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.

A radiographic, histological, and histometric study of endodontic materials.

Silicone tubes filled with CRCS, a calcium hydroxide-based cement, were implanted at two different localizations in the right tibias of white male Wistar rats. A similar number of solid silicone rods were implanted in the left tibias of the same animals and were used as inert controls. Fifteen days after implantation the bone tissue reactions to these materials were evaluated by means of radiographic, histological, and histometric methods. The radiographic and histometric analysis of the tissues around the deep end of the implants showed that the amount of reactional bone formation in contact with CRCS was significantly lower than that observed in contact with the controls (p less than 0.01). Also, the cell counts obtained from the tissues in contact with CRCS were significantly less than those obtained from the controls (p less than 0.01). This methodology appears to be a refined procedure for analyzing the possible toxic effects of endodontic materials in bone tissues. However, we feel that more extensive experiments will be necessary before predicting the long-term results which could be obtained from this experimental model.

A histomorphometric study of tissue interface by laminar implant test in rats.

Study of the implant-tissue interface is one of the fundamental issues in implantology, both odontologic and orthopedic. The characteristics of this interface will influence the success or failure of an implant. The aim of the present study was to evaluate histomorphometrically the capacity of different metals to osseointegrate employing laminar implants of zirconium, titanium, aluminum, and zirconium coated with diamond-like carbon. The experimental model herein allowed for the quantitative evaluation of the tissue-implant interface for different metals. The implants were placed in the tibiae of Wistar rats under anesthesia and allowed to remain in situ for a 30-day period. The interfaces of the zirconium and diamond-like coated zirconium implants exhibited better responses than the interface of titanium implants. Aluminum produced a local toxic effect, evidenced by osteoid formation.

Effect of biphosphonates on abnormal mandibular growth of rats intoxicated with uranium.

Intoxication with uranium induces severe alterations in kidneys and in acute intoxications inhibits bone formation and bone growth. Administered at high doses, uranium leads to death, an event that can be prevented by the administration of ethane-1-hydroxy-1,1 biphosphonate (EHBP), which would presumably exert its effect via its chelating properties. Based on this information, the aim of the present study was to determine the best therapeutic method for treatment using biphosphonates to prevent mandibular growth alterations in animals that survive uranyl nitrate poisoning. Seven groups of Wistar rats weighing 14 g were used as follows: I, untreated control; II, one intraperitoneal injection (IPI) of 2 mg Kg(-1) of body weight of uranyl nitrate (238U). In groups III to VII animals were intoxicated as in Group II and in addition were given a single injection of 10 mg Kg(-1) of ethane-1-hydroxy-1, 1-biphosphonate (EHBP), or 3-amine-1, 1-hydroxypropylene-1, 1 biphosphonate (APD) as follows; Group III, IPI of EHBP given immediately after poisoning (0 h); IV, IPI of EHBP given at 24 h; V, subcutaneous injection (SCI) of EHBP at 0 h; VI, SCI of EHBP at 48 h; VII SCI of APD at 0 h. Survivors were killed on the sixtieth day. Body weight increase, survival rate, and biometric parameters of mandibular growth (Eratalay's method) were studied. Student's "t" test was used for statistical analysis. On day 60 there was only one survivor in Group II and none in Group IV. All animals in the other groups survived. Mandibular growth parameters showed the worst results in Group VII and the best in Group V for which no difference with the controls was observed. These results show that a single subcutaneous injection of EHBP given immediately after uranium poisoning, apart from preventing death, is capable of preventing undesirable alterations in mandibular growth.

Ethane 1-hydroxy-1, 1-diphosphonate (EHDP) counteracts the inhibitory effect of uranyl nitrate on bone formation.

The beneficial effect of ethane 1-hydroxy-1, 1-diphosphonate (EHDP) in restoring the inhibition of bone formation in cases of acute uranium intoxication is presented. Bone formation was studied histomorphometrically in a model of alveolar bone healing. After tooth extraction, 40 rats were divided into 4 groups that received (1) no further treatment, (2) 10 daily intraperitoneal injections of 7.5 mg/kg of body weight of EHDP, (3) an intraperitoneal injection of 2.0 mg/kg of body weight of uranyl nitrate, and (4) the same treatment as was provided rats in groups 2 and 3. The results showed that the healing of bone did not occur in exposed animals, whereas healing in EHDP-treated exposed animals did not differ from that of nonexposed controls. This effect might result from a blocking and/or competitive action of EHDP and/or the stimulation that EHDP elicits at the doses and in the administration period studied.

Chronodynamic evaluation of the stages of osseointegration in zirconium laminar implants.

Osteogenesis occurs throughout all stages in life, due to both bone turnover and reparative processes. Thus, osseointegration (OI) can be described as the final step in a cascade of processes involved in bone healing in relation to implants. Ten groups of 5 Wistar rats each (mean = 90 g b.w.) were used. Under ether anesthesia a zirconium laminar implant was placed in the tibia following the method previously described by our laboratory (Cabrini et al Imp Dent 2:264-7, 1993). The animals were killed at Ohs, and 1, 2, 3, 6, 7, 10, 12, 14, and 60 days post-implantation. Tibiae were resected, radiographed and processed for their embedding in methyl methacrylate. Three sections, perpendicular to the major axis of the tibia, were obtained per implant and histologic and histomorphometric studies were carried out. Volume occupied by blood clot, woven bone, percentage of OI and OI bone tissue thickness, were determined. Histologic and histomorphometric studies as function of time revealed: a) at 6 days the presence of non-osseointegrated woven bone around the device is evident increasing in volume from 7 to 10 days post-implantation, and disappearing from day 12 to 14., b) at 14 days after implantation lamellar bone formation on the surface of the zirconium implants (OI) is noticeable. Additional bone growth is observed after 60 days. This study enables quantification of peri-implant reparative process response in an unloaded, necrotic trabeculae free model showing, in the different phases of the osseointegration process, the role of the blood clot and of the appearance and disappearance of woven bone and the final stages of osseointegration. Further investigation will allow comparison of results obtained under the effect of local and/or systemic factors that might affect osseointegration.

Enlargement of periosteocytic lacunae associated to mechanical forces.

Demineralization of bone has been linked to the action of osteocytes via the process of osteocytic osteolysis. The aim of this study was to assess the influence of mechanical forces produced during orthodontic movements on the osteocytic lacunae. Orthodontic movements were achieved employing a device constructed "ad hoc" for rats that exerts a force of approximately 70 gr. The experimental animals and the corresponding controls were killed 48 and 96 hours after the onset of the experiment. Histologic sections oriented along the bucco-palatine axis were employed to measure the area of osteocytic lacunae to infer information on volume in keeping with standard stereological concepts. Regions alongside resorption areas of cortical bone and resting areas of palatine bone were evaluated. Osteocytic lacunae associated to erosive surfaces were rounded and rose markedly in area after the application of the orthodontic force (58.4 +/- 6 mm2). Elongated lacunae were present in relation to resting areas (24.8 +/- 2 mm2). The present study shows an association between the increase in size of osteocytic lacunae and the resorption fronts induced by the application of orthodontic forces. This finding would suggest that the osteocyte would participate in the resorption process of bone submitted to pressure.

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.

An original orthodontic appliance for experimental mesial movements in rats.

The design of an original appliance to achieve mesial movement of the first upper molar on one side in rat is presented. The appliance is constructed in 0.4 mm stainless steel wire forming a parallelepiped 3 mm in width in its anterior sector, 4 mm in width in its posterior sector and 12 mm in length in its lateral branches. In addition a central longitudinal bar is welded to the structure. The lateral branches of the appliance slide freely through the tubes welded to the palatal aspect of the molar bands, cemented in turn to both first molars. A wire 2 mm in length is welded in the anchorage area to the mesial side of the molar tube at 3 mm from the anterior end of the appliance, which acts as a butt. A pre-formed nickel-titanium open coil spring 0.23 mm thick, lumen 0.60 mm and 5.00 mm in length is placed distal to the molar tube. The spring is compressed by 1 mm of its original length, the final force being approximately 50 g. The movement achieved was measured on plaster casts obtained from pre and postoperative impressions, and afforded values of 0.250 mm +/- 0.790 mm on the active side and 0.012 +/- 0.011 mm on the passive side. The histologic studies showed an extensive erosive area on the pressure side of the alveolar wall. The appliance presented will be useful to achieve models of experimental movements of only one molar towards mesial with a force of known magnitude.

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.

Use of Zircalloy to induce bone regeneration. A 2-year follow-up study.

In a previous study we analyzed the behaviour of Zircalloy 4 particles employed in patients with significant loss of periodontal bone support which threatened the permanence in situ of the tooth. We herein present the results obtained after regular examination over a 2-year period of 6 patients submitted to this treatment. Our results reveal that all patients exhibited excellent gingivo-periodontal health with an increase in clinical insertion of 7 mm +/- 1 and a marked reduction in motility from 3 to 1. None of the cases featured added inflammation or expulsion of the implant material. In one of the cases it was possible to study the histology of the area by punch biopsy with a disposable needle (Sherwood-Medical). Analysis of the sample revealed the presence of bone tissue in intimate contact with implanted metallic particles of Zircalloy 4, producing osseointegration. This osseointegration would lead to filling of the periodontal bone defect. Macrophages loaded with metallic particles were found in the vicinity of particles which were not osseointegrated. These features may correspond to superficial areas related with soft tissue as observed in our study on alveolar filling in rats with the same type of particles.

Bone growth is impaired by uranium intoxication.

Acute and chronic uranium intoxication leads to the inhibition of bone formation and impaired bone modeling and remodeling. As these are processes directly involved in bone growth the aim of this paper is to present a biometric study of bone growth--tibiae and mandibles of rats intoxicated with uranium. Wistar ratios weighing 60-80 g were used as follows, a) one intraperitoneal injection (IPI, 2 mg/Kg of body weight)) of uranyl nitrate; b) 30 daily applications on the dorsal skin of aliquots of a mixture of U308, concentrated at 2% and at 4%--percutaneous absorption(PA)-. Tibia and mandible length were smaller in both experimental groups than in their respective controls. Some of the mandibular parameters were lower in intoxicated animals than their controls which in turn results in the alteration of the mandibular shape. We conclude that impairment in bone growth can be achieved by uranium intoxication.

A method for the quality control of osseointegration in endosseous implants.

Metallic dental and orthopedic implants are essential therapeutic tool. The biologic success of an implant involves intimate contact between the implant and vital bone tissue, an event which has been termed osseointegration. The aim of the present study was to study the biocompatibility and biomechanic properties of different implant materials. Zircalloy 4 metallic cones (1.8 mm base x 2 mm height) were implanted in the diaphysis of the tibiae of 6 Wistar rats (90 g body weight) under i.p. Ethyl Urethane anesthesia. The animals were killed 30 days postimplantation, the tibiae were resected, and radiographed. One side chosen at random was used for the biomechanical study; whereas the other implant of each animal was processed for embedding in methyl-methacrylate. The bond of the implanted material to bone tissue was monitored immediately after resecting the tibiae by applying an extraction force to the base of the cone via a device designed ad hoc connected to a testing machine. Histological and radiographic analyses revealed the presence of bone tissue in contact with the implant surface. A force of about 35 g was necessary to separate the implant from the bone tissue. The system proposed renders possible the study of biocompatibility in histologic terms and in terms capacity to bond to bone tissue and could be a valuable research tool and the basis for quality control of all types of material, metallic or otherwise, used in endosseous implants.

Early steps in bone resorption in experimental periodontitis: a histomorphometric study.

A histomorphometric study of the initial steps in the process of bone resorption of inflammatory origin is presented. The study was performed in a model of experimental periodontitis, in which plaque formation is induced by means of a cotton thread ligature placed around the neck of the first molar of Wistar rats. The treated animals were killed in groups after 24, 48, 72 and 96 hours. An equivalent number of untreated rats were employed as controls. After routine histologic processing mesiodistal sections were obtained. The histologic studies showed the sequence of events of the inflammatory process and bone resorption. The histomorphometric studies showed the concomitant increase in erosive areas and number of osteoclasts with the highest values at 72 hours. At 96 hours both parameters decreased. The results are discussed in the light of those afforded by other experimental models, in which bone resorption is induced by orthodontic forces. In these models the increase in the resorptive areas precedes the increase in the number of osteoclasts. The existence of two different mechanisms for the initiation of bone resorption, one which involves the action of an inflammatory environment and the other which results from the action of mechanical forces, is proposed.

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.

An experimental model to study implant corrosion.

Corrosion phenomena would appear to play a decisive role in metallic implant long term behaviour. This study presents a method to correlate results obtained in "in vivo" and "in vitro" studies on materials used for metallic implants. Samples of titanium and copper immersed in saline solution (pH 7.4 and 5.2) were used for the "in vitro" study. Implants of these same materials were placed in rat tibiae following the method previously described by Cabrini et al. The animals were killed 14 days post-implantation, the tibiae were resected, radiographed and embedded in acrylic resin. Polarization curves revealed high corrosion in copper implants and low corrosion in titanium implants. It is important to point out that the titanium samples suffered slightly higher corrosion when immersed in a lower pH medium (pH 5.2), as in chronic inflammatory processes. A passive film was found on titanium samples as opposed to the strong corrosion observed in the copper implants. Microscopy revealed osseointegration around titanium implants and a severe inflammatory reaction with abscesses surrounding the copper implants. The method presented herein would allow to correlate "in vivo" and "in vitro" studies on corrosion in different implant materials and establish their relation with cell response.

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.

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.