diagnostic purposes and immunofluorescence detection of epithelial differentiation markers. RESULTS: 4NQO-treated K14-CreER(tam) /Klf4(flox/flox) mice (Klf4KO 4NQO) showed a significant weight loss and developed more severe dysplastic lesions than control mice with 4NQO (P < 0.005). The Klf4KO 4NQO showed a tendency to higher incidence of oral SCC and a marked keratinization pattern in dysplasias, in situ carcinomas and SCC. Also, tongues derived from Klf4KO 4NQO mice exhibited reduced terminal differentiation as judged by cytokeratin 1 staining when compared with 4NQO-treated controls. CONCLUSIONS: Klf4 ablation results in more severe dysplastic lesions in oral mucosa, with a tendency to higher incidence of SCC, after chemical carcinogenesis. We show here, in a context similar to the human carcinogenesis, that absence of Klf4 accelerates carcinogenesis and correlates with the absence of cytokeratin 1 expression. These results suggest a potential role for KLF4 as a tumor suppressor gene for the tongue epithelium.

Migration of titanium dioxide microparticles and nanoparticles through the body and deposition in the gingiva: an experimental study in rats.

The aim of this experimental work was to evaluate deposition of titanium dioxide (TiO2 ) microparticles and nanoparticles, which could originate from titanium bioimplants, in the gingiva. Wistar rats were injected intraperitoneally (i.p.) with a suspension of TiO2 particles of different sizes (150, 10, or 5 nm). The rats were killed 12 months post-injection, and the buccal and lingual gingivae were resected and evaluated using light and scanning electron microscopy. Energy-dispersive X-ray spectroscopy (EDS) was used to confirm the presence of titanium in deposits of microparticles and nanoparticles, and the concentration of titanium in tissues was measured using inductively coupled plasma-mass spectrometry (ICP-MS). Histological examination showed that all experimental groups exhibited agglomerates, in the gingiva, of titanium particles of micrometer size range, with no associated inflammatory response. Higher concentrations of titanium traces were shown, by ICP-MS, in both buccal and lingual tissues of all experimental groups compared with their matched controls. Titanium concentrations were significantly higher in the buccal gingiva than in the lingual gingiva, and after injection with 5-nm particles than with 10-nm particles in both localizations. Titanium microparticles and nanoparticles deposit in the gingiva, and mostly on the buccal side. Gingival deposition of titanium could be considered a tissue indicator of tribocorrosion processes of titanium bioimplants.

Impairing squamous differentiation by Klf4 deletion is sufficient to initiate tongue carcinoma development upon K-Ras activation in mice.

Oral squamous cell carcinoma (SCC) is among the most prevalent cancers in the world and is characterized by high morbidity and few therapeutic options. Like most cancers, oral SCC arises from a multistep process involving alterations of genes responsible for balancing proliferation and differentiation. Among these, Krϋppel-like factor 4 (Klf4) suppresses cell proliferation and promotes differentiation and thus helps to maintain epithelial homeostasis. However, the prevailing role of Klf4 in maintenance of normal homeostasis in oral epithelium has not been established in vivo. Here, we used an inducible oral-specific mice model to selectively ablate Klf4 in the oral cavity. We generated K14-CreER(Tam)/Klf4 (f/f) mice that survived to adulthood and did not present overt phenotype. However, histologically these mice showed dysplastic lesions, increased cell proliferation and abnormal differentiation in the tongue 4 months after induction, supporting a homeostatic role of Klf4 in the oral epithelia. Furthermore, by breeding these mutants with a transgenic line expressing at endogenous levels K-ras (G12D), we assessed the role of disrupting differentiation gene programs to the carcinogenesis process. The K14-CreER(TAM)/K-ras (G12D)/Klf4 (-) (/-) mice rapidly develop oral SCC in the tongue. Thus, our findings support the emerging notion that activation of differentiating gene programs may represent a barrier preventing carcinogenesis in epithelial cells harboring oncogenic mutations, and thus that molecules acting upstream and downstream of Klf4 may represent components of a novel tumor-suppressive pathway.

Therapeutic Efficacy, Radiotoxicity and Abscopal Effect of BNCT at the RA-3 Nuclear Reactor Employing Oligo-Fucoidan and Glutamine as Adjuvants in an Ectopic Colon Cancer Model in Rats.

Boron neutron capture therapy (BNCT) is based on the preferential uptake of 10B compounds by tumors, followed by neutron irradiation. The aim of this study was to assess, in an ectopic colon cancer model, the therapeutic efficacy, radiotoxicity, abscopal effect and systemic immune response associated with (BPA/Borophenylalanine+GB-10/Decahydrodecaborate)-BNCT (Comb-BNCT) alone or in combination with Oligo-Fucoidan (O-Fuco) or Glutamine (GLN), compared to the "standard" BPA-BNCT protocol usually employed in clinical trials. All treatments were carried out at the RA-3 nuclear reactor. Boron biodistribution studies showed therapeutic values above 20 ppm 10B in tumors. At 7 weeks post-treatment, the ratio of tumor volume post-/pre-BNCT was significantly smaller for all BNCT groups vs. SHAM (p < 0.05). The parameter "incidence of tumors that underwent a reduction to ≤50% of initial tumor volume" exhibited values of 62% for Comb-BNCT alone, 82% for Comb-BNCT+GLN, 73% for Comb-BNCT+O-Fuco and only 30% for BPA-BNCT. For BPA-BNCT, the incidence of severe dermatitis was 100%, whereas it was significantly below 70% (p ≤ 0.05) for Comb-BNCT, Comb-BNCT+O-Fuco and Comb-BNCT+GLN. Considering tumors outside the treatment area, 77% of Comb-BNCT animals had a tumor volume lower than 50 mm3 vs. 30% for SHAM (p ≤ 0.005), suggesting an abscopal effect of Comb-BNCT. Inhibition of metastatic spread to lymph nodes was observed in all Comb-BNCT groups. Considering systemic aspects, CD8+ was elevated for Comb-BNCT+GLN vs. SHAM (p ≤ 0.01), and NK was elevated for Comb-BNCT vs. SHAM (p ≤ 0.05). Comb-BNCT improved therapeutic efficacy and reduced radiotoxicity compared to BPA-BNCT and induced an immune response and an abscopal effect.

Paget's disease of the jaws: Histopathological features of a series of 31 cases.

The aim of the present study was to analyze the histopathological features of Paget's disease of the jaws observed in a series comprising 31 cases. The study comprised all cases of Paget's disease of the jaws filed in the archives of the Surgical Pathology Laboratory of the Oral Pathology Department, School of Dentistry, University of Buenos Aires, between 1960 and 2018. Their microscopic features were evaluated, and available clinical data and radiographic studies were analyzed. Paget's disease of the jaws accounted for 0.05% of retrieved oral-maxillofacial pathologies. Microscopically, all cases showed lamellar bone trabeculae with the characteristic mosaic pattern. Twenty cases (64%) showed osteoblastic-osteoclastic activity, and all showed areas of necrosis. Cemento-osseous trabeculae were observed in 15 cases (48%), and cementicles were observed in 13 (42%). Osteomyelitis was seen in 11 cases (35%), all of which showed cemento-osseous trabeculae with a mosaic structure, sclerosis and necrosis, and chronic inflammation with abscess formation. Mean age was 61 years (44-85 years); 19 cases were women. Localization was the maxilla in 13 cases (42%), and the disease involved other skeletal bones in five cases. To our knowledge, this is the largest series of Paget's disease of the jaws reported to date. Paget's disease is infrequent in the jaws and has distinct histopathological features that not only differ from those observed at other skeletal sites but also require differential diagnosis from other pathologies affecting the jaws exclusively.

El objetivo del presente trabajo fue analizar las características histopatológicas de la enfermedad de Paget en los maxilares en una serie de 31 casos. El estudio comprendió todos los casos de enfermedad de Paget de los maxilares provenientes del Laboratorio de Patología Quirúrgica de la Cátedra de Anatomía Patológica de la Facultad de Odontología de la Universidad de Buenos Aires, entre 1960 y 2018. Se evaluaron las características microscópicas y se analizaron los datos clínicos y estudios radiográficos disponibles. La enfermedad de Paget en los maxilares representó el 0,05% de las patologías buco-maxilofaciales. Microscópicamente, todos los casos mostraron trabéculas óseas laminares con el característico patrón en mosaico. Veinte casos (64%) mostraron actividad osteoblástica-clástica y todos los casos mostraron necrosis focal. En 15 casos (48%) las trabéculas presentaron aspecto cemento-óseo y en 13 casos (42%) se observaron cementículos. Once casos (35%) presentaron cuadros osteomielíticos y todos ellos mostraron trabéculas cemento-óseas con estructura en mosaico, esclerosis y necrosis, e infiltrado inflamatorio crónico con formación de abscesos. La media de edad fue 61 años (44-85 años), y 19 fueron mujeres. Trece casos (42%) se localizaron en maxilar superior y 5 casos presentaron compromiso de otros huesos. A nuestro entender, esta es la serie más grande de enfermedad de Paget en los maxilares reportada hasta la fecha. La enfermedad de Paget es poco frecuente en los maxilares y presenta características histopatológicas que además de ser diferentes a las observadas en otros sitios del esqueleto plantean el diagnóstico diferencial con otras entidades que se presentan exclusivamente en los maxilares.

Extranodal oral non-Hodgkin's lymphomas. A retrospective study of 40 cases in Argentina.

A retrospective study was conducted of extranodal oral Non-Hodgkin's Lymphomas diagnosed at the Surgical Pathology Laboratory of the School of Dentistry at Buenos Aires University, Argentina, between 1985 and 2004. The 40 cases found represent 0.2% of the oral biopsies diagnosed during that time and 4.6% of malignant neoplasias. Overall mean age of patients was 49.4 years, and frequency was greater in males. 80% affected soft tissues. Prevalent location was gingival, followed by palate. Intraosseous cases were more frequent in mandible (75%) than in upper maxilla. 100% of the cases were phenotype B, with a higher frequency of high-grade aggressiveness. The most common histological type was Diffuse Large Cell Lymphoma. 60% of the Plasmablastic Lymphomas in the series came from HIV+ patients. Evolution time prior to consultation was 1 to 3 months in 57.7% of the cases.

Biokinetics and tissue response to ultrananocrystalline diamond nanoparticles employed as coating for biomedical devices.

Although Ultrananocrystalline diamond (UNCD) has been proposed as a coating material for titanium biomedical implants, the biological effects and toxicity of UNCD particles that could eventually detach have not been studied to date. The biokinetics and biological effects of UNCD compared to titanium dioxide (TiO2 ) nanoparticles was evaluated in vivo using Wistar rats (n = 30) i.p. injected with TiO2 , UNCD or saline solution. After 6 months, blood, lung, liver, and kidney samples were histologically analyzed. Oxidative damage by membrane lipidperoxidation (thiobarbituric acid reactive substances-TBARS), generation of reactive oxygen species (superoxide anion- O2-), and antioxidant enzymes (superoxide dismutase-SOD, catalase-CAT) was evaluated in lung and liver. Histologic observation showed agglomerates of TiO2 or UNCD in the parenchyma of the studied organs, though there were fewer UNCD than TiO2 deposits. In addition, TiO2 caused areas compatibles with foci of necrosis in the liver and renal hyaline cylinders. Regarding UNCD, no membrane damage (TBARS) or mobilization of enzymatic antioxidants was observed either in lung or liver samples. No variations in O2- generation were observed in lung (Co: 35.1 ± 4.02 vs. UNCD: 48 ± 9.1, p > 0.05). Conversely, TiO2 exposure caused production of O2- in alveolar macrophages and consumption of catalase (p < 0.05). The studied parameters suggest that UNCD caused neither biochemical nor histological alterations, and therefore may prove useful as a surface coating for biomedical implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2408-2415, 2017.

Paget's disease of the jaws: Histopathological features of a series of 31 cases / Enfermedad de Paget en los maxilares. Características histopatológicas de una serie de 31 casos

ABSTRACT The aim of the present study was to analyze the histopathological features of Paget's disease of the jaws observed in a series comprising 31 cases. The study comprised all cases of Paget's disease of the jaws filed in the archives of the Surgical Pathology Laboratory of the Oral Pathology Department, School of Dentistry, University of Buenos Aires, between 1960 and 2018. Their microscopic features were evaluated, and available clinical data and radiographic studies were analyzed. Paget's disease of the jaws accounted for 0.05% of retrieved oral-maxillofacial pathologies. Microscopically, all cases showed lamellar bone trabeculae with the characteristic mosaic pattern. Twenty cases (64%) showed osteoblastic-osteoclastic activity, and all showed areas of necrosis. Cemento-osseous trabeculae were observed in 15 cases (48%), and cementicles were observed in 13 (42%). Osteomyelitis was seen in 11 cases (35%), all of which showed cemento-osseous trabeculae with a mosaic structure, sclerosis and necrosis, and chronic inflammation with abscess formation. Mean age was 61 years (44-85 years); 19 cases were women. Localization was the maxilla in 13 cases (42%), and the disease involved other skeletal bones in five cases. To our knowledge, this is the largest series of Paget's disease of the jaws reported to date. Paget's disease is infrequent in the jaws and has distinct histopathological features that not only differ from those observed at other skeletal sites but also require differential diagnosis from other pathologies affecting the jaws exclusively.

RESUMEN El objetivo del presente trabajo fue analizar las características histopatológicas de la enfermedad de Paget en los maxilares en una serie de 31 casos. El estudio comprendió todos los casos de enfermedad de Paget de los maxilares provenientes del Laboratorio de Patología Quirúrgica de la Cátedra de Anatomía Patológica de la Facultad de Odontología de la Universidad de Buenos Aires, entre 1960 y 2018. Se evaluaron las características microscópicas y se analizaron los datos clínicos y estudios radiográficos disponibles. La enfermedad de Paget en los maxilares representó el 0,05% de las patologías buco-maxilofaciales. Microscópicamente, todos los casos mostraron trabéculas óseas laminares con el característico patrón en mosaico. Veinte casos (64%) mostraron actividad osteoblástica-clástica y todos los casos mostraron necrosis focal. En 15 casos (48%) las trabéculas presentaron aspecto cemento-óseo y en 13 casos (42%) se observaron cementículos. Once casos (35%) presentaron cuadros osteomielíticos y todos ellos mostraron trabéculas cemento-óseas con estructura en mosaico, esclerosis y necrosis, e infiltrado inflamatorio crónico con formación de abscesos. La media de edad fue 61 años (44- 85 años), y 19 fueron mujeres. Trece casos (42%) se localizaron en maxilar superior y 5 casos presentaron compromiso de otros huesos. A nuestro entender, esta es la serie más grande de enfermedad de Paget en los maxilares reportada hasta la fecha. La enfermedad de Paget es poco frecuente en los maxilares y presenta características histopatológicas que además de ser diferentes a las observadas en otros sitios del esqueleto plantean el diagnóstico diferencial con otras entidades que se presentan exclusivamente en los maxilares.

Impact through time of different sized titanium dioxide particles on biochemical and histopathological parameters.

Due to corrosion, a titanium implant surface can be a potential source for the release of micro (MPs) and nano-sized particles (NPs) into the biological environment. This work sought to evaluate the biokinetics of different sized titanium dioxide particles (TiO2 ) and their potential to cause cell damage. Wistar rats were intraperitoneally injected with 150 nm, 10 nm, or 5nm TiO2 particles. The presence of TiO2 particles was evaluated in histologic sections of the liver, lung, and kidney and in blood cells at 3 and 12 months. Ultrastructural analysis of liver and lung tissue was performed by TEM, deposit concentration in tissues was determined spectroscopically, and oxidative metabolism was assessed by determining oxidative membrane damage, generation of superoxide anion (O2(-)), and enzymatic and non-enzymatic antioxidants. TiO2 particles were observed inside mononuclear blood cells and in organ parenchyma at 3 and 12 months. TiO2 deposits were consistently larger in liver than in lung tissue. Alveolar macrophage O2(-) generation and average particle size correlated negatively (p < 0.05). NPs were more reactive and biopersistent in lung tissue than MPs. Antioxidant activity, particularly in the case of 5 nm particles, failed to compensate for membrane damage in liver cells; the damage was consistent with histological evidence of necrosis.

Exfoliative cytology and titanium dental implants: a pilot study.

BACKGROUND: Oral exfoliative cytology is a diagnostic method that involves the study of cells exfoliated from the oral mucosa. Ions/particles released from metallic implants can remain in the peri-implant milieu. The aim of the present study is to assess the presence of metal particles in cells exfoliated from peri-implant oral mucosa around titanium dental implants. METHODS: The study comprised 30 patients carrying titanium dental implants, who had neither a metallic prosthesis nor metal restorations in neighboring teeth. Individuals undergoing orthodontic therapy and those who had oral piercing were also excluded from the study. The study sample included patients with and without peri-implantitis. Cytologic samples of the peri-implant area were collected. Samples of the marginal gingiva on the contralateral side of the implant were taken from the same individuals to serve as control. Cytologic analysis was performed using light microscopy. Titanium concentration was determined using inductively coupled plasma-mass spectrophotometry. RESULTS: Metal-like particles were observed inside and outside epithelial cells and macrophages in cytologic smears of peri-implant mucosa of both patients with and without peri-implantitis. No particles were found in the control cytologic samples. The concentration of titanium was higher in the peri-implantitis group compared with the group without peri-implantitis; no traces of titanium were observed in controls. CONCLUSIONS: Regardless of an inflammatory response, ions/particles are released from the surface of the implant into the biologic milieu. Exfoliative cytology is a simple technique that may be used to detect metal particles in cells exfoliated from the peri-implant mucosa.

Oral mucosa tissue response to titanium cover screws.

BACKGROUND: Titanium is the most widely used metal in dental implantology. The release of particles from metal structures into the biologic milieu may be the result of electrochemical processes (corrosion) and/or mechanical disruption during insertion, abutment connection, or removal of failing implants. The aim of the present study is to evaluate tissue response of human oral mucosa adjacent to titanium cover screws. METHODS: One hundred fifty-three biopsies of the supra-implant oral mucosa adjacent to the cover screw of submerged dental implants were analyzed. Histologic studies were performed to analyze epithelial and connective tissue as well as the presence of metal particles, which were identified using microchemical analysis. Langerhans cells, macrophages, and T lymphocytes were studied using immunohistochemical techniques. The surface of the cover screws was evaluated by scanning electron microscopy (SEM). RESULTS: Forty-one percent of mucosa biopsies exhibited metal particles in different layers of the section thickness. Particle number and size varied greatly among specimens. Immunohistochemical study confirmed the presence of macrophages and T lymphocytes associated with the metal particles. Microchemical analysis revealed the presence of titanium in the particles. On SEM analysis, the surface of the screws exhibited depressions and irregularities. CONCLUSIONS: The biologic effects seen in the mucosa in contact with the cover screws might be associated with the presence of titanium or other elements, such as aluminum or vanadium. The potential long-term biologic effects of particles on soft tissues adjacent to metallic devices should be further investigated because these effects might affect the clinical outcome of the implant.

Nucleolar organizer regions (AgNOR) and subepithelial vascularization as field cancerization markers in oral mucosa biopsies of alcoholic and smoking patients.

OBJECTIVE: The aim of this study was to show that variations in nucleolar organizer regions (AgNOR) and the increase in subepithelial vascularization could reveal changes related to markers of field cancerization in alcoholic and smoking patients who have not yet expressed clinical or histological malignant lesions. STUDY DESIGN: Quantitative variations in epithelial AgNOR and in the vascularization of the underlying connective tissue were assessed by image analysis in histologically normal biopsy specimens from alcohol drinkers and smoking patients (DS). AgNORs were evidenced by silver staining and vessel walls were labeled by immunohistochemical demonstration of the CD34 antigen. Samples of oral mucosa of nonalcoholic, nonsmoking patients (NDS) obtained during surgical procedures served as controls. Eight parameters related to number, volume, and shape of nuclei and AgNORs, and 4 parameters related to number and diameter of vascular sections were evaluated. Differences between DS and NDS groups were statistically evaluated by means of ANOVA test and posterior Bonferroni comparisons. RESULTS: The morphometric analysis revealed more irregular-shaped AgNORs in the superficial and suprabasal layers of the oral mucosa of DS patients. The suprabasal layers also exhibited a significantly larger number of AgNORs. The normal oral mucosa of DS patients exhibited a greater vascular density, with predominance of small-caliber blood vessels underlying the basement membrane. CONCLUSION: The variations in AgNOR and epithelial vascularization would be practical biomarkers to evaluate changes underlying the augmented risk of cancerization in oral mucosa.