Qualitative autoradiography with polycarbonate foils enables histological and track analyses on the same section.

Neutron autoradiography is an imaging methodology that enables analysis of the spatial distribution of heavy ion emitters in a given material. In particular, it allows localization of (10)B in a tissue section put in contact with a nuclear track detector. Boron imaging is essential when considering boron neutron capture therapy as an option for treating cancerous tumors. A description of the autoradiography method is presented together with specific characteristics and technical details developed in our laboratory. We propose a new mounting technique to compare autoradiography images with the same section that gave rise to the latent tracks. The solid state nuclear track detector is polycarbonate, because it can be processed rapidly to obtain the autoradiographic results. It is a transparent material, which allows visualization of the sections mounted on it. Tissue can be removed easily and background is minimal.

10B concentration evaluation in autoradiography images by optical density measurements.

The quantification and analysis of the tracks forming the autoradiography image of a tissue section is essential for the measurement of particle emitter concentration and distribution (e.g. (10)B) in the sample. Conventional counting techniques cannot be used when track density is high because of track overlapping. A densitometry supported by image analysis method suitable for these cases has been developed. Optical density measurements obtained for boron solutions of known concentrations showed a linear behavior in the range of concentrations under consideration.

Reactive lesions of peri-implant mucosa associated with titanium dental implants: a report of 2 cases.

The aim of this study was to report 2 novel clinical cases of reactive lesions of the peri-implant mucosa associated with titanium dental implants where metal-like particles were observed histologically. In both cases, the lesions were diagnosed as epulis, based on clinical evidence. Extirpation biopsies were carried out. Case 1 was diagnosed as pyogenic granuloma and case 2 as peripheral giant cell granuloma. The presence of metal-like particles in the tissues suggests that the etiology of the lesions might be related to the corrosion process of the metal structure. This is the first case of pyogenic granuloma to be reported in association with dental implants. All clinical cases of soft tissue lesions associated with implants should be reported to contribute to the understanding of the etiology and pathogeny of these lesions.

Local effect of titanium implant corrosion: an experimental study in rats.

The aim of this study was to evaluate histologically the biological effect of pitting corrosion and to contribute clinically relevant data on the permanence of titanium metal structures used in osteosynthesis in the body. Commercially pure titanium laminar implants (control) and commercially pure titanium laminar implants with pitting corrosion (experimental) were implanted in the tibiae of rats. At 14 days post-implantation the animals were killed. The tibiae were resected, fixed, radiographed and processed for embedding in methyl methacrylate. Percentage of bone-implant contact and peri-implant bone volume were evaluated. The histological study of the titanium implants submitted to pitting corrosion showed scarce bone-implant contact, it was only present in the areas with no pitting and/or surface alterations. There was a statistically significant lower percentage of bone-implant contact in the experimental group (6%+/-4) than in the control group (26%+/-6) (p<0.001). Products of corrosion in the peri-implant bed, especially around the blood vessels and areas of bone marrow in the metal-tissue interface, were observed. The microchemical analysis of corrosion products revealed the presence of titanium. The adverse local effects caused by pitting corrosion suggest that titanium plates and grids should be used with caution as permanent fixation structures.

Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate.

Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 microM) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1-100 microM UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1-100 microM UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage.

Effect of ethane-1-hydroxy-1,1-bisphosphonate (EHBP) on endochondral ossification lesions induced by a lethal oral dose of uranyl nitrate.

A 350 mg/kg body weight (b.w.) oral dose of uranyl nitrate (UN) caused 100% mortality in mice three days after administration, due to resulting kidney lesions. Mortality decreased 50% after an oral (o) or subcutaneous (sc) dose of bisodic etidronate (EHBP). Given that bone is also a target organ for uranium (U) in acute intoxication, the aim of this work was to study the effect of exposure to a lethal oral dose of UN on endochondral ossification, and the latter's response to EHBP treatment. One hundred male Balb/c mice weighing 25 g were assigned to one of ten groups. Group I served as control. Group II received a lethal 350 mg/kg b.w. oral dose of UN by gavage. Groups III, IV, VII, and VIII received an equal dose of UN immediately followed by a single 500 mg/kg oral dose of EHBP in groups III and VII and a single 50 mg/kg subcutaneous dose of EHBP in groups IV and VIII. Groups V and IX only received a single 500 mg/kg oral dose of EHBP, and groups VI and X received a single 50 mg/kg subcutaneous dose of EHBP. The animals in groups II, III, IV, V, and VI were sacrificed 48 h after the onset of the experiment, whereas those in groups VII, VIII, IX, and X were killed at 14 days. Histologic and histomorphometric studies were performed on the femurs to determine growth cartilage width, bone volume, and metaphyseal bone activity. Our results showed that all growth cartilage and metaphyseal bone histomorphometric parameters were significantly lower in animals exposed to UN at 48 h than in controls. EHPB administration was found to prevent this condition at 48 h reaching similar values to those of controls. Although histomorphometric values did not reach control values at 14 days, they were higher than those of animals exposed to UN at 48 h not treated with EHBP. It is noteworthy that these values also decreased in animals only receiving EHBP at 14 days. Our results show that EHBP effectively ameliorates the adverse effects of a lethal dose of UN on endochondral ossification.

Biodistribution of p-borophenylalanine (BPA) in dogs with spontaneous undifferentiated thyroid carcinoma (UTC).

Human undifferentiated thyroid carcinoma (UTC) is a very aggressive tumor which lacks an adequate treatment. The UTC human cell line ARO has a selective uptake of BPA in vitro and after transplanting into nude mice. Applications of boron neutron capture therapy (BNCT) to mice showed a 100% control of growth and a 50% histological cure of tumors with an initial volume of 50 mm(3) or less. As a further step towards the potential application in humans we have performed the present studies. Four dogs with diagnosis of spontaneous UTC were studied. A BPA-fructose solution was infused during 60 min and dogs were submitted to thyroidectomy. Samples of blood and from different areas of the tumors (and in one dog from normal thyroid) were obtained and the boron was determined by ICP-OES. Selective BPA uptake by the tumor was found in all animals, the tumor/blood ratios ranged between 2.02 and 3.76, while the tumor/normal thyroid ratio was 6.78. Individual samples had tumor/blood ratios between 8.36 and 0.33. These ratios were related to the two histological patterns observed: homogeneous and heterogeneous tumors. We confirm the selective uptake of BPA by spontaneous UTC in dogs and plan to apply BNCT in the future.

Renal function in mice poisoned with oral uranium and treated with ethane-1-hydroxy-1,1-bisphosphonate (EHBP).

Exposure to uranium is a risk for the workers involved in uranium mining, purification, and manufacture, principally by its ingestion or inhalation. It is also a risk for the population at large in case of intake of contaminated water or food. Uranium induces nephropathy that is characteristic of heavy metals, which can lead to death. The toxic effects of uranium can be prevented by a biphosphonate, ethane-1-hydroxy-1,1-bisphosphonate (bisodic etidronate), administered orally or subcutaneously. Employing bisodic etidronate, our laboratory obtained satisfactory results in terms of survival in adult mice, adult rats, and suckling rats. The aim of the present study was to evaluate the efficacy of bisodic etidronate for preventing renal dysfunction induced by a lethal dose of uranyl nitrate, employing serum levels of urea and creatinine as end-points. Two experiments were performed over different time periods, i.e., Experiment A: 48 h, Experiment B: 14 d. Each experiment was performed with 4 groups of 20 male Balb/c mice each, 25 g average body weight. Three of these groups received 350 mg kg(-1) of body weight of uranyl nitrate by gavage (forced oral administration). Two of the three exposed groups were treated with bisodic etidronate either by gavage in a dose of 500 mg kg(-1) body weight or with a subcutaneous injection of 50 mg kg(-1) body weight. The fourth group served as control. Survivors of the experimental groups were sacrificed at the end of the experiment by overdose of inhalation anesthetic (ether). The kidneys were routinely processed for histological analysis. Blood samples were taken by cardiac puncture to assess urea and creatinine serum levels. Urea and creatinine serum levels were markedly lower at 48 h in exposed animals treated with bisodic etidronate than in untreated exposed animals. On day 14 these values in exposed and treated animals did not differ significantly from control values. The renal function of animals treated with orally or subcutaneous bisodic etidronate that survived uranyl nitrate exposure was markedly improved compared to the controls of untreated exposed animals at 48 h. At 14 days, treatment with bisodic etidronate averted renal damage. At this time, the histologic study of kidneys showed images of tissue recovery. These results suggest that the use of EHBP may be of great value in reducing the renal damage.

A quantitative method to evaluate corrosion products in tissues.

The use of odontological or orthopedic metal implants requires the availability of techniques to estimate tissue response to the corrosion processes. In previous experimental studies we showed the deposition of corrosion products not only locally (Olmedo et al., Implant Dent 2003; 12: 75-80) but also systemically (Olmedo et al., J Mater Sci: Mater in Medic 2002; 13: 793-796) in organs such as liver, spleen and lung. The aim of the present study was to propose a method to quantitatively assess the tissue deposits of the corrosion products of the materials used to manufacture implants. The samples (liver and lung) were embedded in paraffin, and the histological sections were submitted to thickness standardization. The quantitative evaluation of the deposits was performed in an MPM-800 (Carl Zeiss)* microscope. The light microscopy images were digitalized and then analyzed employing the DNA-IBAS-Kontron software that allows for the identification and evaluation of cells loaded with corrosion products (objective 20x). The following end-points were assessed: total field area, number of deposits of corrosion products, partial and total area of the deposits, and the ratio between volume of the deposits and tissue volume. The method proposed serves to quantitatively evaluate, at light microscopy level, the deposition of corrosion products in tissues.

A quantitative method to evaluate corrosion products in tissues

The use of odontological or orthopedic metal implants requires the availability of techniques to estimate tissue response to the corrosion processes. In previous experimental studies we showed the deposition of corrosion products not only locally (Olmedo et al., Implant Dent 2003; 12: 75-80) but also systemically (Olmedo et al., J Mater Sci: Mater in Medic 2002; 13: 793-796) in organs such as liver, spleen and lung. The aim of the present study was to propose a method to quantitatively assess the tissue deposits of the corrosion products of the materials used to manufacture implants. The samples (liver and lung) were embedded in paraffin, and the histological sections were submitted to thickness standardization. The quantitative evaluation of the deposits was performed in an MPM-800 (Carl Zeiss)* microscope. The light microscopy images were digitalized and then analyzed employing the DNA-IBAS-Kontron software that allows for the identification and evaluation of cells loaded with corrosion products (objective 20x). The following end-points were assessed: total field area, number of deposits of corrosion products, partial and total area of the deposits, and the ratio between volume of the deposits and tissue volume. The method proposed serves to quantitatively evaluate, at light microscopy level, the deposition of corrosion products in tissues.

A quantitative method to evaluate corrosion products in tissues.

The use of odontological or orthopedic metal implants requires the availability of techniques to estimate tissue response to the corrosion processes. In previous experimental studies we showed the deposition of corrosion products not only locally (Olmedo et al., Implant Dent 2003; 12: 75-80) but also systemically (Olmedo et al., J Mater Sci: Mater in Medic 2002; 13: 793-796) in organs such as liver, spleen and lung. The aim of the present study was to propose a method to quantitatively assess the tissue deposits of the corrosion products of the materials used to manufacture implants. The samples (liver and lung) were embedded in paraffin, and the histological sections were submitted to thickness standardization. The quantitative evaluation of the deposits was performed in an MPM-800 (Carl Zeiss)* microscope. The light microscopy images were digitalized and then analyzed employing the DNA-IBAS-Kontron software that allows for the identification and evaluation of cells loaded with corrosion products (objective 20x). The following end-points were assessed: total field area, number of deposits of corrosion products, partial and total area of the deposits, and the ratio between volume of the deposits and tissue volume. The method proposed serves to quantitatively evaluate, at light microscopy level, the deposition of corrosion products in tissues.

Selective uptake of p-borophenylalanine by undifferentiated thyroid carcinoma for boron neutron capture therapy.

Undifferentiated thyroid carcinoma (UTC) lacks an effective treatment. Boron neutron capture therapy (BNCT) is based on the selective uptake of 10B-boronated compounds by some tumors, followed by irradiation with an appropriate neutron beam. The radioactive boron originated (11B) decays releasing 7Li, gamma rays and alpha particles, and these latter will destroy the tumor. In order to explore the possibility of applying BNCT to UTC we have studied the biodistribution of BPA. In in vitro studies, the uptake of p-10borophenylalanine (BPA) by the UTC cell line ARO, primary cultures of normal bovine thyroid cells (BT), and human follicular adenoma (FA) thyroid was studied. No difference in BPA uptake was observed between proliferating and quiescent ARO cells. The uptake by quiescent ARO, BT, and FA showed that the ARO/BT and ARO/FA ratios were 4 and 5, respectively (p < 0.001). In in vivo studies, ARO cells were transplanted into the scapular region of NIH nude mice, and after 2 weeks BPA (350 or 600 mg/kg body weight) was injected intraperitoneally. The animals were sacrificed between 30 and 150 minutes after the injection. With 350 mg, tumor uptake was highest after 60 minutes and the tumor/normal thyroid and tumor/blood ratios were 3 and 5, respectively. When 600 mg/kg body weight BPA were administered, after 90 minutes the tumor/blood, tumor/normal thyroid, and tumor/distal skin ratios for 10B concentrations per gram of tissue were approximately 3, showing a selective uptake by the tumor. The present experimental results open the possibility of applying BNCT for the treatment of UTC.

Use of correction procedures in ploidy analysis of oral carcinomas.

Ploidy analysis is an aid in the diagnosis and evaluation of prognosis of tumors. Image analysis is a relatively simple technique to assess ploidy that can be carried out with accessible equipment. However, it requires the use of accurate technical procedures to avoid methodological errors that may bias the measurements. We previously developed two procedures that are simple to apply in routine work and serve to correct the errors derived from the measurement of large nuclei that are not fully contained in the thickness of the section and those derived from non-specific background readings. In the present study we applied these corrections to the retrospective ploidy analysis of a series of 67 patients with oral carcinoma with a follow-up time of 18 months. Thirty-four patients were alive at the end of the study, 33 were deceased. The ploidy values and the malignancy indices corresponding to the deceased and live patients with TNM stage III and IV carcinomas at the time of biopsy were significantly different. There were no significant differences in ploidy values between live and deceased patients with TNM stage I and II at the time of biopsy. The corrections improved the sensitivity of the method and thus the statistical significance of the data. These data suggest that the method proposed may be of use to estimate lesion evolution, in particular in patients with advanced oral squamous cell carcinomas.

Role of crevice in the occurrence of hemoglobin in saliva.

The aim of the present study was to analyze the role of the gingival-periodontal component in the presence of blood in saliva. We studied a population of 184 adult patients composed of 101 totally edentulous patients with no oral mucosa lesions and 83 dentulous patients with clinically healthy gums. Saliva samples obtained by spontaneous salivation were collected in sterile containers. Qualitative and quantitative evaluation of each of the samples was performed. 67% of the dentulous patients tested positive for hemoglobin in saliva. None of the patients in the totally edentulous group exhibited hemoglobin in saliva. These data suggest that the periodontal condition is an essential factor for the presence of hemoglobin in saliva.

Use of correction procedures in ploidy analysis of oral carcinomas

Ploidy analysis is an aid in the diagnosis and evaluation of prognosis of tumors. Image analysis is a relatively simple technique to assess ploidy that can be carried out with accessible equipment. However, it requires the use of accurate technical procedures to avoid methodological errors that may bias the measurements. We previously developed two procedures that are simple to apply in routine work and serve to correct the errors derived from the measurement of large nuclei that are not fully contained in the thickness of the section and those derived from non-specific background readings. In the present study we applied these corrections to the retrospective ploidy analysis of a series of 67 patients with oral carcinoma with a follow-up time of 18 months. Thirty-four patients were alive at the end of the study, 33 were deceased. The ploidy values and the malignancy indices corresponding to the deceased and live patients with TNM stage III and IV carcinomas at the time of biopsy were significantly different. There were no significant differences in ploidy values between live and deceased patients with TNM stage I and II at the time of biopsy. The corrections improved the sensitivity of the method and thus the statistical significance of the data. These data suggest that the method proposed may be of use to estimate lesion evolution, in particular in patients with advanced oral squamous cell carcinomas.

Role of crevice in the occurrence of hemoglobin in saliva

The aim of the present study was to analyze the role of the gingival-periodontal component in the presence of blood in saliva. We studied a population of 184 adult patients composed of 101 totally edentulous patients with no oral mucosa lesions and 83 dentulous patients with clinically healthy gums. Saliva samples obtained by spontaneous salivation were collected in sterile containers. Qualitative and quantitative evaluation of each of the samples was performed. 67

of the dentulous patients tested positive for hemoglobin in saliva. None of the patients in the totally edentulous group exhibited hemoglobin in saliva. These data suggest that the periodontal condition is an essential factor for the presence of hemoglobin in saliva.

Role of crevice in the occurrence of hemoglobin in saliva.

The aim of the present study was to analyze the role of the gingival-periodontal component in the presence of blood in saliva. We studied a population of 184 adult patients composed of 101 totally edentulous patients with no oral mucosa lesions and 83 dentulous patients with clinically healthy gums. Saliva samples obtained by spontaneous salivation were collected in sterile containers. Qualitative and quantitative evaluation of each of the samples was performed. 67

of the dentulous patients tested positive for hemoglobin in saliva. None of the patients in the totally edentulous group exhibited hemoglobin in saliva. These data suggest that the periodontal condition is an essential factor for the presence of hemoglobin in saliva.

Use of correction procedures in ploidy analysis of oral carcinomas.

Ploidy analysis is an aid in the diagnosis and evaluation of prognosis of tumors. Image analysis is a relatively simple technique to assess ploidy that can be carried out with accessible equipment. However, it requires the use of accurate technical procedures to avoid methodological errors that may bias the measurements. We previously developed two procedures that are simple to apply in routine work and serve to correct the errors derived from the measurement of large nuclei that are not fully contained in the thickness of the section and those derived from non-specific background readings. In the present study we applied these corrections to the retrospective ploidy analysis of a series of 67 patients with oral carcinoma with a follow-up time of 18 months. Thirty-four patients were alive at the end of the study, 33 were deceased. The ploidy values and the malignancy indices corresponding to the deceased and live patients with TNM stage III and IV carcinomas at the time of biopsy were significantly different. There were no significant differences in ploidy values between live and deceased patients with TNM stage I and II at the time of biopsy. The corrections improved the sensitivity of the method and thus the statistical significance of the data. These data suggest that the method proposed may be of use to estimate lesion evolution, in particular in patients with advanced oral squamous cell carcinomas.

Effect of total body irradiation on peri-implant tissue reaction: an experimental study.

The aim of the present study was to evaluate the osseointegration process under total body irradiation conditions (LD 50/30). Twenty Wistar rats (mean body weight: 90 g) were used. Under ethyl urethane intraperitoneal anesthesia (1 g/kg body weight), the animals were irradiated with a single 700 cGy dose (linear accelerator 6 Mev photons). Four days post irradiation, a titanium laminar implant was placed in the left tibia of each rat. Antibiotic therapy (ceftriaxone) was administered daily post implantation, to prevent infection by radiation. Fourteen days post implantation, the animals were killed by ether overdose. The tibiae were resected, radiographed and processed for embedding in methyl methacrylate. The results showed impaired osteogenesis and absence of osseointegration in experimental tibiae. This could be due to a direct action of total body irradiation on osteogenesis precursor cells. This effect would impair bone formation involved in peri-implant osseointegration processes in this experimental model.

An experimental model to study intrusive forces in rats.

We have developed an experimental model for the study of the response of the periodontal ligament and bone to intrusive orthodontics forces in rats. Thirty-five Wistar rats, body weight 250 gr., were grouped as follows: control (GI), 48 hs (GII), 96 hs (GIII), 7 days (GIV). A steel band was cemented to the first upper right molar with a welded spring running to the occlusal surface of the second molar to exert a vertical force of 15 gr. The left side of treated animals was also used as control. The animals were sacrificed at the corresponding treatment times. Radiographs of the left and right halves of the jawbones were taken with an ultrafine grain industrial film. Seven measurements of the width of the periodontal ligament were taken. The data reveal narrowing of the periodontal ligament at the peri-apex and the furcation area for the animals in Group II. Conversely, Group III and IV animals exhibited widening of the periodontal ligament, particularly in the animals submitted to the force for 7 days. Statistical analysis of the data by Student's paired t test showed that the difference for the latter group was statistically significant (p < 0.05). The data for the horizontal measurements do not afford conclusive results. The present results confirm the value of the model to study the response to vertical intrusive forces.