In addition, the complex showed a B+V-type crystalline structure, which confirmed that the interaction modes between the starch and tea polyphenol include hydrogen bonding and hydrophobic interactions. Moreover, the appearance of an irregular sponge network structure of the complex further supported the interactions between the starch and tea polyphenol. This study provides a theoretical basis for the development of functional foods using Chinese yam starch.Interactions between food components have a positive impact in the field of food science. In this study, the effects of tea polyphenol on the structural and physicochemical properties of Chinese yam starch using autoclave-assisted pullulanase treatment were investigated. X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, rapid visco analysis, differential scanning calorimetry, and the 3,5-dinitrosalicylic acid method were applied in this study. The results showed that the Chinese yam starch–tea polyphenol complex formed a structural domain with higher thermal stability along with lower pasting viscosities than native starch. The in vitro digestibility of Chinese yam starch decreased with the addition of the tea polyphenol, and the amount of resistant starch content in the complex was 56.25 ± 1.37%, significantly higher than that of native starch ( Food products contain complex components such as water, carbohydrates, proteins, and lipids. Different molecular interactions between different food components are ubiquitously present. These interactions usually have dual effects on the quality and functionality of foods, which are hot topics in scientific research circles . The intPolyphenols, as natural substances with multiple biological activities, have demonstrated health-promoting properties. The phenolic hydroxyl groups in polyphenols may interact with starch noncovalently, such as through hydrogen bonding, hydrophobic interactions, and electrostatic forces . At the Various studies have demonstrated that the content of amylose is a key factor in forming complexes with ligands, and that amylopectin has a much weaker complexing ability with ligands due to its highly branched structure and steric hindrance . It is wThere are no reports in the last decade on autoclave-assisted pullulanase treatment for the preparation of Chinese yam starch–tea polyphenol complexes. Therefore, a Chinese yam starch–tea polyphenol complex was prepared using autoclave-assisted pullulanase treatment, and the effects of tea polyphenol on the structural and physicochemical properties of Chinese yam starch were investigated in this study. The pasting viscosities, thermal properties, in vitro digestibility properties, crystallinity, micromorphology, and the interactions between molecules of the complex were characterized using a variety of modern technologies. The data obtained contribute to providing a theoretical basis for the development of functional foods using Chinese yam starch.Chinese yams (Anxi middle lobe variety) were provided by Fujian Shange Agricultural Comprehensive Development Food Co., Ltd. . Based on the results of our previous experiments, the amylopectin content of Chinese yam starch was 69.72 ± 0.499%.Pullulanase  was obtained from Shanghai Macklin Biochemical Co., Ltd. . Tea polyphenols  were obtained from Shanghai Titan Technology Co., Ltd. , BR. All other chemical reagents used in this study were of analytical grade.Firstly, the unusable parts of fresh yam tubers had been removed and then mixed with pure water to make a fresh Chinese yam slurry. The fresh Chinese yam slurry was cleaned with two times the volume of pure water and anhydrous ethanol to clarify the top clear liquid. The sediment obtained using this method was the Chinese yam starch. The Chinese yam starch was dried in an oven  at 40 °C to a constant weight and filtered through a 100-mesh screen.The production steps for the Chinese yam starch–tea polyphenol complex (CYS–TPC) were as follows: a Chinese yam starch suspension  was pregelatinized and cooled to 50 °C, and then pullulanase  was added to the sample for enzymolysis . Then, the sample was subjected to a boiling water bath (10 min) to deactivate the enzymes. Tea polyphenol  was added to the sample and processed in an autoclave . The autoclave treatment was carried out at 105 °C for 30 min. Next, the treated sample was refrigerated  and washed with anhydrous ethanol (repeated three times) to remove uncomplexed tea polyphenol. Then, the sample was freeze-dried  at approximately −80 °C for 24 h before milling and filtering through a 100-mesh screen.The production steps for the Chinese yam starch (I) were the same as those of the CYS–TPC samples, but pullulanase and tea polyphenol were replaced with equal volumes of distilled water. This sample was named CYS(I).The production steps for the Chinese yam starch (II) were the same as those of the CYS–TPC samples, but the tea polyphenol was replaced with an equal amount of distilled water. This sample was named CYS(II).The viscosity characteristics of native and modified starches during gelatinization were determined using a rapid visco analyzer . A sample suspension of 8% was prepared and equilibrated at room temperature for 24 h. The test conditions were as follows: Firstly, the suspension was held at 50 °C for 1 min, heated to 95 °C within 5 min, held at 95 °C for 11 min, cooled to 50 °C within 4 min, and subsequently held at 50 °C for 3 min. The paddle speed was 960 rpm during the first 10 s to disperse the sample, and then the paddle speed was set at 160 rpm during the measurement.The energy change in the samples during gelatinization was measured via differential scanning calorimetry . The sample and purified water were added to the liquid crucible at a ratio of 1:2, and then left to equilibrate at room temperature for 24 h. The test conditions were as follows: the equipment was heated from 20 °C to 120 °C at a speed of 20 °C/min, and the onset, peak, and completion temperatures were obtained. Afterward, the gelatinization enthalpy was calculated according to the peak area.The measurement method for the content of the free glucose in the starch was as follows : the staRDS) and slowly digestible starch (SDS) were measured after incubation for 20 min and 120 min, respectively. The resistant starch (RS) was the undigested starch after 120 min of incubation. The hydrolysis rate  of starches and the content of RDS, SDS, and RS in the starch were calculated according to the following equations:G(t) is the amount of glucose in the starch sample after t min of digestion, mg; G is the amount of glucose in the standard curve, mg; V is the volume of solution removed from the digestion system, mL; 331 is the total volume of the digestion system, mL; A is the amount of glucose contained in the solution removed from the digestion system, mg; FG is the amount of free glucose contained in the original starch sample, mg; 0.9 is the conversion factor of starch and glucose; W is the amount of the starch sample on a dry basis; and t is the response time, h.The in vitro digestibility of the native and modified starches was determined based on the method described by Khurshida et al.  with a sA smartLab X-ray diffractometer  was used to determine the crystallinity of the sample powder. The operating parameters used were as follows: the target was Cu-Kα radiation, and the scanning range was 3–40° with a scanning speed of 5°/min.–1 with 4 cm−1 resolution at room temperature (about 25 °C) using a diffuse reflectance accessory. Samples were diluted with KBr (1:100 v/v) before acquisition, and the background value from pure KBr was acquired before the samples were scanned.The vibrational spectra of the samples were determined using a Perkin Elmer FTIR spectrometer . The spectra were recorded with 25 scans in the wavenumber range 400–4000 cm−6 mbar) with an accelerating voltage of 3.00 keV.The morphologies of the starch granules were observed at 1000× and 10,000× magnifications using an SEM . The powder was fixed to the SEM stub with double-sided tape. The floating powder was blown off, and the remaining powder was sprayed twice with gold. The entire operation was performed under vacuum conditions . The pasting properties of native and modified starches are listed in H of 15.47 J/g. The modified starches exhibited a lower To, Tp, Tc, ΔT, and ΔH value in comparison with CYS. A high ΔT value has been related to a high degree of crystallinity, which produces structural stability and causes the starch granules to be more resistant to gelatinization. The ΔH value mainly reflects the energy required to break the double helix structure of starch during gelatinization [T value of the four samples was CYS–TPC > CYS(II) > CYS > CYS(I), which made clear that the relative crystallinity of CYS–TPC was the highest, indicating that CYS–TPC did not gelatinize easily and was more resistant to enzymatic hydrolysis. Compared to CYS, the lower ΔH value of the modified starches might be because of their slack amylopectin double helices, and, thus, less enthalpy was required for their pasting process [H value of CYS(II) was significantly lower than that of CYS(I), which was probably due to the further destruction of the double helix structure of amylopectin during pullulanase treatment [H value of CYS–TPC was slightly higher than CYS(II), indicating the interaction between starch and tea polyphenol stabilized the double or single helix structure, thus enhancing thermal stability [H [A significant variation in thermal properties between native and modified starches was obtained, as shown in nization . The ord process . In contreatment . The ΔH tability . This miility [H .HR, %) of CYS(I) at 20 min and 120 min was also significantly higher than that of CYS. The RS content of CYS(II) slightly increased compared to that of CYS(I). The reason might be related to the rearrangement of short amylose produced after pullulanase treatment. The broken starch chains realigned with the amorphous region, which reduced the number of attack sites for enzymes, enhancing the resistance to digestion and contributing to a greater RS content [HR, %) at 20 min and 120 min. The resulting hydrolysis rate  might have been related to the treatment conditions or the composition of starch and might also depend on the starch molecular chain length distribution, which should be further investigated. CYS–TPC showed the highest RS content (56.25 ± 1.37%), whereas the lowest RDS content (22.92 ± 0.14%) illustrated that tea polyphenol effectively resisted the digestion of starch. Furthermore, the hydrolysis rate  of CYS–TPC at 120 min was 45.08 ± 1.72%, lower than that of the other starches. This result might be attributed to the interaction between the starch and tea polyphenol, which led to a higher degree of aggregation of the starch molecules, Making it difficult for enzymes and water to enter the starch granules. Furthermore, tea polyphenol may bind with the active sites of the starch digestion enzymes, causing competitive inhibition [The in vitro digestive properties of the native and modified starches are presented in  content . Howeverhibition .The XRD patterns and relative crystallinity of the native and modified starches are shown in −1 were associated with O-H stretching and hydrogen bonds. The weaker intensity of the absorption peak at 3419 cm−1 reflected a lower content of O-H and higher content of hydrogen bonds [The FT-IR spectra of native and modified starches are presented in en bonds . Compareen bonds .−1 and 1022 cm−1 (R1047/1022) and the ratio between 1022 cm−1 and 995 cm−1 (R1022/995) of native and modified starches are summarized in R1047/1022 reflects a higher starch crystallinity. A smaller value of R1022/995 reflects a more ordered double helix structure of the starch [R1047/1022 values of the modified starches increased significantly, and the highest R1047/1022 value was in CYS–TPC, indicating that CYS–TPC had a more ordered crystalline structure. In particular, the R1047/1022 values of native and modified starches were ordered as CYS–TPC > CYS(II) > CYS(I) > CYS, which was consistent with the conclusion of the XRD analysis. Except for CYS(I), the R1022/995 values of CYS(II) and CYS–TPC were significantly lower than that of CYS, indicating that the double helix structure of CYS(I) was destroyed by high temperature, but the effect of the pullulanase and tea polyphenol might play a positive role in enhancing the double helix structure [R1022/995 value of CYS–TPC was lower than that of CYS(II), demonstrating that a more perfect crystalline structure formed with the interaction of tea polyphenols and starch.The ratio between 1047 cme starch . Comparetructure . FurtherThe morphological observation of native and modified starch granules scanned via SEM is shown in This study provides a theoretical basis for further research on the functional characteristics of the Chinese yam starch–tea polyphenol complex as a resistant starch. This study investigated the influences of tea polyphenol on pasting, thermal, in vitro digestive, and structural properties of Chinese yam starch using autoclave-assisted pullulanase processing. Tea polyphenol exhibited an inhibitory effect on the viscoelasticity of Chinese yam starch but had a positive role in enhancing thermal stability. Furthermore, tea polyphenols might interact with starch through non-covalent interaction, such as hydrogen bonds and hydrophobic interactions, according to the Fourier infrared spectroscopy and X-ray diffraction results. In particular, hydrogen bonding was speculated to be the main force of interaction between Chinese yam starch and tea polyphenol, which led to CYS–TPC presenting a B+V-type crystalline structure. Furthermore, the number of the RS fractions in the resulting CYS–TPC was effectively enhanced through the addition of tea polyphenol, causing the complex to show a lower digestion efficiency. In terms of its microscopic morphology, CYS–TPC was observed to be more porous with a looser microstructure and irregular cavities, which revealed that the starch gel structure was destroyed by the presence of tea polyphenol. However, the CYS-TPC in the study underwent several gelatinization treatments during preparation, which might significantly affect the physicochemical and structural properties of the CYS-TPC. Based on this situation, the degree of gelatinization in the physicochemical and structural properties of CYS-TPC should be further explored to supplement the shortcoming of this study. Additionally, in order to further improve the research content, future work is required to investigate the edible safety of CYS-TPC and the biological activity function of tea polyphenols within the complex.


Histologically, stratification of epithelial cells was formed without evidence of cytotoxic damage and the epithelial thickness measured was the same for all model tissues. The excellent biocompatibility of nanozirconia, as evidenced by the multiple endpoint analyses of the 3D-OMM, may indicate the potential of its clinical application as a restorative material.Over the years, advancement in ceramic-based dental restorative materials has led to the development of monolithic zirconia with increased translucency. The monolithic zirconia fabricated from nano-sized zirconia powders is shown to be superior in physical properties and more translucent for anterior dental restorations. Most in vitro studies on monolithic zirconia have focused mainly on the effect of surface treatment or the wear of the material, while the nanotoxicity of this material is yet to be explored. Hence, this research aimed to assess the biocompatibility of yttria-stabilized nanozirconia (3-YZP) on the three-dimensional oral mucosal models (3D-OMM). The 3D-OMMs were constructed using human gingival fibroblast (HGF) and immortalized human oral keratinocyte cell line (OKF6/TERT-2), co-cultured on an acellular dermal matrix. On day 12, the tissue models were exposed to 3-YZP (test) and inCoris TZI (IC) . The growth media were collected at 24 and 48 h of exposure to materials and assessed for IL-1β released. The 3D-OMMs were fixed with 10% formalin for the histopathological assessments. The concentration of the IL-1β was not statistically different between the two materials for 24 and 48 h of exposure ( In clinical applications, 3 mol % yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP) are widely utilized. This is because the yttria-partially stabilized tetragonal zirconia polycrystalline (Y-TZP) showed better mechanical properties and superior resistance to fracture than other conventional dental ceramics . The Y-TNanoparticles can exist between 1 and 100 nm ,5, and eSo far, toxicological studies on zirconia nanoparticles are limited, and the results are controversial. A study by Laiteerapong et al.  reportedApart from the cytotoxicity testing, it is also essential to investigate the immunogenic potentials of dental materials. Dental materials may provoke local inflammatory reactions of the native gingiva with the induction of pro-inflammatory cytokines. Cytokines are small-sized proteins, normally present in a minimal quantity in body fluids. However, the concentrations can amount to 1000-fold when associated with trauma or inflammation . CytokinThe effects of zirconia nanoparticles on the inflammatory response of the human gingival mesenchymal stromal cells (hG-MSCs) were investigated by Nemec et al . They diWith the exponentially increasing clinical use of nanoparticles , there iNanosized zirconia powders in this study were processed through a combined consolidation method, colloidal slip casting and cold isostatic pressing (CIP) to form zirconia dental blocks . Conside2O3 . The primary particle size of the nanopowder was approximated at 20 nm. Briefly, The Y-TZP nanopowder suspension was subjected to the slip-cast method followed by a cold isostatic pressing (CIP) method to produce the green bodies. Subsequently, the green bodies were pre-sintered at 1200 °C to produce the 3-YZP [Two types of 3 mol% yttria-stabilized tetragonal zirconia were used in this experiment: (1) Sirona inCoris TZI (IC) , which acted as control; (2) newly developed nanozirconia (3-YZP), prepared from tetragonal zirconia polycrystalline (Y-TZP) nano powder partially stabilized with 3 mol% of Yhe 3-YZP . Both ziThe surface roughness of the materials was evaluated using the atomic force microscope (AFM)  in contact mode. Six samples were used to determine the surface roughness of the materials. In brief, five random points were chosen in each sample, and these values were averaged.®, Plymouth, MA, USA).Before the experiments, all specimens were cleaned with an acetone solution for 20 min in an ultrasonic bath and immersed in the ascending concentrations of ethyl alcohol  each for 5 min. The zirconia specimens were also sterilized on both surfaces under ultraviolet radiation for 20 min  and human gingival fibroblast cells (HGF) (passage 4–5) used in this study were courtesy of Professor Dr Chai Wen Lin under her research grant supported by the Ministry of Higher Education High Impact Research Grant (UM.C/625/HIR/MOHE/DENT/05). This study received approval from the Research and Ethics Committee, Secretariat of Research and Innovation, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM PPI/111/8/JEP-2020-618).The OKF6/TERT-2 cell is an immortalized epithelial cell line processed by the forced expression of the telomerase. This cell line was chosen because it has been proven to resemble the primary oral keratinocytes . The cel®  was added to the tissue, to separate the connective tissue from the epithelium. The separated tissues were minced into small pieces of 1 mm × 1 mm in size using a scalpel blade in separate Petri dishes and transferred to culture flasks. The growth media in the flasks were changed every 2 days and subcultured once they had attained 70% confluency.The primary human gingival fibroblast cells were isolated from healthy gingival tissues obtained from the crown lengthening surgery. The primary cell was extracted from the gingival tissue using an explant technique. The Dispase5 HGFs and 5 × 105 OKF6/TERT-2 was co-cultured onto the basement membrane side of the rehydrated acellular dermal membrane . The 3D-OMM was developed based on the modification from the protocols developed by Chai et al. . A mixtu® Costar® Snapwell™ Insert, Corning Life Sciences, Corning, NY, USA). The membrane was rehydrated in a 5 mL Dulbecco’s phosphate buffer saline  for 30 s and then immersed in a 5 mL DMEM for 15 min. The 3D-OMMs were fabricated inside 12 mm diameter inserts with a 0.4 µm pore size and a 1 × 108 pore density/cm2 in a six-well plate. The seeded membrane was submerged in 3 mL of K-SFM and incubated for five days. On the sixth day, the tissues were raised at the air-liquid interface (ALI) to promote epithelial differentiation. The cells were allowed to be stratified further for up to seven days in the incubator, with the media changed every two days. On day 12, the samples (3-YZP and IC) were placed on top of the models. Prior to cell seeding, the membrane was cut into a round shape with a 12 mm diameter to fit into a 12 mm ring insert (CorningThe expression of pro-inflammatory cytokine (IL-1β) from cell culture supernatants of unexposed, 3-YZP-exposed and IC-exposed models were measured using an ELISA kit. Human IL-1β ELISA kit from Abcam PLC, Cambridge, UK  was used. The cell culture media were collected at 2 time points, after 24 h and 48 h of exposure.For the assay procedure, all the reagents, controls and samples were prepared based on the manufacturer’s instructions. The optical density was measured using a multi-plate reader  at 450 nm and correction at 570 nm. The concentration of IL-1β was expressed as pg/mL and compared with a standard curve provided by the manufacturer.Histological analyses of the 3D-OMMs were performed after 72 h of contact. The models were fixed in a 10% formalin solution for 24 h, processed for histological sections via dehydration in a graded series of ethanol concentrations (70–100%) and embedded in a paraffin block. Vertical sections of 5 µm thickness were cut and stained with hematoxylin and eosin staining (H&E staining).The resultant histology sections were evaluated for evidence of cytotoxic epithelial damage using the inverted microscope  at 20× and 40× magnification. The damage was manifested as the loss of the normal cell’s morphology, such as the separation of the epithelium from a scaffold and the presence of the pyknotic nuclei. Two examiners were assigned to examine the histological appearance of the models.B, Olympus Soft Imaging Solutions GmbH, Imaging Software, Muenster, Germany) at 20× magnification. A linear scale was placed perpendicular to the histology image. The distance was taken from the basement membrane’s basal surface to the epithelium’s uppermost keratinized layer. Measurements were taken at five random points per section, and these values were averaged.The thickness of the epithelial layers of all models was measured using the imaging software  between the two types of zirconia. The individual components of each sample and the surface roughness values of two zirconia discs are tabulated in p = 0.892). It is interesting to note that from this chart , followed by 3-YZP-exposed (115.34 ± 41) and IC-exposed (102.84 ± 41.2). The differences between these groups were not statistically significant, Welch’s F  =0.68, Surface roughness typically plays an essential role in cell attachment and proliferation ,28. In oThe 3-YZP and IC had acceptable tetragonal phase stability. The X-ray diffraction (XRD) test revealed a negligible amount of monoclinic zirconia after sintering ,11,32. ARecently, studies have incorporated the pro-inflammatory mediator’s assessment for cytotoxic changes in cells that directly interact with dental materials ,39,40. TThe in vitro studies of pro-inflammatory mediators among zirconia restorations, especially utilizing the 3D-OMMs, are limited. In line with our result, a study by Özen et al. , who invPresently, there is no direct evidence that an increased IL-1β released after exposure to ceramic indicates inflammatory reactions in vivo. The ceramics’ immunogenic potential was mainly reported in human clinical studies by estimating the inflammatory mediators in the gingival crevicular fluid (GCF) ,48,49. TIn this current research, we assessed the toxicity of the 3-YZP by placing it directly onto the uppermost surface of the oral mucosal models as an experiment by Moharamzadeh et al. . HistoloOur analysis of epithelial thickness revealed that the epithelial thickness was the highest for the untreated models, followed by the 3-YZP-exposed and IC-exposed models. However, the one-way Welch ANOVA failed to demonstrate the statistically significant difference between these groups. Furthermore, the reduction in the epithelial thickness of the model correlated with the increased rate of cell apoptosis . Hence, With the increasing clinical use of nanoparticles, there is a need to identify risks concerning human health . CurrentThe in vitro biocompatibility assessment is crucial for the clinical validation of dental materials, which allows the evaluation of many samples simultaneously. The evidence regarding the biocompatibility of zirconia on monolayer cells has been discussed extensively in the literature. The use of 3D-OMM could serve as transition testing between monolayers, to preclinical testing, where mucotoxicity and genotoxicity may resemble those in vivo due to multiple cellular interactions that could be observed. Nevertheless, the evidence of zirconia toxicity on oral mucosal equivalent is limited. Using the 3D-OMM would allow the investigation of the inflammatory mediators and the histological evaluation of the changes in soft tissue in response to the toxicity of dental nanomaterials. Additionally, the tissue culture assays will provide a controllable and repeatable method of assessment. Hence, the findings reported from the analysis of the oral mucosal model in this study are more clinically applicable than the monolayer cell culture.However, this study also had some limitations. The histological analysis of the models was assessed by more than one investigator who was not blinded to the nature of the treatment the tissue had received. This could have potentially caused bias. Furthermore, only one pro-inflammatory cytokine was reported in this current research. The potential use of other interleukins, such as IL-6 , IL-8 anIn addition to that, the nanoparticle’s size, shape, surface area, agglomeration state, chemical composition and surface charge may influence their toxicity and biological responses. Among the various microscopy method for physicochemical characterization, the scanning electron microscope, transmission electron microscope (TEM) and atomic force microscope are frequently used. With this regard, TEM can be used to evaluate the permeation effects of the cell in contact with nanomaterials. Meanwhile, single-particle ICP-MS can be used for information on the size and size distribution of nanoparticles or the number of leachable elements from the nanozirconia. Even though both the zirconia were stabled following sintering, the oxide bonds of the ceramic could be damaged by the ageing or LTD of the zirconia. A study had shown that some yttrium ions were leaching out from the commercial zirconia in the corrosion test . NeverthWithin the limitations of this study, the excellent biocompatibility of the 3-YZP, as shown from the biological endpoint analyses of the 3D-OMM, may indicate the potential of its clinical application as a dental restorative material. It was found that 3-YZP supported oral fibroblasts and keratinocytes’ cellular attachment and proliferation. There was no cytotoxic damage to the human oral mucosal equivalent, as demonstrated histologically with the preservation of the epithelium layer. However, the 3-YZP had the potential to modulate the secretion of IL-1β, similar to the currently used zirconia (IC).


Sepiolite clay is a natural filler particularly suitable to be used with polysaccharide matrices , increasing their attractiveness for a wide range of applications, such as packaging. Herein, the effect of the processing  and of the sepiolite filler amount on the microstructure of starch-based nanocomposites was investigated by SS-NMR (solid-state nuclear magnetic resonance), XRD (X-ray diffraction) and FTIR (Fourier-transform infrared) spectroscopy. Morphology, transparency and thermal stability were then assessed by SEM (scanning electron microscope), TGA  and UV–visible spectroscopy. It was demonstrated that the processing method allowed to disrupt the rigid lattice structure of semicrystalline starch and thus obtain amorphous flexible films, with high transparency and good thermal resistance. Moreover, the microstructure of the bio-nanocomposites was found to intrinsically depend on complex interactions among sepiolite, glycerol and starch chains, which are also supposed to affect the final properties of the starch–sepiolite composite materials. Starch is a biopolymer composed of two D-glucose homopolymers: amylose, characterized by linear chains based on α-D1-4)-glucan bonds, and amylopectin, which is essentially a highly branched amylose through α-D(1-6) links -4-glucan. Starch 4, giving the chemical formula [Si12O30Mg8 (OH)4(OH2)4 nH2O]. In sepiolite, the edges are shared with the neighboring ones, resulting in a “checkerboard” pattern with the remarkable formation of tunnels , XRD (X-ray diffraction), FTIR (Fourier-transform infrared) spectroscopy, and SEM (scanning electron microscope). Thermal stability and transparency of the composite films were investigated by TGA  and UV–Vis spectroscopy, respectively.A yuca starch powder originated from Colombia was used. Glycerol  was also analyzed.First, 2.5% f) were prepared by adding 40% w/w of glycerol with respect to starch before proceeding with gelatinization as described above.Plasticized starch films (YGw/w with respect to starch), sepiolite was suspended in water and mixed overnight with a magnetic stirrer at room temperature prior to starch and glycerol addition. The suspension was then stirred at 70 °C for 30 min to achieve gelatinization. The film-forming solution obtained in this way was cast onto a polystyrene Petri dish and oven-dried at 40 °C. The obtained films were about 50 µm thick. Samples labels and compositions are reported in To prepare nanocomposite film samples with different filler contents  were manufactured without glycerol and were analyzed only by 29Si CPMAS NMR.In addition, two film samples of starch and sepiolite  400WB spectrometer operating at a proton frequency of 400.13 MHz under the following conditions for cross polarization magic angle spinning (CPMAS) experiments: n et al. . Line shd-spacing in sepiolite composites, XRD was also carried out in asymmetric scattering with a fixed incident angle of 1.5°, with the other parameters unchanged. In this case the film was fixed to the sample holder by a small piece of modeling clay to avoid misalignment. The asymmetric scattering was necessary to eliminate any reflections that would have come from the modeling clay.Long-range order was analyzed by means of XRD analyses performed on a Rigaku  DMAX III diffractometer in Bragg–Brentano geometry, equipped with a Cu source (λ = 1.54056 Å) in the following conditions: 2θ range from 2° to 45°, steps of 0.05° and 3 s counting time. Crystallinity was evaluated following the methodology proposed by Lopez-Rubio et al. . In orde2 > 0.98.Profile fitting analysis of XRD patterns were performed with Origin Pro 2018. Results were considered acceptable with R−1 range, with 4 cm−1 resolution. The crystalline-sensitive region of the spectra 1200–800 cm−1 was isolated, baseline corrected with a straight line and fitted with Gaussian peaks; then, the area ratio of 1040 cm−1/1016 cm−1 peaks was calculated.Changes in conformational order were investigated by Fourier-transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode on a Nicolet iS50 spectrometer , equipped with a Smart iTX-Diamond ATR accessory. Spectra were averaged over 32 scans in the 4000–550 cm2 > 0.98.Profile fitting analysis of FTIR-ATR spectra was performed with Origin Pro 2018. Results were considered acceptable with RUV–Vis spectroscopy was carried out with a JENWAY 6850 UV/Vis spectrophotometer .SEM analyses were performed by a Vega TS5136 XM Tescan  microscope in a high-vacuum configuration. The electron beam excitation was 30 kV at a beam current of 25 pA, and the working distance was 12 mm. In this configuration, the beam spot was 38 nm. The samples were applied on carbon tape onto an aluminum substrate and covered with gold coating.2 flux. The materials were equilibrated at 30 °C for 15 min and then heated from 30 to 1000 °C at the rate of 10 °C min−1.The TGA was performed using a Mettler Toledo  TGA/DSC1 STARe system under NThe film-forming solution was prepared by gelatinization, a process in which the suspension of starch granules in water undergoes the action of temperature and the swelling of amorphous regions, resulting in destruction of crystalline domains . Through13C CPMAS NMR spectra of pristine starch powder (Yp), starch film (Yf) and plasticized starch film (YGf) samples are reported in The p and Yf and YGf film samples. Yp was characterized by a shoulder at 102.9 ppm attributed to a V-type polymorph [f and YGf film presented a completely different shape of C1 site, typical of amorphous starches [The most interesting sites are C1 and C4, as they are involved in glycosidic bonds, and they are the most sensitive to starch conformations, since they are defined by the geometry of glycosidic linkages ,26. As aolymorph ,26 and tolymorph ,25. Fromstarches ,26.f sample presented glycerol resonances, namely CH2-OH at 63.8 ppm and the CH-OH at 72 ppm, which is overlapped to C2,3,5 signals of starch [The line shape of C2,3,5 substantially changed as the components merged together into a broader and less resolved signal. It is worth noting that the C4 site, the intensity of which relates to the amorphous part of starch , increasf starch ,28.p at 2θ 9.97°, 11.24°, 14.95°, 17.10°, 17.80°, 23.00° and 26.42°, with the V-type reflection at 2θ 19.94° [p pattern  and starch macromolecules [2 stretching vibrations were clearly visible with peaks at 2925 (C-H asymmetric stretching) and 2884 cm−1 (C-H symmetric stretching). In the 1500–1200 cm−1 range, the overlapped bands of CH2 bending and some COH vibrations are found. The most interesting region is the one delimited by the dashed rectangle in −1), characteristic of the C-O vibrations in COH and COC groups of starch. Particularly, peaks at 1150, 1125 (barely visible in processed starches) and 1103 cm−1 are related to C-O, C-C and C-OH stretching modes, while signals at 1076, 1040, 1016, 992 and 926 cm−1 are due to C-O-H bending and CH2-related modes [−1 are related to crystalline domains, while the peak at 1016 cm−1 is related to the amorphous regions [Comparing the FTIR-ATR spectra of Y samples , the effolecules . The CH2ed modes ,32. Preced modes . Howevered modes . In deta regions ,33.p, a shoulder was clearly visible at 1045 cm−1, while for film samples it was much less pronounced, with a shift toward 1040 cm−1. Moreover, for film samples, the 1016 cm−1 peak increased in intensity with respect to the 992 cm−1 peak. These differences suggested structural modifications as a consequence of processing.In Yp is reported in −1 peaks, which can alter their intensity ratio [p and film samples with or without fillers. Results of 1040/1016 peak ratio (p (1.77), Yf (0.80) and YGf film (1.01), in line with starch amorphization, as a consequence of film processing.An FTIR profile-fitting analysis was carried out to better highlight the peaks attributed to crystalline and amorphous structures . Uniform and transparent films were obtained (f) was compared with a starch film (Yf) and a plasticized starch film (YGf). In the visible range, the effect of the addition of glycerol or sepiolite is negligible. In YG15Sf, an absorbance peak below 300 nm is detected, due to sepiolite Mg-O-Si bonds [Starch-based nanocomposite films were prepared by adding sepiolite in different contents . The sepiolite clay filler, with its characteristic needle-like shape, is well-dispersed in the starch matrix, and its original dimensions are retained in the composite films. However, some agglomerates could be detected when 15% of sepiolite was introduced , but the peak became broader in the composite films with a reduction in intensity compared to the YGf sample. The proportion between the C6 area and glycerol peak remained almost constant among the composite samples; however, the broadening of the glycerol CH2-OH peak might indicate less chain mobility or increased anisotropy compared to YGf film, suggesting glycerol–sepiolite interactions [The microstructure of the nanocomposite films was investigated by SS NMR, XRD and FTIR-ATR analyses. The ractions .n describes the SiO4 unit with n Si-O-Si oxo-bridges. Briefly, sepiolite structure is characterized by four types of silicon atoms: one Q2 and three Q3. The three well-resolved resonances accounting for Q3 Si atoms correspond to their different position  were manufactured without glycerol to study the shape of the Si1 peak. The 29Si CPMAS NMR spectra of the composites, reported in Driven by these hypotheses, two film samples of starch and sepiolite  reflections at 19.8° in the film samples, which appears higher than in pristine sepiolite.13C CPMAS NMR results suggest the occurrence of the onset of filler-preferred orientation in the bio-composites. This could be due to interactions with starch leading to sepiolite arrangement into the matrix, as recently reported by some of us for sepiolite–rubber nanocomposites [A similar behavior is shown by the (080) peak at 26.3°. It should be mentioned that Chivrac et al.  attributmposites .f , Si-O (1057 and 1000 cm−1), SiOH (976 cm−1) and MgOH (689 cm−1) [−1 increased with increasing of sepiolite content (it was three times higher in the YG15Sf film than the YG3Sf film). This might be explained by the increasing presence of Si-OH bond vibration, possibly confirming that some signals were hidden by starch vibrations.From FTIR-ATR analyses, the spectra of the composites resulted very similar to the one of YGf . Profilea ratios  were com5 and T20, corresponding to the temperature at which the sample weight losses are 5% and 20%, respectively, and the residual weights.The results of the thermogravimetric analysis performed on the film samples are shown in f, showed two main weight loss events, the first close to 100 °C, due to loss of adsorbed water, and the second around 300 °C, attributed to the decomposition of starch. The glycerol containing films showed a reduced amount of adsorbed water. However, an additional weight loss event, between 150 °C and 260 °C, appeared, lowering the thermal stability of the plasticized films YGf with respect to the non-plasticized starch film Yf, as demonstrated by the lower T20 . These different behaviors confirm the existence of complex interactions between sepiolite, glycerol and starch chains. Finally, the residual weights are consistent with the inorganic fraction of the samples, increasing with the filler amount.The non-plasticized unfilled starch film sample, Yower T20 . Such beIn this study, the effect of starch processing  and the impact of different filler amounts on the starch microstructure were evaluated through SS-NMR, XRD and FTIR-ATR techniques. While the crystallinity of the pristine starch was about 45% during processing, after gelatinization, starch became amorphous and remained amorphous also after the addition of glycerol and fillers. The obtained bio-nanocomposite films were transparent in the visible range, and the filler was uniformly distributed in the starch matrix. In the bio-nanocomposites, a strong interaction between plasticizers and starch hydroxyl groups was highlighted by FTIR-ATR analysis. The polysaccharide interacted also with sepiolite, inducing a preferential orientation of the filler along crystallographic planes {0k0}, as suggested by XRD analyses. Moreover, glycerol interacted with sepiolite by partially substituting zeolitic water present inside sepiolite’s channel. While plasticization with glycerol decreased the thermal stability of starch, the addition of sepiolite allowed recovering of the initial thermal resistance. Furthermore, the transparency of the composite films in the visible range was not affected by the highest amount of sepiolite filler. The present study demonstrates the existence of complex interactions between sepiolite, glycerol and starch chains that must be totally understood to tune the final properties of the composite materials.


Bacillus and Acinetobacter, two dominant microbial genera in Daqu, produce enzymes and organic acids that influence the Daqu quality. However, there are no rapid analytical methods for detecting Bacillus and Acinetobacter. We designed primers specific to the genera Bacillus and Acinetobacter to perform genetic comparisons using the 16 S rRNA. After amplification of polymerase chain reaction using specific primers, high-throughput sequencing was performed to detect strains of Bacillus and Acinetobacter. The results showed that the effective amplification rates for Bacillus and Acinetobacter in Daqu were 86.92% and 79.75%, respectively. Thus, we have devised and assessed a method to accurately identify the species associated with Bacillus and Acinetobacter in Daqu, which can also hold significance for bacterial typing and identification.Daqu is a spontaneous, solid-state cereal fermentation product used for saccharification and as a starter culture for Chinese Baijiu production.  Bacillus and Acinetobacter play important roles. Bacillus can hydrolyze proteins and starch . Primer synthesis was completed by Sangon Biotech (Shanghai) Co., Ltd.Daqu samples were obtained from mature Daqu at the Shandong Lanling Fine Wine Co., Ltd. production plant. Five Daqu samples from five different points in each room were taken, crushed, mixed as parallel samples , placed DNA was extracted using the FastDNA SPIN Kit for Soil. First, the solid distillate was lysed, and 500 mg of soil was added to the Lysing Matrix E tube (Lysed DNA) and mixed with the reagents. The mixture was mixed in the FastPrep apparatus for 40 s at a speed setting of 6.0. The supernatant was separated by centrifugation, and 250 μL of PPS was added. The supernatant was separated by centrifugation and the DNA was eluted by adding l mL of the Binding Matrix Supension, while the DNA binding matrix was left in place. Subsequently, we removed the supernatant and transferred 500 μL the mixture to the SPINTM Filter, which was centrifuged and air-dried. Finally, 50 μL of DES was added to elute the DNA (the reagents mentioned in this section are included in the FastDNA SPIN Kit for Soil).A total of 500 mg of Daqu was placed in a sterilized 2 mL tube, 1× PBS solution was added, and the mixture was shaken and mixed. The sample was centrifuged at 13,000 RCF for 3 min at 25°C, and the top layer was discarded. The 2 mL centrifuge tube was inverted onto blotting paper for 1 min until no liquid was further removed. The first round of PCR amplification was performed after genome extraction using the FastDNA SPIN Kit for Soil Gene Extraction. We used 16 S V3 to V4 zone  universaBacillus subtilis 16 S rRNA was used as the template. 16 S rRNA is relatively conserved in structure and function and has changed relatively little during evolution. Although the 16 S rRNA gene sequence is relatively conserved, there are still differences in the 16 S rRNA gene sequence in different microorganisms. In total, 100 16 S rRNA genes of Bacillus were searched using NCBI GenBank, and multiple sequence matching was performed using Clustalw to identify gene regions that are highly conserved in the 16 S rRNA of Bacillus. Using the identified gene region as a template, Clustalw was used to design primer pairs specific for Bacillus. The sequence of this primer pair was: F: 5′-GTC TGT AAC TGA CGC TGA GGC-3′, R: 5′-GCG ATT ACT AGC GAT TCC A-3′. The primers were used to amplify the Daqu DNA.Acinetobacter 16 S rRNA was used as the template. NCBI GenBank was used to search for 100 16 S rRNA genes of Acinetobacter, and multiple sequence comparisons were conducted using Clustalw to identify highly conserved gene regions in Acinetobacter encoding 16 S rRNA. Using the identified gene region as a template, specific Acinetobacter primer pairs were designed using Clustalw. The primer pair sequences used were: F2: (5′-ATG TGA AAT CCC CGA GCT T-3′) and R2: (5′-AGT TTG TCA CTG GCA GTA TCC T-3′). Two specific primers with high homology in Bacillus and Acinetobacter, but with little homology in non-Bacillus and non-Acinetobacter with similar sequences, were used for amplification. .Bacillus primers used were F: 5′-GTC TGT AAC TGA CGC TGA GGC-3′, R: 5′-GCG ATT ACT AGC GAT TCC A-3′, and Acinetobacter primer pairs included F2: 5′-ATG TGA AAT CCC CGA GCT T-3′, R2: 5′-AGT TTG TCA CTG GCA GTA TCC T-3′, which were used to process and analyze Illumina MiSeq high-throughput sequencing data from Bacillus and Acinetobacter, respectively.The PCR amplified sample (10 µL) was analyzed on a 2% agarose gel along with the DL 2000 Plus DNA Marker. The Vazyme Gel Extraction Kit was used to recover the PCR amplification products. The Firmicutes (82.67%), Proteobacteria (13.22%), Actinobacteria (7.06%), and Bacteroidetes (1.65%). Firmicutes were the dominant group of bacteria in the macrophyte samples (The community structure of the Daqu samples at the phylum level was determined based on taxonomic information of the species obtained from the different microbes within the sample . At the  samples .Bacillus (69.61%), Weissella (34.33%), Lactobacillus (22.07%), Thermoactinomyces (19.9%), and Acinetobacter (11.56%). Bacillus was the dominant genera in the Daqu samples. The protein and starch in Daqu can be decomposed by Bacillus, and the aromatic substances in Daqu mainly originate from its action.The community structure of the Daqu samples at the genus level was analyzed from the taxonomic information of the species in the samples . In the Bacillus and Acinetobacter were 626 and 536 bp, respectively, as shown in Bacillus and Acinetobacter were specific.DNA was extracted from Daqu samples. The PCR amplification products of The amplified bands were purified using a Vazyme Gel Extraction Kit, and the purified products were sent to Sangon Biotech (Shanghai) for sequencing using the Illumina MiSeq PE300 platform and for high-throughput data processing and analysis.Bacillus mostly contains macromolecules and produces proteases, amylases, and cellulases. We designed specific primers for Bacillus, performed specific primer PCR to amplify 16 S rRNA from only one microorganism, and verified the specificity of these primers using high-throughput sequencing . All six Bacillus species, namely, B. velezensis (82.07%), Bacillus paramycoides (2.89%), Bacillus licheniformis (1.6%), Bacillus sp. (0.25%), Bacillus coagulans (0.086%), and Bacillus ginsenggisoli (0.022%), belonged to the phylum Firmicutes, with Bacillus velezensis being the dominant strain.The results in quencing . We idenAcinetobacter was the dominant bacterial group in Daqu. We designed specific primers for Acinetobacter, performed PCR to selectively amplify the 16 S rRNA, verified the specificity of these primers after amplification, and conducted high-throughput sequencing, as shown in Acinetobacter by high-throughput sequencing and the resulting sequences were uploaded to NCBI database (PRJNA985236). These five species were classified as Proteobacteria at the phylum level and Acinetobacter at the genus level. The five species were A. baumannii (74.34%), Acinetobacter calcoaceticus (3.17%), Acinetobacter septicus (1.01%), Acinetobacter sp. GeSchum1Abdr4 (0.94%), and Acinetobacter bereziniae (0.29%), with Acinetobacter baumannii being the dominant strain.As shown in Bacillus and Acinetobacter) were selected for further high-throughput sequencing. We found that Bacillus detected in Daqu included six species: B. velezensis (82.07%), B. paramycoides (2.89%), B. licheniformis (1.6%), Bacillus sp. (0.25%), B. coagulans (0.086%), and B. ginsenggisoli (0.022%). Bacillus species are protein hydrolase and amylase producers , A. calcoaceticus (3.17%), A. septicus (1.01%), Acinetobacter sp. GeSchum1Abdr4 (0.94%), and A. bereziniae (0.29%) via high-throughput sequencing. Acinetobacter, the dominant bacterial genus in white wine barley, oxidizes glucose to produce acetic acid, which is one of the main flavor components in Baijiu. Acinetobacter, which is widely distributed in nature, is an aerobic, power-negative, gram-negative Coccobacillus that has been the focus of several research studies in the medical field  and R2 (5′-AGT TTG TCA CTG GCA GTA TCC T-3′) for Acinetobacter are useful for high-throughput second-generation sequencing analysis. This method can be used to detect more types of Acinetobacter and better understand their role as dominant strains in Daqu.In this study, we identified five species of al field . Actinobibiotics . ActinobClostridium 16 S rRNA using specific primers and determined the genus specificity of the bacteria, enabling distinction between strains. By ensuring genus specificity, data can be improved, especially for 16 S rRNA-based typing of bacteria , R (5′-GCG ATT ACT AGC GAT TCC A-3′) and Actinobacter F2 (5′-ATG TGA AAT CCC CGA GCT T-3′), R2 (5′-AGT TTG TCA CTG GCA GTA TCC T-3′). The results showed that the prevalence of Bacillus and Acinetobacter in Daqu was 86.92% and 79.75%, respectively.bacteria . Liu et Bacillus, additional information for different species can be obtained, along with a more accurate analysis of the species level. Our present study demonstrates that this method can be used as a rapid detection method when designing specific primers for other species. Relative to high-cost flow cytometry and long-cycle bacterial sequencing, specific primer PCR can amplify the required strains in a short time at a low cost and achieve 79.75% accuracy, making it a widely applicable method.In summary, we performed PCR using specific primers to identify the species of various microorganisms and obtain nucleotide sequences of the bacteria, demonstrating an important method for bacterial typing. Compared with traditional 16 S rRNA taxonomic sequencing and high-throughput sequencing with specific primers, the method used in this study can reveal more phylum and genus classes. Therefore, for specific microbial genera such as


The purpose of this study was to evaluate the differences in the patient-reported functional outcomes, and graft failure in revision ACL reconstruction using quadriceps tendon (QT), Hamstring tendon (HT) and bone-patellar tendon-bone (BPTB) autografts.Between 2010 and 2020, 97 patients who underwent revision ACL reconstruction  met the inclusion criteria. Pre-injury and at 2-year postoperatively patients were evaluated for patient-reported functional outcomes; Lysholm knee score, Tegner activity level and VAS  for pain; and graft failure. Patient-reported outcomes and graft failure were compared between the QT, HT and BPTB groups. The patients with graft failure were not included for outcome analysis at 2-years of follow-up.All three revision groups with QT, HT and BPTB autograft did not differ significantly in terms of age, sex, time from injury to surgery, concomitant injuries and single-stage or double-stage procedures (n.s.). No significant difference was found in the pre-injury patient-reported outcome; Lysholm knee score, Tegner activity and VAS for pain (n.s.) between the three groups. At the 2-year follow-up functional outcomes improved in all three groups and all the patients returned to pre-injury activity level; however, no significant difference was found in functional outcomes at the 2-year follow-up between the three groups (n.s.). Graft failure occurred in 4 (10%), 5 (19%) and 3 (10%) patients of QT, HT and BPTB groups, respectively. However, the rate of failure did not differ significantly between groups.All three autografts  demonstrated satisfactory patient-reported outcomes in revision ACL reconstruction. Compared with QT and BPTB grafts, HT graft showed a higher tendency for failure rates. With the increasing incidence of revision ACL reconstruction, surgeons should be aware of all the available graft options. The findings of this study will assist the surgeons in the graft selection for revision ACL reconstruction.Level III. Various patient-related factors such as age, sex, and tibial slope; surgeon-related factors such as graft size, graft type, and tunnel placement; other factors such as activity level, concomitant injuries, and timing of return to sport are associated with retear rates after ACL surgery , 7. GrafHamstring tendon (HT) and bone-patellar tendon-bone (BPTB) are the two most commonly used grafts and both grafts provided satisfactory outcomes , 16, 29.In recent years, Quadriceps tendon (QT) graft has gathered enthusiasm due to lower donor site morbidity than HT and BPTB, lower failure rate than HT, greater mean cross-sectional area compared to BPTB and greater load to failure compared to other grafts , 20, 28.However, as far as the authors’ knowledge, no study is available that compares the functional outcome and graft failure with QT, HT and BPTB autograft in revision ACL reconstruction. Thus, the purpose of this study was to evaluate the differences in the patient-reported functional outcomes, and graft failure in revision ACL surgery using QT, HT and BPTB autografts. The hypothesis was that all three grafts would have similar functional outcomes but higher graft failure would be associated with HT compared to QT and BTBP in revision ACL reconstruction. The findings of this study will assist the surgeons in the graft selection for revision ACL reconstruction.The study was performed at Gelenkpunkt—Sports and Joint Surgery, FIFA Medical Centre of Excellence and approved by the ethics committee of the Medical University of Innsbruck (AN2015-0050). Prospectively collected data were obtained from an ACL registry. Patients were included in the study if they fulfilled the following inclusion criteria: revision ACL reconstruction using QT, HT, BPTB autograft; age 18–55 years; and a minimum of 2-year follow-up. The exclusion criteria were: primary ACL reconstruction; contralateral knee injuries; utilization of allograft or graft other than QT, HT, BPTB; inflammatory arthritis or osteoarthritis; less than 2-year of follow-up and conditions that might interfere with the standard postoperative rehabilitation protocol.Pre-operative CT (computed tomography) scan with 3D reconstruction was performed for all the patients to determine femoral and tibial tunnel positions, tunnel widening and tunnel convergence. The present study followed the classification for ACL revision published by Fink et al. . Based oIn most cases, a rectangular femoral tunnel was used . BesidesBetween 2010 and 2020, 97 patients who underwent revision ACL reconstruction  met the inclusion criteria. All surgeries were performed by two fellowship-trained knee specialists. Patients were specifically asked to fill out the questionnaire considering their pre-injury state (just before failure of primary reconstructed ACL), during the first week of revision surgery for the baseline functional scores. Similarly, patients were evaluated at a 2-year follow-up for Lysholm knee score, Tegner activity level and VAS  for pain; and graft failure. Patient-reported outcomes and graft failure were compared between the QT, HT and BPTB groups. The patients with graft failure were not included for outcome analysis at 2-years of follow-up.α level with p = 0.05, a power of 80%, and an effect size of 0.20 it was estimated that 63 subjects would be needed in order to detect a statistically significant difference in Lysholm knee score. The sample size calculation was performed with the use of the G-Power software .A priori power analysis was performed to determine the appropriate sample size for the study. Considering an p value less than 0.05 was indicative of statistically significant differences. The statistical analysis was performed with XLSTAT (Addinsoft SARL) software packages.Data were retrieved and organized using an Excel sheet . Categorical variables were expressed in numbers and percentages (%). Continuous variables were expressed by average and standard deviation (SD). The normal distribution of variables was verified through Shapiro–Wilk test. Variables were not normally distributed therefore; nonparametric tests were used for the comparison of variables. Specifically, the Mann–Whitney test was used for unpaired samples, when analyzing the variables between pre-injury level and follow-up in each group. The Kruskal–Wallis test was used to analyze continuous variables between groups. The chi-square statistic test was used to determine differences in nominal data between groups, with Yates correction applied when the frequency of observations was below 5. A Demographic details and characteristics of the study population can be found in Table . All thrp = 0.01 and p = 0.04). Although improvement was noted in Lysholm score in either QT and HT groups, it did not reach statistical significance at a 2-year follow-up (n.s.) . Similarly, VAS for pain also improved and reach to pre-injury level in all three groups. No significant difference was noted in VAS at 2-year compared to the pre-injury level (n.s.) in QT and HT groups. However, in BPTB groups VAS improved significantly at 2-year follow-up compared to pre-injury level. Graft failure occurred in 4 (10%), 5 (19.2%) and 3 (9.7%) patients of QT, HT and BPTB groups respectively. However, the rate of failure did not differ significantly between groups (n.s).The most important findings of this study were that after revision of ACL reconstruction satisfactory improvement was noted for patient-reported functional outcomes  in all the groups. No difference was found in functional outcomes between the three groups at the final follow-up. All the patients returned to the pre-injury activity level. A tendency for higher graft failure was noted with HT (19.2%) than QT (10%) or BPTB (10%) grafts. To the best of the author’s knowledge, this is the first study comparing all three autografts; QT, HT and BPTB for revision ACL reconstruction.The incidence of ACL reconstruction is increasing over the past few decades. Excellent results after primary ACL reconstruction allow the patients to participate in highly demanding activities such as jumping, cutting, deceleration, and pivoting sports . These aIn the present study, the Lysholm, Tegner activity levels and VAS for pain scores improved and reached pre-injury levels in all three groups. No significant difference was noted in patient-reported outcomes between the three groups at a 2-year follow-up. Mouarbes et al.  noted noInterestingly, a significant improvement from pre-injury to final follow-up was seen in the BPTB group. In their recent study, Yumashev et al.  also fouThe choice of graft in revision ACL reconstruction may be limited based on previously used grafts and it depends on various factors such as age, level of sports activities, previous tunnel position, tunnel enlargement, graft used in primary ACL surgery and surgeon’s preference , 21. GraGraft choice influences graft failure . MulticeThe current study found a higher tendency of graft failure with HT than with QT and BPTB autograft, although the graft failure rate was not significant between the 3 groups. Both QT and BPTB have a higher graft diameter and strength compared to an HT graft which may explain the higher failure rate of HT grafts . In theip < 0.001) in 2015–2020 compared to 2010–2014.QT autografts are used far less commonly than HT and BPTB grafts . One of In the current study, graft failure was similar in QT and BPTB groups. In their meta-analysis, Riaz et al.  reportedThere are a few limitations of the present study. The first and the most important limitation is the small sample size, that underpowered to identify any difference in functional outcomes and graft failure. Revision ACL reconstruction is a less frequently performed procedure than primary reconstruction. Second, this was a retrospective analysis of patient-reported subjective outcome measures; however, all data were collected prospectively. A prospective study considering objective scores along with subjective scores should be conducted.The clinical relevance of the present study lies in the fact that the incidence of revision ACL reconstruction is increasing and surgeons should be aware of all the available graft options. QT autograft is the least studied and least used graft compared to other grafts, especially for revision ACL reconstruction. Many surgeons do not even consider the QT as a possible graft option when discussing with the patients. Promising clinical results continues to emerge concerning the viability of QT autograft in revision ACL reconstruction. Based upon the findings of this study surgeon can counsel and advise the patient regarding the graft choice for revision ACL reconstruction.All three autografts  demonstrated satisfactory patient-reported outcomes in revision ACL reconstruction. Compared with QT and BPTB grafts, HT graft showed a higher tendency for failure rates. With the increasing incidence of revision ACL reconstruction, surgeons should be aware of all the available graft options. The findings of this study will assist the surgeons in the graft selection for revision ACL reconstruction.


Social media use has exploded in popularity over the past decade with over 1.5 billion users on Facebook and 320 million users on Twitter. The aim of this study was to analyze the use of social media by orthopaedic journals and determine whether a relationship exists between social media followers and journal impact factor.The Clarivate Analytics Impact Factor tool was used to identify all orthopaedic journals with a 2022 impact factor of greater than 1.5. We then conducted a query on Instagram, Twitter, LinkedIn, and Facebook to determine which programs had pages on each platform.P = 0.003). When analyzing each social media platform independently, we found a significant correlation between the number of Facebook and Twitter followers and journal impact factor .Seventeen journals were included across all orthopaedic subspecialties. Of the 17 journals, 14 (82.4%) had a Facebook page, eight (47%) had an Instagram page, 15 (88.2%) had a Twitter account, and 8 (47%) had a LinkedIn profile. When compiling the number of followers by social media platform, Twitter had the most , followed by Facebook , Instagram , and LinkedIn . We found a significant correlation between the number of social media followers and journal impact factor (Pearson correlation coefficient [PCC] = 0.67; We have shown a notable association between the number of social media followers and a journal's impact factor. With the increasing shift toward online distribution, orthopaedic journals may use our data when evaluating their social media strategy to maintain and potentially increase their exposure and potentially their impact factor. We then conducted a query on Instagram, Twitter, LinkedIn, and Facebook to determine which journals had pages on each platform. Once the social media accounts were identified, we used the first post to provide us with the start date for the account. In addition, we calculated the number of posts and noted the most recent post on the platform. This was repeated for each journal, independently. All social media websites were accessed on November 9, 2022. The data were then compiled into Microsoft Excel. Analyses were conducted using Minitab, and significance was set at Seventeen journals were included across all orthopaedic subspecialties Table . Of the P = 0.003) (Table P = 0.13). When analyzing each social media platform independently, we found a significant correlation between the number of Facebook and Twitter followers and journal impact factor . We did not find a significant correlation between the number of Instagram or LinkedIn followers and journal impact factor .We found a significant correlation between the number of social media followers and journal impact factor (Pearson correlation coefficient (PCC) = 0.67; The aim of this study was to determine whether there was a correlation between an orthopaedic journal's impact factor and their activity on social media. We did find a statistically significant correlation between impact factor and the number of social media followers.10 Journals with high impact factors publish articles that are cited more often than journals with lower impact factors.11 The journal impact factor is calculated by taking into account the average number of times articles from the journal are cited over 2 years.11 It is specifically calculated by dividing the number of citations by the total number of articles published in the previous 2 years.11 The greater exposure a journal has, the more likely their articles will be cited, increasing the journal's impact factor. Our results support this because there is a positive correlation between the number of followers on social media and a journal's impact factor. This is likely because of the increased exposure the journal receives because of a larger social media following.A journal's impact factor is used to sort or rank it by relative importance.5 They found that social media influence was highly concordant with academic productivity as measured by the h-index.5 Our findings are similar as we found that a larger number of social media followers are associated with higher journal impact factors.A recently published study identified the top 100 social media influencers within orthopaedics and related their social media influence to academic influence.1 published a study aimed to determine which types of online activities are most prevalent in orthopaedics, to identify potential factors associated with higher counts of online mentions and standard citations, and to explore a complementary approach to measuring overall scholarly influence on the basis of online activity and citations. They found that online scholarly influence in orthopaedics is dominated by activity on Twitter and Facebook and is associated with a longer time since publication, higher journal impact factor, less risk of bias, and higher author h-index values. In this study, the authors included randomized controlled trials and did assess individual journals independently. Regardless, our findings are similar because Facebook and Twitter are the main sites used by orthopaedic journals and an increased number of followers on those platforms are associated with a higher journal impact factor.Evaniew et alThe main strength of our study is its novelty. To our knowledge, this is the first and only study in the literature to evaluate social media activity by medical journals. Furthermore, we are the first study to associate the number of followers on social media with an increased journal impact factor. The major weakness of our study is, although our findings are statistically significant, a journal's impact factor is likely a result of many factors, not just a following on social media.In conclusion, we detail the use of social media by the top 17 orthopaedic journals across multiple social media platforms. In addition, we have demonstrated a statistically significant correlation between the overall number of social media followers and a journal's impact factor. Specifically, an increased number of followers on Facebook and Twitter are correlated with a higher impact factor. With the increasing shift toward online distribution, orthopaedic journals may use our data when evaluating their social media strategy to maintain and potentially increase their exposure and potentially their impact factor.


Currently, there is a lack of diagnostic methods for liver fibrosis in veterinary medicine. Two-dimensional shear wave elastography is a non-invasive diagnostic technique widely used in human medicine. However, there is a limited amount of relevant research in veterinary medicine, with only a few studies available. These studies suffered from small sample sizes, and the results of different experiments contradict each other. The aim of this study was to measure the range of liver stiffness in healthy dogs and to investigate the factors influencing it. We found that liver stiffness in healthy dogs was 3.96 ± 0.53 kPa, which was influenced by anesthesia and measurement site. These findings provided a theoretical basis and data support for its application in veterinary clinical practice. At the same time, the determination of health values contributed to the subsequent study of two-dimensional shear wave elastography in dogs.(1) Background: Two-dimensional shear wave elastography (2D-SWE) is a non-invasive method widely used in human medicine to assess the extent of liver fibrosis but only rarely applied to veterinary medicine. This study aimed to measure liver stiffness in healthy dogs and investigate the factors that impacted 2D-SWE measurement. (2) Methods: In total, 55 healthy dogs were enrolled and subjected to 2D-SWE measurements before and after anesthesia administration. Post-anesthesia 2D-SWE measurements and computerized tomography (CT) scans were obtained. (3) Results: The liver stiffness range in healthy dogs was 3.96 ± 0.53 kPa. In a stratified analysis based on confounding factors, liver stiffness was influenced by measurement site and anesthesia, but not by sex. No correlation was observed between liver stiffness and weight or liver CT attenuation. (4) Conclusions: 2D-SWE is feasible for liver stiffness measurement in dogs. Anesthesia and measurement site are sources of variability. Therefore, these factors should be considered while recording 2D-SWE measurements. Our data on liver stiffness in healthy dogs can serve as the basis for future studies on 2D-SWE to assess pathological conditions in dogs. Liver stiffness is one of the most important indicators of liver disease, as changes in liver stiffness often accompany the progression of liver disease . StudiesFor this reason, a non-invasive imaging diagnostic technique, elastography, was first introduced in human medicine. Elastography includes strain elastography (SE), acoustic radiation force impulse imaging (ARFI), transient elastography (TE), point shear wave elastography (p-SWE), and 2D-SWE. The type of force application is one of the crucial factors in the categorization of elastography, and it can be further divided into two categories, quasistatic and dynamic excitation methods, based on the method of force application. In the quasistatic excitation method, pressure is applied to the target tissue using a transducer or physiological motion to induce deformation, as in SE. On the other hand, the dynamic excitation method generates stimulation either mechanically on the body surface or using acoustic radiation force internally, as in TE and 2D-SWE ,5,6.2D-SWE is a promising technology to measure liver stiffness. As one of the earliest elastography techniques, SE can only provide qualitative or semi-quantitative measurements of tissue stiffness. It assesses the elasticity of the target tissue by comparing it to the surrounding healthy tissue. The application of acoustic radiation force has made it possible to quantitatively measure tissue stiffness values. TE, as a shear wave imaging technique, can only measure the average hardness along a single measurement line. In contrast, 2D-SWE not only allows quantitative measurement of liver stiffness but also allows the placement of multiple regions of interest (ROI) in the target area, thereby expanding the measurement range and providing more representative data. 2D-SWE integrates conventional ultrasound and color-coded tissue stiffness in real time to effectively avoid the inclusion of non-targets, thereby producing more reliable results. 2D-SWE allows real-time placement of the ROI under the guidance of conventional ultrasound, avoids blind inspection, and provides improved accuracy while reducing operational difficulties. Studies have shown that an operator can be considered a 2D-SWE expert after 300 cases of conventional ultrasound experience or more than 50 cases of elastography experience. Moreover, the use of 2D-SWE is not limited by the presence of ascites. 2D-SWE is an excellent diagnostic imaging technique and offers promising clinical applications in veterinary medicine ,8,9.2, where ρ is the tissue density, defined as 1.00 kg/m3 [As a form of shear wave imaging, 2D-SWE directly measures the physical quantity of the shear wave velocity itself. The process of performing 2D-SWE measurements typically begins through the application of an acoustic radiation force to multiple points within a larger ROI. The arrival time of shear waves generated at each focal point along a transverse line is then determined. This collected information is used to calculate the shear wave velocity , which is then automatically converted to Young’s modulus  in most cases using the equation E = 3ρC00 kg/m3 ,11,12.The measurement results are encoded by the ultrasound machine into color images that can be overlaid on B-mode grayscale images or displayed side-by-side with B-mode images. The transparency and color display scale can be adjusted. By placing the ROI at the desired location, quantitative statistical data of Young’s modulus of the target tissue, such as mean, standard deviation, minimum and maximum values, can be obtained ,13.Liver biopsy is still considered as the gold standard for diagnosis of liver diseases, but its invasive nature can lead to complications such as bleeding, pain, and other complications . ImagingThere is limited published literature on the use of 2D-SWE in veterinary medicine and only a few articles have investigated its feasibility and repeatability in measuring canine liver stiffness. Recently published 2D-SWE studies in veterinary medicine have failed to fully investigate confounding factors such as measurement site, state of anesthesia, and the effect of spontaneous breathing, and to measure liver stiffness in healthy dogs. Further, the results of these studies are contradictory, owing to small sample sizes ,23,24.Herein we aimed to measure liver stiffness in a sufficient number of healthy dogs to establish a range of healthy values and to aid in the diagnosis of liver fibrosis and other liver diseases in dogs. Identifying an appropriate range of liver stiffness values in healthy dogs will facilitate future research on the use of the 2D-SWE technique in dogs. To this end, we determined the feasibility of 2D-SWE to measure liver stiffness in 55 healthy dogs and investigated the effects of several factors on 2D-SWE measurements. Previous studies have shown that 2D-SWE had good applicability and accuracy in diagnosing liver disease in human medicine ,25. In aFifty-five healthy dogs  were included in this study. There were 19 mixed-breed dogs, 28 spaniels, 1 German shepherd, 1 labrador, 3 reindeers, and 2 beagles, with an average age of 3 years (range 1–4 years) and a median weight of 13 kg (range 4.5–32.5 kg). All dogs were deemed to be healthy, as per physical examination, routine blood tests, blood biochemistry , abdominThis study was approved by the Ethics Committee of Nanjing Agricultural University.Liver stiffness in dogs was measured using 2D-SWE in the awake state. Intravenous anesthesia was administered, and 2D-SWE measurements and abdominal CT scans were performed. Liver stiffness and CT attenuation were measured six times on each side of the liver lobes when 2D-SWE measurements and CT scans were completed. All CT scans and 2D-SWE measurements were performed in the same anesthesia event.2D-SWE measurements were obtained for each experimental dog in both the awake and anesthetized states.Prior to examination in the awake state, dogs were fasted for more than 12 h, and 2D-SWE measurements were performed using a Philips ultrasound scanner  and a c5-1 probe (1–5 mHz), as previously described ,27,28,29To maintain uniformity in measurement results, the ultrasound parameters for 2D-SWE were set to the system’s default configuration and no adjustments were made during the measurement process. The dual-screen mode of 2D-SWE was selected on the ultrasound machine, which displayed the confidence map on the left and the stiffness map on the right. The confidence map converted the confidence level of the measurements into different colors, which were overlaid on the ultrasound greyscale map. Green was regarded as highly reliable data, while red indicated unreliable data. The stiffness map represented tissue stiffness, with blue and red indicative of the lowest and highest stiffness, respectively . The absThe 2D-SWE measurements were conducted in the left lateral and right lateral positions to measure the liver stiffness of the left and right lobe, respectively. Measurements were repeated with different frames and ROIs until six valid observations were obtained. The mean values of SWEs were representative of the liver modulus values.2 were continuously monitored.All animals were anesthetized with an intravenous dose of xylazine hydrochloride (0.85 mg/kg), tiletamine (4 mg/kg), and midazolam (0.2 mg/kg). During the anesthesia, heart rate, blood pressure, oxygen saturation, and end-tidal COA Hitachi CT scanner was used with the following scan parameters: slice thickness of 1.25 mm to 2.50 mm, tube voltage of 120 kV, tube current of 200 mA, rotation time of 0.75 s, pitch of 1.0625, and collimation of 1.25 × 16.Animal positioning and CT scan results are shown in t-tests.Data were presented as mean ± standard deviation (SD) and analyzed using independent samples p = 0.05), 90% power). Four healthy dogs were used in the pre-experimentation stage. The mean difference in liver stiffness values for the experimental dogs before and after anesthesia was 2.71 kPa (4.05 kPa vs. 6.76 kPa), with standard deviations of 104% and 141% for liver stiffness before and after anesthesia, respectively. Based on this, PASS 15 calculated that a sample size of 15 animals would result in >90% power using an independent sample t-test with a 5% type 1 error. The sample size was determined by pre-experimentation, experimental design, and conventional numerical settings using PASS 15  Microsoft Corp., Redmond, WA, USA). All measurements were expressed as Young’s modulus, and the data were analyzed by the Shapiro–Wilk test. Correlation analysis was used to determine the relationship between weight and liver stiffness. A value of p > 0.05). However, the liver stiffness was significantly higher in the left lobe than in the right lobe in the anesthesia state (p < 0.01).The liver stiffness in healthy dogs was 3.96 ± 0.53 kPa. We compared the liver stiffness between the left and right lobes in the awake and anesthesia states. . No signp < 0.01 for the right liver lobe, p < 0.01 for the left liver lobe). The color changes in the stiffness map reflect the impact of anesthesia on liver stiffness in healthy dogs. After anesthesia, the color of the stiffness map shifted from a uniform light blue to a mixture of blue and green .Sex did not significantly affect liver stiffness in healthy dogs in either the awake or anesthesia states . No signp > 0.05 for the awake state, p > 0.05 for the anesthesia state).We conducted a correlation analysis to explore the effect of weight on liver stiffness . The resp = 0.637 for the left liver lobe, p > 0.05 for the right liver lobe, and p >0.05 for the entire liver).We determined the correlation of liver CT attenuation with liver stiffness . No signIn the present study, we investigated the applicability of 2D-SWE to assess liver stiffness in healthy dogs. The effects of anesthesia, measurement site, sex, and weight on the results were investigated, and the correlation between liver CT attenuation and liver stiffness was explored. The aim was to measure liver stiffness in a sufficient number of healthy dogs to establish a range of healthy values and to aid in the diagnosis of liver fibrosis and other liver diseases in dogs.Unlike humans, dogs were not cooperative during examinations. Anesthesia is occasionally necessary in veterinary medicine to perform liver 2D-SWE. Tamura et al. identified motion as an important limitation in 2D-SWE measurements in dogs . 2D-SWE In our study, the liver stiffness of anesthetized dogs was significantly higher than that of awake dogs. This result is similar to a previously published study but different from the results of another study ,23. ThisIn our study, the stiffness of the right liver lobe was slightly higher than that of the left lobe, but there was no significant difference. Interestingly, a significant difference was observed in stiffness between the right and left liver lobes in the anesthesia state. Consistent with the above speculation, we believe that this observation was related to changes in the liver blood perfusion. The anesthesia protocol used in this study might lead to differential changes in perfusion between the left and right liver lobes. The comparison of the changes in the liver stiffness before and after anesthesia made it clear that the right lobe experienced a greater increase in stiffness than the left lobe. Given the relatively poor distensibility of the liver capsule, we speculate that this phenomenon is attributed to the greater increase in the blood perfusion to the right lobe. Unfortunately, there are no reported studies on this topic. Given the time constraints and other factors, we were unable to thoroughly investigate this issue, and it warrants future study.The results of this study indicated that sex had no significant effect on liver stiffness in healthy dogs, and that body weight was not significantly correlated with liver stiffness. Thus, our results are applicable across different sexes and body weights of dogs. In clinical practice, there is no need for concern about the influence of these two factors on measurement results. However, it is important to note that the dogs included in this experiment had a BCS of 3, which eliminated the effect of obesity on the results. The applicability of these results to obese dogs, which are commonly encountered in clinical settings, needs to be confirmed. Unfortunately, there are no published reports investigating the effect of obesity on 2D-SWE measurements in canine livers, and further studies are warranted to investigate this.The lack of correlation between liver CT attenuation and liver stiffness indicated that 2D-SWE technology cannot be replaced by CT. In addition, 2D-SWE has several advantages over existing clinical imaging diagnostics in veterinary medicine. First, it offers high sensitivity and specificity and has been considered one of the preferred non-invasive imaging techniques for the clinical assessment of liver fibrosis in human medicine ,37,38. SThe present study has some limitations. First, the proportion of large dogs in this study was low, which may have influenced our findings. Therefore, the results of this study may not apply to large dogs. Second, all the data were measured by a single imaging physician which did not allow for the assessment of inter-observer variability. Third, the dogs in this study were not histologically examined to determine the health status of the liver, and this was presumed by physical examination, blood test, CT scan, and ultrasound. However, the dogs were relatively young, and no clinical signs or abnormalities of illness were found.Here we demonstrated the applicability of 2D-SWE to measure dogs’ liver stiffness with high reproducibility. Liver stiffness was measured in 55 healthy dogs, and the results provided statistical support for the use of 2D-SWE in veterinary medicine. The range of liver stiffness in healthy dogs established herein will be useful in the diagnosis of liver fibrosis and other liver diseases in dogs. In addition, our study lays the foundation for future studies using 2D-SWE to diagnose pathological conditions that increase liver stiffness in dogs.Anesthesia was deemed as a source of variability in liver stiffness. However, the effect of anesthesia on the liver stiffness of healthy dogs might be related to the anesthesia protocol used. The measurement site also affected 2D-SWE values and this effect was also, at least in part, related to the state of anesthesia. Therefore, these two factors should be interpreted and considered while recording 2D-SWE measurements in dogs.


This study evaluated the graft success rate and hearing outcomes of the inside-out raising mucosal-tympanomeatal flap technique for the repair of large marginal perforations.Prospective case series.The study enrolled patients with large marginal perforations who underwent endoscopic cartilage myringoplasty with the inside-out raising mucosal-tympanomeatal flap technique. The graft success rate, hearing outcomes, and complications were evaluated at 12 months postoperatively.p = 0.001). The functional success rate was 85.4% (41/48). None of the patients experienced worsened sensorineural hearing loss or graft-related complications, such as graft lateralization, significant blunting, and graft medialization, during follow-up.In total, 48 patients with large marginal perforations were included. 81% of the population had large perforation, 14.6 had subtotal and total perforation was seen in 4.2%. The mean operation time was 38.6 ± 7.1 min. At 12 months postoperatively, the graft success rate was 89.6% (43/48). The mean air-bone gap was 25.6 ± 5.2 dB preoperatively and 16.5 ± 4.1 dB at 12 months postoperatively, with significant differences between these values (Endoscopic cartilage-perichondrium myringoplasty for the repair of large marginal perforations using the inside-out raising mucosal-tympanomeatal flap technique was associated with satisfactory graft outcomes and minimal complications. In general, myringoplasty for repairing large perforations requires raising the mucosal-tympanomeatal flap to reinforce the graft , 2. HoweThe study protocol was approved by the Institutional Ethical Review Board of Yiwu Central Hospital, Yiwu, Zhejiang, China . Informed consent was obtained from all participants.This prospective case series enrolled consecutive patients with chronic large marginal perforations who underwent the cartilage-perichondrium graft technique with inside-out elevation of a mucosal-tympanomeatal flap. The inclusion criteria were as follows: marginal perforations with the absence of tympanic membrane (TM) remnant on at least one side of the perforation; large perforations covering more than one-fourth of the TM area; patient age of 18–75 years; involvement of the malleus handle; dry ear for at least 3 months prior to surgery; a positive Valsalva maneuver; sensorineural hearing loss with a need for TM reconstruction to prevent middle ear infection due to water entry into the ear. We excluded patients with a need for revision surgery, a need for ossicular chain reconstruction, middle ear cholesteatoma or inflammation, or fungal otitis externa. Marginal perforation was defined as the absence of any TM remnant on at least one side before surgery or after de-epithelializing the margins. Perforations were categorized as large , subtotal , and total (more than three-fourths of the TM area). All surgeries were performed by the same surgeon. All patients underwent temporal bone high-resolution computed tomography preoperatively. Operative time was determined from graft harvesting to EAC packing.Audiometric data were collected before and 12 months after surgery. Pure-tone averages (PTAs) were determined using the findings at 500, 1,000, 2,000, and 3,000 Hz for both air conduction and bone conduction. For most patients, the threshold at 4,000 Hz was used to interpolate the threshold at 3,000 Hz, following the recommendations of the Hearing Committee of the American Academy of Otolaryngology–Head and Neck Surgery. Pre- and postoperative air-bone gaps (ABGs) were calculated by subtracting air-conduction PTAs from bone-conduction PTAs. ABG closure was determined by subtracting the preoperative ABG from the postoperative ABG.The patient was positioned in the supine position with the head inclined upward at 30°, facing the opposite side of the surgeon. Video equipment with a camera system  was placed on the opposite side of the surgeon. All patients underwent myringoplasty with a cartilage-perichondrium graft using a 0° rigid endoscope  under total intravenous anesthesia.We developed an inside-out approach for cases where no TM remnant exists, eliminating the need to raise a mucosal-tympanomeatal flap through an EAC incision. The medial EAC skin and annulus were elevated, beginning approximately 2 mm away from the mucosa of the tympanic sulcus using an inside-out approach to raise the mucosal-tympanomeatal flap at least 2 mm off the bone. This approach is also referred to as antidromic raising of the mucosal-tympanomeatal flap. This procedure only involved the use of a microscopic cross-cutting knife and did not require any other techniques or surgical tools.Cartilage, consisting of a one-sided perichondrium graft, was harvested from the ipsilateral tragus. The cartilage, along with the perichondrium, was harvested to match the perforation size. The perichondrium was elevated circumferentially, with its pedicle attached to the cartilage. The cartilage was approximately 1 mm larger than the perforation margins  that had been soaked in antibiotic ointment. The perichondrium-cartilage composite graft was inserted trans-perforation, with the cartilage graft placed medial to the TM remnant and bone annulus. The notch in the graft accommodated the malleus, whereas the perichondrium graft was positioned lateral to the malleus handle and bone annulus, tucked inside the mucosal-tympanomeatal flap Fig. . The EACPatients were discharged on the day following the surgery. All patients received a course of antibiotics (amoxicillin) postoperatively. Postoperative follow-up appointments were scheduled in the outpatient department at 2 and 4 weeks and 3 and 12 months. At each follow-up visit, endoscopic examination was performed to assess the graft status. Patients were also asked about altered taste, vertigo, and tinnitus. A postoperative ABG of ≤ 20 dB was considered a functional successful outcome. Graft success was defined as an intact graft without perforation, retraction, lateralization, significant blunting, or medialization.t-test. P-values < 0.05 were considered indicative of statistical significance.Statistical analyses were performed using SPSS software . Data are expressed as means with standard deviations or numbers with percentage. Differences between preoperative and postoperative ABGs were analyzed using the paired-samples In total, 48 patients  with a unilateral large marginal perforation were included in the study. The perforation affected the left ear in 21 (43.8%) patients and the right ear in 27 (56.2%) patients. The mean duration of perforation was 19.7 ± 5.1 years. Of the 48 patients, 81%, 14.6%, and 4.2% had large, subtotal, and total perforations, respectively. Sensorineural hearing loss was absent in 45 (93.8%) patients, whereas 3 (6.2%) patients aged > 65 years had sensorineural hearing loss. The mean operation time was 38.6 ± 7.1 min.Postoperative middle ear infection developed in 2 4.2%) patients, leading to residual perforation at 1 month postoperatively. Furthermore, a small residual perforation was observed in 3 (6.3%) patients without middle ear infection at 3 months postoperatively, whereas no cases of residual perforation or re-perforation were observed at 12 months postoperatively. The graft success rates were 95.8%, 89.6%, and 89.6% (43/48) at 1, 3, and 12 months postoperatively. Figure .2% patiet-test p = 0.001). The ABG was < 10 dB in 11 (22.9%) patients, 10–20 dB in 30 (62.5%), and > 20 dB in 7 (14.6%), corresponding with a functional success rate of 85.4% (41/48).Audiological testing was performed in 48 patients at 12 months postoperatively, which showed no change in the sensorineural threshold. The mean ABG was 25.6 ± 5.2 dB preoperatively and 16.5 ± 4.1 dB at 12 months postoperatively, with significant differences between these values  at 12 months postoperatively. Our results demonstrated successful repair of large marginal perforations using the antidromic raising mucosal-tympanomeatal flap technique, with no need for additional EAC skin incisions. The graft success rate achieved with this technique is comparable to that of other techniques for the repair of large marginal perforations that require raising a mucosal-tympanomeatal flap via an EAC skin incision. Shoman et al.  demonstrThe primary concerns in this study were the operation time and complications. The mean operation time was 38.6 ± 7.1 min, which was significantly shorter than other techniques involving the raising of a tympanomeatal flap via an EAC incision. Plodpai et al.  demonstrFourth, we did not evaluate the relationships between graft success, functional success (ABG), and perforation size due to the unequal number of patients with perforations of different sizes .Endoscopic cartilage-perichondrium myringoplasty with inside-out elevation of mucosal-tympanomeatal flap is associated with satisfactory graft outcomes and minimal complications during the repair of large marginal perforations.