Rapid synthesis of drug-encapsulated films by evaporation-induced self-assembly for highly-controlled drug release from biomaterial surfaces.

Infection at the surgical site for dental implants results in failed procedures, patient pain, burdensome economic impact, and the over-prescription of prophylactic antibiotics. Mesoporous silica films as coatings for implants may provide an ideal antimicrobial drug storage and local release vector to the site of infection, however traditional drug loading techniques result in insufficient drug load and short-term release kinetics. In this work, we have applied a method to use a surfactant-antimicrobial drug octenidine dihydrochloride (OCT) as a template for mesostructured silica, to demonstrate silica-OCT composite films. The films are synthesized by evaporation induced self-assembly (EISA) and we explore the effects of synthesis parameters on porous film structure, OCT incorporation, and OCT drug release rates. Drug micelle incorporation into the silica mesostructure was highly dependent on silica precursor pre-reaction to form silica oligomers before film spin-casting. The OCT drug concentration of the synthesis solution dictated the time required for effective incorporation (without phase separation), with total loading in the film of up to 90% by mass. The OCT content in the films was found to directly determine the timescale of drug release, from 2 to 8 h for a single layer film. The total release timescale was increased by the addition of multiple layers of OCT-silica films to nearly 2 weeks. Drug release from films completely inhibited Streptococcus mutans (UA159) growth, while drug-free porous silica films showed no increase in bacterial growth over non-porous control. These OCT-silica films have a significant potential to store and release antimicrobial drugs from dental implant surfaces.

Esterases affect the physical properties of materials used to seal the endodontic space.

Materials used to seal the endodontic space are subjected to enzymatic degradative activities of body fluids and bacteria. OBJECTIVES: To assess effects of simulated human salivary, blood and bacterial esterases (SHSE) on physical properties of typical restorative material and root canal sealers. METHODS: Specimens of set methacrylate-based resin composite (BisfilTM2B; RC), calcium-silicate sealer (EndoSequence®; BC) or epoxy-resin sealer (AH-Plus®; ER) were tested after up to 28Days exposure to phosphate buffered saline (PBS) or SHSE, using ANSI/ADA-57:2000 and ISO-6876:2012. RESULTS: Regardless of media, microhardness increased with time for BC remained unchanged for ER and decreased for RC (p < 0.05). SHSE moderated the increase for BC compared to PBS (28.0 ± 4.8 vs. 38.1 ± 7.9 KHN) at 7Days, and enhanced the decrease for RC at 7Days (55.6 ± 7.1 vs. 66.3 ± 6.5 KHN) and 28Days (52.3 ± 9.2 vs. 62.6 ± 8.5 KHN). Compressive strength was enhanced only for BC by either media. BC expanded with time for both incubation conditions; SHSE moderated the expansion compared to PBS at 7Days (0.026 ± 0.01% vs. 0.049 ± 0.007%). Shrinkage of ER was similar for both incubation media and was lower than that for RC (p < 0.05). Shrinkage of RC was enhanced by SHSE compared to PBS at 7Days (0.5 ± 0.07% vs. 0.38 ± 0.08%). Weight loss was lowest for ER and highest for BC (p < 0.05). It was enhanced by SHSE compared to PBS for BC at 28Days (2.40 ± 0.2 vs. 2.96 ± 0.19 W L%), and for RC at 7Days (0.54 ± 0.09 vs. 0.80 ± 0.1 W L%). SIGNIFICANCE: Simulated body fluids and bacterial esterases affected the physical properties of test materials, suggesting potential impacts on sealing ability and resistance to bacterial ingress, and tooth strength ultimately affecting their clinical performance.

Endodontic pathogens possess collagenolytic properties that degrade human dentine collagen matrix.

AIM: To measure collagenolytic protease activity from Enterococcus faecalis and Micrococcus luteus and their ability to degrade human dentinal collagen. METHODOLOGY: Proteases activity of E. faecalis ATCC 29212, ATCC 47077 and M. luteus towards generic and specific human matrix metalloproteinase (MMP) substrates was measured using a fluorimetric assay. The ability of the bacteria to degrade dentinal collagen was tested by quantifying the amount of hydroxyproline released into the media following incubation of the bacteria or heat-inactivated bacteria (HIN) with demineralized human dentine samples for 24 h and by scanning electron microscopy (SEM). Multifactorial anova and Tukey's post hoc test were used to analyse the data (P < 0.05). RESULTS: All strains had MMP-like activities, but with different substrate affinity; E. faecalis ATCC 29212, ATCC 47077 and M. luteus had the greatest affinity towards MMP-8 (7.75 ± 0.88 µmol L-1 /3 × 106 CFU), MMP-9 (33.86 ± 5.16 µmol L-1 /3 × 106 CFU) and generic MMP (26.08 ± 4.48 µmol L-1 /3 × 106  CFU), respectively. The amount of hydroxyproline released from demineralized dentine was similar (P > 0.05) for the three strains (range 1.8 ± 0.17 to 2.38 ± 0.39 µg 50 µL-1 ) and was significantly higher (P < 0.001) compared to their HIN counterparts (0.61 ± 0.22 µg 50 µL-1 ). SEM revealed increased collagen network degradation after incubation with bacteria versus HIN. CONCLUSIONS: Endodontic pathogens possess collagenolytic protease properties that enable them to degrade dentinal collagen, potentially compromising the restoration-tooth and sealer-tooth interfaces. These collagenolytic protease properties could facilitate the migration of pathogenic bacteria into the root canal system and explain in part their role in root canal infections.

In Vivo Biodegradation of bisGMA and Urethane-Modified bisGMA-Based Resin Composite Materials.

The aim of this study was to compare the levels of in vivo chemical degradation sustained by bisphenyl-glycidyl-dimethacrylate (bisGMA)-based and urethane-modified bisGMA-based resin composites. A cohort of 58 patients was recruited for the study. Human salivary esterase activity (HSDE) was measured for each patient prior to restoration placement. Class V or III composite restorations without occlusal contacts were placed in adult patients using a 3-step adhesive (Scotchbond MP, 3M) and 1 of 2 resin composites: a traditional bisGMA-based (Z250; 3M) ( n = 28) or a urethane-modified bisGMA-based composite (TPH Spectra, Dentsply) ( n = 30). Patients followed a 2-min rinse (saline containing 20% ethanol) protocol before, immediately after, and 7 days after restoration placement. The rinse samples were analyzed for the presence of bisphenol A (BPA) and bishydroxypropoxyphenylpropane (bisHPPP), a bisGMA breakdown product, using high-performance liquid chromatography in combination with mass spectrometry. The overall mean ± standard error (SE) HSDE activity was 23.4 ± 1.9 U/mL, with no statistical difference between the Z250 (22.6 ± 2.8 U/mL) and TPH (24.1 ± 2.1 U/mL) groups ( P = 0.69). BPA was not detected from any rinse samples. BisHPPP was detected from both composites only in rinse samples immediately after resin composite placement (0.59 µg/mm2 ± 0.16 and 0.68 µg/mm2 ± 0.16 for Z250 and TPH, respectively, P = 0.767). There was no statistically significant correlation between HSDE and amount of bisHPPP obtained from the saliva for the Z250 group ( r = 0.071, P = 0.723), TPH group ( r = 0.266, P = 0.155), and both groups combined ( r = 0.080, P = 0.549). Conventional commercial resin composite materials used in the current study did not release any detectable amount of BPA and only showed detectable levels of bisHPPP for a short term after placement, suggesting that hydrolytic consumption of any available resin substrate is fast and the generated products are rapidly diluted below the detection level limit (<20 ppb) in the oral cavity. This short-term release of bisHPPP was not significantly affected by material type or esterase level in the saliva. Knowledge Transfer Statement: This clinical study demonstrated that the duration and degree of biodegradation of 2 representative formulations of resin composites was limited in both duration and amounts of detectable matrix derived degradation products. No significant level of potential biohazards was released following the application of the resin composites. The results of this study can help oral care professionals address concerns from their patients about possible health issues regarding the application of resin composite restorative materials.

Cariogenic bacteria degrade dental resin composites and adhesives.

A major reason for dental resin composite restoration replacement is related to secondary caries promoted by acid production from bacteria including Streptococcus mutans (S. mutans). We hypothesized that S. mutans has esterase activities that degrade dental resin composites and adhesives. Standardized specimens of resin composite (Z250), total-etch (Scotchbond Multipurpose, SB), and self-etch (Easybond, EB) adhesives were incubated with S. mutans UA159 or uninoculated culture medium (control) for up to 30 days. Quantification of the BisGMA-derived biodegradation by-product, bishydroxy-propoxy-phenyl-propane (BisHPPP), was performed by high-performance liquid chromatography. Surface analysis of the specimens was performed by scanning electron microscopy (SEM). S. mutans was shown to have esterase activities in levels comparable with those found in human saliva. A trend of increasing BisHPPP release throughout the incubation period was observed for all materials and was more elevated in the presence of bacteria vs. control medium for EB and Z250, but not for SB (p < .05). SEM confirmed the increased degradation of all materials with S. mutans UA159 vs. control. S. mutans has esterase activities at levels that degrade resin composites and adhesives; degree of degradation was dependent on the material's chemical formulation. This finding suggests that the resin-dentin interface could be compromised by oral bacteria that contribute to the progression of secondary caries.

Biodegradation of resin-dentin interfaces increases bacterial microleakage.

Bis-GMA-containing resin composites and adhesives undergo biodegradation by human-saliva-derived esterases, yielding Bis-hydroxy-propoxy-phenyl-propane (Bis-HPPP). The hypothesis of this study is that the exposure of dental restorations to saliva-like esterase activities accelerates marginal bacterial microleakage. Resin composites (Scotchbond, Z250, 3M) bonded to human dentin were incubated in either buffer or dual-esterase media (pseudocholinesterase/cholesterol-esterase; PCE+CE), with activity levels simulating those of human saliva, for up to 90 days. Incubation solutions were analyzed for Bis-HPPP by high-performance liquid chromatography. Post-incubation, specimens were suspended in a chemostat-based biofilm fermentor cultivating Streptococcus mutans NG8, a primary species associated with dental caries, for 7 days. Bacterial microleakage was assessed by confocal laser scanning microscopy. Bis-HPPP production and depth and spatial volume of bacterial cell penetration within the interface increased with incubation time and were higher for 30- and 90-day PCE+CE vs. buffer-incubated groups, suggesting that biodegradation can contribute to the formation of recurrent decay.

Retreatment efficacy of the Epiphany soft resin obturation system.

AIM: To assess the efficacy of retreatment of canals filled with the Epiphany System with and without solvent, with particular reference to the extent of canal enlargement during retreatment. METHODOLOGY: Sixty roots with canals prepared to apical size 45 were embedded in resin blocks and sectioned vertically. Digital micrographs of canal walls were captured. Roots were re-assembled and filled with Epiphany/Resilon (experimental) or gutta-percha/AH Plus (control). After 8 weeks, canals were retreated to size 45 with or without chloroform, and the time recorded. Roots were split, imaged, re-assembled, retreated to size 55, split and imaged. Root-filling residue, traced at three canal levels, was expressed as percentage of canal surface. RESULTS: Residue percentage was greater (t-test, P < 0.01) in the experimental group than in the control. Most residue in all specimens was in the apical third (ANOVA, P < 0.01). Chloroform and enlargement to size 55 decreased residue in both groups (t-test, P < 0.01). Retreatment time was longer in the experimental group (P < 0.05), and reduced by chloroform in both groups (P < 0.05). CONCLUSIONS: The Epiphany System was retreatable with and without chloroform, with lesser efficacy than gutta-percha and AH Plus sealer.

Influence of silanated filler content on the biodegradation of bisGMA/TEGDMA dental composite resins.

It has been shown that an increase in the content of nonsilanated submicron colloidal silica filler particles within dental composites resulted in the release of more bis-phenol-A diglycidyl dimethacrylate (bisGMA)-derived product, bis-hydroxy-propoxyphenyl propane, following incubation with cholesterol esterase (CE). This work further investigates the enzyme-catalyzed biodegradation of fine composite resin systems, containing silanated micron-size irregular glass fillers, commonly used in clinical restorations. Model composite resin samples (10 or 60% weight fraction silanated barium glass filler, 1 mum average particle size) based on bisGMA/triethylene glycol dimethacrylate (TEGDMA) were incubated in buffer or buffer with CE (pH = 7.0, 37 degrees C) solutions for 32 days. The incubation solutions were analyzed using high-performance liquid chromatography, UV spectroscopy, and mass spectrometry. Both groups were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. In contrast with previous findings for nonsilanated submicron filler systems, the higher filler containing composite showed an increase in its stability with time, following exposure to esterase and when compared to the lower filler content material. As well, the 60% filler composite leached less unreacted monomer TEGDMA. Since the model composite resins studied here were identical and only the filler content varied, the differences in biostability could be specifically associated with the relative amount of resin/filler distribution. The clinical use of different materials in varied dental applications (ranging from fissure sealant to tooth-colored highly filled materials) must consider the potential for different degradation profiles to occur as a function of filler content.

The influence of resin chemistry on a dental composite's biodegradation.

Previous work reported that commercial dental composite resins containing a urethane-modified bisGMA (bisphenylglycidyl dimethacrylate)/TEGDMA (triethylene glycol dimethacrylate) (ubis) based monomer system showed a 10-fold reduction in the release of a bisGMA-derived product, bishydroxypropoxyphenyl propane (bisHPPP), as compared with that found for bisGMA/TEGDMA (bis) based composites after incubation with cholesterol esterase (CE). Unfortunately, these materials also differed substantially in filler type and content, making it impossible to directly relate any specific parameter to the differences in biodegradation levels. By controlling for filler content and type, the current study will seek to probe the biomolecular interactions between composite resin chemistry and esterase activity in order to help explain the observed differences in biodegradation levels between the ubis and bis resin systems. After 32 days of incubation, buffer and CE solutions were analyzed for degradation products using high-performance liquid chromatography, UV spectroscopy, and mass spectrometry. Both materials were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. In the CE groups, the ubis system showed a 2.6- to 86-fold reduction (dependent on the product) in the amount of isolated products relative to the bis system (p < 0.01). Scanning electron microscopy data also demonstrated the relative stability of the ubis system and X-ray photoelectron spectroscopy analysis showed a higher content of the ester bond at the surface of the bis samples. Fourier transform infrared data showed that both resins had similar conversions. Because both systems were identical except for their monomer systems, it was concluded that changes in biostability were associated with chemistry. Crosslinking, hydrophobicity, and solubility all relate to ubis's pro-stability.

Mutual influence of cholesterol esterase and pseudocholinesterase on the biodegradation of dental composites.

It has been demonstrated that human saliva contains cholesterol esterase (CE)- and pseudocholinesterase (PCE)-like hydrolase activities. While PCE has been shown to preferentially degrade triethylene glycol dimethacrylate (TEGDMA) and its derivatives, CE has a greater catalytic effect on the breakdown of bis-phenol-A-diglycidyl dimethacrylate (bisGMA) components in composite dental resins. The current study seeks to determine if there is a mutual influence between the different esterases with respect to the biodegradation of resin composite. Photopolymerized model composite resin samples (containing 60% by weight fraction of silanated barium glass filler) based on bisGMA/TEGDMA (bis) or urethane-modified bisGMA/TEGDMA/bisEMA (ubis) monomers were incubated in buffer, CE and/or PCE solutions (pH=7.0, 37 degrees C) for 8 and 16 days. The incubation solutions were analyzed for degradation products using high-performance liquid chromatography, UV spectroscopy and mass spectrometry. In the bis system, higher amounts (p<0.05) of a bisGMA derived product, bishydroxy-propoxyphenyl-propane (bisHPPP), were detected in the combined enzyme group as compared to the sum of the two individual enzyme groups. In the ubis system, similar comparisons showed that higher levels (p<0.05) of bisHPPP were detected in the combined group at 8 days while higher amounts (p<0.05) of a bisEMA derived product, ethoxylated bis-phenol A, were detected in the combined group at 16 days. The study concluded that CE and PCE act synergistically to increase the biodegradation of both composite resin materials.

Salivary esterase activity and its association with the biodegradation of dental composites.

Pseudocholinesterase (PCE) and cholesterol esterase (CE) can hydrolyze bisphenylglycidyl dimethacrylate (bisGMA) and triethylene glycol dimethacrylate (TEGDMA) monomers. This study will test the hypothesis that enzyme activities showing CE and PCE character are found in human saliva at levels sufficient to hydrolyze ester-containing composites important to restorative denstistry. The study also seeks to ask if the active sites of CE and PCE with respect to composite could be inhibited. Photo-polymerized model composite resin was incubated in PCE and CE solutions, in the presence and absence of a specific esterase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Incubation solutions were analyzed for resin degradation products by high-performance liquid chromatography (HPLC), UV spectroscopy, and mass spectrometry. Saliva was found to contain both hydrolase activities at levels that could degrade composite resins. PMSF inhibited the composite degradation, indicating a material hydrolysis mechanism similar to the enzymes' common function.

Biodegradation of a dental composite by esterases: dependence on enzyme concentration and specificity.

Studies have shown that inflammatory (cholesterol esterase, CE) and salivary (pseudo-cholinesterase, PCE) enzymes can cause the breakdown of bisphenol-A diglycidyl dimethacrylate (bisGMA) and triethylene glycol dimethacrylate (TEGDMA) components from composite resins. Based on the above consideration, it was desired to show how CE- and PCE-catalyzed hydrolysis of resin components was dependent on the enzymes' concentration and to determine their distinct specificities (if any) towards resin components. Photopolymerized model composite resin samples (60% weight fraction silanated barium glass filler) based on bisGMA and TEGDMA monomers (55/45 weight ratio of the matrix, respectively) were incubated with PBS and either 0.01, 0.05, 0.1 or 1 unit/ml of CE or PCE for 16 days (pH 7.0, 37 degrees C). Incubation solutions were analyzed by high-performance liquid chromatography (HPLC), UV spectroscopy and mass spectrometry. The composite samples were characterized by scanning electron microscopy (SEM). Degradation rates of bisGMA and TEGDMA monomers were assessed. The results showed that CE had a greater specificity towards cleaving bisGMA while PCE showed a greater specificity towards TEGDMA. A strong enzyme concentration dependence was observed which suggests that the level of degradation products generated for a material will depend on the esterase make-up of an individual's saliva in combination with the specific formulation of monomer components used.

Interactions between resin monomers and commercial composite resins with human saliva derived esterases.

Cholesterol esterase (CE) and pseudocholinesterase (PCE) have been reported to degrade commercial and model composite resins containing bisphenylglycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA) or the latter in combination with urethane modified BisGMA monomer systems. In addition, human saliva has been shown to contain esterase like activities similar to CE and PCE. Hence, it was the aim of the current study to determine to what extent human saliva could degrade two common commercial composite resins (Z250 from 3M Inc. and Spectrum TPH from L.D. Caulk) which contain the above monomer systems. Saliva samples from different volunteers were collected, processed, pooled, and freeze-dried. TEGDMA and BisGMA monomers were incubated with human saliva derived esterase activity (HSDEA) and their respective hydrolysis was monitored using high performance liquid chromatography (HPLC). Both monomers were completely hydrolyzed within 25 h by HSDEA. Photopolymerized composites were incubated with buffer or human saliva (pH 7.0 and 37 C) for 2, 8 and 16 days. The incubation solutions were analyzed using HPLC and mass spectrometry. Surface morphology characterization was carried out using scanning electron microscopy. Upon biodegradation, the Z250 composite yielded higher amounts of BisGMA and TEGDMA related products relative to the TPH composite. However, there were higher amounts of ethoxylated bis-phenol A released from the TPH material. In terms of total mass of products released, human saliva demonstrated a greater ability to degrade Z250. In summary, HSDEA has been shown to contain esterase activities that can readily catalyze the biodegradation of current commercial composite resins.

The effects of 17 beta-estradiol on chondrocyte differentiation are modulated by vitamin D3 metabolites.

Both 17 beta-estradiol (17 beta) and the vitamin D metabolites, 1,25-(OH)2D3(1,25) and 24,25-(OH)2D3(24,25), regulate endochondral bone formation in vivo and in vitro. The effects of 17 beta are sex-specific and cell maturation-dependent. Similarly, the effects of 1,25 and 24,25 are cell maturation-dependent, with 1,25 affecting growth zone chondrocytes (GC) and 24,25 affecting resting zone chondrocytes (RC). This study examined whether the response of chondrocytes to 17 beta is altered after pretreatment with 1,25 or 24,25. Cells were isolated from the costochondral cartilage of male or female rats. Confluent, fourth-passage GC and RC cultures were pretreated with 1,25 or 24,25, respectively, for 24 or 48 h followed by treatment with 17 beta for an additional 24 h. At harvest, cell proliferation ([3H]-thymidine incorporation), differentiation (alkaline phosphatase specific activity [ALPase]), general metabolism ([3H]-uridine incorporation), and proteoglycan production ([35S]-sulfate incorporation) were determined. 1,25 enhanced the inhibitory effect of 17 beta on [3H]-thymidine incorporation by female GC cells; in contrast, no effect was observed in GC cells obtained from male rats. When male RC cells were treated with 17 beta, [3H]-thymidine incorporation was inhibited; however, when these cells were pretreated with 24,25 for 48 h, 17 beta stimulated [3H]-thymidine incorporation 24,25 had no effect on 17 beta-dependent [3H]-thymidine incorporation by female RC cells. 17 beta stimulated ALPase in female GC cells, but had no effect on male GC cells. 1,25 pretreatment of female GC cells inhibited the stimulatory effect of 17 beta on ALPase, but had no effect on ALPase in male GC cultures. 17 beta had no effect on male RC cell ALPase and stimulated ALPase in female RC cells. This was not affected by pretreatment with 24,25. Pretreatment with 1,25 increased the basal level of sulfate incorporation only in female GC. No effect was found in RC cells. These results indicate that pretreatment of rat costochondral chondrocytes with vitamin D metabolites modulate the effect of 17 beta. Although the effect of vitamin D metabolites alone on these chondrocytes is maturation-dependent and not sex-specific, the influence of preincubation with vitamin D metabolites on the effect of 17 beta is hormone-specific, sex-specific, and maturation-dependent.