A Practical Primer: Raman Spectroscopy for Monitoring of Photopolymerization Systems.

Photopolymerization systems provide compelling advantages for industrial applications due to their fast reaction kinetics, wide selection of monomers for physical property development, and energy-efficient initiation via illumination. These same advantages can present challenges when attempting to monitor these reactions or characterize their resulting polymers; however, Raman spectroscopy can provide the flexibility and resolution needed. In this overview, Raman spectroscopy is compared to common characterization techniques, such as photo-differential scanning calorimetry and infrared spectroscopy, highlighting advantages of Raman spectroscopy. Examples are provided of how Raman spectroscopy has been used to monitor photopolymerizations and to provide insight on the impact of monomer chemistry and processing conditions, as well as paired with other techniques to elucidate physical properties. Finally, practical tips are provided for applying Raman spectroscopy and microscopy in photopolymerization systems.

Differentiating Pseudomonas sp. strain ADP cells in suspensions and biofilms using Raman spectroscopy and scanning electron microscopy.

High quality spectra of Pseudomonas sp. strain ADP in the planktonic and biofilm state were obtained using Raman microspectroscopy. These spectra enabled the identification of key differences between free and biofilm cells in the fingerprint region of Raman spectra in the nucleic acid, carbohydrate, and protein regions. Scanning electron microscopy (SEM) enabled detailed visualization of ADP biofilm with confirmation of associated extracellular matrix structure. Following extraction and Raman analysis of extracellular polymeric substances, Raman spectral differences between free and biofilm cells were largely attributed to the contribution of extracellular matrix components produced in mature biofilms. Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species. Graphical Abstract Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species.

Ethanol-wet bonding and chlorhexidine improve resin-dentin bond durability: quantitative analysis using raman spectroscopy.

PURPOSE: To directly test the effectiveness of ethanol-wet bonding (EW) in improving monomer infiltration into demineralized dentin through quantitative measurement of bis-GMA and TEG-DMA molar concentrations within hybrid layers, and to comprehensively evaluate the effect of EW and chlorhexidine on durability of resin-dentin bonds compared to conventional water-wet bonding (WW). MATERIALS AND METHODS: A three-step etch-and-rinse adhesive (70% bis-GMA/28.75%TEG-DMA) was applied to coronal dentin using a clinically relevant ethanol-wet bonding protocol (EW) or the conventional water-wet bonding (WW) technique. Bis-GMA and TEG-DMA molar concentrations at various positions across the resin/dentin interfaces formed by EW and WW were measured using micro-Raman spectroscopy. The experiment was repeated at the same positions after 7-month storage in phosphate buffer solution containing 0.1% sodium azide. The µTBS and hybrid layer morphology (TEM) of bonding groups with and without chlorhexidine application were compared immediately and after 1-year storage in terms of nanoleakage, collagen fibril diameter, collagen interfibrillar width, and hybrid layer thickness. RESULTS: Specimens bonded with EW showed significantly higher monomer molar concentrations and µTBS throughout the hybrid layer immediately and after storage, providing direct evidence of superior infiltration of hydrophobic monomers in EW compared to WW. Microscopically, EW maintained interfibrillar width and hybrid layer thickness for resin infiltration and retention. The application of chlorhexidine further preserved collagen integrity and limited the degree of nanoleakage in EW after 1-year storage. CONCLUSION: EW enhances infiltration of hydrophobic monomers into demineralized dentin. The results suggest that a more durable resin-dentin bond may be achieved with combined usage of a clinically relevant EW and chlorhexidine.

In vitro enzymatic biodegradation of adhesive resin in the hybrid layer.

Penetration of adhesives into the demineralized dentin surface and their subsequent conversion are critically important to longevity of the adhesive resin (AR)-dentin bond. The durability of the resin-dentin bond is investigated by monitoring the change of adhesive concentration within the hybrid layer (HL) of aged specimens using Raman spectroscopy. Absolute molar concentrations of Bis-GMA and HEMA were measured across the HL of resin-dentin specimens 24 h after photopolymerization and after 24-week storage in one of three media: artificial saliva (SAL), SAL containing cholesterol esterase to attack resin (EST), and SAL containing bacterial collagenase to attack collagen (COL). No significant difference among these groups for both Bis-GMA and HEMA molar concentrations at 24-h storage was found; however, concentrations decreased from the AR to the middle of the HL. Concentrations remained unchanged at any resin-dentin position after aging in SAL. In the HL, concentrations significantly decreased with aging in COL and tended to decrease in EST. While showing potential enzymatic biodegradative effects of endogenous matrix metalloproteinases and salivary esterases, this methodology may also prove to be a valuable assessment of new chemistries and future approaches to improve resin-dentin bond performance. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Quantitative analysis of adhesive resin in the hybrid layer using Raman spectroscopy.

The objective was to determine absolute molar concentration of adhesive resin components in the hybrid layer by establishing methods based on Raman spectroscopy fundamentals. The hybrid layer was treated as a three-component system consisting of collagen and an adhesive resin containing two monomers. Adhesive standard specimens and Raman peak area ratios obtained with a 785 nm excitation wavelength were used to construct separate calibration curves for comonomer relative molar concentration and Bis-GMA absolute molar concentration. As collagen and water had no measurable peaks in the fingerprint region, a dilution coefficient K(j) was defined to describe their impact on Raman peak area and to calculate HEMA absolute molar concentration. Methodology was validated using an analogous system containing acetone/ethanol/water. The absolute molar concentration of Bis-GMA and HEMA decreased 87% and 83%, respectively, from the top quarter to the middle of the hybrid layer. Additionally, less Bis-GMA penetrated the hybrid layer than HEMA, as indicated by the approximately 20% decrease in comonomer molar concentration ratio between the adhesive resin layer and the top half of the hybrid layer. Lack of complete monomer infiltration will further challenge dentin-adhesive bond longevity. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Apparent conversion of adhesive resin in the hybrid layer, Part II: In situ studies of the resin-dentin bond.

Penetration and conversion of adhesives into the hybrid layer (HL) is important to the quality and longevity of the adhesive resin (AR)-dentin bond. In this study, a methodology is developed to examine the degree of conversion and relative HEMA concentration with respect to Bis-GMA using Raman spectroscopy. This methodology will be used in the future reports related to this topic. Conversion in the AR of water-stored resin-dentin samples (84% +/- 3%) agreed well with that measured in commercial adhesive (Comm Adh) resin samples after 24-h water storage (80% +/- 2% from Part 1) and was significantly higher than Comm Adh without water storage (58% +/- 3% from Part 1) (p = 0.0005). Adhesive conversion was not significantly different (p = 0.5036) through the middle of the HL, with a mean of 83% +/- 6%. HEMA mole fraction, relative to Bis-GMA, was significantly higher (p = 0.0028) in the top half of the HL (0.67 +/- 0.03), when compared to HEMA in the AR (0.60 +/- 0.01). HEMA and EDMAB were identified through GC/MS as leachable components in the aqueous 24-h storage media. The effect of this elution explains the change in conversion measurements observed between dry and water-stored conditions, which is more appropriately described as the "apparent" conversion.

Effects of polar solvents and adhesive resin on the denaturation temperatures of demineralised dentine matrices.

OBJECTIVES: To measure the denaturation temperature (Td) of demineralised dentine matrix as a function of infiltration with water vs. polar solvents vs. adhesive resins. METHODS: Small discs of normal dentine were completely demineralised in 0.5M EDTA. Dried demineralised specimens were placed in water, methanol, ethanol, acetone, eta-butanol or HEMA. Additional specimens were infiltrated with Prime&Bond NT and polymerised. All specimens sealed in high-pressure pans and scanned using differential scanning calorimetry (DSC). RESULTS: Demineralised dentine saturated with water showed a Td of 65.6 degrees C that increased with saturation by methanol, ethanol, acetone, eta-butanol or HEMA to 148.5 degrees C. These increases in Td were inversely related to the molar concentration of the solvents and to their Hoy's solubility parameter for hydrogen bonding (delta h, p<0.01), as well as directly related to the cube root of their molecular weights (p<0.001). The presence of adhesive resins also increased the Td of demineralised matrices to even higher values depending if the resin bonded dentine was measured after 24h of water storage (166.8 degrees C) or dry (172.7 degrees C) storage. CONCLUSIONS: Solvents and monomers with low delta(h) values (i.e., 100% HEMA) increase the Td of demineralised dentine above that produced by solvents with higher delta h values such as methanol and water.

Apparent conversion of adhesive resin in the hybrid layer, Part 1: Identification of an internal reference for Raman spectroscopy and the effects of water storage.

Monomer conversion of adhesives in the hybrid layer is important to the quality and longevity of the dentin bond. In this study, degree of conversion and relative co-monomer concentrations of both experimental and commercial adhesive resins were determined using Raman spectroscopy. The objectives were to identify stable Raman scattering peaks to use as internal references and to determine the effect of water storage on measured conversion and co-monomer concentrations. The peak at 605 cm(-1) did not change throughout polymerization and is associated with monomers in both adhesives. This peak was used as an internal reference for conversion and composition calculations before and after water storage. Conversion of the adhesive resins immediately after photopolymerization was approximately 20% lower than that measured after 24-h water storage. HEMA concentration (relative to bis-GMA) in the adhesive resins immediately after photopolymerization was at least 5 wt % higher than that measured after 24-h water storage. Elution of unreacted HEMA provides a reasonable explanation for the "supposed" change in conversion measurements, which is more appropriately described as apparent conversion. This apparent conversion will impact interpretation of physical properties and structure of the polymer, as well as increase the probability of water penetration and its reaction within the hybrid layer.

Effects of exogenous collagenase and cholesterol esterase on the durability of the resin-dentin bond.

PURPOSE: The purpose of this work was to determine microtensile dentin bond strengths (microTBS) of dentin-resin composite bonds after three-month storage in artificial saliva containing either collagenase (COL) or cholesterol esterase (EST). The null hypothesis tested is that the resin-dentin bond strength is equivalent for each storage medium at the tested storage times. MATERIALS AND METHODS: Resin composite was bonded to occlusal dentin, and microTBS specimens were formed and stored in the artificial saliva, COL, EST, or synthetic oil. After 24 h and 12-week storage, microTBS was determined and failure modes were characterized by SEM. The interfacial ultrastructure was evaluated by transmission electron microscopy as unstained and stained sections (phosphotungstic acid/uranyl acetate). Statistical analysis was performed by ANOVA and Weibull survival analyses at the 0.05 level of statistical significance. RESULTS: There were significantly weaker bond strengths after 12 weeks for all experimental storage media (p < 0.001). Artificial saliva containing EST lowered bond strengths to a significantly greater extent than did COL after 12 weeks of storage, while no difference between these groups could be discerned after 24 h. Therefore, the null hypothesis of this experiment is rejected. CONCLUSION: Exogenous enzymatic challenge to resin-dentin bonds decreased bond durability only with EST. However, when further challenges to ideal infiltration of the comonomers into the hybrid layer were carried out using inadequate removal of solvent, additional alterations in hybrid layer ultrastructure were discerned by TEM that may represent different potential degradative processes. The contribution of endogenous enzymatic challenges to the primary degradative process, ie, hydrolysis, is unknown and deserves continued attention.

Denaturation temperatures of dentin matrices. I. Effect of demineralization and dehydration.

The denaturation temperature (T(d)) of dentin collagen in mineralized versus demineralized teeth was examined as a function of dentin age and the extent of dehydration. Using differential scanning calorimetry, T(d) of mineralized dentin was shown to be between 160 degrees C to 186 degrees C, depending on whether it was from young or old dentin that was hydrated or dehydrated, respectively. Demineralized dentin exhibited a T(d) of 65.6 degrees C that increased with dehydration to 176 degrees C. The presence of apatite crystallites or interpeptide bonding increased the T(d) of demineralized matrices. Interpeptide hydrogen bonding seems to stabilize collagen to thermal challenge. Water breaks interpeptide hydrogen bonds making collagen more susceptible to thermal denaturation. Rises in intracanal temperature are unlikely to cause extensive denaturation of mineralized root dentin walls. However, hydrated or partially dehydrated root canal walls that have been partially demineralized with chelating agents or mild acids may be susceptible to thermal denaturation.