Indigofera tinctoria extract (ITLE) along with maltodextrin in different concentrations was encapsulated using a freeze dryer, and some physicochemical properties were measured. Powder containing 30% maltodextrin was selected as the optimal powder for use in ice‐cream production in four levels (0%–1.8%), and some quality parameters were examined. The results showed that with increasing the carrier concentration, moisture content, aw, solubility, a*, and b* of the powders decreased; bulk density, tapped density, and L* did not change significantly; total phenolic content and antioxidant activity of powders also increased significantly (p < .05). Addition of encapsulated ITLE to the ice cream caused a significant decrease in L*, b*, and melting rate, a significant increase in a*, overrun, and hardness of the samples and no change in the viscosity of the ice‐cream mix (p < .05). Ice cream containing 1.2% encapsulated ITLE had higher sensory acceptance than other levels following control ice cream. The results of this study showed that ITLE can be used as a desirable additive in the production as a natural antioxidant and color agent.In this study,  In this study, Indigofera tinctoria extract along with maltodextrin in different concentrations was encapsulated using a freeze dryer, and some physicochemical properties were measured. Final powder was used in ice‐cream production, and some quality parameters were examined. The results of this study showed that Indigofera tinctoria extract encapsulated can be used as a desirable additive in the production as a natural antioxidant and color agent. The first impression of food products is their appearance; therefore, whether they are rejected or accepted is largely determined by their appearance  and Folin–Ciocalteu reagent were prepared from Sigma. Gallic acid was prepared by Merck Company. Maltodextrin (DE of 18–20) was prepared by Foodchem Company (China). Sugar, vanilla, mono‐ and diglyceride, and salep were purchased from a local supplier in Mashhad, Iran. Cream (30% milk fat) and skim milk powder (96% milk solids) were bought from Kalleh Co. , Golshad Co. , respectively.2.2After cleaning manually the leaflets and branches to remove all foreign matter such as dust, dirt, stones, and chaff; to achieve uniform particle sizes, the samples were ground and passed through a 0.3 mm mesh sieve. The extraction process was performed by adding 20 g of plant powder in 200 ml of distilled water at room temperature using a magnetic stirrer at 100 rpm overnight. The mixture was filtered through Whatman #4 , the frozen samples were dried under vacuum for 48 h at −50°C condenser and 20 mbar absolute pressure. After that, the samples were milled three times in a 10 s on 10 s off mode on a grinder.2.42.4.1w) of samples.The moisture content (MC) of the samples was determined by drying them in an oven (UF55 MEMMERT) at 70°C until they reached a constant weight, as per the AOAC standard AOAC, . A water2.4.2g. At 105°C, the supernatant was dried until it achieved a consistent weight. The difference between the weights of the primary and secondary dry materials was used to compute the powder's solubility percentage  was transfered to 100 ml of distilled water and agitated at 600 rpm for 5 min on a magnetic stirrer. After that, the dispersion was centrifuged for 10 min at 4000× 2.4.3m is the weight of the powder (g) and V denotes the volume of the sample (ml).Two grams of sample was carefully put into a measuring cylinder and lightly shook to smooth the powder's surface. Finally, the bulk density was calculated using the following equation:m/V (g/ml)  was used to determine the color of the powder samples. On the basis of our previous studies, we captured images and processed samples , 17% sugar, 0.1% vanilla, 0.4% salep, 0.15% mono‐ and diglyceride (MDG), and 0, 0.6, 1.2, and 1.8% ITLE encapsulated powder . The following steps were used to make the ice‐cream samples: First, a domestic mixer  was used to mix cream and skim milk powder (as providers of milk fat and MSNF) with sugar for 2 min. Other ingredients  were blended together for 5 min in the second step. The mixtures from Steps 1 and 2 were then blended together for 1 min to generate a homogenous system, pasteurized at 80°C for 25 s (HTST), homogenized at 18,000 rpm for 2 min , cooled rapidly to 4°C, and aged at 4°C for 6 h. The ice‐cream mixtures were then frozen for 15 ± 2 min in a batch ice‐cream machine  and hardened for around 24 h in a freezer (−18°C)  and analyzed using the Herschel–Bulkley model:τ0 is the Herschel–Bulkley yield stress (Pa), k stands for the Herschel–Bulkley consistency coefficient (Pa sn), and n shows the Herschel–Bulkley flow behavior index (dimensionless).A rotational viscometer  was used to run rheological tests on the ice‐cream mixtures at a temperature of 25 ± 0.1°C. Prior to measurement, the mixtures were sheared for 800 s at 150 s2.6.2The overrun was computed as follows:2.6.3Thirty grams of ice‐cream samples was placed on a wire rack to melt at 25 ± 1°C. During 60 min, at 5‐min intervals, the weight of molten material was measured. The slope of the linear section of the drained mass‐time graphs was used to determine the melting rate (g/min).2.6.4A texture analyzer  fitted with a conical probe (45°) at room temperature was used to perform penetration test on the ice creams. The penetration depth and speed were considered 15 mm and 2 mm/s, respectively. Hardness was measured as the peak compression force (N) during penetration, and adhesiveness was evaluated as the negative surface area (N s) following withdrawal.2.6.5The ice creams −18°C, 30 g) were coded with three‐digit random numbers and presented to 10 panelists  over the course of two sessions. Quantitative descriptive analysis (QDA) was used to identify the sensory characteristics of the ice‐cream samples  using the Minitab statistical software .The data were analyzed by one‐way analysis of variance . So that with increasing the concentration of maltodextrin, the MC of the samples decreased. The MC of Vasmeh extract powder was in the range of 1.21% to 3.14%  (Table w (0.241) was in the M10 sample, and the lowest one (0.139) was in M30. As it was observed, the aw of encapsulated ITLE ranged from 0.139 to 0.241, which indicates the microbiological stability (aw <0.6) of the dried powders and its long shelf life in suitable packaging and storage conditions. According to the results, with increasing the maltodextrin concentration, the aw of the samples decreased  Table . The higable w 0.41 was in3.1.3p < .05). As the concentration of maltodextrin increased, the solubility of the powders decreased . This finding is similar to that of Goula et al. who reported on tomato powder   Table . With in5) Table . Particl5) Table . Therefo5) Table .Particle size is an effective factor in tapped density; if the particle size is larger, the volume does not change much due to impact, so the tapped density becomes smaller. Because small particles can be placed in empty spaces (pores), smaller particles will have less volume and higher impact density . The encapsulating process produces porous particles that due to the reduction in density and particle compaction, a bright and shiny color is observed in the powders, which with the increase in carrier concentration, the color of the powders tends to white maltodextrin. Increasing the lightness with increasing the maltodextrin concentration is also a result of the white color of maltodextrin, which with increasing the concentration of maltodextrin, the color of the samples tended to white maltodextrin. The results also showed that with increasing the maltodextrin concentration, the value of a* and b* increased significantly (p < .05)  Table . Some re5) Table , Gac fru5) Table , waterme5) Table , and Por3.1.6The results showed that with increasing the concentration of maltodextrin, the TPC in the powders increased. Phenolic compounds are sensitive to high drying temperatures and therefore lowering the temperature in freeze‐drying prevents thermal decomposition or oxidation and consequently preserves the TPC. It has also been reported that dried powders at low temperatures tend to agglomerate due to their higher MC. This is especially true for powders that are highly sticky in nature due to their high sugar content. Agglomeration results in less exposure of powders to oxygen and thus protects phenolic compounds from decomposition   Table . Mahjuri5) Table  and Moha5) Table  also repMahjuri, . The valMahjuri, . The resMahjuri, . Based oOne of the important and influential physical parameters on the quality of ice cream is its melting rate. If the melting rate is too low or too high, it is a defect for ice cream   Table . The higp > .05)  Table . Accordi5) Table , adding 5) Table .3.2.2R2 and low RMSE obtained by fitting the experimental data to the Herschel–Bulkley model confirmed the model's proper fitting, as shown in Table Viscosity is one of the most important parameters to achieve a suitable formulation, select the type of pump for proper transfer, and design of equipment. The viscosity of the ice‐cream mixture affects the texture and quality of the final product, overrun, creaminess, and the rate of mass and heat transfer  of the ice‐cream mixture (Table n) and the highest for the mix containing 1.8% ITLE (17.0 Pa sn). Probably, one of the reasons for the increase in the consistency coefficient of the ice‐cream mix was the increase in dry matter due to the addition of encapsulated ITLE to ice‐ceam mix. Mohammeed et al. stated that the increase in total solids is one of the factors affecting the increase in consistency coefficient  suggested that all of the samples were non‐Newtonian/shear thinning, which was consistent with prior reports . As can be seen, hardness scores decreased with the addition of ITLE (Table Sensory evaluation of the samples was performed in terms of color, coldness, coarseness, hardness, and overall acceptance. The results showed that in all parameters, the control ice‐cream sample had a higher acceptance Table . In the LE Table .4w and solubility, bulk density, and tapped density of powders. A significant increase in sample lightness and a significant decrease in a* and b* parameters were observed with increasing maltodextrin concentration. Also, with increasing the concentration of maltodextrin, the TPC and antioxidant activity of powders increased. In ice‐cream samples, with increasing concentration of ITLE, the lightness of the samples decreased, the a* value increased, and the b* value decreased. Addition of ITLE to the ice‐cream formulation caused a decrease in the melting rate and hardness of the samples, an increase in overrun of ice cream, and the viscosity of the ice cream mix. The results of this study showed that ITLE, with its coloring ability and appropriate antioxidant properties, can be used as a desirable additive in the production of useful products with natural color.In recent years, the tendency to consume functional foods has increased. In addition to high nutritional value, functional products have health‐friendly ingredients for the consumer. Consumption of dairy products is a major part of the diet of people around the world and is considered as an indicator of the development of human societies. Ice cream is one of the most delicious dairy products that can be considered as a substrate for beneficial compounds. The results of this study showed that encapsulation of Vasmeh extract with different concentrations of maltodextrin by freeze‐drying method reduced the MC, aThis research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.Many thanks go to the Research Institute of Food Science and Technology in Mashhad for facilitating the process to conduct this study and providing laboratory equipment for experiments of this research.There is no conflict of interest.All authors have read and agreed to the published version of the manuscript. All authors read and approved the final manuscript.


Robotic-assisted total knee arthroplasty typically necessitates use of tracking pins, which can vary in diameter. Complications such as infections and fractures at the pin-site have been observed, but clarification of the effect of pin diameter on complication is needed. The aim of this study is to compare the pin-related complication rates following robotic-assisted total knee arthroplasty between 4.5 mm and 3.2 mm diameter pins.In this retrospective cohort study, 90-day pin-site complication rates after robotic-assisted total knee arthroplasty were compared between 4.5 mm diameter and 3.2 mm diameter groups. In total, 367 patients were included: 177 with large pin diameter and 190 with small pin diameter. All four pin sites were evaluated using postoperative radiographs. Cases without orthogonal views or visualization of all four pin tracts were noted. Multivariate logistic regression was used to control for age, which differed between the two cohorts.p-value of 0.18. The most common pin-site complication was infection/persistent drainage, found in 1.9% of patients, followed by intraoperative fracture of the second cortex in 1.4%. Intraoperative fracture could not be ruled out in 96 cases due to inadequate radiographic visualization of all pin sites. There was one postoperative pin-site fracture in the large diameter cohort, which required operative fixation.The rate of pin-site complications was 5.6% in the large pin diameter cohort and 2.6% in the small pin diameter cohort, with no statistically significant difference between the groups. The adjusted odds ratio for complications in small compared to large diameter group was 0.48, with a This study demonstrates no statistically significant difference in pin-site complication rates after robotic-assisted total knee arthroplasty between 4.5 mm and 3.2 mm pin diameter cohorts, although there was a trend towards increased intraoperative and postoperative pin-site fractures in the 4.5 mm group. The use of robotic-assisted (RA) total knee arthroplasty (TKA) procedures has rapidly increased in recent years, with the goal of improving limb alignment, component positioning, and soft-tissue balancing . RA deviPrecise and accurate osseous resection and component positioning is critical in order to effectively restore the mechanical axis of the lower extremity or achieve the desired kinematic alignment . PostopeHowever, the addition of any new technology may introduce new problems. In order to fix the navigation trackers to the distal femur and tibia, a total of four temporary navigation pins must be placed. While the placement of pins is generally a benign procedure, there remains a risk for pin-site fracture, pin-site infections, wound complications, neurovascular injury, and pin breakage in patients undergoing RA-TKA. The reported rate of pin-site complications following computer navigated and RA-TKA has ranged between 0.3% to 1.9% , 17, 18.The purpose of this study was to compare pin-related complication rates following RA-TKA between 4.5 mm and 3.2 mm diameter pins during a 90-day follow up period. We hypothesized that the large pin diameter system would result in an increased rate of pin-site complications.Institutional Review Board (IRB) approval was obtained through Columbia University before study initiation (#AAAT8150). This retrospective cohort study compared the 90-day pin-site complication rate between 177 patients with the large pin diameter  system  and 190 patients with the small pin diameter  system . The study included consecutive patients at a single large academic institution undergoing primary robotic-assisted arthroplasty between January 2017 and August 2021. Patients of three senior attending surgeons who used both technologies were included in this study. Choice of RA-TKA system was based on surgeon preference. Revision arthroplasties, arthroplasties that did not utilize either the 4.5 mm or 3.2 mm pin diameter systems detailed above, and patients without 90-days of postoperative follow-up were excluded. The electronic medical record was retrospectively reviewed for demographic data  and perioperative data . Each of these demographic and perioperative data, including total non-pinsite complications , were evaluated as potential confounders in the comparison of the primary outcome of total pin-site complications. When pin-site complications occurred, the type , location , and time to complication were recorded. Pin-site infection/drainage and delayed wound healing included only tibial pin sites, as femoral pin sites were placed through the primary incision. Pin-site infection/drainage included patients with persistent local drainage or erythema necessitating the use of post-operative antibiotics. To evaluate for intraoperative pin-site fractures, immediate postoperative radiographs taken in the post anesthesia care unit (PACU) were reviewed. Some cases were recorded as “inconclusive” if one or more of the four pin tracts was not visualized on the radiograph , or if orthogonal views were unavailable. Radiographs were each independently reviewed by two authors .p < 0.05.Continuous variables were recorded as mean and standard deviation unless otherwise indicated and compared with students t-test. Categorical variables were recorded as count and percentage and examined with logistic regression. Outcomes were analyzed per patient with multivariate logistic regression, controlling for demographic and perioperative parameters that differed between the groups. Significance is set to p < 0.01). There was no significant difference in overall rates of non-pin-site complications between the LPD and SPD systems , or in overall reoperation rate   in this study. There was no difference in sex, BMI, ASA level, or laterality between the cohorts   for pin-site complications in the SPD group was 0.48 (95% CI 0.16 to 1.44), but this finding was not statistically significant (p = 0.18). The most common pin-site complication was infection/persistent drainage   , in which there was no obvious intraoperative fracture identified, but fracture could not be ruled out due to the aforementioned reasons. Removal of these patients from analysis for intraoperative pin-site fracture still demonstrates no significant difference in rate of fracture between the two groups (p = 0.10). One postoperative pin-site fracture was identified, occurring within the LPD group (p = 0.25) , and there was no statistically significant difference between the LPD and SPD cohorts , 1 involved the femur (postoperative pin-site fracture), and 1 was unspecified . Most pin-site complications were either intraoperative or presented within one month postoperatively Table . Age at p = 0.123).There were 5 cases of periprosthetic joint infection (PJI) each in LPD and SPD groups . The rate of PJI did not significantly differ between groups ( = 0.910). The rate of all superficial wound infection, including pin-site and non-pin-site, was 5.1% in the LPD group and 2.1% in the SPD group (p = 0.18). The most common complication was pin-site drainage/infection , of which there were 4 (2.2%) in the LPD group and 3 (1.6%) in the SPD group, with no statistically significant difference between the groups . All cases of local pin-site infection resolved at the pin-site uneventfully with antibiotic treatment.The aim of this study was to compare the rate of pin-site complications between two RA-TKA systems with different diameter tracking pins. While prior studies have suggested that larger pin diameters may increase risk for pin-site complications, this is the first study to directly compare the rate of complications between different sized pins . In the The addition of pin placement to total joint arthroplasty introduces the potential for several complications, including pin-site infection, delayed wound healing at pin sites, damage to neurovascular structures, breakage of pins, and pin-site fracture. Thus far, the current literature has shown these complications are relatively rare. The overall rate of pin-site complications following computer navigated and RA-TKA has ranged between 0.3% to 1.9% , 13, 17.Pin sites introduce stress risers in the bone, and although rare, postoperative pin-site periprosthetic fracture can be a serious complication. Pin placement can also cause intraoperative fracture, more frequently occurring in the far cortex as the pin is tightened into place. In one systematic review, the incidence of postoperative pin-site periprosthetic fracture in RA-TKA was 0.16%; no intraoperative fractures were reported . In a mop = 0.25). This displaced femur fracture, which passed through the proximal femoral pin tract, required open reduction and internal fixation . Of note, these intraoperative fractures were either minor cortical “flecks” or posterior cortex lucencies between the two pin sites, which in some cases were audibly identifiable by the surgeon during final hand-tightening of the pin , as well as the clinical irrelevance of each of our 5 minor cortical fractures, somewhat complicates the reporting of intraoperative pin-site fractures. Nevertheless, this appears to be the first analysis to discuss the prevalence of these intraoperative fractures, which as mentioned previously were frequently accompanied by an audible “crack” during tightening of the pin. Clinicians should continue to be aware of this possible complication, as displaced fractures can occur and require appropriate management [All “inconclusive” cases had no overt intraoperative fracture in all visualized pin sites, but fracture could not be ruled out because one or more pin site was not visualized, or orthogonal views were not available. The high overall rate of “inconclusive” cases  and have not been reported to adversely affect outcomes . Neithern = 96, 26.2%) encountered during our evaluation for intraoperative pin-site fractures. The lack of orthogonal views or inclusion of all 4 pin sites in these cases prevented our ability to completely exclude fracture. Secondly, it is possible that some intraoperative fractures occurred but were not identifiable radiographically, even if appropriate radiographs were available, due to the minimally displaced nature of these fractures . However, there was a trend towards increased intraoperative pin-site fracture  and postoperative pin-site fracture  in the large pin diameter group.Pin-site complications are uncommon following robotic-assisted total knee arthroplasty but should continue to be a consideration during surgical management and follow up. There was no statistically significant difference in complication rates demonstrated in this analysis (5.6% in the 4.5 mm pin diameter group versus 2.6% in the 3.2 mm pin diameter group,


To facilitate this transition, we have developed a rapid in vivo assay for the analysis of the effect of oral tyrosine kinase inhibitors on basal tyrosine phosphorylation of circulating mouse neutrophils. The assay uses a single drop of peripheral blood without sacrificing the mice. Flow cytometry using intracellular staining by fluorescently labeled anti-phosphotyrosine antibodies revealed robust basal tyrosine phosphorylation in resting circulating neutrophils. This signal was abrogated by the use of isotype control antibodies or by pre-saturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids or tyrosine-phosphorylated peptides. Basal tyrosine phosphorylation was dramatically reduced in neutrophils of triple knockout mice lacking the Src-family tyrosine kinases Hck, Fgr, and Lyn. Neutrophil tyrosine phosphorylation was also abrogated by oral administration of the Abl/Src-family inhibitor dasatinib, a clinically used anti-leukemic agent. Detailed dose-response and kinetic studies revealed half-maximal reduction of neutrophil tyrosine phosphorylation by 2.9 mg/kg dasatinib, with maximal reduction observed 2 h after inhibitor administration. Taken together, our assay allows highly efficient analysis of the in vivo effect of orally administered tyrosine kinase inhibitors, and may be used as a suitable alternative to other existing approaches.Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the  Tyrosine kinases phosphorylate tyrosine residues on intracellular proteins, leading to conformational changes, altered enzymatic activity and new protein-protein interaction capabilities of their target proteins . Mammalsin silico screening), chemical synthesis and analysis in various in vitro cellular assays. Most promising candidates then progress to further optimization and analysis in vivo animal models  studies  on the C57BL/6 genetic background were described previously  in a specific pathogen-free facility and transferred to a conventional facility for experiments. Mice of both genders were used at 2–6 months of age. Animals were fed with normal commercial diet (Ssniff S8189-S095). All animal experiments were done at Semmelweis University Department of Physiology and approved by the Animal Experimentation Review Board of Semmelweis University .Where indicated, the mice were treated with 1, 2, 5, 10, 20 or 50 mg/kg dasatinib (Selleckchem) suspended in a 1:2 mucilago:water mixture during the morning by oral gavage. Control mice received vehicle as control. The time of dasatinib or vehicle administration was defined as the 0 time point. Blood samples were collected from the experimental animals immediately before (0 h) as well as 2, 5, 8 and/or 24 h after dasatinib/vehicle administration. All mice were tested once and were not re-used beyond taking multiple blood samples from the same mice within the 24-h timeframe of the experiments in Peripheral blood samples were collected from each mouse during the morning or at the indicated time points after dasatinib/vehicle administration. A drop of blood was taken from each mouse by tail incision, and placed immediately in ice-cold PBS supplemented with 5% FBS and 50 IU/mL heparin.Heparinized blood samples were stained with 0.2 μg/mL PerCP-Cy5.5-conjugated anti-Ly6G antibodies . After 45 min incubation on ice in the dark, the cells were washed and then fixed and permeabilized using the eBioscience Intracellular Fixation and Permeabilization Buffer kit according to the manufacturer’s instructions, also allowing lysis of red blood cells. After washing, the permeabilized cells were stained with FITC-conjugated PY20 anti-phosphotyrosine antibodies (Southern Biotech), FITC-conjugated 4G10 anti-phosphotyrosine antibodies (Millipore) or FITC-conjugated isotype control  antibodies, all at a final concentration of 2.5 μg/mL. After incubation for 45 min on ice in the dark, the cells were washed and re-suspended in FACS lysing solution (BD Biosciences).The specificity of the staining was also confirmed in a competitive assay by pretreatment of the anti-phosphotyrosine antibodies with 4 mM or the indicated concentrations of soluble O-phospho-L-tyrosine (referred to as soluble pTyr) or 4 mM tyrosine-phosphorylated phospho-peptide  for 30 min on ice. Staining was then performed using these antigen presaturated antibodies.DeNovo FCS Express softwares. To eliminate events from non-leukocyte debris  the FSc threshold was increased during flow cytometry without interfering with the putative neutrophil gate. Ly6G-positive cells with forward and side scatter characteristics typical of neutrophils were identified as neutrophils. Fluorescence values reflecting intracellular phosphotyrosine levels within those cells were then determined in the respective channel and expressed as the geometric mean fluorescence intensity (MFI) for the given neutrophil population. Isotype control-stained samples were used to obtain non-specific reference staining intensities. The number of neutrophils was between 500 and 1,500 in the majority of the analyzed samples which represented 18%–30% of all leukocytes.Fluorescence analysis was performed on a BD Biosciences FACSCalibur flow cytometer. Data files were processed by the BD CellQuest and in vitro experiments or from all individual mice from the indicated number of in vivo experiments. Statistical analyses were carried out using one-way ANOVA .For generation of the percent response values, the responses were expressed in percent of the values of the anti-phosphotyrosine-stained wild-type or vehicle-treated samples. Quantitative graphs show mean and SEM from all individual mice from the indicated number of experiments. Percent inhibition values were calculated by subtracting fluorescence values of isotype control-treated samples and then expressed as percentage of wild-type or vehicle-treated samples. ICWe have set up a rapid single-cell-based quantitative assay to test tyrosine kinase inhibitors in experimental mice by intracellular flow cytometry using anti-phosphotyrosine antibodies. To this end, a drop of blood obtained from untreated wild-type mice was stained with fluorescently labeled Ly6G antibodies to stain neutrophils, followed by fixation, permeabilization and intracellular staining using anti-phosphotyrosine antibodies labeled with a different fluorochrome. We have tested two separate anti-phosphotyrosine antibodies (clones PY20 and 4G10) during the experiments, along with an isotype-matched irrelevant control antibody. The overview of the assay protocol is shown in Hck−/−Fgr−/−Lyn−/− mice, and tested the effect of oral administration of the tyrosine kinase inhibitor dasatinib. Overview of those interventions are also shown in In additional experiments, we have also used anti-phosphotyrosine antibodies presaturated with soluble phosphotyrosine amino acids or short tyrosine-phosphorylated peptides, compared wild-type with Cell-surface staining of peripheral blood samples with anti-Ly6G antibodies and intracellular staining with anti-phosphotyrosine antibodies was followed by flow cytometric analysis. Neutrophils were identified based on their forward- and side-scatter characteristics and positive staining for the Ly6G marker. Tyrosine phosphorylation was then determined within this population. The gating strategy is shown in p = 2.1 × 10−11). For a better comparison between a large number of experimental conditions, we have also calculated the normalized fluorescence intensity, expressed in percent of the signal of PY20-treated samples ( samples . This cap = 1.8 × 10−10) and normalized  in our assay system. As shown in rmalized  data of Taken together, our assay is apparently able to detect basal tyrosine phosphorylation of circulating mouse neutrophils, with substantially better signal-to-noise ratio with the PY20 than with the 4G10 antibodies.To simplify the presentation, only fluorescence values normalized to phosphotyrosine antibody-treated samples will be shown in the following sections.p = 1.1 × 10−16). On the other hand, soluble phosphotyrosine amino acids did not affect the supposedly non-specific signal in isotype control-stained samples. Quantification of the normalized fluorescence intensities .Next we used another competition approach to confirm the specific binding of the anti-phosphotyrosine antibodies to their cognate antigens. In these experiments, the PY20 antibodies have been pretreated with a short (13-amino-acid) tyrosine-phosphorylated phosphopeptide. As shown in 50 of approx. 12 μM (p = 1.2 × 10−15).We have also performed a dose-response analysis of the effect of soluble phosphotyrosine amino acids on the neutrophil tyrosine phosphorylation signal determined using the PY20 antibodies. As shown in p = 3.0 × 10−13). In addition, tyrosine phosphorylation of neutrophils measured with the 4G10 antibody was also strongly reduced by 4 mM tyrosine-phosphorylated phosphopeptide (p = 8.9 × 10−5).We also tested the effect of soluble antigens on the fluorescence signal obtained using the 4G10 anti-phosphotyrosine antibodies. As shown in the histogram in The above competition experiments indicate that our flow cytometric approach is indeed specific for tyrosine phosphorylation within circulating neutrophils.Hck−/−Fgr−/−Lyn−/− mutation also affected tyrosine phosphorylation of circulating neutrophils that have not gone through a lengthy isolation procedure. As shown in Hck−/−Fgr−/−Lyn−/− neutrophils showed a strong, although incomplete, reduction of the fluorescence signal intensity detected by staining with the PY20 anti-phosphotyrosine antibodies. Quantification of the signals . However, the phosphotyrosine signal in Hck−/−Fgr−/−Lyn−/− neutrophils was clearly distinguishable from the signal in isotype control-treated samples and could be further reduced by soluble phosphotyrosine competition .We have also repeated the same experiments with the 4G10 anti-phosphotyrosine antibodies , which aHck−/−Fgr−/−Lyn−/− neutrophils and at the same time provide an independent, genetic validation of our flow cytometric assay. They also suggest the existence of residual tyrosine phosphorylation in Hck−/−Fgr−/−Lyn−/− neutrophils.Taken together, our results indicate reduced intracellular tyrosine phosphorylation in in vivo effect of novel tyrosine kinase inhibitors, thus facilitating the transition of the development of such inhibitors from in vitro to in vivo stages. To validate this approach, we have tested the effect of dasatinib, a clinically used tyrosine kinase inhibitor, in our assay system. 50 mg/kg dasatinib was administered orally to wild-type mice, followed by the flow cytometric analysis of the intracellular tyrosine phosphorylation of neutrophils from a drop of blood taken 2 h later.The long-term aim of our assay development is to allow the rapid analysis of the p = 6.3 × 10−13). Importantly, however, a remaining tyrosine phosphorylation signal could still be observed in samples from dasatinib-treated mice. Quantification of this response .We have also performed similar experiments using the 4G10 anti-phosphotyrosine antibodies. As shown in Taken together, our experiments revealed robust reduction of the intracellular tyrosine phosphorylation of neutrophils 2 h after oral administration of 50 mg/kg dasatinib, indicating that our approach is indeed able to rapidly detect the effect of orally administered tyrosine kinase inhibitors. These results at the same time provide an independent pharmacological validation of our fluorescence-based neutrophil tyrosine phosphorylation assay.Having been able to directly measure the effect of oral dasatinib treatment on intracellular tyrosine phosphorylation of circulating neutrophils, we next decided to test the dasatinib dose-response relationship during such experiments. To this end, dasatinib doses ranging from 1 to 50 mg/kg was administered to wild-type mice by oral gavage, followed by analysis of neutrophil tyrosine phosphorylation from a drop of blood taken 2 h later. The PY20 antibodies showed more robust and intensive fluorescence signal with better signal-to-noise ratio compared to the 4G10 clone see  which prp = 9.2 × 10−12; Dunnett’s post hoc test showed significant reduction at 5, 10, 20, and 50 mg/kg doses compared to anti-pTyr-stained vehicle-treated samples, p = 8.6 × 10−6). Again, the fluorescence signal observed in samples from dasatinib-treated mice could be further reduced by presaturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids, indicating that the remaining fluorescence signal is still caused by intracellular tyrosine phosphorylation of neutrophils.As shown in the histogram in One of the advantages of our assay is the detailed and quantitative analysis of neutrophil tyrosine phosphorylation from a single drop of peripheral blood, without sacrificing the mice. This allows us to follow tyrosine phosphorylation over a period of time, providing information about the kinetics of the biological effects of the inhibitors used. To exploit this opportunity, we have tested neutrophil tyrosine phosphorylation in mice treated with varying dasatinib doses at multiple time points over a 24-h period. Mice were treated with dasatinib doses ranging between 5 and 50 mg/kg and the intracellular tyrosine phosphorylation of neutrophils was tested immediately before, as well as 2, 5, 8 and 24 h after dasatinib treatment. Again, these experiments were only performed using the PY20 anti-phosphotyrosine antibodies. To facilitate the comparison of the data, fluorescence intensities were normalized to values of anti-phosphotyrosine antibody-treated samples of vehicle control mice at each time point.p = 1.3 × 10−13), the inhibitory effect of dasatinib remained very strong at the 5-h (p = 2.2 × 10−3) and still more than half-maximal at the 8-h time points (p = 8.4 × 10−3). However, neutrophil tyrosine phosphorylation returned to normal levels by the 24-h time point (p = 0.61). Decreasing the dose of dasatinib to 20 or 10 mg/kg hardly affected the inhibitory effect at the 2-h time point , whereas the inhibitory effect was significant but less pronounced at the 5 mg/kg dose (p = 4.3 × 10−8). This is in line with the saturation of the effect of dasatinib at the 2-h time point at or above 10 mg/kg dasatinib (see p = 0.22 and p = 0.045), and hardly any inhibition at 5 mg/kg (p = 0.49). At 8 h, only the 50 mg/kg dose showed a substantial inhibitory effect, which was hardly stronger than 50%. Importantly, all fluorescence signals could be practically completely abrogated by pretreatment of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids, indicating the specificity of the signal for tyrosine phosphorylation.As shown in inib see . HoweverTaken together, the above experiments indicate gradually decreasing effects of dasatinib after more than 2 h from oral administration, and at the same time demonstrate the suitability of our assay to follow the effect of oral tyrosine kinase inhibitors in a kinetic manner.Tyrosine kinases are major targets of the development of novel pharmaceuticals for the treatment of a number of various diseases. Their main advantages as therapeutic targets include their critical role in major human diseases, their enzymatic activity allowing easy targeting by small-molecule inhibitors, their diversity providing opportunity for targeting specific biological processes, as well as the very substantial experience in developing small-molecule tyrosine kinase inhibitors. The main medical fields where such inhibitors can be used include the various types of cancer, as well as autoimmune and inflammatory diseases.in vitro analysis of their effects on various cellular functions using cell lines or primary cells. However, those experiments are often unable to predict the in vivo effects of the inhibitors, their permeability across the various biological barriers , their removal from the circulation by degradation or excretion, and their overall in vivo efficacy. Therefore, these characteristics need to be tested using other approaches such as administration of drug candidates to experimental animals. However, measuring drug concentrations in the circulation may be rather difficult and require very unique experience/equipment because of the difficulties of testing specific organic compounds in a complex biological environment. The efficacy of the inhibitors also needs complex and time consuming in vivo disease models. Those in vivo models often rely on the use of a large number of experimental animals and may require large inhibitor quantities which necessitate scaling up the chemical synthesis using entirely new synthesis pathways. Alternatively, some of the in vivo characteristics of the inhibitors  can be approximated by in vitro assays. However, those assays have their own substantial limitations and should also be followed by conventional in vivo assays. Taken together, the transition of the development of tyrosine kinase inhibitors to in vivo analysis is rather difficult and consumes substantial time and resources.An important step of the development of small-molecule tyrosine kinase inhibitors is the in vivo tyrosine phosphorylation of neutrophils as an approximation of in vivo tyrosine phosphorylation in cells of hematopoietic origin. This assay is suitable for the rapid detection and precise quantification of the biochemical effect of orally administered tyrosine kinase inhibitors. It therefore provides an approximation of the transportation of the inhibitors across biological membranes and barriers, it is intrinsically corrected for factors  that affect the biological availability of the given compounds, and it allows an approximate assessment of the biological effect of the inhibitors, at least at the biochemical level. In addition, our approach requires very low inhibitor quantities , therefore it does not require synthesis of large inhibitor quantities, giving the opportunity to test the in vivo effect of a larger number of inhibitors in early preclinical phases. Our assay procedure is also relatively simple and is based on widely available reagents and equipment, therefore no complex assay development is needed. The fact that quantitative results can be obtained in not more than a few hours after oral inhibitor administration may also provide substantial benefit. Finally, the fact that the animals do not need to be sacrificed for the assay procedure allows more efficient use of experimental animals  and a better implementation of the principle of the 3R  of the humane use of experimental animals. Taken together, we believe that the experimental approach described here provides a number of advantages over the conventional steps of the development of tyrosine kinase inhibitor drug candidates and may therefore be a potentially viable addition to the methodological arsenal of the transition from the in vitro to the in vivo stages of drug development, allowing a more time- and cost-efficient approach with a shorter time-to-clinic interval.Our assay system provides substantial advantages over the above scenario. Our experiments test the basal Hck−/−Fgr−/−Lyn−/− mice (in vivo tyrosine phosphorylation of neutrophils.A number of our experiments served for the validation of our approach at different conceptual levels. The use of two different anti-phosphotyrosine antibodies and a suitable isotype-matched control  provided−/− mice  provided−/− mice  indicateOur experiments also revealed the capability of our approach for detailed analysis of the dose-response relationship and kinetics of orally administered tyrosine kinase inhibitors. Indeed, experiments shown in A clear limitation of our assay is that it focuses on general tyrosine phosphorylation within neutrophils, therefore it is only suitable for the analysis of inhibitors with an intended effect on those cells or at least the myeloid compartment. Nevertheless, given the major challenges of treating hematological malignancies and inflammatory diseases, testing tyrosine phosphorylation in neutrophils may be a suitable strategy for the development of a number of drug candidates.Our experiments may also open up avenues of development of further assays based on similar principles. Such improvements may proceed along various different directions. Using antibodies labeling populations other than neutrophils, it would be relatively easy to extend our assay to the analysis of other circulating leukocytes such as B- and T-cells, monocytes, eosinophils or basophils. We should also be able to test intracellular tyrosine phosphorylation of various leukocyte subpopulations, including different subpopulations of B-cells, T-cells and monocytes. With appropriate selection of fluorochromes and multi-channel flow cytometry, such details could also be obtained from the same single samples labeled by a number of different antibodies, optimizing the use of experimental animals. Another possible direction of further assay development would be the use of phosphorylation-specific antibodies of given intracellular substrates. That way, we should be able to test the effect of inhibitors on specific tyrosine phosphorylation pathways rather than on the general tyrosine phosphorylation response. Although such improvements of our assay would be relatively straightforward, they would also open up a number of new methodological and conceptual issues that we felt are beyond the scope of the experimental work described in this paper.in vitro to an in vivo developmental phase. Our assay also has the potential to inspire the development of further conceptually similar assays that may extend well beyond the analysis of drugs targeting overall tyrosine phosphorylation in the myeloid compartment.Taken together, we believe that our experimental approach of flow cytometric analysis of basal tyrosine phosphorylation of neutrophils may provide a useful addition to the current tools of the transition of the development of tyrosine kinase inhibitors from an


Background: As specific flap designs performed for lower third molar extractions usually influence periodontal healing of the adjacent first and second molars, this study aimed to evaluate the periodontal conditions of these sites after 6 months post-surgery. Methods: Forty patients, aged 14–30 years, were included. Surgical extraction of the lower third molar was performed through a flap with papilla detachment  or a trapezoidal flap . Periodontal parameters at the first and second molar sites were assessed for visible plaque index, bleeding on probing, recession, probing pocket depth, and clinical attachment loss before surgery (T0), one month (T1), and six months after extraction (T2). Results: No statistical differences were found for the plaque and bleeding indexes between the two flaps at each observation time and considering both time intervals. For recession, no statistical differences were found between the two flaps considering the final time interval. For probing pocket depth at the second molar site, both techniques registered a significant increase between T0 and T1, followed by a decrease up to T2. For clinical attachment loss, mean values assessed for the first and second molar sites demonstrated evidently increased values between T0 and T1, followed by moderate decreases up to T2. Conclusions: Considering short (T1) and mid-term (T2) follow-ups, a specific flap design does not seem to particularly influence periodontal healing six months after surgery. Extraction of third molars represents a frequently invasive surgical procedure for both hard and soft tissues, particularly in the lower jaw. As the design of the access flap, gingival detachment included, often implicates a considerable damage for the entire periodontium, periodontal healing seems to depend on the chosen surgical technique . AdditioBeing a relevant violation of soft tissues almost inevitable in performing incisions for the surgical removal of third molars, investigations available in the literature ,8 mainlyAlthough comparisons of different types of surgical access revealed conflicting outcomes regarding their precise influence on post-surgical periodontal conditions, the techniques proposed can be summed up and classified  as envelRegarding daily clinical practice, a greater relevance seems to be usually assigned to early evaluations of periodontal healing after third molar surgery, especially for the second molar site, often disregarding late assessment of tissue conditions. Considering that evidence for the stability of effective wound healing ,15,16 isIn the light of these considerations, the aim of this retrospective study was to evaluate six-month periodontal healing of lower first and second molar sites, following soft tissue incisions performed to obtain access for surgical extraction of lower third molars (included or affected by dysodontiasis), using a marginal flap with papilla detachment (DETP) or a trapezoidal flap (TRAP). Periodontal healing was evaluated for all indexes related to the stability of soft tissues, primarily producing results in terms of inflammation (BOP) and periodontal attachment (CAL).-Patients aged between 14 and 30 years;-Patients with ASA (American Society of Anesthesiology categorization) category I ;-Written informed consent obtained from each subject involved in the study, and proper forms regularly completed by parents or other related legal guardians for patients under the age of 18 years;-Pre-surgical assessment of lower third molar level of inclusion and its relationship to the second molar. Third molar positions were categorized using the Pell and Gregory classification  ,17,18,19Patients who were consecutively treated for a lower third molar surgery at the Dentistry and Maxillo-facial Surgery Unit (University of Verona) during 2018 were included in a study undertaken as a retrospective evaluation conducted between March 2022 and July 2023 on the available medical records, enabling assessments of satisfaction of the inclusion criteria and with both 1-month and 6-month follow-ups after extraction. Specifically, the inclusion criteria were as follows:The nature and goals of this study, as well as the anonymity in the scientific use of data, were clearly presented in the consent form. The Declaration of Helsinki and the good clinical practice guidelines for human research were followed during this study’s execution. This retrospective study received approval from the University Institutional Review Board . This study demonstrated adherence to the STROBE checklist recommendations.The surgeon performed both pre-surgical evaluations and consequent surgical procedures, as previously described . Each paWhen a patient had bilateral mandibular third molars, they were both extracted during a single procedure. One minute of 0.12% chlorhexidine mouthwash was taken one hour prior to surgery, and one gr of amoxicillin + clavulanic acid was taken orally 24 h prior. Both plexus block and troncular nerve block  were performed using local anesthesia . To ensure proper wound closure in flap repositing and to allow for initial wound healing, soft tissue incisions had to guarantee a visible section of the epithelial connective tissue and periosteal layers. Additionally, excessive surgical trauma and lacerations of the flap edges had to be avoided. For soft tissue incisions and ensuing subperiosteal dissection, a scalpel blade N.15  was used.As shown in As shown in Regarding the rest of the surgical procedures, in both cases osteotomy was conducted with appropriate bone-cutting instruments ; odontotomy was performed with fissure burs; final extractions were performed using the appropriate levers and physics or conventional forceps. Flap closure was finally performed with Vicryl 3.0 and 4.0 sutures .Post-operative indications and antibiotic prescriptions (875 mg of amoxicillin + 125 mg of clavulanic acid 3 times a day for a week) were given. Patients were checked for suture removal after 7 days, and for follow-up visits after 14 days, one month, and 6 months.A periodontal probe  was used to assess periodontal soft tissues ,23,24 inBoth the first and second molar sites underwent clinical soft tissue examination to measure the average values  of the mesial, central, and distal sites investigated on the buccal/lingual sides. All sites were detected pre-operatively (T0), after 1 month (T1), and after 6 months (T2) and analyzed for each time interval .t” test or Wilcoxon matched-pairs signed-rank test. The comparison of the means among groups was performed using the one-way analysis of variance (ANOVA) or Kruskal–Wallis equality-of-populations rank test. The level of significance was set at 0.05.After creating a database with Microsoft Excel, data analysis was conducted with Stata v.13.0 for Macintosh . The Shapiro–Wilk test was used to evaluate the normality assumptions for continuous data; means and standard deviations were reported for normally distributed data, with the median and interquartile range (iqr) reported otherwise. Absolute frequencies, percentages, and 95% confidence intervals were reported for categorical data. The comparison between the means of continuous variables in different times was performed using the paired Student’s “A total of 40 (17 men and 23 women) patients were finally included in the retrospective evaluation, with an equal distribution of patients (20 and 20) for each technique evaluated. The mean age was 22 (14–30) years. The demographics are reported in Regarding flap design (which was performed according to tooth position), DETP was employed in all teeth of class IA and IIA, three teeth of class IIIA, five teeth of class IB, one tooth of class IC, and one tooth of class IIIC; TRAP was used in the rest of the cases.As during soft tissue examination  no variations at each time interval  were found on the lingual sides, the following average values only considered variations on the buccal sides.The mean overall values assessed for the first and second molar sites  demonstrp = 0.03 and p = 0.02) for a stable mean value of 0.23. Similarly, the TRAP technique registered a statistically significant variation (p = 0.02 and p = 0.03) at both time intervals for a stable mean value of 0.3. From a clinical point of view, these significant values can be considered as moderately relevant for the second molar site in terms of the VPI.For the second molar site see , the oveNo statistical differences were found between the two flaps at each observation time and considering both time intervals a,b.The mean overall values assessed for the first and second molar sites  demonstrp = 0.02 and p = 0.01) at both time intervals for mean values of 0.13 and 0.2. For the second molar site (see p = 0.005 and p = 0.001) for mean values of 0.22 and 0.26. Similarly, the DETP technique registered a statistically significant increase (p = 0.01) at both time intervals for mean values of 0.28 and 0.36. From a clinical point of view, these significant values for the second molar site can be considered as relevant in terms of BOP.For the first molar site see , the TRAsite see , the oveNo statistical differences were found between the two flaps at each observation time and considering both time intervals a,b.The mean overall values assessed for the first and second molar sites  demonstrFurthermore, no statistical differences were found between the two flaps considering the final time interval a,b.p = 0.004 and p = 0.01) for mean values of 0.23 mm and 0.19 mm. Similarly, the TRAP technique registered a statistically significant increase (p = 0.01 and p = 0.03) at both time intervals for mean values of 0.4 mm and 0.33 mm. From a clinical point of view, only these significant values can be considered as relevant for the first molar site in terms of REC.For the first molar site see , the ovep = 0.001 and p = 0.003) for mean values of 0.49 mm and 0.21 mm. Similarly, both techniques registered a statistically significant increase (p = 0.008 and p = 0.003) between T0 and T1, followed by a significant decrease (p = 0.04 and p = 0.02) up to T2 for final mean values of 0.18 mm and 0.25 mm for DEPT and TRAP, respectively. From a clinical point of view, these significant values can be considered as greatly relevant for the second molar site in terms of REC.For the second molar site see , the ovep = 0.001 for both the first and second molar sites).Moreover, eventual significant variations in comparing flaps at each observation time cannot be considered as clinically relevant due to the findings of statistical differences between the two flaps already at T0  for mean values of 0.4 mm and 0.25 mm. Similarly, the DETP technique registered a statistically significant change (p = 0.001 and p = 0.01) at both time intervals for mean values of 0.6 mm and 0.35 mm. From a clinical point of view, these significant values can be considered as relevant for the first molar site in terms of PPD. Moreover, eventual significant variations for the first molar site in comparing flaps at each observation time cannot be considered as clinically relevant due to the findings of statistical differences between the two flaps already at T0.For the first molar site see , the ovep = 0.001) for mean values of 0.8 mm and 0.51 mm. Similarly, both techniques registered a statistically significant increase (p = 0.001) between T0 and T1, followed by a significant decrease (p = 0.02 and p = 0.01) up to T2 for final mean values of 0.53 mm and 0.5 mm for DEPT and TRAP, respectively. From a clinical point of view, these significant values can be considered as greatly relevant for the second molar site in terms of PPD.For the second molar site see , the oveThe mean values assessed for the first and second molar sites  demonstrFurthermore, no statistical differences were found between the two flaps considering the final time interval a,b.p = 0.1) between T0 and T1 compared to TRAP, and a statistically significant increase (p = 0.004 and p = 0.04) at each time interval, for a mean value of 0.53 mm, decreasing finally to 0.3 mm up to T2. For the second molar site (see p = 0.04) between T0 and T1 compared to TRAP, for a mean value of 0.4 mm, slightly decreasing to 0.35 mm up to T2. From a clinical point of view, these significant values can be considered as relevant for the first and second molar site in terms of CAL. Moreover, eventual significant variations for the second molar site in comparing flaps at each observation time cannot be considered as clinically relevant due to the findings of statistical differences between the two flaps already at T0.Statistical differences, both for the first and second molar sites, were found between the two flaps at each observation time. For the first molar site see , the DETsite see , the DETConcerning tooth class, described as the degree of inclusion see , no signConsidering an overall evaluation of soft tissue conditions of the second molar site between T0 and T2 in terms of BOP, PPD, and CAL see , no signA complete evaluation of post-operative conditions after third molar surgery is fundamental to detect eventual inflammatory complications, such as pain, swelling, trismus, infection, and alveolar osteitis, all implying a relevant negative impact on everyday quality of life of patients ,26.As shown by this investigation, which presents a one-month and six-months analysis of data related to the periodontal indexes of PPD, VPI, BOP, REC, PPD, and CAL, regular and proper monitoring of soft tissue conditions  can reprConcerning the evaluation of the adjacent first and second molar sites, a general increasing trend, between T0 and T1, was assessed for all parameters. Furthermore, a general decreasing trend, up to T2, was assessed for REC, PPD, and CAL; the BOP and VPI indexes showed an increasing trend up to T2 instead.Moderately relevant for the second molar site in terms of the VPI;Relevant for the second molar site in terms of BOP;Greatly relevant for the second molar site in terms of REC and PPD;Relevant for the first and second molar sites in terms of CAL.On the other hand, considering overall variations after 6 months , a moderately increasing tendency was observed between T0 and T2, even if not significant for any of the analyzed parameters. From a clinical point of view, the significant values found can be considered as:Delving deeper throughout the presented outcomes, BOP and the VPI were not particularly influenced by a specific flap design during the periodontal healing process following the surgical extraction of the lower third molar. Nonetheless, it should be emphasized that an increased percentage of BOP is usually linked to consistent gingival inflammation; therefore, it is characterized by a relevant concentration of cytokines and inflammatory mediators, which certainly do not facilitate tissue repair processes ,16. SomeAlways regarding TRAP, the authors have assumed that the clinical relevance of greater REC with this technique at the first molar site could be related to the specific design of this flap, which also provides for the release incision at this site, thus interfering with the adherent gingiva and establishing an ideal condition for the development of recession. The two incisions involved in this flap  determine the need for three sites of suture: one at the interdental papilla, between the first and second molars, and two sutures at the level of the second-releasing incision . An areaConcerning PPD, according to studies in the literature, the choice of a specific flap design does not seem to generally influence clinical outcomes in limiting the increase in probing for adjacent elements in a mid-term follow-up ,36,37. AData reported for CAL revealed overall favorable conditions, both for the first and second molar sites, in patients treated with TRAP compared to DETP. Combining these outcomes with the data for REC, mainly worse for TRAP, and PPD results, instead mainly worse for DETP, it can be seen how both time intervals (1 month and 6 months) were necessary to highlight the final stability of soft tissue conditions for both techniques through measurements of CAL.A secondary aspect evaluated in this study was the response of soft tissues between T0 and T2, also according to different pre-operative degrees of inclusion, for the overall sample and comparing techniques: significant differences between groups were not shown by any of the indexes apart from the technique used, as already found in another study by the same research group . The outIn addition, regarding the entity of surgical ostectomy based on the available bone density, no significant differences were found in terms of the BOP, PPD, and CAL of the second molar site between patients 14–25 and >25 years old, evidencing a limited impact of this aspect on six-month soft tissue stability. Despite an age above 25 years having being reported to be associated with a higher number of complications in third molar surgery , 75% of Finally, while a previously published study by the same research group  evaluateThe present study revealed overall stable outcomes of soft tissue conditions after third molar extraction in terms of inflammation and periodontal attachment, apart from the flap performed and the starting tooth degree of inclusion. Even if mostly present in the patients treated with TRAP , the formation of a gingival recession for both elements adjacent to the element extracted does not seem to depend on the chosen flap.Regarding the increase in PPD in the early post-surgical follow-up, the use of a specific flap design does not seem to be particularly important up to six months. We are aware, however, of the need for even longer follow-ups to properly assess periodontal healing.


This study investigates the use of our DP-based ICA denoising method for harmonizing functional MRI (fMRI) data collected from the Autism Brain Imaging Data Exchange II. After frequency-domain and regional homogeneity analyses, two modalities, including amplitude of low frequency fluctuation (ALFF) and regional homogeneity (ReHo), were used to validate our method. The results indicate that DP-based ICA denoising method removes unwanted site effects for both two fMRI modalities, with increases in the significance of the associations between non-imaging variables  and fMRI measures. In conclusion, our DP method can be applied to fMRI data in multi-site studies, enabling more accurate and reliable neuroimaging research findings.Modern neuroimaging studies frequently merge magnetic resonance imaging (MRI) data from multiple sites. A larger and more diverse group of participants can increase the statistical power, enhance the reliability and reproducibility of neuroimaging research, and obtain findings more representative of the general population. However, measurement biases caused by site differences in scanners represent a barrier when pooling data collected from different sites. The existence of site effects can mask biological effects and lead to spurious findings. We recently proposed a powerful denoising strategy that implements dual-projection (DP) theory based on ICA to remove site-related effects from pooled data, demonstrating the method for simulated and  Over thMost neuroimaging studies are conducted within a single research site, with limited capabilities for collecting large sample-size datasets. Smaller sample sizes and lack of harmonization across independent studies present challenges in achieving acceptable reliability and reproducibility of neuroimaging research . As a revia the projection step and other pure noise components are then removed from the data using a second projection procedure. Compared with traditional ICA and ComBat (COMbining BATches), which is a general linear modal (GLM)-derived method based on the empirical Bayes approach  with dual-projection (ICA-DP), was proposed for removal of site effects in multi-site structural MRI data . For ICAapproach , ICA-DP In this paper, we introduce the use of ICA-DP to harmonize fMRI data collected from multiple sites. We apply ICA-DP to the data from Autism Brain Imaging Data Exchange II (ABIDE II) and compare its performance with two other common harmonization methods: ICA and ComBat. To assess the effectiveness of the harmonization methods, we utilize various techniques for visualizing and quantifying site effects before and after denoising. Additionally, we evaluate the denoising methods in terms of their ability to preserve signal effects.2.2.1.We utilized data from Autism Brain Imaging Data Exchange II (ABIDE II) to investigate the impact of site effects on ALFF and ReHo, and the performance of ICA-DP for denoising site effects and preserving signal effects.Neuroimaging data from 1,114 subjects collected by 18 different sites with various scanner manufacturers   were obtIn this study, the site differences are defined as noise variables, and group differences (ASD/HC), age, and sex are regarded as signal variables. The correlation coefficients among these variables are summarized in 2.2.The raw fMRI data were preprocessed with FSL FEAT, including removing the first six volumes, motion correction, and spatial normalization to standard MNI space. Two functional modalities, ALFF and ReHo, were generated from the preprocessed fMRI data with DPABI . For ReH2.3.Two most widely used harmonization methods: ComBat and traditional ICA, were applied in this study to show the performance of ICA-DP on site-effects removal. We now describe the three different strategies below.2.3.1.ComBat is a GLM-derived method based on empirical Bayes approach. The method assumes that the data can be modeled as a linear combination of signal variables and site effects, which includes additive and multiplicative factors:where Finally, ComBat uses an empirical Bayes (EB) framework to get an improved estimate of site effects 2.3.2.ICA is a data-driven strategy that decomposes the data matrix into a set of statistically independent non-Gaussian maps together with associated courses .where S is the spatial map and A is the corresponding courses. Compared with our ICA-DP, we rename the traditional ICA as ICA-SP (single-projection). To preserve the signal effects, ICA-SP method only removes those pure site-related components (related to site effects only), and leaves those mixed components without any process.where In order to eradicate the site effects, we proposed the ICA-DP method in our previous study . Firstlywhere 2.4.For the ComBat-based method, the input p < 0.05, with Bonferroni correction) were classified as pure signal components. Conversely, those solely correlated with the noise variable were identified as pure noise components. Components related to both signal and noise variables were categorized as mixed components. For ALFF, we identified 79, 120, and 166 pure site-related components, and 21, 29, and 34 mixed components; For ReHo, we identified 83, 136, and 179 pure site-related components, and 15, 10, and 11 mixed components. The ICA-SP method exclusively utilized pure noise components to eliminate site effects, whereas the ICA-DP method employed all noise-related components, including mixed ones, for site effects removal. In the ICA-DP approach, both mixed and pure noise components were used to extract noise effects that are considered as integral site-related noise effects and used for removal. Both ICA methods were implemented using widely used software packages for neuroimaging data analysis, namely MATLAB and FSL MELODIC. FSLeyesFor ICA-based methods, the data were decomposed into 100, 150, and 200 independent components. Pearson correlation and Analysis of Variance (ANOVA) were applied to identify signal, noise, and mixed components based on 2.5.We used several strategies to assess the performance of the three different denoising methods in terms of eliminating the site effects and preserving the signal effects. To visualize the site effects, we used t-distributed stochastic neighbor embedding (t-SNE) to observe the distribution of the data points, with a tendency to be clustered by site or not. Group F-test was also used to find the significant differences in ALFF and ReHo for brain regions associated with site differences. It is also important to show whether the methods can preserve the signal effects well. In this study, we used age, sex, and group difference (ASD/HC) as variables of interest. In addition to t-SNE and group-level tests of ASD vs. HC, the Pearson correlation coefficient between median ALFF, ReHo and age was also assessed. For each modality, the median value for each subject was obtained by calculating the median of 100 regions of interest.3.3.1.p < 0.05).3.2.3.2.1.In summary, ICA-DP increased the age effects by detecting more significantly different regions related to age, while ComBat and ICA-SP decreased the age effects with fewer or no significant regions.3.2.2.Similar to the results for age effects, ICA-DP increased the sex effects by detecting more significantly different regions related to sex, while ComBat and ICA-SP decreased the sex effects with fewer or less significant regions.3.2.3.4.In this study, we applied the ICA-DP method to the multi-site harmonization of ALFF and ReHo, and compared it to traditional ICA and ComBat methods for removing site effects and preserving biological variability. The results showed that our ICA-DP method can better remove site effects and preserve physiological signals compared with two other approaches for denoising, ICA-SP, and ComBat.In the non-denoised data, site effects objectively exist in both modalities: 1) original ALFF and ReHo both show a trend of clustering by site , even ifRegarding denoising performance, both ICA-DP and ComBat methods can thoroughly remove site effects: (1) the denoised data no longer clusters by site; F-test results no longer have significantly correlated activation regions with site variables. The traditional ICA can only remove the effects of site effects to some extent and cannot eradicate it, because the traditional ICA method only removes the influence of pure noise components and does not deal with mixed components. If the proportion of mixed components in all components is relatively large, or the site effects in mixed components are relatively apparent, the denoising ability of the traditional ICA method will be greatly discounted; on the contrary, the ICA method will have better denoising effect.In addition to evaluating the performance of the three methods in removing site effects, it is equally important to evaluate their ability to preserve biological signals. To this end, we define age, sex, and group difference (ASD/HC) as variables of interest. The results show that our ICA-DP method effectively removes site effects while also enhancing the examination of biological signals, including the effects of age, sex, and group difference (ASD/HC). The other two methods reduced the examination of these biological effects. Our method’s enhancement of biological signals is due to the fact that for each noise component identified, we first regress out the influence of biological signals and then use it for denoising so that the proportion of physiological signals in the denoised data is relatively large and it is easier to detect brain regions that are related to signals through statistical tests. From another perspective, this might result in other variables, in which we are not interested, not being well preserved . The othIn 2010, Biswal et al. conducted a study on age and sex in a large sample of fMRI data from 35 sites. They also reported site effects. Although they did not remove the site effect in their study and just utilized sites as covariates in a generalized linear model (GLM), they still identified some brain regions that were significantly correlated with age and sex in the ALFF. In some of our results (age and sex effect of ALFF), we also found activation regions that highly overlap with Biswal’s results. Because we used different datasets and different sample sizes, our results are highly overlapping, but not exactly the same. In addition, we believe that if we apply our method to their dataset and remove the site effect with ICA-DP, more similar activation regions related to age and sex will be founded.Regarding the statistical results of group differences between individuals with autism spectrum disorder (ASD) and healthy controls (HC), our research identified similar brain regions that have been highlighted in previous studies. For example, in patients with ASD, increased ALFF in the Temporal Lobe and Frontal Lobe while decreased ALFF in the Occipital Lobe was also found compared to HC . ParticiTo the best of our knowledge, there are only a few studies focused on the harmonization of multiple sites for ALFF and ReHo to reveal their associations with age, sex, and group difference (ASD/HC). Thus, we are cautious in interpreting the results until the same results can be repeated on a large sample dataset from a single center.5.The combination of multi-site MRI data has the potential to increase the statistical power and improve the reliability and reproducibility of neuroimaging research. However, the analysis of MRI data is often confounded by site effects. Removing these site effects is a critical step in the process of multi-site data fusion. In addition, preserving signals of interest is a major concern when applying any denoising strategy. ICA-SP and ComBat reduced associations with age and sex.In contrast, our ICA-DP method has proven to be effective in removing site effects and preserving biological variability. With our ICA-DP method, multi-site fMRI data can be harmonized, thus allowing for more robust and accurate analysis. This approach can significantly enhance the validity of neuroimaging research, and we believe it will be a valuable tool for future studies.http://fcon_1000.projects.nitrc.org/indi/abide/abide_II.html.Publicly available datasets were analyzed in this study. This data can be found at: HX wrote the manuscript with comments from TK, HL, LN, and FC. YZ and DZ downloaded the data and preprocessed them. YH contributed to the guidance of methods. HX carried out the data analysis. All authors read and approved the final manuscript.This work was supported by STI 2030–Major Projects 2022ZD0211500, Science and Technology Planning Project of Liaoning Provincial (nos. 2022JH2/10700002 and 2021JH1/10400049), National Natural Science Foundation of China [grant numbers 91748105 and 81471742], National Foundation in China [grant number JCKY 2019110B009], and National Institutes of Health [NIA RF1 AG078304].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lumbricus terrestrislongitudinal muscle synapses. GABA treatment significantly reduced the fluorescence intensity observed at these synapses, suggesting that GABAergic signaling does indeed have a presynaptic inhibitory mechanism.Earthworm body wall muscle synapses have been suggested to contain both excitatory and inhibitory inputs, and therefore allow for investigation of excitatory/inhibitory signaling in an easily accessible model system. While previous studies have focused on postsynaptic GABAergic inhibitory mechanisms, this study investigated the hypothesis that GABAergic signaling also has presynaptic inhibitory function. This hypothesis was tested by loading synaptogreen C4 dye  into presynaptic vesicles in the presence of GABA at Ainhibitor picrotoxinLumbricus terrestrisbody wall musclesAand GABABreceptors were present at these synapsesEisenia fetida, GABA application had no effect on spontaneous muscle contractions, but caused relaxation of preparations that were precontracted by serotonin. Additionally, this result was replicated during treatment with the GABABagonist baclofen and was not blocked by the GABAAantagonist bicuculline. The authors suggested that perhaps the inhibitory effect observed was by a presynaptic mechanismLumbricus terrestris. To test this hypothesis, we loaded the styryl dye synaptogreen C4  into presynaptic vesicles at longitudinal muscle synapses in the presence of exogenously applied GABA. The styryl dyes have been previously shown to stain presynaptic axon terminalsA) and metabotropic (GABAB) receptors in vertebrate organismsDrosophilaAreceptor activity, or possibly the opening of potassium channels as a downstream effect of a GABABreceptor mediated second messenger cascade. The contributions of the different GABAergic receptors to this presynaptic inhibitory mechanism can be investigated by loading synaptogreen in the presence of agonists (or antagonists) specific for the individual receptors and measuring changes in fluorescence intensity.The balance of excitatory and inhibitory signaling is critical for normal nervous system function. Earthworm body wall muscle synapses have long been suggested to contain both excitatory and inhibitory inputs, and therefore allow for investigation of excitatory/inhibitory signaling in an easily accessible model system. Previous studies have largely focused on postsynaptic GABAergic inhibition at earthworm body wall muscle synapses. Investigations in the late 1960’s demonstrated that application of GABA caused hyperpolarization of the muscle, and that inhibitory potentials were blocked by the GABA Lumbricus terrestrisfrom Carolina Biological Supply. Earthworms were pinned ventral side down to a dissecting tray. Posterior to the clitellum  worms were bisected longitudinally, the tissue spread and pinned, and digestive structures were removed. Throughout this process, the tissue was thoroughly rinsed with “normal” solution containing: 25 mM NaCl, 26 mM Na2SO4·10H2O, 6 mM CaCl2, 1 mM MgCl2·6H2O, 4 mM KCl, 2 mM Tris, 55 mM sucrose, and pH 7.3-7.4, similar to the recipe as found inEarthworms were purchased asLumbricus terrestris, Carolina Biological Supply Synaptogreen C4, Biotium, Inc. SCAS, Biotium, Inc.


There is ample evidence that higher accuracy can be achieved in thoracolumbar pedicle screw placement by using spinal navigation. Still, to date, the evidence regarding the influence of the use of navigation on the screw diameter to pedicle width ratio remains limited.The aim of this study was to investigate the implications of navigation in thoracolumbar pedicle screw placement not only on screw accuracy, but on the screw diameter to pedicle width ratio as well.In this single-center single-surgeon study, 45 Patients undergoing navigated thoracolumbar pedicle screw placement were prospectively included. The results were compared with a matched comparison group of patients in which screw placement was performed under fluoroscopic guidance. The screw accuracy and the screw diameter to pedicle width ratio of every screw were compared between the groups.Screw accuracy was significantly higher in the navigation group compared to the fluoroscopic guidance group, alongside with a significant increase of the screw diameter to pedicle width ratio by approximately 10%. In addition, both the intraoperative radiation dose and the operating time tended to be lower in the study group.This study was able to show that navigated thoracolumbar pedicle screw placement not only increases the accuracy of screw placement but also facilitates the selection of the adequate screw sizes, which according to the literature has positive effects on fixation strength. Meanwhile, the use of navigation did not negatively affect the time needed for surgery or the patient's intraoperative exposure to radiation. •Matched comparison of navigated and fluoroscopy-guided pedicle screw placement.•Accuracy was significantly higher with navigation compared to fluoroscopic guidance.•In both groups, none of the breaches resulted in postoperative morbidities.•Screw diameter to pedicle width ratio was significantly higher using navigation.•No significant differences found for operating time and patient radiation dose. HoweverIn recent years, there has been a growing interest in the use of modern technology based to improve the accuracy of pedicle screw placement in minimally invasive spine surgery, such as 3D imaging-based navigation and robotic assistance . The avaThe aim of this study was to investigate the implications of navigation in thoracolumbar pedicle screw placement not only on pedicle screw accuracy, but on the SD/PW ratio as well.2In this study, we prospectively collected data for all procedures of thoracolumbar pedicle screw placement using intraoperative 3D imaging guided navigation performed by a single senior spine surgeon in our institution between August 2021 and Mai 2022. The study was approved by the ethics committee responsible (application number 2021–16061). All patients gave informed consent, and the procedures were in compliance with the 1964 Helsinki Declaration and its subsequent amendments.The results were compared with a control group of fluoroscopy-guided procedures performed in our institution performed in our institution between 2018 and 2022. To allow for reasonable comparison, an extensive matching process was carried out: For each navigated procedure performed, the institutional database was filtered for similar procedures performed under fluoroscopic guidance. Criteria were the number of levels instrumented (with deviations by one level considered acceptable), the area of the spine  and additional procedures performed . If several matches were available, the type of approach (percutaneous/open), the number of screws placed, indication for surgery, patient age and BMI were used in the order given as secondary criteria to find the best match. All procedures included in the fluoroscopy group were performed by spine surgeons with a similar level of experience as the surgeon who performed the procedures included in the navigation group.In both groups, preoperative screw planning was performed using a DICOM viewer  by measuring pedicle diameter and possible screw length in the computed tomography (CT) performed beforehand. In the fluoroscopy group, all screws were placed according to the preoperative screw planning. In the navigation group, intraoperative adjustments of the screw diameter and length were possible at the discretion of the surgeon according to the visualization of the screw in the correct trajectory.For all procedures, patients were placed on a radiolucent carbon surgical table. In the navigation group, after preparation, draping and mounting of the patient array on a spinous process, a registration scan was performed using a latest generation mobile 3D C-arm cone beam computed tomography . During the 30 s scan acquisition, ventilation was paused resulting in higher quality of both imaging and navigation accuracy. The 3D datasets were then transferred to the Pulse platform  for navigated pedicle screw placement. The implant systems used for dorsal stabilization in the navigation group were Reline MAS for percutaneous and Reline Open for open procedures . The detailed screw placement technique with the navigation system used in this study has been published recently . An intrIn the fluoroscopy group, screw placement was performed under fluoroscopic guidance using a mobile 3D C-arm CBCT . Yamshidi needles were inserted into the pedicle under fluoroscopic control in both anteroposterior and lateral directions, then wires were passed through them, over which the screws were inserted. Systems used for dorsal stabilization in the fluoroscopy group were Reline MAS (33 cases), Reline Open (2 cases) or Precept . Exceptionally, in one of the cases included in the control group, the system Viper 2  was used. All systems used include cannulated polyaxial titanium screws, which were placed in the technique described above.Screw accuracy was assessed in postoperative CT reconstructed in the axial, sagittal and coronal planes at average slice thickness of 2.0 mm by an independent experienced investigator according to Gertzbein-Robbins grading system . Grades Using the electronic patient chart and the dose report, the operating times (in minutes), intraoperative fluoroscopy time (in seconds) and intraoperative Dose Area Product  were cot-test was used to compare both groups, while Wilcoxon matched-pairs signed rank test was used as nonparametric alternative. For contingency testing, Fisher's exact test was used.Statistical analysis was performed using Prism 9 . Kolmogorov–Smirnov test was used to check for normal distribution of data. Descriptive statistics are shown as means ± standard deviation for continuous variables and absolute and relative numbers for nominal and ordinally scaled variables. For normally distributed continuously scaled data, a paired U test. The significance level was set at p < 0.05.The mean screw pedicle ratio in both groups was compared using Mann-Whitney 3A total of 45 patients with 379 screws, of which 361 were placed using 3D navigation, were included in the study group. The fluoroscopy group likewise consisted of 45 patients, with a total of 365 screws placed. A detailed comparison of patient and procedure characteristics for the two groups is displayed in While the fluoroscopy group was significantly younger than the study group, there were no significant differences in gender distribution, body mass index (BMI) or the percentage of percutaneous and open procedures in the groups. There was also no significant difference in the indication for surgery, the number of fused segments, the number of screws placed, and the additional surgical interventions performed.The intraoperative dose area product tended to be lower (p = 0.68), and the fluoroscopy time was significantly lower in the navigation group .In the navigation group, in 16 of the 45 cases included, an intraoperative 3D scan was performed after screw placement to allow for control of implant position. In two cases, the 3D scan resulted in revision of a single screw at levels T3 and L5.The final screw accuracy as measured in postoperative computed tomography was 91.4% in the navigation group and 86.3% in the fluoroscopy group  and 3 grade D perforations (0.8%) were found. Except for 2 grade C perforations at levels (at levels T2 and T8), all screws with relevant perforations occurred on the lateral side of the pedicle. All but one screw with grade C or grade D perforations showed a so-called ‘in-out-in configuration’. Relevant pedicle breaches on levels T4 to T7 and T11 to L2 accounted for 80.6% of all grade C and D breaches.The assessment of screw accuracy in the fluoroscopy group found 50 screws with pedicle breaches corresponding to grade C to grade E. Of these screws, four screws showed a medial breach  and 46 screws showed a lateral perforation (40 grade C and 6 grade D). Of the 50 screws, 42 showed an ‘in-out-in configuration’ while 8 screws showed a parapedicular and paravertebral screw course. In the case with the exceptional use of a different implant system, the four screws placed were assessed as Grade A.None of the pedicle breaches reported in the present study resulted in neurological deficits, accordingly, no revision surgery due to screw misplacement was performed.In the navigation group, postoperative complications were seen in five patients (11.1%), of which four were cases with open surgery: Two cases with non-infected wound-healing disorders (4.4%), two cases with pull-out of a single screw each on levels T11 and L2 in osteoporotic patients despite primary cement augmentation (4.4%) and one case with postoperative hematoma (2.2%). In all five cases, revision surgery was performed. In the pullout-cases, extended re-instrumentation was performed.In the fluoroscopy group, postoperative complications were seen in two patients (4.4%), of which one had had open surgery. Both patients showed postoperative hematoma for which revision surgery was performed. There was no significant difference regarding the occurrence of postoperative complications between the groups (p = 0.434).In the navigation group and the fluoroscopy group, all complications occurred within six weeks after the initial surgical procedure. Except for implant removal, after a mean follow-up time of 16 ± 3 months in the navigation group and 27 ± 9 months in the fluoroscopy group, there were no additional re-admissions for further complications.2) between the mean SD/PW ratio and the rate of relevant perforations  navigation . Even moNumerous studies have investigated different factors that influence the biomechanical properties of screws used for spinal fixation. The size, design, entry point and the trajectory of the screw as well as bone mineral density (BDM) have been named relevant factors . When deThe increase in screw diameter has been identified as the most important factor in achieving higher pullout strength  The use In their meta-analysis, Solitro et al. listed the recommendations of various studies regarding the suggested screw diameters, showing considerable differences. While some authors advocated the so-called “pedicle fit and fill” theory , others Due to the lack of proper guidelines or standards, many spine surgeons choose the appropriate screw size based on their experience or based on patient size, which may in some cases lead to the selection of screws that are significantly too small or too large . While tStill, in retrospect, the limits may have been pushed with some of the screws placed in the navigation group, especially in the thoracic spine. In a trial by Korkmaz et al. it was shown that the pullout strength without pedicle perforation was 70% higher than with a high-grade lateral pedicle perforation. Correction of an initially laterally perforating screw with subsequent complete intrapedicular placement was shown to be biomechanically significantly advantageous, as a large portion of the lost pullout strength is regained . Ye et aMatsukawa et al. demonstrated in their study that screw diameter had a greater effect on resistance to screw pullout and flexion-extension loading, while greater screw length, commonly measured as %depth of the vertebral body, proved to be advantageous in lateral bending and axial rotation loading . Liu et Overall, in screw planning, spine surgeons have to deal with the trade-off between avoiding relevant perforations while maximizing screw diameter and length as well as the amount of cortical layer grip. Unfortunately, the literature suggests that even the use of preoperative computed tomography to determine screw diameters may also lead to inadequate screw selection . When coOur results show that even in a small sample, navigated pedicle screw placement has advantages in terms of a significantly higher SD/PW ratio with the corresponding biomechanical implications for dorsal stabilization, while ensuring the high accuracy of screw placement especially in minimally invasive screw placement. Recent studies suggest that the use of robotic-assisted techniques could potentially optimize these results even further .The results of the present study should be interpreted within the confines of study limitations. Due to the study being a review of single-surgeon, single-center data with a limited number of cases and little clinical follow-up data, the generalizability of the results is limited. However, an extensive matching process was performed, and the results of the navigation group were compared to similar fluoroscopy-guided procedures performed in our institution. Furthermore, the data available in the literature support the results presented above. Future studies should investigate whether the increase in SD/PW ratio enabled by navigation and/or robotic assistance contributes to a better clinical outcome.5In conclusion, this study was able to show that navigated thoracolumbar pedicle screw placement not only increases the accuracy of screw placement but also facilitates the selection of the adequate screw sizes, which according to the literature has positive effects on fixation strength. Meanwhile, the use of navigation did not negatively affect the time needed for surgery or the patient's intraoperative exposure to radiation.Conception and design of the study: PAG, JF, SYV. Data Acquisition: EM, JG, FZ. Analysis and Interpretation of Data: EM, SYV. Writing of the manuscript: EM, JG. Revision of the manuscript: FZ, PAG, JF, SYV. All authors have read and approved the final version of the manuscript.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The study was reviewed and approved by the responsible Ethics Committee (application number 2021–16061). All patients provided verbal and written consent. All procedures were performed in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.All data and statistics are available on request from the corresponding author.PAG reports a relationship with Siemens Healthineers that includes: consulting or advisory and travel reimbursement. JF reports a relationship with Siemens Healthineers that includes: consulting or advisory and travel reimbursement.The research group received grants/has grants pending and technical support from Siemens Healthineers  and Nuvasive Inc. . The funders had no involvement in the study design, collection, analysis, and interpretation of data, writing of the manuscript, or decision to submit the manuscript for publication.