Effects of soft-drinks and remineralising treatment on teeth assessed by morphological and quantitative X-ray investigations.

AIM: To morphologically and chemical-physically analyse both the surface and the subsurface of enamel undergoing soft- drink demineralisation and remineralisation treatment. MATERIALS AND METHODS: Fifteen human premolars were split and immersed in saline or three popular soft drinks, as demineralising agent, 15 minutes per day, for seven days at room temperature. Half of drink-processed teeth was then treated with casein phosphopeptide-amorphous calcium phosphate, as remineralising agent, for an additional seven days. The surface morphology was evaluated by stereomicroscope and scanning electron microscope (SEM). Teeth were then re-embedded and sectioned, and analysed under SEM and X-ray microprobe. RESULTS: Drink-processed teeth showed root pigmentation, opacification and deterioration of the superficial enamel. The enamel surface resulted greatly furrowed after drink processing, and apparently restored after remineralising treatment. However, in tooth sections, SEM showed always a subsurface demineralisation of dentine and enamel, in particular at the cementoenamel junction, also after reminalising treatment. The remineralising agent produced a partial remineralisation of the subsurface enamel, sometimes statistically significant, but not in hydroxyapatite stoichiometry. CONCLUSION: Soft-drink erosion impaired not only the surface but also the subsurface enamel. The applied remineralising treatment, yielding some effects on surface and subsurface enamel reversing basically the decalcification process.

Cytopathological and chemico-physical analyses of smears of mucosa surrounding oral piercing.

OBJECTIVE: The aim of this comparative study was to analyze cytopathologically and chemico-physically the mucosa surrounding oral piercing to correlate results with adverse tissue signs. MATERIALS AND METHODS: The tongue superficial mucosa of 15 young subjects (control group) and the superficial mucosa surrounding oral piercing of 15 young subjects (test group, TG) were smeared on slides, Papanicolaou stained and analyzed under the optical microscope. Some smears were prepared for (back-scattered) scanning electron microscope (SEM) and X-ray microanalysis to study piercing fragments. RESULTS: Smears of TG displayed a variable extent of bacterial cytolysis of epithelial cells, fungi, hyperkeratosis, parakeratosis, granulocyte infiltration, calcium formations and bacterial flora; the four last statistically significant (P < 0.05). Foreign bodies surrounded by keratinocytes were detected under both light and SEM. X-ray microanalyses highlighted piercing alloy aggression, ion release and an inverse gradient of ion concentration inside keratinocytes. CONCLUSIONS: The pathological findings in smears correlated with adverse effects of oral piercing. Ion release may be related to direct toxic effects and belated reactions because of metal sensitization. A strict regulation of piercing is warranted.

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage.

BACKGROUND AND PURPOSE: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. EXPERIMENTAL APPROACH: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP--MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. KEY RESULTS: All shocked rats treated with saline died within 30-35 min. Treatment with NDP--MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. CONCLUSIONS AND IMPLICATIONS: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.

Histomorphometric study of bone reactions during orthodontic tooth movement in rats.

The biological response to orthodontic tooth movement has generally focused on reactions within the periodontal ligament (PDL), whereas less attention has been paid to the behavior of neighboring bone. The purpose of the study was to describe the influence of orthodontic force on bone surrounding the displaced tooth and the adjacent, untreated teeth. Bone changes in relation to treatment time and different sites were investigated. A mesial tipping of the left maxillary first molar was obtained from 54 adult male Wistar rats. Oxytetracycline was injected subcutaneously 48 h before killing, which took place after 4, 7, or 14 days. The maxilla was fixed in paraformaldehyde and embedded undecalcified in methylmethacrylate. A set of thick horizontal sections was taken from the cervical, intermediate, and apical levels of the roots. The sections were microradiographed and analyzed microscopically under bright-field and fluorescent illumination. Bone fraction and PDL width was measured using a Zeiss Videoplan device equipped with an overlay system. New bone formation was detected by oxytetracycline labels. The analysis showed a consistent, significant decrease of the alveolar bone fraction around both displaced and adjacent teeth at all treatment times. Apposition, indicated by the tetracycline uptake, was found on the periosteal side of the treated hemimaxilla and, after 14 days, also on the surface toward which the tooth was moving and around the adjacent teeth. These results suggest that a time rather than a space relationship exists between bone resorption and formation and that the whole hemimaxilla reacts to the mechanical challenge, resembling the regional acceleratory phenomenon (RAP) observed in other circumstances.

Long-term behaviour of active glasses in sheep mandibular bone.

Granules of a glass (A) prepared according to Hench's formula and a new vitreous material for biological applications (AKRA 15) were used for repair of bone defects in the dental field. The behaviour of these materials implanted in holes drilled in sheep's mandibular bone was examined 4, 8, 12 month after implantation. Microradiographic analyses, optical and scanning electron microscopic observations, and X-ray microprobe evaluations were carried out using undecalcified, methacrylate-embedded sections of the jaw containing the granules. After one year the granules of A disappeared, but not important bone growth was observed also in the holes containing AKRA 15. SEM and microprobe showed: disappearance of Na and Si ions at different stages; increase of P and Ca up to 4 month and then decrease, but in different ways in the two glasses; unexpected appearance of K ions after 4 month only in AKRA 15.

Short-term behaviour of two similar active glasses used as granules in the repair of bone defects.

The bioactivity of two similar vitreous materials used in the form of granules of 'critical' size was investigated in bone defects in jaws of two sheep. The granules consisted of Hench's Bioglass and another glass with the same chemical composition made in Italy. Two months after implantation, the sheep were killed and elemental analyses carried out on sections of the embedded jaws. The microanalyses for both the glasses showed a diffusion from the granules towards the surrounding tissue of silicon and sodium, and an inverse diffusion (from the surrounding tissue towards the granules) of calcium and phosphorus. The degradation for the Italian glass was slower than for the Bioglass. No significant osteoinduction was seen after that time at the interface of the glass granules or in the bone pocket.

Behaviour of tricalcium phosphate and hydroxyapatite granules in sheep bone defects.

Granules of hydroxyapatite (HA) and tricalcium phosphate (TCP) were implanted in separate holes drilled in mandibular bone of sheep to check the bone growth and in vivo behaviour of the materials. The experiment was performed in three sheep, killed respectively at 4, 8, 12 month. Samples of bone with the materials were explanted, microradiographed and sectioned to evaluate the interface under optical and electron scanning electron microscope. The hole, left empty as a reference, showed no full repair; whereas 4 month after implantation the TCP granules induce total repair of the hole. HA granules crumbled and no new bone induction was seen even 12 month after implantation.

Neuroprotective effect of gamma-hydroxybutyrate in transient global cerebral ischemia in the rat.

The effect of gamma-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia was studied in the four vessel occlusion rat model. In saline-treated animals, 30 min ischemia caused a massive loss of neurons in the hippocampal CA1 subfield (normal neurons: 14%, 5%, 23% and 30% on the 3rd, 10th, 15th and 65th day after ischemia, respectively). gamma-Hydroxybutyrate - 300 mg/kg intraperitoneally (i.p.) 30 min before or 10 min after arteries occlusion, followed by 100 mg/kg i.p. twice daily for the following 10 days - afforded a highly significant protection (normal neurons on the 3rd, 10th, 15th and 65th day after ischemia: 88% and 91%, 80% and 80%, 91% and 90%, 72% and 71% in rats receiving the first dose before or after arteries occlusion, respectively). The ischemia-induced sensory-motor impairment was significantly attenuated in rats receiving the first dose of gamma-hydroxybutyrate before arteries occlusion. Finally, the ischemia-induced impairment in spatial learning and memory, evaluated starting 27 days after the ischemic episode, was significantly attenuated by gamma-hydroxybutyrate, either injected first at 30 min before or 10 min after arteries occlusion. Lower doses of gamma-hydroxybutyrate had no significant effect. In conclusion, these results indicate that gamma-hydroxybutyrate provides significant protection against both histological and behavioral consequences of transient global cerebral ischemia in rats.

Morphometric study of collagen maturation in chick compact bone.

An ultrastructural-morphometric study was carried out on the process of osteoid maturation in growing surfaces of parallel-fibered chick bone. The aim was to investigate the distribution, size and amount of collagen fibrils (CFs), as well as the proteoglycan (PG) content, throughout the osteoid seam and in the adjacent bone. The results show that the organic components secreted by osteoblasts undergo complete maturation inside the osteoid seam only. Proceeding from the secreting plasma membrane of osteoblasts (osteoidogenic surface) towards the mineralizing surface, we found that CFs gradually increase in diameter but not in number per surface unit. As a consequence, the proportion of osteoid seam occupied by CF increases too, at the expense of the interfibrillar substance. PG content also decreases inversely in this direction. In the adjacent bone, CF size and density do not change significantly with respect to the mature osteoid close to the mineralizing surface.

Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling.

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.

PLGA microspheres for oral osteopenia treatment: preliminary "in vitro"/"in vivo" evaluation.

The aim of this work was to prepare and to evaluate "in vitro"/"in vivo" microspheres based on poly(D,L-lactide-co-glycolide) copolymers containing ipriflavone, for the local treatment of oral bone loss. The first objective was the preparation and "in vitro" characterization of ipriflavone loaded microspheres, by emulsion/solvent evaporation method. Process parameters such as drug:polymer weight ratio, and molecular weight of copolymers, were also investigated. The second objective was to elaborate a suitable animal model of mandibular osteoporosis, to evaluate the efficacy of these microparticulate drug delivery systems. "In vivo" experiments were carried out on female rats, in which oral osteopenia was induced by gonadectomy and molar avulsion. Morphometric analysis of mandibular segment were carried out to quantify the development of oral osteopenia and the efficacy of drug loaded microspheres. Results showed that ipriflavone loaded PLGA microspheres can be successfully obtained with good "in vitro" characteristics, utilizing the emulsification/solvent evaporation method. "In vivo" experiments revealed that local administration of microspheres produced only mild inflammation on the injection site. Morphometric analyses showed, at the level of the third molar, a slight increase in spongy and total bone mass on rat jaw treated with microspheres with respect to control. Control animals exhibited a scarce degree of osteopenia demonstrating that this animal model is not suitable for this purpose.

Histomorphological analysis of the effect of rigid fixation on growing sutures in the rabbit.

The effect of internal rigid fixation (IRF) on bone growth was studied in an experimental model set up in the rabbit. The frontonasal suture of the right side was surgically bridged by a microplate. As reference for bone growth, four screws were placed symmetrically in the four bony segments including the frontonasal suture on both sides. The suture development was followed dynamically for 40 days on the basis of the position of the screws established radiographically. The rate of bone formation along the sutural bones was evaluated by means of the tetracycline labelling technique. The structure of the newly formed bone and its degree of mineralization were respectively analysed under polarized light and with microradiography. It was demonstrated that IRF prevents growth of the sutural membrane but not of the osteogenic process; as a consequence the constrained sutures soon undergo synostosis. This fact must be taken into consideration when IRF is employed in children in order to avoid delayed removal of the plate irreversibly stopping the growth of the constrained suture.

Immunohistochemical detection of cell-cycle associated markers on paraffin embedded and formalin fixed needle biopsies of prostate cancer: correlation of p120 protein expression with AgNOR, PCNA/cyclin, Ki-67/MIB1 proliferation-scores and Gleason gradings.

Paraffin embedded and formalin fixed needle biopsies of prostate cancer (PC) were used to immunocytochemically detect the p120 nucleolar protein in relation to the Gleason histological gradings (GHG), the labelling indices of proliferating nuclear immunocytochemical markers (PCNA/Cyclin, Ki-67/MIB1) and the argyrophilic nucleolar region (AgNOR) rate. The twenty-six cases of PC (6 from large histological samples and 20 from needle biopsies) were equally distributed into low (< or = 6) or high (> or = 7) GHG groups. The p120 nucleolar protein immunocytochemical reaction was randomly expressed in large histological sections but uniformly distributed without gaps in needle biopsy sections. Only on the latter were quantitative values of PCNA/Cyclin (23.2 in low and 45.3 in high GHG), Ki-67/MIB1 (13.8 in low and 43.3 in high GHG) and AgNOR (5.0 in low and 7.5 in high GHG) related to those of p120 nucleolar protein (0.8 in low and 3.8 in high GHG). The values of all these cell cycle markers increased from low to high GHG of PC, all four reaching high statistical significance between the two groups (ANOVA-two tailed p < 0.0001). The PCNA/Cyclin index showed a higher positivity than the Ki-67/MIB1 index in PC with low GHG but not in PC with high GHG. In conclusion, paraffin embedded and formalin fixed PC needle biopsies exhibit a higher diagnostic PCNA/Cyclin than Ki-67/MIB1 index for cases presenting differentiated features, whereas p120 nucleolar protein detection seems to be a suitable marker of poorer outcome of PC.

Histomorphological and chemico-physical analyses of the mineral matrix of micropetrotic human bone.

Micropetrotic areas of human bone were analyzed with reference to their distance from blood vessels and to the age of the subjects. Undecalcified bone sections were treated with various solvent, soaking and etching substances to establish the nature of the material occluding the osteocytic canalicular cavities, and were examined by means of microradiographic and microdurimetric methods and X-ray microanalysis to evaluate the degree of mineralization in the bone matrix. Since it was only after strong etching with HCl that the canalicular network became visible under light and scanning electron microscopy, it is clear that the debris filling the canalicular network consists almost entirely of mineral substance. The degree of mineralization of micropetrotic bone is high because it is always a more mature type of bone, but the mineral content of the matrix and the Ca/P ratio do not differ significantly from those of neighbouring bone where the canalicular network is fully pervious.

A reabsorbable tubular chamber for the treatment of large diaphyseal bone defects. Experimental study in rabbits.

Twenty-five rabbits were used to test the healing of diaphyseal defects by means of a reabsorbable tubular chamber. In 20 animals a 10 mm gap was created in the left radius and bridged with a poly-L-lactide chamber (experimental side). The same defect was created on the right radius, remaining untreated (control side). In the remaining 5 animals, the right radius was left intact and they were used only for biomechanical tests at 12 months. X-rays and histomorphometry showed no bone repair at the control side while the inner surface of the chamber was progressively lined by neoformed bone (75% of the normal cortical thickness). Biomechanical data demonstrated that elastic modulus and ultimate stress were not significantly different in both experimental and intact radii. The chamber, by preventing the passage of soft tissues and creating a close space for intrinsic repair factors, seems to be a suitable device for the repair of long bone defects.

Jawbone enlargement using immediate implant placement associated with a split-crest technique and guided tissue regeneration.

Five patients with sufficient vertical bone height but insufficient bone width for implant placement were chosen for treatment with a split-crest technique combined with guided tissue regeneration. The surgical technique involved splitting the alveolar ridge longitudinally in two parts, provoking a greenstick fracture. A chisel was then used to make a fine cut and spread apart the two cortical plates. Implants were then placed. Implants and defects were covered with expanded polytetrafluoroethylene membranes. Biometrical examination showed a gain in bone width, varying between 1 and 4 mm; maxillary sites showed greater ridge enlargement. Histologic examination showed regeneration of bone tissue between the two portions of the split crest. This membrane technique could be effective and predictable for horizontal ridge augmentation associated with immediate implant placement.

A comparative study of the effectiveness of e-PTFE membranes with and without early exposure during the healing period.

This study compared clinical, microbiologic, and morphostructural aspects of the healing process in clinical cases treated with expanded polytetrafluoroethylene membranes placed in fresh extraction sockets, which did or did not show early membrane exposure. The examination was focused particularly on morphology of the microbic population and its ability to penetrate membrane pores. Results of the study suggest that use of the membranes is effective in the treatment of fresh extraction sockets with osseointegrated implants; that the membranes are highly biocompatible with gingival tissues; and that early exposure of the membranes during healing hinders the effectiveness of guided tissue regeneration in periimplant tissues.