Enrico Modena

Environmental Scanning Electron Microscopy Connected with Energy Dispersive X-ray Analysis and Raman Techniques to Study ProRoot Mineral Trioxide Aggregate and Calcium Silicate Cements in Wet Conditions and in Real Time

INTRODUCTION ProRoot mineral trioxide aggregate (MTA) and calcium silicate cements are able to set in a moist environment. The aim of the study was to examine the surface structure and composition of a cement paste under wet conditions and in real time during setting by environmental scanning electron microscopy connected with energy dispersive x-ray analysis (ESEM-EDX) and micro-Raman techniques. METHODS White ProRoot MTA and experimental white tetrasilicate cement (wTC) and wTC containing bismuth oxide (wTC-Bi) were studied. Cement disks were analyzed 10 minutes after powder-liquid mixing (freshly prepared samples) and after immersion in Dulbecco phosphate-buffered saline at 37 degrees C for 24 hours (24-hour-aged samples). RESULTS Freshly prepared wet cements at ESEM-EDX exposed an irregular surface (displaying calcium, silicon, aluminum, chlorine reflexes, and bismuth traces in MTA and wTC-Bi) with needle-like and cubic-hexagonal shaped crystals. Aggregates of spheroidal Ca-P-rich crystals (spherulites) appeared on the surface of 24-hour-aged samples. The starting unhydrated powders displayed the typical Raman bands of Portland cement components: alite, belite, and calcium sulfate (only as anhydrite in MTA and as both anhydrite and gypsum in wTC and wTC-Bi). MTA powder showed higher amount of calcium carbonate and lower quantities of anhydrite and higher crystallinity of the silicate component, leading to a slower hydration reaction. Products/markers of hydration reactions were present on fresh samples; ettringite formed on the surface of all the cements; calcium hydroxide (portlandite) was detected only on the surface of wTC, but no conclusion can be drawn on wTC-Bi and MTA because of the interference of bismuth oxide. Calcium phosphate and calcite/aragonite bands were detected on all 24-hour-aged cements; portlandite was no longer detected on wTC. CONCLUSIONS ESEM and micro-Raman are powerful and suitable techniques to investigate endodontic calcium silicate hydrated cements in real time and in their humid state without inducing artifacts by sample preparation. The formation of apatite spherulites on calcium silicate cements might have clinical relevance.

Development of the foremost light-curable calcium-silicate MTA cement as root-end in oral surgery. Chemical–physical properties, bioactivity and biological behavior

AIM An innovative light-curable calcium-silicate cement containing a HEMA-TEGDMA-based resin (lc-MTA) was designed to obtain a bioactive fast setting root-end filling and root repair material. METHODS lc-MTA was tested for setting time, solubility, water absorption, calcium release, alkalinizing activity (pH of soaking water), bioactivity (apatite-forming ability) and cell growth-proliferation. The apatite-forming ability was investigated by micro-Raman, ATR-FTIR and ESEM/EDX after immersion at 37°C for 1-28 days in DPBS or DMEM+FBS. The marginal adaptation of cement in root-end cavities of extracted teeth was assessed by ESEM/EDX, and the viability of Saos-2 cell on cements was evaluated. RESULTS lc-MTA demonstrated a rapid setting time (2min), low solubility, high calcium release (150-200ppm) and alkalinizing power (pH 10-12). lc-MTA proved the formation of bone-like apatite spherulites just after 1 day. Apatite precipitates completely filled the interface porosities and created a perfect marginal adaptation. lc-MTA allowed Saos-2 cell viability and growth and no compromising toxicity was exerted. SIGNIFICANCE HEMA-TEGDMA creates a polymeric network able to stabilize the outer surface of the cement and a hydrophilic matrix permeable enough to allow water absorption. SiO(-)/Si-OH groups from the mineral particles induce heterogeneous nucleation of apatite by sorption of calcium and phosphate ions. Oxygen-containing groups from poly-HEMA-TEGDMA provide additional apatite nucleating sites through the formation of calcium chelates. The strong novelty was that the combination of a hydraulic calcium-silicate powder and a poly-HEMA-TEGDMA hydrophilic resin creates the conditions (calcium release and functional groups able to chelate Ca ions) for a bioactive fast setting light-curable material for clinical applications in dental and maxillofacial surgery. The first and unique/exclusive light-curable calcium-silicate MTA cement for endodontics and root-end application was created, with a potential strong impact on surgical procedures.

Biointeractivity‐related versus chemi/physisorption‐related apatite precursor‐forming ability of current root end filling materials

Commercial root end filling materials, namely two zinc oxide eugenol-based cements [intermediate restorative material (IRM), Superseal], a glass ionomer cement (Vitrebond) and three calcium-silicate mineral trioxide aggregate (MTA)-based cements (ProRoot MTA, MTA Angelus, and Tech Biosealer root end), were examined for their ability to: (a) release calcium (Ca(2+) ) and hydroxyl (OH(-) ) ions (biointeractivity) and (b) form apatite (Ap) and/or calcium phosphate (CaP) precursors. Materials were immersed in Hanks balanced salt solution (HBSS) for 1-28 days. Ca(2+) and OH(-) release were measured by ion selective probes, surface analysis was performed by environmental scanning electron microscopy/energy dispersive X-ray analysis, micro-Raman, and Fourier transform infrared spectroscopy. IRM and Superseal released small quantities of Ca(2+) and no OH(-) ions. Uneven sparse nonapatitic Ca-poor amorphous CaP (ACP) deposits were observed after 24 h soaking. Vitrebond did not release either Ca(2+) or OH(-) ions, but uneven nonapatitic Ca-poor CaP deposits were detected after 7 days soaking. ProRoot MTA, MTA Angelus, and Tech Biosealer root end released significant amounts of Ca(2+) and OH(-) ions throughout the experiment. After 1 day soaking, nanospherulites of CaP deposits formed by amorphous calcium/magnesium phosphate (ACP) Ap precursors were detected. A more mature ACP phase was present on ProRoot MTA and on Tech Biosealer root end at all times. In conclusion, zinc oxide and glass ionomer cements had little or no ability to release mineralizing ions: they simply act as substrates for the possible chemical bonding/adsorption of environmental ions and precipitation of nonapatitic Ca-poor ACP deposits. On the contrary, calcium-silicate cements showed a high calcium release and basifying effect and generally a pronounced formation of more mature ACP apatitic precursors correlated with their higher ion-releasing ability.

Biomimetic calcium-silicate cements aged in simulated body solutions. Osteoblast response and analyses of apatite coating

PURPOSE Calcium-silicate cements have been recently proposed for application in dentistry as root-end filling and root-perforation repair materials. The aim of this study was to investigate the effect of ageing of experimental calcium-silicate cements on the chemistry, morphology and in vitro bioactivity of the surface, as well as on osteoblast viability and proliferation. METHODS Two experimental cements (wTC-Bi, containing bismuth oxide and wTC), mainly based on dicalcium-silicate and tricalcium-silicate, were prepared and tested for their bioactivity after soaking in Dulbeccos phosphate buffered saline (DPBS), used as simulated body fluid. Human marrow stromal cells (HMSC) were seeded on the cements maintained in DPBS for 5 hr (non-aged group), 14 and 28 days (aged group). Cell viability was assessed by the Alamar blueTM test and morphology by scanning electron microscopy (SEM) at different time endpoints. The surface of the soaked cements was analyzed by environmental scanning electron microscopy or SEM coupled with energy dispersive X-ray microanalysis (ESEM/EDX or SEM/EDX respectively) and the micro-Raman technique. RESULTS The ESEM/EDX results showed a uniform surface composed of CSH hydrogel (mainly derived from the hydration of belite and alite) on both non-aged cements. Micro-Raman spectroscopy revealed the presence of calcium carbonate, anhydrite, ettringite, alite and belite. The SEM/EDX data showed an irregular calcium-phosphate multi-layered biocoating with many sharp and protruding crystals on both the aged cements. Micro-Raman spectroscopy revealed crystalline apatite and calcite. The osteoblast response results showed that both the experimental cements exerted no acute toxicity in the cell assay systems. The non-aged samples promoted greater cell growth. SEM showed cells well spread and adherent to the non-aged materials. A reduced number of attached cells was noticed on the aged cements. Bismuth oxide-containing cement allowed a reduced cell viability suggesting some cytotoxic effects. However, the thick biocoating formed on the 28-day aged samples lowered the deleterious effect of bismuth oxide on cell growth. Actually, micro-Raman spectroscopy revealed progressive bismuth oxide depletion on the wTC-Bi surface, due to the increased thickness of the apatite deposit. CONCLUSIONS The study demonstrated that (1) these materials support osteogenic cells growth and may induce early bone formation, (2) the ageing in DPBS reduced the growth of HMSC, but eliminated the deleterious effect of the bismuth oxide on cell growth. In conclusion, the experimental cements have adequate biological properties to be used as root-end/root repair filling materials or pulp capping materials.

Retrieval analysis of three generations of Biolox® femoral heads: Spectroscopic and SEM characterisation

Wear and osteolysis continue to be the major reasons for revision surgery in Total Hip Arthroplasty. The introduction of the ceramic-on-ceramic bearings eliminates the problem of polyethylene wear debris. During the last 40 years, three generations of Biolox® ceramics were developed (Biolox®, Biolox® forte, and Biolox® delta), each better than the previous from the density, grain size and purity point of view. We conducted a retrieval study on 15 Biolox® femoral heads (Biolox®, Biolox® forte, and Biolox® delta) articulating against liners of the same type. Surface properties and residual stress were assessed using SEM, fluorescence and Raman spectroscopy. At SEM examination, the Biolox® delta retrievals showed a lower wear than the previous generations. The fluorescence measurements suggested different wear mechanisms in the three sets of retrievals. Micro-cracking was predominant in Biolox®, while in Biolox® forte and Biolox® delta a wider range of residual stress values was observed upon wear. Surface polishing was observed only in Biolox® and Biolox® forte. Raman spectroscopy of Biolox® delta femoral heads showed a progressive improvement in material composition. Wear induced a tetragonal to monoclinic transformation. The Raman results on the retrievals, here reported for the first time, allowed to validate the in vitro ageing protocols proposed in the literature to simulate the effects of the in vivo wear.

Fluoride-containing nanoporous calcium-silicate MTA cements for endodontics and oral surgery: early fluorapatite formation in a phosphate-containing solution

AIM To test the chemical-physical properties and apatite-forming ability of experimental fluoride-doped calcium silicate cements designed to create novel bioactive materials for use in endodontics and oral surgery. METHODOLOGY A thermally treated calcium silicate cement (wTC) containing CaCl(2) 5%wt was modified by adding NaF 1%wt (FTC) or 10%wt (F10TC). Cements were analysed by environmental scanning electron microscopy with energy-dispersive X-ray analysis, IR and micro-Raman spectroscopy in wet conditions immediately after preparation or after ageing in a phosphate-containing solution (Dulbeccos phosphate-buffered saline). Calcium and fluoride release and pH of the storage solution were measured. The results obtained were analysed statistically (Tukeys HSD test and two-way anova). RESULTS The formation of calcium phosphate precipitates (spherulites) was observed on the surface of 24 h-aged cements and the formation of a thick bone-like B-type carbonated apatite layer (biocoating) on 28 day-aged cements. The rate of apatite formation was FTC>F10TC>wTC. Fluorapatite was detected on FTC and F10TC after 1 day of ageing, with a higher fluoride content on F10TC. All the cements released calcium ions. At 5 and 24 h, the wTC had the significantly highest calcium release (P<0.001) that decreased significantly over the storage time. At 3-28 days, FTC and F10TC had significantly higher calcium release than wTC (P<0.05). The F10TC had the significantly highest fluoride release at all times (P<0.01) that decreased significantly over storage time. No significant differences were observed between FTC and wTC. All the cements had a strong alkalinizing activity (OH(-) release) that remained after 28 days of storage. CONCLUSIONS The addition of sodium fluoride accelerated apatite formation on calcium silicate cements. Fluoride-doped calcium silicate cements had higher bioactivity and earlier formation of fluorapatite. Sodium fluoride may be introduced in the formulation of mineral trioxide aggregate cements to enhance their biological behaviour. F-doped calcium silicate cements are promising bone cements for clinical endodontic use.