Mannitol (1% w/v) hindered NC enlargement on lyophilization. FT-IR negated the chemical interaction of NC components. DSC and XRD were performed to verify the crystalline state. DSN dissolution enhancement was attributed to the nanometric rod-shaped NCs, the high surface area, and the improved wettability. CS insolubility and its diffusion layer may explain controlled DSN release from CS-PLX188 NCs. CS-PLX188 NCs were more stable than PLX188 NCs, suggesting the significance of the combined electrostatic and steric stabilization strategies. The superiority of CS-PLX188 NCs was indicated by the significantly regulated biomarkers, pathological alterations, and inducible nitric oxide synthase (iNOS) expression of the hepatic tissue compared to DSN suspension and PLX188 NCs. Permeation, mucoadhesion, and cellular uptake enhancement by CS may explain this superiority.Diosmin (DSN) exhibits poor water solubility and low bioavailability. Although nanocrystals (NCs) are successful for improving drug solubility, they may undergo crystal growth. Therefore, DSN NCs were prepared, employing sonoprecipitation utilizing different stabilizers. The optimum stabilizer was combined with chitosan (CS) as an electrostatic stabilizer. NCs based on 0.15%  Approximately 70–90% of active pharmaceutical compounds encounter poor water solubility, which diminishes their bioavailability . NanocrySteric stabilizers prevents crystal growth by adsorbing onto the particle surface and acts as a barrier that hinders particle aggregation. Thermodynamic driving forces for adsorption on hydrophobic drug surfaces are absent in the case of hydrophilic polymers such as hydroxypropyl methylcellulose (HPMC) and methylcellulose, but present in amphiphilic polymers such as poly vinyl pyrrolidone (PVP) and poloxamers (PLXs) . TherefoElectrostatic stabilization is based on the formation of repulsive Coulomb forces between the charged colloidal particles. They include anionic surfactants such as sodium dodecyl sulfate (SDS) and charged polymers such as chitosan (CS) . CS is aDiosmin (DSN)  is a natural flavonoid glycoside derived from hesperidin by dehydrogenation . It is uThe low solubilizing capacity of most lipids and surfactants towards DSN reflects its rigid nature, being insoluble in water and most organic solvents . To enhaTherefore, the aim of this study was to improve DSN dissolution and delivery after oral administration. To fulfill this target, DSN NCs were developed, combining steric and electrostatic stabilization strategies aiming to attain enhanced NC stabilization and therapeutic efficacy. In vitro evaluation of the selected NCs was attempted. Furthermore, the effects of the selected DSN NCs on the therapeutic efficacy against ferrous sulfate-induced liver damage in rats were investigated through serum and tissue biomarker assessments, histopathological examinations, and the immunohistochemical localization of inducible nitric synthase (iNOS). 2O2), and ferrous sulfate were purchased from Adwic, EL Nasr Pharmaceutical Chemicals Co., El Khanka, Egypt. Serum alanine amino transferase (AST), aspartate aminotransferase (ALT), albumin (ALB) and total bilirubin (TB) kits were purchased from Diamond Diagnostics, Holliston MA, USA. The serum gamma glutamyl transferase (GGT) kit was merchandized from Analyticon, Lichtenfels Germany. The serum lactate dehydrogenase (LDH) kit was procured from Biosystems, Barcelona, Spain. The serum total cholesterol (TC) kit was acquired from Spinreact, Girona, Spain. The serum alkaline phosphatase (ALP) kit was obtained from Biodiagnostic, Giza, Egypt. 3,3′-diaminobenzidine tetrahydrochloride  was obtained from Vector Laboratories Inc., Burlingame, CA, USA. The tissue-inducible nitric oxide synthase (iNOS) primary antibodies were procured from Proteintech, Rosemont, IL, USA. All other chemicals were of fine analytical grades.Diosmin (DSN) was a kind gift from Sigma Pharmaceuticals Industries, Quesna, Egypt. Poloxamer 188 (PLX188), reduced glutathione (GSH), hepatic malondialdehyde (MDA) kits were purchased from Sigma-Aldrich Co., St Louis, MO, USA. Poly vinyl pyrrolidone K25 (PVP-K25) and sodium dodecyl sulfate (SDS) were supplied by Sedico Pharmaceutical Company, Cairo, Egypt. Low-molecular-weight chitosan  was kindly donated by Primex, Siglufjörður, Iceland. Dimethyl sulfoxide, glacial acetic acid (99%), sodium hydroxide, sodium carboxymethyl cellulose, formalin, hydrogen peroxide (Hw/v (w/v was obtained by probe sonication at an 80% amplitude for 5 min (data not shown). Accordingly, NC dispersions were then sonicated using a probe sonicator  attached to a homogenizer  at 80% amplitude for 5 min and the temperature was kept constant using an ice bath [DSN nanocrystals (NCs) were prepared through procedures that combined the bottom-up technique of antisolvent precipitation and top-down method of probe sonication ,44. A scw/v . The resv/v acetic acid to obtain three solutions of different concentrations . Five milliliters of each CS solution was diluted with double-deionized water containing the selected stabilizer at its optimum concentration to a final volume of 25 mL as an aqueous phase to obtain different concentrations of CS . The produced dispersions were kept on a magnetic stirrer  for 1 h.CS-based NCs of DSN were prepared by employing the above-mentioned procedure with a modification. Firstly, CS was dissolved in 0.1% The average diameters of the prepared NCs were determined by dynamic light scattering technique . An aliquot of the prepared samples was taken, properly diluted (1:100) with double-deionized water, and analyzed for particle size, polydispersity index (PDI) and ζ-potential. All measurements were performed in triplicate and the results are expressed as the average value ± standard deviation (SD).The morphology of the selected NCs with and without CS was examined using a transmission electron microscope  operating at an accelerating voltage of 160 kV. Each sample was properly diluted (1:10) and sonicated for 2 min. One drop was added on a Formvar-coated copper grid . The excess material was removed with a filter paper. After complete drying at room temperature, the image was captured.The surface properties of DSN powder and the lyophilized selected NCs, either with or without CS, were examined using scanning electron microscopy (SEM) . Samples were mounted on a metal stub using double-sided adhesive carbon tapes. They were coated with a gold layer and monitored under SEM at 30 kV.−1.FT-IR spectra of DSN powder, the selected stabilizer, CS, the physical mixtures (PMs) of DSN and the selected stabilizer with or without CS, as well as the lyophilized NCs based on the stabilizer alone or combined with CS, were recorded using an FT-IR spectrophotometer . The samples were homogenized with KBr to then be compressed into discs and scanned at the range of 4000–400 cmThe thermal behavior of the above-mentioned samples was assessed employing differential scanning calorimetry (DSC) . Five milligrams of each sample was placed into an aluminum crucible and sealed using an aluminum lid by a sealing machine. Then, they were heated from 35 °C to 350 °C at a rate of 10 °C/min under constant dry nitrogen purging at 20 mL/min. Indium with a purity of 99.99% and a melting point of 156.6 °C was used to calibrate the temperature. X-ray diffractometry (XRD) patterns of the above-mentioned samples were obtained  The diffraction patterns were recorded in a step-scan model at a voltage of 45 kV and a current of 9 mA in a range of 3° to 55° (2θ) with a step size of 0.020°.Lyophilization is recommended to attain long-term stability and easy handling. Due to the high energy, NCs can easily be subjected to crystal growth; thus, water removal is required to protect against this growth . After lw/v) was added as a cryoprotectant to the examined PLX188 NC dispersion either with or without CS before freezing at −80 °C [−2 mbar pressure . The lyophilized powders were kept in a desiccator for characterization. Particle size analysis of the lyophilized powders after reconstitution with double-deionized water was carried out, and the results were compared to those of the freshly prepared nanosuspensions as well as those of the lyophilized powders of the selected nanosuspensions without the addition of mannitol.Mannitol has been presented as a promising cryoprotectant because it crystallizes around the nanoparticles, creating a glassy/vitreous coat that protects them from the mechanical stress of ice crystals, thereby preventing aggregation . Moreovet −80 °C ,45,49. LFor drug content determination, the accurately weighed (0.01 g) lyophilized powder was well mixed with 10 mL 0.2 N sodium hydroxide and filtered through a 45 µm Millipore filter to remove any undissolved material such as CS. The filtrate was properly diluted and analyzed spectrophotometrically  at 268 nm against a blank of the corresponding lyophilized plain NCs that was treated the same to eliminate interference by components other than the drug, if any. The experiment was performed in triplicate and the average drug content (%) and SD were calculated.a = 9.39–10.12); hence, it exhibits poor solubility in an acidic medium (pH 1.2) and a neutral (pH 7.4) buffering system [2h), mean dissolution time (MDT), dissolution efficiency (DE) and similarity factor (f2) were calculated based on the literature [Dissolution studies were carried out using USP apparatus II (paddle method) . It has been reported that DSN exhibits pH-dependent solubility  value.To describe the release mechanism of DSN from the selected CS-based NCs, in vitro release results were analyzed according to different kinetic models, including zero-order, first-order  and Higust log t . The kinStability studies were performed using the lyophilized DSN NCs based on the selected stabilizer either alone or in combination with CS during a storage period of three months at room temperature (25 ± 2 °C). The samples were stored in screw-capped amber glass containers to be reconstituted in double-deionized water and then examined initially and monthly regarding particle size, PDI, ζ-potential, and percentage drug content, as previously discussed.All animal handling and procedures were performed in compliance with the U.S. National Institute of Health Guide for the Care and Use of Laboratory Animals . The protocol was approved by the Ethical Committee of Faculty of Pharmacy, Mansoura University, Egypt . Animals were kept under regular 12 h light/12 h dark cycles at a temperature of 25 ± 1 °C and a relative humidity of 55 ± 5%. Free access to a standard laboratory food and water was allowed.Sixty male Wistar albino rats weighing 200–220 g were divided into ten groups (six rats per each group). Intragastric gavage was utilized to enable oral administration. In addition to the pretreatment regimen, after treatment, one rat was followed to assess the possibility of potentiated uptake of the examined NCs, particularly the positively charged ones incorporating CS by overexpressed Kupffer cells in the inflamed hepatic tissue . Therefow/v was used as a suspending agent of DSN suspension to allow a uniform dose following shaking. Therefore, rats of normal and positive control groups received 1% w/v Na CMC orally for 10 consecutive days to examine its effects on the liver, if any. Oral pretreatment with 50 mg/kg DSN suspended in 1% w/v Na CMC or an equivalent amount of DSN NCs based on either the selected stabilizer alone or combined with CS was accomplished for 10 consecutive days. Rats of all groups, except normal controls, were intraperitoneally (i.p) injected with ferrous sulfate at a dose of 30 mg/kg [Sodium carboxymethylcellulose (Na CMC) at a concentration of 1% 30 mg/kg  on the 9w/v Na CMC orally for 10 consecutive days. All groups except the normal control were injected i.p with ferrous sulfate at a dose of 30 mg/kg [w/v Na CMC orally at a dose of 50 mg/kg for 10 consecutive days, starting from the 2nd day after the induction dose. An equivalent amount of DSN NCs based on the chosen stabilizer either alone or together with CS was orally given to the fourth and fifth groups, respectively, starting from the 2nd day after the induction dose.Similarly, rats of normal and positive control groups received 1% 30 mg/kg  on the 1Blood and tissue samples were collected according to a method described in the literature . On the Blood samples were allowed to coagulate at room temperature to then be placed in a water bath maintained at 37 ± 0.5 °C for 10 min and centrifuged at 10,000 rpm for 20 min to separate the clear sera . The serx). GSH content in the liver homogenates was determined according to a previously adopted method [X production was evaluated in the liver homogenates [One gram of the liver median lobe was homogenized with 5 volumes of isotonic ice-cooled normal saline for preparation of the 20% liver homogenate. The resultant homogenates were employed for the determination of oxidative stress biomarkers, including hepatic malondialdehyde (MDA) and reduced glutathione (GSH), as well as inflammatory biomarkers and nitrate/nitrite production (NOd method . The hepd method . Additioogenates .v/v) buffered formalin solution for 24 h. The fixed samples were washed, dehydrated by ascending grades of ethyl alcohol, cleared in xylene, and embedded in a melted paraffin wax in a hot air oven (56 °C). Paraffin blocks were divided into two sets of sections. Each was made 5 μm thick using a microtome and picked up on a clean glass slide. At the time of staining, the first set of the paraffin sections was deparaffinized, rehydrated in descending grades of ethyl alcohol and routinely stained with hematoxylin and eosin (H&E), in accordance with a reported method [Hepatic tissue samples were fixed in 10%  for 15 min at room temperature. This was followed by boiling with 0.01 M sodium citrate buffer (pH 6) for 20 min in a microwave oven to promote immunoreactivity and counteract the antigenicity loss that might happen due to some epitopes of formalin-fixed paraffin-embedded tissues. The slides were allowed to cool and washed four times with phosphate-buffered saline (pH 7.2). Primary antibodies (dilution 1:100) were added and the tissue sections were incubated at room temperature for 60 min in a humidity chamber, which was a covered Petri dish and a filter paper moistened with water and fixed on the dish bottom to prevent the specimens drying. After that, incubation with the biotinylated secondary antibodies was allowed for 10 min and the enzyme conjugate was added to the specimens to then be incubated for 10 min. Finally, the sections were rinsed with Tris-buffered saline and the immunoreaction was visualized using 3,3′-diaminobenzidine tetrahydrochloride (DAB). Sections were washed for 10 min under running tap water, and then counterstained with Mayer’s hematoxylin. Staining intensity of the positively stained cells was evaluated using a digital camera  mounted on a light microscope . The intensity of the immunohistochemical staining as an indication of the extent of iNOS activity was scored as follows: 0, negative; 1, weak; 2, moderate; and 3, strong [The second paraffin section set was used to immunohistochemically evaluate iNOS activity in the hepatic tissue. Paraffin sections were deparaffinized using xylene for 30 min and then rehydrated in descending grades of ethanol. The blockage of endogenous peroxidase activity was attained by incubation in 3% hydrogen peroxide . The results were statistically analyzed at the significance level of w/v to 0.15% w/v resulted in smaller nanoparticles. A greater increase in PVP K25 concentration beyond 0.15% w/v produced significantly larger nanoparticles. The increase in PLX188 concentration to 0.3% w/v insignificantly affected the particles size, although a higher content (0.6% w/v) of this stabilizer significantly enlarged the particles. Regarding SDS, a further size reduction was recorded on the increase in SDS concentration to 0.3% w/v; however, significant particle enlargement was noticed beyond such a concentration (0.6% w/v). In other words, the optimum stabilizer concentration that produced the smallest particle size was 0.15% w/v for both PVP K25 and PLX188, and 0.3% w/v for SDS. These results can be explained on the basis that at a low concentration, the stabilizer would be insufficient to completely cover the NC surface and decrease the surface free energy; hence, aggregation could be expected. At the optimum stabilizer concentration, the adsorbed sterically stabilizing layer would have the sufficient thickness and provide a stable NC dispersion [The results of particle size and PDI analyses are listed in spersion ,63. Beyospersion ,65. Morespersion .w/v was selected as a steric stabilizer of DSN NCs. Hence, the ζ-potential of PLX188 NC dispersions was measured . These results can be explained on the basis that the ζ-potential decreased with the increase in the stabilizer layer thickness due to the outward shift of the slipping plane at which the zeta potential was measured; thus, the charge density was much smaller than that on the surface, resulting in lower recorded ζ-potential values [w/v of this stabilizer may counteract NC aggregation and enlargement, and hence account for the smallest size recorded for these NCs among the examined dispersions.Significantly smaller NCs were obtained with PLX188 than that recorded when PVP K25 or SDS were used at the same concentration. Among the studied stabilizers, the smallest particle diameter (350.20 ± 1.79 nm) was produced when PLX188 was used. Accordingly, PLX188 at a concentration of 0.15% measured . The resl values ,69. The w/v resulted in significant NC enlargement when compared to those prepared using PLX188 (0.15% w/v) alone. These results can be attributed to the partial coverage of DSN NCs by CS. However, the increase in CS concentration to 0.04% w/v significantly lowered the particle size again, possibly due to the complete coverage of the particles surface by CS and the higher electrostatic stability, as indicated by the significantly greater ζ-potential values compared to the 0.02% w/v CS-based NC dispersion that could strongly counteract NC aggregation, and hence, the particle enlargement. Accordingly, a rise in CS content to 0.06% w/v insignificantly affected ζ-potential, possibly due to the complete coverage of the particle surface that was previously accomplished when this polymer was used at 0.04% w/v. However, such increments in the CS concentration imparted significantly larger NCs. The subsequent size increase can be explained by multilayer CS accumulation on the particle surface [Based on the above-mentioned results, PLX188 was combined with CS as an electrostatic stabilizer aiming to provide more stable DSN NCs as well as an enhancement of DSN delivery due to the expected permeation, mucoadhesion and cellular uptake potentiation capabilities of CS. CS at a concentration of 0.02%  surface . Successw/v PLX188 combined with 0.04% CS, because CS incorporation did not significantly affect NCs size, although increased their ζ-potential when compared to PLX188 NCs. The resultant positive ζ-potential might augment the interaction with the negatively charged cellular membranes; hence, their intracellular uptake [In light of the results of particle size and ζ-potential analysis, the selected DSN NCs dispersion to be further investigated was that based on 0.15% r uptake . Moreover uptake . These rr uptake . The morphology of the selected PLX188 and CS-PLX188 NCs examined using TEM is illustrated in TEM images revealed NCs with diameters smaller than that estimated by DLS. It has been reported that DLS measures the hydrodynamic diameter of the particles including the polymer(s) and the hydration layer; hence, larger particle diameters were recorded . AdditioThe surface morphology of DSN powder and its selected NCs, employing PLX188 either alone or combined with CS, is presented in −1 due to more than one O–H aromatic bond stretching as well as an absorption band at 1661 cm−1, corresponding to aromatic ketonic carbonyl stretching (C=O vibration) [−1 [−1, assigned to –OH and –CH3, respectively [−1 due to hydroxyl and amine group stretching vibrations. It also exhibited an absorption peak at 2877 cm−1 which may be attributed to C–H stretching. The peaks at 1656, 1030 and 1077 cm−1 corresponded to –C=O of the residual acetyl group, C–O and C–H groups, respectively. The band at 895 cm−1 was due to the β- glucoside bond in CS [FT-IR spectroscopy was applied to assess the possibility of the interaction between DSN, PLX188, and CS. bration) ,41. The ion) [−1 . PLX188 ectively . CS exhind in CS . The chaDSC thermograms of the previously mentioned samples under FT-IR are presented in w/v) were measured after reconstitution with double-deionized water and are listed in w/v mannitol. On the other hand, those lyophilized without the addition of mannitol encountered a significant increase in the particle size and PDI when compared to the corresponding dispersions, either before or after lyophilization, employing mannitol as a cryoprotectant. These results confirmed that the addition of 1% w/v mannitol counteracted the particle aggregation of both types of NC dispersions on lyophilization. The size and PDI of the lyophilized powders of the selected DSN–PLX188 and DSN–PLX188–CS NCs with and without the addition of mannitol , and the results are graphed up to 60 min A and 8 ho PLX188 . GeneralN powder . The disN powder ,77. The N powder . 2 = 0.9973) was obtained in the case of the Higuchi model versus 0.9498 and 0.9877 for zero and first orders, respectively. Thus, in vitro release of DSN from its PLX188–CS NCs can be explained by the Higuchi model, suggesting a diffusion-controlled drug release. In accordance, the Fickian mechanism  was found to describe the drug release from these NCs, verifying the diffusion-controlled drug release [Regarding DSN–PLX188–CS NCs, the highest coefficient of determination  markers when compared to normal rats. Significantly reduced ALB serum levels and GSH tissue contents relative to normal rats were observed in the positive control rats. DSN was able to significantly diminish the levels of these markers (except ALB and GSH) relative to the positive control in both models, possibly due to its antioxidant and anti-inflammatory capabilities that maintain membrane permeability . MoreoveConcerning PLX188 NCs of DSN, the serum and the tissue levels of the studied biomarkers (except ALB and GSH) were significantly lower than those recorded for the positive control and for most of the biomarkers relative to the drug suspension. The higher efficacy of DSN–PLX188 NCs over the drug against the liver damage can be explained by the increased drug solubility due to the nanometric rod-shaped NCs and the improved wettability, as well as P-glycoprotein inhibition and membrane interaction capabilities of PLX188 which could, together, enhance DSN bioavailability [x expression in the hepatic tissue when compared to PLX188 NCs. In addition to the augmented drug solubility, CS mucoadhesive and permeability-enhancing properties, as well as the expected electrostatic interaction with the negatively charged cellular membranes, may account for the superiority of its NCs over that based on PLX188 alone and the drug suspension [The DSN–PLX188–CS-NC-administered group encountered significantly regulated levels of all or most of the studied biomarkers in both regimens when compared to those determined for the positive control as well as the rats that received the DSN suspension or PLX188 NCs. Moreover, there was an insignificant difference between the rats that were administered DSN–PLX188–CS NCs and normal controls regarding all biomarker levels. These results reflect the superiority of DSN–PLX188–CS NCs over the drug suspension and its PLX188-based NCs. The potentiation of DSN efficacy against liver injury by its formulation into PLX188–CS NCs was evident in the post-treatment model, as indicated by the significant reduction in NOspension . Additiospension . Moreovespension .Regarding DSN–PLX188–CS-NC-pretreated and post-treated groups  and narrow size distribution (PDI = 0.23 ± 0.01) on lyophilization on the addition of mannitol (1% w/v). The stabilizers were physically adsorbed on DSN NC surfaces without a chemical interaction or drug amorphization, as revealed by the solid-state characterization. The nanometric rod-shaped NCs, the increased surface area, and the improved drug wettability can together account for the enhanced DSN dissolution from the selected NCs. DSN–PLX188–CS NCs were more stable than those based on PLX188 alone up to the third month at room temperature, suggesting the significance of the combined electrostatic and steric stabilization strategies. Assessment of therapeutic efficacy of the selected DSN–PLX188–CS NCs was performed; the results revealed their superiority, particularly in the post-treatment model, as indicated by the significantly regulated serum and tissue levels of most of the examined biomarkers as well as the significantly reduced pathological alterations and iNOS expression in the hepatic tissue in comparison with the positive control, DSN suspension and DSN–PLX188 NCs. The prevalence of DSN–PLX188–CS NCs was verified by the insignificant differences from normal rats regarding the above-mentioned parameters. Therefore, DSN–PLX188–CS NCs can be defined as a promising drug delivery system for improving DSN dissolution and therapeutic efficacy against ferrous-sulfate-induced hepatic injury in rats. The optimized NCs were based on a combination of 0.15%


Valerianella locusta “Vit” salad. Primary metabolites, assimilatory pigments as well as secondary metabolites were monitored during 1 week of refrigerated storage. Home-grown lamb's lettuce exhibited highest levels of total sugars, total organic acids, vitamin C, and total phenolic content as well as enhanced levels of most individual phenolic compounds and chloroplast pigments. Locally produced samples of lamb's lettuce also contained high levels of analyzed bioactive components. All samples retained most bioactive components during the entire period of refrigerated storage. The results underline the instability of vitamin C during refrigerated storage of lamb's lettuce and pinpoint this parameter as being the most affected by storage.Lamb's lettuce is a popular winter salad, often grown in private vegetable plots, small local farms or in intensive vegetable production. It is usually marketed as a ready-to-eat produce in supermarkets. The aim of the study was to evaluate the changes in biochemical composition and degradation of bioactive compounds during consumer-relevant time of home-grown and store-bought  Valerianella locusta L. Betcke or lamb's lettuce is a common minimally processed ready-to-eat vegetable. It can be used either as a leafy salad, as an ingredient in salad mixes or in more complex culinary dishes  “Vit” was chosen as a model plant as it is rich in phenolic compounds, highly perishable and readily available year-round. Moreover, a steady increase of consumer demand for this pre-packed produce can be registered in Europe  were purchased at three different supermarkets in Ljubljana, Slovenia, at the first day of their commercial life (T0), 24 h after packaging on March 26th 2018 . All supermarket-bought samples had the same expiry date, were field-grown in an organic production and one of them (SM1) was advertised as a locally grown product. On the same day (March 26th 2018), organic home-grown (HG) lamb's lettuce was collected at a private garden in Lendava, Slovenia  and transferred immediately to the laboratory facility at Biotechnical faculty in Ljubljana. The cultivation practice for home-grown lamb's lettuce followed the same technological practices applied in large-scale organic vegetable production. All samples were a dark green short leaf “Vit” commercial variety preferred by European consumers and often cultivated on a garden scale. Each treatment was combined into a single batch containing 0.500 kg lamb's lettuce. Prior to analyses, the rosettes were washed in distilled water and gently tapped dry with tissue paper. A portion of each treatment/batch was subjected to immediate analyses on day 1 (T0). To mimic common domestic use, the rest of the batch was stored in plastic bags (4 L) with a zipper, deposited in a refrigerator at 4°C and reopened and analyzed after 2 days (T2), 4 days (T4), and 7 days (T7). On each sampling date, five individual portions (each containing seven rosettes) were ground with liquid nitrogen and all chemical analyses were performed on this source material (n = 5).Several bags  of ready-to-eat lamb's lettuce (n = 5) per treatment and sampling; each repetition consisted of several rosettes. Primary metabolites were analyzed on a high-performance liquid chromatography (HPLC) system  with injection volume of 20 μl and flow rate maintained at 0.6 ml min−1. Sugar separation was carried out on a Rezex RCM-monosaccharide column from Phenomenex (Ca+ 2%) at 65°C (300 × 7.8 mm). Double distilled water was used as a mobile phase, total run time was 30 min, and a refractive index (RI) detector was used for carbohydrate detection as described by Weber et al. (−1. A Rezex ROA-organic acid [H+ (8%)] column from Phenomenex (300 × 7.8 mm) was used as described by Mikulic-Petkovsek et al. (Rosettes (1.5 g) were homogenized in 2 ml of double distilled water with an Ultra-Turrax T-25 (Ika-Labortechnik). Extraction was performed at room temperature with constant stirring for 30 min . Samplesr et al. . Analysek et al.  and Webek et al. . The elu−1 fresh weight (FW). Total sugar content, total organic acids content and sugar/organic acid ratio were calculated as sums (or ratios) of all detected sugars or organic acids.Corresponding external standard curves were used for quantification of primary metabolites and all compounds were expressed in mg gm-phosphoric acid and for the analysis of DHA with 2 ml of ice-cold 6% m-phosphoric acid containing 4 mM EDTA. Extraction was performed with homogenisation on a shaker for 30 s.Vitamin C was determined by separately detecting the amount of L-ascorbic acid (AA) and L-dehydroascorbic acid (DHA) according to the method described by Helland et al. , with soA similar HPLC method as for other organic acids was utilized for the quantification of ascorbic. However, it was operated at room temperature and at a different wavelength (245 nm). The identification and quantification were achieved with comparison of the retention time and corresponding external standard curves.DHA was calculated by subtracting the ascorbic acid content in reduced extract from that of the non-reduced extract. Vitamin C content was defined as the sum of AA and DHA contents.n = 5) per treatment and sampling date; each repetition included several rosettes. Rosettes (1.5 g) were chopped and extracted with 2 ml methanol containing 3% (v/v) formic acid in an iced ultrasonic bath for 1 h as described by Mikulic-Petkovsek et al.  column operated at 25°C was used. The elution solvents were (A) 3% acetonitrile with 0.1% formic acid in double distilled water (v/v/v) and (B) 3% double distilled water with 0.1% formic acid in acetonitrile (v/v/v). Samples were eluted according to the linear gradient described by Weber et al.  coupled with mass spectrometer with a diode array detector at 280 nm (derivatives of hydroxycinnamic acids) or 350 nm  were used for identification of individual phenolics as described by Weber et al. . Spectrar et al. . The injn scanning from m/z 115 to 1,500. Operating conditions were previously reported by Weber et al. . For compounds lacking standards, quantitation was carried out using comparable compounds. Therefore, luteolin-pentosylhexoside, kaempferol-3-O-rutinoside, genistin, hesperidin and diosmetin were quantitated in equivalents of luteolin-4'-O-glucoside.Phenolics were identified on a mass spectrometer with electrospray ionization (ESI) operating in negative ion mode using full scan data-dependent MStion SM, . QuantifTotal phenolic content (TPC) was determined using Folin-Ciocalteau (FC) reagent (five replicates per sampling date) as described by Mikulic-Petkovsek et al. . Gallic The extraction of chloroplast pigments from lamb's lettuce (1.5 g) was performed with 2 ml ice-cold acetone under dimmed light as described by Sircelj and Batic . The ext18 column at 25°C and flow rate was maintained at 1 ml min−1. The gradient was as follows: from 10 to 70% B in the first 18 min, then linearly to 70% B to 22 min and returning to the initial conditions to the end of the run. Mobile phase A was acetonitrile, double distilled water and methanol  and mobile phase B was acetone with ethyl acetate .Analysis of individual carotenoids was carried out on a HPLC system with a DAD detector at 450 nm. Separation of samples was achieved on a Gemini Cm/z 70 to 650 and argon was used as collision gas. Data acquisition was performed using Xcalibur 2.2 software. Identification was achieved with mass spectra scans, fragmentation, retention times and spectral properties of target compounds .The following standards were used for determination of sugars and organic acids: sucrose, fructose and glucose, as well as citric, malic, oxalic, tartaric and fumaric, ascorbic acid from Fluka Chemie ; shikimic and quinic acid from Sigma-Aldrich Chemicals . Standards for phenolic compounds were acquired from Sigma-Aldrich Chemicals  and neoxanthin, violaxanthin, antheraxanthin and β-cryptoxanthin from DHI LAB Product . Methanol for the extraction of phenolics was obtained at Sigma-Aldrich Chemicals. The chemicals for the mobile phase were HPLC-MS grade acetonitrile, sulphuric acid and formic acid from Sigma-Aldrich Chemicals. Water for the mobile phase was double distilled and purified with a Milli-Q system .P-values of <0.05 were considered statistically significant.Data were statistically analyzed with the program Statgraphics Centurion XVII  using one-way analysis of variance (ANOVA). Differences between samples, separately for each sampling date, were estimated using the Duncan multiple range test. V. locusta leaves during 1-week refrigerated storage were studied for the first time. In all samples analyzed in our study glucose was present in highest amounts, followed by fructose and significantly lower levels of sucrose  compared to SM1 sample, which was second richest in this group of primary metabolites. Significantly lower levels of TS were recorded in SM2 and SM3 samples and both supermarket-bought samples also demonstrated a comparable sugar turnover during refrigerated storage . Althougt et al.  measuredt et al. . Decreast et al. . Similart et al.  and turnt et al.  and the t et al.  who moniV. locusta leaves albeit several were only present in traces. All samples analyzed in the present study contained highest amounts of citric and malic acids and lower levels of fumaric, oxalic, quinic, shikimic and tartaric acids (V. locusta samples followed by a decrease on day 7 (V. locusta rosettes) corresponds to the report of Tsuchida et al.  in vegetables during postharvest handling procedures and storage has been reported in other papers, for instance on spinach  of fresh samples was determined in SM1 sample, followed by home-grown V. locusta rosettes . The values were in range with previously reported data on lamb's lettuce , two flavones , an isoflavone (genistin) and a flavanone (hesperidin). Hydroxycinnamic acids (particularly 5CQA) represented the most abundant phenolic class in V. locusta, regardless of sample origin or duration of refrigerated storage , two flavonols .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.


Debridement, antibiotics, and implant retention (DAIR) is the recommended treatment for acute hematogenous periprosthetic joint infection (PJI) after total knee arthroplasty (TKA). However, DAIR is associated with a high percentage of unsuccessful outcomes. Since 2007, direct intra-articular antibiotic infusion, which can provide a high concentration of intra-articular antibiotic, has been used in combination with DAIR to improve treatment outcomes among patients in our institution. This study aimed to assess the outcomes of DAIR combined with direct intra-articular antibiotic infusion in patients who presented with acute hematogenous PJI after TKA.We reviewed the data of all patients diagnosed with acute hematogenous PJI after primary TKA (from 2008 to 2015) who received DAIR combined with direct intra-articular antibiotic infusion.n = 3 knees) had a well-functioning, non-infected prosthesis 6 and 10 years after the procedure. Two patients (n = 2 knees) had re-infection 2 and 5 years after surgery, and they required two-stage revision. None of the patients were lost to follow-up. Finally, 13 (86.6%) of 15 infected knees were successfully treated with this method.In total, 15 knees in 12 patients were semi-urgently treated with this method. The mean follow-up time was 93.3 (minimum: 56) months, and the longest follow-up time was 11 years. Two patients (DAIR combined with direct intra-articular antibiotic infusion is an effective treatment for acute hematogenous PJI after TKA. Prosthetic joint infection (PJI) is a serious complication that occurs after primary total knee arthroplasty (TKA), with incidence rates ranging from 0.5 to 2%. Moreover, it is a major cause of revision surgery after TKA, with an incidence rate of 25% in all cases , 2. AccoStaphylococcus aureus infection, was 94%. Hence, a high intra-articular antibiotic concentration is important for the treatment of PJI. Moreover, the intra-articular antibiotic concentration was 10,000 times greater than the minimal inhibitory concentration and 750 times higher than that of the intravenous route, with an intra-articular half-life of 3.22 h. The trough levels of treatment once a day remained above the minimal inhibitory concentration in the joint fluid even 24 h after injection [To improve the outcomes of PJI, several studies have used direct intra-articular antibiotic infusion to increase the concentration of intra-articular antibiotics –10. A prnjection , 12.In 2007, we developed a treatment protocol for our institution to improve the outcomes of PJI treatment particularly in cases of acute infection after primary TKA. To decrease bacterial load and the risk of mature biofilm formation caused by delayed treatment, semi-urgent surgeries are performed immediately after the patient’s condition improved. Moreover, high-dose intra-articular antibiotic infusion via an intra-articular catheter was adopted, and it was used in combination with standard DAIR. Therefore, the current study aimed to assess the treatment outcomes of high-dose intra-articular antibiotic infusion and DAIR.This study was approved by the institution review board (COA 045/57). All procedures were performed in accordance with the relevant guidelines. Then, we retrospectively reviewed data collected from the medical records from July 2008 to July 2015. Our inclusion criteria included all patients with primary TKA who had acute hematogenous infection according to the Tsukayama classification  and who 3 and percentage of polymorphonuclear cell (PMN) (> 80% of neutrophil); and positive joint fluid culture result. A diagnosis of PJI was made only if 3 of 4 criteria were met. Plain radiograph images were meticulously reviewed to identify new osteolysis around the implant, which could be a sign of chronic PJI. If suspected, standard two-stage revision will be performed, and the patient will not be included in this study. According to the institution’s protocol, most patients with PJI were diagnosed within 12 h after admission to the hospital. Subsequently, DAIR and insertion of an intra-articular catheter could be performed within the next 12 h.Other than the clinical symptoms and signs of knee infection, PJI was mainly diagnosed using the laboratory investigation criteria, which were as follows: elevated serum erythrocyte sedimentation rate  and serum C-reactive protein (CRP) levels (> 10 mg/L); synovial fluid of the knee was analyzed in all cases; white blood cell (WBC) count at > 3000 cell/mmThe surgery started with standard open arthrotomy debridement and removal of polyethylene tibial insert. Debridement and initial irrigation with sterile saline solution (≥5 l) were cautiously performed; then, implantation with a new polyethylene liner was conducted. A catheter was placed into the knee joint, and the wound was closed normally Fig. . We chosThe first dose of intra-articular antibiotic was started in the operating room after wound closure based on previous culture or synovial fluid gram staining findings. The suction drain was used in all cases, and it was clamped for 4 h after surgery before removal within 48 h according to the standard protocol. After the removal of suction drain, antibiotic was infused daily via the catheter. Vancomycin  100 mg diluted with 10 mL of sterile water was injected on the first day; then, the dose was increased to 100 mg daily, with a maximum at 500 mg. The local effect of vancomycin was observed until the patient was discharged. The duration of intra-articular infusion was 3 weeks. The function of the intra-articular catheter was checked daily before antibiotic administration. If there were any leakage or if obstruction is suspected, the catheter will be removed. Intravenous antibiotic was administered for 6 weeks. The type of antibiotic is based on culture sensitivity results or the presence of bacteria in gram staining. However, the duration of intravenous antibiotic treatment was reduced to 3 weeks and 1 week in 2011 and 2014, respectively. From 2014 onwards, all patients with positive culture results were discharged early, and they were instructed to go to the outpatient department daily for the infusion of intra-articular antibiotics for 3 weeks. Oral antibiotics were administered at a duration of 12 weeks according to current guideline . The choAfter 3 weeks, the double-lumen catheter was removed. All patients were assessed for clinical signs of infection, and blood samples were collected for the evaluation of serum ESR and serum CRP levels weekly for 4 weeks, monthly for 3 months, and then every 3 months for 1 year. After 1 year, all patients were scheduled for annual follow-up or telephone consult in case they find it difficult to visit. The plain radiograph image of the knee was evaluated at 3 months and 1 year after DAIR. If there were clinical signs indicative of recurrent PJI, all laboratory results were evaluated to identify any infections. In cases of re-infection, two-stage revision was performed. Demographic, clinical, and laboratory data were collected. Treatment failure was defined using the following criteria: (1) clinical signs such as a non-healed wound with or without sinus tract, wound drainage, and persistent pain; (2) need for any surgical interventions for infection; (3) PJI-related mortality; and (4) need for long-term discontinuation of antibiotic treatment (> 6 months).2. Six (50.0%) patients had diabetes mellitus. None of patient receive antibiotic-loaded cement in primary TKA. The baseline characteristics and ASA physical status of all patients are summarized in Table From July 2008 to July 2015, 15 knees in 12 patients were diagnosed with acute hematogenous infection after TKA. Three (25%) patients presented with bilateral knee infection. Moreover, there were 3 men and 9 women, with a mean age of 65.9 (range: 48.0–82.0) years, and the mean body mass index was 27.7 (22.9–42.0) kg/m3. In addition, the culture was positive for Streptococcus group A. The mean ESR level was 92.7 (60–155) mm/h, and the mean serum CRP level was 173.4 (14–381) mg/L. The mean synovial WBC count was 52,736  cell/mm3, and the mean PMN percentage was 96.1% (82–100%). In total, five knees including two with methicillin-resistant Staphylococcus aureus, two with Streptococcus group A, and one with Streptococcus group B infections had positive culture results. All patients diagnosed with PJI at the time of surgery met at least three criteria.The mean interval from the index TKA was 29.9 (range: 2–70) months. Only one patient underwent primary index TKA within 3 months. The patient had PJI, as evidenced by a high serum ESR (67 mm/h) and serum CRP level (381 mg/L). The PMN percentage was 94%, and the synovial WBC count was 65,700 cell/mmThe mean time from symptom onset to surgery was 107.2 (range: 24.0–240.0) h. The serum CRP level significantly decreased and normalized as early as 23 days, with an average of 71.4 days after surgery. The average duration of IA injection was 17.5 (range: 7–24) days. All patients were successfully treated based on this protocol, and they did not require further surgery. The average length of hospital stay was 36.7 (range: 4–66), and the mean follow-up time was 93.3 (range: 56–133) months. All clinical data are shown in Tables After catheter removal, all patients had minimal drainage in the catheter site for a few days, and none had prolonged drainage (> 5 days). There was no complication in the catheter site and catheter-related events. All patients regained their previous activity level. Thereafter, oral antibiotic was administered for 12 weeks.n = 3 knees) patients had an intact prosthesis after 6 and 10 years. Two patients with negative culture results (n = 2 knees) developed symptoms of loosening and underwent two-stage revision. A patient with colon cancer who is receiving chemotherapy first presented with re-infection, which developed to fungal PJI during the course of chemotherapy. However, the patient was symptom-free for 2 years after DAIR. Moreover, a patient with diabetes developed septic loosening after 5 years of DAIR. Finally, 13 (86.66%) of 15 infected knees were successfully treated with this technique.None of the patients were lost to follow-up in this study. Two (PJI has been an increasing concern in arthroplasty practice nowadays. Since the average age of the population is increasing, a higher proportion of elderly patients will undergo TKA. With the presence of concomitant diseases such as diabetes, chronic kidney disease, and cancer, treatment could decrease the immune response of patients who undergo TKA, making them more susceptible to infection , 15. TheGenerally, PJI treatment is chosen based on duration of symptoms, which indicated biofilm formation. In case of acute PJI, such as acute postoperative and acute hematogenous, the duration of infection is < 4 weeks. This will then lead to the formation of immature biofilms. In this group, DAIR was the recommended treatment. However, chronic PJI is correlated with mature biofilm formation, which requires implant removal for a thorough debridement of a mature biofilm . The earDAIR was the treatment of choice for acute PJI because it was a strategic and economic approach. Although two-stage revision arthroplasty had a higher success rate, the overall treatment cost is significantly higher, and re-infection still occurs after the procedure. With consideration of economic burden, recent studies have found that DAIR is more cost-effective than two-stage revision arthroplasty –18. HoweS. aureus organisms [The efficacy of infection eradication can locally affect failure to maintain an adequate level of antibiotic locally. Some antibiotics, including beta-lactams, have a lower intra-osseous concentration than others. The sub-therapeutic level of beta-lactams could cause biofilm formation in rganisms . A higherganisms , 12. TheRetaining an intra-articular catheter might be associated with catheter-related events, which lead to occult infection and recurrent periprosthetic joint infection. There was no catheter-related event in our study, and it is rarely reported in previous studies. However, there were two cases of treatment failure, which were not correlated to the use of a catheter.The current study had several limitations. First, we obtained a relatively high proportion of negative culture findings, which reflected the disadvantages of such technique during that period and caused empirical antibiotic treatment about two-third of the cases. However, in our study, the diagnosis of PJI was based on clinical and laboratory studies, which were in accordance with the MSIS criteria 2014 and 2018. All patients diagnosed with PJI met at least three minor criteria. Second, the number of cases was quite small due to the nature of the disease. However, none of the patients were lost to follow-up.DAIR combined with 3-week direct intra-articular antibiotic infusion via a double-lumen catheter is an effective treatment for acute hematogenous infections after TKA. Early debridement and maintenance of high-dose intra-articular antibiotic infusion could be key factors in the eradication of causative organisms, which leads to a successful treatment of PJI type III.


A total of 274 articles was identified, and 11 RCTs (6 direct comparisons of 15 techniques) were included in the NMA, which examined 6 GRT techniques: control, Os, PL, MB, MB + Os, and MB + PL. The MB + Os group compared to the control  and to the MB group  showed statistically significant ORs (p ˂ 0.05). The MB + Os group presented the highest degree of certainly (P-score = 0.93).Several regeneration techniques and materials have been proposed for the healing of bone defects after surgical endodontic treatment; however, the existing literature does not provide evidence on the most recommended techniques or materials. The aim of the present systematic review and network meta-analysis (NMA) is to summarize the clinical evidence on the efficacy of guided tissue regeneration techniques (GRTs). The PRISMA recommendations were followed. Four databases were searched up to December 2021. Randomized clinical trials (RCTs) with a minimum follow-up of 6 months were included. The risk of bias was assessed using the Cochrane Collaboration tool. A fixed effects model and frequentist approach were used in the NMA. Direct GRT technique comparisons were combined to estimate indirect comparisons, and the estimated effect size of the comparisons was analyzed using the odds ratio (OR). Inconsistency was assessed with the Q test, with a significance level of  Bacterial infection plays an important role in establishing pulp tissue inflammation, which may lead to subsequent pulp necrosis and the formation of periapical lesions . The comEndodontic surgery is recommended after unsuccessful retreatment, when retreatment is impossible, or when there is an unfavorable prognosis . SurgicaRecently, guided tissue regeneration (GTR) techniques have been widely used in medicine, including in dentistry, to improve tissue healing. Furthermore, GTR techniques have been recommended as an adjunct to endodontic surgery to promote periapical tissue healing and improve the treatment outcome .Complete periapical healing involves the regeneration of alveolar bone, periodontal ligament cells, and cementum . HoweverNumerous studies reported the clinical effectiveness of GTR techniques to promote healing and improve the outcome of surgical endodontic treatments ,11. HoweNetwork meta-analysis (NMA) extends the principles of meta-analysis to the evaluation of several treatments in a single analysis, comparing multiple treatments simultaneously by combining direct and indirect evidence within an array of randomized controlled trials . It is tThe aim of the present study is to conduct a systematic review and NMA to analyze the influence of GTR techniques on the success rate of surgical endodontic treatment. The null hypothesis (H0) was that GTR techniques do not influence the success rate of surgical endodontic treatment.http://www.prisma-statement.org, accessed on 30 July 2020) guidelines. The review also fulfilled the PRISMA 2009 Checklist [This systematic review and NMA was conducted following the Preferred Reporting Items for Systemic Reviews and Meta-Analyses  question: in adult patients undergoing endodontic surgery (P), does the use of regeneration techniques (I) compared to not applying regeneration techniques (C) influence the success rate (O)? An electronic search was conducted in the PubMed, Scopus, EMBASE, and Web of Science databases. The search covered all of the literature published internationally up to December 2021. The search included the following medical subject heading (MeSH) terms: “apicoectomy”, “periapical surgery”, “endodontic surgery”, “periapical lesion”, “surgical endodontic treatment”, “root-end surgery”, “root-end resection”, “periradicular surgery”, “guided tissue regeneration”, “bone graft”, “bone regeneration”, and “membrane”. The Boolean operators applied were OR and AND. The search terms were structured as follows: ((“apicoectomy”) OR  OR (“endodontic surgery”) OR  OR  OR (“root-end surgery”) OR (“root-end resection”) OR (“periradicular surgery”)) AND ((“guided tissue regeneration”) OR (“bone graft”) OR (“bone regeneration”)) AND ((“membrane”)). Two researchers (R.T. and A.Z.M.) independently conducted the database searches in duplicate. Titles and abstracts were selected by applying the inclusion and exclusion criteria. One researcher (R.T.) extracted data for the relevant variables. The systematic review was carried out by R.T., and two researchers not involved in the selection process (A.Z.M. and J.M.C.) performed the subsequent meta-analysis.The inclusion criteria for the selected studies were as follows: randomized clinical trials (RCTs) that had a minimum follow-up period of at least 6 months; studies that analyzed GTR techniques  or compared GTR techniques with a control treatment; patients that were 18 years old or older; and endodontic surgery procedures that were used to treat apical and/or apical-marginal lesions. No restrictions were placed on the year of publication or language.The exclusion criteria for the selected studies were as follows: systematic or bibliographic reviews, clinical cases, case series, retrospective studies, and editorials and studies, including patients younger than 18 years.The following data were extracted from each study by independent reviewers (S.H.M. and J.M.A.): author and year of publication, title, journal in which the article was published, sample size (n), follow-up time, measurement procedure, type of GTR technique, success rate, periapical reduction, and bone density. The success of healing was analyzed according to the radiographic criteria established by Rud et al.  and MolvThe risk of bias in the selected studies was assessed using the Cochrane Collaboration tool for methodological quality assessment of clinical trials . This to2 statistical index [The meta-analysis was carried out using a random effects model to estimate the success rate of endodontic surgery with and without GTR techniques, along with the confidence intervals. Heterogeneity among the combined studies for each treatment group was assessed using the Ial index , which dal index . The resp < 0.01, and a net heat plot [Direct treatment comparisons were combined with a fixed effects model in a frequentist NMA to estimate indirect comparisons. The estimated effect size of the comparisons was analyzed by the OR. The inconsistency of studies included in the NMA was assessed with the Q test , with a eat plot .Direct comparisons were performed using a NETWORK graph, and treatments were ranked on a scale of 0 to 1 using a P-score measuring the degree of certainty and indicating whether one treatment was superior to another .Publication bias was analyzed using the trim and fill adjustment method for funnel plot asymmetry. In this analysis, each study was represented by a point, and the effect size and standard error were represented on the X-axis (logit transformed proportion). If there were no significant differences between the initial and adjusted estimates, the publication bias was considered to be low. R software was used with the Metaprop and Netmetaprop statistical packages to perform the meta-analysis.https://refworks.proquest.com/reference/upload/recent/, accessed on 14 August 2020). After reading the titles and abstracts, an additional 130 articles were eliminated, leaving 90 articles; a further 55 articles were rejected because they did not fulfil the inclusion criteria: they did not include complete success rate data, did not use in vivo patient data, or presented a minimum follow-up time of less than 6 months. Finally, 11 articles were included in the qualitative and quantitative synthesis because they included all of the required data and variables :2 = 0%) estimated a significant OR of 3.53 with a 95% confidence interval between 1.33 and 9.33. The remaining comparisons do not produce a significant OR  were carried out with the data obtained from the eleven selected RCTs. The meta-analysis of combined studies comparing MB-Os versus control .Eleven RCTs (sixteen pairs of comparisons) were included in a frequentist NMA examining six GRT techniques  to analyze their influence on the success of healing after endodontic surgery. The data were combined with a fixed effects model (Mantel–Haenszel method). The nodes represent treatments, and the lines connecting the nodes are the six direct comparisons included in the NMA .p < 0.05). Direct comparisons (6/15) are highlighted in gray, and indirect comparisons are uncolored. Only two statistically significant ORs were found (in bold) (p < 0.05). The probability of obtaining a successful result was 3.67 times greater in the MB + Os group than in the control group (p < 0.05). The success of healing was 3.47 times greater in the MB + Os group than in the MB group (p < 0.05). The remaining comparisons among the groups do not show significance (p > 0.05)  .The ranking of the GTR techniques was performed according to the P-score, which measures the degree of certainty and indicates whether one alternative is superior to the others. The P-score is measured on a scale of 0 to 1. The MB + Os group presents the highest P-score (0.93), followed by MB + PL (0.60) and PL (0.53) .p = 0.589). The net heat plot , the present NMA satisfied the assumption of transitivity, indicating that there were no systematic differences among the compared techniques other than the GTR techniques being compared [p < 0.05) for the comparison between membrane plus bone grafting and endodontic surgery alone indicates that the success rate of this combination was almost four times higher than that of surgery without an adjunct GTR technique.Since the NMA did not show heterogeneity or inconsistency , and more effective than platelet-rich plasma techniques. However, the membrane plus bone graft combination was 3.6 times more successful than membrane only.The success rate of the combined membrane plus bone graft was 3.7 times higher than that of endodontic surgery alone (control). Parmar et al. (2019) reported a nonsignificant radiographic reduction in periapical bone defects regenerated using a resorbable collagen membrane. Complete periapical healing was observed in the control group, with rates of 60 to 80% and 53.3 to 73.3% of those of the membrane group, depending on the radiodiagnostic technique .p ˃ 0.05) radiographic reduction in periapical bone defects after endodontic surgery. Additionally, they did not recommend the use of resorbable membranes for bone defects with four walls that are confined to the apical region [p ˃ 0.05) in bone density at 6 months after surgery between the two types of membranes.Marin-Botero et al. (2006) also reported that polyglactin-910 resorbable membranes had little influence on the complete healing of periapical bone defects after endodontic surgery (40%) compared with the control treatment (60%) . Garret l region . Santamap ˃ 0.05) among the membrane alone, platelet-rich plasma alone, and the two combined [p ˃ 0.05) in the clinical and radiographic reduction in periapical bone defects after endodontic surgery using platelet-rich plasma techniques with respect to the control group [These results suggest that GTR techniques using membranes do not contribute to increased periapical bone regeneration regardless of the membrane type . Howevercombined . Dhiman ol group .p ˃ 0.05) in radiographic reduction between the two bone graft materials [p ˃ 0.05) difference between the two bone graft materials at 6-month follow-up [Most authors reported that bone grafts stimulate bone defect healing and prevent adjacent soft tissue collapse ,37,38,39ollow-up . Howeverollow-up .p ˂ 0.05) at short-term follow-up (9–24 months), but no statistically significant difference (p ˃ 0.05) at long-term follow-up (33–48 months) [p ˃ 0.05) after endodontic surgery when using resorbable collagen membrane and bovine-derived hydroxyapatite bone graft material for through-and-through lesions [Other bone graft materials have been used as adjuncts to GTR techniques in endodontic surgery. Pantchev et al. (2009) retrospectively analyzed the clinical and radiographic outcomes of a synthetic bioactive glass material used as a bone graft after endodontic surgery. They found a statistically significant difference ( months) . It is mlow-up 9– months,  lesions  and four lesions . Tobon e lesions .In addition, the wound healing scales and indices used in oral surgery do not capture the relationships between outcome parameters; therefore, Hamzani et al. (2018) proposed a novel scale that allows the assessment of wound healing phases . RecentlA limitation of this systematic review and meta-analysis is the possibility that not all articles related to the selection criteria were identified, although the risk was decreased because three databases were searched. In addition, most of the studies were of poor quality, according to the Cochrane Collaboration tool . FurtherWithin the limitations of this study, it was found that GTR techniques increased the success rate of endodontic surgery. The use of bone grafts plus membranes as an adjunct to surgical endodontic treatment promoted complete periapical bone healing, with a higher success rate, and improved the prognosis of endodontic surgery. Therefore, we recommend the use of bone grafts plus membranes as a GTR technique in endodontic surgery.


FREM2 gene has been shown in glioblastoma progression. Here we reconstructed the FREM2 molecular pathway. We assessed the biomarker capacity of FREM2 expression and its pathway as the overall survival (OS) and progression-free survival (PFS) biomarkers. We used 566 glioblastomas (GBM) and 1097 low-grade gliomas (LGG) to test these biomarkers. FREM2 molecular pathway was a better biomarker than FREM2 gene expression. It could robustly discriminate between GBM and LGG. High FREM2 pathway activation level was associated with poor overall survival (OS) in LGG, and low progression-free survival in LGG and GBM. FREM2 pathway activation level was also a poor prognosis biomarker for OS and PFS in LGG with IDH mutation, for PFS in LGG with wild type IDH and mutant IDH with 1p/19q codeletion, in GBM with unmethylated MGMT, and in GBM with wild type IDH.Gliomas are the most common malignant brain tumors with high mortality rates. Recently the role of the FREM2 gene has a role in glioblastoma progression. Here we reconstructed the FREM2 molecular pathway using the human interactome model. We assessed the biomarker capacity of FREM2 expression and its pathway as the overall survival (OS) and progression-free survival (PFS) biomarkers. To this end, we used three literature and one experimental RNA sequencing datasets collectively covering 566 glioblastomas (GBM) and 1097 low-grade gliomas (LGG). The activation level of deduced FREM2 pathway showed strong biomarker characteristics and significantly outperformed the FREM2 expression level itself. For all relevant datasets, it could robustly discriminate GBM and LGG . High FREM2 pathway activation level was associated with poor OS in LGG (p < 0.001), and low PFS in LGG (p < 0.001) and GBM (p < 0.05). FREM2 pathway activation level was poor prognosis biomarker for OS (p < 0.05) and PFS (p < 0.05) in LGG with IDH mutation, for PFS in LGG with wild type IDH (p < 0.001) and mutant IDH with 1p/19q codeletion(p < 0.05), in GBM with unmethylated MGMT (p < 0.05), and in GBM with wild type IDH (p < 0.05). Thus, we conclude that the activation level of the FREM2 pathway is a potent new-generation diagnostic and prognostic biomarker for multiple molecular subtypes of GBM and LGG.Gliomas are the most common malignant brain tumors with high mortality rates. Recently we showed that the  Gliomas account for ~30% of all CNS tumors and 80% of all malignant brain tumors ,2. In thOther types of gliomas (grade 1–3) are typically called low-grade gliomas (LGG), while some authors refer to LGG only grade 1–2 gliomas and other authors refer to LGG diffuse low-grade and intermediate-grade gliomas (grade 2–3) . In thisIDH mutation and MGMT promoter methylation are associated with better survival in gliomas [IDH mutation had better overall survival  and progression-free survival  [IDH LGGs (1.7 years) is between OS for wtIDH GBMs (1.1 years) and mutated IDH GBMs (2.1 years) [MGMT promoter methylation was associated with longer PFS and OS in GBM patients without therapy, and with better OS in GBM patients treated by DNA-alkylating agents such as temozolomide [MGMT promoter is also a favorable predictor of PFS in LGG treated with neoadjuvant temozolomide [Furthermore, molecular markers can serve as predictors for the survival of glioma patients. Robust biomarkers such as  gliomas ,10,11. A< 0.001) . Median zolomide ,14. In tFREM2 (FRAS1 Related Extracellular Matrix 2) gene product was statistically significantly associated with GBM [FREM2 encodes an integral membrane protein with multiple chondroitin sulfate proteoglycan element (CSPG) repeats and Calx-beta domains that mark sodium-calcium exchanger activity, which is used to expel calcium from cells. The expression of FREM2 is higher in GBM cell lines than in normal astrocytes [FREM2 gene and protein expression levels are higher in GBM stem cells compared to conventional GBM cell lines [FREM2 expression was found in LGGs compared to healthy brain tissues, and in GBMs compared to LGGs [Recently, using agnostic proteomic screening we found that with GBM ,16. FREMtrocytes . Also, Fll lines . This trFREM2 gene expression and its linkage with survival using the major publicly available datasets of LGG and GBM RNA sequencing profiles. We also reconstructed the FREM2 molecular pathway using the human interactome model and found that it had a significantly better performance as the OS and PFS biomarker of gliomas. To this end, we used three literature and one experimental RNA sequencing datasets collectively covering 566 glioblastomas (GBM) and 1097 low-grade gliomas (LGG). The activation level of deduced FREM2 pathway showed strong biomarker characteristics and significantly outperformed the FREM2 expression level itself. For all relevant datasets, it could discriminate GBM and LGG . High FREM2 pathway activation level was associated with poor prognosis in LGG and GBM and several LGG or GBM subtypes. Thus, we conclude that the activation level of the FREM2 pathway is a potent new-generation diagnostic and prognostic biomarker for multiple molecular subtypes of GBM and LGG.In this study, we analyzed MGMT methylation statuses were obtained for GBM samples from the report [IDH mutation statuses were extracted for GBM and LGG samples from SNV data (vcf files) from the GDC Data Portal. Molecular subtype classifications were obtained from Ceccarelli M. et al. [http://gliovis.bioinfo.cnio.es/, acceded on 7 August 2021).Overall and progression-free survival data were extracted from the clinical description on the GDC Data Portal for 591 GBM and 510 LGG samples . RNA seqe report  and LGG e report . IDH mut. et al.  and the IDH mutation status, MGMT methylation status, and patient age information were extracted from the clinical description for 137 GBM and 172 LGG samples from the CGGA database; dataset id: mRNAseq_325 [http://gliovis.bioinfo.cnio.es/, accessed on 7 August 2021).Overall survival data, Aseq_325 ,21. RNAsIDH mutation status, MGMT methylation status, and patient age information were extracted from the clinical description for 237 GBM and 420 LGG samples included in CGGA database; dataset id: mRNAseq_693) [http://gliovis.bioinfo.cnio.es/, accessed on 7 August 2021).Overall survival data, seq_693) ,23. RNAshttps://www.laek-rlp.de/ausschuesse-kommissionen/ethikkommission/, accessed on 17 April 2021). Clinical data were obtained for every patient including diagnosis, IDH mutation status, MGMT promoter methylation status, type of therapy, and time to progression . For some experimental patients (12/16), two or more tumor samples were included in the analysis that were obtained surgically from different regions of the same tumor. Written informed consents for using excess tumor tissue for research purposes were obtained from all the patients. Tumor samples were coded and processed for RNAseq anonymously and in accordance with the approval by the UMM Institutional Review Board and ethics committee approval No. 837.178.17 (11012) granted to the UMM Clinic for Neurosurgery by the Rhineland Palatinate Chamber of Physicians  is an aggregated quantitative and qualitative characteristic of changes in expression levels for genes participating in a certain molecular pathway [pPAL is PAL for pathway p, nCNR is the case-to-normal ratio, the ratio of gene n expression level in a sample understudy to an average level in the control group; ARR (activator/repressor role) is a Boolean value that depends on the function of this gene product in pathway p. ARR values were defined as follows: −1 when the product of n inhibits p; 1 when n activates p; 0 when n has an ambiguous or unclear role in the pathway; 0.5 or −0.5, when n is rather p activator or inhibitor, respectively. We used an averaged-by-gene-expression tumor sample as the control. PAL values calculated for all the samples investigated are available in  pathway ,31,32. PWatermelon multisection method [We used principal component analysis to assess the compatibility of glioma gene expression data from a different dataset. Also, quality measuring of hierarchical clustering was applied to CGGA datasets for the same goal. This was performed by the n method  that rett-test were used as the measure of biomarker quality in comparison of low-grade glioma with glioblastomas.ROC AUC value and p-value. Hazard ratios were calculated in the univariate and multivariate Cox model to assess survival differences in comparative groups.Overall and progression-free survival was assessed by Kaplan-Meyer analysis. The statistical significance of survival differences was measured with a log-rank test We used three major publicly available glioma datasets with clinically annotated RNA sequencing profiles  [IDH mutation statuses were extracted [Specifically, the TCGA dataset included 153 GBM , and 505 LGG samples , and overall survival (OS), progression-free survival (PFS) , MGMT prsamples) , and IDHxtracted .mRNAseq_325 and mRNAseq_693 [IDH mutation, and MGMT methylation status information were available for both datasets. CGGA_693 contained RNAseq profiles for 237 GBMs  and for 420 LGGs .CGGA database contained two large RNAseq glioma datasets with internal IDs Aseq_693 ,21,22,23CGGA_325 included profiles for 137 GBMs  and 172 LGGs .IDH mutation, and MGMT promoter methylation information was collected for each patient, but no OS information was available.The experimental dataset included 39 tumor samples, which were collected from 16 patients with primary GBM . Some patients (12/16) have several tumor samples which were obtained from different regions of the same tumor. RNAseq profiles were deposited in Gene Expression Omnibus (GEO) database under accession number GSE139533. PFS, The expression data from the datasets under investigation were all processed using the Illumina platform but were obtained with different library preparation kits and protocols. Thus, all datasets had a different number of sequenced genes . We normalized raw counts by DESeq2 software  and inspWatermelon multisection method [In addition, hierarchical clustering of samples from two batches of the CGGA project (CGGA_325 and CGGA_693) showed that samples were clustering by batch ID rather by glioma type (LGG or GBM). This was also quantitatively measured by the n method  that retThus, we didn’t combine CGGA_325 and CGGA_693 expression data and used them as the two independent literature datasets.Using a collection of published molecular pathways as the knowledge base of molecular interactions, we built a human interactome model—the graph, where the nodes are genes/gene products, and the edges are known pairwise connections between the elements of molecular pathways. Visualization of the model was performed using Gephi software, ForceAtlas2 algorithm  Figure .Molecular architectures of 1180 different pathways were used. Gene composition and nodes interactions of the pathways were extracted and cataloged. We combined all pathway graphs based on the coinciding genes/gene products. The obtained interactome graph consists of 7152 nodes (genes/gene products) with 298,824 molecular interactions. The following types of interactions were considered: “activation”, “compound”, “inhibition”, “phosphorylation”, “dissociation”, “repression”, “dephosphorylation”, “binding/association”, “ubiquitination”. The graph has a low density (0.01) with an average vertex degree of 42. All genes included form a connected network, i.e., there is an at least undirected path between every pair of genes/gene products involved.FREM2 protein. FREM2 is included in the “extracellular matrix” node from five pathways considered: “Akt_Signaling_Pathway”, “ERK_Signaling_Pathway”, “ILK_Signaling_Pathway”, “MAPK_Signaling_Pathway”, and “PTEN_Pathway”. There were several downstream interactions with FREM2, but no upstream interactions were cataloged  were calculated for all three pathway types, for all tumor samples in every dataset, and were defined PAL1, PAL2, and PAL3, correspondingly. PAL positively reflects activation of a molecular pathway, where the absolute value of PAL reflects the extent of a pathway up/down regulation, and sign (+/-) of a PAL shown overall upregulation or inhibition of a pathway, respectively .FREM2 expression level) were connected with specific glioma conditions. To this end, we used the ROC AUC value as the measure of biomarker quality. The area under the ROC curve (AUC) is frequently used for scoring molecular biomarkers in oncology [t-test, p-value, and ROC AUC can answer two different questions: whether a metric under consideration is differentially regulated, and whether it can serve as a good biomarker.We then measured if these metrics , and for FREM2 expression levels (p < 4.46 × 10−4), we detected significant differences between LGG and GBM for all the relevant datasets tested  biomarker. To this end, we performed Kaplan-Meier analysis and calculated p-values of the log-rank test separately for GBMs and LGGs for all relevant datasets . Thus, we obtained groups with high-level samples (FREM2 variable was higher than its median) and low-level samples  in each dataset or subgroup.Where possible, we also analyzed tumor molecular subgroups classified according to either FREM2 expression, PAL1-3) on different sets of glioma samples  with the survival characteristic in 33% of the comparisons with average log  −1.18. In turn, PAL1-3 were effective in 39.6%, 39.6%, and 37.5% of the cases, with average lg −1.43, −1.51, and −1.47, respectively.Thus, we performed a total of 48 comparisons for each possible biomarker under investigation  and at the same time had the lowest average q-value for the statistical tests . For furFREM2 pathway PAL2 with other well-known survival predictors: MGMT methylation and IDH mutation status , and one experimental GBM dataset for PFS. We used FREM2 PAL2 and IDH mutation showed very good comparable performance for both OS and PFS analysis  and GBM , with a mean correlation of −0.31  for PFS prognosis inside four LGG subgroups: with wtIDH, mutant IDH, mutant IDH with 1p/19q codeletion (p < 0.05) and methylated MGMT, and in GBM subgroups with wtIDH and with unmethylated MGMT promoter. This further confirms the independent utility of the PAL2 biomarker in gliomas.We also detected good effectiveness of PAL2 (FREM2 pathway PAL2 effectiveness was confirmed in two or more available datasets as OS or PFS predictor (i) for OS in LGG, (ii) for OS in LGG with methylated MGMT, (iii) for OS in LGG with unmethylated MGMT, (iv) for OS in LGG with IDH mutation; (v) for PFS in GBM. We used p-value without FDR correction in In total, we identified five glioma conditions where redictor : (i) forhazard ratio values from univariate Cox models and built Kaplan-Meyer plots  using the expression profile of each sample separately, and (ii) using averaged expression profile for every patient. In both types of the analysis, high PAL2 values were associated with the poor prognosis on PFS in GBM patients . For FREM2 pathway PAL2 values  we detected a significant difference between mesenchymal and other subtypes for all relevant datasets tested , but AUC was less than 0.7 for most of the comparisons. Thus, FREM2 gene expression cannot serve robust biomarker for GBM molecular subtypes , and lower PFS in LGm3 and LGm4 (p < 0.01).Likewise, we explored how . et al.  and stro. et al. . Only thectively ). HigherFREM2 molecular pathway, which activation is strongly associated with unfavorable prognosis in glioma patients. We have tested three algorithmically built agnostic variants of this pathway and selected the best version in 34 independent comparisons. This survival predictor capacity of FREM2 pathway activation was robust and comparable with IDH mutation status, but significantly superior to MGMT methylation status. Moreover, FREM2 pathway activation level (PAL) could effectively predict survival within the subgroups with different IDH mutation statuses: OS in IDH mutant LGG, and PFS in IDH mutant GBM, in LGG with wtIDH, and LGG with mutant IDH. Furthermore, activation levels of the FREM2 pathway were significantly higher in GBM than in LGG samples.We report here a new reconstructed SPRY1 gene, another potential glioma biomarker that showed comparable characteristics to FREM2 in the previous study [FREM2 expression (SPRY1 pathway hasn’t been reconstructed and investigated yet, and we plan that this will be a matter of our further studies.Expression of us study , was alspression . HoweverFREM2 is associated with mesenchymal differentiation in gliosarcoma because it was strongly overexpressed in mesenchymal compared to glial tumor areas. [FREM2 gene expression was demonstrated in gliomas compared to the normal glia, and in GBM compared in LGG [FREM2 implication in cancers.As reported previously, r areas. . In addid in LGG ,16,48. WFREM2, namely FRAS1 and FREM1, were recently reported as cancer-related genes. In particular, knockout of FRAS1 inhibits proliferation of gastric cancer cells through caspase activity increment and cell cycle arrest both in vitro and in vivo [FREM1 in breast cancer is associated with a favorable prognosis and high-level immune infiltration status [However, other genes of the same gene family as  in vivo . Conversn status .FREM2 pathway members we detected strongly upregulated expression of integrin family members, filamin, and several types of membrane receptors associated with the poor survival prognosis. Thus, we propose that these components of the FREM2 pathway are important actors of glioma pathogenesis and could be regarded as the possible new targets for next-generation molecular therapeutics.Among the reconstructed FREM2 pathway can help to find and validate additional molecular targets that could be affected in a combinational therapy of malignant gliomas.Indeed, integrins were previously mentioned as the potential targets of GBM therapy because of their major role in tumor invasion and strongly differential expression ,52,53. UMGMT promoter methylation and 1p/19q codeletion. In older patients presenting with wtIDH glioblastoma, the presence of MGMT promoter methylation predicts a positive response to therapy and longer survival [Next-generation sequencing technologies allowed us to get a better insight into the molecular biology of GBM, but also to identify new disease-specific changes and molecules. However, despite extensive research, the life expectancy of GBM patients has not significantly improved in decades. Primary GBMs are ranked first among cancer types in years of life lost—on average 20.1 years compared to 11.8 years for lung cancer and 6.8 years for prostate cancer . Currentsurvival ,56,57. Csurvival ,59. Predsurvival  and implsurvival . Howeversurvival . Thus, tsurvival .FREM2 pathway can be used for adjustment of treatment schedule that will result in a better quality of life of patients and optimization of costs. Owing to the large sample size used in this study, we believe that the FREM2 pathway shows the potential to be easily implemented in routine practice after validation in clinical settings.Because of its robustness and survival predictor capacity, the IDH mutation status, and superior to MGMT methylation status in most of the comparisons, we propose FREM2 pathway activation as a novel robust predictor of unfavorable prognosis of glioblastoma patients.This biomarker holds the potential to be used as a predictive biomarker of high potential benefit for the patients. On that account, because it was found robust, comparable to