The paper presents the results of tests of the stiffness modulus according to the 4PB-PR method of low-noise asphalt mixtures with the addition of rubber granulate (RG). Mixtures of this type are characterized by an increased air void content (about 10–25%). This causes a rapid bitumen oxidation, which results in oxidative hardening, contributing to a faster deterioration of the properties of the mixtures. This means that binders of appropriate quality should be used in the process of producing asphalt mixtures, which will provide the mixtures with sufficiently high technical properties. The tested asphalt mixtures are differentiated according to the type of bitumen modifiers: styrene–butadiene–styrene copolymer (SBS) and crumb rubber (CR). The article presents the tests results of the stiffness modulus using the 4PB-PR method. This test has a high correlation with regard to “in situ” tests. The research proved that each of the modifiers used increased the stiffness modulus of low-noise asphalt mixtures. Replacing the mineral aggregate with 30% RG leads to a tenfold decrease in the stiffness modulus. In the entire range of analyzed temperatures, mixtures with the use of modifiers show higher values of the elastic component of the stiffness modulus, as evidenced by lower values of the phase angle. The 20th century was characterized by the rapid development of road traffic . During The quality of the raw material used in the distillation process of the crude oil has a huge impact on the properties of road bitumen, which was used in the production of asphalt mixture. Unfortunately, in recent years there has been a trend to refine light crude oil (increasing fuel profit), which has reduced the distillation residue from 20% to 10% or less. For this reason, the quality of industrially produced bitumen does not meet the requirements for use in “quiet” pavements, which has been confirmed by investigations carried out by many scientific centers ,9,10. ThCR obtained from scrap tires was first used in the 1960s by engineer Charles McDonald. It is a well-known additive to bitumen binders. The process of bitumen modification with CR is a complex process and depends on many factors, such as mixing time and temperature, number of rotations during mixing, method of grinding rubber and its particle size, method of adding, chemical composition of binder, and rubber ,15. In tSBS copolymer is the most commonly used modifier for bitumen binders. It began to be used on a large scale as bitumen modifier in the 1970s of the 20th century . SBS copRecently, composites have been increasingly used in place of single bitumen additive. One of them is the polymer-rubber-asphalt binder SBS/CR. Due to the simultaneous modification, such a composite is able to eliminate the disadvantages and strengthen the advantages characteristic of a single modifier, e.g., only SBS or only CR . In the It is necessary in the design process to properly select the technical parameters of asphalt mixtures in order to construct durable, safe, and characterized-by-low-maintenance-costs road pavements . One of However, the influence of rubber granulate (RG) on changes in stiffness modulus by the 4PB-PR method of “quiet” mixtures has not yet been investigated, which is an important aspect in the design of this type of pavement.The aim of the research presented in this paper is to analyze the effect of the type of modification of bitumen binders with SBS polymer and CR and the addition of RG (1/4 mm fraction) on the change of technical parameters (stiffness modulus and phase angle) in the four-point bending study with prismatic samples 4PB-PR of low-noise asphalt mixtures.Chapter 2 presents the characteristics and properties of the materials used for the designed asphalt mixtures  and mineral-rubber-asphalt mixtures SMA8 LA (10% RG), SMA8 LA (20% RG), SMA8 LA (30% RG). The grain size distribution , binder content, air void content, and density of mixtures are given. The basics and principles of determining the stiffness modulus and phase angle according to the four-point bending with prismatic samples (4PB-PR) are also discussed.Chapter 3 presents and discusses the results of tests of stiffness modulus 4PB-PR for conventional asphalt mixtures and results of tests of stiffness modulus for asphalt mixtures with the addition of RG, change of the phase angle Φ at variable temperature and frequency for conventional asphalt mixtures and change of the phase angle Φ at variable temperature and frequency for asphalt mixtures with the addition of RG. A discussion of the results in comparison with the findings of other researchers presented in the literature is also provided.−Bitumen 50/70 (reference);−Bitumen 50/70 modified with copolymer SBS (5%) (SBSM-5);−Bitumen 50/70 modified with CR (10%) (CRM-10);−Bitumen 50/70 modified with a combination of copolymer SBS (2%) and CR (10%) (SBSM-2 + CRM-10).Four types of bitumen binders were used for the tests:The technical properties of the modified binders before and after the RTFOT (rolling thin film oven test) technological aging process are presented in Modified binders were obtained in the “wet process”. Kraton D1192 which is a linear triblock polymer with 30% styrene content by mass, was used for the preparation of polymer bitumen. Information on the basic chemical properties of the SBS copolymer is presented in In the mixtures with increased flexibility, part of the aggregate in the “dry process” was replaced with RG with a grain size of 1/4 mm. The sieve analysis of the granulate is presented in Bitumen with a penetration of 50/70 was used for the production of modified binders. Bitumen was heated to a temperature of 180 °C. A measured amount of modifier  was gradually added to the bitumen and mixed for 1 h at a constant speed of 700 rpm . Then, uBefore being added to the mix, RG was preheated, which caused a swelling effect and a “slight” devulcanization of the rubber to allow the binder to bond with the rubber more effectively. In mineral-rubber-asphalt mixtures, part of the mineral aggregate was replaced with RG in the amount of 10%, 20%, and 30% in relation to the mixture volume in order to improve their flexibility.The following asphalt mixtures and low-noise mineral-rubber-asphalt mixtures were used for laboratory tests: porous asphalt (PA8), stone mastic asphalt (SMA8), stone mastic asphalt reducing tire/road noise (SMA8 LA), and stone mastic asphalt reducing tire/road noise with 10% (SMA8 LA [10% RG]), 20% (SMA8 LA [20% RG]), and 30% (SMA8 LA [30% RG]). The mixtures were prepared in a laboratory mixer which set up a 3-dimensional counter-rotating mixing process with an eccentric rotary agitator. Before compacting, the mixtures were spread into a thin layer and for 2 h subjected to thermostating in a dryer at the temperature of 145 ± 5 °C with the reference binder and at 155 ± 5 °C for the modified binder. Such a procedure made it possible to bring the mixture preparation process closer to the conditions in an asphalt plant and is required by the standard . The samThe binder content, air void content, grain size composition, and density of the designed asphalt mixtures are presented in Particle size distribution of tested mixtures  indicateThe stiffness of asphalt mixtures has a significant impact on the stress level generated in the structural layers of the road surface by traffic loads and temperature changes during operation. These stresses determine the resistance to fatigue and cracking of the pavement. The stiffness modules by the four-point bending method were tested according to . The loaThe value of the stiffness modulus is defined as the absolute value of the complex modulus, |E *|, determined from the dependency between the stress and the deformation at time t. Characteristic for viscoelastic materials is the shift of the stress curve in relation to the deformation curve by the value of the phase angle, Φ .Perfectly elastic bodies are characterized by a phase angle, Φ = 0°. In viscous bodies, the phase angle fluctuates around 90°. For visco-elastic bodies, the angle value ranges from 0° to 90°.E1) and the imaginary (E2) component:The stiffness modulus consists of the real (γ—shape coefficient (1/m);F—vertical force (kN);z—specimen deflection (m);Φ—phase angle (deg);μ—mass coefficient (kg);ω—angular frequency (rad/s).The real component is determined according to the Equation (2):The imaginary component is calculated according to the Dependency (3):L—distance between outer supports (m);B—sample width (m);H—sample height (m);M—sample mass (kg);m—mass of the moving parts of the device (kg);l—distance between places of sample loading (m);A—distance between the outer clamp and the next inner clamp (m);x—position of the probe for measuring beam deformation (m).The mass and shape coefficients are calculated according to the Formulas (4)–(7):εB—phase angle of the approximate function of the strain value (rad);tB—phase angle of the approximate function of the stress value (rad).Phase angle is defined as the phase difference between stress and strain and is determined by Equation (8):The results of testing the stiffness modulus with the 4PB-PR method at different frequencies at temperatures 5 °C, 15 °C, and 25 °C are shown in The results of the 4PB-PR stiffness modulus tests presented in this study confirm the previous research results described in publications ,46,47,48Due to the fact that asphalt mixtures are materials with visco-elastic properties, their technical features should be presented as a function of temperature change. The stiffness modulus of asphalt mixtures significantly decreases with increasing test temperature. This phenomenon results in a reduction in load-bearing capacity and load-carrying capability. Inverse changes are observed when the test temperature is decreased: the value of stiffness modulus begins to increase rapidly. This indicates that there is an increase in thermal stresses in the visco-elastic material. When the thermal stress value exceeds the tensile strength of the material, the initiation of low-temperature cracking occurs.When analyzing the results presented in Considering the type of mixture, the highest values of the stiffness modulus were obtained at 5 °C at the frequency of 20 Hz for the mixture SMA8 with SBSM-2 + CRM-10 and CRM-10 binder . On the other hand, the lowest values of modulus under the same test conditions were obtained for the mixture SMA8 LA (30% RG) with bitumen CRM-10 and SBSM-2 + CRM-10 . It is a tenfold decrease in the value of the modulus in relation to the SMA8 LA mixture without the addition of RG . It is worth emphasizing that the addition of another 10% of RG to the SMA8 LA mixture causes a decrease in the modulus value by approximately 50% in practically the entire range of analyzed temperatures.The lowest (favorable) temperature sensitivity  in the group of mixtures without the addition of RG achieved the mixtures with SBSM-5 and SBSM-2 + CRM-10 binder . On the Testing the stiffness modulus at variable frequencies of load application in the range from 0.5 Hz to 20 Hz allows for predicting the behavior of asphalt mixtures at variable vehicle speeds. The conducted tests proved that the temperature and the amount of RG have a significant influence on the change of the range of modulus values obtained at different load frequencies. The lowest discrepancies were obtained at 5 °C for mixtures without RG, the highest—at 25 °C for mixtures with 30% RG. This proves that at 5 °C the phase angle is much lower  than at 25 °C . Low valThe second degree polynomial was used to describe the changes in the stiffness modulus values:The statistical analysis of the obtained results was started with the significance test using the ANOVA analysis of variance (STATISTICA software). The results of this analysis are presented in p-value is lower than the assumed significance level α = 0.05 . Analyzing the square term referring to the type of binder (Type of Binder (Q)) and the factor describing the interaction of the binder type and frequency (2L·4L), no significant influence was found on the values of the stiffness modulus determinations. The values describing the parameters of the regression model are summarized in Based on the analysis of the parameters, it can be clearly stated that the temperature, type of mixture and binder, and frequency are important factors affecting the stiffness modulus, because the 2adj = 76%, which proves the correct adoption of the model.On the basis of the analysis, it was observed that the value of the corrected determination coefficient was RTM—type of mixture: SMA8 = 105, PA8 = 106, SMA8 LA = 107, SMA8 LA (10% RG) = 108, SMA8 LA (20% RG) = 109, SMA8 LA (30% RG) = 110;TB—type of binder: 50/70 = 101, SBSM-5 = 102, CRM-10 = 103, SBSM-2 + CRM-10 = 104;Temp—test temperature;F—frequency.The developed model of changes in the stiffness modulus can be presented using the following Relationship (10):The obtained regression model (Equation (10)) allows us to predict with good precision (about 76%) the stiffness modulus of the analyzed mixtures with SBS and crumb rubber modified binder .The graphical interpretation of the 4PB-PR stiffness modulus change as a function of the type of mixture and binder is shown in The changes in the phase angle Φ for the individual mixtures at different temperatures and frequencies are presented in In the literature, there are few studies of the stiffness modulus tested using the 4PB-PR method. In addition, the authors of these publications hardly analyze the very important parameter, which is the phase angle, Φ. According to ,50, the When analyzing the obtained results of the phase angle, Φ, on the basis of the stiffness modulus tests using the 4PB-PR method presented in The type of mixture does not significantly affect the value of the phase angle, Φ, in the low frequency range of 0.5 Hz, 1 Hz, 5 Hz and temperatures of 5 °C and 15 °C . The greChanges in this parameter at higher frequency ranges (10 Hz and 20 Hz) and test temperatures (25 °C) are significantly influenced by the type of binder, the amount of RG addition and the type of mixture.The comprehensive description of the dependence of the phase angle, Φ, of asphalt mixtures with modified binder was made using the second degree polynomial:The first stage of model evaluation was to perform the significance test using ANOVA. The results of this analysis are presented in p-value related to them is lower than the assumed significance level α = 0.05. This relationship is not only observed with the square expression describing the influence of temperature. The existence of interactions between the type of mixture and the temperature, which affects the analyzed parameter , is of significant importance.The comprehensive statistical analysis of the parameters listed in The developed regression model of the dependence of the phase angle, Φ, in terms of the type of mixture and binder, temperature, and frequency are summarized in 2 adj = 92%, which proves the correct adoption of the model.Based on the analysis, it can be concluded that the value of the corrected determination coefficient equals RTM—type of mixture: SMA8 = 105, PA8 = 106, SMA8 LA = 107, SMA8 LA (10% RG) = 108, SMA8 LA (20% RG) = 109, SMA8 LA (30% RG) = 110;TB—type of binder: 50/70 = 101, SBSM-5 = 102, CRM-10 = 103, SBSM-2 + CRM-10 = 104;Temp—test temperature;F—frequency.The developed model of the analyzed change in the phase angle, Φ, can be presented using the Equation (12):The obtained regression model (Equation (12)) allows to predict with very good precision (about 92%) the stiffness modulus of the analyzed mixtures with SBS and crumb rubber modified binder .The graphic interpretation of the change of the phase angle as a function of the type of mixture and binder is shown in The type of modifier has a significant impact on the stiffness modulus of all analyzed asphalt mixtures, increasing its values in the entire range of analyzed temperatures from 5 °C to 25 °C. The highest values of the stiffness modulus were obtained at 5 °C and at the frequency of 20 Hz for the mixture of SMA8 with the binder SBSM-2 + CRM-10 and CRM-10 . On the other hand, the lowest values of modulus under the same test conditions were obtained by the mixture SMA8 LA (30% RG), with bitumen CRM-10 and SBSM-2 + CRM-10 .The addition of rubber granulate significantly lowers the stiffness modulus. It was found that the addition of 30% rubber granulate reduces the value of the stiffness modulus tenfold. For example: SMA8 LA mixture with bitumen CRM-10 and SBSM-2 + CRM-10 , SMA8 LA (30% RG) (stiffness modulus-951 MPa and 964 MPa).Replacing the mineral aggregate by another 10% rubber granulate in the SMA8 LA mixture causes a decrease in the stiffness modulus by about 50% in the entire range of analyzed temperatures.Mixtures SMA8, PA8, SMA8 LA with the reference bitumen 50/70 had the highest (unfavorable) temperature sensitivity. The highest temperature sensitivity in mixtures, in which part of the aggregate was replaced with rubber granulate, was achieved by the mixtures SMA8 LA (10% RG and 20% RG) with asphalt 50/70 and SMA8 LA (30% RG) with the SBSM-5 binder. The SMA8 LA (10% RG) mixture with bitumen SBSM-5, SMA8 LA (20% RG) mixture with SBSM-2 + CRM-10 binder and SMA8 LA (30% RG) with CRM-10 binder showed the lowest sensitivity to temperature changes.The type of binder, the temperature, and the frequency show a significant influence on the change of the phase angle, Φ.Mixtures with the use of modified binders show higher values of the elastic component of the stiffness modulus  in the entire range of tested temperatures and frequency of loading. This fact proves the greater resistance of these mixtures to accumulation and deformation in a pavement compared to mixtures with conventional binders.According to the analysis of the obtained test results, the optimal solution from a practical point of view is to use a binder simultaneously modified with 2% of SBS and 10% of crumb rubber for the production of low-noise asphalt mixtures. Mixtures SMA8 LA with the addition of 10% of rubber granulate are a good solution for applications in a climate where the average annual temperature is between 5–10 °C.Based on the analysis of technical parameters obtained in the four-point bending test with prismatic samples of stone mastic mixtures SMA8, stone mastic asphalt reducing tire/road noise SMA8 LA and porous asphalt PA8 with SBS copolymer-modified binders, crumb rubber modified binders, and binders simultaneously modified with SBS and crumb rubber, the following conclusions were made:


Streptococcus mutans and Porphyromonas gingivalis. The biocompatibility of the material surface was evaluated using human gingival fibroblasts. Our study shows that the zirconia surface was successfully modified with silver nanoparticles by using the ion-implantation method. The surface modification remained stable, and the silver-ion elution was below 1 ppm after one-month of storage. The modified surface can effectively eliminate bacterial growth, while the normal gingiva’s cell growth is not interfered with. The results of the study demonstrate that a silver-ion-implanted zirconia surface possesses good antibacterial properties and good biocompatibility. The surface modification using silver-ion implantation is a promising method for future usage.Peri-implant lesions, such as peri-implant mucositis and peri-implantitis, are bacterial-derived diseases that happen around dental implants, compromising the long-term stability and esthetics of implant restoration. Here, we report a surface-modification method on zirconia implant abutment using silver linear-beam ion implantation to reduce the bacterial growth around the implant site, thereby decreasing the prevalence of peri-implant lesions. The surface characteristics of zirconia after ion implantation was evaluated using energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and a contact-angle device. The antibacterial properties of implanted zirconia were evaluated using  Porphyromonas gingivalis, an anaerobic bacterium and an opportunistic pathogen, has been identified as a pathogen around peri-implant. It has been recognized as a late colonizer that adheres to the extracellular matrix formed by early colonizers such as Streptococcus mutans [Dental implantation is now widely applied as a reliable method to restore missing human teeth. Successful implants rely not only on solid integration with the surrounding bone tissues (osseointegration), but also on soft-tissue integration. A robust soft-tissue integration can act as an intact seal, preventing oral bacterial invasion, as well as providing good esthetics . When ths mutans . These bs mutans ,8.The management of peri-implant lesions is difficult and costly. The commonly used non-surgical approaches include scaling, sand-blasting, laser treatment, and localized/general antibiotic administration; the surgical approaches include periodontal flap surgery, guided tissue regeneration, and other regeneration therapies . If thesRecent studies revealed that silver poses little biocidal threat to normal cells when at a nano-sized state ,17. The Streptococcus mutans and Porphyromonas gingivalis, and the biocompatibility was evaluated using human gingival fibroblasts. This study provides a viable surface-modification method on zirconia abutments to prevent peri-implant lesions.With the development of dental materials, zirconia has gradually become a proper substitute for titanium in implant dentistry. With its ivory color, zirconia implant abutments are believed to be more advantageous than titanium abutments, especially in the esthetic zone . MeanwhiTwenty-four yttrium-stabilized zirconia disks  with 15 mm diameter and 2 mm thickness were designed and milled by computer-aided design and computer-aided manufacturing (CAD/CAM) process. An 800-grit SiC abrasive paper was used to polish the disks until a unified roughness height of 0.1 μm was achieved. Then, the zirconia disks were ultrasonically cleaned using absolute ethanol and deionized water, each for 20 min. They were naturally dried and stored at room temperature before silver-ion implantation.14 ions/cm2, 1 × 1015 ions/cm2, and 1 × 1016 ions/cm2, respectively. The implantation energy and nominal dose were determined by preliminary experiments. After ion implantation, the specimens were cooled down to room temperature (25 °C). All specimens were stored at room temperature before use. Before various tests, the disks were sterilized with 75% ethanol for 40 min and washed 3 times using 0.1 M PBS buffer .Prior to silver-ion implantation, the zirconia disks were randomly divided into four groups, with six specimens in each group. One group was left untreated and was used as the negative control. The other three groups were implanted using ion bombardment at 30 keV and a nominal dose of 1 × 10The energy dispersive spectroscopy analysis (EDS) was performed to determine surface element composition. The X-ray photoelectron spectroscopy (XPS)  examination was performed to further determine the surface chemical composition. In all XPS tests, the survey spectra within the range of 0–600 eV were collected and calibrated using C1s peak at 284.6 eV.The surface wettability was evaluated by measuring the contact angle of 1 μL deionized water droplet using a surface-wettability survey device . For each sample, five locations were randomly chosen for the measurement.The zirconia disks with different silver doping were soaked in 10 mL deionized water at 37 °C for 1 day up to 1 month. After the incubation period, the leaching liquid was collected and subjected to inductively coupled plasma mass spectrometry . Silver-ion concentration in the liquid was recorded and recognized as a silver release from zirconia disks.This study was approved by the Institutional Review Board of the Peking University School of Stomatology (PKUSSIRB-201943034). The unstimulated human saliva was collected from 5 healthy volunteers, and human gingival fibroblasts (HGFs) were grown from the biopsies obtained from another 10 healthy periodontal human volunteers during periodontal surgery. All participants signed the written informed consent before the procedure.g for 20 min at 4 °C. After clarification, distilled water was used to dilute the supernatant with a 3:1 ratio v/v. The 25% saliva was then flited with 0.22 μm PES membrane  and stored at −80 °C until use. Prior to the usage, the saliva was immediately thawed at 37 °C and centrifuged again at 1430× g for 5 min. All zirconia disks, including the untreated zirconia, were immersed in the saliva for 2 h at 37 °C.Saliva was collected as previously described . BrieflyStreptococcus mutans  bacteria and gram-negative Porphyromonas gingivalis  bacteria, which were provided by the Institute of Microorganisms, Chinese Academy of Sciences. The bacteria strains were maintained using brain–heart infusion (BHI) agar plate . The incubation condition for S. mutans was the standard cell condition , and the incubation condition for P. gingivalis was the standard anaerobic condition . After the exponential growth phase in the liquid medium, the bacterial cells were collected, centrifuged at 3000× g for 15 min, and washed two times with 0.1M PBS buffer. Bacterial suspensions with different final concentrations were shaken for 30 s  to obtain single cells or pairs, then seeded onto the sample disks for further experiments. Each experiment was run in triplicate and repeated on three separate occasions.The antimicrobial activity of sterilized samples was tested against gram-positive 5 CFU/mL before added onto saliva-coated zirconia disks. After 4 h of contact with disks, 10 μL of bacterial suspension was pipetted as a ‘spot’ onto the BHI agar plates to assess the viability of the bacteria cells. The agar plates were cultivated in standard cultivation conditions and the numbers of bacterial colonies were recorded.The antibacterial efficiency of silver-implanted zirconia was evaluated using a spot assay described by Suppi and Kasemets . Brieflyv/v. The bacteria were seeded to the zirconia surface and cultivated for 24 h, then washed three times with PBS to remove non-adhered cells. A 300 μL mixed staining dilution was added to the zirconia surface. After the staining process, zirconia disks were observed using confocal laser scanning microscopy  at 100-fold magnification. The live cells were stained as green fluorescent and dead cells were stained as red fluorescent.To evaluate the viability of the bacteria after seeding on silver-implanted zirconia, the LIVE/DEAD BacLight Bacterial Viability Kit  was used. Briefly, staining components A (SYTO 9) and B (propidium iodide) were premixed and diluted with PBS at a ratio of 1.5:1.5:1000 8 CFU/mL was added to saliva-coated zirconia disks and cultivated for 24 h. After removing the culture medium, the disks were washed three times with sterile PBS to remove non-attached bacterial cells. To perform the assay, 5 μL of MTT solution was added into 500 μL BHI broth culture medium. The mixed solution was added to the disks and incubated 3 h in the dark in standard cultivation conditions. After discarding the MTT solution, 500 μL DMSO  was added to dissolve formazan crystals formed by viable cells. The optical density of the solution was determined at 570 nm using a microplate reader . Solutions without seeding bacteria were used as blank controls. The MTT assay is based on the cleavage of MTT -2,5-diphenyltetrazolium bromide) into a blue formazan by living cell enzymes. As previously described , the amo8 CFU/mL was added to saliva-coated zirconia disks and cultivated for 48 h to form a biofilm. After discarding the growth medium, the samples were washed 3 times using PBS and fixed for 20 min at 37 °C using 2.5% glutaraldehyde. The crystal violet solution  was used to stain the fixed biofilm by 10 min incubation at room temperature. The unbound dye was washed with gently running deionized water, and the bound dye was extracted using absolute ethanol. The amount of biofilm was measured at an optical density of 570 nm using a microplate reader. The background staining was corrected by subtracting the mean value for CV bound to negative controls.The total amount of biofilm formed on the zirconia disks was evaluated by using crystal violet (CV) assay. The bacterial suspension with a final concentration of 1 × 103) and placed in 35 mm cell-culture dishes containing Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . Fibroblasts were obtained by trypsinization of the primary outgrowth of cells and were maintained and routinely passaged in 10 cm dishes in DMEM with 10% FBS and 1% antibiotic–antimycotic solution at 37 °C under a humidified atmosphere of 5% CO2 in 95% air. Cells in the third to sixth passage were used for the experiments.Primary HGFs were grown from tissue explants. Briefly, healthy gingival tissue obtained from patients who underwent periodontal surgery (crown-lengthening surgery) was cut into pieces  was added to each well, followed by a 2 h incubation time at 37 °C. The supernatant was then transferred to 96-well plates and subjected to the optical density test using a microplate reader at 450 nm wavelength.The cell proliferation on sample disks was evaluated by a quantitative colorimetric cell-counting kit-8 assay . Briefly, the HGF cells were seeded onto the disks at a density of 10The experimental data were obtained by three repeated experiments performed in triplicate. One-way analysis of variance (ANOVA) was used for comparisons among different groups. The level of significance was set at 0.05. The confidence level was set as 95%. Data analyses were performed using SPSS statistics software .16 ions/cm2 show different chemical elements on the surface. As shown in 16 ions/cm2 will lead to a 0.62% silver atomic percentage increase on the zirconia surface.The EDS results of the untreated zirconia and the silver-implanted zirconia at a nominal dose of 1 × 103/2) and 368.04 eV (Ag 3d5/2) corresponds to metallic silver. These results confirm the successful implantation of silver nanoparticles to the zirconia surface.The XPS results  further The results of surface wettability are shown in For all liquid samples stored for 1, 3, 5, 7, 10, 14, 17, 21, 25, and 30 days, the silver-ion concentration is less than 0.1 ppb, which is barely detectable by ICP-MS. This result confirms that silver-implanted zirconia disks are stable, with little silver-ion elution to the surrounding environment.P. gingivalis, a spot analysis was performed. As shown in P. gingivalis.To first evaluate the bactericidal ability of silver-implanted zirconia against red-complex pathogen P. gingivalis’s viability on different zirconia surfaces. There are mainly viable  bacterial cells on untreated zirconia surfaces. As the silver implant dose becomes higher, the number of viable bacteria cells decreases, and the number of inviable bacteria cells increases. The inviable bacterial cells were stained red by propidium iodide. For the gram-positive S. mutans bacteria, the same trend can be observed. As shown in To further evaluate the bactericidal ability of silver-implanted zirconia, the LIVE/DEAD staining assay was performed. P. gingivalis was inhibited on the silver-implanted zirconia surface after a one-day cultivation. The group of 1016/cm2 showed the least-adhered bacterial load on the surface among all groups. As the cultivation time extends, the same trend can be observed (fewer bacteria are adhering to the zirconia surface as the implantation dose increases).The MTT colorimetric assay was utilized to evaluate the bacterial adhesion to untreated zirconia disks or silver-implanted zirconia disks. As shown in S. mutans grows faster than P. gingivalis, the MTT assays were performed 12 h after seeding to the sample surfaces. The results in S. mutans‘s adhesion was also interfered with on the silver-implanted zirconia surfaces. This adhesion-inhibition effect also shows a dose-dependent pattern, with fewer bacteria adhering to high implant-dose groups. Collectively, these results show that silver-implanted zirconia attenuates bacterial adhesion for both gram-positive and gram-negative pathogens. An enhanced inhibition can be seen as the implantation dose becomes higher.Since P. gingivalis, a total reduction in biofilm mass can be observed after a two-day cultivation. The zirconia disks with nominal doses of 1015/cm2 and 1016/cm2 accumulate fewer biofilms than the 1014/cm2 group. Indeed, after a four-day cultivation, there are no significant differences in the biofilms formed between the 1014/cm2 group and the untreated group. However, the 1015/cm2 and 1016/cm2 groups still show a biofilm-inhibition effect after a 4-day cultivation. This result indicates that, after a certain cultivation time, the group with an implant dose of 1014/cm2 might eventually lose its anti-biofilm formation ability.The biofilm formation on silver-implanted zirconia surfaces is shown in S. mutans on the zirconia disks shows a more unified trend within 3 days of seeding bacteria to the sample surface. As shown in 15/cm2 and 1016/cm2 groups also show a good anti-biofilm-formation ability. The crystal violet results reveal that the decrease in biofilm formation can be observed on silver-implanted zirconia for both gram-positive and gram-negative bacteria.The biofilm formed by To evaluate the cytotoxicity of silver-implanted zirconia, the CCK-8 colorimetric assay was performed. As shown in 3/2) and 368.04 eV (Ag3d5/2) corresponds to metallic silver, indicating the nanoparticles are metallic silver. The ICP-MS test shows that silver nanoparticles remain stable and have little ion release (the leaching of silver ions is under the detection limit of 0.1 ppb even after 1 month of immersion). The low leaching property indicates silver ions will not accumulate in the tissues around implants; otherwise, it would result in cytotoxicity or other side effects.Implant-associated infection has been widely researched due to its high prevalence. Until now, most researchers and clinicians believed it was bacterially derived, thus lots of researchers have focused on the surface modification of implant materials to improve their antimicrobial efficacy. The surface modification of implant materials should show a great antimicrobial effect without releasing antibiotic agents at a fast rate, while being non-hazardous to the surrounding living human cells. Although many antimicrobial coatings have been presented, most of them are prone to degradation in the oral environment quickly. The method of silver nanoparticles embedded into zirconia by ion implantation is presented in this article. Using EDX, silver can be detected on the surface of zirconia. In the XPS results, the Ag3d doublet at 374.05 eV  uptSoft-tissue connection is crucial to implant dentistry. Ideal dental implant bio-materials require effective soft-tissue integration for long-term stability after implantation. A sound soft-tissue seal on the implant-abutment site can not only inhibit unwanted soft-tissue recession and marginal bone loss, but also resist bacterial invasion. Following implant surgery, HGFs are involved in the formation of connective tissues around the implant site. The CCK-8 proliferation assay indicates that silver nanoparticles show no obvious damage to HGFs’ proliferation. An effective HGF attachment to implant abutment plays a critical role in soft-tissue connection, and a strong attachment starts with a undisturbed initial proliferation. At the same time, the antimicrobial effect of silver-implanted surfaces can further enhance soft-tissue integration. The silver-implanted zirconia surface can provide an antibacterial effect without obvious cytotoxicity; however, as many cells are involved in the soft-tissue wound-healing process, more experiments should be carried out in the future to evaluate their response to the silver-implanted zirconia.Staphylococcus aureus and other red-complex pathogens, should be studied further. Moreover, mono-culture in vitro studies are sometimes at odds with clinical reality. To avoid this, further in vivo studies to mimic clinical situations are required. Furthermore, the long-term release of silver ions and their safety for human beings need to be carefully evaluated. The release of silver ions in a different medium  should also be evaluated in the future. The possible mechanical property changes (such as surface hardness or the Young’s modulus) after silver-ion implantation should also be investigated before its clinical usage.Taken together, the results suggest that nanostructured surfaces with silver nano-particles incorporated in zirconia may provide a better choice for dental implant abutments than traditional ones. Silver nanoparticles show a promising bactericidal ability that will be effective in the oral environment. However, only two pathogens were evaluated in this study. More pathogens, such as P. gingivalis and S. mutans. It also shows no harm to human gingival fibroblast proliferation. Silver-implanted zirconia shows promise for both preventing peri-implant lesions and forming peri-implant soft-tissue attachments in clinical application.In this work, we present an effective surface-modification technique for zirconia dental implant materials. Silver was implanted into zirconia with a nanostructured surface. Within the limitations of this in vitro study, the results suggest that silver-implanted zirconia has a strong antimicrobial effect against oral microorganisms, including


Background: Fingertip injuries are very common and require a stable and durable cover. The end result after reconstruction must be a painless finger with good aesthetic appearance. Skin loss in fingertip, with or without partial loss of pulp fat, is often managed with a split-thickness skin graft, which causes a poor aesthetic result due to color mismatch of the grafted skin in the glabrous volar skin of fingertip. The full-thickness and partial-thickness skin graft harvested from palmar skin provide color match but may cause donor site morbidity in the form of painful scar or contour deformity. Harvest of the second layer from the palm (intermediate part of dermis) allowed the first layer  to be reposed over the remaining dermis in palm, thus allowing closure of donor defect without any tension. This technique provides a good color match for the primary defect, along with reduced donor site morbidity.Aim: The study aims to analyze the outcomes of second-layer palmar graft (SLPG) in patients with fingertip injuries.Materials and methods: The retrospective study was conducted in January 2012 on 40 patients who underwent SLPG.Result: The graft take was 100% in 36 patients with an average static two-point discrimination (2PD) of 6 mm. The average cosmetic visual analog score for the donor area was 100 and recipient site was 80.Conclusion: The SLPG is a good surgical procedure for reconstructing fingertip defects, providing excellent aesthetic appearance and optimal function. The reconstruction of fingertips depends on the size of the defect and the structures lost. Gonzalez et al.  stated tThe retrospective study was conducted in January 2012 at the Institute for Research and Rehabilitation of Hand and the Department of Plastic Surgery at Government Stanley Hospital, Chennai. The patients who underwent SLPG between July 2010 and June 2011 were recruited for the study. Patients who underwent other surgeries in addition to SLPG were excluded. A total of 49 patients were recruited, of which nine patients were lost for follow-up. The patients were operated on by multiple surgeons at different levels of expertise from consultants to first-year trainees. For this study, patient demographics, mode of injury, size of the defect, assessment of graft take, and duration of healing were recorded from the patients' records. Graft take was assessed by its adherence to the wound bed, and complete reepithelialization was considered as the endpoint of healing. At the time of final follow-up, graft site sensation was assessed by static two-point discrimination (2PD), and the patients’ scars at the donor site and the graft site were assessed using the cosmetic visual analog scale rated between 0 and 100 with 0 being the worst scar and 100 being the best scar . PatientSurgical techniqueThe surgery was done under axillary block or digital block with local anesthesia infiltration for the donor site. The anesthesia was decided after discussion with the patient and taking the patients' comfort into consideration. All patients underwent the procedure as outpatients, and none needed hospitalization.The wound was debrided, and the injured finger was thoroughly washed. A template of the defect was taken. The first layer of palmar graft (epidermis with part of dermis) was raised from the hypothenar region by a manual dermatome and kept in continuity as a proximal-based flap Figure . A templCare was taken while placing the dermal graft so that the deeper part of the graft was in contact with the wound bed and the graft was not placed upside down. To avoid the upside-down placement of the graft, the harvested graft was placed facing upward in a saline gauze in a bowl. The graft was anchored with intermittent nonabsorbable sutures or tied-over dressing Figure .The first layer of the graft was repositioned back and sutured Figure . ParaffiOf the total 40 patients, 34 were males and six were females. Age group analysis showed that most of the patients were in the age group of 21-30 years  was injured. The size of the defects varied from 1 cm x 1 cm to 2 cm x 2 cm, and they were confined to volar aspect of terminal phalanx region. There was an associated distal phalanx fracture in 15 patients (37.5%), but none of the defects had exposed bone.Of the 40 patients, 10 patients underwent surgery under axillary block, and 30 patients underwent surgery under digital block with local infiltration. The average duration of this surgery was 20 minutes (range: 15-30 minutes). There was partial graft loss in four patients, which healed with dressings. None of the patients needed secondary surgical procedures. The epithelialization time was between three and four weeks. None of the patients had problem with respect to donor site healing or donor site hypersensitivity. The average cosmetic visual analog score for the donor site was 100, whereas the average cosmetic visual analog score for the recipient site was 80. The late follow-up photos of recipient site and donor site are shown in Figures 2 without exposed barebone or tendon, healing by secondary intention is considered [The goal of treatment of any injured fingertip should be the restoration of a stable interface for object manipulation while looking as normal as possible . When a nsidered . The othIn 1961, Moynihan  summarizPatton 1969) advocated the reconstructive option of split-thickness skin graft from the hypothenar area based on their 20-year experience of usage of the procedure . The don69 advocaSLPG, described as glabrous dermal grafting by Wu et al.  and keraThe skin that lines the palm  of a humSchenck et al.  stated tThis study had a number of limitations. First, being a retrospective study, the selection bias for the patients undergoing the procedure could not be determined. Second, the sample size was small. A prospective randomized comparative study with split-thickness skin graft and other procedures can yield more meaningful conclusions.SLPG from the hypothenar region is a safe technique to resurface the fingertip injuries with good aesthetic outcomes, especially in terms of a color match. It has the advantage of donor site in the vicinity and primary closure of donor site defect. Also, the healing of donor and recipient sites is aesthetically and functionally good.


In the Western world, the successful introduction of insect consumption may need awareness of insect ingredient benefits, and consumers’ anticipation of insect-based foods with sensory quality is crucial. The objective of this study was to develop protein-rich nutritional chocolate chip cookies (CCC) from cricket powder (CP) and analyze their physicochemical, liking, emotions, purchase intent, and sensory properties. The CP additions levels were 0%, 5%, 7.5%, and 10%. Chemical composition, physicochemical, and functional properties were analyzed using individual and mixed CP and wheat flour (WF). The proximate composition of CP mainly consisted of ash (3.9%), fat (13.4%), and protein (60.7%). In vitro protein digestibility of CP was 85.7%, whereas the essential amino acid score was 0.82. The CP inclusion significantly impacted the WF functional and rheological properties in all given incorporation levels in flour blends and doughs. The incorporation of CP produced darker and softer CCC, resulting from the effect of the CP protein. Adding 5% of CP did not impact the sensory attributes. Purchase intent and liking improved by using 5% of CP after panelists had revealed the beneficial information regarding CP. Concerning emotion terms, “happy” and “satisfied” significantly decreased while the negative emotion term “disgusted” increased among the highest CP substitute levels (7.5% and 10%) after beneficial information. Overall liking, flavor linking, education, consumption intent, gender, age, and positive emotion term “happy” were significantly assertive predictors affecting purchase intent. Human consumption of insects is known as entomophagy. Entomophagy is not a new trend in some parts of the world, including Africa and Southeast Asia, but it is still unfamiliar to many Western societies . Few WesS. icipe and G. bimaculatus, obtaining protein percentages also higher than 50% [The United Nations expects the world population to increase by 2,000,000 people over the next 30 years and is expected to reach 1,100,000 by the end of the 21st century . Due to than 50% . In addithan 50% . AnotherThe UNAG Farms  provided the cricket (Acheta domesticus) powder and wheat flour. Treatments consisted of three concentrations of cricket powder (0% (control), 5%, 7.5%, and 10%) singly added into chocolate chip cookie formulations made of wheat flour. Color, hardness, water activity, proximal analysis, sensory characteristics, liking, purchase intent, and emotions were evaluated in chocolate chip cookies. The rheology of the protein-rich nutritional chocolate chip cookies (CCC) dough was studied using a farinograph and alveograph. Furthermore, the cricket powder was analyzed to study protein digestibility and amino acid profile. The functional properties such as water absorption capacity, oil absorption capacity, emulsion capacity, foam capacity, foam stability, protein solubility, pasting properties, and particle size distribution of individual and mixed wheat flour and cricket powder were also studied.The experiments were performed in triplicates to study protein digestibility and amino acid profile. The protein digestibility of cricket powder was analyzed using the method of Tinus et al. 2012) . Protein . Proteitography .g for 10 min. The supernatant was then collected into the sample for protein analysis in Kjeldahl digestion (AACC Method 46_13.01) [Protein solubility was analyzed using 1 g of sample dissolved in 10 mL of distilled water in a test tube, and then the solution was placed in a vortex and stirred at 550 rpm for 30 min. The obtained solution was adjusted to pH 7 using 0.1 N NaOH. The suspension was centrifuged in a 10 mL test tube at 4500× 6_13.01) . ProteinSimilarly, the water absorption index (WAI) was studied by preparing the sample, as described above. The water absorption index (WAI) was calculated by Equation (3):× g for 10 min. The pelleted samples were removed and weighed after centrifugation, and the oil absorption index (OAI) was calculated by Equation (4):For the oil absorption index determination, 1 g of cricket powder was added and mixed with 10 mL of soybean oil. The obtained mixture was then centrifuged in a 10 mL test tube at 1500w/w) was adjusted to pH 7 and was stirred overnight at room temperature. The solution was then whipped using a mixer  at the highest shear level. In a 100 mL graduated cylinder, the foam volume was evaluated at 1 min intervals from 0 min to 30 min. The foaming capacity and foam stability were determined using Equations (5) and (6), respectively:For foaming properties, the cricket powder solution   with sliThe pasting properties of wheat flour and cricket powder were examined following AACC method 61.02.01   by usingThe proximal analysis of wheat flour, cricket powder, and cricket chocolate chips was carried out by the Food Analysis Laboratory facilities of the Zamorano University, Valle del Yeguare, San Antonio de Oriente Municipality, Francisco Morazán Province, Honduras, including ash (AACC method 08_01.01), fat (AACC Method 30_20.01), moisture (AACC method 44_01.01), carbohydrate , and protein (AACC Method 46_13.01) .The cricket cookie dough rheological determinations were measured in triplicates by alveograph, coupled with an alveolink integrator–recorder  and with a farinograph . The ISO 27971  methodology  was usedIngredients used to make chocolate chip cookies containing 85% solids cricket powder (CCC) were wheat flour (300 mg/g), chocolate chips (200 mg/g), margarine (160 mg/g), sugar (130 mg/g), salt (25 mg/g), baking soda (4 mg/g), vanilla extract (6 mg/g), water (65 mg/g) and partial substitutions  of cricket protein. For chocolate chip cookies preparation, margarine and sugar were placed in the bowl in a stand mixer  and blended for 5 min using medium-high speed. Later, the baking soda, salt, whey cricket powder, and flour were put through a sieve and mixed at low speed for 5 min. The vanilla extract was mixed and added to the mixture, and the chocolate chips were combined and mixed at low speed for 2 min . The douThe color was analyzed using a colorimeter , including the L*, a*, b*, Chroma, Hue angle, and browning index values. The water activity (aw) of cricket chocolate chip cookies was measured using an aw meter . The hardness of the cookies was determined using a Texture Analyzer  through a 3-point bending test using a 3-point bending rig, trigger force of 30 g, and load cell of 45 kg. The textural properties were studied using a pretest speed of 1.6 mm/s, test speed of 2.2 mm/s, and post-test speed of 12 mm/s with a distance of 10 mm .® [n = 180) from UNAG  participated in the sensory analysis using partitioned sensory booths. Panelists were given four chocolate chip cookie samples with a cup of water  and unsalted crackers . The questionnaire was developed and made using Compusense® five software , and all samples were enumerated using 3-digit random numbers with a counterbalanced design . Panelists were introduced and instructed to evaluate color, aroma, flavor, texture, and overall linking (OL) on a 9-point hedonic scale. With tasting, purchase intent (PI) was examined on a “yes/no” scale, and emotions were analyzed using a 5-point intensity scale. Consumers were presented and introduced to the following beneficial information (HBI) statement(s): “This sample contains cricket powder, and it provides essential nutrients such as high content of protein (87.7% digestible) and sufficient essential amino acid and environmental benefits. According to statistics, poultry and pork require 385 and 340 megajoules of fossil fuel input per kilogram for protein output. On the other hand, cricket protein only requires 120 megajoules. Moreover, insects emit lower levels of greenhouse gases than cattle and use much less water than vertebrate livestock.” Once more, consumers evaluated overall liking, emotion intensities, and PI after receiving HBI.This study was approved by the Honduran Association of Physicians-Nutritionists (ASOHMENU) with form # AS-ASHOMENU-005-2022. Emotional terms taken from the EsSense Profile®  were scrt-test was used to determine differences in OL and emotion responses before and after the panelist acquired HBI. Logistic regression analysis (LRA) was conducted to analyze the effects of sensory characteristics and emotional terms on PI.All statistical analyses were analyzed using SPSS 16 software . A one-way analysis of variance (ANOVA) and post hoc Tukey test (α = 0.5) were used in sensory characteristics, emotional responses, and all physicochemical evaluations. The McNemar test was used to determine significant differences in PI before and after HBI. Multivariate analysis of variance (MANOVA) and descriptive discriminant analysis (DDA) was used to determine overall product differences across samples . A dependent Gryllus bimaculatus and Acheta domesticus, respectively. The protein digestibility could be mainly affected by insect sex [The in vitro test indicated 87.7% protein digestibility  for the sect sex , insect sect sex , and prosect sex . For thesect sex  and Stonsect sex  have repsect sex . Even thp < 0.05) the water absorption capacity, foaming capacity, foaming stability, protein solubility, and emulsion capacity, while the oil absorption capacity was not affected. The increase in protein solubility with the addition of CP to WF may be associated with CP containing more albumins than WF. CP reported up to 3.15% of albumins  [The functional properties of individual WF and cricket powder are shown in ., 2019) , while W., 2019) . Not sur., 2019) , foaming., 2019) , and emu., 2019) . In our ., 2019) .Tenebrio molitor). A high level of final and setback viscosity could lead to a high level of retrogradation [Concerning pasting properties , the pasradation .The proximal analysis of individual wheat flour, cricket powder, and chocolate chip cookies is presented in The farinograph and alveograph test results of chocolate cookie dough are shown in −4). On the other hand, the inclusion of cricket powder showed a decrease in the degree of softening (DSS) (61.5 to 51.7 UB). Not surprisingly, the increase in DS, DDT, and DT and the decrease in DSS with cricket powder incorporation could be related to the cricket powder protein content. This phenomenon could be associated with the slowdown of gluten hydration and development caused by the interaction between cricket protein and wheat protein [In insect protein, variations in water absorption could be due to the amino acid composition related to hydrophilic and hydrophobic groups . When in protein ,45. In oConcerning dough extensibility (DE) and index of swelling (SI), the addition of cricket powder showed a decline in the DE (87.5–77.1 mm) and SI (20.7–17.5 mm) values. DE and SI are related to gluten and starch content in flour blends . The incp < 0.05) lower hardness when compared to control samples. Another study corroborates that adding Mealworm powder (Tenebrio molitor) also lowers the hardness of crackers [The hardness of the CCC showed a marked effect on the cricket powder incorporation . The CCCcrackers . The harOur results from the proximal analysis confirmed this statement . SimilarTenebrio molitor) in crackers. Significant differences in Chroma and ∆E values were found among the levels of cricket powder addition, suggesting that consumers can easily distinguish samples. For water activity, cricket powder inclusion did not affect the aw values. Commonly, protein could play a role in binding the water and reducing aw, but the amounts of cricket powder used in this study did not impact the aw values. The aw values of CCC were low (<0.17), meaning that spoilage bacteria do not have optimal conditions in this food matrix [In our study, cricket protein most likely reacted with the carbohydrates present in the CCC formulation. Similarly, a* and b* values had the same pattern as L* values  and alsod matrix .Texture liking scores of all CCC varied from 6.19 to 6.05. Compared with the control CCC, the CCC with 10% of CP had a remarkably lower hardness (7721.07 vs. 1945.93 g force) , which mp < 0.05) influenced OL scores only for CCC made, with 5% of CP giving a slight increase (from 6.05 to 6.59) (p < 0.05) different in PI. A study [The HBI has given to consumers significantly (to 6.59) . There w A study  also rep A study . Neverth A study .p < 0.05) decline for the positive emotions of “happy” and “satisfied” occurred when it was revealed the presence of CP, whereas “disgusted” significantly (p < 0.05) increased after tasting.The emotion scores of CCC made with different levels of CP are shown in Gurdian et al. (2021)  also dedp < 0.05) differences among all samples based on sensory characteristics and emotion terms , whereas color  provided the lowest values among sensory properties to overall product differences. Insect flavors can differ depending on species and processing technique, and crickets had reported to provide a meat-like flavor [The Wilks Lambda test indicated that there were overall significant . In other words, positive emotions were more suitable to show overall product differences than negative emotions . OccasioThe CP incorporation in chocolate cookies was analyzed by ascertaining the effect of OL, consumption intent, education, gender, age, hedonic responses, and emotion terms on PI (before and after HBI) . Gender,Acheta domesticus) powder to chocolate chip cookies to improve their nutritional profile. The influence of cricket protein on the sensory characteristics of chocolate chip cookies was more noticeable for flavor. As a result, flavor improvement is the proposed strategy when increasing the acceptability and purchase intent. The positive emotion scores were higher than the negative emotion scores. Nevertheless, positive emotion scores decreased while negative emotion scores increased after HBI was revealed and given to the customers. Cricket protein intrinsic characteristics should be adjusted or masked instead of showcased to improve familiarity. Preference mapping techniques and conjoint analysis should be used in future research for a better understanding of consumer behavior should be addressed.It was accentuated by adding cricket (


By applying an electric field and controlling the voltage applied to the hydrogel the mechanical properties of the hydrogel can be changed. The voltage can directly change the stress–relaxation properties of the hydrogel, and after the voltage was applied for hundreds of seconds, a gradient stiffness was generated along the direction of the electric field. The value of gradient stiffness can be regulated by charge. With the increase of charge, the stiffness of the hydrogel near the anode increases, while the stiffness of the hydrogel near the cathode decreases. Experiments show that the electro-regulation method allows dynamic manipulation of the material properties of the hydrogel with ionic crosslinks. Dynamic electro-regulation of hydrogels is a powerful tool that will promote the study of the dynamic microenvironments and lead to a better understanding of the fundamental phenomena of the extracellular matrix.Hydrogels are promising biomaterials which provide bionic environments to study the effect of stiffness. A hydrogel with dynamically changeable stiffness can be used to further understand the dynamic biological processes such as embryonic development, tumorigenesis,  A reversible electro-regulation process was developed which can dynamically regulate the stiffness gradient of hydrogels (10 to 30 kPa). For example, it is reported that a substrate with nearly 30 kPa stiffness can induce osteogenic differentiation of mesenchymal stem cells (MSCs) while a softer substrate can enhance myogenic and neurogenic differentiation.5 Therefore, many researchers have focused on the control of the stiffness of the culture environment.Hydrogels are network polymeric materials with high water contents. Due to their biocompatibility and biodegradability, hydrogels are considered as promising materials which can mimic the native extracellular matrix.6 However, it is known that the biological processes are dynamic, and the stiffness of the ECM is with gradient properties and changeable.7,8 The gradient properties of stiffness commonly exist in the extracellular environment.9 Natural stiffness gradients occur in both pathological process and physiological process. For example, the myocardial infarction can induce the environment to have a gradient of nearly 8.7 kPa mm−1, and myocardium can induce the environment to have a gradient of nearly 0.6 kPa mm−1.10 Besides, studies on the mechanical properties of the ECM reveals that microenvironment dynamically change its matrix stiffness and significantly influence the biological processes such as embryonic development, healing and tumorigenesis.11 Dynamic changes in ECM stiffness can provoke changes in cell behaviour.12 For example, the time of ECM stiffening could induce the human mesenchymal stem cells into adipocytes or osteogenic.8 To mimic the formation of adult myocardium, the continuous increase of tissue stiffness of hydrogels also makes the pre-cardiac cells form more muscle fibres.13Most studies were based on the substrate with certain modulus or with constant mechanical properties throughout the culture period.14 Some tunable hydrogels were made to degrade hydrolytically,15 and later engineered with degradation sites tailored to specific proteases.16 However, these methods mostly were in a passive and uncontrolled manner, and it is not easy to realize both dynamic soften (low to ∼10 kPa) and stiffen (up to ∼30 kPa).17Methods to control the hydrogels with gradient and dynamic stiffness are important to mimic the natural ECM environment. However, most methods initially controlled the mechanical property of hydrogels by changing polymer concentration or chain length, and the mechanical properties kept constant throughout the culture period. Degradation offers a means to soften gels over time. Degradable linkers have been utilized for controlled degradation and the hydrogels became collapse.Thus, the main aim of the present study was to develop (i) a substrate with a well-defined stiffness gradient along the designated direction using a simple technique; and (ii) a dynamic tuning method to time-dependant control the stiffness of the gradient hydrogels. Alginate hydrogel is adopted in our system as model material because its high biocompatibility and it can easily be molded by addition of divalent cation. The stiffness of alginate hydrogel can be controlled by the calcium concentration, and can be regulated/modulated by moving the calcium ions. Based on these properties, an electro-regulation method is proposed to control the stiffness gradient for the alginate hydrogel. The hydrogel can be softened or stiffened. Besides, this method has the ability to time-dependently control the trend of softening or stiffening. The speed of softening or stiffening can be controlled by the voltage, and the value of softening or stiffening can be precisely controlled by controlling the conduction time. Finally, the application of electro-regulation allows for dynamic control of the hydrogel stiffness and generates precise gradient hydrogels.d-(+)-gluconic acid delta-lactone (Ca-EDTA/GDL) crosslinked alginate was prepared. This slow-releasing alginate hydrogel can slowly release calcium ions and the hydrogel spent 10 hours to reach a stable and homogeneous stiffness. After that, the stress–relaxation behaviours of alginate hydrogel with or without electric filed were observed.To reduce the individual difference of hydrogels after gel formation, ethylenediaminetetraacetic acid calcium disodium salt hydrate and The stress–relaxation behaviour provides information of the dynamic stress with a constant strain applied. By using the universal testing machine, electrochemical workstation and platinum electrode, the stress–relaxation curves with different voltage were obtained. A cylinder-shaped gel was placed between two impermeable rigid plates and the electrode was positioned on the both sides of the hydrogel. A strain quickly applied to the gel while the force applied to the sample was 4.5 kPa. The voltage was applied after the strain was applied to the gel. Subsequently, the strain is held constant, and the stress was recorded as a function of time. The hydrogels applied with different voltage exhibited different stress–relaxation behaviours, as shown in τ  of each curve was shown in Fig. 1(f)τ shows that the curve induced by a higher voltage decreased faster than the curve induced by a lower voltage. With the increase of voltage, τ decreased more sharply. The result indicates that the voltage applied to the hydrogels can directly change the mechanical properties of the hydrogels, and voltage value and durations are both critical to the change of mechanical properties. By change the applied voltage, the speed of the change of the mechanical properties can be controlled. The results indicated that the decay speed of the loading curve can be controlled by the voltage.An exponential decay equation was used to fit the curve after the voltage applied on the sample. The Aimed at studying the gradient distribution of the hydrogels, each sample was cut into three sections after the electric field treatment  as shown in The first goal of the present work is to generate a gradient stiffness, and the second goal is that the stiffness can be dynamically controlled. Therefore, the current and electric charge which is related to time was considered here. While the voltage was applied to the alginate hydrogel, a current was formed between the electrodes. The value of the current is recorded as shown in versus the charge was performed. By recording the influence of the charge on mass loss, as shown in The charge indicates the movement of the ions in hydrogels, and the changed structure of the hydrogel could be indicated by the mass loss. The mass loss induced by electric charge mostly due to hydrogel collapse. More ionic bond was broken, more mass loss was observed. An experiment to plot of the weight of the mass loss Aimed at further studying the structure change induced by charge, the compression modulus for each section was recorded with different charge values . Same as previous mentioned, 1 presents the section near the anode, 2 is the middle section and 3 present the section near the cathode. The results are shown in vs. time was shown in An experiment for reversible regulation study was performed, and the he results are shown in 18 Another gradient hydrogel which was regulated by nanocomposite had the same limitation.19 A hydrogel with light-triggered liposomes was developed to overcome this limitation, however, the modulus range of dynamic tuning was below 2 kPa which was lower than the modulus in native extracellular matrix.20 Therefore, the importance of our results is that these limitations are overcame by developing a gradient stiffness hydrogel with appropriate modulus and dynamic tuning properties.Our results shows that the hydrogels had a gradient stiffness (range from 10 kPa to 30 kPa) which was similar to the modulus in native extracellular matrix, and the stiffness was dynamically regulated by time and electric charge. The process was reversible and controllable. It is reported that a gradient hydrogel (modulus from 1 kPa to 25 kPa) was fabricated by freezing method, however, the modulus could not be dynamically controlled after gelling.In summary, an electro-regulation method which can dynamically control the stiffness of the hydrogels with ionic crosslinks was proposed. By applying the electric field and controlling the voltage applied to the hydrogel, the mechanical properties of the hydrogel can be time-dependent changed. A gradient stiffness of the hydrogel was generated along the direction of electric field. The initial stiffness of the hydrogel was 20 kPa, and the stiffness could increase to 30 kPa or decrease to 10 kPa in different areas. By controlling the voltage and the duration, the charge can be time-dependent controlled, and any stiffness between 10 kPa and 30 kPa can be dynamically tuned.There are no conflicts to declare.RA-008-C7RA11382J-s001


Numerous studies, including randomized controlled trials (RCTs), have been published on the optimal graft choice for primary anterior cruciate ligament (ACL) reconstruction.To review existing studies to investigate whether advances in orthopaedics have affected revision rates after primary ACL reconstruction.Systematic review; Level of evidence, 4.The PubMed database was searched from inception to December 31, 2020, using the PRISMA  guidelines. Patient series, observational studies, clinical trials, and registry-based studies investigating primary ACL reconstruction were included, as were high-quality RCTs from an additional study. The minimum required follow-up time for inclusion was 1 year. The primary outcome measure was the pooled prevalence of revision ACL reconstruction. The effect of the year the surgery was performed on revision rates was evaluated with metaregression analysis. All graft types were analyzed simultaneously, and all analyses were repeated separately for each graft type.Overall, 330 articles with 52,878 patients were included, with a median patient age of 28 years . The primary ACL reconstructions were performed between 1969 and 2018. At a median of 2.3 years of follow-up, the overall revision rate was 3.14% ; it was 2.71%  for hamstring autografts, 2.38%  for bone–patellar tendon–bone (BPTB) autografts, and 5.24%  for other graft types. For hamstring grafts, the revision rate increased over time (year of surgery), with a 0.0434  increase effect in the logit-transformed scale for every additional year. There was a slight decrease in revision rates for BPTB  and other graft types  over time; however, confidence intervals for BPTB included the zero change.Based on this systematic review and meta-analysis, ACL reconstruction is a reliable procedure with overall low historical revision rates. BPTB autograft had the lowest revision rate and a slightly decreasing trend of failures during the past 45 years, although both BPTB and hamstring autografts are reliable graft choices. In recent decades, the incidence of ACL injuries has increased by up to 40%. Increased knowledge of the physiology behind ACL tears and patient-specific differences in prognosis have provided us with a better understanding of options for both treatment and effective postoperative rehabilitation. Furthermore, shared decision making plays a key role in deciding how to treat an ACL tear to achieve the optimal results. ACL reconstruction is the standard for achieving an optimized function, and most patients can return to their preinjury activity level after surgery.Anterior cruciate ligament (ACL) reconstruction is one of the most common orthopaedic procedures. In the early years, the patellar tendon was the gold standard for reconstruction. Other biological graft origins as well as synthetic materials have been researched for decades, but optimal synthetic materials have not been found, as many of them have been associated with high failure rates. Surgical techniques have since evolved a great deal because of the increased volume of ACL reconstructions. Today, ACL reconstruction is a common orthopaedic procedure that often leads to good patient-reported outcomes. However, revision surgery after primary ACL reconstruction is needed for various reasons, including reinjury, technical errors in surgery, or instability, with graft failure being the most severe complication.Arthroscopically assisted ACL reconstruction gained popularity in the 1980s, but it was initially considered a challenging and complex procedure.More than 16,000 articles can be found in the PubMed database by searching “ACL reconstruction.” Of these studies, more than half have been published in the past decade. ACL graft type has been a widely researched topic. Research has also focused on different surgical techniques, such as graft fixation methods, different surgical techniques, and optimal timing of surgery. Research has an effect on our treatment methods and options to treat the torn ACL, and currently the treatment for torn ACL may be highly individualized. Research has also affected the worldwide trends in ACL surgery, and currently the torn ACL is usually reconstructed with the use of either hamstring or patellar tendon as a graft.At the patient level, medical research should have a positive effect on the treatment results. For a single patient, the increasing number of studies during the past few decades should be seen as a better prognosis . The revision rate is one of the most important outcomes of ACL reconstruction. To the best of our knowledge, previous studies have not investigated whether revision rates after primary ACL reconstruction have evolved during the recent decades.The aim of the current study was to investigate whether advances in orthopaedics have affected revision rates after primary ACL reconstruction. Our hypothesis was that the revision rates have decreased within the period studied. The following search strategy was used: (anterior cruciate ligament or acl or ligamentum cruciatum anterius), (reconstruction* or repair* or surgery* or operation* or reconstructive or graft*), and (fail* or reoperatio* or revision* or re-operation* or retear*). No filters were used. Our review was performed according to the PRISMA  2020 checklist.The PubMed database was searched from inception to December 31, 2020. As a supplementary search, we included randomized controlled trials (RCTs) from the 2017 meta-analysis of Kay et al.http://www.rayyan.ai), and duplicates were removed. A study was eligible for our analysis if the following inclusion criteria were fulfilled: (1) the focus of the study was primary ACL reconstruction, (2) it was a clinical or registry-based study, and (3) graft ruptures and/or revisions were reported. All the records from the searched database were screened, and abstracts of those articles were assessed by 2 authors (V.T.P. and A.R.). Records meeting the inclusion criteria were selected for the eligibility assessment. After full-text reading, a study was excluded if ≥1 of the following criteria were met: (1) follow-up <12 months; (2) follow-up >10 years; (3) mean patient age <15 years; (4) failures or revisions not reported; (5) non-English language; (6) based on large national registries or similar ; or (7) size of the study was <10 patients. Furthermore, if the study included a cohort that had been used in other studies, we included the cohort with the longest or representable follow-up. Studies that were based on national registries or similar were excluded because of the cohorts they might have included that overlapped with those of other, smaller studies with better quality, such as RCTs. The risk of bias assessment was not assessed, because all the studies that met the inclusion criteria and passed the eligibility assessment were included.Records from the database search were imported to a free online systematic review platform . The extracted data included the study characteristics, such as title, authors, publication year, and study setting . For each study, we collected data on the first year and last year of the included surgeries, duration of follow-up, mean patient age, and number of primary reconstructions. The outcomes included the number of revisions. Other recorded variables were the graft type used in the primary reconstruction and definition of failure. The reason for revision surgery after primary reconstruction was considered the definition of failure. The extracted definitions were graft rupture and graft failure. If only the number of revisions was reported, it was categorized as “revision count.” We only included the number of revisions performed, although not all graft failures were necessarily revised, because the choice to undergo revision surgery is ultimately based on patient preference. However, our aim was to investigate revision rates rather than failure rates.Graft type used in primary reconstruction was categorized into one of the following categories: hamstring autografts, bone–patellar tendon–bone (BPTB) autografts, or other graft types. Hamstring autografts included all reconstructions that used the semitendinosus and/or gracilis graft. Other graft types included synthetic grafts  and autografts other than hamstring or BPTB. If the graft used in primary reconstruction was reported without any specific origin, it was categorized as “other.” Categorization based on surgical techniques  or autograft and allograft was not conducted, and therefore all the groups might have included both. Study arms were formed based on the subgroups reported in the original article.The primary pooled outcome measure was the prevalence of revision ACL reconstruction. This was calculated by dividing the total number of revisions by the total number of primary reconstructions included in the study. Explanatory variables included in the metaregression were follow-up time, year the surgery was performed, and age at surgery. The year of surgery was calculated as the mean of the first and last years of surgery within each study.I2 statistic. The fixed-effects model was not used because of inherent patient-specific differences and variability in prognosis. Logit transformation was used to calculate the overall proportions. Metaregression analysis was used to assess whether revision rates decreased as a function of time (year of surgery). A maximum-likelihood estimator was used. If any of the explanatory variables were not reported, the study was not included in the metaregression analysis. The effect of the year of surgery on the revision rate was presented in a scatterplot with a fitted regression line. The weight of the individual studies in the metaregression analysis was presented as the third variable. Finally, we conducted sensitivity analyses based on the duration of the follow-up period, by dividing the follow-up time into different durations and repeating the graft type–stratified metaregression analysis for each of them. All analyses were performed using the meta package from R .All graft types were analyzed simultaneously, and subgroup analyses were conducted separately for each graft type. The proportion of revision ACL reconstruction was determined for each study, and pooled revision rates were reported for each subgroup with 95% CIs. The random-effects model was used because of the high heterogeneity among the analyzed studies. Heterogeneity was assessed using the  and the most recent study was published in 2021 and included primary reconstructions performed in 2018. A flowchart of study inclusion is shown in In total, 3920 articles were found to be eligible for screening. After screening and full-text reading, 193 articles were included. In addition, 137 RCTs from the additional study were included, resulting in a total of 330 articles. The earliest included study was published in 1984 and included primary ACL reconstructions performed as early as 1969,At a median of 2.29 years of follow-up, the overall pooled revision rate was 3.29%  for all analyzed study arms. The pooled revision rates for the hamstring and BPTB graft types were 2.71%  and 2.38% , respectively. For other graft types, the revision rate was 5.24% . The most common reason for revision surgery was graft rupture (52.1%) .A total of 460 study arms with 47,653 patients were included in the metaregression analysis: 260 study arms with 20,896 patients for hamstrings, 98 study arms with 7368 patients for BPTB, and 83 study arms with 5541 patients for other graft types. The graft type was unknown in the remaining 13,848 patients and 19 study arms. The year of surgery influenced revision rates, as a 1-year increase in the surgery year had a 0.0077  increase effect in the logit-transformed scale to the revision rate ; howeverSensitivity analyses were performed separately for each graft type. The 2- to 4-year follow-up cohort was the largest in every graft type, including 152 study arms with 11,424 patients with the hamstring graft, 56 study arms with 3686 patients with the BPTB graft, and 58 study arms with 3910 patients with other graft types. BPTB results were more imprecise, and in both 2-year and 2- to 4-year cohorts the effect of the operational year to the revision rates was an increase. The results for other graft types were similar, except for the 2-year cohort, where year of surgery had an increase effect. With the hamstring graft, the results were similar to those observed in the analysis with all follow-up durations. The effect of year of surgery remained the same, with the exception of the 4- to 8-year follow-ups . , the overall revision rate was still very low  for a common orthopaedic procedure.To the best of our knowledge, this is the first study to investigate revision rates in this way. Our primary aim was to investigate how previous advances in orthopaedics have affected the revision rates after primary ACL reconstruction. Our systematic review and meta-analysis demonstrated an overall pooled revision rate of 3.14% with a median follow-up of 2.3 years. This outlines the fact that ACL reconstruction is a reliable procedure with historically low revision rates. Although we found that the revision rates increased between surgeries performed with hamstring autograft, the most used graft in ACL reconstruction, in 1969 and 2018 At the patient level, the risk of failure is affected by multiple factors, such as graft type, age, and body mass index. Graft type has been a widely investigated topic, as the revision rate is associated with graft choice. BPTB is reported to have the lowest revision rate. The risk for graft rupture and/or revision surgery after BPTB reconstruction has been reported to be between 1.3% and 4.0% in the 5-year follow-up period. For reconstruction with hamstrings, these risks have been reported to be between 2.7% and 15.0% in the same period. In this study, patients who received BPTB autograft in primary reconstruction had a 2.38% risk for revision surgery, with a median follow-up of 2.2 years. For hamstring autografts, we observed a 2.71% revision rate, with a median follow-up of 2.1 years, which is slightly inferior to BPTB. From the perspective of the individual patient, the minor difference in the average risk for revision surgery between the BPTB and hamstring might not be crucial when deciding the treatment of the torn ACL for an optimal result.Previous meta-analyses have reported revision rates ranging from 2.80% to 7%. For hamstring grafts, the revision rates have increased, but are very low for a common orthopaedic procedure, with excellent patient-reported outcomes. It is a positive finding that revision rates have remained very low throughout the history of ACL reconstruction, which makes it a reliable procedure for everyday practice. By choosing the age at the time of surgery and follow-up time as other confounding factors besides the operational year, we successfully investigated the effect of the operational year on the observed revision rates. We also found a positive correlation between patient age at surgery and decreased revision rates, which has been previously reported in the literature. We did not conduct subgroup analyses based on sex, as the current consensus seems to be that different revision rates are not affected by sex. Overall, both BPTB and hamstring can be considered reliable graft choices for ACL reconstruction.Failure after primary ACL reconstruction is a very important factor for an individual patient. Based on the previously published literature, as well as our analysis, ACL reconstruction with BPTB graft has very low reported revision rates through the history of ACL reconstruction. found that from 2000 to 2015, the annual incidence of ACL reconstruction increased by 43%, from 54.0 to 77.4 per 100,000 in the Australian population. However, the overall incidence of ACL revisions has increased by 127%, or by 5.6% per year. Hence, the incidence of revision surgery has increased more than the incidence of primary reconstructions. With the increased knowledge and development of new surgical techniques, this might be considered a surprising finding. Moreover, with the increased amount of literature on ligamentous knee injuries, we might assume that the surgical techniques have also evolved in revision surgeries, and therefore we are able to reoperate on more patients than in the beginning of the 21st century. Also, the increased knowledge on the sources of failure might have changed our indication for reoperation. Our analysis showed that revision rates have remained low with no upward trend. Especially the revision rates after primary reconstruction with BPTB have been low throughout the history of ACL surgery. The strongest and most significant effect between operation years and revision rates was after reconstructions with the hamstring as a graft, where 1 year effected a 0.0434 increase in the risk of revision surgery on a logit-transformed scale. But still, the revision rates for hamstring graft were very close to those observed in the BPTB group, and the increasing trend of the revision rates might be partly explained by the changing indications on the revision surgeries. Due to the systematic review and meta-analysis, our study had a more heterogeneous cohort, as compared with that of Zbrojkiewicz et al; with a sample size of almost 50,000 patients, we managed to produce unique information on the progress of the revision rates.A 2018 study published by Zbrojkiewicz et al reported that for a patient with an acute ACL tear, rehabilitation, combined with an optional delayed reconstruction, results in similar outcomes in the 2-year period, as compared with the early ACL reconstruction surgery. During the 2-year follow-up, only 23 (39%) of these patients underwent delayed reconstruction due to self-reported symptomatic instability caused by insufficient ACL and positive pivot shift. In patients with an early ACL reconstruction, the results were slightly inferior compared with those of the patients with rehabilitation only or with those of the patients who had undergone delayed ACL reconstruction. If nonoperative treatment results in similar or even superior outcomes than early reconstruction, it might be possible that the demographics of the patients who are treated surgically have changed and the preoperative risk for revision is greater than it has been before. This may have affected the observed failure rates.Our findings might also be partly explained by the changing indications for surgical treatment of primary ACL ruptures. Currently, with increasing knowledge and development of surgical techniques, we might choose to operate on more risky patients, which might not have been operated on some time earlier. A 2010 RCT by Frobell et al When planning future research, it is necessary to evaluate the most crucial questions. As the number of previously published studies is massive, we can truly ask how much more research is needed, for example, on graft choice and graft fixation techniques. We postulate that we should focus more on patient-specific differences on the ACL surgery, and how they affect the outcome after surgically treated ACL injury. In addition to the surgical variables, it is worth focusing on nonoperative treatment, as it has been shown to result in outcomes similar to those of early reconstruction.ACL surgery has many surgical variables that must be optimized based on the estimated revision rate or risk for any other complications. However, patients and surgeons might not share the same priorities related to ACL reconstruction and treatment decisions.Our study has several limitations. The year of the operation was calculated as the mean based on the operational years, which might not be the exact truth. However, with our sample size of more than 300 studies, differences in the exact time of operation may not have been that significant. The same method was used for every study included in our analysis. Another limitation of our study was that not all studies reported the exact reason for failure. For example, the definition of graft failure may include graft ruptures. We have reported the definitions of failure as did the original author from the analyzed article. The third limitation of our study is that in some studies, primary ACL reconstruction failed, but not all failures were revised. We included the number of revision surgeries performed in our analysis. Finally, the choice to undergo revision surgery is based on a single patient’s preferences. However, our aim was to investigate revision rates rather than failure rates. The fourth and main limitation in our study is a rather heterogenic categorization between graft origins. However, we do believe that our results simplify how ACL surgery has developed throughout our 45-year study period. In particular, the “other” group includes very different graft types, but as stated before, we believe that this categorization simplifies how the previous advances in orthopaedics have affected the revision rates in ACL surgery.The strength of this study is the sample size; thus, the number of not-revised failures is not that significant. It would have barely produced any difference in our results.Based on our systematic review and meta-analysis, ACL reconstruction is a reliable procedure with overall historically low revision rates. Our meta-analysis revealed that revision rates are comparable with those of the most used graft types, with hamstrings slightly inferior to BPTB. Overall, both BPTB and hamstring autografts are reliable graft choices for ACL reconstruction.http://journals.sagepub.com/doi/suppl/10.1177/23259671221110191Supplemental Material for this article is available at Click here for additional data file.Supplemental Material, sj-pdf-1-ojs-10.1177_23259671221110191 for Revision Rates After Primary ACL Reconstruction Performed Between 1969 and 2018: A Systematic Review and Metaregression Analysis by Rasmus J. Liukkonen, Ville T. Ponkilainen and Aleksi Reito in Orthopaedic Journal of Sports Medicine