Physicochemical and mechanical characterization of a fiber-reinforced composite used as frameworks of implant-supported prostheses

ObjectivesTo characterize the physicochemical and mechanical properties of a milled fiber-reinforced composite (FRC) for implant-supported fixed dental prostheses (FDPs).MethodsFor FRC characterization, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction, Fourier-transformed infrared spectrometry, simultaneous thermogravimetric analysis and differential scanning calorimetry were performed. For fatigue testing, 3-unit FRC frameworks were fabricated with conventional (9 mm² connector area) and modified designs (12 mm² connector area and 2.5 mm-height lingual extension). A hybrid resin composite was veneered onto the frameworks. FDPs were subjected to step-stress accelerated-life fatigue testing until fracture or suspension. Use level probability Weibull curves at 300 N were plotted and the reliability for 100,000 cycles at 300, 600 and 800 N was calculated. Fractographic analysis was performed by stereomicroscope and SEM.ResultsThe FRC consisted of an epoxy resin (∼25%) matrix reinforced with inorganic particles and glass fibers (∼75%). Multi-layer continuous regular-geometry fibers were densely arranged in a parallel and bidirectional fashion in the resin matrix. Fatigue analysis demonstrated high probability of survival (99%) for FDPs at 300 N, irrespective of framework design. Conventional FDPs showed a progressive decrease in the reliability at 600 (84%) and 800 N (19%), whereas modified FDPs reliability significantly reduced only at 800 N (75%). The chief failure modes for FRC FDPs were cohesive fracture of the veneering composite on lower loads and adhesive fracture of the veneering composite at higher loads.SignificanceMilled epoxy resin matrix reinforced with glass fibers composite resulted in high probability of survival in the implant-supported prosthesis scenario.     

Validation of a dental pain screening questionnaire in a semi‐urban hospital setting in South India

Aims : To assess the validity of modified DePaQ items in correctly classifying dental pain patients into three groups of common dental conditions when compared against clinically‐determined classification; generate a model of classification equations from a sub‐population of the sample; and assess the validity of the model in predicting group membership of new dental pain cases. Methods : Consecutive adult hospital dental patients aged 18 years and older attending with a dental pain complaint were diagnosed by a dentist. Before treatment commenced a self‐complete questionnaire was completed. The examining dentist was blinded to the questionnaire responses. The sample was randomly split into two groups (RS1 and RS2). Discriminant analysis was carried out on RS1 to develop a model for classifying new dental pain cases into three groups. This model was used to classify dental pain cases in RS2. Results : Of the 311 questionnaires distributed, 306 (98.4%) were returned. Of the 205 Group 1 cases diagnosed clinically 186 (90.7%) were correctly predicted as belonging to that group by M‐DePaQ. For 54 clinically diagnosed Group 2 cases 44 (81.5%) were correctly predicted, and for 32 Group 3 cases, 26 (81.3%) were correctly predicted. The model of classification equations derived from RS1 were capable of correctly classifying 85%, 82% and 79% of clinically‐derived classification for Group 1, 2 and 3 respectively. Conclusions : This study has demonstrated that the M‐DePaQ is valid in classifying dental pain patients into three groups of common dental conditions. Classification equations generated from a sub‐group of the sample provided a valid statistical model for classifying dental pain cases from the same setting.     

The Influence of Precondensation Mercury Content and Mulling on the Transverse Strength of Amalgams Condensed After a Delay

The purpose of this study was to investigate the influence of changing the precondensation mercury content (initial mercury content) on the final strength of amalgams when the condensation was delayed. Another purpose was to study the effect on the transverse strength of mulling the amalgam mix after the delay. The material consisted of ten different brands of alloy. Three different alloy-mercury ratios were used with each alloy brand; Mix I about 50% Hg, Mix II about 54% Hg and Mix III about 59% Hg. Rectangular amalgam test pieces, measuring 2 × 2 × 12 mm, were condensed after a 5 minutes delay by hand using a load of about 17 kg/cm². The transverse strength test was performed after one week using three point loading. The five minutes delay of the condensation reduced the strength of the amalgams by 1 to 42 per cent depending on the brand of alloy and precondensation mercury content. The three preamalgamated amalgams were affected less by the delay than the other amalgams. Increasing the precondensation mercury content reduced the effect of the delay on the final strength. The «mulling» of the amalgam mix also decreased the effect of the delay. It was concluded that a moderate excess of initial mercury gives the dentist a longer condensing time thus allowing him to perform the condensing procedure with care.     

下颌第一磨牙种植固定桥的受载分析

目的 观察下颌第一磨牙种植固定桥的位移和应力分布规律。方法 采用了三维有限元法进行受载分析。结果 发现第二双尖牙的最大位移值较大;由于固定桥的支架作用,垂直分散载荷下天然基牙和种植基牙的位移均值之间差异无显著性。基牙颈部为应力集中区,第二双尖牙根尖的应力较小。集中载荷加于基牙时,位移和应力值明显增加。结论 提示应注意末端种植基牙的载荷和咬合状态,该设计是临床可使用的特殊固定桥。     

Luting glass ceramic restorations using a selfadhesive resin cement under different dentin conditions

Objectives

The aim of this study was to investigate the bond strength of ceramic restorations luted using a self-adhesive resin cement (RelyX Unicem, 3M ESPE) under different dentin conditions.

Material and Methods

In the experimental groups, ceramic restorations were luted to bovine incisors with RelyX Unicem under the following conditions: [Dry dentin]: surface was dried using air stream for 15 s; [Moist dentin]: excess dentin moisture was removed with absorbent paper; [Bonding agent]: Clearfil SE Bond (Kuraray) self-etching adhesive system was previously applied to dentin. In the Control group, cementation was done using an etch-and-rinse adhesive (Excite DSC) and Variolink II resin cement (Ivoclar Vivadent). Photoactivation of the resin cements was performed with UltraLume LED 5 unit (Ultradent). The restorations (n=5 per group) were sectioned into beams and microtensile testing was carried out. Data were subjected to ANOVA and Tukey''s test (p<0.05). Failure modes were classified under Scanning Electron Microscopic (SEM) (×120 magnification).

Results

The bond strength was dependent on the moisture status of the dentin. Bond strength in the "dry dentin group" was significantly lower than that of all other groups, which showed similar results. A predominance of mixed failures was detected for the control group, while a predominance of adhesive failures was observed for the "bonding agent" and "dry dentin" groups. The "moist dentin" group presented predominantly cohesive failures within the luting material. The previous application of a self-etching adhesive showed no significant effect.

Conclusions

Only excess dentin moisture should be removed for the cementation of ceramic restorations with self-adhesive resin cements.     

Preparation and evaluation of a BisGMA-free dental composite resin based on a novel trimethacrylate monomer

ObjectiveThe use of the BisGMA as base monomer in dental composites has been questioned because of bisphenol A (BPA) is used as raw material in its synthesis, and BPA possess estrogenic potential associated to several health problems. This study describes the synthesis of the trimethacrylate tris(4-hydroxyphenyl)methane triglycidyl methacrylate (TTM) monomer and evaluate its effect when used as base monomer in the formulation of experimental photopolymerizable composite resins.MethodsThe TTM monomer was synthesized by a nucleophilic acyl substitution. Its chemical structure was confirmed via ¹H and ¹³C NMR spectroscopy and FTIR spectroscopy. Experimental composite resins were formulated by mixing TTM, triethyleneglycol dimethacrylate (TEGDMA) and inorganic fillers. A BisGMA/TEGDMA based composite resin was prepared and used as control to compare several physicochemical properties. Cell viability assay was used for cytotoxicity evaluation.ResultsTTM was successfully synthesized with quantitative yields. The results showed that the TTM-based composite resin had similar values of flexural strength, elastic modulus, degree of conversion and polymerization shrinkage than the control (p > 0.05). Water sorption and solubility were statistically significantly higher than the control (p < 0.05), however they complied the requirements stablished by the ISO 4049. Finally, this study shows there were no statistically significant differences for the biocompatibility outcomes (p = 0.345).SignificanceTTM monomer could be potentially useful in the formulation of BisGMA free composite resins, which could mean to minimize the human exposure to BPA.     

Adhesive bonding of resin composite to various titanium surfaces using different metal conditioners and a surface modification system

Objective

This study evaluated the effect of three metal conditioners on the shear bond strength (SBS) of a prosthetic composite material to cpTi grade I having three surface treatments.

Material and Methods

One hundred sixty eight rivet-shaped specimens (8.0x2.0 mm) were cast and subjected to polishing (P) or sandblasting with either 50 mm (50SB) or 250 mm (250SB) Al₂O₃. The metal conditioners Metal Photo Primer (MPP), Cesead II Opaque Primer (OP), Targis Link (TL), and one surface modification system Siloc (S), were applied to the specimen surfaces, which were covered with four 1-mm thick layers of resin composite. The resin layers were exposed to curing light for 90 s separately. Seven specimens from each experimental group were stored in water at 37ºC for 24 h while the other 7 specimens were subjected to 5,000 thermal cycles consisting of water baths at 4ºC and 60ºC (n=7). All specimens were subjected to SBS test (0.5 mm/min) until failure occurred, and further 28 specimens were analyzed using scanning electron microscope (SEM) and X-ray energy-dispersive spectroscopy (EDS). Data were analyzed by 3-way ANOVA followed by post-hoc Tukey''s test (α=0.05).

Results

On 50SB surfaces, OP groups showed higher SBS means than MPP (P<0.05), while no significant difference was found among OP, S, and TL groups. On 250SB surfaces, OP and TL groups exhibited higher SBS than MPP and S (P<0.05). No significant difference in SBS was found between OP and TL groups nor between MPP and S groups. The use of conditioners on 250SB surfaces resulted in higher SBS means than the use of the same products on 50SB surfaces (P<0.05).

Conclusion

Sandblasting associated with the use of metal conditioners improves SBS of resin composites to cpTi.     

Selective serotonin reuptake inhibitors as a risk factor for dental implant failure: a retrospective clinical study

The trend in recent decades in Europe and the United States points to an exponential increase in the consumption of antidepressant drugs and, in particular, selective serotonin reuptake inhibitors (SSRIs). This retrospective study aimed to investigate whether there is an association between SSRI intake and dental implant (DI) failure or survival and, secondarily, to investigate the influence of other systemic and local factors. This retrospective cohort study was done in accordance with the the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for observational studies. A total of 170 patients received 573 DIs between 2014 and 2020.The reported DI failure rate was 6.11% (n = 35 DIs). Of these 18.31% failed in patients treated with SSRIs while 4.38% failed in patients who were not prescribed SSRIs (p < 0.001). Specifically, use of these drugs was associated with a hazard ratio rate of DI failure that was 4.53 times higher (95% CI: 1.93 to 10.61), and in the multivariate analysis, a 3.70 times higher adjusted risk was found. A lower DI survival rate at 90 months’ follow up was also observed in these patients compared with those not taking them (84.30% vs 96%, respectively; p = 0.00014). With the limitations of the present study it can be affirmed that there is a relation between the intake of SSRIs and DI failure, as well as a lower survival rate in these patients.     

下颌骨形态对种植体-骨界面应力分布影响的研究

目的　模拟单个下颌磨牙缺失的种植修复 ,分析不同下颌骨形态对种植体 骨界面应力分布的影响。方法　采用三维有限元法 ,根据下颌骨测量资料建立不同颌骨截面形态的种植修复模型并进行分析。结果　不论垂直载荷或斜向载荷 ,不同颌骨形态模型骨界面应力分布规律及应力值差异均无显著性。其中Von Mises应力最大差异为 6 4% ,压应力最大差异为 2 8% ,拉应力最大差异为 6 2 %。结论　在有限元研究中将下颌骨形态简化为规则形态是合理的     

Performance of crowns cemented on a fiber-reinforced composite framework 5-unit implant-supported prostheses: in silico and fatigue analyses

ObjectiveTo characterize the biomechanical performance of fiber-reinforced composite 5-unit implant-supported fixed dental prostheses (FDPs) receiving individually milled crowns by insilico and fatigue analyses.MethodsEighteen implant-supported five-unit fiber-reinforced composite frameworks with an individually prepared abutment design were fabricated, and ninety resin-matrix ceramic crowns were milled to fit each abutment. FDPs were subjected to step-stress accelerated-life testing with load delivered at the center of the pontic and at 2nd molar and 1st premolar until failure. The reliability of the prostheses combining all loaded data and of each loaded tooth was estimated for a mission of 50,000 cycles at 300, 600 and 900 N. Weibull parameters were calculated and plotted. Fractographic and finite element analysis were performed.ResultsFatigue analysis demonstrated high probability of survival at 300 N, with no significant differences when the set load was increased to 600 and 900 N. 1st and 2nd molar dataset showed high reliability at 300 N, which remained high for the higher load missions; whereas 1st premolar dataset showed a significant decrease when the reliability at 300 N was compared to higher load missions. The characteristic-strength of the combined dataset was 1252 N, with 1st molar dataset presenting higher values relative to 2nd molar and 1st premolar, both significantly different. Failure modes comprised chiefly cohesive fracture within the crown material originated from cracks at the occlusal area, matching the maximum principal strain location.SignificanceFive-unit implant-supported FDP with crowns individually cemented in a fiber-reinforced composite framework presented a high survival probability. Crown fracture comprised the main failure mode.     

Effect of a multi-layer infection control barrier on the micro-hardness of a composite resin

Objective

The aim of this study was to evaluate the effect of multiple layers of an infection control barrier on the micro-hardness of a composite resin.

Material and Methods

One, two, four, and eight layers of an infection control barrier were used to cover the light guides of a high-power light emitting diode (LED) light curing unit (LCU) and a low-power halogen LCU. The composite specimens were photopolymerized with the LCUs and the barriers, and the micro-hardness of the upper and lower surfaces was measured (n=10). The hardness ratio was calculated by dividing the bottom surface hardness of the experimental groups by the irradiated surface hardness of the control groups. The data was analyzed by two-way ANOVA and Tukey''s HSD test.

Results

The micro-hardness of the composite specimens photopolymerized with the LED LCU decreased significantly in the four- and eight-layer groups of the upper surface and in the two-, four-, and eight-layer groups of the lower surface. The hardness ratio of the composite specimens was <80% in the eight-layer group. The micro-hardness of the composite specimens photopolymerized with the halogen LCU decreased significantly in the eight-layer group of the upper surface and in the two-, four-, and eight-layer groups of the lower surface. However, the hardness ratios of all the composite specimens photopolymerized with barriers were <80%.

Conclusions

The two-layer infection control barrier could be used on high-power LCUs without decreasing the surface hardness of the composite resin. However, when using an infection control barrier on the low-power LCUs, attention should be paid so as not to sacrifice the polymerization efficiency.     

IPS-Empress 2 玻璃陶瓷结构及性能的研究

目的 研究新型IPS-Empress 2牙科高强度陶瓷的显微结构和机械性能。方法采用原子力显微镜、扫描电子显微镜和X射线衍射仪，分析IPS-Empress 2的显微结构和晶相，用三点弯曲实验和压痕法测试其弯曲强度和断裂韧性。结果IPS-Empress 2玻璃陶瓷主要由二硅酸锂晶体和磷酸锂晶体组成，二者形成相互交错的三维网络式结构；这种玻璃陶瓷在热压铸前后晶体相保持不变，其三点弯曲强度和断裂韧性分别为300MPa和3．1MPam^1/2。结论IPS-Empress 2玻璃陶瓷的高强度和韧性与高含量的二硅酸锂晶体、相互锁结的网络结构和裂纹偏转有关。     

氟化物涂膜的临床应用

氟化物的防龋作用较为明显，目前临床上应用的各种氟化物剂型多有其缺点，严重影响了氟化物的临床应用。氟化物涂膜作为一种局部应用氟化物缓释剂型，由于其独特的剂型特点，在防龋、治疗牙本质过敏症和预防正畸过程中牙釉质白斑的发生等多个方面都有着不可替代的优点，因此在许多国家氟化物涂膜已取代了氟化物凝胶的应用。本文就氟化物涂膜的临床应用情况及功能特点作一简要综述。     

The influence of a novel propolis on mutans streptococci biofilms and caries development in rats

A flavonoids-free Brazilian propolis (type 6) showed biological effects against mutans streptococci and inhibited the activity of glucosyltransferases. This study evaluated the influence of the ethanolic extract of a novel type of propolis (EEP) and its purified hexane fraction (EEH) on mutans streptococci biofilms and the development of dental caries in rats. The chemical composition of the propolis extracts were examined by gas chromatography/mass spectrometry. The effects of EEP and EEH on Streptococcus mutans UA159 and Streptococcus sobrinus 6715 biofilms were analysed by time-kill and glycolytic pH drop assays. Their influence on proton-translocating F-ATPase activity was also tested. In the animal study, the rats were infected with S. sobrinus 6715 and fed with cariogenic diet 2000. The rats were treated topically twice a day with each of the extracts (or control) for 5 weeks. After the experimental period, the microbial composition of their dental plaque and their caries scores were determined. The results showed that fatty acids (oleic, palmitic, linoleic and stearic) were the main compounds identified in EEP and EEH. These extracts did not show major effects on the viability of mutans streptococci biofilms. However, EEP and EEH significantly reduced acid production by the biofilms and also inhibited the activity of F-ATPase (60-65%). Furthermore, both extracts significantly reduced the incidence of smooth surface caries in vivo without displaying a reduction of the percentage of S. sobriuns in the animals' plaque (P < 0.05). However, only EEH was able to reduce the incidence and severity of sulcal surface caries (P < 0.05). The data suggest that the cariostatic properties of propolis type 6 are related to its effect on acid production and acid tolerance of cariogenic streptococci; the biological activities may be attributed to its high content of fatty acids.     

Dental specialty,career preferences and their influencing factors among final year dental students in Saudi Arabia

Objective

The purpose of this study was to investigate evolving trends in dental post graduate specialty preferences and career aspirations among final year dental students in Saudi Arabia.

The use of autologous bone for augmentation procedures leads to low prevalence of peri-implantitis–a retrospective study over a 20-year period

The aim of this study was to compare the prevalence of peri-implantitis in implants inserted into pristine bone (control) to implants where autologous bone was used for grafting procedures (study).All patients who underwent implant surgery during a 20 years interval by one maxillofacial implant surgeon and received a prosthodontic rehabilitation afterwards were eligible for inclusion in the study. Periimplant bone resorption and periimplant disease were assessed.Of 421 patients 384 (91.2%) patients responded to a recall after having been treated over a 20-year period by one maxillofacial surgeon and several dentists. A total of 110 patients had 239 implants in pristine bone, and 274 patients had 607 implants placed in combination with autologous bone grafting procedures. Mean time in function was 74 months (range 15–236 months). In all, 342 implants (34.8%) were in function for longer than 7 years. A total of 64 implant sites (7.6%) in 39 patients (10.2%) showed signs of peri-implant mucositis. In addition, 17 implants (2.0%) in 14 patients (3.6%) revealed signs of peri-implantitis, of which five implants were in the control group (2.09%) whereas 12 implants were in the study group (1.98%), with no statistically significant difference (p = 0.8405). More than half of the patients with peri-implantitis had a history of periodontitis. Three implants were lost due to peri-implantitis and four implants failed for other reasons, resulting in an overall success rate of 99.2% in 846 implants.ConclusionsWithin the limitations of the study it seems that the use of autologous bone still is a relevant option when performing augmentation procedures because of the low prevalence of peri-implantitis.     

Effects of light-curing time on the cytotoxicity of a restorative composite resin on odontoblast-like cells

This in vitro study evaluated the cytotoxicity of an experimental restorative composite resin subjected to different light-curing regimens.

Methods

Forty round-shaped specimens were prepared and randomly assigned to four experimental groups (n=10), as follows: in Group 1, no light-curing; in Groups 2, 3 and 4, the composite resin specimens were light-cured for 20, 40 or 60 s, respectively. In Group 5, filter paper discs soaked in 5 μL PBS were used as negative controls. The resin specimens and paper discs were placed in wells of 24-well plates in which the odontoblast-like cells MDPC-23 (30,000 cells/cm²) were plated and incubated in a humidified incubator with 5% C_O2 and 95% air at 37ºC for 72 h. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). The data were analyzed statistically by Kruskal-Wallis and Mann- Whitney tests (p<0.05).

Results

In G1, cell metabolism decreased by 86.2%, indicating a severe cytotoxicity of the non-light-cured composite resin. On the other hand, cell metabolism decreased by only 13.3% and 13.5% in G2 and G3, respectively. No cytotoxic effects were observed in G4 and G5. In G1, only a few round-shaped cells with short processes on their cytoplasmic membrane were observed. In the other experimental groups as well as in control group, a number of spindle-shaped cells with long cytoplasmic processes were found.

Conclusion

Regardless of the photoactivation time used in the present investigation, the experimental composite resin presented mild to no toxic effects to the odontoblast-like MDPC-23 cells. However, intense cytotoxic effects occurred when no light-curing was performed.     

Effect of prosthodontic planning on lateral occlusion scheme: a comparison between conventional and digital planning

Recently, digital wax-up is proposed as a tool to aid prosthodontic planning. However, there are no data about the effect of prosthodontic planning on lateral occlusion scheme.

Objective

: This study aims to evaluate the impact of conventional and digital prosthodontic planning on lateral occlusion scheme.

Material and Methods

: Dental models of 10 patients were collected. All models had Angle Class I occlusion and were undergoing prosthodontic treatment that would influence the lateral occlusion scheme. Each set of models had received both conventional wax-up and digital wax-up. In relation to the lateral occlusion scheme, the following variables were evaluated: the prevalence of the different lateral occlusion scheme, number of contacting teeth and percentage of each contacting tooth. Four excursive positions on the working side were included: 0.5, 1.0, 2.0 and 3.0 mm from the maximal intercuspation position.

Results

: The lateral occlusion scheme of the two wax-up models was subjected to alterations following excursion. There was a tendency for the prevalence of canine-guided occlusion to increase and for the prevalence of group function occlusion to decrease with increasing excursion. The number of contacting teeth was decreasing with the increasing magnitude of excursion. For the 0.5 mm and 1.0 mm positions, the two wax-ups had significantly greater contacts than the pre-treatment models, while at the 2.0 mm and 3.0 mm positions, all the models were similar. For all models, canines were the most commonly contacting teeth, followed by the teeth adjacent to them. No difference was observed between the two wax-ups in relation to the number of contacting teeth.

Conclusion

: Although the prosthodontic planning had influenced the pattern of the lateral occlusion scheme and contacts, there was no difference between the conventional and digital prosthodontic planning.     

Interfacial adaptation of a calcium aluminate cement used in class II cavities,in vivo

The aim of this in vivo study was to evaluate the interfacial marginal adaptation of a calcium aluminate cement, Doxadent (DD), and to compare it intra-individually with a resin composite, Tetric Ceram/Syntac Single-Component (TC/SS), in Class II cavities. Sixteen Class II box-shaped, enamel-bordered cavities were prepared in eight premolars scheduled to be extracted after 1 months service for orthodontic reasons. The interfacial marginal adaptation (internal surfaces) of the restorations was evaluated by a quantitative scanning electron microscope analysis using a replica method. DD showed a statistically significant, lower degree of gap-free adaptation to enamel compared with TC/SS: 84% vs. 93%. To dentin, DD showed a significantly better adaptation than TC/SS: 72% vs. 49%. A high frequency of enamel fractures perpendicular to the margins was observed for the DD restorations, which may be explained by an expansion of the calcium-aluminate cement. It can be concluded that DD showed a better adaptation to dentin while TC/SS showed a better adaptation to enamel. The dimensional changes of DD have to be investigated before clinical use can be recommended.     

Effect of a Ca(OH)2 solution and a chlorhexidine based detergent on the microbial activity of human carious teeth

Occlusal carious lesions from human molars, preserved in continuous humidity after extraction, were removed using conventional clinical techniques. Bacteriological samples were taken after rinsing the cavity with water only, after experimentally infecting the cavity and after treating uninfected and infected cavities either with a saturated Ca(OH)₂ solution or with a chlorhexidine based detergent. The samples were cultivated on blood agar plates aerobically and anaerobically. Cavities rinsed with water only showed very sparse bacterial growth. After experimental infection the growth was significant, but decreased radically after treatment with the test materials. In order to describe the effect of the two test materials on the microbial enzyme activity in infected dentin, cryostat sections of 10 μm were prepared from undemineralized carious dentin fragments excavated from freshly extracted human teeth. Conventional histochemical techniques were applied to demonstrate the aminopeptidase activity in the sections using N-L-leucyl-2-naphthylamide as a substrate. The aminopeptidase activity of carious dentin was inhibited totally with the Ca(OH)₂ solution, whereas the chlorhexidine based detergent had no effect on the enzyme activity.