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.     

Synthesis,characterization, biodegradation,and drug delivery application of biodegradable lactic/glycolic acid polymers. Part II: Biodegradation

A series of previously-synthesized lactic/glycolic acid polymers (PLGA) with various molar ratios of lactic to glycolic acid and various molecular weights were further studied with regard to their biodegradation behavior, and in particular, the factors affecting the biodegradation rate. The biodegradation of PLGA is affected by many factors including polymer composition, molecular weight, and nature of the incubating media. The biodegradation rate of PLGA containing higher content of lactic acid moiety is lower than those containing a lower content of lactic acid moiety. PLGAs with a higher molecular weight, degrade faster than those with a lower molecular weight, i.e. the molecular weight decreases more rapidly for higher molecular weight PLGAs than their lower molecular weight counterparts. Nature or properties of the hydrolysis/incubating media may have an effect on the biodegradation of PLGAs. A basic medium may slow down the biodegradation of PLGA in comparison with samples in an acidic medium. The rate of pH reduction for the incubating medium can be divided into three deferent phases, giving an inverted S-type pH profile for the non-buffered incubating media.     

Influence of surface morphology and chemistry on the enzyme catalyzed biodegradation of polycarbonate-urethanes

Abstract —Polycarbonate based polyurethanes were synthesized with varying hard segment content as well as hard segment chemistry based on three different diisocyanates,1,6-hexane diisocyanate (HDI), 4,4′-methylene bisphenyl diisocyanate (MDI) and 4,4-methylene biscyclohexyl diisocyanate (HMDI). The surface chemistry and morphology were characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The polymers were incubated with cholesterol esterase (CE) in a phosphate buffer solution at 37°C over 10 weeks. XPS results showed that the surface chemistry changed as the size and chemistry of the hard segment varied within the materials. AFM images exhibited distinctive surface morphologies for all polymers, and this was particularly apparent with changes in the hard segment chemistry. The results showed that the surface of HDI polymers consisted of relatively stiff rod-like structures, which corresponded to the soft segment domains. Polymers with a higher HDI content exhibited a dense top layer containing a relatively higher hard segment component, covering the sub-surface matrix of rod like structures. The MDI based polyurethane had large aggregates on its top surface, which corresponded to the aggregation of harder components. The HMDI based polycarbonate-urethane presented a relatively homogeneous surface where no phase separation could be detected. The relative differences in hard and soft segment content in their surface structure was supported by XPS findings. The analysis of the biodegradation results, concluded that enzyme catalyzed biodegradation within these materials was initiated in amorphous soft segment regions located in the region of the interface between hard and soft segments. A higher hard segment content at the surface contributed significantly to an increase in biostability. The findings provided an enhanced understanding for the role of surface molecular structure in the enzyme catalyzed biodegradation of polyurethanes.     

High-pressure infrared and FT-Raman investigation of a dental composite

Composite resins are often used as filling materials on load-bearing surfaces of teeth. As masticatory stresses can be high, here, we study the effect of pressure on the behaviour of a dental composite. Using a polymerized wafer, the IR and FT-Raman spectra of a zirconia-containing proprietary composite (Z100, 3M, Minneapolis, MN, USA) were recorded. The high-pressure IR spectra were also recorded. Band assignments were made for the main peaks of both organic and inorganic components. Breaks in the pressure dependences (dv/dP) of the organic components were found at 22 kbar. Different pressure dependences for different vibrational modes of inorganic components were also observed. These data suggest that the network structure of the composite is compacted under high pressure and that both the atomic distance and bonding angles in the network are altered.     

Caffeine dependence in rats: effects of exposure duration and concentration

Groups of rats were chronically exposed to a 1.0-g/L caffeine solution for 5, 10, 15 or 20 days. Upon removal of caffeine, rats were given brief exposure to a novel flavour CS (withdrawal CS) followed by 12 days of plain water and then brief exposure to a second flavour CS (neutral CS). Only rats exposed to 20 days of caffeine strongly preferred the neutral CS to the withdrawal CS in a 2-bottle test. In Experiment 2, groups of rats were chronically exposed to caffeine at one of four concentrations (1.0, 0.5, 0.25, or 0.125 g/L) for 21 days, after which withdrawal and neutral CSs were established. Only rats that drank the highest caffeine concentration, 1.0 g/L, preferred the neutral CS to the withdrawal CS. This suggests that long exposure to a strong caffeine solution is required in order to induce dependence in rats such that a CS associated with the withdrawal of caffeine becomes avoided.     

Organic overlayer model of a dental composite analyzed by laser desorption postionization mass spectrometry and photoemission

Some dental composites consist of a polymerizable resin matrix bound to glass filler particles by silane coupling agents. The resin in these composites includes bisphenol A diglycidyl methacrylate (Bis-GMA) as well as other organic components. Silane coupling agents such as 3-(trimethoxysilyl) propyl methacrylate (MPS) have been used to improve the mechanical properties of the dental composites by forming a covalent bond between the glass filler particles and the resin. These resin-glass composites undergo material property changes during exposure to the oral environment, but degradation studies of the commercial composites are severely limited by their chemical complexity. A simplified model of the dental composite has been developed, which captures the essential chemical characteristics of the filler particle-silane-resin interface. This model system consists of the resin matrix compound Bis-GMA covalently bound via a methacryloyl overlayer to amorphous silicon oxide (SiO2) surface via a siloxane bond. Scanning electron microscopy shows the porous characteristic and elemental composition of the SiO2 film, which approximately mimics that of the glass filler particles used in dental composites. LDPI MS and XPS verify the chemistry and morphology of the Bis-GMA-methacryloyl overlayer. Preliminary results demonstrate that LDPI MS will be able to follow the chemical processes resulting from aging Bis-GMA-methacryloyl overlayers aged in water, artificial saliva, or other aging solutions.     

Vicker's hardness and Raman spectroscopy evaluation of a dental composite cured by an argon laser and a halogen lamp

We present the results of the Vicker's hardness test and the use of near-infrared Raman spectroscopy (RS) to measure in vitro the degree of conversion (DC) of a bis(phenol)-A-glycidyl-dimethacrylate-based composite resin, photoactivated by both a halogen lamp (power density=478 mW/cm(2); 8-mm diameter spot) and an argon laser (power density=625 mW/cm(2); 7-mm diameter spot). The degree of conversion was estimated by analyzing the relative intensities between the aromatic C=C stretching Raman mode at 1610 cm(-1) and the methacrylate C=C stretching Raman mode (1640 cm(-1)) on top and bottom surfaces. For the hardness evaluation, the samples were embedded in polyester resin and three indentations with a 50-g load for 10 s were made on the top surface. The higher relative DC values achieved by the photoactivation of a composite resin by the argon laser suggest a better biocompatibility in the bottom surface. The correlation test showed that the higher Vicker's hardness number (VHN) values were associated with higher DC values. The derivative analysis showed a greater curing rate from 5 to 20 s of exposure. The comparison of VHN and DC values with both light sources at each curing time showed that a small change in conversion is related to a large change in hardness. Raman spectroscopy is more sensitive to changes in the first stages of curing reaction than later ones, and the Vicker's hardness assay is more sensitive to changes in the last stages.     

Behavior of human dermal fibroblasts in three-dimensional fibrin clots: dependence on fibrinogen and thrombin concentration

Fibrin sealant products are used in hemostasis and tissue sealing, and potentially as a cell delivery vehicle. In this study, fibrin sealant was evaluated as a delivery vehicle for human dermal fibroblasts. Fibroblast proliferation and migration were assessed in various dilutions of fibrin sealant by changing the fibrinogen and thrombin concentration. Fibroblasts proliferated well within three-dimensional (3-D) fibrin clots consisting of fibrinogen (5-17 mg/mL) and thrombin (1-167 U/mL). These fibroblasts also retained good morphology and growth characteristics after migrating out of the 3-D fibrin clots. Furthermore, using Western blot and fluorescence-activated cell-sorting analysis, we found that the expression of growth factors and interleukins in the entire fibroblast-fibrin construct was dependent on the fibrin sealant formulation. For example, in a formulation in which fibroblasts showed modest proliferation and migration, interleukin 8 was secreted to a lesser extent than in a formulation that supported robust proliferation and migration. To our knowledge, this is the first time that it has been shown that modifying the concentration of fibrinogen and thrombin affects fibroblast behavior within formed 3-D fibrin clots. In addition, some of these formulations present an ideal delivery vehicle for fibroblasts that could be used for the treatment of chronic wounds.     

Carbachol effects on hippocampal neurons in vitro: dependence on the rate of rise of carbachol tissue concentration

Summary Extracellular activity from vestibular nuclei neurons and vertical eye movements were recorded in the alert cat during sinusoidal optokinetic stimulation in the vertical plane at frequencies varying from 0.0125 Hz to 0.75 Hz. Among a population of 96 vestibular units located in and around Deiters' nucleus, 73 neurons (76%) displayed a firing rate modulation which followed the input at the standard parameters of visual stimulation (0.05 Hz; 10.1 deg/s or 9.1 cm/s peak to peak velocity). Two different patterns of modulation were found. In 42 cells (57%) an increase in the firing rate was observed during motion of the visual scene in the downward direction, while 31 neurons (43%) showed the opposite behavior, with an enhanced firing rate during upward movement. The phase of the neuronal responses was close (± 45°) to the velocity peaks (+90°: downward and -90°: upward) of visual scene motion for 65 among the 73 neurons. Mean values of phase was-6.1 ± 19.5° (SD) and -3.2 ± 15.5° (SD) with respect to the +90° and -90° velocity peaks, respectively. In the frequency range 0.0125–0.75 Hz, the phase of the neuronal responses remained almost stable, with only a slight lag which reaches -22° at the 0.25 Hz visual stimulation. The firing rate modulation was found to be predominant at low frequencies (0.0125 Hz–0.25 Hz), with three distinct peaks of modulation occurring either at 0.025 Hz, 0.10 Hz or 0.25 Hz, depending on the recorded cells. Above 0.5 Hz, the cell modulation was very poorly developed or even absent. A gain attenuation was observed in all units, which was more important in cells showing a peak of modulation at 0.025 Hz as compared with the others (-20.7 dB vs -9.6 dB, respectively, in the 0.025 Hz–0.25 Hz decade). The gain of the optokinetic reflex (OKR) progressively decreased from mean values of 0.78 ± 0.15 to 0.05 ± 0.06 in the 0.025 Hz–0.5 Hz frequency range. A close correlation was observed between the OKR slow phase velocity and the modulation of the neuronal responses in the two cell populations with maximal modulations at 0.10 Hz or 0.25 Hz. No correlations were noticed in the third population characterized by a peak of modulation at 0.025 Hz. In all units, the phase of eye movement velocity and of neuronal responses were both related to the velocity of the visual surround motion. These correlations were also found when varying the amplitude of the visual stimulation at a fixed frequency. Saturation was observed in the unit responses at velocities above 68.5°/s. When considering both the gain attenuation in the frequency range and the correlation between firing rate modulation and OKR slow phase velocity, two rather different cell populations can be distinguished: one with neurons peaking at 0.025 Hz (strong gain attenuation; no correlation with OKR velocity) and one with neurons peaking at 0.10 Hz or 0.25 Hz (slight gain attenuation; correlation with OKR velocity). This study points to the influence of visual motion cues on vestibular nuclei unit activity in the low-frequency range. A velocity coding of visual — surround motion in the vertical plane is performed by vestibular neurons. Our results in the alert cat suggest that both retinal (retinal slip) and extraretinal (proprioceptive afferences from eye muscles, efference copy) inputs can be involved in this visually induced modulation of vestibular nuclei neurons.     

改良酶消化法分离培养人乳牙牙髓干细胞

背景：乳牙牙髓干细胞具有极强的增殖活力,可以在短期内获得组织工程需要的细胞量。 目的：比较传统酶消化法和改良酶消化法获取人乳牙牙髓干细胞的成功率及生物学特性。 方法：取无龋滞留乳切牙12 颗,随机数字表法均分为两组,分别应用传统酶消化法和改良酶消化法分离获取乳牙牙髓细胞,在不同代次细胞中检测细胞扩增天数、收获量、生长曲线、倍增时间、克隆形成率、细胞表面特异标记物STRO-1、CD146、CD34 和CD45 的表达。细胞成骨、成脂诱导分化能力及通过检测碱性磷酸酶和牙本质涎蛋白表达水平,判定细胞牙向分化潜能。 结果与结论：两组细胞生长曲线,扩增天数、细胞收获量及细胞倍增时间相比,差异有显著性意义(P <0.05);牙向分化实验结果显示,改良酶消化法碱性磷酸酶和牙本质涎蛋白较传统酶消化法和对照组而言呈强阳性表达。然而,两组在克隆形成率、细胞表面特异标记物及多项诱导分化等方面差异无显著性意义(P > 0.05)。说明改良酶消化法与传统酶消法相比,可在较短时间内完成同源乳牙牙髓干细胞体外扩增培养,可为牙齿再生治疗提供高质量种子细胞,是乳牙牙髓干细胞体外培养的可靠方法。     

Coeliac autoantibodies can enhance transamidating and inhibit GTPase activity of tissue transglutaminase: dependence on reaction environment and enzyme fitness

Modification of the enzymatic functions of tissue transglutaminase (TG2) by anti-TG2 autoantibodies may play a role in manifestations of coeliac disease. Our aim was to evaluate the effect of coeliac autoantibodies on reactions catalysed by TG2 by a systematic biochemical approach, and in relation to observed clinical presentation type. Coeliac antibodies did not have significant inhibitory effect on transamidation/deamidation activity of TG2 as measured by amine-incorporation into solid and immobilised casein and by ultraviolet kinetic assay. In contrast, immunoglobulins from patients with severe malabsorption enhanced the reaction velocity to 105.4-242.2%. This activating effect was dose-dependent, most pronounced with immobilised glutamine-acceptor substrates, and correlated inversely with the basal specific activity of the enzyme and with dietary treatment. A similar activation could be demonstrated also with the TG2-specific fraction of autoantibodies and in transamidation activity assays which use fibronectin-bound TG2 and thereby mimic in vivo conditions. These results suggest that coeliac antibodies may stabilise the enzyme in a catalytically advantageous conformation. GTPase activity of TG2 decreased to 67.0-73.4% in the presence of antibodies raising the possibility that inhibition of GTPase activity may affect cellular signalling in case coeliac autoantibodies would reach intracellular compartments.     

Alterations of the specificity of lectin by chemical modification: IV. Further purification and alteration of the specificity of a snail lectin by chemical modification

The anti-A agglutinin of the snail Otala lactea, the most potent non-immune source of anti-A thus far discovered, was partially purified and studied serologically. Studies were made of the effect of chemical alteration on the specificity. Coupling diazotized p-aminophenyl-α-N-acetyl-[¹⁴C]galactoside to the purified lectin greatly enhanced the agglutinating activity of the lectin for group B erythrocytes, without significantly affecting its activity for A₁, A₂ or 0 (no anti-0 activity) cells. Treatment of the lectin with phenylazo-benzoyl [¹⁴C] (PAB)chloride did not significantly alter its activity or specificity. The anti-B activity conferred by the first of these treatments could be inhibited by sufficient amounts of galactose.     

Effects of postcuring and water sorption on the mechanical properties of composite dental restorative materials

Four commercial dental restorative composites with different filler contents, were tested for the effects of postcuring and water sorption on elastic modulus, compressive strength and ultimate strain. Large variations in mechanical properties were seen; water sorption plasticizes the matrix, causing loss of low molecular weight substances.     

Functional profile of human influenza virus-specific cytotoxic T lymphocyte activity is influenced by interleukin-2 concentration and epitope specificity

The ability of influenza A virus-specific cytotoxic T lymphocytes (CTL) to degranulate and produce cytokines upon antigenic restimulation was studied in four HLA-A*0101 and HLA-A*0201 positive subjects. Peripheral blood mononuclear cells of these subjects were stimulated with influenza A virus in the presence of high or low interleukin (IL)-2 concentrations. CD8(+) T cell populations specific for the HLA-A*0101 restricted epitope NP(44-52) and the HLA-A*0201 restricted epitope M1(58-66) were identified by positive staining with tetramers of peptide major histocompatibility complexes (MHC) (NP-Tm and M1-Tm, respectively). Within these populations, the proportion of cells mobilizing CD107a, or expressing interferon (IFN)-gamma and tumour necrosis factor-(TNF)-alpha upon short-term peptide restimulation was determined by flow cytometry. Independent of IL-2 concentrations, large subject-dependent differences in the mobilization of CD107a and expression of IFN-gamma and TNF-alpha by both NP- and M1-specific T cells were observed. In two of the four subjects, the functional profile of NP-Tm(+) and M1-Tm(+) cells differed considerably. Overall, no difference in the proportion of NP-Tm(+) or M1-Tm(+) cells expressing CD107a was observed. The proportion of M1-Tm(+) cells that produced IFN-gamma (P < 0.05) was larger than for NP-Tm(+) cells, independent of IL-2 concentration. When cultured under IL-2(hi) concentrations higher TNF-alpha expression was also observed in M1-Tm(+) cells (P < 0.05). The IL-2 concentration during expansion of virus-specific cells had a profound effect on the functionality of both M1-Tm(+) and NP-Tm(+) cells.     

The behavior of human mesenchymal stem cells in 3D fibrin clots: dependence on fibrinogen concentration and clot structure

Natural biopolymers such as human fibrin are appealing to tissue engineers, because fibrin is biocompatible, bioresorbable, and essential in normal wound healing. There have been numerous studies to date to develop a fibrin-based injectable cell delivery system, albeit with varying success. We propose that the outcome of fibrin cell delivery can, in part, be attributed to the relative concentrations of fibrinogen and thrombin solutions (i.e., formulations) and the structure of the final 3D fibrin clot. Formulation-dependent proliferation of human mesenchymal stem cells (hMSCs) within 3D fibrin clots was investigated in vitro. Our results indicate that hMSCs are viable in all fibrin sealant formulations investigated, and proliferation rates vary with fibrin formulations. Furthermore, the fibrinogen solution, not thrombin, was found to have a more dominant role on hMSC proliferation, with dilute fibrinogen solutions promoting greater hMSC proliferation. Confocal and electron microscopy reveal formulation dependence on 3D fibrin clot structure, with dilute fibrinogen solutions yielding more open, homogeneous microstructures. This study suggests that the concentrations of fibrinogen and thrombin solutions must be carefully considered for cell delivery because they affect 3D fibrin clot structure and cell proliferation.     

Phagocytosis of yeast by Biomphalaria glabrata: carbohydrate specificity of hemocyte receptors and a plasma opsonin

In vitro phagocytosis of yeast cells by hemocytes of the mollusc Biomphalaria glabrata can occur in the absence of plasma, but is enhanced by opsonization in plasma from certain snail strains. We have investigated the carbohydrate specificity of the hemocyte-bound receptor for phagocytosis and the free plasma opsonin using two dominant carbohydrate components of the yeast cell wall (laminarin and mannan). Phagocytic uptake in the absence of plasma of both untreated and opsonized yeast is strongly inhibited by laminarin, but is unaffected by mannan. In contrast, laminarin does not interfere with opsonization whereas mannan blocks the process, blocking the binding of a 57 kD plasma component as detected by Western immunoblot. These results suggest that the opsonic lectin in plasma is not simply a free form of the hemocyte-bound receptor for yeast phagocytosis.     

Vancomycin area under the curve to minimum inhibitory concentration ratio predicting clinical outcome: a systematic review and meta-analysis with pooled sensitivity and specificity

BackgroundVancomycin is a first-line antibiotic for methicillin-resistant Staphylococcus aureus infections or other Gram-positive infections. The area under the curve (AUC) to minimum inhibitory concentration (MIC) ratio is proposed as a therapeutic drug-monitoring parameter. How well clinical efficacy is predicted by this measure has not been established.ObjectiveDetermine the test performance characteristics (TPC) of AUC:MIC of vancomycin for prediction of positive outcome.Data SourcesPubMed and Ovid Medline (1946 to 2018) and EMBASE (1974 to 2018).Study Eligibility Criteria and Participants: Studies of patients treated with vancomycin for any type of infection in peer reviewed publications. All patient populations were included.InterventionsVancomycin AUC:MIC or AUC was related to patient clinical outcome.MethodsSearches of medical databases using relevant terms were performed. Screening, study reviewing, data extracting and assessing data quality was performed independently by two reviewers. Studies were stratified by type of primary outcome for calculation of pooled sensitivity, specificity and construction of hierarchical summary receiver operating characteristic (HSROC) curves.ResultsNineteen studies including 1699 patients were meta-analysed. Pooled sensitivity and specificity were 0.77 (95% CI 0.67–0.84) and 0.62 (95% CI 0.53–0.71) respectively for the seven studies with primary outcome of mortality and 0.65 (95% CI 0.53–0.75), 0.58 (95% CI 0.48–0.67) for studies with composite or clinical cure outcome (n = 12). HSROC curves suggested considerable heterogeneity. An additional 11 studies were described but could not be included for meta-analysis because data were not available. The majority of these studies (9/11) failed to demonstrate a relationship between AUC:MIC and positive clinical outcome.ConclusionsVancomycin AUC:MIC performance was modest and inconsistent. Analysis was limited by studies without sufficient data; therefore, meta-analytic results may overestimate TPC values. Given this, as well as the lack of standardization of methods, widespread adoption of AUC:MIC as the preferred vancomycin monitoring parameter may be premature.     

Photoprotective activity of melanin preparations from black yeast-like fungus during UV irradiation of human skin: dependence on the concentration

The effect of melanin solutions on the skin exposed to UV irradiation (1050 kJ/m2) depended on its dose and varied from photoprotection (0.005 mg/ml) to photosensitization and phototoxicity (burn, 0.1 mg/ml). These results suggest that doses of melanin preparations should be empirically selected to achieve optimum photoprotective effect.     

Steep concentration dependence and fast desensitization of nicotinic channel currents elicited by acetylcholine pulses,studied in adult vertebrate muscle

Skeletal muscles of adult mice and frogs were dissociated enzymatically and prepared for patch-clamping within less than 6 h. Outside-out patches were superfused with repetitive pulses of acetylcholine (ACh) with switching times of about 0.2 ms. Peak responses were reached within 1 ms. In mouse muscle the average channel conductance was 65 pS and the average open time 1 ms (20° C). Between 1 and 10 M ACh, the peak responses increased proportional to the second to third power of the ACh concentration, and less steeply between 10 and 1000 M ACh. The apparent K _m of the dose-response curve was about 100 M. After the peak, channel opening probability declined with time constants decreasing from about 1 s with 1 M ACh to 15–50 ms with 1 mM ACh. After 100 ms desensitization the channel opening had decreased to less than 1/300 peak value. The rate of desensitization increased with rising temperature, with Q ₁₀ values of 1.7–2.5 between 10 and 30° C. The desensitization characteristics of channels from frog muscle were similar to that from mice. With pulses of 100 M ACh the channels opened with a probability of 0.55, the open probability declining with a time constant of about 60 ms and dropping to less than 0.001 after 300 ms. The results support the view that three binding steps of ACh are necessary for opening of the channel. Desensitization in the presence of high ACh concentrations is slower than the decay of synaptic currents.