In-vivo evaluations of bone regenerative potential of two novel bioactive glasses.

Due to the aging of population, materials able to repair damaged tissues are needed. Among others, bioactive glasses (BGs) have attracted a lot of interest due to their outstanding properties both for hard and soft tissues. Here, for the first time, two new BGs, which gave very promising results in preliminary in vitro-tests, were implanted in animals in order to evaluate their regenerative potential. The new BGs, named BGMS10 and Bio_MS and containing specific therapeutic ions, were produced in granules and implanted in rabbits' femurs for up to 60 days, to test their biocompatibility and osteoconduction. Additionally, granules of 45S5 Bioglass® were employed and used as a standard reference for comparison. The results showed that, after 30 days, the two novel BGs and 45S5 displayed a similar behavior, in terms of bone amount, thickness of new bone trabeculae and affinity index. On the contrary, after 60 days, 45S5 granules were mainly surrounded by wide and scattered bone trabeculae, separated by large amounts of soft tissue, while in BGMS10 and Bio_MS the trabeculae were thin and uniformly distributed around the BG granules. This latter scenario could be considered as more advantageous, since the features of the two novel BG granules allowed for the neo-formation of a uniformly distributed bony trabeculae, predictive of more favorable mechanical behavior, compared to the less uniform coarse trabeculae, separated by large areas of soft tissue in 45S5 granules. Thus, BGMS10 and Bio_MS could be considered suitable products for tissue regeneration in the orthopedic and dental fields.

Effect of self-adjusting file and WaveOne reciprocating file on the filling ability of oval-shaped canals with thermoplasticized gutta-percha.

The aim of the study was to compare the effect of Self-Adjusting Files (SAF) and WaveOne Primary file with syringe and needle irrigation on the filling ability of oval-shaped root canals obturated with thermoplasticized gutta-percha. Twenty-four single root teeth with single oval-shaped root canals were distributed into two experimental homogeneous groups. One group was instrumented and cleansed using the SAF system while in the other group the WaveOne system with syringe and needle irrigation was used. After instrumentation, the roots were filled by Thermafil Obturators and TopSeal sealer. Specimens were transversally sectioned at 2-, 5- and 7-mm levels from the apex and observed under light microscope. The percentage of gutta-percha filled area (PGFA), the percentage of sealer filled area (PSFA) and the percentage of voids area (PVA) were measured for each section, moreover the percentage of completely filled sections was evaluated. At all levels, no significant differences in terms of PGFA, PSFA, PVA and percentage of completely filled canals between groups were obtained (P > 0.05). On the contrary, when the data were pooled, the mean PGFA in the SAF group was 95.8%, whereas it was 93.2% in the WaveOne group (P less than 0.05). The percentage of sections completely filled was 77.8% in the SAF group, and 52.8% in the WaveOne group (P less than 0.05). Overall, the use of the SAF system in oval canals allows to obtain a significantly greater complete filling than the use of the WaveOne system.

Sodium hypochlorite solution penetration into human dentine: a histochemical evaluation.

AIM: The aim of this study was to evaluate ex vivo the penetration depth of sodium hypochlorite solutions, with and without surfactants, into human dentine using a histochemical stain and the bleaching ability of NaOCl. METHODOLOGY: Extracted maxillary central incisors were decoronated and their root canals were shaped to a size 30, .07 taper. Dentinal tubules were stained with 10% copper sulphate solution followed by a 1% rubeanic acid alcohol solution under vacuum. Specimens were irrigated with 5 mL of Niclor 5 (5.25% NaOCl solution) or 5 mL of Hypoclean (5.25% NaOCl solution + surfactants) according to the corresponding treatment group, for 2 min. A transverse section was obtained in the middle portion of the apical, middle and coronal thirds of each tooth. Under microscopic examination, irrigant penetration was evaluated by measuring the following: the bleached circumference of the root canal with respect to the stained circumference; bleached areas, mean and maximum penetration depth. For statistical analysis, Pearson's chi-squared test and one-way anova were used. RESULTS: Only 30% of dentine tubules in the apical portions were stained; therefore, these were excluded from further comparisons. In the middle and coronal thirds, the average bleached circumference of the root canal was 80-99% of the stained circumference and the bleached area was 73757-135107 µm2 . The average penetration depth ranged from 39 to 62 µm and maximum penetration from 74 to 131 µm. For all reported results, no differences within and between groups were observed (P = 1). CONCLUSION: No differences in penetration of irrigants with and without surfactants into dentinal tubules were observed in the middle and coronal thirds of incisor teeth.

Double dye technique and fluid filtration test to evaluate early sealing ability of an endodontic sealer.

OBJECTIVES: The aim of the present study is to evaluate the possible correlation between sealer penetration into dentinal tubules and sealing ability both in presence and absence of smear layer. MATERIALS AND METHODS: Fourteen maxillary central incisors were treated with 5.25 % NaOCl +10 % EDTA to remove the smear layer (SL-free group) or 5.25 % NaOCl without EDTA (SL group). Root canals were filled using #25 Thermafil Obturators with Topseal sealer labelled with 0.1 wt% rhodamine B. Sealing ability was measured as fluid filtration rate with a fluid-flow meter using water supplemented with 0.3 % calcein fluorescent dye. Specimens were sectioned, observed under confocal microscope to co-localize the presence of sealer (rhodamine B labelling) into dentinal tubules and gaps (calcein labelling) into the root canal. The depth of sealer penetration into dentinal tubules and the percentage of sealer penetration around the root canal were measured at 3, 5 and 8 mm from the apex. RESULTS: No significant differences between groups were observed in fluid filtration rate nor in depth of calcein penetration. Sealer penetration depth and percentage into dentinal tubules were not significantly different between groups, except at 8-mm level in absence of smear layer. CONCLUSION: Sealer penetration at 3- and 5-mm levels was not influenced by smear layer while it was significantly reduced at 8-mm level. Fluid filtration rate was not correlated either with depth of calcein penetration nor with sealer penetration into dentinal tubules. CLINICAL RELEVANCE: The sealing ability of Topseal sealer is not affected by presence or absence of smear layer.

The biocompatibility of porous vs non-porous bone cements: a new methodological approach.

Composite cements have been shown to be biocompatible, bioactive, with good mechanical properties and capability to bind to the bone. Despite these interesting characteristic, in vivo studies on animal models are still incomplete and ultrastructural data are lacking. The acquisition of new ultrastructural data is hampered by uncertainties in the methods of preparation of histological samples due to the use of resins that melt methacrylate present in bone cement composition. A new porous acrylic cement composed of polymethylmetacrylate (PMMA) and ß-tricalciumphosphate (ß-TCP) was developed and tested on an animal model. The cement was implanted in femurs of 8 New Zealand White rabbits, which were observed for 8 weeks before their sacrifice. Histological samples were prepared with an infiltration process of LR white resin and then the specimens were studied by X-rays, histology and scanning electron microscopy (SEM). As a control, an acrylic standard cement, commonly used in clinical procedures, was chosen. Radiographic ultrastructural and histological exams have allowed finding an excellent biocompatibility of the new porous cement. The high degree of osteointegration was demonstrated by growth of neo-created bone tissue inside the cement sample. Local or systemic toxicity signs were not detected. The present work shows that the proposed procedure for the evaluation of biocompatibility, based on the use of LR white resin allows to make a thorough and objective assessment of the biocompatibility of porous and non-porous bone cements.

In vivo effect of two different pulsed electromagnetic field frequencies on osteoarthritis.

Osteoarthritis (OA) is a joint pathology characterized by fibrillation, reduced cartilage thickness and subchondral bone sclerosis. There is evidence that pulsed electromagnetic fields (PEMFs) counteract OA progression, but the effect of two different PEMF frequencies has not yet been shown. The aim of this study was to test the effectiveness of PEMFs at two different frequencies (37 and 75 Hz) in a late OA stage in 21-month-old Guinea pigs. After 3 months of 6 h/day PEMF stimulation, histological and histomorphometric analyses of the knees were performed. At both frequencies, PEMFs significantly reduced histological cartilage score, fibrillation index (FI), subchondral bone thickness (SBT) and trabecular number (Tb.N) and increased trabecular thickness (Tb.Th) and separation (Tb.Sp) in comparison to the not treated SHAM group. However, PEMFs at 75 Hz produced significantly more beneficial effects on the histological score and FI than 37 Hz PEMFs. At 75 Hz, PEMFs counteracted cartilage thinning as demonstrated by a significantly higher cartilage thickness values than either those of the SHAM or 37 Hz PEMF-treated groups. Although in severe OA both PEMF frequencies were able to limit its progression, 75 Hz PEMF stimulation achieved the better results.

Ablation of bone cells by electroporation.

Short intense electrical pulses transiently increase the permeability of the cell membrane, an effect known as electroporation. This can be combined with antiblastic drugs for ablation of tumours of the skin and subcutaneous tissue. The aim of this study was to test the efficacy of electroporation when applied to bone and to understand whether the presence of mineralised trabeculae would affect the capability of the electric field to porate the membrane of bone cells. Different levels of electrical field were applied to the femoral bone of rabbits. The field distribution and modelling were simulated by computer. Specimens of bone from treated and control rabbits were obtained for histology, histomorphometry and biomechanical testing. After seven days, the area of ablation had increased in line with the number of pulses and/or with the amplitude of the electrical field applied. The osteogenic activity in the ablated area had recovered by 30 days. Biomechanical testing showed structural integrity of the bone at both times. Electroporation using the appropriate combination of voltage and pulses induced ablation of bone cells without affecting the recovery of osteogenic activity. It can be an effective treatment in bone and when used in combination with drugs, an option for the treatment of metastases.

Gas-phase phenol methylation over Mg/Me/O (Me = Al, Cr, Fe) catalysts: mechanistic implications due to different acid-base and dehydrogenating properties.

This contribution reports about an in situ FT-IR investigation and the catalytic reactivity of Mg/Me(3+) mixed oxides (Me = Cr, Fe, or Al; Mg/Me = 2, atomic ratio) in the gas-phase methylation of phenol with methanol. It is the second of two papers concerning the mentioned systems, and its purpose is twofold: to confute the classic and not accurate theory concerning the reaction mechanism, and to propose a novel interpretation based on the combined use of catalytic tests and in situ molecular spectroscopy. Results here reported highlight that: (i) the reaction mechanism in phenol methylation, when catalysed by basic systems, is not a classical electrophylic substitution, as generally reported in the literature, but proceeds through the formation of formaldehyde as an intermediate, and (ii) the catalytic behaviour in respect to both methanol and phenol reactants is strictly dependent on catalyst features. Although all investigated systems exhibit a basic-type behaviour with regard to phenol, which dissociates to yield an adsorbed phenolate species, the distribution of phenolic compounds obtained with the Mg/Al/O catalyst was that typically observed with acid catalysts, with prevailing formation of anisole when the reaction was carried out below 350 degrees C and of mono and poly-C-alkylated compounds when the reaction temperature was above 350 degrees C. On the contrary, the reactivity shown by both Mg/Fe/O and Mg/Cr/O systems was that reported in the literature as typical of mixed oxides possessing basic features. The extent of methanol decomposition into light compounds was maximum in the case of Mg/Fe/O catalysts, because of the pronounced redox behaviour typical of Fe(3+) species, whereas neither methanol dehydrogenation nor decomposition were ever observed with Mg/Al/O up to 400 degrees C. Reactivity tests and spectroscopic experiments hinted for methanol dehydrogenation to formaldehyde as the first step in the ring-methylation of phenol with Mg/Cr/O and Mg/Fe/O: in that case, o-cresol and 2,6-xylenol were the only reaction products. But, with Mg/Al/O systems, for which no methanol dehydrogenation occurred, the formation of anisole was due to the synergistic effect of stronger basic features and the presence of Lewis acidic sites, that facilitate the reaction between phenol and methanol after activation over the two different types of catalytic sites.

Influence of additives in defining the active phase of the ethylene oxychlorination catalyst.

The understanding, at the atomic level, of the role played by additives (dopants or promoters) in the chemistry of an industrial catalyst is a very complex and difficult task. We succeeded in this goal for the ethylene oxychlorination catalyst (CuCl(2)/gamma-Al(2)O(3)), used to produce dichloroethane, a key intermediate of the polyvinyl chloride chemistry (PVC). Among the most used additives for both fluid and fixed beds technologies (LiCl, KCl, CsCl, MgCl(2), LaCl(3), CeCl(4)) we have been able to highlight that KCl, and CsCl, forming in reaction conditions a mixed phase with CuCl(2), strongly modify the catalyst behaviour. In particular, these additives are able to displace the rate determining step from the CuCl oxidation (undoped catalyst) to the CuCl(2) reduction. This results from the decrease of the rate of the latter reaction, thus the overall activity of the system. For all remaining additives the rate determining step remains the CuCl oxidation, as for the undoped catalyst. These results have been obtained coupling the catalyst activity monitored with a pulse reactor working in both non-depletive and depletive modes with time resolved XANES spectroscopy performed under in operando conditions (i.e. coupled with mass spectrometry). Formation of CuK(x)Cl(2+x) and CuCs(x)Cl(2+x) mixed phases has been proved monitoring the Cu(II) d-d transitions with UV-Vis spectrometer and the CO stretching frequency of carbon monoxide adsorbed on reduced catalyst by in situ IR spectroscopy. Finally, of high relevance is the observation that the fully oxidized catalyst is inactive. This unexpected evidence highlight the role of coordinatively unsaturated Cu(I) species in adsorbing ethylene on the catalyst surface indicating that copper, in the working catalyst, exhibits a (I)/(II) mixed valence state.

Influence of density, elasticity, and structure on ultrasound transmission through trabecular bone cylinders.

The aim of this in vitro study is to evaluate the potentiality of quantitative ultrasound (QUS) to separate information on density, elasticity, and structure on specimens of trabecular bone. Fifteen cylinders of spongy bone extracted from equine vertebrae were progressively demineralized and subjected to QUS, micro computed tomography (muCT), Dual energy X-ray absorptiometry (DXA) at various mineralization levels. Eventually all cylinders underwent a compression test to calculate the Young's modulus. Correlation analysis shows that speed of sound (SOS) is strictly associated to bone mineral density (BMD), Young's modulus, and all muCT parameters except for degree of anisotropy (DA). Fast wave amplitude (FWA) is directly correlated with bone surface and total volume ratio (BS/TV) and trabecular separation (Tb Sp), and inversely correlated with trabecular number (Tb N). Because muCT parameters were strictly correlated to BMD and Young's modulus data, partial correlation analysis was performed between SOS, FWA, and structural and elastic data in order to eliminate the effect of density. SOS was significantly correlated to bone volume and total volume ratio (BV/TV), BS/TV, and Young's modulus, and FWA was significantly correlated to Tb Sp only. These results show that SOS is strongly influenced by volumetric mineral bone density and elastic modulus of the specimen, and FWA is mainly affected by trabecular separation independently on density. Therefore, SOS and FWA are able to provide different and complementary information, at least on trabecular bone samples.

Sympathectomy alters bone architecture in adult growing rats.

Sympathetic nervous system (SNS) fibres and alpha- and beta-receptors are present in bone, indicating that the SNS may participate in bone metabolism. The importance of these observations is controversial because stimulation or inhibition of the SNS has had various effects upon both anabolic and catabolic activity in this tissue. In this study we evaluated the effects of pharmacological sympathectomy, using chronic treatment of maturing male rats with 40 mg of guanethidine/kg i.p., upon various parameters in bone. Double labelling with tetracycline injection was also performed 20 and 2 days before sacrifice. Bone mass, mineral content, density and histomorphometric characteristics in different skeletal regions were determined. Bone metabolic markers included urinary deoxypyridinoline and serum osteocalcin measurements. Guanethidine significantly reduced the accretion of lumbar vertebral bone and of mineral content and density, compared to controls. Femoral bone mineral content and density were also significantly reduced, compared to controls. Histomorphometric analyses indicated these effects were related to a reduction of cortical bone and mineral apposition rate at femoral diaphysials level. Both markers of bone metabolism were reduced in controls as they approached maturity. Guanethidine significantly decreased serum osteocalcin compared to controls, while urinary deoxypyridinoline was unchanged. These data indicate that guanethidine-induced sympathectomy caused a negative balance of bone metabolism, leading to decreased mass by regulating deposition rather than resorption during modeling and remodeling of bone.

Histomorphometric and mechanical analysis of the hydroxyapatite-bone interface after electromagnetic stimulation: an experimental study in rabbits.

We investigated the effect of stimulation with a pulsed electromagnetic field on the osseointegration of hydroxyapatite in cortical bone in rabbits. Implants were inserted into femoral cortical bone and were stimulated for six hours per day for three weeks. Electromagnetic stimulation improved osseointegration of hydroxyapatite compared with animals which did not receive this treatment in terms of direct contact with the bone, the maturity of the bone and mechanical fixation. The highest values of maximum push-out force (F(max)) and ultimate shear strength (sigma(u)) were observed in the treated group and differed significantly from those of the control group at three weeks (F(max); p < 0.0001; sigma(u), p < 0.0005).

Different skeletal regional response to continuous brain infusion of leptin in the rat.

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 microg/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton.

Pulsed electromagnetic fields reduce knee osteoarthritic lesion progression in the aged Dunkin Hartley guinea pig.

An experimental in vivo study was performed to test if the effect of Pulsed Electromagnetic Fields (PEMFs) on chondrocyte metabolism and adenosine A2a agonist activity could have a chondroprotective effect on the knee of Dunkin Hartley guinea-pigs of 12 months with spontaneously developed osteoarthritis (OA). After a pilot study, 10 animals were randomly divided into two groups: PEMF-treated group (6 h/day for 3 months) and Sham-treated group. Microradiography and histomorphometry were performed on the entire articular surface of knee joints used in evaluating chondropathy severity, cartilage thickness (CT), cartilage surface Fibrillation Index (FI), subchondral bone plate thickness (SBT) and histomorphometric characteristics of trabecular epiphyseal bone. The PEMF-treated animals showed a significant reduction of chondropathy progression in all knee examined areas (p<0.05). CT was significantly higher (p<0.001) in the medial tibia plateaus of the PEMF-treated group when compared to the Sham-treated group. The highest value of FI was observed in the medial tibia plateau of the Sham-treated group (p<0.05). Significant lower values were observed in SBT of PEMF-treated group in comparison to Sham-treated group in all knee examined areas (p<0.05). The present study results show that PEMFs preserve the morphology of articular cartilage and slower the progression of OA lesions in the knee of aged osteoarthritic guinea pigs. The chondroprotective effect of PEMFs was demonstrated not only in the medial tibial plateau but also on the entire articular surface of the knee.

Influence of bone tissue density and elasticity on ultrasound propagation: an in vitro study.

Ultrasound (US) waves are mechanical vibrations that are applied to a material--bone tissue--in order to study its properties, that is, density, elasticity, and structure. In this study we evaluated in which way density and elasticity of the spongy bone influenced the transmission of 1.25 MHz US pulses. Twelve cylindrical specimens (diameter, 8 mm; height, 5 mm) excised from phalanxes of pig were decalcified with 0.5 M EDTA for different times (0, 2, and 5 days). During these periods, the samples underwent the following investigations: US transmission, density, and elasticity measurements. To assess the homogeneity of decalcification, the cross-sections of some samples were microradiographed. A detailed analysis of the US signal received was performed using velocity, Fourier analysis, and some parameters typical of signal processing technique. A good correlation was found between US velocity and density (r2 = 0.70); a lower correlation was found between velocity and elasticity (r2 = 0.59). If density and elasticity are considered simultaneously, the correlation with the US velocity improves significantly (r2 = 0.84). Fourier analysis enabled us to observe a shift of the main frequency toward lower values as the decalcification process advanced. We also observed that in the regressions weighted for density, US velocity correlated poorly with elasticity (r2 = 0.16), whereas signal processing parameters maintain a good correlation with elasticity (ultrasound peak amplitude [UPA], r2 = 0.48; slope, r2 = 0.62). In this study, it has been observed that when using a signal processing technique to analyze US pulses, it is possible to identify some parameters that are related in different ways to density and to elastic properties of bone. Our results show the potentiality of US technique to separate information on bone density and elasticity that X-ray-based densitometric methods do not provide.

Morphine coupling to invertebrate immunocyte nitric oxide release is dependent on intracellular calcium transients.

Morphine significantly stimulated invertebrate immunocyte intracellular calcium level increases in a concentration-dependent manner in cells preloaded with Fura 2/AM. Morphine's action was blocked by prior exposure of the cells to the opiate receptor antagonist naloxone. Various opioid peptides did not exhibit this ability, indicating a morphine-mu 3 mediated process. In comparing the sequence of events concerning morphine's action in stimulating both [Ca2+]i and NO production in these cells, we found that the first event precedes the second by 42 +/- 7 s. The opiate stimulation of [Ca2+]i- was attenuated in cells leached of calcium. strongly suggesting that intracellular calcium levels regulate cNOS activity in invertebrate immunocytes.

Morphine and anandamide stimulate intracellular calcium transients in human arterial endothelial cells: coupling to nitric oxide release.

Both morphine and anandamide significantly stimulated cultured endothelial intracellular calcium level increases in a concentration-dependent manner in cells pre-loaded with fura 2/AM. Morphine is more potent than anandamide (approximately 275 vs. 135 nM [Ca]i), and the [Ca]i for both ligands was blocked by prior exposure of the cells to their respective receptor antagonist, i.e., naloxone and SR 171416A. Various opioid peptides did not exhibit this ability, indicating a morphine-mu3-mediated process. In comparing the sequence of events concerning morphine's and anandamide's action in stimulating both [Ca]i and nitric oxide production in endothelial cells, we found that the first event precedes the second by 40+/-8 sec. The opiate and cannabinoid stimulation of [Ca]i was attenuated in cells leeched of calcium, strongly suggesting that intracellular calcium levels regulate cNOS activity.