Fecal samples from 10 individuals were used as the inoculum for fermentation with 10 different starch sources. Butyrate production was heterogeneous across both fecal inocula and starch source, suggesting that a given microbiome is best suited to produce butyrate only from a subset of RS sources that differs between individuals. Interestingly, neither the total amount of RS degraders nor butyrate producers seemed to be limiting for any individual, rather the membership of these sub-populations was more important. While none of the RS degrading organisms were correlated with butyrate levels, Ruminococcus bromii was strongly positively correlated with many of the most important butyrate producers in the gut, though total butyrate production was strongly influenced by factors such as pH and lactate levels. Together these results suggest that the membership of the RS degrader and butyrate producer communities rather than their abundances determine the RS sources that will increase butyrate levels for a given microbiome.One of the primary benefits associated with dietary resistant starch (RS) is the production of butyrate by the gut microbiome during fermentation of this fiber in the large intestine. The ability to degrade RS is a relatively rare trait among microbes in the gut, seemingly confined to only a few species, none of which are butyrate producing organisms. Thus, production of butyrate during RS fermentation requires a network of interactions between RS degraders and butyrate producers. This is further complicated by the fact that there are multiple types of RS that differ in their structural properties and impacts on the microbiome. Human dietary intervention trials with RS have shown increases in fecal butyrate levels at the population level but with individual to individual differences. This suggests that interindividual differences in microbiome composition dictate butyrate response, but the factors driving this are still unknown. Furthermore, it is unknown whether a lack of increase in butyrate production upon supplementation with one RS is indicative of a lack of butyrate production with any RS. To shed some light on these issues we have undertaken an  Increasingly it is being recognized that a healthy gut microbiome is important to overall human health. While it is still an active area of research as to what constitutes a healthy microbiome, a high capacity to ferment dietary fiber to short chain fatty acids is one aspect that is nearly universally agreed upon. Resistant starch (RS) is starch that is able to resist digestion by human enzymes and thereby survive transit to the colon where it can be fermented by the microbial population that resides there . ConsumpBifidobacterium, most prominently B. adolescentis, and the Firmicutes Ruminococcus bromii are the only species that have been conclusively demonstrated to extensively degrade RS  we have performed fermentations with each on a panel of 10 different starches, eight of which are RS sources, spanning RS2, RS3, and RS4. Changes in pH, organic acid production and composition of the microbiome were measured in comparison to a starch-free control. We hypothesized that microbiomes from different individuals would exhibit unique profiles of resistant starches that increase butyrate production. Indeed, the results show stark individuality in the responses to each starch, but with some links to key taxa within the microbiome.Despite the relative ubiquity of RS degraders and butyrate producers within human gut microbiomes, consumption of RS does not universally lead to an increase in butyrate . The rea® 260 resistant starch (HAM2) and VERSAFIBETM 2470 resistant starch (HAM4) were from Ingredion Incorporated , potato starch was from Bob’s Redmill , ActiStar RT tapioca resistant starch was from Cargill  and the tiger nut flour (Tn) was from Organic Gemini . Retrograded potato starch was produced according to a previously published protocol  starch/sterile water suspensions. RS solutions were allowed to sit overnight in the anaerobic chamber before use to remove oxygen. CS was subjected to the same sterilization procedure without the pre-digestion step and Ap was sterilized via autoclave without pre-digestion.All resistant starches were prepared via a pre-digestion step to simulate passage through the small intestine (treatment with pancreatin and amyloglucosidase), followed by non-thermal sterilization. Briefly, 2 g of sample were incubated at 37°C for 16 h in a shaking water bath with 800 mg porcine pancreatin and 900 μL amyloglucosidase (both from Sigma-Aldrich). Samples were rinsed 10 times with ∼30 mL DI water. Supernatant was removed by centrifugation at 3,000 × Digestive enzymes and buffer components were purchased through Sigma-Aldrich. Media components, HPLC consumables and DNA-polymerase were purchased through Thermo Fisher Scientific . DNA extraction kits were purchased from Qiagen . Sequencing supplies were from Illumina .Bifidobacterium adolescentis (ATCC 27255) and Ruminococcus bromii (L2-63) were grown in pure cultures prior to the beginning of fermentations so that they could be supplemented into PS fermentations  to test the impact of adding additional RS degrading organisms to these fermentations. This allows us to test if the numbers of these organisms are a limiting factor in the fermentation. The pure cultures were grown overnight in RUM media containing amylopectin (see below for media composition) to ensure expression of their starch degrading genes. The overnight cultures were diluted with sterile-reduced PBS to an optical density at 650 nm (OD650) of 1.5 and 50 μL of the culture was added to a 4 mL fermentation for a starting OD650 of ∼0.02 in these fermentations.The resistant starch degrading bacteria Fecal samples were obtained through a concurrent study, examining the impact of potato and wheat dishes on cardiovascular markers and the gut microbiome, approved by the Penn State IRB (STUDY00007854). Subjects were between the ages of 25–75 years with BMI between 20 and 40 kg/m2. Subjects were non-smokers, and were free from metabolic and inflammatory diseases such as diabetes, hypertension and colitis. Samples were collected in sterile containers and immediately frozen at the subject’s home. Samples were transported to Penn State on ice within 24 h of collection and further stored at −80°C until needed. The study treatments were found to have only a modest impact on the microbiomes of the participants (unpublished results). The cardiovascular impacts of the treatments have been published .R. bromii. Before use, fecal samples were thawed under anaerobic conditions and diluted 1:3 w/v in pre-reduced PBS. For each inoculum/treatment combination under investigation, 200 μL of diluted fecal slurry was inoculated in triplicate into test tubes with the RUM media supplemented with either a treatment carbohydrate at 1% w/v or a no carbohydrate control (water), for a final volume of 4 mL. In total 11 different fecal inoculum sources (T01-T11) were investigated with a water control, two non-resistant starches (Ap and CS) and eight RS sources . Additionally, fermentations were carried out with PS supplemented with of B. adolescentis or R. bromii (PS_Ba and PS_Rb) as described above. Thus, a total of 13 treatment conditions were used. Note that RS3_Ex and RS3_Wh fermentations were not carried out with T10 and T11 due to an inadequate amount of material. After inoculation, samples were allowed to ferment for 24 h at 37°C, to simulate a colonic fermentation. Samples were then removed from anaerobic conditions, and their pH, fermentation profile, and final community structure were measured, as described below.The RUM media used for these experiments was originally developed  to suppog for 10 min. The supernatant was filtered at 0.2 μm then diluted in 1:1 in 10 mM H2SO4 for a final concentration of 5 mM H2SO4. Thermo Fischer’s Dionex 5000+ series HPLC was used in this experiment, with a 50 mm guard column  and a 300 mm ion exclusion column . Runs were 60 min long at 50°C, 4.5 mL/min flow rate, with no temperature or flow gradients. Organic acids were detected by UV absorbance at 214 nm. Quantification was done with a standard curve of each of acetate, butyrate, formate, lactate and succinate and subtraction of a media blank. It was determined that propionate could not be accurately determined due to co-elution of a media component that largely obscured the signal and could not be separated from propionate without resorting to prohibitively long run times. Differences in organic acid levels were measured between each of the starch containing treatments and the no-starch control. Organic acid data was found to follow a skewed distribution and was subjected to a ln(x+1) transformation to normalize the data prior to statistical testing. Changes in each organic acid for each treatment were analyzed with a linear mixed effects model with inoculum source as a random effect using the lmer function of the lmerTest package . The protocol used was derived from a previous study  with som package  in R. P-rocedure .The pH was measured on a Denver Instrument  Model 250 pH and conductivity meter. Changes in pH were tested as described for the organic acids but without prior transformation.DNA was extracted from one random replicate of each treatment. Qiagen’s Power Fecal DNA Kit was used, and protocol followed according to the manufacturer’s instructions, except that bead beating was carried out on a BeadBeater96  for 5 min at 3,800 rpm. Extracted DNA was diluted 1:10 in sterile water and stored frozen at −20°C before PCR and sequencing. PCR was used to amplify the 16S rRNA V4 region and purity was confirmed with gel electrophoresis. The forward primer used was 505F (5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG GTG YCA GCM GCC GCG GA A-3′) and reverse primer 806R (5′-GTC TCG TGG GCT CGG AGA TCT GRA TAA GAG ACA GGG ACT ACN VGG GTW TCT AAT-3′). PCR run conditions were as follows: 94°C for 2 min, 94°C for 20 s, 56°C for 30 s, 68°C for 40 s, repeat for 30 cycles, then 72°C for 5 min. PCR products were sent to Penn State’s Genomics Core Facility where a second round of PCR was performed to add Illumina adapters. Library quality control was performed on an Agilent Bioanalyzer and confirmed PCR products were normalized by concentration and purified before running on an Illumina MiSeq using v2 reagent kits and 250 × 250 nucleotide paired-end sequencing.https://github.com/darrell25/Teichmann_2020. De-multiplexed sequences were returned from the sequencing core and primer sequences were removed with the program cutadapt (v3.1)  . Qualityt (v3.1) , utiliziDatabase . ChimeriDatabase  and de nDatabase . Blast+ Database  was usedDatabase  with a rAlpha diversity and beta diversity were analyzed at the genus level in R using the phyloseq package  and otheFor alpha diversity, Faith’s phylogenetic diversity, Shannon diversity and inverse Simpson diversity were analyzed. For Faith’s phylogenetic diversity the OTU count data was first rarefied to an even number of reads for each sample by random sub-sampling to 2490 reads (the lowest number in any sample). Diversity was then calculated with the pd function of the picante package . The res2, followed by the calculation of Euclidian distance via the Vegan package3. All beta diversity distance or dissimilarity matrices were ordinated via principle coordinate analysis and plotted to look for treatment-based clustering. Treatment-driven differences in beta diversity were statistically tested via PERMANOVA using the adonis function  was adjusted to 0.95 to match the analysis performed with the other methods. Taxa were considered differentially abundant if they passed the 0.7 threshold of the method (recommended setting). For all methods, P-values were corrected with the Benjamini-Hochberg method . Butyrate producers were limited to those known to produce butyrate through the acetyl-CoA pathway for butyrate from carbohydrates. This included organisms previously reported to produce butyrate from carbohydrates through direct tests  were the most strongly positively correlated with butyrate, particularly for the high butyrate treatments. Eubacterium rectale and Roseburia faecis were also positively correlated in the high butyrate treatments while the Otu00180 F. prausnitzii was also positively correlated in the low butyrate conditions. Neither of the RS degraders were positively correlated with butyrate levels, however, there were a number of strong correlations between the RS degraders and the butyrate producing organisms. R. bromii had strong positive correlations with E. rectale, R. faecis, R. inulinivorans, and the F. prausnitzii OTUs and generally strong negative correlations with members of the Clostridiaceae and C. difficile. In contrast B. adolescentis had fewer connections to the butyrate producers, though with strong positive correlations with Coprococcus eutactus, Anaerostipes hadrus, Blautia faecis, and the Otu00071 F. prausnitzii, mostly in the high butyrate producing treatments. The relationship with A. hadrus may be particularly important as it along with Coprococcus catus and Anaerobutyricum hallii are key taxa that are able to convert lactate that is the main fermentation product of B. adolescentis to butyrate  rich undefined media such as brain heart infusion that provide fairly complete nutritional sources in an attempt to insure all possible requirements are present  or by providing a competing carbohydrate source (mucin). These are, however, important factors for mimicking the gut environment and future work should endeavor to incorporate them into in vitro fermentation experiments.In conducting these experiments we have utilized the RUM media  develope the gut . Media t present , (2) def present  and (3)  present . The RUM systems  where anin vitro metagenome and metaproteome to those found in vivo peaked around 9–24 h, with decreases in the bacterial population after that point . Past studies have examined microbiome stability over time and found that despite daily variations due to diet , gut mic and T05.The Bn condition was the most consistently favorable for increases in butyrate production relative to the no-starch added control . It was E. rectale has very poor activity against PS, moderate activity against CS and very good activity against Ap . For instance, the cell-wall anchored amylase of ainst Ap . Thus, d enzymes , so ther enzymes , suggest enzymes , an inhi enzymes , non-RS rganisms .B. adolescentis, one had only R. bromii and five had both as detected RS degraders  is also the one that had the most robust increases in butyrate production across treatments  did not fare as well, producing increases in butyrate for only Bn and HAM2. Interestingly, it did have the largest increase in succinate of any of the inocula, primarily in the HAM4, RS3_Wh and Ap conditions  can be found below: The studies involving human participants were reviewed and approved by The Pennsylvania State University Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.JT conducted the experiments and performed part of the data analysis and writing. DC conceived of the experiments and performed part of the data analysis and writing. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Critical-sized defects of irregular bones requiring bone grafting, such as in craniofacial reconstruction, are particularly challenging to repair. With bone-grafting procedures growing in number annually, there is a reciprocal growing interest in bone graft substitutes to meet the demand. Autogenous osteo(myo)cutaneous grafts harvested from a secondary surgical site are the gold standard for reconstruction but are associated with donor-site morbidity and are in limited supply. We developed a bone graft strategy for irregular bone-involved reconstruction that is customizable to defect geometry and patient anatomy, is free of synthetic materials, is cellularized, and has an outer pre-vascularized tissue layer to enhance engraftment and promote osteogenesis. The graft, comprised of bioprinted human-derived demineralized bone matrix blended with native matrix proteins containing human mesenchymal stromal cells and encased in a simple tissue shell containing isolated, human adipose microvessels, ossifies when implanted in rats. Ossification follows robust vascularization within and around the graft, including the formation of a vascular leash, and develops mechanical strength. These results demonstrate an early feasibility animal study of a biofabrication strategy to manufacture a 3D printed patient-matched, osteoconductive, tissue-banked, bone graft without synthetic materials for use in craniofacial reconstruction. The bone fabrication workflow is designed to be performed within the hospital near the Point of Care. Importantly, the periosteum-like tissue shell compacted over 3 weeks of in vitro culture to create a coating of collagen, MSCs, and a haMV derived neovasculature around the final printed shape  or honeycomb latticed cuboids (1 cm × 1 cm x 3 cm). Early development work involved the cylinder shapes while all implanted grafts were cuboid, which filled an equivalent space of 3 cc. An internal lattice structure was chosen to promote vessel ingrowth once implanted and avoid formation of necrotic zones throughout the tissue while culturing prior to implantation . Once pred shape . The conue shell .via microCT to assess implant ossification over time. Five of the grafts were explanted at 4 weeks and the remaining 5 were explanted at 8 weeks, a time course consistent with bone fracture repair in rats  rats . Animals in rats . All 5 g in rats . Graft d in rats . Grafts  in rats . In addi in rats . Minor g in rats . There wConfocal imaging of grafts explanted at 4 weeks revealed a dense, heterogeneous microvasculature around and adjacent to the DBM particles of the implant comprised, in part, of human microvessels, visualized using fluorescent UEA-1 lectin, indicating that some of the haMVs present within the periosteum-like shell at the time of fabrication persisted as mature constituents of the implant vasculature through at least 4 weeks . The micAs compared to pre-implantation, the graft tissues within the 4- and 8-week implants contained more collagen, with the matrix appearing considerably denser and more complex as assessed by histology . Further using a standardized compression test . The appantation . Compariantation .in vitro culturing and subcutaneous implantation in a rodent model, the printed bone graft remained viable; became highly vascularized around and within the spaces of the DBM elements, including large vessel supplies; and ossified in situ . Furthermore, the graft integrated well with the surrounding tissue at the implant site without evidence of significant inflammation  and scarring, suggesting this graft to be a viable replacement for critical size bone defects.Bone grafting is the second most common tissue transplantation in the world, preceded only by blood transfusion . Findingin vitro prior to implantation. The synthetic polymers and hydrogels commonly used with DBM in clinical applications do not support these important cellular activities in vitro or in vivo.We chose to use 3D printing with a paste of demineralized bone matrix (DBM), known to promote bone formation when implanted , to formin vitro  ossified to a similar extent, beginning between the 4th and 5th week time point. The one exception began to mineralize at the same 4-week time point but ossified exponentially faster than the other grafts in the remaining weeks. While the overall degree of compaction (flattening of the graft) did not qualitatively change between this graft and the others, it is feasible that compaction occurred faster with this graft. If true, this would indicate that compaction time, not total strain rate, is important for ossification. Whether that graft experienced more compressive loading, perhaps due to a different location on the animal flank, is not clear. If this did happen, though, it suggests that compressive loading during implantation promotes ossification, consistent with what is known about native bone . FurtherWhile each graft had areas of robust ossification at levels similar to the native rat skeleton (as assessed by microCT intensities), full ossification across the entire graft had not occurred after 8 weeks. However, one graft, #687, was clearly progressing faster than the other grafts and indicates, the ossification potential of our approach. Why this one graft progressed quicker than the other grafts is not clear. Comparison of histological sections of each graft did not indicate any obvious differences in cell and/or vascular densities between graft 687 and the other grafts. Possibly the location of graft 687 implant site, bleeding, or post-surgical complications unique to 687 might offer avenues for explanation. However, review of surgical notes and gross images of the implants and implant sites did not offer any insight into possible mechanisms. Furthermore, graft 687 was not anymore compressed in shape and size than the other grafts. One possible explanation is that the vascularized tissue surround of 687 may have been better integrated with the graft core, thereby facilitating a more rapid vascularization and, therefore, quicker ossification. Because a thorough assessment of this possibility involves destructive approaches , we were not able to determine the degree of component integration for each graft. We are currently pursuing ideas as to how to improve these aspects of the graft making process.via microCT. Whether the absence of human cells over time is due to the natural turnover of the human cells (either the MSCs or those comprising the microvessels) or the limited immune system of the nude rats used as recipients attacking the human cells is not clear. Osteocalcin-expressing rat cells were present in the spleens of recipients, consistent with the normal release and circulation of osteocyte precursors from the bone marrow during bone repair, an intended activity in our fabrication strategy.MSCs were used to condition the graft prior to implantation and potentially act as osteocyte precursors to facilitate ossification. Indeed, expression of human osteocalcin, a marker of osteoblasts , in the via tissue banking, to establish a more useful bone graft that, depending on the site of banking, could be employed as a leashed or vascularized free flap. Indeed, the fabricated grafts were fully integrated with the surrounding tissue; highly vascularized; and free of necrosis, fibrosis, or destructive inflammation after implantation in the subcutaneous position. In many respects, an osteo-cutaneous, pedicle tissue flap, whereby the bony component was custom shaped, developed after implantation, mimicking the desired outcome for clinical implementation. Finally, the fabrication strategy for the graft is designed to be employed in the hospital, at the Point of Care, removing challenges of shipping and transport, and maximizing the ability to incorporate autologous cells into the graft.In summary, we present a fabrication strategy for a bone graft free of synthetic elements for use in tissue reconstruction involving space-filling bony defects. A distinctive feature is the use of a unique formulation of DBM to 3D print a customizable graft matched to a patient’s anatomy and defect geometry. This contrasts with other approaches in which decellularized autogenous bone is machined to the desired shape . While aBone marrow-derived mesenchymal stromal cells  were expanded using vendor-provided growth media (RoosterBio). MSCs were used at passage 3 throughout all experiments. Primary, isolated microvessels  were derived from discarded human lipoaspirates following accepted ethical and privacy standards. Each lot of microvessels was subject to quality control testing involving assessment of angiogenic activity. To reduce the effects of donor-to-donor variation, only haMV lots with similar angiogenic potentials were used.A digital model  was used to create a mold with a square casting chamber and a surrounding circular media reservoir. The casting chamber was designed to be 5 mm larger than the design of the final printed object on each side. As previously described Sylgard 184 (Krayden) and SE 1700 (Krayden) were each combined in a 10:1 ratio with their respective base:crosslinker solutions . After tA 15% solution of 300 Bloom Gelatin (Sigma), was prepared by dissolving in 1X PBS and stirring vigorously on high heat. Once dissolved the solution was sterile filtered and allowed to cool to 37degC until use. Fibrinogen (Sigma) was dissolved in 1X PBS to 60 mg/ml and sterile filtered through a 0.22 μm filter. Fibronectin (Sigma) was dissolved at 1 mg/ml and stored in frozen stock solutions until further use. Tranglutaminase (Amazon) was dissolved in culture media at 60 mg/ml by placing the solution into a 37degC water bath for 20 min. Once dissolved the solution was sterile filtered and used within 30 min. Factor 13 (Enzyme Research Labs) and Thrombin (Sigma) were dissolved in sterile 1X PBS and stored in frozen aliquots until use.A 3D printable ink was created by measuring 6.2 ml of powdered Demineralized Bone Matrix particle size 125–500 μm (Essent Biologics), in combination with 10 mls of Gelatin, Fibrinogen, Fibronectin, and MSCs at 7%, 10 mg/ml, 10 μg/ml, 500,000 cells/ml respectively. The ink was mixed and loaded into a sterile 10cc printing barrel. Care was taken to invert the barrel until the ink solidified to ensure no settling of the DBM particles occurred.A digital model of a 1 cm × 1 cm × 3 cm cuboid structure was created using 3D design software . Slic3r 3D was used to add an 18% honeycomb patterned infill. The ink described above was bioprinted  using a 18 GA conical needle (Nordson) at ambient temperature in aseptic conditions. Once printed the graft was transferred to a 3D printed silicone mold. The printed graft was enzymatically crosslinked using a crosslinking solution consisting of transglutaminase, FXIIIa, and Thrombin, 10 mg/ml, 1.4 U/ml, 1 U/ml respective and was diluted into media #1 without VEGF. Care was taken to ensure the entire print object was covered with the enzymatic crosslinker. The graft was placed into the incubator overnight.in vitro culture.The day after printing, a 5 mg/ml solution of collagen type I was prepared as described previously . MSCs (2Eight of the grafts were cultured for 3 weeks. The first week grafts were cultured in Media #1 50:50 base of DMEM/F-12(Fisher):RPMI supplemented with 10 μg/ml Insulin (Sigma 15500), 100 μg/ml transferrin (Sigma T2252), 100 μM putrescene (Sigma P5780), 20 nM progesterone (Sigma 8783), 30 nM sodium selenite (Sigma), 2% Human platelet lysate  and 50 ng/ml VEGF. After one-week grafts were switched over to media #2: RPMI (Fisher supplemented with B27 without vitamin A (Fisher), 2% Human Platelet Lysate , and 50 ng/ml VEGF (Peprotech). Four of the grafts were cultured for 2 weeks in media #2. Media was changed every other day for the first week of culture then every 4 days.All animal studies were performed with approval from the Dartmouth IACUC review committee. A total of ten six-month-old, male, nude rats  were anesthetized with 2.5% isoflurane. Animals were shaved, weighed, and given a 2.5 mg/kg subcutaneous injection of ketoprofen. An approximately 2 cm long, medial to lateral incision was created on the skin of one side of the flank, rostral to the hip. Blunt dissection with scissors created a subcutaneous pocket. The graft was inserted with the open lattice side down and the pocket closed with sutures. This was repeated with ten animals in total, using one graft per animal. The ten surgeries were spread out over a 3-day period.MicroCT scans were performed every week. Animals were anesthetized with isoflurane (2%) then placed on an imaging sled. A nose cone was fitted onto the animal and isofluorane (2%) was continually administered. Animals were positioned prone in the imaging tray placing the graft  upwards. Care was taken to include both the graft and the animal spine in each collected image An eXplore Locus Micro CT Scanner  with a 93 μm resolution in the XY plane and 93 μm slice thickness (z-axis) resolution was used. Imaging parameters were set at 80 kV, 450 μA, and an exposure time of 100 ms. The mineralized volume of grafts was determined from microCT images using standard thresholding features of the 3-Matic Medical 14.0 software . To visually represent mineral formation over multiple weeks, graft mineralization was segmented using thresholding tools, rendered, and spatially registered between weekly scans using the distance to the recipient’s spine. Thresholding values were based on Hounsfield units (HU), a measure of attenuation corresponding to tissue density, with the cut-off determined for each animal based on the HU of the adjacent spine included in the imaging field of view, to clearly separate mineralization from adjacent soft tissues.en bloc confocal imaging. Blood was collected via a heart stick for CBC analysis (Antech Diagnostics). A pneumothorax was performed to euthanize the animal while under anesthesia. Samples from peripheral tissues were collected and placed into either RNA later or 4% PFA. This procedure was repeated at week 8 with the remaining animals.At week 4, five animals were anesthetized using isoflurane (2.5%), shaved, weighed, and imaged. The right jugular was exposed and 500 μl of 2 mg/ml dextran tetramethylrhodamine (Invitrogen D7139) was injected into the jugular and allowed to circulate for 15 min. After 15 min the implant graft was exposed with blunt dissection, photographed, and removed from the animal. Grafts were further photographed and sectioned into three pieces using a razor blade. A third of each graft was placed into buffered 4% paraformaldehyde (PFA) to be used for histology and another third was placed into bone culture media +2% pen/strep +2% AmpB to be used for mechanical testing. The remaining third was cut further into 2 pieces with one piece placed into RNA later solution for PCR and the other into 4% PFA for in vitro incubation for 3 weeks (t = 3); 3) after 3 weeks of in vitro incubation and 4 weeks of in vivo implantation (t = 4) and 4) after 3 weeks of in vitro incubation and 8 weeks of in vivo implantation (t = 8). All samples tested were 1 × 1 × 1 cm sized grafts. Samples were tested prior to tissue fixation. Mechanical properties were characterized by uniaxial quasi-static tests to determine compression modulus, also known as apparent modulus. All tests were performed using an Instron ElectroPuls E3000 (Instron) in displacement control at 1 mm/min. All samples were pre-loaded at 0.1 kN and tested under wet conditions using phosphate-buffered saline (PBS). One test per sample was performed and at least four specimens per condition were tested. Apparent modulus was calculated as the stress/strain ratio obtained at low strain (1%–5%) avoiding the initial non-linear toe region (<1%).Mechanical properties were assessed at different time points during the bone maturing process 1) after bioprinting (t = 0), 2) after Explanted grafts were fixed overnight using 10% Formalin then washed with 1X PBS. Samples were sent out for processing and staining (Saffron Scientific Histology). Briefly, graft pieces were paraffin embedded with the cut section down and 5 µm sections were prepared. Decalcification was not performed. Sections were stained with alizarin Red, Masson’s trichrome, hematoxylin and eosin, and picrosirius red/fast green by Saffron Scientific Histology.Sections of samples were removed from RNALater and incubated overnight with lysis buffer containing 50 μg/ml SDS, 200 μg/ml EDTA, 1.25 mg/ml 1 M Tris-HCL, and 800 μg/ml Proteinase K (Fisher Sci.) at 55°C. Samples were combined 2X with 1 volume of phenol:chloroform:isoamyl alcohol in Phase lock tubes and centrifuged for 10 minutes at 2500 xG. Samples were then resuspended in ethanol with ammonium acetate and centrifuges at 14000 xG for 3 min. The resultant pellets were washed with 70% ethanol and stored at 4°C until use.RNA was isolated using a Qiagen RNAeasy kit. Briefly tissues were removed from RNALater and placed into lysis buffer consisting of Buffer RLT, and β-mercaptoethanol and homogenized on ice using a pro 200 homogenizer. Isolated RNA was pelleted and washed according to the supplied instructions. Isolated RNA was converted to cDNA using Invitrogen Superscript IV VILO Mater Mix Kit (Fisher). Control blanks were made up with each cDNA conversion. Samples and blanks were annealed by incubating at 25°C for 10 min then RNA was reverse transcribed by incubating at 50°C for 10 min followed by an incubation at 85°C for 5 min to inactivate the enzyme. A PCR reaction was run with each sample by incubating the converted cDNA with SYBR select mater mix, and the forward and reverse primers . The PCRFixed samples were further portioned for en bloc staining to visualize the vasculature. Briefly, samples were incubated with 0.25% triton X for 40 min then blocked with 5% BSA followed by incubation with Ulex Europaeus Agglutinin I DyLight® 649  and Griffonia Simplicifolia Lectin I Fluorescein  both at 1:50 dilution in PBS/5% BSA. UEA-1 lectin was used to visualize the vasculature derived from the human microvessels while GSL-1 was used to identify the rodent vasculature. Samples were rinsed with PSB + 0.1% Tween and imaged on a FV1000 Confocal Microscope (Olympus). The analysis performed on each graft are summarized in


Tissue repair after trauma and infection has always been a difficult problem in regenerative medicine. Hydrogels have become one of the most important scaffolds for tissue engineering due to their biocompatibility, biodegradability and water solubility. Especially, the stiffness of hydrogels is a key factor, which influence the morphology of mesenchymal stem cells (MSCs) and their differentiation. The researches on this point are meaningful to the field of tissue engineering. Herein, this review focus on the design of hydrogels with different stiffness and their effects on the behavior of MSCs. In addition, the effect of hydrogel stiffness on the phenotype of macrophages is introduced, and then the relationship between the phenotype changes of macrophages on inflammatory response and tissue repair is discussed. Finally, the future application of hydrogels with a certain stiffness in regenerative medicine and tissue engineering has been prospected. Trauma, infection, tumor, and other diseases can cause tissue damage and have an adverse impact on physiological function . Tissue Recently, the emerging interdisciplinary-tissue engineering integrating materials science and cell biology has made it hopeful to solve the problems of tissue repair. The three elements of tissue engineering include scaffold materials, cytokines, and stem cells. As one of the scaffold materials, hydrogel plays an important role in tissue engineering because of its high water content, hydrophilicity, simulation of extracellular matrix environment , good biin vitro  and M2 . M1 releases pro-inflammatory cytokines, while M2 can promote tissue repair and down-regulate inflammatory response . In the In this review, we introduced the fabrication of hydrogels with different stiffness and the effects of related biophysical cues on cell differentiation and proliferation. At the same time, we described the research of different hydrogels in tissue repair and osteogenic differentiation. We hope to provide new research ideas for regenerative medicine and tissue engineering by analyzing the mechanism of action of different stiffness hydrogels on cells.Biomaterials with different matrix stiffness have been widely used to study cell differentiation and phenotype . At the 2+ ion crosslinking, and hydrogels with different crosslinking degrees can be obtained by changing the crosslinking time and temperature. In this study, the results showed that the stiffness of the hydrogel was significantly improved at high concentrations and long crosslinking time . The final hydrogel was obtained by freeze-thaw operation and ultrasonic dispersion. Compared with pure PVA hydrogels, the mechanical properties of the hydrogels with nanoparticles were enhanced. Different freezing and thawing times may also change the compactness of the hydrogel network and thus change the stiffness of hydrogel. Besides the methods above, the mechanical properties of hydrogels can also be improved by adding nanoparticles into hydrogels. The nanomaterials added into hydrogels include metal nanoparticles, carbon-based nanomaterials, ceramic nanoparticles, and so on . Among toperties . GO is aoperties . In addiDifferent synthesis methods can obtain hydrogels with different stiffness, and the different stiffness of hydrogels led to various cell behaviors. Different stiffness hydrogels can affect the behavior of stem cells, MSCs, fibroblasts , macrophThe change of hydrogel stiffness affects the behaviors of cells , such asIn the process of the above hydrogel stiffness affecting cell behavior, it may regulate cell expression by affecting the expression of related genes and proteins . In humaThe stiffness of hydrogels does not simply directly affect the gene expression of cells in proteins. As one of the components of the assembled cytoskeleton, stress fibers felt and transferred stiffness changes from the external environment during cell growth. And the increase in stiffness can increase the number of stress fibers. 2+. The results shown that integrins interacted with the cytoskeleton, and structural changes in the cytoskeleton inhibited Piezo1 channels, resulting in less Ca2 + entering the cytoplasm. Moreover, Piezo1 with different stiffness has obvious dependent growth behavior , hydrogen peroxide (H2O2), hydroxyl radical (OH·), etc., and ROS certainly plays a role in maintaining related physiological functions in the body. Excessive ROS can lead to oxidative stress  modified (PCL) chitin hydrogel with good mechanical strength. Under inflammatory conditions, BMSCs promoted the transformation of M2 macrophages and inhibited the expression of M1 macrophages-related factors, thereby promoting tissue repair. On the hydrogel platform with dynamic stiffness change, it is also reflected that high stiffness makes M2 type turn to M1 type and has a higher YAP/TAZ nuclear ratio. In addition, a higher YAP/TAZ nuclear ratio is also related to the osteogenic differentiation of BMSCs  play a stabilizing role in late angiogenesis . Therefoof BMSCs . TherefoIn addition, the change of matrix stiffness affects the expression of ROS, and ROS as an upstream signal affects the nuclear factor-κB (NF-κB) pathway, causing the conversion of macrophages between M1 and M2 . At the Hydrogel is a bio-scaffold material with biocompatibility and biodegradability. The stiffness of hydrogel can be adjusted by changing the crosslinking mode, external stimulation, molecular weight, concentration ratio of composite materials, and adding nanoparticles. Hydrogels with different stiffness are promising materials in biomedicine and their stiffness will change the behavior of BMSCs. The higher hardness hydrogel can induce BMSCs to differentiate into osteoblasts while lower hardness hydrogel can induce BMSCs to differentiate into chondrocytes or adipocytes. Recently, a composite hydrogel with nano-silicate was developed. The gradient stiffness of hydrogel can change the morphology and behavior of cells, which has a certain effect on the tissue repair of the cartilage-bone interface. Recent studies have also found that hydrogels with physiological stiffness may also alter the behavior of corresponding cells by affecting the production of oxidative stress ROS, thereby playing a role in tissue repair or inflammatory response.But there are still some problems limiting the application of hydrogels. For instance, the systematic toxicity of added nanomaterials still requires further clinical research. Besides, How the stimuli-responsive hydrogels should be effectively applied to the actual treatment by changing the external conditions is also a problem to be considered in the future. In addition, through in-depth exploration of the relationship between matrix stiffness and ROS, a new hydrogel material may be designed to improve the skin aging process by affecting the production of ROS in the future. At the same time, Combined with their drug-loading function , hydrogeIn addition, the hydrogel types discussed above are carried out in 3D environment, but most of them are carried out in two-dimensional (2D) environment. We all know that the real environment of organ is a complex 3D structure, and it is difficult to obtain the fiber structure characteristics of the real extracellular matrix under 2D conditions. Therefore, the influence of stiffness on cells may be different in 3D or 2D condition. Recently, researchers have designed 3D hydrogels with adjustable stiffness to mimic the natural tissue environment. It’s found that the increase of 3D scaffold stiffness also promotes the proliferation and cartilage differentiation of BMSC, and the change of stiffness also affects the adhesion, migration and viability of cells . This inIn summary, hydrogels with different stiffness can affect the morphology or lineage selection of stem cells/MSCs and macrophages, thereby promoting or inhibiting tissue repair. In the future, researchers can prepare stiffness-adjustable hydrogels for tissue engineering by different methods and use changes in hydrogel stiffness to influence cell lineage selection to promote osteogenic differentiation or angiogenesis. This idea of regulating the cell microenvironment for tissue repair provides a promising strategy for tissue engineering and regenerative medicine.


Zirconia had twice the wear resistance of PEEK . Zirconia was more color stable than PEEK . PEEK had more displacement resistance than zirconia . PEEK offers minimal abrasion, better stress modulation through plastic deformation, and good color stability, which make it a promising alternative to zirconia crown.Recently, polyetheretherketone (PEEK) has been introduced to the dental market as a high-performance and chemically inert biomaterial. This study aimed to compare the wear resistance, abrasiveness, color stability, and displacement resistance of zirconia and PEEK milled crowns. An ideal tooth preparation of a first maxillary molar was done and scanned by an intraoral scanner to make a digital model. Then, the prosthetic crown was digitally designed on the CAD software, and the STL file was milled in zirconia  and PEEK  crowns using five-axis CNC milling machines. The wear resistance, color stability, and displacement resistance of the milled monolithic zirconia with unfilled PEEK crowns using a chewing simulator with thermocyclic aging  were compared. The antagonist wear, material wear, color stability, and displacement were evaluated and compared among the groups using the Wilcoxon–Mann–Whitney U-test. Zirconia was shown to be three times more abrasive than PEEK ( Wear and tribological properties are important in dentistry ,2. ProgrThe discoloration is another significant parameter that needs to be studied extensively. This value of the color stability of the restorative material is evaluated over a prolonged period using a spectrophotometer . The sur2) is currently the most used tooth-colored restorative material in dentistry  consists of polyetheretherketone (PEEK) and polyetherketoneketone (PEKK), and it was recently introduced in dentistry . RecentlThere has been a vast development in digital technologies in dentistry including 3D scanning, designing, and 3D/4D/5D printing ,22,23,24PEEK can be a promising alternative to titanium and zirconium to use in various clinical situations in dental practice due to its high-quality mechanical properties such as favorable elastic modulus, strength, rigidity, and light weight ,27,28,29Although PEEK is widely used at present, there are very limited studies that compared the mechanical properties of PEEK as fixed prostheses with other materials such as zirconia. In addition, no study compared the mechanical properties of zirconia and PEEK using five-axis milling. Thus, this study aimed to compare the wear resistance, abrasiveness, color stability, and displacement resistance of PEEK and zirconia milled crowns using a chewing simulator with thermocyclic aging.n = 12) consisted of milled zirconia crowns  and Group 2 (n = 12) consisted of milled PEEK crowns . Each group was divided into 3 subgroups, as shown in The study was approved by the Scientific Review Board of Saveetha University (SRB/SDMDS09/18/PROSTHO/001). A total of 24 crowns were made, which were divided into two different groups based on two different materials taking reference to sample size from previous studies ,39. GrouTooth preparations were done on a first maxillary molars of the Typodont model  using a taper flat end bur for the buccal and palatal surfaces and were replicated into multiple epoxy resin dies . The tooth preparations had 1.5 mm shoulder margins ideal for an all-ceramic restoration with a clearance of 2 mm occlusally according to the previous study .The teeth were scanned in with an intraoral scanner , and the first maxillary molar crown was digitally designed on the CAD software. From STL files, zirconia and PEEK crowns were created and milled using a 5-axis computerized numeric control (CNC) milling machine  ,42. The Then, the digitally fabricated zirconia and PEEK crowns were polished with polishing paste and a rubber wheel according to the manufacturer’s protocol ,45. The After the fabrication, the crowns were stored at room temperature (25 °C) for at least 1 day, and any possible crack formations were examined. Then, the crowns were sandblasted with 100 µm at 1 bar pressure for 10 s. They were conditioned with 2 layers of bonding agents  and then cured for 20 s using curing light  as recommended by the manufacturer ,47. ThenThe crown samples were prepared in the resin die and mounted with fast-setting high-strength low-expansion plaster . The steatite antagonists  were used to mimic enamel and mounted using acrylic resin onto the aluminum mounts. The steatite balls were of 4 mm width and length. They were kept in cusp to fossa occlusion with the zirconia and PEEK crowns.The chewing simulator  had 4 testing chambers within a thermocycling chamber. They have 2 moving parts: the vertical bar (Z-axis) and the horizontal table (X-axis). The samples were mounted onto the table, which could move back and forth. The antagonists were connected to the vertical bar and moved vertically. The load of 5 kg weights was created and applied to the samples by the antagonists. The antagonist samples of 4 mm diameter  were held by the vertical bar and the horizontal bar held the force sensors in order to evaluate the force applied at each cycle. The load was added to each rod to increase the masticatory load on each sample to simulate the oral environment. Thermocycling was done from 4 to 60 °C and each cycle consisted of one cold and one hot cycle, and the filling time of the chamber was kept for 12 s ,49. ChewThe zirconia and PEEK samples were pretested by a 3D laserscanner , and the color graph was obtained. The surface wear of the samples was analyzed and recorded following the chewing simulator cycles with the thermocycling process. The vertical sensor resolution is 0.8 µm; thus, fine movements and cracks were detected. The samples were inserted onto the self-centering mount. The measurement fields were defined by a built-in camera, and the images were exported as jpeg files to be evaluated .A digital spectrophotometer  has been used to determine the precise and reliable shade matching for natural teeth and ceramic restorations. It was also used to evaluate the color stability of the crowns before and after thermocycling and the chewing simulator. The digital spectrophotometer measured the wavelength range from 400 to 700 nm and used LED technology, which was unaffected by the environment.Instron Ltd., Buckinghamshire, UK) capable of dynamic and static testing was used to evaluate the displacement of the crowns. The samples were mounted on the epoxy resin dies  and placed onto the platform of the universal testing machine. The zirconia and PEEK crowns were tested before and after the wear test and thermocycling process. They were subjected to a load of 2500 Ncm−1. The displacement of the material was observed in mm2 , material wear (mm2), color stability (ΔE), and displacement (mm2) were done using the Kolmogorov–Smirnov test, Levene test, and Shapiro–Wilk test, and the data were not normally distributed. As the variables were not normally distributed, non-parametric tests using Wilcoxon–Mann–Whitney U-tests were used to compare the values among the groups. The significant level was set at p value = 0.05.All the data obtained were analyzed using the SPSS . Normality tests for the antagonist wear (mmp < 0.001), as the zirconia caused three times more antagonist wear than PEEK, as shown in Wear of the steatite antagonist caused by zirconia compared to PEEK showed a significant difference (p < 0.001), as shown in p < 0.001).Similarly, the zirconia crowns were more wear-resistant than PEEK crowns over 120,000 cycles in the chewing simulator (p < 0.001) when the zirconia and PEEK crowns underwent wear and aging in the chewing simulator, as shown in p < 0.001). The color stability of thermocycled and worn zirconia was better than that of PEEK crowns (p < 0.001)  .p < 0.001) following thermocycling and wear in the chewing simulator (p < 0.001). Furthermore, PEEK crowns showed significantly (p < 0.001) more resistance to displacement than zirconia crowns following thermocycling and wear in the chewing simulator , maintained higher color stability , and offered the least displacement  compared with crowns made of PEEK. These findings are similar to the previous studies [Although various new biomaterials are introduced for biomedical applications, PEEK has been widely used for dental applications ,55,56,57 studies ,59,60,61This study used standardized steatite balls, since it is a standard antagonist used for wear simulation studies . The mod−1 [In this study, the thermocycling temperature in the chewing simulator was simulated to match the oral environment. It was variable between −10 and 60 °C . This wa−1 . The mov−1 . The occ−1 ,68. The The process of sintering was carried out at 1450 °C for 60 min, as recommended by the previous studies ,70. ThisZirconia can be polished or glazed, and it has been shown that polishing produces minimal wear compared to glazed zirconia ,71,72,73In this study, strict environment and lighting protocol were maintained as reported in the previous study . A calibThe displacement in millimeters at a force of 2500 kN was evaluated for the PEEK and zirconia samples . This waThe evaluation of the wear can be observed using various contact and non-contact profilometers such as True Definition (TD) from the 3M ESPE and 3D laserscanner (LAS-20) from SD MEchatronik . AlthougThere are certain limitations to this study. Enamel was not used as an antagonist due to errors in standardization, the wear pattern was simulated only in the buccolingual direction, the circular movement of mastication was not replicated, and distilled water was used in the simulator instead of artificial saliva. Various confounding factors in this study can be the type of antagonist used, thermocycling process, characteristics of the chewing simulator, accuracy of the milling machine, and effect of the environment on the color stability. Although the surface wear can be studied from various techniques such as scanning electronic microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) , we usedThe present report tested only wear, color stability, and displacement. It could be interesting in the future to test and compare PEEK materials also for flexural strength , synergiZirconia is currently the most used tooth-colored restorative material, and PEEK is used as an alternative to zirconium in dentistry. Within the limitation of this study, the crowns made of zirconia produced three times more antagonist wear, maintained higher color stability, and offered the least displacement compared with crowns made of PEEK. The PEEK crowns showed minimal abrasion, better stress modulation through plastic deformation, and good color stability, which makes it a promising alternative to zirconia for fabrication of the crown. The clinician can choose the material for the fabrication of dental prostheses according to the application considering its properties, advantages, and limitations.


ClinicalTrials.gov. A total of 126 articles were yielded by the search, out of which four randomized controlled trials (RCTs) were included according to our eligibility criteria and irrelevant articles were excluded. The intervention group received PRP application to the wound bed before applying the graft while the control group received treatment with conventional fixation procedures (sutures and staples). We estimated the pooled odds ratio with a 95% confidence interval (CI). The total number of participants in the analysis was 460. The participants had wounds of varying etiology. Post-operative graft loss and hematoma formation were taken to be the primary and secondary outcome measures, respectively. Thirty-four participants suffered graft loss of differing extent in the control group while three participants suffered graft loss in the intervention group. The pooled odds ratio for graft loss was 0.15  signifying that PRP use decreased the odds of graft loss by 85%. For our secondary outcome measure, 440 participants were studied. Forty-four participants suffered hematoma formation in the control group versus 11 in the intervention group. The pooled odds ratio for hematoma formation was calculated as 0.21  signifying that PRP use decreased the odds of hematoma formation by 79%. PRP appears to significantly impact graft survival, and further studies are needed to strengthen the evidence base for its use in split-thickness skin grafting.Split-thickness skin grafting is a very popular technique of wound closure, especially for large wounds. The success rate of a split-thickness skin graft (STSG) has consistently been in the range of 70-90%. Multiple techniques have been introduced to further improve graft survival, for example, the use of autologous platelet-rich plasma (PRP), thrombin gel, platelet-rich fibrin matrix, and negative pressure wound therapy. We evaluated the impact of PRP use on the survival of STSG through a meta-analysis. We conducted the analysis in accordance with Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) protocol and performed a literature search using the following databases: PubMed, Cochrane, and  Split-thickness skin grafting is a technique where cutaneous tissue is transferred from one region of the body to the other. Split-thickness skin graft (STSG) consists of a full epidermis and partial dermis harvested from a donor site which then heals on its own. STSGs are most often used to cover large defects because their donor sites are able to heal better. A portion of the dermis that is left behind after harvest of STSG helps in the regrowth of new skin at the donor site .Since grafts do not contain a blood supply of their own, the wound bed that they are applied on should be clean, healthy, and well-vascularized to improve the graft take. Several studies have reported the split-thickness graft takes to be around 70-90%. A number of ways have been tried to improve the take, for example, bolstering with the help of negative pressure wound therapy, hypertonic glucose with negative pressure wound therapy, antimicrobial dressing combined with negative pressure wound therapy, and autologous platelet-rich plasma (PRP) -4.Several authors have reported using PRP to prepare a wound bed before placing the graft. To completely establish the role of PRP in preventing graft loss, it was felt that there is a need to perform a meta-analysis of studies exploring the benefits of PRP in STSG application.Data source and searchesClinicalTrials.gov from the date of inception of each database to April 2021. The following search terms with Boolean operators were used: platelet-rich plasma AND split-thickness skin graft, platelet-rich plasma AND wound bed preparation, split-thickness skin graft AND wound bed preparation. The reference lists of all eligible studies were also checked for any additional relevant articles.The meta-analysis was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) protocol Figure . We carrStudy selection and eligibility criteriaThe search led to the identification of 126 articles. The titles and abstracts of the articles identified by the search were independently reviewed by two authors against the eligibility criteria  method. For the detection of publication bias, we used direct observation of funnel plot symmetry, Egger’s regression asymmetry test, and Begg’s rank correlation methods. In the evaluation of publication bias using the Egger’s and Begg’s tests, P<0.05 is considered to be statistically significant. Funnel plots were used for the assessment of publication bias by graphical inspection.A forest plot was used to illustrate the analysis of the data. We evaluated heterogeneity among studies using the Q statistics and IRStudio version 1.0.136  and “Meta” R package  were used for the meta-analysis.ResultsPublication and small-study biases were assessed through the generation of a funnel plot and assessment for asymmetry. Egger’s linear regression test and Begg’s correlation test were used to investigate any suspected asymmetry. From the overall result of the meta-analysis, the central estimate was plotted , and the 95% confidence intervals (CI) were drawn  to form the funnel or inverted V. If there was any publication bias, then the studies would not be equally distributed within the inverted V Figures  and 3.Egger’s test and Begg’s correlation test were used to detect potential publication bias . There was no considerable publication bias as detected by Egger’s tests and Begg’s correlation test.Graft Loss2=38%; p=0.184; Figure The results of our meta-analysis of four studies indicate a pooled odds ratio of 0.15  and hematoma formation (reported in three of the included studies). The study by Dhua et al. reported post-operative graft loss and hematoma formation in a graphical representation in which data were recorded for post-operative day 7, day 14, and day 21 [According to our analysis, it appears that the addition of PRP to wound bed before application of STSG reduces the rate of graft loss and hematoma formation. However, our study is not without its limitations. First of all, an insufficient number of studies have been carried out addressing the topic of concern. Furthermore, the authors have only taken into consideration the studies in the English language. Moreover, there were differences with regard to methods of PRP preparation among the studies. In order to clearly distinguish the role of PRP in split-thickness skin grafting, further high-quality RCTs should be performed. It is recommended that a uniform method of PRP production be used in RCTs on this topic along with the recruitment of a patient population with similar wound etiology. These considerations will make comparison among studies more reliable.Recent years have also seen the development of a new generation of platelet concentrates, for example, platelet-rich fibrin matrix , plateleSTSGs are one of the many important ways of treating chronic wounds. It has long been desirable to find appropriate modifications to improve its survival. In this study, we analyzed the data available to investigate the benefits of PRP in split-thickness skin grafting. Our study considered two outcome measures: post-operative graft loss and hematoma formation. The use of PRP decreased the odds of graft loss by 85% and the odds of hematoma formation decreased by 79%. Thus, it can be concluded that PRP improves outcomes for STSGs. The authors acknowledge that a limited number of RCTs have been conducted addressing the research question and further studies should be carried out to improve the quality of additional meta-analyses on this topic.


Background and Objectives: Whole body vibration is widely used to enhance muscle performance, but evidence of its effects on the tendon stiffness of the knee extensor tendon in stroke remains inconclusive. Our study was aimed to determine the difference in patellar and quadriceps tendon stiffness between hemiparetic and unaffected limbs in stroke patients and to investigate the immediate effect of whole body vibration on tendon stiffness. Materials and Methods: The patellar and quadriceps tendon stiffness of first-ever hemiplegic stroke patients was evaluated with elastography to compare the differences between hemiparetic and unaffected limbs. After one 20 min session of whole body vibration exercise in the standing position, tendon stiffness was again measured to evaluate the immediate effects of whole body vibration on tendon stiffness. Results: The results showed no significant differences in the tendon stiffness of the patellar and quadriceps tendons between hemiparetic and unaffected limbs. However, significant associations were found between the tendon stiffness of the patellar and quadriceps tendons and knee extensor spasticity on the hemiparetic side . There were no significant changes in tendon stiffness after a single session of whole body vibration. Conclusions: In conclusion, knee extensor tendon stiffness in hemiparetic limbs is positively correlated to the degree of knee extensor spasticity in stroke patients. However, a single session of whole body vibration does not alter tendon stiffness. Decreased tendon stiffness has been noted in older people and paralytic patients due to spinal cord injury ,2. The mWeakness is one of the cardinal symptoms of patients with stroke. A study by Hunnicutt et al. showed that the knee extensor strength of hemiplegic limbs was 36% lower than that of nonparetic limbs . The bioWhole body vibration (WBV) has been applied to improve muscles strength, power, and flexibility . HoweverWe hypothesized that unloading due to knee extensor weakness in a stroke population leads to decreased knee extensor tendon stiffness. The objectives of this study were to investigate changes in knee extensor tendon stiffness in a stroke population using shear wave elastography and to evaluate the immediate effect of WBV on the tendon stiffness of a hemiplegic stroke population.This study was conducted on participants with first-ever hemiplegic stroke. The inclusion criteria were as follows: the subjects (1) have an age of 20 years or older, (2) have experienced their first hemiparetic stroke, (3) were more than three months from the onset of stroke, (4) were able to stand with or without a device, and (5) could cooperate with the evaluation. The exclusion criteria were as follows: the subjects could not have (1) experienced recurrent stroke, (2) experienced stroke onset within three months, (3) received knee surgery before, (4) a limited range of motion of the knee joint, (5) acute arthritis attack, (6) lower extremity fracture in recent six months, (7) weakness due to other neuromuscular diseases, (8) an inability to cooperate with the evaluation. The study was approved by the Institutional Review Board of our institute. All subjects were given and signed informed consent before participation.The knee extensor spasticity of the hemiparetic limb of all participants was evaluated using the Modified Ashworth scale (MAS), which has six degrees , and transformed to 0, 1, 2, 3, 4, and 5 for further statistical analysis. Knee sonography was performed to assess the thickness and mechanical properties (tendon stiffness and shear wave velocity) of the patellar and quadriceps tendons. Then, participants received 20 min of WBV exercise in the standing position , which wThe grayscale sonography and elastography of the patellar and quadriceps tendons were performed with an Acuson S2000 ultrasound system  equipped with 9L4 (4–9 MHz) linear array ultrasound transducer. The room temperature was controlled at 25 °C.Before testing, participants rested in the supine position for five minutes to ensure the mechanical properties of quadriceps and patellar tendons were evaluated at resting unloaded status. The measurement was performed with the knee flexed at 30 degrees . We usedThen, we shifted to elastography to assess tendon stiffness with shear wave velocity. Before measuring shear wave velocity, we checked the quality map to make sure there were no red areas that indicated poor quality in the region of interest b. The mep-value < 0.05 was regarded as statistically significant.Descriptive data were reported as means and standard deviations. The tendon thickness and stiffness were analyzed by the Wilcoxon rank sum test for comparisons between hemiparetic and unaffected sides. The correlation between tendon thickness, stiffness, change after WBV, and knee extensor spasticity was analyzed using Spearman’s correlation. Tendon thickness and stiffness were analyzed with the Wilcoxon signed rank test for comparisons of before and after WBV exercise in hemiparetic and unaffected sides, as well as the change in mean differences of the hemiparetic and unaffected sides before and after WBV exercise. We used SAS  for all analyses. A A total of 11 participants were enrolled. The basic sociodemographic data and clinical characteristics of the study population are shown in p = 0.044)  .p > 0.05) and the quadriceps tendon  in the hemiparetic side. In the unaffected side, smaller changes in the stiffness of the patellar tendon  and the quadriceps tendon  were observed (After WBV exercise, no statistically significant change was observed in patellar and quadriceps tendon stiffness in the hemiparetic or unaffected sides. However, there were trends of decreased stiffness in the patellar tendon (mean change: −0.36; observed .p > 0.05 for the patellar tendon; mean change of −0.24 and p > 0.05 for the quadriceps tendon; The mean differences between the patellar and quadriceps tendon stiffness of the hemiparetic and unaffected sides were observed to be decreased after WBV exercise, although they did not reach statistically significant levels (mean change of −0.36 and The knee extensor spasticity was not correlated to tendon stiffness change after WBV exercise in the hemiparetic side  or the cTo the best of our knowledge, this is the first study to analyze the mechanical properties of the patellar and quadriceps tendons of the hemiparetic and unaffected limbs of individuals with stroke.Patients with hemiplegic stroke have motor impairment, which leads to reduced loading over affected lower limbs. Previous research has shown lower Achilles tendon stiffness in the affected legs of patients with stroke ,19,20,21In this study, there were no significant differences in the tendon stiffness of the patellar and quadriceps tendons between hemiparetic and unaffected limbs. This finding was inconsistent with previous studies, which reported change in Achilles tendon stiffness in stroke patients ,19,21. HRegarding the effect of WBV on tendon stiffness, a previous animal study by Sandhu and colleagues showed that WBV training for five weeks resulted in increased flexor carpi ulnaris tendon stiffness . HoweverA limitation of this study was its small sample size of enrollment. Moreover, only one session of WBV exercise was performed, and the longitudinal effect was not evaluated. Additionally, we did not evaluate functional performance metrics, such as walking speed or gait pattern, as outcome measures after single bout of WBV exercise. Further long-term investigations of the longitudinal effects of vibration exercise on changes in knee extensor tendon properties and improvements in functional performance are necessary.In conclusion, there were no significant differences in the mechanical properties of the knee extensor tendon between hemiparetic and unaffected limbs. However, the tendon stiffness of hemiparetic limbs was positively correlated to knee extensor spasticity. After a single session of WBV exercise, no significant changes in tendon mechanical properties were observed in either hemiparetic or unaffected limb. However, a trend toward decreased tendon stiffness in hemiparetic limbs have been caused by the immediate effect of WBV in reducing spasticity. Further investigations with increased sample sizes are necessary to determine the biomechanical effects of hemiplegic limbs on the knee extensor tendon’s properties and the long-term effects of WBV on the knee extensor tendon’s properties.


Using a set of modern spectral and microscopic methods , the phase state, elemental composition of the nanocomposites and their nanomorphological characteristics have been investigated. It was found that nanocomposites are formed as sulphur particles with the shape which is nearly spherical dispersed in the polysaccharide starch matrix with a pronounced tendency to cluster into ring formations. Water solubility and stability of the obtained nanoparticles is ensured by sorption of starch macromolecules on the surface of sulphur nanoparticles, with the thickness of the stabilising shell in a range of 10‐171 nm. In vitro experiments were carried out to study the anti‐microbial activity of the obtained sulphur nanocomposite (1.6% S) using the propidium iodide fluorescent dye staining method and the diffusion method. It showed that the water solution of the starch‐capped sulphur nanoparticles at the concentration of 6.25 µg/ml had a pronounced anti‐phytopathogenic activity against the potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus.Water‐soluble, stable nanoparticles of elemental sulphur with a size of 9‐52 nm have been synthesised using the stabilising potential of starch. Sulphide anions were used as sulphur precursors that were generated earlier from the bulk powder sulphur in the base‐reduction system NaOH‐N One of the possible ways to provide the stability of the synthesized nanoparticles is their inclusion in the stabilising shell, which prevents aggregation of nanoparticles in the solution [Chaudhuri RG and Paria S, Salem N, et al., Seitzhan T, et al., Kouzegaran VJ and Farhadi K report on the successful application of Triton X‐100, sodium dodecyl sulphate, cetyl trimethylammoniumbromide, The aqueous extract of Melia azedarach leaves , sulfanol, sodium ligninsulphonate, sodium salt of polynaphthalene sulphonic acid, water soluble polymer sodium carboxymethylcellulose, saponin extracted from A and Phylum Bracteatum as effective stabilizers of S0NPs [0NPs hinders standardisation and reproduction of the target nanomaterials due to heterogeneity of the chemical composition of the natural raw material, which depends strongly on the source and extraction method. Here, the use of naturally available polysaccharide, starch, as a stabilising matrix is proposed. Constant composition, cheapness, water solubility and stabilising potential determine the prospects of its application in the synthesis of biocompatible, water‐soluble nanomaterials [0NPs in the starch matrix are currently missing. Inclusion of S0NPs in the starch shell will produce nanocomposites combining the biological properties of S0NPs  with water solubility of starch. In addition, the method for the preparation of S0NPs from elemental sulphur powder, which is offered, can significantly reduce the cost of their production and get rid of a number of toxic reaction products. These water‐soluble, low‐toxic nanomaterials can be used in biology, medicine, agriculture, and in particular to fight the bacterial phytopathogen Clavibacter michiganensis sepedonicus (Cms) that causes potato ring rot [Cms. The known anti‐microbial properties of S0NPs against Cms bacteria allow suggesting that the water‐solubility of S0NPs determined by the starch macromolecule shell will increase their bioavailability.Powder sulphur and its colloidal solutions have been actively used for several centuries as effective anti‐microbial , anti‐furganisms . The imprganisms . At the articles . Howeversolution . Varioussolution . In partof S0NPs , 21, 22.aterials . So, theaterials , 29, 30.ring rot . Each yering rot . To dateCms.This article presents the results of the development of an effective method for the synthesis of a number of water‐soluble nanocomposites consisting nanoscale sulphur particles capped by polysaccharide starch matrix, followed by a detailed study of their structural and nanomorphological characteristics and testing of their bactericidal activity against bacterial phytopathogen 22.1In this work, water‐soluble starch from Sigma Aldrich, powder sulphur, sodium hydroxide and hydrazine hydrate (64%), ethyl alcohol (96%) were used. All reagents were purchased from Vekton (Russia) and were used as received without additional purification.2.22H4·H2O. So, in a three‐necked flask equipped with a reflux condenser and a thermometer, 1.0 g of NaOH and 2 ml of N2H4·H2O were placed upon constant stirring, after which the temperature of this reaction medium was brought to 70°C and 0.5 g of elemental bulk‐sulphur was added under an argon atmosphere. The synthesis time was 15 min. Next, an aliquot of the obtained reaction mixture containing S2‐‐anions (Vol. 200‐700 µL) was added to the previously obtained starch solution (1.0 g starch in 25 ml water) and stirred at heating (40°C) for 20 min. The isolation of nanocomposites and their purification from impurities was performed by precipitation with a four‐fold excess of EtOH, followed by repeated washing with ethanol and air drying at room temperature. Passivation of nanoparticles’ surface and their aggregative stability were achieved through its interaction with polar hydroxyl groups of starch. Elemental analysis: Starch ‐ C 42.42%, H 6.60%, O 50.98%; Starch/S0NPs (0.57% S) ‐ C 41.66%, H 5.74%, S 0.57%, O 52.03%; Starch/S0NPs (0.7% S) ‐ C 41.43%, H 6.29%, S 0.67%, O 51.61%; Starch/S0NPs (1.6% S) ‐ C 41.22%, H 6.60%, S 1.58%, O 50.60%. Yield of nanocomposites Starch/S0NPs: 89%‐98%%.Starch‐capped sulphur nanoparticles were synthesised by oxidation of sulphide anions generated from the elemental bulk sulphur in the base‐reduction system NaOH‐N2.32.3.1TEM measurements were performed on a Leo 906 E microscope operated at an accelerating voltage of 120 kV. The size distribution of nanoparticles was determined by statistical processing of TEM microphotographs.2.3.20NPs were recorded on a Perkin Elmer Lambda 35 spectrophotometer in a quartz cuvette .The optical absorption spectra of aqueous solutions of starch and nanocomposites Starch/S2.3.30NPs was determined by a Thermo Scientific Flash 2000 CHNS analyser and by X‐ray energy dispersive microanalysis with a Hitachi TM 3000 scanning electron microscope equipped with a SDD XFlash 430‐4 X‐ray detector.The elemental composition of starch and nanocomposites Starch/S2.3.4X‐ray diffraction study was carried out on a Bruker D8 ADVANCE X‐ray diffractometer under monochromatised Cu‐Kα radiation mode Locked Coupled. The exposure time was 1 s for the phase analysis, and 3 s for the cell parameters and coherent lengths.2.3.5oζ/ηs, where ε and εo are the dielectric permittivity of the solvent and vacuum, respectively. Each measurement was carried out 3 times, and the results were averaged.DLS measurements were carried out by using the Photocore Compact‐Z equipment at 25° ± 0.1°C. A software package DynaLS v2.0 (ALANGO) was used to calculate the correlation time distributions. Scattering angle was 90°, laser 654 nm, power 20 mV. The solutions were prepared at least 5 h before the measurements. In order to dispose dust, the scattering cells were washed with benzene, vacuumed, and filled with dust‐free air. The concentration of solutions was 0.4%. The polyelectrolyte velocity ν under external electric field E was measured. The electrophoretic mobility μE = ν/E was converted into the ζ‐potential  by the Smoluchovsky equation μE = εε2.40NPs was studied using Cms cultures. The Ac 1405 strain was derived from the All‐Russian collection of microorganisms of the G.K. Scriabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences. Bacteria were cultivated in a liquid medium consisting of peptone (10 g/L), yeast extract (5 g/L), glucose (5 g/L), sodium chloride (5 g/L). In the experiment, 10 ml of bacterial suspension grown during 3 days was used, to which 100 µL 0.1% aqueous solutions of starch or Starch/S0NPs nanocomposite (1.6% S) were applied pre‐filtered through a sterile syringe filter (Minisart NML) with a pore diameter 0.2 µm or the same volume of sterile water. The final concentration of sulphur in the bacterial suspension was 6.25 µg/ml. The bacteria were incubated together with the administered samples for 24 h, after which their viability was determined using a vitally fluorescent dye of propidium iodide at a final concentration of 7.5 µM. Cell viability was assessed by counting the dead cells coloured by propidium iodide in red and correlating the resulting number with the total number of cells in the field of view. At least 15 random fields of vision were used to count cells. The images were obtained using inverted fluorescent microscope Zeiss Axio Observer for microscopic analysis of bacterial cells after they were coloured by propidium iodide. The statistical processing of the obtained data was carried out using the STATISTICA 6.1 licence application package .The bactericidal activity of starch‐capped S0NPs (1.6% S), the bactericidal activity against Cms was studied using the agar diffusion method. Sterile Petri dishes were placed on a strictly horizontal surface and a nutrient medium consisting of peptone (10 g/L), yeast extract (5 g/L), glucose (5 g/L), CaCO3 (5 g/L), microbiological agar (10 g/L) of 20 ml was poured into them to create an optimal layer thickness equal to 4 ‐ 5 mm. A thick layer of agar was sown with 50 µL of Cms bacterial suspension for 1 day and swabbed with a spatula until the microorganisms were evenly distributed over the entire surface of the Petri dish. Excess suspension was completely removed and dried for 30 min. Then holes (d = 6 mm) were drilled ("wells") at a distance of 2.5 cm from the centre of the Petri dish and at equal distances from each other, which were then filled with the objects under study ). Then, the Petri dishes were placed in the thermostat at 37°C without turning them over, strictly horizontally to form round areas. The results were evaluated after 5 days by the diameter of growth delay areas around the 'well', including the diameter of the 'well' itself. In some cases, oppressed areas were oval. In such cases, we were measured the largest and smallest zone diameters and calculated the average. The absence of delay of microbial growth around the 'well' indicates that this microorganism is not sensitive to testing drug. The experiment was carried out in 3‐fold reproducibilityIn addition, for the obtained nanocomposite Starch/S33.10NPs was carried out in an aqueous starch solution via mild oxidation  of sulphide ions pre‐generated by the reduction of powder elemental sulphur in the base‐reduction system ‘NaOH‐N2H4·H2O’ in an inert atmosphere. Sulphur atoms formed by the oxidation of sulphide‐anions coalesce with each other to afford S0NPs, which pass through all the main stages of nucleation, growth and maturation [Synthesis of Sturation . The sta2− ratio. It was found that the reduction reaction of elemental sulphur without alkali leads to formation of H2S. After introduction of S2−ions into the aqueous starch solution due to their low dissociation ability (pKa = 6.89), they are partially eliminated from reaction medium or oxidised with the formation of sulphur atoms that rapidly aggregate with each other without stabilisation, followed by sulphur sedimentation. This hinders the control of nanomorphological and structural characteristics of the resulting nanocomposites.The sulphur content in the nanocomposites varied between 0.57% and 1.6% by changing the starch/S2S and NaHS. Sodium hydrosulphide dissociates more easily (pKa = 12.91) than H2S and therefore dissolves in the reaction medium providing contact of the sulphide‐anions with dissolved oxygen acting as their oxidant. These features ensure favourable conditions for the gradual, controlled formation of elemental sulphur atoms, their coalescence in the nuclei of a new phase, growth and maturation and, finally, the generation of starch‐capped S0NPs.The use of alkali, in this case, is necessary for the directed production of sodium sulphide, which is easily hydrolysed in the starch aqueous solution to HAccording to the data of transmission electron microscopy, the nanocomposites are formed as sulphur nanoparticles dispersed in the starch matrix, whose shape is near to spherical Figure . Their a2−/Starch" ratio is accompanied by increase of average size of the nanoparticles and their polydispersity. So, the increase of quantitative S0 content in the starch matrix from 0.57% to 1.58% augments the average size of the nanoparticles from 19 to 35 nm, respectively. Due to the fact that the concentration of the formed nuclei of new phase in the volume of the reaction medium is constant, the increase in concentration of sulphur precursor at constant concentration of a stabilizer promotes the gradual growth of the formed nuclei and maturation of nanoparticles with their increasing size. The enhancement of the ionic strength of the solution due to addition of Na+ ions into the reaction medium and the alteration of pH value change its rheological characteristics. Starch macromolecules reshape their conformation in the presence of higher amounts of sodium ions [2‐‐2ē→S0) to the surface of growing particle. Appearance and upsurge of this kinetic barrier at a higher viscosity of the reaction medium increase the stochastic growth of the nanoparticles and, correspondingly, augment their polydispersion degree.The stabilising potential of starch is determined by the presence of hydroxyl groups in its macromolecules. Presumably, starch hydroxyl groups interact (by means of the formation of intermolecular bonds) with uncompensated sulphur states having a charge on the surface of nanoparticles . This provides surface passivation and prevents further aggregation of the nanoparticles. It has been found that the increase of "Sium ions . This in0 [0  of 13.5 and 85.0 nm in a ratio of 1:3 Figure .0NPs to the starch composition leads to a decrease in percentage of a fast fraction (0.219) and a small increase in Rh particles to 16.0 nm, whereas the content of slow particle fraction with Rh 90.0 nm increases to 0.780. The observed increase in the percentage of a slow fraction is probably due to the preservation of globular structure of starch granules owing to the absence of alkaline or thermal degradation of starch granules in the process of nanocomposite synthesis. At the same time, the introduction of 1.58% S0NPs into the starch composition decreases Rh values to 9.5 and 43.4 nm for the fast and slow fraction, respectively, and their ratio being 0.125:0.876. Presumably, such a decrease of Rh particles in the solution of nanocomposite is caused by alkaline degradation of starch in the process of sulphur nanocomposite synthesis due to the introduction of previously synthesised sulphide anions as Na2S into the reaction medium. Hydrolysis of sodium sulphide augments pH of the reaction medium and initiates the alkaline degradation of starch granules with amylose release and depolymerisation of its macromolecules [0NPs, whose surface is covered by amylose or amylopectin macromolecules. Taking into account the size of S0NPs (9‐26 nm)  starch grains correspond to a slow fraction of particles with Rh 85.0 nm. Aqueous solutions of the nanocomposites are also characterised by the presence of two particle fractions, but the average size of Rh, as well as the ratio of these fractions significantly changes. Thus, addition of 0.57% Solecules . Besides) Figure  determin0‐containing nanocomposites was evaluated by determining the ζ‐potential of their aqueous solutions. At the same time, ζ‐potential was established by the method of electrophoretic light scattering under the conditions of combination of two measurement modes: PALS  and ELS (electrophoretic light scattering). In the ELS mode, the autocorrelation function of the Doppler signal has a characteristic type of attenuation cosine μEv‐velocities of charged particles in an electric field with voltage E.Electrophoretical mobility of the particles is converted into ζ ‐potential using the Smolukhovsky's theory and application of corrections for different thicknesses of the double electric layer Eq .(2)μEζ ‐potential.μE‐electrophoretic mobility.ε‐dielectric permittivity.η‐viscosity.0). The obtained values indicate that nanocomposites are sufficiently stable.It is found that ζ‐potential of the initial aqueous solution of starch and all nanocomposites obtained based on it varies from −9 mV  to −20.8 mV (for the nanocomposite with 1.58% S3.23.2.1Cms (Strain Ac 1405) on their interaction with starch‐stabilised S0NPs has been evaluated. The study was carried out by means of light and fluorescent microscopy for microscopic analysis of bacterial cells after colouring them with propidium iodide. At the same time, the fluorescent staining by this dye is typical only for dead cells of Cms due to the free passage of propidium iodide molecules through their damaged plasmatic membrane and its interaction with the nitrogen bases of intracellular nucleic acids [Cms cells in the field of view allows estimating the degree of anti‐phytopathogenic action of the investigated nanocomposite. It is revealed that nanocomposite Starch/S0NPs has an inhibiting effect on the viability of Cms cells, causing their death, morphology changes, and partial incystation. Figure Cms culture (a), and Cms cultivated together within 24 h with Starch/S0NPs nanocomposite  increase in Cms dead cells, while the exposition of Cms with a solution of Starch/S0NPs nanocomposite results in a massive death of Cms cells, up to 25% after their 24‐h co‐incubation. The image of Cms cultures obtained by means of fluorescence microscopy  can be considered as a supposed mechanism of anti‐microbial activity of S0NPs. The result of this interaction may be a red‐ox reaction of available negatively charged S2− ions on the nanoparticles’ surface with organic compounds in the cultural medium, with subsequent formation of either sulphides  or thiocarboxylic acids. Involvement of these sulphur‐containing compounds in cell metabolism of the Cms bacteria and their interaction with the surface of the bacterial cell can lead to inactivation of a number of enzymes, components of cell membranes, damage to DNA, increased oxidative stress and finally a programed cell death [The ability of sulphur  to interact with environmental components  of tools to control the potato ring rot and other agricultural plant diseases caused by the Cms bacteria.Thus, an available, environmentally friendly method has been developed for the synthesis of water‐soluble aggregate‐stable sulphur nanoparticles capped by starch. The resulting starch‐capped sulphur nanoparticles are characterised by high stability  and expressed anti‐phytopathogenic action against


The use of unmodified starch in frozen foods can cause extremely undesirable textural changes after the freeze-thaw process. In this study, using cyclodextrin glucanotransferase (CGTase) and branching enzymes, an amylopectin cluster with high freeze-thaw stability was produced, and was named CBAC. It was found to have a water solubility seven times higher, and a molecular weight 77 times lower, than corn starch. According to the results of a differential scanning calorimetry (DSC) analysis, dough containing 5% CBAC lost 19% less water than a control dough after three freeze-thaw cycles. During storage for 7 days at 4 °C, bread produced using CBAC-treated dough exhibited a 14% smaller retrogradation peak and 37% less hardness than a control dough, suggesting that CBAC could be a potential candidate for clean label starch, providing high-level food stability under repeated freeze-thaw conditions. Bread is one of the most widely consumed foods in the world and baking is one of the oldest known cooking technologies ,2,3. NovDough can be damaged during frozen storage due to ice crystal formation ,15,16. IVarious additives have been suggested to improve the quality of frozen dough ,15,19,20Starch is commonly used in various food products . HoweverIn this study, we hypothesized that the form of modified starch with many short branches could represent a material with the potential to reduce the release of water molecules from dough, thus helping to maintain the quality of the dough during freeze-thaw storage. Therefore, we prepared an enzymatically modified dough and investigated its applicability to bread baking.w/v) was prepared in 50 mM sodium acetate buffer . After preheating at 60 °C for 5 min, the starch solution was incubated while stirring with cyclodextrin glucanotransferase  and the branching enzyme (0.000001872 BEU/mg substrate) at 60 °C for a specific time, to produce CGTase and branching enzyme-treated corn starch (CBAC). Cyclodextrin glucanotransferase  and a branching enzyme (Branzyme) were purchased from Novozymes  and Isoamylase derived from Pseudomonas sp. was purchased from Megazyme . One KNU-CP of CGTase was defined as the amount of enzyme that breaks down 5.26 g starch per hour according to Novozyme’s standard method for determining alpha-amylase. One BEU was defined as the quantity of enzyme that causes a decrease in absorbance at 660 nm of an amylose-iodine complex of 1% per minute under standard conditions . After the enzyme reaction, enzyme mixtures were boiled for 20 min to halt enzyme activity.A corn starch suspension . The precipitates were suspended in distilled water with the same relative ratio as the CBAC starch solution, and then boiled for 5 min to ensure complete dissolution. The samples were frozen at −40 °C before freeze-drying using an Operon instrument .The liquid form of CBAC starch prepared using the above method was lyophilized to attain a powder form. First, the oligosaccharides produced from the starch during the enzymatic treatment were removed using ethanol. The CBAC was precipitated using two volumes of ethanol at −25 °C and collected by centrifugation (5500× w/v) was prepared using distilled water. The starch was left in distilled water for 30 min to hydrate sufficiently. Dimethyl sulfoxide (DMSO) was then added to the starch suspension to achieve a 1% (w/v) concentration. After boiling for 1 h at 100 °C, the mixture was stirred overnight at room temperature. The sample and ethanol were mixed at a ratio of 1:6 and the mixture was centrifuged to remove the supernatant. The precipitate was dispersed in boiling water and boiled for 30 min with continuous stirring. Finally, the starch solution (2 mg/mL) was filtered for analysis using a 5-μm syringe filter . The weight-average molecular weight (Mw) and Mw distribution curves were determined using a size exclusion chromatography–multiangle laser light scattering-refractive index detector (SEC-MALLS-RI). The SEC separation was performed with a high-performance size exclusion chromatograph  including a degasser, autosampler, pump , guard column , and SEC column , which were connected to a MALLS detector  and a refractive index detector . The column was kept at 55 °C. The flow rate of the mobile phase (water) was 0.6 mL/min. The calculation of Mw was performed using Astra 472 software (Wyatt Technology) with the Berry extrapolation method and a dn/dc value of 0.185 mL/g.Starch suspension  operated at a wavelength of 550 nm. The standard curve covered a concentration range of 0.1%–0.5% (w/v). The results are expressed as the mean and standard deviation (SD) of three measurements per sample.The CBAC powder was mixed with distilled water and supersaturated. The suspensions were boiled for 5 min at 100 °C. The samples were then cooled to 20 °C and the supernatant was collected via centrifugation at 11,200× w/w) and a CBAC concentration of 5% (w/w), respectively. After that, the ingredients for the dough were kneaded for 30 min and the first fermentation proceeded for 120 min at 30 °C. After the first fermentation, the dough was sealed inside a polyethylene bag, frozen-stored for 22 h at −25 °C, and thawed for 2 h at 30 °C. This cycle was repeated three times. After the three freeze-thaw cycles, the dough was fermented again for 100 min at 30 °C and the bread was baked for 40 min at 190 °C. The bread was left to cool for 3 h and then sealed with a polyethylene bag and stored in a refrigerator at 4 °C.The dough was prepared for a white bread mix  and a bread maker  was used. The control dough was prepared using only 376 g of wheat flour, 4 g of yeast, and 220 mL of water. The CLS  group and CBAC group doughs were treated with modified starches to achieve a CLS concentration of 10% . The changes in the textural properties of each bread loaf were analyzed after 7 days of storage at 4 °C. The breads were measured by cutting four 20-mm-thick slices. Twice-repeated compression tests were conducted on the bread slices using a texture profile analyzer equipped with a 25 N load cell. The bread slices were compressed to a thickness of 12 mm (60%) with a 50-mm probe at a speed of 60 mm/min. The crumb firmness and elastic recovery were calculated based on the force-distance curves generated by two cycles of compression. The results are expressed as the mean and SD values of three measurements per bread slice.Bread staling was determined using a DSC 214 Polyma system  after 7 days of storage at 4 °C. The DSC was evaluated by making measurements directly on breadcrumbs without further pretreatment. Breadcrumbs were obtained from the center of the loaves after the aging process. The bread samples (10 mg) were weighed and sealed in aluminum pans. An empty pan was used as a reference. The pans were heated from 20 to 90 °C at a heating rate of 5 °C/min. Retrogradation was determined as the enthalpy, calculated based on the area under the endothermic peak located between 40 and 70 °C.The freezable water in the dough was monitored with the Polyma DSC system using a previously reported method ,9. Five p < 0.05 taken to indicate statistical significance.All measurements were repeated at least three times for each sample. The Kruskal–Wallis H test was used to test for significant differences among samples. All analyses were performed using SPSS software  with The structural characteristics of freeze-thaw stable starch were analyzed via HPAEC. Compared to the corn starch, in CBAC the relative amounts of side chains below DP 10 and in the range of DP 28 to DP 47 increased. Consequently, the relative amounts of the other chain lengths in CBAC  decreased . Both CBIn w of starch was determined according to the enzyme reaction time using the SEC-MALLS-RI system. The Mw of corn starch before the enzyme reaction was 1.62 × 108 Da. After the CGTase and branching enzyme treatment, the Mw of starch decreased to 2.09 × 106 Da. After a 1-h reaction, the Mw remained constant . However, there was no significant difference in volume between the bread made with CLS and the control bread.To investigate the effect of modified starch on freeze-thaw stability, CLS and CBAC were added to bread dough, respectively. The fermented doughs were subjected to the freeze-thaw process three times, as described in the Material and Methods, and the bread was then baked. As shown in In the same bread loaves, textural changes were measured using TPA after storage for 7 days at 4 °C. The differences in the bread samples due to the type of starch used are shown in The retrogradation rate of the bread was also analyzed using DSC . The degDSC can be used to study the influence of freeze-thaw treatments on the thermodynamic properties of dough and bread ,7,9,15. w distributions of modified starches  with cyclodextrin glucanotransferase (CGTase) and branching enzymes. It showed a water solubility that was seven times higher and a Matically .