At present, people more actively pursuing biodegradable-based food packaging to lower the environmental problems of plastic-based packaging. Starch could become a promising alternative to plastic because of its properties . This review article is focused mainly on the impact of the properties of starch-based biodegradable films, such as their thickness, morphology, and optical, water-barrier, mechanical, oxygen-barrier, antioxidant, and antimicrobial properties, after the incorporation of additives, and how such films fulfill the demands of the manufacturing of biodegradable and edible food-based film with preferable performance. The incorporation of additives in starch-based films is largely explained by its functioning as a filler, as shown via a reduction in water and oxygen permeability, increased thickness, and better mechanical properties. Additives also showed antimicrobial and antioxidant properties in the films/coatings, which would positively impact the shelf life of coated or wrapped food material. In past decades, edible coatings received a great deal of attention as a promising approach to preserving fresh food products by reducing water loss, controlling respiration, improving glossiness, and inhibiting microbial growth during postharvest storage. The synthesis of common, reliable, and all-purpose coating solutions is a challenge for researchers because of the huge mechanical and physicochemical properties of the biopolymer. Edible coatings are generally derived from various biopolymers, e.g., starch, cellulose, protein, lipid, etc. .Starch biopolymer has attracted great attention to its film-forming properties because of its low cost, abundance, and organoleptic , optical , and barrier properties (carbon dioxide and oxygen permeability) . For filAccording to current research, adding additives and other active ingredients to starch polymers could enhance their water barrier properties. Plasticizers improve the stability and strength of polymers and are utilized to form effective edible coatings and films . Most stStarch is a natural, biodegradable polymer that can be utilized to form beneficial food packaging substances. Researchers have studied it extensively because of its large quantity, low cost, edible nature, and biodegradability. Many raw agricultural food items, such as wheat, rice, corn, beans, and potatoes, contain starch, which is a form of natural biopolymer ,20. AccoAmylose and amylopectin are the two major components found in starch, and other elements, such as lipids and proteins, can be found in trace quantities in starch granules. Amylose is a linear chain polymer with a molecular weight of 20 to 800 kg/mol made up of 1,4 anhydroglucose units. Many granular starches contain between 20 and 25% of this substance ,24. AmylTwo methods are often used to make films from starch: the dry method and the wet method. Starch is heated above its glass transition temperature in the dry phase method, which uses thermoplastic properties by extrusion. The starch is then plasticized with low water content. The thermoplastic starch requires a plasticizer, particularly glycerol, to reduce the phase-transition temperature  to be lower than the degradation temperature . In the A food additives was described as any compound that is intentionally added in the manufacturing, processing, treatment, preparation, packing, transport, and holding of food to perform a technological function according to the Codex Alimentarius , which is recognized as the international standard. The use of food additives enhances the finished product’s consistency and extends the shelf life of foods on store shelves. Additives should be used in a defined limit that does not negatively impact health or give an allergic reaction to the customer. By implementing the Codex Alimentarius, the FAO/WHO (2017) and the European Union  defined the criteria under which the food industry can utilize food additives .The major focus of this review is on the impact of various additives on the physical, water-barrier, biodegradability, mechanical, antioxidant, and antibacterial properties of edible starch-based films.Staphylococcus aureus, Campylobacter, Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli by rupturing the plasma membrane and causing intracellular material to flow [Various essential oils and extracts from plants have shown many beneficial effects in starch-based films and coatings . Essenti to flow ,40. Desp to flow ,41. Inco to flow . It also to flow  and help to flow . The dev to flow . The essPenicillium spp., Candida spp., Salmonella, and S. aureus spp. [The incorporation of chemicals into films showed the highest elongation at break, opacity, rigidity, and solubility  and increus spp. . The streus spp. . Chemicaeus spp. . The reaeus spp. . Some cheus spp. . Differe2 and CO2) [Pigments are generally rich in antioxidants and therefore used as additives to improvement in the properties of films . They aland CO2) ,68. Preband CO2) . Shahramand CO2)  found thand CO2) . Increasand CO2) . Mango aand CO2) . DetailsThe impact of various types of additives on the physical, water barrier, biodegradability, mechanical, antioxidant, and antibacterial properties of edible starch films are discussed in the following section.The thickness of edible starch films influences their physical and mechanical properties. The thickness of the film has a strong relationship with the permeability properties of the film ,75. The Lycium ruthenicum extract (LRE) was significantly thicker that with added betacyanin-rich red pitaya peel extract (RPPE), and the film with added RPPE showed a denser inner structure [Garcinia atroviridis leaf) extract to the cassava starch film had no massive impact on the film thickness because of the small amount of extract used in the film [Cordia verbenacea extract combined with a pregelatinized starch film resulted in the desired flavonoid content, even though the thickness of the films was 0.060 mm. Similar changes were observed in the moisture present in the films [Eriobotrya japonica Lindl. leaf the extract to the film solution (4% w/w), which increased the solids content in the film [E. faecium and P. acidilactici and used in the corn starch film. The resulting films showed thicknesses ranging from 180 to 200 μm, with no significant differences between them [The incorporation of 6% cotton CNC  in a corn starch film improved the thickness of the film by 22% as compared with the control film . Becausetructure . The addthe film . Chakravthe film  reportedthe film . A high he films . Film thhe films . The banthe film . The incthe film . Incorpoeen them .4) and glycerol added to corn starch can damage the internal crystal structure of starch; affect its binding capacity and thermodynamic properties; and enhance its gelatinization [Film qualities such as water vapor permeability, mechanical properties, and optical properties are all influenced by the microstructure of the film . Sodium nization ,83. The nization  reportednization . Comparenization . SEM imaIn comparison, control films and starch/gelatin films with 1% GAL extract showed a similar XRD pattern, indicating a more crystalline structure . There wOptical properties are important in determining the capability of edible starch films to be utilized in food. These properties protect food against light exposure, particularly UV radiation-caused food deterioration. The light transmittance of a film in the range of 200–800 nm can be used to determine its transparency. Furthermore, studies have determined that the transparency value of the film is defined as the absorbance of the film at 600 nm divided by the thickness, which is referred to as “opacity” by most researchers ,86.Pedocococcus pentosaceus and Lactobacillus plantarum, respectively). In CS/CMC/PPE-2%, transmittance was increased by 6.28% over that of CS/CMC (carboxymethyl cellulose) film [E. faecium and P. acidilactici. When compared with control films, bacteriocin-immobilized crystalline nanocellulose (BIN) reduced the transmission rate by 14–16%, and the incorporation of BIN in films decreased the transmission rate by 27–29% [When LRE and RPPE were added to cassava starch film, different colors (pink and purple) appeared, which were imparted by varied anthocyanin and betacyanin content. Other studies reported similar colors in films rich in anthocyanins or betacyanins . The UV se) film . Controly 27–29% .The permeability of a film is influenced by its chemical structure, degree of crystallinity, cross-linking degree, polarity, density, polymerization, and molecular weight, with extra plasticizers ,10. MoreBiodegradability studies simulates the breakdown conditions that occur in the environment because of soil microflora activity . BiodegrDuring the first 15 days, the starch–chitosan–nanoclay bionanocomposite films degraded quickly. They then degraded slowly and regularly for the next 60 days. The corn starch–chitosan–nanoclay (control) film decomposed quicker than the other bionanocomposite films, as it lacked an antibacterial component . In an aThe stress range of edible starch films is directly determined by their mechanical characteristics in the actual use of food packing. The elongation at break (EB) and tensile strength (TS) of a film are the common factors that represent its maximum stress strength and flexibility . The intBitencourt et al.  studied Edible starch films have significant antioxidant and antibacterial effects and prevent packaged food from deteriorating. They act as an important barrier to the surface food and effectively inhibit the growth of spoiled bacteria in packaged foods .Free radicals can cause food to degrade and nutritional content to deteriorate. Extracts consisting of phenolic hydroxyl groups, anthocyanins, and betacyanins, which have H-donating capacity, have shown high free radical scavenging activity in films and coatings . AntioxiE. coli and fungi  [S. aureus, E. coli). Films containing crystalline nanocellulose (CNC) made from cotton linters had the lowest activity against microorganisms, whereas those containing bacteriocins had the maximum activity. BIN in the films reduced bacterial activity against microorganisms by up to 17% as compared with films without bacteriocins, whereas it increased significantly by three times as compared with films with CNC [Eugenia uniflora L.) leaf extract (PE) and natamycin (NA) improved radical scavenging activity in cassava starch films from 7.68% to 86.20%. Pitanga extract contains phenolic and pigment compounds that may have contributed to the film’s increased antioxidant capability. When compared with films without additions, natamycin alone improved antioxidant activity because of its compositional impact [LRE and RPPE have considerable antioxidant and antimicrobial properties, which helped in improving the properties of casava starch film, because anthocyanin phenolic hydroxyl groups might bind to free radicals and provide phenolic hydrogen atoms . Notablys niger) . The ants niger) . The pres niger) . This cos niger) . Aqueouss niger) . When ans niger) . A consis niger) . The CARs niger) ,110. Cors niger) . Films ws niger) . Corn stwith CNC . The addl impact .Starch films have good oxygen barrier characteristics, because amylose and amylopectin have highly ordered hydrogen-bonded network structures containing crystalline and noncrystalline regions in alternating layers, leading to more permeability with the mixing of other additives ,112.The plasticizing impact of water content and glycerol-influenced oxygen permeability showed greater values with more water content and glycerol. Glycerol increased the space within and between starch components while decreasing interchain hydrogen bonding, allowing starch chains to glide and the substance to elongate ,113. TheConsumer demand for healthy and safe meals has prompted scientists to look for more natural alternatives to better food quality and safety. Since edible components have been accepted as additives by the European Commission (EC) and the Food and Drug Administration (FDA) and are now more likely to be used in and on food items rather than as synthetic preservatives  by incorCitrus limon, Rosmarinus officinalis, and Vitis vinifera essential oils into packing materials reduced the formation of biogenic amines in fresh fruit [Lippia gracilis Schauer to preserve guavas (Psidium guajava L.) after 10 days of storage at 25 °C. In this study, blends of chitosan, cassava starch, and essential oils were shown to be more efficient at inhibiting the development of most bacteria [The addition of sh fruit . de Aquish fruit  studied bacteria . The oveEvangelho et al.  studied Escherichia coli and Salmonella typhimurium decreased with the addition of EO on fresh spinach leaves.Issa et al.  studied Radha et al.  studied Caetano et al.  showed tZataria multiflora. Compared with the film without EO, the film with EO showed positive results in sensory physiochemical parameters after storage [Amiri et al.  studied  storage .The major goal of this review was to present the recent and innovative work in the development of starch-based films with additives such as essential oils, chemical agents, pigments, and other edible compounds. Starch types, time and temperature during film formation, cobiopolymers, plasticizers, and storage conditions affect the properties of the starch film. Additives are very easy to obtain and have shown highly antibacterial and antioxidant properties, which can improve the shelf life of food. However, the drawbacks of lower water vapor barrier, mechanical strength, and thermal stability remain the main challenges that must be solved before starch-based films can compete in the food packaging industry. Simultaneous incorporation of additives is a viable strategy partially compensating for the aforementioned loss of characteristics. In view of the composite films that have been developed, the compatibility of additives with the biopolymer matrix can also help to improve the results in future studies. Another attractive feature to consider is the absence of comprehensive studies on the stability of additives in terms of their antioxidant, antibacterial, and other properties in film. Furthermore, standardization of the additives potentially impacting material consistency in film manufacture needs to be investigated.


To compare bond strength of resin cements with and without 10-Methacryloyloxydecyl Dihydrogen Phosphate (MDP) to zirconia and evaluate effect of thermocycling on bond strength.Standardised test specimens were fabricated as per ADA specification 131. Each Zirconia specimen was mounted in autopolymerzing acrylic resin material. The specimens were divided into 2 groups: Group 1 – specimens bonded with resin cement containing 10-MDP and Group II - specimens bonded with resin cement without 10-MDP. Forty samples of resin cement cylinders were prepared with dimensions of 6mm height and 4mm diameter in line with ADA specification 27 were cured onto the zirconia surface of 10mm x10mm x5mm using customised moulds. Specimens from each cement group were further divided into 2 subgroups: Subgroup A – Specimens that were not thermocycled and Subgroup B – Specimens that were thermocycled. Specimens were then subjected to tensile bond testing by using a Universal testing machine, the data were analysed using independent sample t test for bond strength and paired t test for effect of thermocycling. Statistical analysis used: Data was subjected to normalcy test (Shapiro-wilk test). Data showed normal distribution. Hence parametric test paired t test were applied. Paired t test revealed that the thermocycling affected the bond strength to zirconia. The highest bond strength was achieved for the resin cement with 10-MDP before thermocycling, whereas the lowest bond strength values were recorded for resin cement without 10-MDP after thermocycling. Resin cement with 10-MDP showed superior bond strength to Zirconia than resin cement without 10-MDP. Adhesive failure was predominant at Zirconia and resin cement interface. Thermocycling had a significant effect on the bond strength of resin cements to zirconia, showing decreased bond strength. Key words:10-MDP, Tensile Strength, Zirconia. Ceramics were ﬁrst used successfully in dentistry in 1774, at a time when tooth coloured replacements were fabricated from ivory, bone, wood, animal teeth, or expensive extracted teeth obtained from human “donors.” None of these biologically derived products was stable toward oral ﬂora or corrosive components of food and saliva. Ceramics immediately solved the issues of stained and decayed dentures /Computer-Aided Manufacturing (CAM), has made digital dentistry increasingly favoured over the past decade. CAD/CAM systems are continuously developed and upgraded in prosthetic dentistry, and in association with zirconium oxide, is used primarily for the restoration of single crowns and fixed partial dentures (FPDs) and, dental implantology, as abutments or implants .et al. reported that conventional luting failed to reduce fracture resistance of Zirconia-ceramic restoration. Surface treatment had no impact in relation with the adhesion  to bonding agents (primers or cement) appears to reinforce bond strength to zirconia because P-O-Zr chemical bonds are formed between MDP and zirconia . It was Presently 10-MDP is the most widely-used phosphate ester monomer applied for the coupling of Y-TZP. Chemical bonding between 10-MDP and Y-TZP has been substantially studied. MDP has been incorporated into various bonding and luting products, such as primers, adhesives, and resins  as its constituents against the regular resin cement to zirconia and evaluate the impact of thermocycling on bond strength.This -Fabrication of Standardised test specimens:A wax pattern was prepared with dimensions of 10x10x05 mm using modeling wax. Later a mould was prepared using medium body elastomeric impression material. Die stone was poured onto to the prepared surface. The final block was sandpapered and transferred to the extra oral scanner. A Zirconia block  was used for the study. Forty Zirconia specimens were machined from a CAD/CAM Milling System .-Surface treatment of the Zirconia blocks.The forty specimens were surface treated with sand blasting.•Preparation of Samples of Group 1: Panavia 2.0 Surface of 20 Zirconia specimens were bonded with resin cement with 10-MDP. Equal amounts of ED Primer II A&B were incorporated. Later applied to the fabricated specimen. Equal amounts of A&B mixed for 20 seconds. Mixture of this paste was applied into the customized molds.•Preparation of Samples of Group 2: Maxcem Elite Surface of 20 Zirconia blocks was bonded with resin cement without 10-MDP. The dual barrel syringe with auto mix tips and angulated dispensing tips were used to deliver the desired amount of the cement.-Fabrication of test specimens:All the 40 Zirconia blocks were embedded in acrylic resin  blocks (10 mm length × 10 mm breadth × 5 mm height) with the surface treated side of block exposed. To fabricate standardized resin cement cylinders which were bonded onto the ceramic surfaces, customized clear vinyl plastic tubes, 4 mm in diameter and 6 mm in height were used. To facilitate the testing of tensile bond strength between Zirconia and resin cement, Orthodontic wire of 0.6 mm diameter was placed through the molds. Resin cement was slightly overfilled in the molds and was then positioned firmly on the Zirconia bonding sites. The resin cement cylinders were then polymerized using a light-polymerizing unit, with the light angulated approximately 45 degrees from the junction of the Zirconia bonding sites and resin cement cylinders. Two cycles of 20 seconds light polymerization were carried around the circumference of the composite resin cylinder to strengthen the bond.-Thermocycling of the samples:Each group was subdivided into 2 subgroups A & B of 10 Samples each. After cementation, 10 samples of each group were subjected to thermocycling in two different thermal baths with temperature maintained at 5ºC and 55ºC using distilled water. A temperature regulating button and thermometer was used to record temperature fluctuation. Each sample was exposed to thermocycling for a period of 15s at 5ºC and 55ºC with 15s interval between each cycle and total of 5000 Temperature cycles.Testing of specimens:Each specimen was mounted on the lower fixture of a universal testing machine , and the orthodontic wire was connected to the upper fixture , P is the maximum force (N), and A is the interfacial area (mm2) and date recorded in computer.-Site of Tensile Failure:For all the test specimens, the interface where fracture occurred was classified as either cohesive or adhesive in nature. The test specimen which fractured at the interface was examined under magnification to determine whether the fracture was adhesive or cohesive. A layer of resin cement present on the Zirconia surface after fracture was inferred as cohesive failure. From the total samples of 40, adhesive failures Fig.  were eviIn this study, tensile strength of 20 resin cement with 10-MDP and 20 without 10-MDP samples, (10 before thermocycling and 10 after thermocycling) were tested. .Aesthetically driven approach has caused dental professionals and patients to opt for the use of metal‑free restorations in prosthodontics. The substantial advancement associated with leucite, lithium disilicate, zirconia, and alumina‑reinforced ceramics has allowed substitution of metallic infrastructures in diverse clinical situations, due to their high flexural and compressive strength ,12. The P > 0.05) difference in mean tensile bond strength was observed.The results in this study showed that the mean bond strength of Panavia 2.0 bonded to Zirconia with and without thermocycling was, respectively, 15.331 MPa and 12.222 MPa. The mean bond strength of Maxcem bonded to Zirconia with and without thermocycling was, respectively, 8.165 MPa and 3.893 MPa. While comparing two resin cements, it was observed that a statistically significant (Panavia 2.0 resin cement presented highly significant results than Kerr Maxcem. The disparity in the bond strength observed could be due to difference in the chemical composition of the two types of cement used. Panavia 2.0 is composed of methacrylate monomers containing phosphoric acid groups which are all frequently used cross‑linkers in adhesive systems . PhosphoThe impact of silane incorporated in a universal multimode adhesive might be limited. In general, so‑called universal primers/adhesives achieve more durable bonding to zirconia than to lithium disilicate . ThermalThe results showed that the difference between the mean tensile bond strength of Zirconia with Panavia 2.0 and Maxcem Elite with and without thermocycling was statistically significant difference. Failure analysis revealed that failures were predominantly adhesive nature in the resin cements Fig. .in vitro studies is that the oral conditions cannot be simulated. Subjecting the specimens to active loading in artificial saliva prior to testing may closely resemble intraoral conditions with respect to hydrolytic degradation of the bond due to pH changes of saliva and the effect of temperature change in the oral cavity. Furthermore, other clinically relevant factors such as conFiguration of crown preparation, dentinal tubular fluid movement, pulpal pressure, remaining dentin thickness, and type of dentin should be considered when testing adhesive systems in vitro (The main disadvantage of such in vitro ,18.Considering the limitations of this study, conclusion can be summarised as,1. The bond strength of resin cements with 10-MDP as its constituent is much better with zirconia than that of without 10-MDP.2. Thermocycling has significant effect on the bond strength between resin cements with and without 10-MDP and zirconia, decreasing the bonding efficiency of the resin cements.


We appreciate the positive feedback that Zhou GP and his colleagues provided on our article, “A Novel Strategy for Preventing Posttransplant Large-For-Size Syndrome in Adult Liver Transplant Recipients: A Pilot Study” . Their aIt is crucial to match donor and recipient sizes appropriately to prevent Large-for-Size Syndrome (LFSS). A valuable idea presented by Zhou et al. is the incorporation of graft morphological parameters, particularly the anteroposterior (RAP) vertical distance and the longest horizontal distance, into the LFSS indicator . By combin vitro, the ex vivo measurement value is smaller than the actual one in vivo. For a closer match between in vitro and in vivo measurement values, we propose to combine several transplant centers and develop a new calculation formula with a large sample size.The point is how to measure the morphological parameter of graft using an appropriate method. As of today, computed tomography (CT) scan is the most accurate method to measure the right RAP vertical distance and the largest horizontal distance of grafts in living donor liver transplantation (LDLT) . HoweverPaterno et al. recently proposed a new solution, “bilateral marginal costotomy,” for rescuing a liver transplant recipient from severe graft compression caused by bilateral narrow rib cages after temporary abdominal closure failed . Yet, th


Newly formed tissue resembling native elastic arteries with concentric layers composed of smooth muscle cells, collagen, and elastin was found in the implanted polycaprolactone-based grafts. Moreover, the inner layer of the graft was seen to have developed structural similarities to the regular aortic wall. The grafts appeared to be well tolerated, and no severe adverse reaction was recorded with the exception of one case of cartilaginous metaplasia close to the junctional suture.The development of an ideal vascular prosthesis represents an important challenge in terms of the treatment of cardiovascular diseases with respect to which new materials are being considered that have produced promising results following testing in animal models. This study focuses on nanofibrous polycaprolactone-based grafts assessed by means of histological techniques 10 days and 6 months following suturing as a replacement for the rat aorta. A novel stereological approach for the assessment of cellular distribution within the graft thickness was developed. The cellularization of the thickness of the graft was found to be homogeneous after 10 days and to have changed after 6 months, at which time the majority of cells was discovered in the inner layer where the regeneration of the vessel wall was found to have occurred. Six months following implantation, the endothelialization of the graft lumen was complete, and no  According to the World Health Organization, more people die annually from CVDs than from any other cause. A large number of patients suffer from vascular damage, thus resulting in the need for bypass surgery. Since limitations remain concerning the replacement of small diameter vascular grafts, the need and demand for the development of more suitable grafts are increasing day by day. While the materials currently employed are commercially fabricated from inert polymers such as expanded polytetrafluorethylene (ePTFE) or polyethylene terephtalate, known as Dacron, research is increasingly being focused on biodegradable materials, since they have been found to possess several advantages over their inert counterparts. Biodegradable polyester poly at the beginning of the healing process. It is believed that three layers are formed following regeneration. The studies listed above investigated cell density or cellularization of implanted grafts in the whole thickness of the material. Stereological assessment based on the division of the graft into three layers enables localization of the cells into specific regions and could be helpful in quantification of regeneration in terms of cell distribution within these virtual layers.The aim of this article is to submit a proposal for the assessment of the healing characteristics of an implanted prosthesis based on a detailed evaluation of nanofibrous PCL vascular grafts in vivo in a rat animal model. The study was designed so as to provide proof of a low molecular weight nanofibrous PCL vascular graft concept that has not yet been tested in vivo. Histological processing and a stereological evaluation were employed so as to describe the regeneration of a blood vessel prosthesis in an experimental animal model.ε-caprolactone)  were obtained via the electrospinning of a 16% (w/w) solution of PCL dissolved in chloroform/ethanol/acetic acid (8:1:1 v:v:v). The solution was charged at 20 kV and ejected through a 22-gauge needle  at a constant rate of 2.54 mL h−1. The fibers were collected on a rotating tubular 304 stainless steel mandrel at a rotational speed of 250 rpm. The needle tip was placed 15 cm above the mandrel. The tubes were removed from the collector following electrospinning and placed under vacuum conditions for 2 h so as to allow for the evaporation of any residual solvent. The samples were sterilized via immersion in 70% ethanol for 10 min prior to implantation.Vascular grafts  produced from poly(n = 100).The fiber morphology was evaluated by means of field emission scanning electron microscopy (FESEM) on a Hitachi S-4700 . The samples were sputter coated with platinum/palladium on a Hummer 6.2 sputter coater  to a thickness of 5 nm and observed at low and high magnification. The fiber diameter was measured using NIH Image J software  board . NIH guidelines for the care and use of laboratory animals (NIH Publication #85-23 Rev. 1985) were fully observed. Seven Sprague Dawley rats received an abdominal aorta replacement graft with an internal diameter of 1.65 mm. The rats were anesthetized via the inhalation of 2.1% isoflurane in oxygen. A midline laparotomy incision was then performed so as to isolate the abdominal aorta. Side branches from a 10 mm long segment of the artery between the renal and femoral bifurcations were tied off so as to prevent collateral blood flow from within the segment. The proximal and distal ends of the segment were clamped and the artery severed near to the midpoint of the 10 mm segment. A microdose (~10 μL) of 200 units/mL heparin was administered to the exposed ends of the artery. The polymeric grafts were sutured to the exposed ends of the artery using an end-to-end anastomotic technique with 10-0 nylon sutures (15−20 stitches at each end). After suturing both ends of the graft to the native vessel, blood flow was re-established, and the pulsation of the distal artery was observed for the demonstration of blood flow. The abdomen was closed using sutures for the muscle and staples for the skin. The rats recovered from the anesthesia and were provided with food and water ad libitum. No anticoagulation treatment was administered to the rats.n = 3) and 6 months (n = 4), the rats were sacrificed by increasing the inhaled isoflurane concentration from 2.1 to 5.0% and then puncturing the diaphragm followed by the removal of the heart after the rats had been deeply anesthetized. The graft was collected with the host distal and proximal ends of the artery. The samples were embedded in freezing medium , snap frozen in liquid nitrogen, and stored at −80 °C prior to cryo-sectioning.After 10 days (n = 3), and the long-term response was assessed 6 months (n = 4) following implantation. The tissue blocks were cut so as to provide approx. 10 µm-thick histological sections with a section plane parallel to the long axis of the aorta , where Q is the number of nuclei profiles counted, and A is the estimated reference area of the section through the graft. In order to assess the relative distribution of the nuclei profiles within the graft in more detail, three virtual sublayers  were arbitrarily defined within the graft , each of which had an equal thickness comprising exactly one third of the local graft thickness while respecting all the irregularities of the local graft borders. The inner, middle, and outer sublayers did not consist of the actual morphological layers of the graft but rather ideal thirds that were used for the assessment of the homogeneity of the distribution of the nuclei profiles across the whole thickness of the graft. Finally, the numbers of nuclei profiles per unit area of the individual layers were added together and related to the total cross-sectional area of the whole graft profile so as to allow for the calculation of the mean density of the nuclei profiles per whole sample. Two micrographs were taken for analysis purposes from each aorta, and in total 33,548 nuclei profiles were counted.With respect to the quantification of cell density within the graft wall, a cross-section of the graft was evaluated employing stereological techniques. The inner  and outer  linear border profiles were highlighted in each micrograph using the Ellipse software Multiline tool. Whereas the inner border was always clearly visible, the outer border sometimes also contained connective tissue with adjacent blood vessels that did not belong to the graft but were harvested together with the graft. The outer graft border was therefore defined as the most external continuous part of the graft. The thickness of the graft was estimated using the Ellipse software LocalizeInWall2 module  as the mf(graft), describing the relative distance of the profiles in the radial direction across the graft 1f = d/(1 + d2d), where d1 is the distance of the nuclei profiles from the outer  graft border, and d2 is the distance of the nuclei profiles from the inner  graft border. The value f equaled 0 on the outer border and 1 on the inner border of the graft. In order to eliminate a potential edge effect [The relative position of the nuclei profiles within the graft was assessed using an arbitrarily defined function, e effect  and the e effect ,33.The grafts were found to be composed mostly of thin fibers with a mean ± standard deviation of 183 ± 150 nm in diameter. The majority of fiber diameters was found to be between 100 and 150 nm, as seen in The PCL vascular grafts exhibited excellent surgical handling and suture retention properties during implantation. No significant blood leakage was observed following the restoration of blood flow and pressure. At both assessed time points, i.e., 10 days and 6 months, all the implanted grafts remained patent without exhibiting any signs of aneurysm formation.f = d1/(d1 + d2) . Six months following implantation, however, signs of regular aortic layers with concentric layers of elastin, vascular smooth muscle cells, and collagen were observed .The host inflammatory reaction was relatively mild with concern to all the samples. After 10 days, multinucleated foreign-body giant cells (FBGCs) were discovered associated principally with the stitches between the aortic wall and the grafts . After 6Six months following implantation, signs of cartilaginous metaplasia were observed in a quarter of the PCL vascular grafts. The location of this transformation was determined as being in proximity to the stitches between the aorta junction and the graft .Small diameter single-layer biodegradable polycaprolactone vascular grafts with an inner diameter of 1.65 mm and a length of 2 cm were produced via the needle electrospinning technique for implantation purposes. The nanofibrous structure was composed of tiny fibers of around 180 nm in diameter with bead-like structures, as shown in tunica intima, tunica media) were quantified by the stereological method compared to planimetry [Stereological assessment provides objective unbiased quantification of specific parameters. Stereological approaches were utilized in tissue engineering of bone ,14, cartanimetry .tunica intima, tunica media, and tunica adventitia, and the implantation of the polymeric prosthesis was conducted so as to lead to the regeneration of these three layers in vivo. It was decided to distinguish between the adluminal (inner), middle, and abluminal (outer) parts in order to be able to quantify the regeneration process in different parts of the prosthesis. At the earlier investigated time point of 10 days, the grafts had been infiltrated homogeneously throughout the graft thickness with around 2500 cell nuclei profiles per mm2 , which was explained by the rapid cellularization of the graft by immune cells that were able to penetrate into the whole graft thickness of 744 ± 56 μm due to the high degree of porosity of the nanofibrous structure. No preference with respect to outer or inner site cell origin was detected. After 6 months of healing, the cell distribution had changed, i.e., the inner layer was seen to have become more densely cellularized (2976 ± 353 cell nuclei profiles per mm2). Moreover, the cell number was observed to have decreased with distance from the lumen of the graft . The regeneration of the graft wall was observed to progress from the inner site as evinced by the specific staining, as discussed further. The thickness of the graft had decreased slightly after 6 months to 686 ± 62 μm, which could be explained by the partial degradation of the polymer. Penetrated cells, especially immune cells, produce enzymes that may act to quicken the degradation rate of polycaprolactone: firstly, the PCL is degraded via the non-enzymatic hydrolytic cleavage of ester bonds, which leads to increased crystallinity and a decrease in the molecular weight of the polymer, followed by the enzymatic degradation of the PCL in the macrophages [However, the regeneration of vascular graft has never been evaluated by stereological methods based on our best knowledge. Wang et al. analyzed the cellularization of a vessel wall in distinct layers of 100 μm thickness . Since tvasa vasorum in electrospun PCL grafts in the first months following implantation with further regression [The endothelization of the graft lumen was observed after 6 months. Endothelial cells proliferated through the anastomosis as depicted in the assessment of the early stage see . Since tgression ,23.The implantation of biodegradable polymers serves to provide a temporary scaffold that is replaced by an extracellular matrix (ECM) of the final tissue during the remodeling phase. The process commences with the infiltration of the scaffold by host cells, which is followed by the synthesis of a typical tissue ECM. In the case of the implanted vascular grafts, smooth muscle cells penetrated the graft and synthesized the collagen and elastin fibers in the same way as in native vessels, as can be seen in The immunological response to the implanted biomaterial was as anticipated. The grafts were observed not to be surrounded by a collagen capsule that might potentially have limited the healing response and the remodeling phase. Only a small number of foreign body giant cells were found in the vicinity of the stitches and the outer part of the implanted nanofibrous grafts. Since polycaprolactone is a biodegradable polymer, it was assumed that the rat immune system cooperated with the material and “allowed” the initiation of the healing and remodeling phase, as suggested by the specific staining of the cells and the ECM.After 6 months in vivo, one of implanted grafts showed signs of cartilaginous metaplasia, as demonstrated in The overall assessment of a nanofibrous-based polycaprolactone vascular graft provided herein indicates that such a material is able to regenerate the native vessel wall in rats, as can be seen in vasa vasorum within the graft. Concentric layers of smooth muscle cells producing collagen and elastin were found under the endothelium, resembling the native vessel wall architecture. No severe immunological reaction was detected around the implanted biomaterial. The adverse effect of cartilaginous metaplasia close to the anastomoses was detected in one of the implanted grafts (25%).The study provides new insight into the vessel remodeling process and the assessment of nanofibrous low molecular weight polycaprolactone vascular grafts. The grafts remained patent and mechanically stable for 6 months following implantation. Cellularization of graft was assessed by a novel stereological approach. It was found that the graft wall had become homogeneously penetrated by cells after 10 days in vivo. The regeneration of the vessel wall was observed at the inner site where the majority of cells was present after 6 months. The prosthesis lumen was endothelialized after 6 months without the detection of


IEAtlas reanalyzed publicly available mass spectrometry-based HLA immunopeptidome datasets against our integrated benchmarked non-canonical open reading frame information. The current IEAtlas identified 245 870 non-canonical epitopes binding to HLA-I/II allotypes across 15 cancer types and 30 non-cancerous tissues, greatly expanding the cancer immunopeptidome. IEAtlas further evaluates the immunogenicity via several commonly used immunogenic features, including HLA binding affinity, stability and T-cell receptor recognition. In addition, IEAtlas provides the biochemical properties of epitopes as well as the clinical relevance of corresponding genes across major cancer types and normal tissues. Several flexible tools were also developed to aid retrieval and to analyze the epitopes derived from non-coding regions. Overall, IEAtlas will serve as a valuable resource for investigating the immunogenic capacity of non-canonical epitopes and the potential as therapeutic cancer vaccines.Cancer-related epitopes can engage the immune system against tumor cells, thus exploring epitopes derived from non-coding regions is emerging as a fascinating field in cancer immunotherapies. Here, we described a database, IEAtlas ( T-cell based cancer immunotherapies engage the immune system to recognize and eliminate tumor cells, and the recognition of tumor cells by natural killer T cells relies on presentation of tumor peptides by human leukocyte antigen (HLA) molecules. HLA-bound peptide sequences on the target cell surface are usually called ‘epitopes’, while their parent proteins are referred to as ‘antigens’ . There aRecent evidence highlights the importance of non-canonical epitopes, which can also elicit anti-tumor immune responses and mainly originated from translation products of transcripts from putative non-protein-coding regions, such as from long non-coding RNAs (lncRNAs), pseudogenes and untranslated regions (UTRs) of coding genes . AlthougTo date, mass spectrometry (MS) has enabled direct identification and quantification of all HLA-presented peptides, defined as the immunopeptidome . SeveralOn the other hand, the MS data of the HLA immunopeptidome are scattered among innumerable published articles, which is inconvenient for researchers exploring novel candidates for immunotherapy. Currently, several databases have been developed for a canonical immunopeptidome derived from proteins, such as IEDB 3.0 , HLA Lighttp://bio-bigdata.hrbmu.edu.cn/IEAtlas), a novel database that allows the exploration and visualization of the atlas of HLA-presented immune epitopes derived from non-coding regions  . Files wAccording to previous studies, presentation and recognition features were used to assess the immunogenicity of non-canonical epitopes via three key parameters, i.e. major histocompatibilty complex (MHC) binding affinity, MHC binding stability and T-cell recognition probability ,28.In terms of presentation features, binding affinity was predicted by NetMHCpan (v.4.1) for HLA-I and by NetMHCIIpan (v.4.0) for HLA-II with default settings Figure  29). St. St29). –16 were defined as immunogenic epitopes.In addition, the recognition features were assessed by the multistate thermodynamic model . We hypoT-cell reactive epitopes had a significantly higher hydrophobicity score and lower polarity score compared with non-reactive epitopes ,35. Whathttp://cancergenome.nih.gov/) and 17 382 samples across 30 normal tissues from GTEx  as personalized immunotherapy targets could generate an efficient and safe antitumor immune response as long as they are not expressed in normal tissues . The TSArom GTEx . Differees <0.05 . MoreoveThe server backend of IEAtlas was constructed and accessed based on Java Server Pages with Tomcat container (v.6.0). The MySQL database (v.5.5.48) was used to document and manage all the metadata in IEAtlas. HTML, JavaScript and CSS code constituted the web frontend of IEAtlas. The visualization of all analysis results and multiple statistical tables was supported by jQuery (v.3.3.1), Datatable (v.1.10.25) and ECharts (v.5.5.1) plugins. The R framework (v.3.6.3) was performed for statistical analyses. IEAtlas has been tested on several popular web browsers, including Google Chrome (preferred), Firefox and Apple Safari browsers.Currently, IEAtlas has re-analyzed public MS-based immunopeptidome datasets from 1444 cancer samples covering 15 cancer types and 1303 non-cancerous samples of 30 tissues Figure . Based oTo provide a convenient way for users to investigate the functions of epitopes and genes of interest, IEAtlas has also developed five flexible tools. Considering the strong tumor/tissue specificity of epitopes, IEAtlas allows users to access each epitope entry in cancer or normal tissues of interest by quick searching or query options. In addition, multiple visualization results are offered for facilitating comprehension of their roles in cancer immunity.IEAtlas allows users to explore the comprehensive atlas of HLA-presented immunogenic epitopes derived from non-coding regions, and further identify the potential immunotherapy targets. The user-friendly web interface of IEAtlas allows users to query, browse, visualize and download data Figure . On the Entering the detailed page for each epitope, the users can obtain extensive information about that epitope and the other epitopes produced by the same parent gene Figure . First, in vitro in melanoma  analysing the available datasets of the HLA immunopeptidome to expand the current atlas of non-canonical epitopes; (ii) constructing a more general resource for ncORF regions by integrating newly generated omic datasets about translation; and (iii) incorporating the information on experimentally defined T-cell epitopes. These additions are anticipated to enhance the efficiency of IEAtlas and it will be an important database for investigating the immunogenic capacity of non-canonical epitopes and their potential as therapeutic cancer vaccines. More rigorous investigation of these non-canonical immune epitopes would lead to a profound understanding and provide new insights into cancer immunotherapy, which should be prioritized in future perturbation studies.http://bio-bigdata.hrbmu.edu.cn/IEAtlas).IEAtlas is an open resource for HLA-presented immune epitopes derived from non-coding regions (gkac776_Supplemental_FileClick here for additional data file.


Erosive acids might create surface flaws and deteriorate the mechanical properties of CAD-CAM materials. This invitro study aimed to investigate the effect of simulated gastric HCl and extrinsic erosive acids on surface microhardness and fracture toughness of CAD-CAM materials.3) were prepared from 4 different CAD-CAM dental materials (n = 100/group); monolithic zirconia , lithium disilicate ceramic , nanohybrid resin composite  and polymer-infiltrated glass network . Specimens from each material type were further subdivided into 5 groups (n = 20) according to the erosive media applied . Specimens were immersed for 24 h in an incubator at 37 ℃, then ultrasonically cleaned in distilled water and air-dried. Half of the specimens were tested for Vickers microhardness (VHN) at parameters of 500 gf for 10 s, while the rest of the specimens underwent 3-point flexure till fracture. Fractured surfaces were examined under a scanning electron microscope (SEM) for fracture toughness (KIC) calculation using the quantitative fractographic analysis method. Data collected were statistically analyzed using two-way analysis of variance (α = 0.05) after verification of data normality and homogeneity of variances.400 bar-shaped specimens (17×4×2 mmIC values were found among Ceramill Zolid HT+ groups followed by IPS e.max CAD and Grandio Blocs regardless of the erosive media employed. Erosive media significantly reduced the VHN and KIC in Vita Enamic specimens compared to the rest of the material types.Erosive media created surface flaws that lowered the surface microhardness of the material and initiated the fracture pattern under different loads according to material type. The material type was a more predominant factor than erosive media that affected either the microhardness or the fracture toughness of CAD-CAM dental materials. The highest VHN and KAll CAD-CAM materials used, except Vita Enamic, showed high resistance against the erosive acids indicating higher longevity of the material in patients frequently exposed to either extrinsic or intrinsic acid. Dental erosion can be defined as an irreversible loss of tooth enamel and/or dentin due to frequent exposure to erosive acids of non-bacterial origin . These aRestorative treatment of dental erosion will depend on the stage at which patients seek dental advice . Early mThe revolutionary innovations in CAD-CAM dental materials such as resin composites, glass–ceramics and zirconia have been accompanied by a significant increase in their application in restorative and esthetic dentistry owing to their more precise fit, high mechanical properties, and ultimate reproduction of the natural appearing of teeth –14. The 3) using a precision diamond cutting machine . Zirconia blocks were sectioned at higher dimensions of (20.9 × 4.9 ×  2.5 mm3) to compensate for a later volumetric sintering shrinkage of approximately 20% to reach similar dimensions of other CAD-CAM specimens. Zirconia bars were then sintered at 1450 °C for 9 h in a Zirconia sintering furnace . In the firing cycle, the temperature increased gradually from room temperature to sintering temperature of 1450 °C in 30 min. Sintering temperature was retained by the furnace for 9 h after which it started to descend in a cooling phase till 1140 °C in 8 min. The furnace then opened gradually while the temperature kept descending till 540 °C in 14 min, which represented the end of the firing cycle. Specimens were then taken out of the furnace and left to cool down to room temperature. IPS e.max CAD specimens underwent crystallization at 840 °C in a porcelain firing furnace  following the manufacturer’s recommendations. In the sintering cycle, the temperature was raised from room temperature to 830 °C at a rate of 90 °C/min. The temperature was then further increased to 840 at a rate of 30 °C/min and retained at this degree for 7 min before automatic cooling down to 710 °C representing the end of the sintering cycle. Specimens were then taken out and left to cool till reaching the room temperature. The dimensions and perpendicularity of faces of the tested bars were checked and verified by measuring the length, width and thickness of specimens at 4 separate points placed equally from each other along the bar using a digital caliper . Specimens that showed similar measurement outcomes at the four different points were included in the study. All bars were then polished with metallographic silicon carbide paper at a sequence of (600- 800- 1000- 1200-grit) and underwent ultrasonic cleaning in distilled water for 10 min.A total of 400 bar-shaped specimens were obtained by sectioning 4 types of CAD-CAM materials: high-translucent zirconia  (HZ), lithium disilicate glass–ceramic  (EC), PICN  (VE), and nano-hybrid resin composite  (RC). All CAD-CAM blocks, except zirconia, were sectioned into targeted dimensions of (17 × 4 × 2 mmLab-prepared simulated gastric acid (0.113 wt% HCl in deionized water) .Fresh orange juice (obtained from freshly squeezed orange fruit). The main acid components in orange juices are citric, malic and ascorbic acids.12.5% alcohol-containing white wine . White wines contain erosive acids such as lactic acid, succinic acid, acetic acid, and tartaric acids.Phosphoric acid-based sugar-containing fizzy drink (Coca-Cola®).Artificial saliva served as a control group. An artificial saliva was prepared according to the following formula : 1 L of The pH of the tested erosive media was measured immediately before the insertion of the specimens in the acid-containing glass tubes, using a pH meter device , before the insertion of the bars.One hundred bars from each material were then assigned, according to exposure to different media, into five groups of 20. The bars were inserted into one of 5 of the following media:A 100 ml of each of the media was used to fill glass bottles into which the bars were inserted and then covered with lids. The bottles were kept in an incubator  at 37 °C for 24 h under constant slow shaking of 70 rpm , 23. Thi2, P is the indenter load in kg and d is the diagonal length of the impression in mm.The hardness of studied groups (n = 10/subgroup) was evaluated at room temperature using Vickers microhardness tester  with a load of 500 gf for 10 s. Five indentations were placed 0.5 mm apart from each other in a horizontal direction across the tested specimen (UNI EN ISO 6507) . The twoThe average of 5 VHN readings was counted as one for each specimen. According to the Standard Test Method for Vickers Indentation Hardness of Advanced Ceramics ASTM, any resulted indentation, shows ragged chipped or spalled edges, asymmetric diagonal lines, and a shift in the location of the tip of the indentation was excluded.Tested bars from each material group (n = 10/subgroup) underwent a 3-point flexural strength testing using a universal testing machine . A brass-metal custom-made device  was madeIC) can be calculated through the quantitative fractographic analysis method [IC is the fracture toughness in MPa m1/2, Y is the geometry constant = 1.24 [Fracture toughness . The normality of data obtained was checked for all the variables using the Shapiro–Wilk test. All variables showed normal distribution, so mean and standard deviation (SD) were calculated, and parametric analysis was adopted. Two-way analysis of variance (ANOVA) was used to compare the effect of different erosive media and different CAD-CAM materials as two independent variables on surface microhardness and fracture toughness of CAD-CAM materials.Simulated gastric HCl pH was found to be 1.2, while the white wine, orange juice, Coca-Cola®, and artificial saliva were found to be 3.3, 2.7, 3.9, and 7, respectively.P = 0.00) on the measured Vickers microhardness number and fracture toughness. The greatest influence was for the material  followed by the interaction effect  while erosive media had the lowest effect (ηP2 = 0.251 and 0.322). Generally, the highest VHN was found in Ceramill Zolid HT+ followed by statistically significantly lower VHN in IPS e.max CAD, and Grandio Blocs, while Vita Enamic showed the lowest significant VHN regardless of the erosive media. In Ceramill Zolid HT+ group, zirconia saliva showed the highest statistically significant (P = 0.00) microhardness number compared to the rest of the groups (1342.9 ± 6.9). No significant difference was found in microhardness between zirconia HCl and zirconia orange groups. Significantly higher VHN were found in zirconia wine and zirconia Coca-Cola®. In IPS e.max CAD groups, erosive media did not significantly influence the VHN of the specimens. In Vita Enamic groups, HCl erosive media significantly deteriorated the microhardness of the material compared to the rest of the media, while all media had no significant influence on the VHN in all Grandio Blocs groups.Descriptive statistics of mean Vickers microhardness number and fracture toughness are presented in Table IC was found (P = 0.03). HCl had the highest significant impact on the KIC of Vita Enamic specimens followed by orange juice and Coca-Cola® while white wine showed the least effect.Fracture toughness was the highest in Ceramill Zolid HT+ groups followed by IPS e.max CAD and Grandio Blocs, while the Vita Enamic groups showed the lowest values regardless of the erosive media used. Erosive media did not significantly change the fracture toughness of zirconia. Similarly, for Grandio Blocs and IPS e.max CAD except for HCl-immersed specimens in the IPS e.max CAD group; a significant reduction in KScanning electron microscope images of the fractured surfaces of CAD-CAM specimens showed the largest defect surface flaws on Vita Enamic 187–210 µm) followed by Grandio Blocs and IPS e.max CAD  while zirconia specimens showed the smallest defect sizes (36–41 µm) , in agreement with a previous study . GrandioFew studies investigated the effect of gastric HCl on hybrid ceramics , 35. TheThe continuous development of zirconia material has enabled its use as a whole restoration rather than being a core substructure for all-ceramic restorations . MonolitFracture toughness can be defined as the material’s resistance to crack propagation . It is oTo the authors’ knowledge, there are no previous studies that investigated the effect of erosive media on different CAD-CAM materials. Zirconia showed the highest resistance to the propagation of acid-induced surface defects compared to lithium disilicate and other CAD-CAM materials used. The effect of simulated gastric acid HCl on zirconia has been previously studied and no significant changes in the material mechanical properties were reported , in agreThe current study is an in-vitro model to simulate an erosive exposure and its effect on the microhardness and fracture toughness of four CAD-CAM dental materials. This simulation may not accurately reflect the erosive environment in the clinical situation in which other factors might affect the severity of the erosive acids such as the buffering capacity of saliva. Therefore, the results of this study are only applicable to the tested conditions. Further long-term in-vivo studies are recommended to draw a clearer image of the CAD-CAM materials’ survival in different erosive media.IC values followed by IPS e.max CAD, Grandio Blocs, while Vita Enamic scored the lowest significant values.Zirconia had the highest VHN and KExcept for Vita Enamic, different erosive media did not significantly affect the VHN and fracture toughness of zirconia, IPS e.max CAD and Grandio Blocs.Based on the findings of this in-vitro study, the following conclusions were drawn:


Recombinant tissue plasminogen activator (rtPA) has a short half-life, and additional hemorrhagic transformation (HT) can occur when treatment is delayed. Here, we report the design and thrombolytic performance of 3 via a top-down fabrication.The rmDPPs consisted of a biodegradable polymeric matrix, rtPA, and SPIONs and were synthesized in vitro blood clot lysis, the rmDPPs with magnetoacoustic stimuli could not enhance the lytic potential compared to the rmDPPs without stimulation. Furthermore, although the reduction of the infarcts in vivo was observed along with the magnetoacoustic stimuli in the rmDPPs, more enhancement was not achieved in comparison with the rtPA. A notable advantage of rmDPPs was shown in delayed administration of rmDPPs at poststroke. The late treatment of rmDPPs with magnetoacoustic stimuli could reduce the infarcts and lead to no additional HT issues, while rtPA alone could not show any favorable prognosis.The rmDPPs could be concentrated at the target site with magnetic attraction, and then the rtPA could be released under acoustic stimulus. Therefore, we named that the particles had magnetoacoustic properties. For the The rmDPPs may be advantageous in delayed treatment of thrombotic patients.The online version contains supplementary material available at 10.1186/s40824-022-00298-y. The occlusion of blood vessels supplies abnormal oxygen and nutrients into surrounding occluded tissues, which can induce several diseases, such as ischemic/hemorrhagic stroke, coronary infarction, and pulmonary embolism. Conventional thrombosis therapies mainly consist of mechanical thrombectomy and treatment with intravenous (IV) thrombolytic agents. However, thrombectomy is not only technically limited to specific thrombotic patients but can also cause embolism, vasospasm, and increased traumatic injury in vessels –7. As anll occur –11.Recently, drug carriers have been actively developed to overcome the inherent limitations of rtPA. Improvement of targeting potential in drug carriers through surface modification offers promising lytic potential –15. Howein vivo compared to lipid-based microbubbles and can be modified to respond to stimuli [Sonothrombolysis that utilizes ultrasound (US) to mechanically lyse blood clots has been used as an alternative technique for treating thrombotic diseases. US is noninvasive, has high spatial resolution, and can stimulate a deep territory compared to other external stimuli. In particle-mediated sonothrombolysis, the efficacy of sonothrombolysis can be accelerated with microbubbles through a cavitation phenomenon –28. Howe stimuli , 35. The stimuli –38.The safety of rtPA delivery can be improved by tuning the degree of drug exposure allowed by the carriers. Most rtPA-containing carriers have been designed to either have exposed rtPA on the outside of the matrix through chemical conjugation or have nonspecific release when in conditions of pathological thrombosis –41. The i.e., perpendicular to the direction of flow) [in vitro compared to the rtPA alone  was also effective and did not show any HT complications  –48. Acouone Fig. . Howeverion Fig. . Delayedns Figs. .Fig. 1ScrtPA was purchased from Boehringer Ingelheim . The commercially available rtPA was provided with L-arginine as a stabilizer. Poly  acid , poly  , dichloromethane (270997), chloroform (C2432), hexane (139386), thrombin from bovine plasma  and N,N’-Dicyclohexylcarbodiimide , iron(III) chloride hexahydrate (F2877), 1-Octadecene (O806), oleic acid (364525), RIPA buffer (R0278), phosphatase inhibitors , Rose Bengal , 3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5’,5”-disulfonic acid disodium salt (P4272), L-ascorbic acid (A92902), TraceCERT iron standard for ICP (43149), 2,3,5-triphenyl tetrazolium chloride , 0.5 % Triton X-100 (T8787), Brij L23 solution (B4184), hemoglobin bovine (H2500), and Drabkin’s reagent (D5941) were provided by Sigma-Aldrich . Sodium oleate (00057) was purchased from TCI . N-Hydroxysuccinimide , n-Tetracosane (42462), ammonium acetate (A647), 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindodicarbocyanine Perchlorate , and acetic acid (A38) were provided by Thermo Fisher Scientific . Protease inhibitors (11836170001) were purchased from Roche . Polydimethylsiloxane  184 silicon Elastomer Kit was purchased from Dow Corning . Isopore Membrane Filter  was poured into the master template  was used to assess the size and zeta potential of the particles. A Multisizer  was utilized for accurate size measurement of the rmDPPs since the shape of the particles was not homogeneously spherical. The zeta potential evaluation was performed using a Zetasizer . All particles were dispersed in DW at a 0.2 mg/mL concentration for size and zeta potential measurements.Morphological information of rmDPPs was assessed using scanning electron microscopy  and bright-field microscopy . 10 Element distribution was analyzed by energy-dispersive X-ray spectroscopy (EDS) to validate whether both rtPA and SPIONs were successfully localized within the polymeric structure. EDS analysis was performed simultaneously with SEM imaging.v/v). 10 The colloidal stability of rmDPPs was assessed through size variation. The test was performed in both phosphate-buffered saline (PBS) fetal bovine serum (FBS)  was quantified using spectroscopic measurements. It was necessary for rtPA to be conjugated with a fluorescent substance to quantify the drug loading spectroscopically; therefore, rtPA was covalently bonded with Rhodamine B (RhoB-rtPA). A more detailed procedure for chemical bonding is described in Supplementary Fig. m is the mass.Recruiting efficacy with magnetic attraction was evaluated using a 0.42 T magnet. rmDPPs (1 mg/mL) were exposed to the magnet for 60 min, and recruited masses near the magnetic field were assessed at 30 min intervals. Attracted rmDPPs were collected through the withdrawal of supernatant DW and freeze-dried to quantify the mass. Recruiting efficacy toward the 0.42 T magnet was calculated as:The magnetic properties of rmDPPs were evaluated at 5 in vitro and in vivo magneto-sonothrombolysis, respectively. Both UT and FT were customized to have the same resonance frequency at 1.5 MHz. The acoustic setup consisted of a function generator , linear power amplifier , transducer, and oscilloscope . Mechanical pressure from the transducer was delivered into a calibrated needle-type hydrophone  as sampled electrical signals, which were further processed to calculate acoustic distribution and intensity (AI). Acoustic parameters, such as fundamental frequency (FF), tone-burst duration (TBD), pulse repetition frequency (PRF), and duty cycles (DC), were regulated using the function generator.An unfocused transducer (UT) and focused transducer (FT) were employed for in vitro and in vivo magneto-sonothrombolysis experiments. The IThe hydrophone was moved in 3-axis spatial volume using a robotic platform  to precisely describe the acoustic features of transducers under acoustic stimulus. The movement of the platform was controlled through MATLAB. Acoustic characterization was performed in degassed water. The UT and FT were each characterized at a focal depth and geometrical focus: 100 mm and 13 mm, respectively. Acoustic distribution in both UT and FT was examined in the lateral, vertical, and axial directions. Depending on the various electrical inputs from the function generator, the AI of both UT and FT transducers was characterized with the 1.5 MHz FF, 100 1515 m/s . The I\dTwo approaches for testing the acoustic responsiveness of rmDPPs were validated. First, 200 i.e., mixture consisting of normal saline and assay reagents) at each investigated time.Second, an enzymatic activity assay was performed with the US stimulus. This assay can confirm the ability of rtPA to activate plasminogen to plasmin, releasing a yellow para-nitroaniline (pNA) chromophore in solution. Overall enzymatic assay was performed with the diluted solution following the protocol of manufacture. The rmDPPs were synthesized with different loading amounts of rtPA: 0, 0.1, 0.5, 1, and 2 mg. The activity of native rtPA was also evaluated to validate whether the acoustic stimulus toward the rmDPPs could release the cargoes more. The particles treated with/without US were reacted through an enzymatic kit. The investigations were maintained for 120 min with 30 min intervals, and enzymatic activity was quantified by measuring the OD at 405 nm. The result was presented as accumulative rtPA enzymatic activity relative to the control , rtPA, or rmDPPs was added to the top of the vertical channel. To evaluate the magnetic recruitment potential of the rmDPPs, a 0.42 T neodymium magnet was placed at the bottom of the channel. The channels subjected to US stimulus were sonicated for 10 min. The polymerized gels were carefully withdrawn from the channel after 24 hrs to quantify the magnetoacoustic potential of rmDPPs. Quantification was performed by using NIH ImageJ software with binary image processing.A slightly-modified fibrinolytic gel model was developed to examine the magnetoacoustic potential of rmDPPs for targeted and tunable rtPA release . An agarin vitro blood clots. Briefly, the rat was anesthetized with a mixture of oxygen and isoflurane, followed by a 5 mL syringe with 21 G into the left ventricle. Then, 20 U of thrombin (1 kU/mL) and 70 i.e., control), rtPA, or rmDPPs equal to 10 i.e., the dissolution of hemoglobin) was quantified by spectroscopy at 415 nm after 60 min.All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology (KIST-2021-06-073) and performed with appropriate recommendations. Blood was obtained from Sprague-Dawley rats weighing 280 g by the heart puncture method to fabricate in vivo, near-infrared carbocyanine fluorescent (NIRF) DiD-loaded rmDPPs , depending on the weight of the mouse. A mixture of Rose Bengal (15 mg/mL) and thrombin (80 U/kg) was injected into the IV route. To induce acute ischemic stroke, the mice were transcranially irradiated by a 532 nm green dot laser onto the area covering motor 1 and broad somatosensory cortex for 10 min. We prepared two in vivo experiments. First, we compared the thrombolytic potential of rmDPPs with that of rtPA through treatment at 20 min poststroke . Second, we compared the potential of rmDPPs with that of rtPA at 3 hrs poststroke . The control mice were injected with 200 A platelet-rich photothrombotic model was established for the fication . BrieflyImmunoblotting for fibrinogen was performed to examine whether fibrinogen was decreased in the ipsilateral hemisphere depending on the magneto-sonothrombolysis treatment with rmDPPs. Briefly, the brain was divided into the contralateral and ipsilateral hemispheres, and each hemisphere was homogenized with the mixture consisting of RIPA buffer, phosphate inhibitors, and protease inhibitors using a hand-held homogenizer  on ice. A Bradford assay was employed to quantify the total protein concentration in each hemisphere. The target protein was visualized by HRP-reactive chemiluminescence reagents and quantified using a blotting imaging system . The antibodies used for immunoblotting were rabbit anti-fibrinogen and mouse anti-Intracerebral HT was quantified using a cyanmethemoglobin assay as previously described elsewhere . First, in vivo magneto-sonothrombolysis. The collected brains were fixed in 4 % paraformaldehyde for 1 day at room temperature, followed by coronal sectioning using a cryostat . Five sections were obtained at intervals of 200 We evaluated the histological safety of acoustic stimulus and hematological toxicity of rmDPPs. A total of 4 mice were prepared for hematoxylin and eosin (H &E) staining to validate histological safety of acoustic stimulation. Acoustic parameters were the same as those used for Behavioral testing was performed to examine the recovery of locomotor function after rmDPPs treatment. We examined the locomotor function using a rotarod . All mice were trained and recorded for their behavioral features prior to the photothrombotic stroke  indicating statistical significance.All plotting and statistical analyses of data were performed using GraphPad Prism software. All data are presented as the mean ± standard deviation (SD). The Shapiro-Wilk test confirmed the normality of the data, and significant differences between the data were compared using unpaired t test and one-way analysis of variance (ANOVA) with i.e., DPPs that did not contain any substance) were additionally fabricated to compare their physicochemical properties with those of rmDPPs. According to Multisizer measurements, the average sizes of DPPs and rmDPPs were 2.87 and 2.83 The SPIONs were observed to be a spherical and 10.70 nm in size by the TEM images , which was approximately 46.85 % lower than that of rtPA (p<0.001). This might be related to the limited exposure of rtPA from the polymeric matrix in rmDPPs. The 54.69 and 59.01 % gel degradation was observed with magnetic and acoustic stimulus alone, respectively . This indicates that the sole external stimulus with rmDPPs could not significantly improve the lysis of the gel. However, the +M+US group showed an approximately 88.97 % of the gel lysis. The lytic potential in the +M+US group was approximately 1.73-, 1.63-, and 1.51-fold higher compared to that in the None, +M, and +US groups, respectively  were acquired and presented as normalized radiant efficacy. First, we investigated the basic pharmacokinetics of the freely circulating rmDPPs. The circulating particles mainly accumulated in the lung . Recent observation demonstrated that thrombin added with RB (T+RB) could produce the blood clot with an appropriate fibrin ratio and platelets . We firsvia the blood-brain barrier (BBB) disruption. Although additional HT in the ipsilateral hemisphere was detected in the delayed rtPA-treated group, the +M+US group showed low HT in the hemisphere. Intracerebral HT was quantified using a cyanmethemoglobin assay. The cyanmethemoglobin standard curve was regressed to y=0.0008x+0.0416 (Fig. Figure 416 Fig. f. Consis416 Fig. g. The HTFigure This study proposes a method to relieve infarcts without side effects such as intracerebral HT in the ischemic photothrombotic model while dissolving blood clots using magnetoacoustic rmDPPs that limit the inherent thrombolytic function of rtPA as much as possible.We demonstrated that the SPIONs and rtPA were successfully loaded within the polymeric structure Fig. . Since tin vivo might be safe (Table We checked that the rtPA release from the rmDPPs could be feasible using acoustic stimulus within a relatively short time compared to the natural hydrolysis of the PLGA Fig. b. EspecitPA Fig. d. HowevetPA Fig. . Furtherentclass1pt{minimaConventionally, relevant investigations in acoustic-assisted drug delivery have employed the microbubbles consisting of gases and lipids , 57. AltIn the aspect of the design of thrombolytic carriers, the interaction between nanoparticles and blood clots in the bloodstream might be complicated in comparison with the rmDPPs. The particles with over 500 nm size tend to marginate toward the vascular wall due to the gravitational forces , 63. It culation , 65. It articles . Additioarticles –48. Howein vivo (Fig. The ability to localize drug carriers at the lesion location prior to selective drug release is also important consideration for successful thrombolysis. Especially, the thrombolytic drug carriers could penetrate within the blood clot structure to achieve high thrombolytic efficacy. Several reports adopted the fibrinolytic gel model or showed penetrated drug carriers as real images , 67. Ournjection . Therefoin vitro human artery model demonstrated that the SPIONs having large size and reducing repulsive force from the surface coating could achieve high accumulative efficacy at the same magnetic gradient [Although targeting of either thrombolytic agents or drug carriers could expect a high quality of prognosis in thrombotic patients, the relevant clinical trials have not been sufficiently performed. Regarding our best knowledge, the various magnetic particles challenged to the clinical phase have been utilized for the contrast agents in magnetic resonance imaging. Since the intensities of the magnetic field applied to the human might be required stronger to target magnetic particles in the desired area, more sophisticated translational investigations should be needed for developing these drug delivery carriers. A recent investigation using gradient . Howeverin vivo investigations (Figs. in vivo were highly consistent with the previous work [Generally, the RB-mediated photothrombosis model induces a poor lytic tendency toward rtPA. Recent observations have indicated that the co-injection of RB with a typical coagulation factor, thrombin, can prompt thrombosis that is reactive to rtPA administration . This sins Figs. -9. Furthous work .Although the infusion of rtPA at 20 min poststroke was effective in relieving the infarcts, the circulating rmDPPs could not be functional to reduce the infarcts, presenting a similar level of infarcts to the control Fig. . This milostazol , 75. Thein vitro examination. Although the infarcts were reduced in the rmDPPs with magnetoacoustic stimuli, there was no significant enhancement over the rtPA. Interestingly, the HT complications were observed in the delayed treatment of the drug, whereas the rmDPPs with magnetoacoustic stimuli did not induce any severe HT. However, the fact that comparable thrombolytic potential of rtPA could be fulfilled in the rmDPPs using the multiple stimuli might still limit the usage of the system in the conventional thrombotic patients. Nonetheless, these discoidal drug delivery systems encapsulated the rtPA into the matrix is apparently appropriate in delayed thrombotic patients to provide successful thrombolysis and reduction of side effects. Future research is expected to develop and examine drug carriers containing various drugs, such as anti-coagulant and anti-platelet drugs, to enhance drug potential.In summary, we proposed that rmDPPs with magnetoacoustic features are effective thrombolytic carriers for thrombolysis without any HT complication. The particles can be directed to the thrombosis-induced site, and the on-demand release of the drug can be done by the acoustic stimulus. The release mechanism is thought to be the physical disruption of the particles. In contrast to conventional spherical nanoparticles, the pharmacokinetic properties of rmDPPs are advantageous for traveling through the bloodstream. The direct loading of rtPA into the PLGA matrix limited the therapeutic potential of the drug. As a limitation of this work, the rmDPPs with magnetoacoustic stimuli could not enhance the blood clot lysis potential compared to those without stimulation for Additional file 1: Supporting Information.


Correction to: European Journal of Nuclear Medicine and Molecular Imaging10.1007/s00259-022-05821-wThe publisher regrets that the Tables The original article has been corrected.