Nonanastomotic aneurysmal degeneration of a great saphenous vein graft is an unusual condition, despite the common use of this conduit in arterial reconstructions. Vein grafts are at risk of degenerative changes, but the real cause remains unknown. Postoperative graft surveillance with duplex ultrasound scanning is important for maintaining patency of the venous graft. We present a rare case of chronic limb ischemia due to partial thrombosis of an aneurysm of the great saphenous vein graft. True nonanastomotic aneurysmal degeneration of great saphenous vein grafts is an unusual condition, despite the common and widespread use of this conduit in arterial reconstructions.A 67-year-old male patient presented to the emergency department with pain of the right lower extremity at rest, which started 24 hours earlier. The patient had a history of arterial hypertension and infrainguinal right saphenous vein bypass 11 years ago, due to severe claudication (Rutherford 3 category). For 2 months, the patient again suffered severe claudication, but he did not seek medical advice.Physical examination revealed no palpable peripheral pulses and an ankle-brachial index of 0.49. Duplex ultrasound scanning and computed tomography angiography recognized the presence of aneurysmal degeneration of the proximal and middle thirds of the graft, with thrombus, turbulence, and elevated velocities consistent with luminal narrowing at the middle of the graft.Aneurysmectomy  and comPostoperative recovery was uneventful, without infection or hematoma development. The patient was discharged on the third postoperative day with palpable peripheral pulses. At the 6-month follow-up, the patient remains asymptomatic, with an ankle-brachial index around 0.96 and no sign of claudication or wound complication.Although true nonanastomotic aneurysms of an infrainguinal vein graft are rarely reported in the literature, a recent publicationAneurysmal dilatations in vein grafts may rupture or develop thrombosis, causing hemorrhage or acute limb ischemia that require surgical treatment on an urgent basis. However, open repair of aneurysmal vein grafts can be challenging because of extensive scar tissue that leads to a high rate of wound infection. Despite the difficulties of reoperation, open repair remains the cornerstone of treatment in these cases. According to published literature, endovascular treatment has been used in two cases to treat dilated venous bypass grafts,Vein graft surveillance is the key to long-term success of infrainguinal bypasses. Postoperative graft surveillance with a program of ankle-brachial index determination and duplex ultrasound scanning is important for maintaining patency. Lesion progression may indicate early intervention for the rescue of the venous grafts.In conclusion, true nonanastomotic aneurysms of an infrainguinal vein graft are rare, and their etiology remains unknown. These can lead to graft thrombosis, rupture, distal embolization, and skin ulceration due to continuous pressure. Therefore, a graft surveillance program of ankle-brachial index determination and duplex ultrasound scanning is mandatory, as early diagnosis and intervention can minimize the potential late complications.


Due to the obvious differences in the DRX rate at various temperatures, the piecewise DRX kinetics equations are proposed to predict the DRX behavior. At the same time, a mathematical model for predicting the grain dimension and the grain growth behavior is established. To further analyze the DRX behavior and the changes in grain dimension, the hot deformation process is simulated. The developed grain-growth equation as well as the piecewise DRX kinetics equations are integrated into DEFORM software. The simulated DRX features are consistent with the test results, indicating that the proposed DRX kinetics equations and the established grain-growth model can be well used for describing the microstructure evolution. So, they are very useful for the practical hot forming of P/M superalloy parts.The dynamic recrystallization (DRX) features and the evolution of the microstructure of a new hot isostatic pressed (HIPed) powder metallurgy (P/M) superalloy are investigated by hot-compression tests. The sensitivity of grain dimension and DRX behavior to deformation parameters is analyzed. The results reveal that the DRX features and grain-growth behavior are significantly affected by deformation conditions. The DRX process is promoted with a raised temperature/true strain or a reduced strain rate. However, the grains grow up rapidly at relatively high temperatures. At strain rates of o.1 s Owing to their excellent anti-corrosion/fatigue properties and strengths, powder metallurgy (P/M) superalloys are widely applied for manufacturing the turbine disks of aero-engines ,2. HowevDuring hot deformation, the microstructures can be refined and the metallurgical defects can be efficiently removed. Up until now, the material flow characteristics ,9 and miXdrx) are quantitatively calculated. The piecewise DRX kinetics equations and grain-growth model are established to predict the DRX and grain-growth behaviors under different deformation conditions. Then, the developed models are integrated into DEFORM software. Finally, the DRX features and microstructure evolution during hot compression are simulated.Though the deformation mechanisms as well as the microstructure evolution of HIPed P/M superalloys have been reported, accurate models for predicting the DRX features and grain-growth behavior are still rarely reported. In this work, the hot-compression experiments of a novel HIPed P/M superalloy are conducted. Then, the flow features, DRX mechanism, and grain features of an advanced P/M superalloy are systematically investigated. The grain dimension and DRX volume fraction  for the novel P/M superalloy are listed in 4 and 90 mL CH3CH2OH at a temperature of −31 to −26 °C as well as a voltage of 23 V [Xdrx and grain size were accurately calculated by MTEX5.7.0.The schematic plot of hot-deformation experiments is illustrated in  of 23 V . The EBS−1. This is caused by heterogeneous deformation or local deformation heating or cracking [cracking ,35.a and b and average orientation of grain a. According to the GOS distribution of the complete DRX sample and GOS division principle [The grain-orientation spread (GOS) method can reflect the orientation gradient within grains and evaluate the DRX degree in the deformed alloy . Here, trinciple , the gra−1, respectively, the GOS distribution at the true strain of 0.22, 0.51, and 0.92 is demonstrated in Xdrx increases. Meanwhile, the average grain dimension is reduced from 7.18 to 4.21 μm when the strain increases from 0.22 to 0.92, whereas the mean DRX grain dimension (ddrx) increases. At 0.22 (Xdrx (15.56%) is low. However, the average grain size is large, which is attributable to the high dislocation density and the low-deformation storage energy. The original GBs are gradually covered by DRX grains and the mean GOS and KAM rapidly decrease when the true strain is 0.51. In addition, the Xdrx increases significantly. Although the DRX grains grow up, the average grain size decreases to 4.39 μm because of the annihilation/rearrangement of dislocations and the migration of GBs [When the temperature and strain rate are 1110 °C and 0.1 s At 0.22 a, a massn of GBs ,43,44. W−1 to 0.1 s−1, the average KAM and GOS increase but the Xdrx decreases. Meanwhile, tiny DRX grains are found and the mean grain dimension decreases because the large strain rate can produce high strain energy and accelerate the development of substructures with a mass of dislocations. It is conducive to DRX nucleation [Xdrx and fine grains at a high strain rate [Xdrx at three strain rates are higher than 95%, which indicates the DRX is complete.cleation . In addiain rate . The gra−1, the relationship between temperature and GOS is depicted in Xdrx increase when the temperature is raised. At low temperatures  is also expressed as:For Based on the measured rheological stresses, the Generally, Xdrx with the true strain at 1140 °C/0.001 s−1. The value of Xdrx is small in the initial incubation stage. After this incubation period, the DRX is accelerated and finally tends to be stable. The −ε curve a. Figurerameters , i.e.,Xdrx=Xdrx. Also, the comparisons between the calculated and experimental Xdrx are represented. To validate the precision of traditional DRX kinetics equations, the average absolute relative error (AARE) and correlation coefficient € are calculated, i.e.,iE is the measured value and iP shows the calculated one. Meanwhile, the measured mean value is expressed as E and the calculated mean value is defined as P. In Xdrx is much higher than the experimental value and the R between experimental and predicted Xdrx is only 0.15. Meanwhile, the predicted ′ phases  and enouIn In Xdrx with the strain. Also, the comparisons between the calculated and experimental Xdrx are represented. The calculated values are very close to the experimental ones. To confirm the precision of the proposed piecewise DRX kinetics equations, R and AARE are calculated. The AARE value is 2.3% and the R value is 0.992. Hence, the DRX behavior of the present HIPed P/M superalloy during hot compression can be accurately described by the proposed piecewise DRX kinetics equations.ddrx) in the stable deformation stage is listed in ddrx. Generally, ddrx is connected with the compression parameters, which is evaluated through Sellars’s empirical equation [ddrx is determined as:The DRX grain size (equation ,51. Hencddrx. The calculated AARE value is 2.5% and the R is 0.991. Meanwhile, the mean grain dimension is demonstrated as:X represents the DRX volume fraction. Similarly, Xdrx/grain-size dimension will be counted. If Xdrx and grain size are outputted. In order to simulate DRX behavior and grain-dimension evolution in this HIPed superalloy during hot compression, the proposed piecewise DRX kinetics equations and grain-growth model are integrated into the DEFORM software through the development of the subprogram. The program flowchart to simulate the DRX behavior and grain size is illustrated in The evolution of the equivalent strain at diverse deformation parameters is demonstrated in Xdrx at diverse deformation parameters is displayed in Xdrx in the large deformation zone increases with a reduced strain rate or a raised temperature. They are 80%, 97%, 85.9%, and 78.1% at 1110 °C/0.1 s−1, 1140 °C/0.1 s−1, 1110 °C/0.01 s−1, and 1110 °C/1 s−1, respectively. These predicted values well agree with the test ones.The distribution of the predicted −1, 1140 °C/0.1 s−1, 1110 °C/0.01 s−1, and 1110 °C/1 s−1, which coincide with the simulated results. The simulated Xdrx and grain sizes in various deformation regions at 1110 °C/0.1 s−1 are quantitatively analyzed and are displayed in 2 and P3 in Xdrx and average grain size at different positions with deformation time. The comparisons of the experimental/simulated Xdrx and average grain size at 1110 °C/0.1 s−1 are shown in The DRX features and grain-growth behavior for a novel P/M superalloy are systematically studied. The piecewise DRX kinetics equations and grain-growth model are developed. The important conclusions are summarized:1. The DRX behavior and grain features are sensitive to deformation parameters. The raised temperature/true strain can increase the volume fraction of DRX and the mean dimension of DRX grains. As the temperature is reduced or the true strain is raised, the mean grain dimension declines. Increasing the strain rate reduces the DRX volume fraction and mean grain size. Moreover, the main DRX mechanism of the novel P/M superalloy is DDRX.2. Piecewise DRX kinetics equations are proposed to predict DRX behavior in forming processes. The correlation coefficient of them is 0.992 and the average absolute relative error is 2.3%. Also, an accurate model is established to describe the grain-growth behavior during hot deformation.3. The piecewise DRX kinetics equations and the grain growth equation are embedded into the DEFORM software using a secondary development method and the DRX features and grain-growth behavior in hot-compression processes are simulated. The simulated results show that the proposed piecewise DRX kinetics equations and grain-growth model can accurately depict the microstructure evolution of this novel P/M superalloy during hot deformation.


Tendons play a crucial role in the musculoskeletal system. In humans, tendon injuries, especially chronic tendinopathy, are very common and the patellar tendon is a frequent location for tendinopathy or injuries. The biomechanical characteristics of the patellar tendon, such as elasticity and stiffness, are of paramount importance and constitute major outcome measures in research studies. We aimed to assess whether the stiffness of the healthy ovine patellar tendon changes with age and weight in a population of normal animals. Sixty-eight ‘patella-patellar tendon-tibial tuberosity’ units from thirty-four Ile-de-France ewes of body mass 65 to 95 kg, euthanized for reasons other than musculoskeletal diseases, underwent a tensile test providing a measure of the tendon stiffness. Animals were sorted into three categories of age . We found a positive but not significant correlation between age category and stiffness . There was a significantly positive correlation between weight and stiffness . In conclusion, the study characterized biomechanical properties of healthy tendons, provided useful reference values, and established the basis for future biomechanical tests on healing tendons in sheep. The most appropriate sheep population for those future studies would be non-overweight young adults presenting with no lameness. Tendons play a crucial role in the musculoskeletal system. In humans, tendon injuries, especially chronic tendinopathy, are very common in occupational and athletic settings, and in the elderly population. They may be associated with significant morbidity and abnormal joint movement, and are an important cause of musculoskeletal disabilities or pain.In particular, patellar tendon injuries occur either naturally as chronic tendinosis, partial/full-thickness tendon tears, acute avulsion fractures , from the Ovine Research Center of the University of Namur, were used. Their age ranged from 2 to 10 years, and their weight from 65 to 95 kg. The experimental protocol 10150MU was approved by the local ethical committee for animal welfare. All animals had no history of hindlimb lameness. They were assessed by palpation and observation. No swelling or pain of the stifle was observed. Animals were not lame before euthanasia.Animal were sorted into three categories of age .Animals were sacrificed by intravenous administration of pentobarbital (150 mg/kg). Both hind limbs were transected at the level of the mid-femur within 1 hour of death. Soft tissue, including skin and muscles were removed. The patellar tendon and its attachments on the patella and the tibial tuberosity were carefully dissected and observed to confirm the absence of lesions or abnormalities.The joint capsule, collateral ligaments and cruciate ligaments were transected. The knee joint was disarticulated, and the tibial tuberosity and patella were transected at 1.5 cm from their respective tendon attachments. Bone attachments were preserved so as not to affect the tendon insertion areas that are fundamental for mechanical load-transfer.Each harvested specimen consisting of an intact ‘patella-patellar tendon-tibial tuberosity’ unit was individually wrapped in moistened gauze (with 0.9 % w/v NaCl), sealed in plastic bags, individually identified by a number and stored at −20°C.Four-mm thick osteochondral slabs in a coronal plane were obtained , before being processed for histology.Histology is used as the gold standard to assess the articular cartilage of a joint, joint deterioration and joint ageing .The OARSI recommendations were used for histological scoring of the cartilage , a uni-axial tensile test was performed on patellar tendons following an in-house validated procedure . As the tendons had been manipulated and then stored at −20° C before the biomechanical tests, a series of 10 pre-conditioning stress-relaxation procedures was performed to ensure fibers alignment before the tensile testing. As recommended in the ASTM D638 standard , Kolmogorov-Smirnov and Shapiro-Wilk tests were applied. Non-parametric tests were used, due to the non-normality of the data. The Wilcoxon signed-rank test was used to compare observations that were not independent (difference in patellar tendon stiffness between left and right hindlimbs). One limb was randomly chosen within each pair and data obtained from that limb  were used for analysis. Correlations between patellar tendon stiffness and other variables  were assessed by Spearman correlation coefficient. Data were collected in Microsoft Excel and analysed using Graph Pad Prism 8. A p-value below 0.05 was considered to indicate a statistically significant difference.Among the 272 (4x68) histological slices, none presented artefacts or inadequate coloration that may have prevented correct interpretation. We found no significant difference in limb histological total scores between left and right limbs (p = 0.93).The histological scores  were, however, different between regions of interest: medial tibial condyle , lateral tibial condyle , medial femoral condyle  and lateral femoral condyle . Among the total number of lesions, we found 36.2% in the medial femoral condyle, 6.8% in the lateral femoral condyle, 37.9% in the medial tibial condyle and 15.5% in the lateral tibial condyle. Among the histological abnormalities, beside the total score, structural defects and tidemark increased the most with age and weight.There was a significant positive correlation between body weight and histological score , and between age and histological score .Our tests confirmed that the patellar tendon has the typical ‘region’ behavior, with a non-linear one (toe-region) followed by a linear region preceding the eventual damaging of the fibers and consequent failure of the tendon .Figure The mean stiffness value of the 68 tendons was 52.44 N/mm .We found a positive but not significant correlation between stiffness and age  . We alsoIn research studies, the biomechanical characteristics of the tendon, like elasticity and stiffness, represent major outcome measures. Due to the biomechanical similarity of the ovine stifle joint and the human knee  in our patellar tendons. Our results are quite similar to those found in a previous ex-vivo study (46–65-80 N/mm) performed in ovine patellar tendon . Several of these components may be involved in tendon stiffness changes with ageing.Two different studies hypothesized that tensile stiffness changes in tissue are most likely due to changes in higher collagen structures rather than stiffness increasing at the fibril level . The level of AGEs was higher in aged mice compared to younger ones  in tendons. Decorin regulates the assembly of collagen I which is the primary structural unit and transmits mechanical force  than that of young TDSCs , and other cytokines expressed in obese individuals generally indicates a pro-inflammatory state (Battery and Maffulli Recently, shear wave ultrasound elastography has been evaluated to assess the mechanical properties of muscles and tendons, including stiffness, based on the speed with which induced shear waves propagate in the tissue of interest. The effect of hyperlipidemia on the patellar tendon stiffness has been investigated by this method; and a moderate positive statistically significant correlation was found between patellar tendon shear wave velocity and low-density lipoprotein independently of body mass index (Torgutalp et al. Those various and contradictory results indicate that different variables should be considered to assess the effect of weight in a species, such as the sheep, including biomarkers of lipid metabolism. This should be taken into account in future studies. One other limitation of the current study is that we measured the stiffness over the entire bone-tendon-bone unit whilst regional variations in stiffness may occur.However, in the scope of an ovine model of patellar tendon, this study showed that the stiffness of the healthy ovine patellar tendon increases significantly with weight. It also increases, but not significantly, with age.Even if the parameters taken into consideration were only a part of the huge amount to address when dealing with soft tissues, this study achieved promising results in terms of characterization of biomechanical properties of healthy tendons, providing useful reference values and establishing the basis for future biomechanical tests on healing tendons in sheep. The most appropriate sheep population for those future studies would be young, non-overweight adults presenting with no lameness. Non-invasive techniques such as shear wave ultrasound elastography warrant investigation.


Starch is affected by several limitations, e.g., retro-gradation, high viscosity even at low concentrations, handling issues, poor freeze–thaw stability, low process tolerance, and gel opacity. In this context, physical, chemical, and enzymatic methods have been investigated for addressing such limitations or adding new attributes. Thus, the creation of biomaterial-based nanoparticles has sparked curiosity. Because of that, single nucleotide polymorphisms are gaining a lot of interest in food packaging technology. This is due to their ability to increase the mechanical and water vapor resistance of the matrix, as well as hide its re-crystallization during storage in high-humidity atmospheres and enhance the mechanical properties of films when binding in paper machines and paper coating. In medicine, single nucleotide polymorphisms (SNPs) are suitable as carriers in the field of drug delivery for immobilized bioactive or therapeutic agents, as well as wastewater treatments as an alternative to expensive activated carbons. Starch nanoparticle preparations can be performed by hydrolysis via acid hydrolysis of the amorphous part of a starch molecule, the use of enzymes such as pullulanase or isoamylase, or a combination of two regeneration and mechanical treatments with the employment of extrusion, irradiation, ultrasound, or precipitation. The possibility of obtaining cheap and easy-to-use methods for starch and starch derivative nanoparticles is of fundamental importance. Nano-precipitation and ultra-sonication are rather simple and reliable methods for nanoparticle production. The process involves the addition of a diluted starch solution into a non-solvent, and ultra-sonication aims to reduce the size by breaking the covalent bonds in polymeric material due to intense shear forces or mechanical effects associated with the collapsing of micro-bubbles by sound waves. The current study focuses on starch nanoparticle manufacturing, characterization, and emerging applications. Many plants generate starch as a source of stored energy in the form of polysaccharides. Starch is a natural, renewable, and biodegradable polymer. It is the second most abundant biomass material and is a carbohydrate storage product found in all chlorophyll-containing plants such as corn, potato, rice, wheat, and barley. If it is extracted from a plant, it is referred to as “native starch,” whereas undergoing one or more modifications  to achieve specific properties categorizes it as “modified starch” . Starch Zea mays L., are one of the primary food crops and a highly unique plant. Maize starch accounts for approximately 80% of the world’s starch production, and it is extracted from corn pieces (content 64–80%) using a wet method. Corn starch is utilized in a wide range of meals and uses. Basic maize starches contain traces of protein (0.35 percent), fat (0.8 percent), ash, and more than 98 percent of two polysaccharides, amylose and amylopectin (carbohydrates). Starch is derived from plant sources that are insoluble in water and is in the form of granules at room temperature [Starch is derived from many crops and is categorized according to its botanical source, which includes cereals, legumes, vegetables, and roots/tubers. Cereal starches derived from maize, whose scientific name is perature .Starch granules vary in size and form, with the majority falling in the range of 2–100 mm, with maize’s size ranging between 2 and 30 µm and its shape round and polyhedral. Whereas lentil has a size ranging between 6–37 and 6–32 µm and their shape is oval, spherical, and elliptical, sweet potato’s size ranges between 1 and 100 µm and has shapes that are oval, spherical, and round. Moreover, tapioca’s size ranges between 5 and 45 µm and its shape is spherical/lenticular. Furthermore, rice’s size is less than 20 µm and ranges between 2 and 8 µm, while its shape is polygonal and angular. In wheat, the size ranges between 10 and 30 µm and is disc-shaped. Faba bean has an irregular or oval shape and the size ranges between 20 and 48 µm [Nanoparticles are solid or colloidal particles with sizes ranging from 1 to 100 nm. The size of the particles influences the chemical reactivity, flowability, opacity, stability, and material strength of many materials . NanoparThere has recently been renewed interest in the creation of biomaterial-based nanoparticles. According to research, a wide range of organic and edible nanoparticles may be made from food-based components such as polysaccharides, lipids, proteins, minerals, and surfactants . The mosThe preparation methods of starch nanoparticles (SNPs) can be classified into three categories: (1) Hydrolysis ; (2) renewal/micro perception; and (3) physical methods .3O+1 from easily penetrating the areas. Second, acid hydrolysis of a glucosidic link may cause the D-glucopyranosyl portion to change shape . If the sugar conformation is inhibited by the crystalline structure, this transition will be impossible. Winarti et al. [2SO4 in hydrolysis resulted in a higher yield and larger particle size. In contrast to HCL, which increased the particle size by increasing the hydrolysis duration, the data showed that a high H2SO4 hydrolysis duration decreases particle size. Furthermore, they stated that the yield of SNPs had dropped as a result of the protracted hydrolysis process, which caused excess starch components to dissolve in the acidic medium, lowering the yield of SNPs. Wang et al. [Acid hydrolysis has been used for years to prepare SNPs using mild acids and temperatures below starch’s gelatinization threshold. Mineral acids including oxalic acid, sulfuric acid, and hydrochloric acid are employed, although citric acid has also been used, at temperatures ranging from 25 to 55 °C . The acii et al.  conducteg et al.  treated g et al.  stated tg et al.  reported2O solution was utilized as the solvent system. The gelatinized starch solution was chilled before being dropped into various amounts of 100% ethanol. QSNPs had a lower particle size of 166.25 nm and a 26.62% higher loading capacity. Through hydrogen bonding, starch nanoparticles (SNPs) interacted with quercetin. Because of their tiny particle sizes and high loading capacities, QSNPs were more successful in protecting and prolonging quercetin bioactivity. Nanoparticles are spontaneously formed when the organic phase is dropped or added in a one-shot manner to the aqueous phase [Two miscible solvents are used to manufacture nanoparticles via the nanoprecipitation method: One as a good solvent  and the other as a non-solvent for the chemical that will form the particle . The requirements for the nanoprecipitation procedure are the preparation of an organic phase and a non-solvent phase, commonly referred to as the aqueous phase, both of which ensure the entire solubility of all initiating components. SNPs have been made from a variety of botanical sources, including maize, tapioca, sweet potato, potato, and pea starch, by adding ethanol drop-wise to a weak solution of gelatinized starch while stirring. Several factors influence the properties of SNPs, including the starch content, surfactant addition, and the ratio of starch solution to non-solvent employed in the precipitation. Surfactants boost the crystallinity of SNPs according to researchers, which is due to the surfactant’s capacity to infiltrate the starch interior cavity of the single-helix portion. Jiang et al.  conducteus phase . The benus phase . It has us phase . It invous phase . The prous phase . SeveralMilling is an environmentally friendly method of producing starch nanoparticles that may directly influence the physicochemical properties of starches, resulting in more amorphous nanoparticles. Lu et al.  investigThis technique can break big molecules into tiny pieces and cleave glycosidic-bonds, therefore gamma radiation can be employed to generate starch nanoparticles. The method involves stirring starch and boiling water to make a homogeneous paste, then irradiating the suspension with gamma rays, which produces active free radicals that cause the starch to hydrolyze. Cleavage occurs in the amorphous portions of starch rather than the crystallite regions, resulting in fragmentation. The gamma radiation approach is remarkably similar to the starch acid hydrolysis method in this regard. The nanoparticles produced by this process typically have a diameter of less than 100 nm. Furthermore, due to the huge number of OH groups on their surface, these nanoparticles contain nano-crystal aggregates, which become firmly connected by hydrogen bonding, resulting in rapid thermal deterioration . Bashir g for 15 min. The obtained precipitate was freeze-dried for three days at 60 °C [Electro-spraying is a novel method for creating nanoparticles from various biopolymers . This method is based on the premise that an electric field bends droplets in the micrometer and nanometer range. A Coulomb force is created in the droplets by applying an electric field, which simulates cohesive forces and Coulomb forces. The cohesive forces release surface tension, resulting in the production of nano-droplets. The electro-spray principle is based on the ability of an electric field to distort the droplet interface and produce droplets in the micro-meter or nano-scale range, depending on the parameters regulated. When the Coulomb force is applied, it is capable of overcoming the droplet’s cohesive force, which manifests in surface tension, allowing it to be free to create nano-droplets. These variables include system characteristics such as the solution flow rate, applied electric potential, collector distance, solution viscosity, conductivity, surface tension, molecular weight, and polymer concentration . Then, eat 60 °C .The chemical, pharmaceutical, food, and biotechnology sectors all use high-pressure homogenization. Changes in the products, as well as the particles, colloids, or macromolecules that make up the products, may occur throughout this therapy. The starch slurry is passed through a micro-fluidizer, which can be intensified by external pressure sources, external mechanical pumps, integrated mechanical micro-pumps, or electro-kinetic mechanisms, resulting in the breakage of the starch slurry. The persistent pressure of 207 MPa was maintained. The starch PS was reduced from 3–6 µm to 10–20 nm without any change in crystal structure or thermal stability of starch granules . The preThe addition of a solution in which the starch is diluted into an aqueous phase in the presence of ultrasonic homogenization reduces the size of the particles formed as a result of shear forces or mechanical effects produced by the collapse of microbubbles that form sound wafers ,43,44. TUltrasonic atomization is a potential method for producing liquid micro-droplets using ultrasonic radiation. Two primary theories have been proposed in the droplet formation mechanism to explain the thin vibrating liquid film breakdown during ultrasonic atomization: Capillary waves and the cavitation mechanism . CapillaOther options for acid hydrolysis include the use of enzymes to de-branch starch chains, which has been performed with enzymes such as Beta-amylase, Alpha-amylase, glucoamylase, and pullulanase. Alpha-amylase is an endo-amylase that causes random cleavage of internal Alpha-1, 4 bonds, whereas Beta-amylase is an exo-amylase that causes cleavage of alternating Alpha-1, 4 bonds from non-reducing ends, which is referred to as hydrolysis rather than de-branching ,51 PulluAcid hydrolysis is a straightforward and easy-to-control method of producing nanoparticles. However, selective hydrolysis is a rather slow process that results in a poor yield. Another disadvantage is the high concentration of hydroxyl groups, which form aggregates due to the supramolecular interactions of starches. This propensity makes SNPs unsuitable for industrial use. Enzymatic hydrolysis is a non-hazardous and ecologically acceptable technique. The regeneration settings were adjusted to regulate the size and form of the SNPs. The process, however, is not complete when the enzyme activity drops. In the food industry, enzymatic hydrolysis of starch nanoparticles is frequently employed. In comparison to acid hydrolysis, ultrasonic treatment is faster, easier to handle, and does not need several rinses because no solvent is employed. The drawbacks of this approach are crystalline structure distortion and, depending on the angle of X-ray diffraction, amorphous or poor crystallinity structure. The applications of SNPs produced using ultrasonic techniques are quite diverse. Depending on the use, the initial starch should be of varying purity. The combination of hydrolysis and ultrasonic treatment resulted in a higher yield (78%) as compared to acid hydrolysis alone, which generated just 15%. The drawbacks of the technique are that SNPs were not sufficiently stable and inflated readily when sonicated, leading to starch aggregation formation . To emplDifferential scanning calorimetry (DSC) is a thermal analysis technique that determines how much energy a sample absorbs or emits as a function of temperature. A DSC equipment diagram, entropy, enthalpy, and special heat determination may all be used to determine the temperature and quantity of heat flow in a sample . Liu et w/w starch/CNP, and 30 percent w/w starch/CNP films. TGA analysis was carried out on a 20 mg sample film at a rate of 10 °C/min in the temperature range of 25–600 °C. TGA determines how much weight each sample loses throughout a temperature range of 25 to 600 °C.Thermo-gravimetric analysis (TGA) is a thermal analysis technique that measures the mass of a sample over time as the temperature varies. It is used to assess a sample’s thermal stability. This study involves knowledge of physical, but also chemical, phenomena. The temperature or time curve, also known as the thermogravimetric curve, displays data on the change in mass with the temperature/time of the sample using TGA as a graph/curve. A derivative plot of the TGA curve, termed DTG, displays the rate of mass change and the rate of mass loss against the temperature curve. Changes in sample mass can occur as a result of processes such as evaporation, dryness, desorption or adsorption, sublimation, and thermal breakdown. These mass shifts are evidenced by phase alterations in the TGA curve or DTG curve peaks . Shapi’IDifferential thermal analysis (DTA) is a technique quite similar to differential calorimetry scanning thermoanalytics. In DTA, the analyte undergoes thermal cycles, thus all variations during the analysis are recorded ,71. GardDynamic mechanical analysis (DMA) is a method for studying the kinetic characteristics of a sample by estimating strain or stress. It is used to measure polymer materials of various kinds utilizing various deformation modes. There is tension, compression, dual bending of cantiles, three-point bending, and shear modes, and the optimum type should be chosen based on the sample form, modulus, and measurement purpose . OchigboParupudi et al.  investigAFM enables three-dimensional simulation and analysis, and individual particles and particle classes may be solved . AFM is Transmission electron microscopy (TEM) technology uses an electron beam to image a nanoparticle sample, providing far better resolution than light-based imaging methods . The besScanning electron microscopy is a typical technique used for observing a material’s microstructure and morphology (SEM). SEM modalities for material characterization  include X-ray mapping, secondary imaging of electrons, backscattered imaging of electrons and electron channels, and Auger electron microscopy. SEM may be used to analyze and interpret micron- or nanometer-scale data . An elec−1 between 4000 and 400 cm−1. The FTIR spectrum of CAP-IRSNPs demonstrated that CAP and IRSNPs successfully interacted. In comparison to free CAP, the distinctive peak of CAP-IRSNPs was pushed to a lower wavenumber, indicating a strong intermolecular hydrogen bond.Fourier transform infrared (FTIR) spectroscopy provides helpful information on the structure’s functional groups by studying the vibrant frequencies of the chemical bonds. The molecules’ vibratory activation intensity is between 1013 and 1014 Hz, which corresponds to infrared light. This enables IR spectroscopy to examine and perform quantitative and qualitative investigations on self-assembled functional groups arranged on nano-paragon surfaces . The benX-ray diffraction (XRD) is a useful technique for studying nano-materials . The intensities obtained using XRD can give quantitative and precise information on nuclear structures at interfaces . Nano-maNuclear magnetic resonance (NMR) is a strong non-destructive analytical technique that uses magnetic field exposure on excited nuclei. It is an electrical phenomenon caused by the unique magnetic properties of particular atomic nuclei . NMR is The size distribution of tiny particles in suspension and/or polymers in a solution can be determined using dynamic light scattering (DLS), a physics method. To investigate temporal changes in DLS, the amplitude or photon self-correlation technique  is commonly utilized. Fluids, such as condensed polymer solutions, are also tested using DLS ,103. Thi3CO) are added, the free hydroxyl groups in the branched chains of the starch polymers react to form a particular ester. When starch is esterified, reactive reagents including anhydrous acetic acid, vinyl acetate, and octenyl succinic anhydride (OSA) are used in the presence of an alkaline catalyst 2, Na2CO3).The use of polymeric nanoparticles in drug delivery systems has attracted much interest because of their unique properties. The medium on which the medication is loaded, entrapped, encapsulated, or bonded is known as the nanoparticle matrix ,108. SNPw/w), glycerol , and oil (2 g/L) were used to make composite films. Gelatinized suspensions (20 mL) were then placed into Petri plates just after film-forming dispersion had been heated in a microwave (2350 W) for 30 to 60 s while being mixed continuously. The film-forming solution-coated plates were then dried for 8 to 10 h at 50 °C in an air-circulating oven. Then, the Petri plates’ dry films were peeled away, which proved that the composite film degraded more quickly, proving the starch nanoparticles improved the biodegradability of the films. The SNPs had an impact on the composites’ ability to degrade more quickly because, in soil, water diffuses into the polymer sample, inflating it and accelerating decomposition through an increase in microbial growth. Chavan et al. [Nanotechnology’s critical role in food packaging is regarded as the greatest commercial use in the food industry . In recen et al.  reported−1 compared to 50 and 40 mol g−1 when unmodified starch was used [Adsorption is thought to be the most promising strategy for treating wastewater, with its straightforward operation process, low cost, and reduced sludge formation. Owing to their unique qualities, such as high adsorption strength, greater surface area, and chemical stability, nanomaterial adsorbents have gained considerable attention in this area . After cwas used ,130,131.was used ,133. Gadwas used  investigFood processing and preservation need a variety of emulsion stabilizers. As emulsion stabilizers in meals, different particles such as hydrophobic silica, carbon nanotubes, latex, bacterial cellulose nano-crystals, and micro-fibrillated cellulose have been employed. SNPs have gained popularity as emulsion stabilizers in recent years. It was discovered that combining a small quantity of an SNP suspension with water and paraffin produced a stable emulsion. The suspension was stabilized for more than two months after SNP was added at a concentration of more than 0.02 percent by weight. However, phase separation occurred when the suspension was heated for 2 h at 80 °C . In addiHaaj et al.  reportedBecause of their enormous potential as nano-carriers in many bio applications such as medicine, cosmetics, and food, nanoparticles have recently seen a significant increase in terms of scientific research. SNPs have a distinct advantage over other types of nanoparticles owing to their excellent biocompatibility and flexibility, since there are a variety of possible synthesis techniques that allow for the customization of final shape characteristics such as size and size distribution. SNPs were created utilizing a variety of synthesis techniques using starch from various sources. In most cases, the manufacturing technique, as well as the kind of starch used, will influence the final characteristics of the nano-particles. Maize starch is one of the most widely utilized types of starch for SNP synthesis, either alone or in combination with other starch such as potato, pea, or faba bean, and has been proven to result in tiny particle sizes. SNPs with small sizes may be obtained using both nano-precipitation and ultrasonic techniques.


Here, we developed a new generation of collagen-binding EVs, by chemically conjugating a collagen-binding peptide SILY to EVs (SILY-EVs), which were designed to bind to collagen in the extracellular matrix (ECM) and form an EV-ECM complex to improve EVs' Methods: SILY was conjugated to the surface of mesenchymal stem/stromal cell (MSC)-derived EVs by using click chemistry to construct SILY-EVs. Nanoparticle tracking analysis (NTA), ExoView analysis, cryogenic electron microscopy (cryo-EM) and western-blot analysis were used to characterize the SILY-EVs. Fluorescence imaging (FLI), MTS assay, ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to evaluate the collagen binding and biological functions of SILY-EVs in vitro. In a mouse hind limb ischemia model, the in vivo imaging system (IVIS), laser doppler perfusion imaging (LDPI), micro-CT, FLI and RT-qPCR were used to determine the SILY-EV retention, inflammatory response, blood perfusion, gene expression, and tissue regeneration.Results:In vitro, the SILY conjugation significantly enhanced EV adhesion to the collagen surface and did not alter the EVs' biological functions. In the mouse hind limb ischemia model, SILY-EVs presented longer in situ retention, suppressed inflammatory responses, and significantly augmented muscle regeneration and vascularization, compared to the unmodified EVs.Conclusion: With the broad distribution of collagen in various tissues and organs, SILY-EVs hold promise to improve the therapeutic efficacy of EV-mediated treatment in a wide range of diseases and disorders. Moreover, SILY-EVs possess the potential to functionalize collagen-based biomaterials and deliver therapeutic agents for regenerative medicine applications. First, DBCO groups were introduced to the EV surface by linking EVs with DBCO-sulfo-NHS. The NHS groups were conjugated to the amine groups present on the surface proteins of EVs to form covalent bonds. Secondly, the DBCO groups on the EVs were then combined with azide-SILY to form stable triazole linkages using copper-free click chemistry. To assess the SILY conjugation qualitatively, PKH67-labeled EVs were conjugated to the TAMRA (a red dye) labeled azide-SILY with or without DBCO-sulfo-NHS. The results showed that the TAMRA and the PKH67 merged efficiently in the group with DBCO-sulfo-NHS. In contrast, almost no TAMRA and PKH67 merged in the group without DBCO-sulfo-NHS . The results indicated the SILY was successfully conjugated to the EV surface by using the conjugation approach established in this study. ExoView tetraspanin kit assay was used to further quantificationally measure the SILY conjugation efficiency on EVs. The results showed the SILY conjugation efficiency on EVs was about 70% .MSC-derived EVs have shown promising regenerative potential, including promoting angiogenesis and suppressing inflammation and cell apoptosis Figure ), which indicated the SILY conjugation approach did not affect the size distribution of the EVs. The cryo-EM results exhibited intact structure and similar round morphology of both the EVs and SILY-EVs , confirming the SILY conjugation approach maintained the integrity of EV structure. The results of the BCA assay showed no significant difference in the protein content between the same number of EVs and SILY-EVs, indicating the SILY conjugation did not alter the protein content of EVs . The western-blot results showed both EVs and SILY-EVs expressed the typical EV markers, such as CD9, CD63, Alix and TSG101, and did not express endoplasmic reticulum marker calnexin , which indicated the SILY conjugation approach designed in this study did not alter the expression of typical EV markers.The EVs and SILY-EVs were characterized by using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-EM), BCA assay and western-blot analysis. The NTA results showed that the EVs had diameters of 119.6-133.6 nm and the SILY-EVs had diameters of 114.2-147.6 nm . To avoid the effects of the DiD dye background, we further evaluated the cellular uptake of EVs or SILY-EVs using a mixture of DiD-labelled (10%) and non-labelled (90%) EVs or SILY-EVs, and the signal of EVs or SILY-EVs untaken by the HECFCs was significantly decreased, compared to the all DiD-labelled (100%) EVs or SILY-EVs , which was consistent with the previous results.To evaluate cellular uptake of EVs and SILY-EVs, DiD-labeled EVs or SILY-EVs were incubated with human endothelial colony forming cells (HECFCs) that is crucial for vascularization in vitro time-course experiments to evaluate the kinetics of EVs and SILY-EVs internalized in HECFCs. After another 48-h culture in the medium without EVs and SILY-EVs, both the EVs and SILY-EVs internalized in HECFCs decreased, and there was no significant difference between EVs and SILY-EVs . These results demonstrated the SILY conjugation approach did not alter the degradation rate of EVs internalized in cells.We further performed the Figure S4), confirming the SILY conjugation approach did not affect the EVs' biological functions.Tube formation assay, cell proliferation assay and Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to further evaluate the effects of the SILY conjugation approach on EVs' biological functions. The results showed no significant difference on HECFC tube formation, HECFC proliferation and expression of pro-angiogenic and inflammatory genes . Besides type I collagen, type III collagen is the second most abundant collagen in the body Figure S5). Based on the attachment of EVs or SILY-EVs on the type I collagen surface, we further evaluated the effects of the collagen surfaces decorated with EVs or SILY-EVs on biological functions. In the cell survival study, the ischemic simulated environment was set up as basic endothelial basal medium-2 (EBM-2) with 1% fetal bovine serum (FBS) and low concentration growth factors at 1% O2, 37◦C and 5% CO2. In the in vitro ischemic simulated environment, the collagen surface decorated with SILY-EVs significantly improved HECFC viability  and survival  and suppressed HECFC apoptosis by decreasing the expression of Caspase 3 and Caspase 9 , compared to the collagen surface decorated with EVs. Additionally, the collagen surface decorated with SILY-EVs significantly improved Ki67 expression in HECFCs  and HECFC proliferation , compared to the collagen surface decorated with EVs. Moreover, the expression of angiogenic genes, including KDR and TIE2, was significantly increased in the HECFCs cultured on the collagen surface decorated with SILY-EVs, compared to the HECFCs cultured on the collagen surface decorated with EVs . In the inflammatory response study, compared to the collagen surface decorated with EVs, the collagen surface decorated with SILY-EVs significantly decreased the expression of Th1-related gene IFN-γ and increased the expression of Th2-related gene IL10  in lipopolysaccharide (LPS)-stimulated human macrophage-like THP-1 cells. These results demonstrated that the SILY conjugation increased the number of EVs attached to the collagen surface, resulting in enhanced angiogenic capacities and suppressed inflammatory responses. Hence, this SILY conjugation approach holds promise to promote EV regenerative functions in vivo by improving EV retention.To evaluate the binding ability of SILY-EVs to collagen, we coated the culture surface with type I collagen and investigated the attachment of EVs or SILY-EVs on the collagen surface via imaging and NTA. Both the results of imaging and NTA showed that the SILY conjugation significantly improved EV attachment on the type I collagen surface . We further evaluated the retention of EVs or SILY-EVs on the ischemic hind limb tissue sections after incubating for 1, 3 or 7 days. The results showed the retention of SILY-EVs was significantly higher on the ischemic hind limb tissue via binding to collagen at day 1, 3 and 7, compared to the EVs, and the numbers of both EVs and SILY-EVs were lower with time .Before EVs were transplanted into the mouse hind limb ischemia model, we performed the In Vivo Imaging System (IVIS) to visualize the presence of the EVs in the entire body of the animals. IVIS results showed that on day 1 after injection, the signal in the EV group decreased sharply at the injection site, whereas the signal in SILY-EV group persisted. On day 7 after injection, the signal in the EV group was almost undetectable, whereas the signal was still visible in the SILY-EV group . These results demonstrated that SILY-EVs possessed longer retention in vivo, compared to the unmodified EVs. This longer retention of SILY-EVs may lead to improved therapeutic efficacy in disease treatment. Moreover, we did not find any injected EVs in other internal organs.Subsequently, the EVs or SILY-EVs were transplanted into the mouse ischemic hind limb via intramuscular injection to evaluate the retention of EVs or SILY-EVs at different time points. To characterize the EV biodistribution after intramuscular administration, whole animal imaging was conducted using the Figure ). To assess the effect of SILY-EVs on vascular remodeling in the mouse ischemic hind limbs, mice treated with PBS, EVs or SILY-EVs were injected with a contrast media, Microfil MV-120, into the left ventricle on day 21 after surgery. Large and branching vessels in the ischemic limbs were detected using micro-CT imaging . The total vessel volume in the mouse ischemic limbs in both the EV treatment group and SILY-EV treatment group were significantly improved, compared to that in the PBS treatment group. Furthermore, the SILY-EV treatment significantly enhanced the total vessel volume compared to the EV treatment . These results demonstrated that SILY-EVs promised greater capability to promote vascular remodeling and blood reperfusion in the ischemic disease model compared to conventional unmodified EVs.To further determine the effects of SILY-EVs on the ischemic disease, EVs or SILY-EVs were intramuscularly injected into the mouse ischemic hind limb on day 1 after surgery. After the injection of EVs and SILY-EVs, all animals were closely monitored for up to 21 days to comprehensively evaluate for safety, especially toxicity and tumorigenicity. Daily visual inspection of the animal's condition includes mortality, clinical presentation, body weight, and consumption of food. No obvious toxicity and tumorigenicity was observed in any animals during the entire study period. The blood perfusion was evaluated by using Laser Doppler perfusion imaging (LDPI). Although both EV and SILY-EV treatments increased blood perfusion compared to the PBS treatment on days 7, 14, and 21 after delivery, blood perfusion in the SILY-EV treatment group was significantly improved compared to the EV treatment group on days 14 and 21 after surgery . CCR7+ M1 macrophages were dramatically increased in the PBS and EV treatment groups and were attenuated in the SILY-EV treatment groups . CD163+ M2 macrophages were significantly higher in the SILY-EV treatment group, compared to the other two groups . Although both the EV treatment group and SILY-EV treatment group improved the M2/M1-like macrophage ratio, the SILY-EV treatment significantly and substantially promoted the macrophage polarization from the M1 to the M2, compared to the EV treatment and PBS treatment . Additionally, we further determined the gene expression related to inflammatory response, we found SILY-EV treatment significantly decreased the expression of Th1-related gene IFN-γ  and increased the expression of Th2-related gene IL10 , compared to the EV and PBS treatments. All of these results demonstrated that compared to the EV treatment, SILY-EV treatment had a greater impact on suppressing the inflammatory response at the injure area and promoting M2 polarization of macrophages, which are vital in promoting tissue regeneration and healing.The role of macrophages in tissue healing and repair is correlated with their ability to polarize between pro-inflammatory and anti-inflammatory phenotypes Figure ), reduction of centrally located nuclei  and collagen deposition  in the SILY-EV treatment group, compared to the EV and PBS treatment groups, suggesting that SILY-EVs possess a greater capacity for muscle regeneration compared to EVs. Moreover, we further determined the gene expression related to muscle regeneration, we found SILY-EV treatment significantly increased the expression of Myoz1  and Myoz3 , compared to the EV and PBS treatments.Having established improved retention, and enhanced polarization of macrophages to an M2 phenotype, we next evaluated the effects of SILY-EVs on ischemic muscle tissue repair. Histologic analysis confirmed significant myofiber augmentation in size  and arterioles , compared to the EV and PBS treatments. In addition, we further determined the expression of angiogenic genes, ANG II and PECAM1, in different treatment groups, and the results showed the SILY-EV treatment significantly increased the expression of ANG II  and PECAM1 , compared to the EV and PBS treatments. These data indicate that the SILY-EVs possess a better regenerative potential on revascularization compared to the EVs. Overall, although EVs were positive regulators in post ischemic recovery in the mouse hind limb ischemia model, the SILY-EVs showed stronger healing efficacy.To evaluate the effects of SILY-EVs on revascularization, we performed CD31 staining for capillaries and α-smooth muscle actin (α-SMA) staining for the arterioles. In agreement with LDPI measurements, the results confirmed that the SILY-EV treatment significantly improved the density of capillaries  via bound to the collagen in ECM to form the stable EV-ECM complex, resulting in significantly improved therapeutic efficacy in the hind limb ischemia model, compared to the locally injected free EVs .Although the emergence of EVs has revealed promising potentials in various tissue repair, the applications of free EVs still face many challenges, especially their rapid clearance Figure Figure S4). Moreover, this engineering strategy is suitable for conjugating multiple molecules with disparate characteristics to EVs derived from different media, and the densities of molecules can be controlled by adjusting the ratio of the reactants. In future studies, as we further develop SILY-EVs towards clinical translation, we will further perform a detailed toxicological evaluation at the endpoint, which will include full clinical evaluation such as hematology, blood chemistry, organ weights, and full pathology, including gross and histopathology on all tissues and organs. We will also collect the blood to evaluate systemic inflammation after the treatment. Furthermore, a sham-operated group should be included in the future studies, which could be a control group to further interpretate the rationale of the hind limb ischemia model established in this study.Compared to many previous EV surface engineering approaches In this study, the SILY-EVs locally injected into the injured site could contribute to sustained tissue repair in a variety of ways. Firstly, SILY-EVs bound to collagen in ECM could still interact directly with the surrounding cells via EV-cell fusion. Secondly, SILY-collagen binding is a non-covalent molecular interaction-based binding. Therefore, there will be a dynamic release of EVs from ECM collagen which will then interact with the surrounding cells. Lastly, EV cargos, e.g., miRNAs and proteins, can be released from EVs which can directly interact with the cells in the local environment. Moreover, since collagen is distributed within most tissues Collagen-based biomaterials have been widely used in clinical applications, and the EVs have also been developed to functionalize the biomaterial scaffolds in the tissue regeneration field Currently, the isolation of EVs is a very laborious process, especially the isolation of MSC-derived EVs To address the current challenge of the locally injected EVs, in this study, we developed a new generation of collagen-binding EVs, SILY-EVs, that possessed stronger binding affinity to collagen and presented longer retention and higher therapeutic efficacy in the mouse hind limb ischemia model, compared to the free EVs. The SILY-EVs do not only hold promise to improve therapeutic efficacy of EV-based treatments in clinical applications, but also provides an innovative way to advance the EV applications in tissue engineering and regenerative medicine fields including functionalizing collagen-based biomaterial and delivering therapeutic agents.◦C, 5% CO2. PMSCs were used between P3 and P5 for all experiments. ECFCs were isolated from human umbilical cord blood (HECFCs) as we previously described MSCs were isolated from early gestation placental chorionic villus tissue (PMSCs) as we previously described 4 cells/cm2 in tissue culture-treated T175 flasks in 20 mL of EV-depleted FBS containing D5 medium for 48 h at 37◦C, 5% CO2. Conditioned medium was collected and sequentially centrifuged at 300 g for 10 min, 2,000 g for 20 min, and passed through a 0.22 mm filter. Then, the medium was concentrated using Amicon Ultra Centrifugal Filter Units with a 100-kDa MW cutoff (Sigma), transferred to thick wall polypropylene tubes (Beckman Coulter), and centrifuged at 8,836 g using the SW28 rotor and L7 Ultracentrifuge (Beckman Coulter). The supernatant was transferred to fresh tubes, centrifuged at 112,700 g for 90 min, and the pellet was resuspended in PBS (HyClone) and spun again at the same speed and time. The final pellet was resuspended in 10 mL of PBS per T175 flask and stored in aliquots at -80◦C.EVs were isolated from PMSCs as described in our previous study ◦C for 12 h. The unincorporated peptides were removed by three washing steps on 100-kDa ultrafiltration tubes, and the SILY-EVs were resuspended in PBS. All the reactions of this conjugation approach were performed in neutral aqueous buffers without catalysts to void EV damage.For the SILY-EV preparation, SILY was conjugated to the EV surface by using click chemistry To assess the SILY conjugation qualitatively, TAMRA labeled SILY-azide (Lumiprobe Co.) was conjugated to the PKH67 (System Biosciences) labeled EVs with or without DBCO-sulfo-NHS linker. The conjugated EVs were applied to coverslips and imaged by fluorescence microscopy (Nikon) with a 60 objective.◦C when not in use and allowed to warm to room temperature prior to use. Chips were pre-scanned using the provided protocol to identify any previously adhered particles during manufacturing. TAMRA-labeled SILY-EVs were diluted to a concentration 5 × 108 particles/mL in the provided incubation solution, and 35 μL of sample were carefully pipetted onto the center of the chip. Samples were incubated on the chip overnight at room temperature. Following incubation, 1 mL of incubation solution was added and shaken at 500 rpm for 3 min at room temperature. 750 μL of incubation solution was removed and 750 μL of fresh solution was added, and the chip was once against shaken for 3 min. This step was repeated two more times to allow for thorough washing of the chip. After the last wash, 750 μL of incubation solution was removed and 250 μL of fluorescently labeled primary antibody CF647-anti-CD63 (clone: H5C6) was added (1:500 dilution in blocking solution). Chips were incubated with the antibodies for 1 h at room temperature while being shaken at 500 rpm. After incubation, chips were washed with 500 μL of incubation solution twice and with 750 μL rinse solution three times. Chips were washed once in DI water and carefully dried on an absorbent paper. Once fully dried, the chips were scanned using the ExoView® R100 (Nanoview Biosciences) for data acquisition.ExoView tetraspanin kit assay was used to quantificationally measure the SILY conjugation efficiency on EVs. ExoView kits (NanoView Biosciences) were used according to the manufacturer's protocol. All chips were stored at 48 - 1 × 109 particles/mL. The diluted EVs or SILY-EVs were analyzed using the NanoSight LM10  equipped with a 404-nm laser and sCMOS camera. Three 90-s videos were recorded for each sample at camera level 12 using NTA v.3.0 software. Data were consistently analyzed with a detection threshold of 3 and screen gain of 10.The concentration and size distribution of EVs and SILY-EVs were characterized by using NTA. EVs or SILY-EVs were diluted in triple-filtered (0.22 μm) MilliQ-water to reach the appropriate concentration range of 1 × 1011 EVs or SILY-EVs/mL solution was applied to the carbon side of the EM grid, blotted for 5 seconds, and then plunge-frozen into precooled vat of liquid ethane with Vitrobot Mark MkIII (FEI). Vitrified samples were then imaged with the Glacios Cryo Transmission Electron Microscope equipped with K3 Direct Electron Detector and acquired with SerialEM software .The structure and morphology of EVs and SILY-EVs was characterized by cryo-EM. Briefly, Carbon EM grids (Ted Pella Inc.) were glow discharged at 30 mA, 30 s . 4 μL of 1 × 1010 EVs or SILY-EVs were resuspended in RIPA buffer (Sigma-Aldrich) with protease inhibitors cocktail (Sigma-Aldrich), and then sonicated for 5 min. Protein contents were measured using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's instruction.The protein contents of EVs and SILY-EVs were measured by using BCA assay. 5 × 10◦C overnight. Secondary antibody mouse IgGκ-HRP was diluted at 1:5000 in 5% milk in PBS and incubated at 1 h at room temperature with shaking (Santa Cruz Biotechnology). Proteins were visualized with Supersignal West Pico PLUS chemiluminescent substrate (ThermoFisher) using a KwikQuant imager (Kindle Biosciences).The expression of EV typical markers on EVs and SILY-EVs were determined by using western-blot analysis. EV or SILY-EV pellets were lysed in RIPA lysis buffer containing protease inhibitor cocktail (Roche). 2 μg of protein per lane was loaded on a 4-12% graded Tris-glycine SDS-polyacrylamide gel (Invitrogen), then the proteins were transferred to a 0.22 μm PVDF membrane (Life Technologies). Membranes were blocked in 5% milk in PBS with 0.05% Tween-20 for 1 h at room temperature with shaking, and then incubated with primary antibodies anti-ALIX, anti-TSG101, anti-CD9 (Millipore), anti-CD63 (Thermo Fisher Scientific) and anti-calnexin  at 1:1000 dilution in 5% milk in PBS at 410 EVs or SILY-EVs for 1 h. Excess DiD was removed using 100-kDa ultrafiltration tube. 1 × 109 DiD-EVs or DiD-SILY-EVs were added to each well and incubated with the HECFCs grown in µ-Slide 8 Well (ibidi) for 4 h or 16 h. In the control groups, a mixture of DiD-labelled (10%) and non-labelled (90%) EVs or SILY-EVs were incubated with the HECFCs for 4 h or 16 h. Media with DiD-EVs or DiD-SILY-EVs was aspirated, and the cells were fixed in 4% Paraformaldehyde  for 20 min. After washing with PBS, the cells were blocked in 1% Bovine Serum Albumin  for 1 h at room temperature and incubated overnight with primary antibody anti-CD31 (Abcam) at 1:100 dilution in 1% BSA at 4◦C. After being washed with PBS 3 times, the tissue was incubated with the relevant secondary antibody (Life Technologies) at 1:500 dilution in PBS for 1 h at room temperature, and then nuclei were stained with 4',6-diamidino-2-phenylindole . After being washed with PBS 3 times, the images were captured using the Zeiss Observer Z1 microscope. Quantification of the number of cells with internalized DiD-EVs or DiD-SILY-EVs was performed using the ImageJ software (NIH). For the in vitro time-course experiments of EVs or SILY-EVs internalized in HECFCs, 1 × 109 DiD-EVs or DiD-SILY-EVs were added to each well and incubated with the HECFCs grown in µ-Slide 8 Well for 16 h. Media with DiD-EVs or DiD-SILY-EVs was replaced with media without DiD-EVs and DiD-SILY-EVs. The HECFCs were cultured for another 48 h, then the cells were fixed in 4% PFA for 20 min and stained with DAPI. The images were captured using the Zeiss Observer Z1 microscope. Quantification of the number of cells with internalized DiD-EVs or DiD-SILY-EVs was performed using the ImageJ software.EVs and SILY-EVs were labeled with DiD dye (ThermoFisher Scientific). DiD at a concentration of 2 μM was incubated with 1 × 104 cells/well and cultured in 100 μL of EBM-2 medium (Lonza) with EVs or SILY-EVs at 1 × 109 particles/mL and incubated at 37◦C and 5% CO2 for 6 h. Images were taken using the Carl Zeiss Axio Observer D1 inverted microscope. The total segment length was quantified using ImageJ Angiogenesis Analyzer tool.HECFCs were seeded onto growth factor reduced Matrigelcoated (Corning) in 96-well plates at 1 × 103 cells/well and cultured in 100 μL of EGM-2 medium with EVs or SILY-EVs at 1 × 109 particles/mL and incubated at 37°C and 5% CO2 for 5 d. Cell proliferation was assessed using CellTiter 96® AQueous One Solution Cell Proliferation Assay  according to the manufacturer's instructions.HECFCs were seeded in 96-well plates at 2 × 10◦C were 40 cycles of 95◦C, 10 s, for denaturation, 55◦C, 10 s, for annealing and 72◦C, 30 s, for elongation. GAPDH was used as the reference gene for calculations. Data were analyzed by the delta-delta cycle threshold (Ct) method. Primer sequences used in this study are listed in Table S1.RNA extraction from cells or tissue was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions, and cDNA was synthesized using Superscript II Reverse transcriptase (ThermoFisher Scientific). PCR was performed using the Biorad CFX96 Real-Time PCR Detection System (BioRad Laboratories) machinewith the SsoAdvanced SYBR Green Supermix (Bio-Rad). Amplification conditions after an initial denaturation step for 90 s at 95◦C for 10 min. Excess dye was removed by centrifuging at 16,000 g for 3 min at 4◦C. The EV pellets were resuspended in 500 μL of cold PBS, placed on ice for 5 min, and centrifuged again at 16,000 g for 3 min at 4◦C. Washes were repeated for a total of 3 times. After the final wash, labeled EVs were resuspended in 500 μL of PBS.EVs were labeled with PKH67. Briefly, PKH67 was diluted 1:500 in PBS and added to EVs. The sample was mixed gently by flicking the tube and incubated at 37◦C. Then, the wells were washed three times with PBS, and 5 × 106 PKH67-labeled EVs or SILY-EVs suspended in PBS was added into each well respectively and incubated for 1 h at 37◦C and 5% CO2. Then the wells were washed three times with PBS, and the attached EVs were imaged using a Carl Zeiss Axio Observer D1 inverted microscope. Quantification of images was performed using the ImageJ software. To further evaluate the EV attachment by using NTA, the wells in a 6-well cell culture plate were coated with 1.5 mL of 100 μg/mL type I collagen or type III collagen and incubated for 1 h at 37◦C. Then, the wells were washed three times with PBS, and 2 mL 5 × 107/mL EVs or SILY-EVs suspended in PBS was added into each well respectively and incubated for 1 h at 37◦C and 5% CO2. The solution in each well was collected, and the remaining EVs or SILY-EVs were measured by using NTA.For the EV attachment assay, the wells in a 48-well cell culture plate were coated with 150 μL of 100 μg/mL type I collagen (Millipore) or type III collagen (Millipore) and incubated for 1 h at 377/cm2 and incubated for 10 min at 37◦C and 5% CO2, then the wells were washed 3 times with PBS. Human THP-1 monocytes were differentiated to macrophage-like THP-1 cells by stimulating with phorbol-12-myristate-13-acetate  as described in the previous study 4 macrophage-like THP-1 cells/well activated with 100 ng/mL LPS (Sigma-Aldrich) were seeded in the collagen coated wells decorated with EVs or SILY-EVs respectively. After incubation for 12 h, cells were collected for evaluation of the gene expression related to inflammatory responses by using RT-qPCR assay as the procedure described above.EVs or SILY-EVs was added into the collagen coated wells of the 48-well cell culture plate at a density of 1 × 107/cm2 and incubated for 10 min at 37◦C and 5% CO2, then the wells were washed 3 times with PBS. For cell viability assay, 1 × 104 HECFCs/well were seeded in the collagen coated wells decorated with EVs or SILY-EVs respectively. The ischemic simulated environment was set up as EGM-2 with 1% fetal bovine serum (FBS) and low concentration growth factors  at 1% O2, 37◦C and 5% CO2 as described previously 4 HECFCs/well were culture in the collagen coated wells decorated with EVs or SILY-EVs respectively in ischemic-mimicking hypoxic environment for 5 d and determined using MTS according to the manufacturer's instruction. For caspase 3 and caspase 9 assay, the cells were cultured in ischemic-mimicking hypoxic environment for 6 h, then lysed and analyzed by using a Caspase 3 Assay Kit and Caspase 9 Assay Kit  according to the manufacturer's instruction. Fluorescence (ex 380 nm/em 450 nm) was measured using a SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices).EVs or SILY-EVs was added into the collagen coated wells of the 96-well cell culture plate at a density of 1 × 107/cm2 and incubated for 10 min at 37◦C and 5% CO2, then the wells were washed three times with PBS. For HECFC proliferation assay, 2 × 103 HECFCs/well were seeded in the collagen coated wells decorated with EVs or SILY-EVs respectively and cultured for 5 d and determined using MTS assay according to the manufacturer's instruction. For Ki67 staining, 2 × 103 HECFCs/well were seeded in the collagen coated wells decorated with EVs or SILY-EVs respectively and cultured for 5 d, and the cells were fixed in 4% PFA for 20 min. After being washed with PBS, the cells were blocked in 1% BSA and incubated overnight with primary antibody anti-ki67 (Abcam) at 1:100 dilution in 1% BSA at 4◦C. After being washed with PBS 3 times, the tissue was incubated with the relevant secondary antibody at 1:500 dilution in PBS for 1 h at room temperature, and then nuclei were stained with DAPI. After being washed with PBS 3 times, the images were captured using the Zeiss Observer Z1 microscope. The number of Ki67 positive cells was quantified by using ImageJ software. For evaluation of pro-angiogenic gene expression, 2 × 104 HECFCs/well were seeded in the collagen coated 48-well plate decorated with EVs or SILY-EVs respectively. After incubation for 12 h, the HECFCs were collected for RT-qPCR assay as the procedure described above.EVs or SILY-EVs was added into the collagen coated wells of the 96-well cell culture plate at a density of 1 × 10◦C. After being washed with PBS 3 times, the tissue was incubated with the relevant secondary antibody (Life Technologies) at 1:500 dilution in PBS for 1 h at room temperature, and then nuclei were stained with DAPI. 1 × 107 EVs or SILY-EVs labeled with DiD were incubated with the mouse ischemic hind limb tissue sections stained with CD31 and DAPI for 1 h at room temperature, followed by washing with PBS 3 times. The images were captured using the Zeiss Observer Z1 microscope. Six views were captured from each slice, and six slices from different mice were used in this study. Quantification of the number of EVs or SILY-EVs attached on the collagen was performed using the ImageJ software.Mouse ischemic hind limb tissue sections were blocked in 1% BSA for 1 h at room temperature and incubated overnight with primary antibody anti-collagen I (Abcam) at 1:100 dilution in 1% BSA at 47 EVs or SILY-EVs labeled with DiD were incubated with the mouse ischemic hind limb tissue sections stained with CD31 and DAPI for 1 h at room temperature, followed by incubating with PBS at 37◦C. The images were captured at day 1, 3 and 7 using the Zeiss Observer Z1 microscope. Quantification of the number of EVs or SILY-EVs attached on the collagen was performed using the ImageJ software.To further evaluate the retention of EVs or SILY-EVs on the ischemic hind limb tissue sections at different time points, 1 × 10All animal experiments were approved by the Institutional Animal Care and Use Committee at University of California Davis. Female mice  were obtained from the Jackson Laboratory. Under anesthesia, mice were subjected to unilateral hind limb ischemia surgeries as previously described 11/mL DiD-labeled EVs or SILY-EVs was intramuscularly injected into four different sites around the hind limb ischemic region in the previous studies To evaluate the retention of EVs and SILY-EVs in the mouse hind limb ischemia model, the mice were randomly assigned to two groups: (1) EVs (n = 6) and (2) SILY-EVs (n = 6). EVs or SILY-EVs were labeled with DiD, and 100 μL 1 × 1011/mL EVs or SILY-EVs was intramuscularly injected into four different sites around the hind limb ischemic region as described above.To evaluate the effects of EVs and SILY-EVs on inflammatory response, revascularization and muscle repair in the mouse hind limb ischemia model, the mice were randomly assigned to three groups: (1) PBS (n = 6); (2) EVs (n = 6); (3) SILY-EVs (n = 6). 100 μL 1 × 10For the blood perfusion assessment, the ischemic limb-to-nonischemic limb blood perfusion ratio was measured weekly up to 3 weeks using a Laser Doppler perfusion imager (Moor Instruments), which provides noninvasive measurement of the blood flow by determining the Doppler frequency shift for light reflected off the moving red blood cells. Briefly, the mice under anesthesia were secured on a monochromatic surface, and an area was scanned from the lower abdomen to the end of the toes. Color images were obtained, and the hindlimb perfusion ratios were determined by comparing the perfusion of the injured hind limbs and the contralateral normal hind limbs. The outcomes were blindly assessed.The mice were euthanized at day 21, and a catheter was inserted into the left ventricle of the mice. The mouse vasculature was flushed with PBS supplemented with 100 U/mL heparin sodium (Fresenius Kabi) and then 4% PFA. One to two milliliters of Microfil MV-120  was injected into the left ventricle to increase contrast of vasculature. The leg muscles from both limbs were separately collected and imaged with a micro-CT imaging system  using a voxel size of 10.5 mm, X-ray source voltage of 55 kVp, and 145 mA current. The micro-CT images were analyzed using a matching three-dimensional segmentation algorithm of thresholding.◦C. Cryosections (thickness = 6 μm) were prepared. For histological analysis, the sections were stained by H&E and Masson's trichrome (Thermo Fisher Scientific) according to the manufacturer's instructions respectively. For immunohistochemistry staining, the tissue sections were incubated with different primary antibodies anti-CCR7 (Abcam), anti-CD163 (Abcam), anti-CD68 (Abcam), anti-CD31 (Abcam), or anti-α-SMA (Abcam) at 1:100 dilution in 1% BSA overnight at 4°C. After being washed with PBS 3 times, the tissue sections were incubated with the relevant secondary antibody at 1:500 dilution in PBS for 1 h at room temperature, and then nuclei were stained with DAPI. After being washed with PBS 3 times, the images were captured using the Zeiss Observer Z1 microscope. The images were analyzed by using ImageJ software.All mice were euthanized at day 21, and the hind limbs were harvested and trimmed to dissociate out the upper part of ligation. Tissues were immersed in 4% PFA for 48 h at 4t test was used for post- analysis. The values of P < 0.05 indicates significant difference between samples in comparison.All the outcomes in this study were assessed blindly. Graphs and statistical analysis were performed using Prism. For multiple-sample comparison, analysis of variance (ANOVA) was used to evaluate whether a significant difference existed between groups with different treatments, and a multiple comparison procedure Holm's Supplementary figures and table.Click here for additional data file.


Thin-walled beams are increasingly used in light engineering structures. They are economical, easy to manufacture and to install, and their load capacity-to-weight ratio is very favorable. However, their walls are prone to local buckling, which leads to a reduction of compressive, as well as flexural and torsional, stiffness. Such imperfections can be included in such components in various ways, e.g., by reducing the cross-sectional area. This article presents a method based on the numerical homogenization of a thin-walled beam model that includes geometric imperfections. The homogenization procedure uses a numerical 3D model of a selected piece of a thin-walled beam section, the so-called representative volume element (RVE). Although the model is based on the finite element method (FEM), no formal analysis is performed. The FE model is only used to build the full stiffness matrix of the model with geometric imperfections. The stiffness matrix is then condensed to the outer nodes of the RVE, and the effective stiffness of the cross-section is calculated by using the principle of the elastic equilibrium of the strain energy. It is clear from the conducted analyses that the introduced imperfections cause the decreases in the calculated stiffnesses in comparison to the model without imperfections. Thin-walled cross-sections have been used in structural engineering for decades ,2,3. TheRecently, due to the increasing popularity of energy from renewable sources, but also due tothe geopolitical situation in Europe, solar-power photovoltaics have gained more interest. Of three main sources, wind, hydro, and solar power, the solar source is the most rapidly developing. In 2008, electricity generated from solar power was 1% in total, while in 2020, the value was 14% [The main advantage of thin-walled sections is that their application allows for the optimal use of the construction material . The relApart from all of the above-mentioned advantages, the biggest disadvantage of the thin-walled cross-sections is their vulnerability to imperfections, both material and geometric. The imperfection may lead to local  or globaIf a structure design is very complex, a three-dimensional finite element method (FEM) model is required to take into consideration the full integrity of the structure and the direct transmission of the loads onto lower substructures. Using FEM and creating a full 3D model for very complex structures would not only be laborious but also very time-consuming in terms of modeling time and computational cost . In suchIn the literature, there are various homogenization methods. One approach uses the equivalent of the deformation energy ,19,20,21Typical homogenization techniques are taking into consideration a periodic part of the structure or body. In the calculation, usually the geometry and the material are idealized, i.e., they have no imperfections. Since, for thin-walled structures such as a Z or C profile, even small loading may cause initial instability, so taking imperfections into account when applying the homogenization technique can be crucial for the correct determination of the actual stiffness of the structure, e.g., in the case of the flexural buckling of purlins.This concern was the motivation to conduct numerical research in this direction in the article. The main purpose of this work is to quantify the reduction in the effective stiffness of a thin-walled beam, taking into account local imperfections due to compression, bending, or shear via the numerical homogenization technique. It is also indicated herein which buckling mode may be representative to be used while taking into account imperfections instead of computing buckling modes for all load-cases separately.The Materials and Methods chapter consists of four sections. In the first section, the general workflow is described, while in the second section, the mathematical details of the homogenization method used are summarized. In the third section, the buckling analysis from an algorithmic point of view is presented, while in the fourth section, the representative volume element (RVE) models of the Z-profile used in the study are shown. The Results chapter contains the outcomes of the numerical homogenization of the reference Z-profile case and its counterparts for the same beam section with varied imperfections. The comparison to the reference case is also included. In the Discussion and Conclusions chapters, the results of the comparison are commented on, and the final outlines are presented.This article continues the work on homogenization presented in our previous papers. However, in this case, a thin-walled open section is analyzed, taking into account the geometrical imperfections of the section. In order to correctly introduce imperfections to the model, in the first step, displacements resulting from different buckling modes of the cross-section are introduced into the model, taking into account the selected boundary conditions. Then, the influence of these imperfections on the stiffness drops, which are determined by the numerical homogenization method, is checked. Eventually, a form of imperfection was chosen that was the most representative.In this study, the influence of the geometric imperfection due to particular deformation mode on the mechanical characteristics of the beam section was computed. The scheme illustrating the study workflow is presented in ABDR matrix [Then, by using this method, the selected Z-profile see  without R matrix  ui=Hix, y, z coordinates refer to the coordinates of all points to which the stiffness matrix of the entire RVE model has been condensed.It is worth noting that the above matrix applies to every node Hk=HeIf The buckling problem solved by the finite element method consists of two steps: pre-buckling static analysis and nominal buckling analysis. In the first step, the global stiffness matrix is computed, The nodal displacements are represented by In this article, the thin-walled cold-formed Z-profile that has been subjected to different types of buckling are analyzed. The influence of geometric imperfections on the local change of the effective stiffness of the Z profile was investigated using the numerical homogenization method, which is described in For this purpose, the RVE of the Z profile with lengths of 100 mm, 150 mm, and 200 mm was built. The cross-section of the beam was modeled as a shell structure with the shape and dimensions shown in The reference model, i.e., without geometric imperfections, was built as described above and no load nor boundary conditions were applied. On the other hand, in models with imperfections, different deformation modes were enforced to cause the cross-section buckling due to compression, bending about to the horizontal and vertical axis and shearing in the plane of the web and flanges separately. The displacements were applied at the reference points shown in For compression-induced cross-section buckling, three elongation lengths of RVE  were analyzed. The buckling in compression was obtained by applying the displacements along the z axis at the reference points. The enforced displacement at point RP-1 was 0.5 mm, and, at point RP-2, it was −0.5 mm. Additionally, in both points, the displacements in the direction of the x axis, the y axis, and the rotation in relation to the z axis were blocked . For the remaining cases, the RVE elongation was assumed to be 100 mm.While considering buckling in bending about the horizontal axis, two cases were analyzed due to the unequal width of the flanges: (i) the tension of the upper part of the cross-section and (ii) the tension of the lower part of the cross-section. In both cases, the displacements in the reference points along the x axis, y axis, and z axis and rotations in the y axis and z axis were assumed to be 0. In contrast, the rotation about the x-axis for (i) case was 0.5 radians at point RP-1 and −0.5 radians at point RP-2, respectively. For (ii) case of bending about the horizontal axis, the rotation about the x axis for RP-1 was −0.5 radians and for RP-2 was equal to 0.5 radians.The buckling for bending about the vertical axis was also considered for two load cases: (i) the tension of the right part of the cross-section and (ii) the tension of the left part of the cross-section. For variant (i), the rotation around the y axis in the reference point (RP-1) was assumed to be 0.5 radians and in RP-2 −0.5 radians. For variant (ii), the rotation around the y axis was assumed to be −0.5 radians in RP-1 and 0.5 radians in RP-2. The remaining displacements were blocked at the reference points in two cases.The next model considered the shear buckling in the plane of the web. For such buckling, two cases were analyzed due to the type of applied load/displacement: (i) the shear of the cross-section in the plane of the web and (ii) the shear in the plane of the flanges. In the model for (i) case, the translational displacements along the x axis and z axis, as well as the rotations around the x and z axes were blocked; the displacements along the y axis were assumed to be 0.5 mm at RP-2 and −0.5 mm for RP-1. However, for (ii) case, the displacements at both reference points were assumed to be equal to 0, except for rotation around x axis, which was equal to −0.5 radians. In the case of shear buckling, in the plane of the flanges, a displacement along the x axis was 0.5 mm at RP-2 and −0.5 mm at RP-1. Displacements along y and z axes and rotations around y and z axes were assumed to be 0.This section presents the results obtained from numerical analyses for a thin-walled Z-type profile without holes and with rounded corners. The calculations examined the influence of buckling on the change in the local stiffness characteristics of the beam. Moreover, it was analyzed to what extent the elongation of the profile and the size of imperfections affect the reduction in individual stiffness. The elongation was from 100 mm to 200 mm, and the size of imperfections was from 0 to 5 mm. The size of the imperfections is here the maximal displacement assumed for RVE due to buckling. The analyses were conducted for compression and bending in relation to the horizontal and vertical axis of the cross-section and shear in two planes, vertical and horizontal.First, the reference stiffness was computed and presented in The changes in stiffness due to compression for the Z-type profile was computed for a fixed mesh with a size of 5 mm and a variable elongation from 100 mm to 200 mm. The influence of imperfection size on the effective stiffness was also investigated.The individual stiffness drops depending on the elongation and imperfection size for buckling modes 1 and 2 are presented in For a better illustration of the results due to buckling caused by compression, the decrease in compressive stiffness On the other hand, the buckling modes 1 and 2 for a Z-profile with an elongation of 150 mm and a decrease in compressive stiffness  the tension of the upper part of the cross-section and (ii) the tension of the lower part of the cross-section, because the cross-section considered has no axis of symmetry. For the Z-type cross-section with 100 mm elongation, the influence of the imperfection size on individual stiffness was investigated. The stiffness reduction due to bending for the (i) case is presented in Buckling mode 1 for a Z profile with 100 mm elongation due to bending about the horizontal axis and tension of the lower part of the cross-section (case (ii)) is shown in Buckling due to bending about the vertical axis was considered for two cases: (i) the tension of the right part of the cross-section and (ii) the tension of the left part of the cross-section. The influence of the size of an imperfection caused by bending on the decrease of individual stiffness for a Z-type cross-section with an elongation of 100 mm was analyzed. The stiffness reduction for a Z-profile with 100 mm elongation due to bending about the vertical axis for the case (ii), depending on the value of the imperfection, are presented in In Buckling in shear was analyzed for two variants of load. The shear of the cross-section in the plane of the web was labeled as case (i), while the shear in the plane of the flanges was labeled as case (ii). For these shear variants, the influence of the imperfection size and the method of implementing shear on the change of the stiffness of the Z-profile with 100 mm elongation were investigated.The decrease in stiffness due to buckling from shearing in the plane of the flanges, i.e., case (ii), the shearing case for the Z profile with an elongation of 100 mm, depending on the imperfection value, is presented in In The systematic numerical studies of homogenization adopted on an cold-formed unsymmetric beam profile to obtain its representative stiffnesses allows a comparison of the results between different buckling modes used, as well as the size of imperfections applied, or the method used to determine a particular deformation.While analyzing buckling due to compression, see  summary  for the When analyzing buckling due to bending about the horizontal axis, two cases were considered, that is, the top flange in tension and the lower flange in tension. The upper flange has a 60 mm width, while the lower one has a 40 mm width. Such a width difference resulted in obtaining different modes; in case (i), the extremes were achieved in the lower flange, and in case (ii), the extremes were obtained in the upper flange. The stiffness reductions of When analyzing buckling due to bending about the vertical axis, two cases were considered, that is, the left stiffener in compression and the right stiffener in compression. Both stiffeners have the same height, i.e., 20 mm. Difference in flanges width resulted in obtaining different modes; see When analyzing buckling due to shearing, two cases were considered, that is, shearing in the In this paper, we presented a methodology on how to compute the deteriorated characteristics of a beam section due to several types of imperfections. The methodology requires solving several buckling problems, with different loads and numerical homogenization ,21 for eThe conclusions presented applies to the cross-sections analyzed. To adopt those findings to another types of cross-section, verification simulations are recommended to be performed. Another limitation is that the buckling results depend on the ratio between the dimensions of flanges/webs and its extrusion, while considering a RVE approach.In the paper, a methodology for numerically determining the deteriorated properties of a beam section due to imperfections was presented. Thin-walled Z-type beams with variable elongation and a different load pattern were analyzed. The analyses conducted use a method of numerical shell-to-beam homogenization by using the principle of the elastic equilibrium of the strain energy.In particular, this paper shows what kind of local imperfections deteriorate the effective stiffness of the cross-section, which so far has not been taken into account. The results presented in the study directly help to build engineering intuition without conducting complex finite element computations. Moreover, the algorithm proposed may serve to compute the effective stiffnesses for other cross-sections, such as a C or Sigma profile. In this research paper, first, the homogenization method was used to calculate the mechanical parameters of an undeformed beam (reference case). Next, in the second part, the buckling analyses for the RVE model were subjected to various types of loading . Finally, the obtained buckling modes were used to calculate the individual stiffness drops. In the end, an alternative, faster, and simplified approach was proposed that gave satisfactory results compared to the full methodology.


A new year is just around the corner, as always bringing new challenges for all of us. Our journal is no exception. However, this time the challenge is even greater. The new CAPES classification for journals, which in principle would not trouble any national journal, has changed its assessment criteria for graduate programs coupled to a new classification system for Brazilian journals, especially those from surgical medical fields, stretching demands farther than we had antecipated. Standards have risen, and risen too much. It is important to raise the bar on standards, push us to reach a higher level of efficiency and excellence. Nonetheless, too high a demand may force us to give up. So much so that it was published in the State of São Paulo newspaper on July 6, 2009, under the title “Ranking Places Brazilian Scientific Journals Under Risk of Extinction”. In a formidable editorial published in the Clinics Journal :721-4) its Chief Editor - Mauricio Rocha e Silva even suggests the motto “Down with the New Qualis! Bring Back Realism!” and aptly shows the mismatch between what he calls CAPES exercise of numerology and the reality  faced by science in Brazil. It sure is worth checking out.Back to our talk at the beginning, by itself, without the changes in the criteria for graduate programs, the New Qualis would not be troublesome. At first glance, our journal which went from A  to B3 would have its grade lowered. However, it is not that simple. Let me explain: imagine that people would have their heights classified into A, B and C. Class A would group those individuals taller than 2m; B would be those between 1.5 and 2m and C would be those below 1.5m. Now, let's say that at a given time they decide to change this classification to A, B, C and D. Class A would be those individuals taller than 2.5m; B from 2m to 2.5m; C from 1.5 to 1.99m and D those shorter than 1.5m. Now, those individuals who were 1.6m tall and before were class B became class C. This does not mean they became shorter in size, just that they were assigned a different letter, which represents the same height range they had before, with another letter. The problem will only surface if they decide that those individuals from class B or higher would have rights to a certain privilege. Well, that is exactly what is happening to Brazilian scientific journals, especially the medical ones.Therefore, our journal did not drop in classification. It was Medline material and continues being so. Nonetheless, to be Medline material alone is no longer attractive to the eyes of our graduate programs, which need to maintain a good standard for CAPES. And I repeat: the standards have risen beyond what is reasonable.Obviously, Brazilian Medical Science can not bend facing such long horizons. It is quite the contrary. We are sure bound for troubled waters; nonetheless, as it happened during other troubled times, it is also certain that we will overcame this new and huge challenge. Our battle now happens in two fronts: one trying to join forces against a journal classification system and programs light years ahead of our reality; and the other is in search of the best quality possible. If ISI is what they want, it is ISI we'll give'em!