Roberto Di Lenarda

Immunohistochemical identification of MMP-2 and MMP-9 in human dentin: Correlative FEI-SEM/TEM analysis.

Matrix metalloproteinases (MMPs) are a family of peptidases trapped within mineralized dentin matrix and involved with degradation of the extracellular matrix components in hybrid layers and caries. Despite their identification through indirect evidences and biochemical assays, MMP-2 and -9 have not been localized within the human dentin extracellular organic matrix. Thus, this study aimed to assess the localization and distribution of MMP-2 and -9 in human dentin organic matrix by employing a correlative field emission in-lens-scanning electron microscopy (FEI-SEM) and transmission electron microscopy (TEM) immunohistochemical approach. Dentin specimens were submitted either to a preembedding or to a postembedding immunolabeling technique using primary monoclonal antibodies anti-MMP-2 and anti-MMP-9 and exposed to a secondary antibody conjugated with gold nanoparticles. MMP-2 and -9 labelings were identified in the demineralized dentin matrix as highly electron-dense gold particles dispersed on the collagen fibrils. Correlative FEI-SEM/TEM observations confirmed that MMP-2 and MMP-9 are endogenous components of the human dentin organic matrix and revealed the three-dimensional relationship between these proteinases and the collagen fibrils, showing that both antibodies yielded a similar labeling pattern. In conclusion, the results of the study contribute to reveal distinct distribution pattern of gelatinases and support the hypothesis that these enzymes are intrinsic constituents of the dentin organic matrix after decalcification.

Contraction stress of low-shrinkage composite materials assessed with different testing systems

OBJECTIVES The contraction stress of a silorane-based material and a new low-shrinkage nanohybrid composite were compared to three conventional dimethacrylate-based resin composites using two different measuring systems. It was hypothesized that the silorane-based material and the low-shrinkage nanohybrid composite would exhibit lower contraction stress than dimethacrylate-based composites irrespective of measuring system. METHODS The materials tested were Filtek Silorane LS (3M ESPE), Venus Diamond (Heraeus Kulzer), Tetric EvoCeram (Ivoclar Vivadent), Quixfil (Dentsply DeTrey), and Filtek Z250 (3M ESPE). Shrinkage stress was assessed using a stress-strain analyzer consisting of two opposing attachments, one connected to a load sensor and the other fixed to the device, or a system fixed to a universal testing machine with an extensometer as a feedback system. All specimens were light-cured with 20 J/cm(2); the contraction force (N) generated during polymerization was continuously recorded for 300 s. Contraction stress (MPa) was calculated at both 40 s and 300 s. Data were statistically analyzed by three-way ANOVA and Tukeys post hoc test (alpha=0.05). RESULTS Venus Diamond exhibited the lowest stress under both experimental conditions. Stress values scored as follows: Venus Diamond<Tetric EvoCeram<Filtek Silorane LS<Quixfil<Filtek Z250 (p<0.05). Stress values measured with the stress-strain analyzer were significantly lower than those measured with the universal testing machine with feedback. SIGNIFICANCE The hypothesis was partially rejected because only Venus Diamond exhibited the lowest stress values among the tested materials. Contraction stress was higher for all composites when measured in a test system with a feedback. This study confirms that simply reducing the shrinkage does not ensure reduced stress development in composites.

Effects of residual ethanol on the rate and degree of conversion of five experimental resins

OBJECTIVES This study examined the extent of ethanol retention in five comonomer blends of experimental methacrylate-based dental adhesives, containing (10, 20, or 30 wt.%) ethanol, after solvent evaporation, as well as observing the effect of residual ethanol and exposure duration on degree of conversion (DC). The null hypothesis that was tested was that residual, unevaporated ethanol has no effect on the rate or extent of DC of polymerized adhesive resins. METHODS A known mass of each mixture was placed in glass wells and evaporated for 60s. The mass of the mixtures before and after evaporation was measured, allowing calculation of the gravimetric ethanol loss/retention. RESULTS The concentration of retained ethanol increased significantly with ethanol concentration (p<0.01): 1.1-1.9 mole/L for 10% ethanol/90% comonomers, 2.2-3.5 mole/L for 20% ethanol, and 2.6-3.7 mole/L for 30% ethanol/70% comonomers. As ethanol is evaporated from solvated comonomer mixtures, the molar concentration of comonomers increases, reducing the vapor pressure of the remaining ethanol. Thus, the fractional loss of ethanol solvent decreases as the comonomer concentration increases. The DC of 10, 20, and 30 wt.% ethanol blends increased with ethanol concentration in four of the five experimental resins (p<0.05), increasing by 30-45% when 10 or 20 wt.% ethanol was added to neat resins, regardless of exposure duration. Depending on the resin system, inclusion of 30% ethanol lowered DC at 20s but increased DC after 40-60s of light exposure. SIGNIFICANCE Since 10 and 20 wt.% ethanol-resin blends increased the DC of solvated resins by 30-45% over neat resins, the test null hypothesis is rejected. Even with prolonged evaporation, 4-9% residual ethanol concentration can remain in 90/10 (wt./wt.) comonomer-ethanol mixtures. This is thought to be because comonomers lower the vapor pressure of ethanol. This amount of residual ethanol facilitates DC but lowers the rate of polymerization.

Adhesive performance of a multi-mode adhesive system: 1-Year in vitro study

OBJECTIVES The aim of this study was to investigate the adhesive stability over time of a multi-mode one-step adhesive applied using different bonding techniques on human coronal dentine. The hypotheses tested were that microtensile bond strength (μTBS), interfacial nanoleakage expression and matrix metalloproteinases (MMPs) activation are not affected by the adhesive application mode (following the use of self-etch technique or with the etch-and-rinse technique on dry or wet dentine) or by ageing for 24h, 6 months and 1year in artificial saliva. METHODS Human molars were cut to expose middle/deep dentine and assigned to one of the following bonding systems (N=15): (1) Scotchbond Universal (3M ESPE) self-etch mode, (2) Scotchbond Universal etch-and-rinse technique on wet dentine, (3) Scotchbond Universal etch-and-rinse technique on dry dentine, and (4) Prime&Bond NT (Dentsply De Trey) etch-and-rinse technique on wet dentine (control). Specimens were processed for μTBS test in accordance with the non-trimming technique and stressed to failure after 24h, 6 months or 1 year. Additional specimens were processed and examined to assay interfacial nanoleakage and MMP expression. RESULTS At baseline, no differences between groups were found. After 1 year of storage, Scotchbond Universal applied in the self-etch mode and Prime&Bond NT showed higher μTBS compared to the other groups. The lowest nanoleakage expression was found for Scotchbond Universal applied in the self-etch mode, both at baseline and after storage. MMPs activation was found after application of each tested adhesive. CONCLUSIONS The results of this study support the use of the self-etch approach for bonding the tested multi-mode adhesive system to dentine due to improved stability over time. CLINICAL SIGNIFICANCE Improved bonding effectiveness of the tested universal adhesive system on dentine may be obtained if the adhesive is applied with the self-etch approach.

Bone Scintigraphy and SPECT/CT of Bisphosphonate-Induced Osteonecrosis of the Jaw

Endovenous bisphosphonate therapy seems associated with osteonecrosis of the jaw. The aim of this study was to evaluate the additional diagnostic value of hybrid SPECT/CT in 99mTc-methylene diphosphonate 3-phase bone scintigraphy of osteonecrosis of the jaw in bisphosphonate-treated patients. Methods: We studied 15 patients (12 women and 3 men) with extraoral tumors affected by lytic bone metastases and multiple myeloma. All patients were previously treated with intravenous bisphosphonates (zoledronic acid) for 1–3 y, were negative for dental disease at clinical examination, and had suspected osteonecrosis of the jaw. All 15 patients underwent panoramic x-ray orthopantomography, CT or MRI (or both), microbiologic examination, 3-phase bone scintigraphy, and SPECT/CT of the maxillary region. Results: Three-phase bone-scintigraphy showed increased perfusion and an increased blood pool in 9 of 12 and 10 of 12 patients, respectively; at the metabolic phase, SPECT was positive in all patients and showed abnormal hyperactivity in the maxilla of 2 patients, in the mandible of 9 patients, and in both the mandible and the maxilla of 4 patients. Hybrid SPECT/CT was of particular value in 8 of 15 patients, allowing discrimination of the osteonecrotic core from nearby hyperactivity due to viable bone. Whole-body scintigraphy showed remote and multiple metastases in all patients. Orthopantomography showed nonspecific bone rarefaction in all patients but was not able to aid diagnosis of osteonecrosis of the jaw. CT and MRI showed anomalies in all symptomatic patients: CT was helpful in evaluating both cortical and trabecular bone aspects, and MRI was able to detect soft-tissue involvement but not cortical bone destruction. Conclusion: In appropriately selected oncology patients treated with bisphosphonates, an increased uptake of 99mTc-methylene diphosphonate in maxillary bones may suggest probable osteonecrosis of the jaw. In such cases, SPECT/CT may be of value in increasing the diagnostic accuracy of bone scanning, providing a precise functional anatomic correlation for the definition of the extent of disease.

Degree of conversion of Filtek Silorane Adhesive System and Clearfil SE Bond within the hybrid and adhesive layer: An in situ Raman analysis

OBJECTIVES To examine the degree of conversion (DC) of the adhesive interfaces created by Filtek Silorane Adhesive and Clearfil SE Bond using micro-Raman spectroscopy. METHODS The adhesives were applied on human dentin in accordance with manufacturers instructions. Specimens were cut to expose the bonded interfaces to the micro-Raman beam (Ranishaw InVia; laser wl 785 nm). Raman spectra were collected along the dentin/self-etching primer/adhesive interface at 1 microm intervals. The relative intensities of bands associated with mineral (P-O functional group at 960 cm(-1)) and adhesive (C-C-O group at 605 cm(-1)) components within the bonded interface were used to detect monomer penetration into the dentin matrix and to calculate the degree of conversion (C=C at 1640 cm(-1) as reaction peak, C-C-O at 605 cm(-1) as reference peak). Data were statistically analyzed with two-way ANOVA. RESULTS DC of Filtek Silorane Adhesive was 69+/-7% in the adhesive layer, increasing (p<0.05) to 93+/-5% in the primer and 92+/-9% in the hybrid layer. Clearfil SE Bond showed a DC of 83+/-3% in the hybrid and 85+/-3% in the adhesive layer. Thus, Filtek Silorane Adhesive showed a higher DC than Clearfil SE Bond in the hybrid layer (p<0.05), but a lower DC in the adhesive (p<0.05). SIGNIFICANCE As high DC is a fundamental pre-requisite for the stability of the bond over time, this study supports the hypothesis that optimal stability of Filtek Silorane Adhesive can be obtained. However, further research is needed to investigate the mechanical properties of the hybrid layer created by Filtek Silorane Adhesive and its long-term stability.

Push‐out stress for fibre posts luted using different adhesive strategies

The influence of thermocycling on the bond strength of fibre posts cemented with different luting approaches was investigated. A total of 84 human incisors were selected for the study. Sixty teeth were assigned to one of the following adhesive/cement combinations for push-out bond-strength evaluation: group 1, XP Bond/CoreXFlow + DT Light-Post; group 2, Panavia F 2.0 + Tech 21; or group 3, RelyX Unicem + RelyX. Bonded specimens were cut into 1-mm-thick slabs and either thermocycled (40,000 cycles) or stored in artificial saliva (control specimens) before push-out bond-strength testing. Additional specimens were processed for quantitative interfacial nanoleakage analysis. Thermocycling decreased the bond strength in specimens of groups 2 and 3, but did not affect the specimens from group 1. No difference was observed among luting approaches in control specimens. Thermocycling resulted in increased silver nitrate deposition (i.e. interfacial nanoleakage) in all groups. Within the limitations of the study, the use of an etch-and-rinse adhesive in combination with a dual-cure cement to lute fiber posts is the most stable luting procedure if compared with a self-etch resin-based cement or a self-adhesive cement, as assayed by thermocycling of the bonded specimens.

Effect of carbodiimide (EDC) on the bond stability of etch-and-rinse adhesive systems.

OBJECTIVE Recent studies supported the use of protein cross-linking agents during bonding procedures to inactivate endogenous dentin proteases, preventing dentin collagen degradation thus improving bond durability. The aim of this study was to evaluate the effect of a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-containing conditioner on the stability of the adhesive interface created by two etch-and-rinse adhesives. METHODS Human dentin was etched with 35% phosphoric acid, treated with 0.3M EDC-containing conditioner followed by a three-step or a two-step etch-and-rinse adhesive. Adhesives were applied to control specimens without EDC pre-treatment. Specimens were subjected to microtensile bond strength test and pulled to failure after 24h or 1 year of storage and interfacial nanoleakage expression was evaluated and quantified by light microscopy. Additionally, to investigate endogenous dentin matrix metalloproteinase activity a zymographic assay was performed on protein extracts obtained from phosphoric-acid-etched dentin powder with or without EDC treatment. RESULTS The use of the EDC-containing conditioner did not affect immediate bond strength to dentin but contributed to preserve the bond strength after 1 year (p<0.05) for both tested adhesives. No difference was found in the interfacial nanoleakage expression that increased after aging irrespective from the treatment. EDC pre-treatment inhibited dentin endogenous MMPs as assayed with the zymography. SIGNIFICANCE In conclusion, the results of the study provide proof that EDC can produce long-term inactivation of MMPs in acid-etched dentin matrices contributing to bond strength preservation over time. Future studies are needed to support the use of EDC in vivo.

Management of dental extraction in patients undergoing anticoagulant treatment. Results from a large, multicentre, prospective, case-control study.

Following favourable results from a previous study, a large, multicentre, prospective, case-control study was performed to further assess the incidence of bleeding complications after dental extraction in patients taking oral anticoagulant therapy (OAT). Four hundred fifty-one patients being treated with warfarin who required dental extraction were compared with a control group of 449 non-anticoagulated subjects undergoing the same procedure. In the warfarin-treated group, the oral anticoagulant regimen was maintained unchanged, such that the patients had an International Normalised Ratio ranging between 1.8 and 4, and local haemostatic measures (i.e. fibrin sponges, silk sutures and gauzes saturated with tranexamic acid) were adopted. All the procedures were performed in an outpatient setting. Seven bleeding complications occurred in the OAT group and four in the control group; the difference in the number of bleeding events between the two groups was not statistically significant (OR=1.754; 95% CI 0.510 - 6.034; p=0.3727). No post-operative late bleeds requiring hospitalisation and/or blood transfusions were recorded, and the adjunctive local haemostatic measures were adequate to stop the bleeding. The results of our protocol applied in this large, multicenter study show that dental extractions can be performed easily and safely in anticoagulated outpatients without any modification of the ongoing anticoagulant therapy, thus minimising costs and reducing discomfort for patients.

A systematic review of therapeutical approaches in bisphosphonates-related osteonecrosis of the jaw (BRONJ)

OBJECTIVES The clinical management of bisphosphonate-related osteonecrosis of the jaw (BRONJ) remains controversial. Since universally accepted guidelines have not been released yet, clinicians usually chose the type of treatment according to position papers based on expert opinion, or on empirical experience. The aim of this systematic review is to identify different therapeutical approaches for BRONJ that have been described in literature and to describe their effectiveness. MATERIALS AND METHODS A Medline via Pubmed and Scopus database literature search was conducted and all publications fulfilling the inclusion and exclusion criteria were included in eligibility assessment. The full texts of 146 retrieved articles were then screened and 40 studies were included in the quality assessment process. RESULTS After quality assessment, 22 full text articles were selected for the final review. 14 articles out of 22 were screened for stage-related outcomes. The overall outcome results and results for every disease stage were the highest when patients were treated with extensive surgery or extensive laser assisted surgery.