Delayed onset of muscle soreness (DOMS) and its physiological consequences influenced an individual's adherence to an exercise routine.  This study aimed to evaluate the efficacy, safety, and tolerability of TurmXTRA® 60N (WDTE60N) on DOMS compared to placebo in recreationally active healthy subjects. n = 15) or placebo . Study treatments were initiated 29 days before the eccentric exercise and were continued for 4 days after the exercise. The primary endpoint was the change in pain intensity measured by the visual analog scale (VAS) at the end of study treatment (at 96 hours after eccentric exercise) from baseline (measured immediately after exercise).  This randomized, double-blind, placebo-controlled parallel-group study enrolled 30 healthy and recreationally active subjects (average age: 28.23 ± 4.20 years) and randomized them to receive WDTE60N . Well-being status was assessed using the adapted version of the Hooper and MacKinnon questionnaire and was calculated as individual and cumulative scores of the domains  that demonstrated improvement in all domains and in overall well-being in the WDTE60N group compared to the placebo group (p < 0.0001). Serum lactate dehydrogenase (LDH) was significantly lower in the WDTE60N group compared to the placebo group .  The VAS score indicated that subjects from the WDTE60N group reported significantly less pain after eccentric exercise compared to the placebo group (AUC Intake of WDTE60N before and after eccentric exercise significantly reduced subjective perception of muscle soreness and serum LDH activity and increased the psychological well-being in recreationally active subjects. Delayed onset muscle soreness (DOMS) is a type of ultrastructural muscle damage, that is, caused by high-intensity eccentric muscle contractions or unaccustomed forms of exercise . The symƘB) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways that ultimately reduce pain sensitivity and DOMS [Curcumin is a natural polyphenolic substance that constitutes 77% of the total curcuminoids present in the turmeric rhizome. It gives a distinctive yellow color to the spice turmeric. Owing to its numerous physiological effects, since ancient times, it has been involved in traditional Indian and Chinese medicine. Evidence demonstrates its beneficial effects in treating cancer, cardiovascular health, arthritis, and Alzheimer's dementia indicates its anticarcinogenic, anti-inflammatory, antioxidant, and neuroprotective properties . Curcumiand DOMS . It is hand DOMS .p < 0.0001). Furthermore, the results for the time taken for 80 m fast-paced walk test and 9-step stair-climb test with WDTE60N intake were also satisfactory.One of the factors that limit its widespread use is—its poor oral bioavailability, however, this can be overcome by administering very large doses (approximately 6–8 g) of turmeric powder or 1500–2000 mg of turmeric extract standardized to 95% for gaining optimal effects. However, long-term administration of such high doses of curcumin is discouraged as they can cause gastrointestinal side effects  and poor compliance . TurmXTRThus, this randomized controlled trial was designed to assess the efficacy, safety, and tolerability of TurmXTRA® 60N (WDTE60N) compared to placebo on DOMS in recreationally active subjects. We hypothesized that consistent use of a single daily dose of 250 mg TurmXTRA® 60N during pre- and post-period of eccentric exercise reduces DOMS and muscle damage.CTRI/2021/12/038892). The study is reported in accordance with the CONSORT 2010 reporting guideline . The study was conducted following the pertinent requirements of the Declaration of Helsinki , Good Clinical Practices for Clinical Research in India 2005, New Drugs and Clinical Trials Rules 2019, ICH E6 (R2), Guidance on Good Clinical Practice, and with ICMR's National Ethical Guidelines for Biomedical and Health Research Involving Human Participants-2017. All subjects were thoroughly informed about the study before their enrollment, and before obtaining their signed informed consent forms. This study was registered with the Clinical Trial Registry of India  Healthy, moderately active (regular aerobic exercise for at least 150 min per week for past 3 months), nonsmoker subjects without any known musculoskeletal pathology; (3) Subjects vaccinated (two doses) for Coronavirus disease 2019 (COVID-19); (4) Female subjects of childbearing potential were included only if they agreed to use a medically acceptable form of birth control; (5) Subjects willing to sign the informed consent and comply with the study procedure; (6) Subjects willing to abstain from using analgesics/nonsteroidal anti-inflammatory drugs during the treatment period; and (7) Subjects, whose screening test results were within the normal range or were not considered clinically significant by the principal investigator. However, the subjects were excluded from the study if female subjects were pregnant, breastfeeding, or were planning to get pregnant; subjects had COVID-19 symptoms; subject had known allergy to any compound present in the study product; subject was regularly involved in strength training; subjects had a history or frequently uses over-the-counter nonsteroidal anti-inflammatory drugs and/or analgesic agents; subjects with high alcohol intake (>2 standard drinks per day) or use recreational drugs , or have nicotine/caffeine dependence; subjects with a history/presence of chronic illness including but not limited to psychiatric , cardiovascular, pulmonary, neurological, gastrointestinal, hepatic, renal, metabolic, and ophthalmologic disorders, or with human immunodeficiency virus infection and acquired immune deficiency syndrome, or those taking regular prescription pharmacological agents; subjects who participated in any other trials involving investigational or marketed products within 30 days prior to the screening visit; subjects who underwent surgery or trauma affecting the knee in the past; subjects with a history or presence of any neurological or muscular disorders that may affect muscle strength; and subjects regularly taking multivitamins/herbal and/or turmeric supplements/any other wellness product.The present study was conducted on 30 healthy and recreationally active individuals. The subjects were included if they met the following inclusion criteria: (1) Male and female subjects (ages 18–35 years) had a body mass index of 18–30 kg/mn = 15) or the placebo group (n = 15) using the block randomization on day 1. The randomization schedule was generated using SAS version 9.4  by an independent statistician and it was made available to the study personnel responsible for packaging, labeling, and blinding the investigational product. The subjects were enrolled by the study investigator. Blinding was performed via a computer-generated process. The study team statistician was responsible for the blinding process. The randomization numbers were preprinted as stickers and were affixed on every bottle, and this was the only way to identify one kit from the other. Investigator assigned treatment kits to the enrolled subjects as per the randomization schedule. The subjects were instructed to administer the study treatment (either a capsule containing 250 mg water-dispersible turmeric extract ((WDTE60N) or placebo) once daily, orally with adequate water after breakfast. The subjects were required to administer the study products for 33 days (a preexercise period of 29 days and a postexercise period of 4 days). They were also asked to maintain a subject diary that would include all the details regarding their daily intake of study products and concomitant medication (if any). Dosing compliance was assessed by reviewing each subject diary at every visit.The study was conducted at the Arogyavardhini Ayurvedic Center in Bangalore, Karnataka, India. All subjects were screened and evaluated 7 days before administering study treatments. Screening evaluation included anthropometry measurements, vital sign assessment, physical examination, medical and medication history , urine pregnancy test for female subjects with childbearing potential, electrocardiogram, laboratory investigations for liver function, renal function, hematology, fasting blood glucose, and human immunodeficiency virus infection. All subjects were given a COVID-19 screening questionnaire and those, who fulfilled all the eligibility criteria were randomly allocated to two treatment groups, the WDTE60N group (Subjects performed eccentric exercise (squatting) on Day 29. Squatting involves quadriceps muscles to undergo eccentric contractions in the downward phase of the movement. According to eccentric exercise protocol, each subject was required to perform one set of squats (measured as 15 squat cycles) in one minute and they performed this for 15 minutes to complete 225 squat cycles. However, they could stop the exercise if they were unable to continue  or when the investigator felt that the exercise was sufficient to induce DOMS.The primary efficacy endpoint was an improvement in pain intensity from baseline (immediately after eccentric exercise) to the end of the treatment (at 96 hours after eccentric exercise).The pain was assessed by asking the question, “what is the intensity of your current pain?” and the response was recorded on a visual analog scale (VAS) that had a 10 cm line with “no pain” on one end and “worst imaginable pain” on the other end. This was measured immediately before, immediately after (baseline), and at 12, 24, 48, 72, and 96 hours after eccentric exercise.The secondary efficacy endpoints of the study included improvement in the domains of fatigue, sleep quality, general muscle soreness, stress level, mood, and overall well-being from baseline (immediately after exercise) to the end of treatment (at 96 hours after exercise) that were evaluated based on the adapted version of the Hooper and MacKinnon questionnaire. The adapted version was used for ease of administration in the study.Self-reported ratings of well-being may be efficient in monitoring overtraining and recovery. The Hooper and MacKinnon questionnaire quantified and qualified the well-being relative to fatigue, sleep quality, general muscle soreness, stress, and mood . This quIn the present study, the well-being of subjects was assessed using the adapted version of the Hooper and MacKinnon questionnaire immediately before, immediately after (baseline), and at 12, 24, 48, 72, and 96 hours after eccentric exercise.Changes in serum creatine kinase (CK) and lactate dehydrogenase (LDH) from baseline (immediately before eccentric exercise) to the end of the treatment (at 96 hours after eccentric exercise), and the muscle damage markers (serum CK and serum LDH activity) were measured immediately before (baseline), and at 24, 48, 72, and 96 hours after eccentric exercise.The subject's and physician's global assessment of therapy was assessed at the end of treatment (at 96 hours after eccentric exercise) .The safety assessment of the study products was based on the adverse events reported by changes in vital parameters, laboratory investigations , and the general well-being of subjects.This was an exploratory study and the sample size was calculated based on the published literature . Considet-test was used for intra-treatment group comparison depending on the normality distribution of continuous. The study involves efficacy evaluation of the test product in DOMS, a condition in which the pathogenesis is known to start-attain peak-and decline over a period of time after performing an unaccustomed eccentric muscular activity, hence the assessment of product efficacy cannot be significantly established at any single time point after eccentric exercise. Therefore, the area under the curve (AUC) was utilized to describe the span of the efficacy parameters over time after the DOMS induction. A p value of <0.05 was considered statistically significant. Statistical analysis was performed using SAS version 9.4 .Subjects were included in the per-protocol population if they completed their study treatment without any major protocol deviations and had treatment compliance of at least ≥80%. The results were presented as mean ± standard deviation for continuous variables and counts and percentages for categorical variables. Kolmogorov–Smirnov test was performed to assess the normality distribution of the data. The unpaired n = 15) or the placebo group (n = 15). All subjects completed the study  were screened, enrolled, and randomly allocated to either the WDTE60N group  when evaluated immediately before exercise.p=0.0320), 24 hours , 48 hours , 72 hours  and 96 hours  after exercise (0–96h) was significantly lower in the WDTE60N group compared to the placebo group  . The VAS score was significantly lowered in the WDTE60N group compared to the placebo group at 12 hours   . Howeverp=0.0025) and 96 hours  after the exercise in the WDTE60N group compared to the placebo group.The fatigue score was significantly higher at 72 hours , 48 hours , 72 hours  and 96 hours  after exercise.The WDTE60N group reported significantly higher scores at 24 hours , 72 hours , and 96 hours  after exercise in the WDTE60N group compared to the placebo group.There was a significantly higher general muscle soreness score at 48 hours , 48 hours , 72 hours , and 96 hours  after exercise.The scores for stress levels were significantly higher in the WDTE60N group compared to the placebo group at 24 hours , and 72 hours  after exercise.Compared to the placebo group, the WDTE60N group showed significantly higher mood scores at 48 hours  and 72 hours  and 96 hours  after exercise.The overall well-being scores were significantly higher at 48 hours . Serum CK concentrations peaked at 48 hours (152.9 ± 52.4 IU/L) but decreased at 72 hours (125.9 ± 41.2 IU/L) and 96 hours (138 ± 53.6 IU/L) after eccentric exercise in the WDTE60N group. Contrastingly, in the placebo group, there was a decrease in serum CK concentration initially at 24 hours and 48 hours followed by an increase at 72 hours and 96 hours after exercise. A decrease in the mean AUC0–96h serum CK was observed in the WDTE60N group compared to the placebo group. However, the difference between both treatment groups at different time points and in AUCs was statistically insignificant.There was no statistically significant difference in the preexercise serum CK concentration between the WDTE60N group and the placebo group  and 96 hours  compared to those in the placebo group. A statistically significant decrease in the mean AUC0–96h serum LDH concentration was observed in the WDTE60N group compared to the placebo group  . Similarly, the physician's global assessment of therapy showed significantly higher scores for the WDTE60N group compared to the placebo group .The subject's global assessment of therapy in both WDTE60N and placebo groups was 4.00 ± 0.0 and 2.00 ± 0.0, respectively  in subjects receiving WDTE60N than placebo; significantly decreased serum LDH concentrations but not serum CK concentration with consistent WDTE60N supplementation before and after eccentric exercise; and significantly higher ratings for WDTE60N in the subject's and physician's global assessment of therapy. Furthermore, the study treatments were well tolerated by all subjects, and no serious adverse events were reported during the study period.p < 0.05) within 12 hours of eccentric exercise and indicated faster recovery. Similarly, an assessment of the well-being status using the adapted version of the Hooper and MacKinnon questionnaire confirmed less muscle soreness in subjects receiving WDTE60N (p < 0.05). Moreover, overall well-being and other aspects such as fatigue, mood, sleep quality, and stress were also significantly improved after exercise in subjects, who received WDTE60N than those, who were administered a placebo. The impact of WDTE60N on stress levels was as per a previous study. Sciberras et al. reported a higher number of subjects with “better than usual” scores in the subjective assessment of psychological stress during the training day when subjects received curcumin supplementation before training [Recently, phytotherapy has received attention in medicine globally. Considering their tremendous therapeutic benefits and a wide margin of safety, herbal products have been increasingly used by different diseased and healthy populations –26. Sevetraining . Furthertraining . Thus, tp > 0.05). Results from several studies have warranted the need to evaluate the effect of curcumin supplements on muscle damage markers to determine the correlation between frequent/higher dosing with a decrease in muscle damage markers. A study evaluated the effects of a curcumin and piperine combination on the recovery kinetics after exercise-induced muscle damage and reported no effect on serum CK concentration [Serum CK concentration increased after eccentric exercise as it is well distributed in the muscle tissue and is released into the circulation according to the loss of sarcolemmal integrity as a result of the mechanical stress of eccentric exercise or metabolic causes (glycogen depletion). Similarly, increased serum LDH concentrations indicate cell damage as LDH present in the cytoplasm catalyzes the reversible process of pyruvate to lactate under anaerobic conditions. Thus, an increase in serum CK or LDH activities are indirect markers for muscle damage. In this study, serum CK and LDH concentrations increased and attained the highest levels at 48 hours after exercise in both treatment groups indicating that DOMS was a clinical condition with peak clinical implications within 72 hours of eccentric exercise. Serum LDH concentration significantly lowered in the WDTE60N group compared to the placebo group at 72 and 96 hours after exercise. Similarly, serum CK activity was also reduced in subjects, who received WDTE60N compared to placebo  and decreases lactate accumulation, which is demonstrated by significantly lowered serum LDH activity without any major safety concern in recreationally active subjects. However, the effect of WDTE60N administration on serum CK levels needs to be investigated in a larger population.


A study cohort of three patients with PD, 29 with atypical parkinsonism , and a control group of 25 patients with normal striatal metabolism is available. Standardized uptake value ratios (SUVR) are extracted from the striatum, and the caudate and the putamen separately. SUVRs are compared among the study groups. In addition, hemispherical and caudate-putamen differences are evaluated in atypical parkinsonisms. Striatal hypermetabolism is detected in patients with PD, while atypical parkinsonisms show hypometabolism, compared to the control group. Hemispherical differences are observed in CBD, MSA and DLB, with the latter also showing statistically significant caudate–putamen asymmetry (p = 0.018). These results indicate disease-specific metabolic uptake patterns in the striatum that can support the differential diagnosis.Neurodegenerative parkinsonisms affect mainly cognitive and motor functions and are syndromes of overlapping symptoms and clinical manifestations such as tremor, rigidness, and bradykinesia. These include idiopathic Parkinson’s disease (PD) and the atypical parkinsonisms, namely progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), multiple system atrophy (MSA) and dementia with Lewy body (DLB). Differences in the striatal metabolism among these syndromes are evaluated using [ Neurodegenerative diseases are a group of diseases characterized by the progressive degeneration or death of neurons and are a major cause of mortality and morbidity. Distinctions can be made at a molecular level and the associated syndromes present different clinical manifestations with characteristic symptoms and affect specific cognitive or motor functions . However123I]FP-CIT SPECT (DaTSCAN) or [18F]fluorodopa PET imaging has shown clinical use for discriminating neurodegenerative parkinsonisms from other types such as vascular or drug-induced parkinsonism, or essential tremor [123I]IBZM SPECT, and lately [18F]fluorodeoxyglucose ([18F]FDG) PET imaging), where PD shows increased and atypical parkinsonism decreased uptake FDG PET imaging is one of the main tools used in the evaluation of neurodegenerative diseases, especially for the differential diagnosis of Alzheimer’s disease (AD) [18F]FDG PET has shown to be useful and with distinct patterns FDG PET imaging for the differential diagnosis of PD and atypical parkinsonism.In this paper, the metabolic activity of the striatum in patients with PD and atypical parkinsonism is studied. The overall activity compared to a control group is evaluated and patterns of intrastriatal differences in atypical parkinsonisms are analyzed. Currently, metabolic imaging is used in clinical routine practice for the differential diagnosis based on cortical uptake values, but the striatum is not interpreted. The aim of the study was to evaluate the value of semi-quantitative analysis of the striatum in [18F]FDG PET/CT scan at the Department of Nuclear Medicine of the Hospital Universitario 12 de Octubre, Madrid, Spain, between 2015 and 2021 were eligible for the study cohort. Patients with potential diagnoses of PD and atypical parkinsonism ) in neurological and nuclear medicine reports were included. All reports were reviewed by experienced specialists in neurology and nuclear medicine to confirm the diagnoses or exclude the patients. PD patients with visually and Z-score-based normalized striatal metabolism in [18F]FDG PET images were excluded.Patients that underwent a [18F]FDG PET scans, (3) [18F]FDG PET scans with at least one cortical region presenting hypometabolism defined as a Z-score below −2, and (4) [18F]FDG PET scans with cortical regions presenting a positive or very high Z-score. Z-scores were obtained using the CortexID Suite .Additionally, a control group of patients with normal striatal metabolism was defined from patients that underwent amyloid PET/CT imaging between 2014 and 2021 at the Department of Nuclear Medicine of the Hospital Universitario 12 de Octubre, Madrid, Spain. The following exclusion criteria were defined: (1) cognitive stages different than Mild Cognitive Impairment (MCI) and without any movement disorder symptoms, (2) patients without [18F]FDG injection, patients underwent a 6 h fasting period, and were advised to drink water and urinate before image acquisition. Images were acquired with a Biograph 6 True Point PET/CT scanner  or with a Discovery MI DR PET/CT scanner , with both scanners being equivalently calibrated. A mean dose of 207 ± 59 MBq of [18F]FDG was injected intravenously. Images were acquired between 20 and 81 min after radiotracer injection and scan duration was 30 min. Reconstructed images had a matrix size of 336 × 336 × 55 and voxel size of 1.01821 × 1.01821 × 3 mm3.Prior to [18F]FDG PET images are preprocessed using SPM12 (http://www.fil.ion.ucl.ac.uk/spm/ (accessed on 1 December 2022)) and segmentation and quantification is performed in MATLAB  ..18F]FDG 18F]FDG uptake among PD, atypical parkinsonisms and patients with visually normal striatal uptake, as well as among the specific atypical parkinsonisms, are evaluated by one-way ANOVA or Kruskal–Wallis tests and post hoc pairwise comparisons with Bonferroni adjusted α values. Additionally, hemispherical differences of metabolic activity in the caudate and putamen, as well as differences between these two regions per hemisphere, are analyzed by paired t-tests or Wilcoxon signed rank tests for each atypical parkinsonism-specific group. Lastly, multinomial logistic regression is used based on caudate and putamen SUVRs to model the diagnosis of PD, atypical parkinsonism, and control, as well as the diagnosis of the different types of atypical parkinsonism. Statistical analysis is performed using SPSS software version 26.00 , and p-values < 0.05 are considered statistically significant.Quantitative variables are represented as mean ± standard deviation (SD). The normality of the distributions is tested using the Shapiro–Wilk test. Differences in striatal [18F]FDG PET images, a total of eight patients were removed. Three of these eight patients were transferred to the control group due to no diagnosis of a neurodegenerative disease, and five were excluded due to diagnoses of other diseases like AD, aphasia, or primary lateral sclerosis. In addition, five patients diagnosed with PD with normalized striatal metabolism due to medication at the time of the image acquisition were excluded. Finally, the study cohort consisted of 32 patients, 3 with PD and 29 with atypical parkinsonism. Specifically, 10 patients with PSP, 6 with CBD, 2 with MSA, 7 with DLB and 4 NC were included. Regarding the control group, a total of 25 patients were included after applying the previously defined exclusion criteria to an initial database of 175 patients. Patient demographics are summarized in The study cohort consisted initially of a total of 44 patients diagnosed with PD or atypical parkinsonism. After reviewing the medical records and FDG PET has also been shown to better discriminate atypical parkinsonisms than postsynaptic [123I]IBZM SPECT imaging FDG PET scans were available for patients with PD or atypical parkinsonism. In addition, only patients with atypical clinical manifestations, i.e., doubtful cases, were transferred for [18F]FDG PET imaging, resulting in a small study cohort. The control group was also composed not of healthy cognitively normal subjects but patients with normal striatal uptake but who may present cortical metabolic and cognitive alterations. In addition, the patients of the control group were significantly younger than those of the PD and atypical parkinsonism groups (Age (years): 63.84 ± 8.23 vs. 73.28 ± 8.45, p < 0.001), which may be expected due to the diagnoses of MCI in the control group. We also specifically excluded those patients from the control group with any movement disorder symptoms when presenting normal striatal uptake, considering symptomatology to be a more important criterion than age when defining both groups. While age-related effects on metabolic activity and [18F]FDG distribution have been shown in subcortical regions such as the striatum, it is more pronounced in cortical regions and has been observed to be more stable until 60–70 years in some regions, corresponding approximately to the age bracket of our study [18F]FDG PET images of PD patients.Regarding the limitations of this study, the limited number of patients composing the final study group needs to be mentioned. On one hand, only three patients could be included with a diagnosis of PD after excluding those with normalized striatal metabolism after levodopa treatment. In the case of the atypical parkinsonisms, the same occurred for the MSA group. Due to the retrospective nature of the study and the importance of DaT imaging in the diagnosis, only a few FDG PET imaging, PD patients showed hypermetabolism compared to a control group, while hypometabolism was characteristic in atypical parkinsonisms. In addition, some differences in striatal metabolism were found for different types of atypical metabolism. Striatal metabolism was able to accurately predict the differential diagnosis.PD and atypical parkinsonisms are neurodegenerative diseases characterized by alterations to the DaT activity in the striatum. By semi-quantitatively analyzing [


Bacillaceae and Pseudomanadaceae, and two white-rot fungi, Trametes versicolor and Agrocybe aegerita. Results have highlighted that the tannin polymer did not inhibit the growth of tested bacteria and even favored the growth of Bacillaceae without extra glucose. The growth of both fungi was enhanced by mimosa tannin and its polymer at low concentrations (<1%), while concentrations above 10% had a growth-inhibiting effect, which was slightly less strong for the polymer compared to the tannin against Trametes versicolor. These findings highlighted that tannin–furanic polymers can be tolerated by certain microorganisms at low concentration and that their inhibitory effect is similar or slightly lower than that of the pristine tannin extract.Tannins are well-known to protect plants from bacteria and fungi, but nothing is known about its effects on microorganisms once they are copolymerized. Therefore, a study was conducted to evaluate the effect of a tannin–furanic polymer in comparison with industrial mimosa tannin extract on the in vitro growth of two strains of bacteria,  Combined with physical protection mechanisms, superior plants also synthetize specific molecules to protect themselves against biotic and abiotic attack, known as extractives . It is kThe possibility to polymerize bio-molecules to higher molecular mass and high water resistance promoted the tannin extract as a possible sustainable precursor for bio-plastics and, hence, highly attractive for research in material science. In particular, the tannin–furanic polymers were produced by combining the tannin extract with another bio-based resource, furfuryl alcohol, in a polycondensation reaction occurring in an acid environment . The pol−1 K−1) and good fire resistance properties (self-extinguishes)   and 200 μL MgSO4 [1 mol∙L−1]  were added into a flask and filled with water to 100 mL, serving as the minimum medium (MM).For the isolation of bacteria, a phosphate-buffered saline  and 800 mL of a standard medium (SM) of HTrametes versicolor and Agrocybe aegerita collected in local forests and stored in a protected dark environment in the laboratory of the TESAF department of the University of Padua. The reactivation of the mycelia was conducted by pouring them into a water–agar substrate for 4 weeks.For the fungal test, agar from Agar Technical BD Difco was used in a 14 wt% water solution as the substrate for the Petri dishes. The inoculated fungi were 3 standard tannin–furanic foam (TFF), which were kept in a litter and in a compost heap of the Botanical Garden of the University of Marburg for 21 days. After exposure, foam blocks were transferred into 20 mL of phosphate-buffered saline and sonicated for 7 min in order to detach bacteria from the foam. Then, 1, 10, 20 and 40 µL of the resulting suspension was transferred to agar plates containing 20 mL of the standard medium (SM). After 3 days, individual colonies were picked and isolated on a separate agar plate. Two strains from the families Bacillaceae and Pseudomanadaceae were identified: The 16S rRNA gene  was amplified and sequenced (forward and reverse) with two universal primers ALer1_341f (5′-CCT ACG GGA GGC AGC AG-3′) and Buniv_970r (5′-CCG TCA ATT CMT TTG AGT TT -3′) with standard PCR-conditions . After purification with the MSB Spin PCRapace kit , the PCR-products were sent to Eurofins Genomics  for sequencing. Raw sequences of the 16S rRNA genes are available at the NCBI Sequence Read Archive (SRA) under the BioProject accessions SAMN26875395 (Bacillaceae) and SAMN26875394 (Pseudomonadaceae).Blocks of tannin–furanic foams (TFF) were exposed to a compost heap to find bacteria colonies more likely to tolerate or even degrade the TFP. For bacterial growth assays bacterial strains were isolated from blocks of 15 × 15 × 15 mmBacterial growth was investigated in the minimum media (MM) with the addition of 1% MT or TFP containing either non or 0.1% glucose. To these solutions and suspensions bacteria were added at an initial optical density (OD) of OD = 1 and incubated at 36 °C in 96-well plates . Bacterial growth was tracked for 6 days in a plate reader  that measured the OD in each well at an interval of 30 min.A 14% water–agar solution was prepared and sterilized at 120 ± 1 °C for 15 min. While still warm (~40 °C), the water–agar solution was homogeneously distributed applying 10 mL to each of the 144 Petri dishes (d = 75 mm). Then, 30 mL of solutions of mimosa tannin (MT) and suspensions of tannin–furanic polymer (TFP) with water were prepared at different concentrations (wt%): 0.1%; 1%; 10%; 20%.0.5 mL of solution (MT) or suspension (TFP) were applied on each Petri dish containing hardened water–agar and quickly distributed on the surface with a glass spreader. Eight Petri dishes were plated for each formulation. The added solution was dried at room temperature for one day before inoculation. Control Petri dishes were prepared without adding anything else on the 10 mL of water–agar solution.The inoculation was performed by laying a mycelium plug of the fungus 7 mm in diameter in the center of the Petri dishes. Eight repetitions for each fungal strain were conducted for every concentration. Summarizing the design of experiments: two substrates (MT and TFP), four concentrations (and one control), two fungi and eight repetitions for a total of one-hundred and forty-four tests.The whole set of experiments was maintained at room temperature and constantly monitored. Periodic checks of the fungal development were performed by tracking the area of fungal growth after 4, 7, 10, 12, 14, 17, 20, 26 and 32 days. The diameters per each colony were measured along three axes, subtracting the diameter of the original mycelium plug.Statistical analyses on bacterial growth was performed using R Statistical Software, version 3.6.0 . The max−1. All data firstly underwent the Shapiro–Wilk’s Normality Test. The daily growth rates of the fungi, per each additive and concentrations, were then elaborated by non-parametric Kruskal–Wallis (p < 0.05) to evaluate the presence of significant differences among groups, followed by multiple comparisons between groups using Dunn’s Test (p < 0.05).The analyses of the data collected on white-rot fungi growth was performed using R Statistical Software, version 3.6.0. The R/CRAN Packages used were “tidyverse”, “ggpubr” and “rstatix” ,30,31,32The exposure of bacteria and white-rot fungi to mimosa tannin (MT) powder and to grinded tannin–furanic polymer (TFP) allowed to monitor the effect of these substances on the growth of these microorganisms. After tracking the optical density for the different formulations, the carrying capacity k was calculated for comparing microbial growth characteristics.Bacillaceae in the presence of tannin and tannin–furanic powder, with and without extra glucose, is presented as the carrying capacity.In Without glucose, the strain showed a significant increase in growth for the tannin–furanic polymer, comparable to that of glucose alone (more than two-fold). MT and TFP in combination with glucose were not growth inhibitory and even increased significantly for TFP. This bacterium seems to be able to take advantage of TFP with an additional food source and even solely from its polymerized macromolecule, whereas MT did not show any influence on bacterial growth.Pseudomonaceae is reported. Here, different trends are observable. Compared to the control, the presence of MT and TFP does not enhance the growth of bacteria when no glucose is added. Conversely, when glucose is added, the presence of MT and TFP significantly enhances the growth of the Pseudomonaceae strain compared to glucose alone.In Bacillaceae), which seems to be able to exploit the polymer as a carbon source as it does with glucose.The bacterial test showed, that in both species, tannin without glucose resulted in no growth enhancement, whereas the addition of glucose could trigger a synergistic effect, and the bacteria grew better than with glucose alone. Similar observations were made for TFP  of tannin and tannin–furanic polymers tend to allow a faster growth of the mycelium, while at high concentrations (10%), the growth is lower or even completely inhibited. In particular, it can be noticed that the inhibitory effect against T. versicolor, according to its daily growth rate, is reported in The observed effect of MT and TFP after 7 days for the strain Trametes versicolor. Low doses up to 1% have a growth-enhancing effect and show no inhibition.In these graphics, the growth effect for small concentrations of MT a and TFPA. aegerita which grows slowly and could therefore be comparatively monitored until day 17, was slightly different  feeding and (ii) hindering effect.Trametes versicolor and bacteria of the family Pseudomonaceae and Bacillaceae, were reported to degrade condensed tannins or catechin, the basic constituent of condensed tannins [The feeding effect occurs when the microorganism can exploit the molecule to recover energy, for instance, hydrolysable tannin can be reinserted in the citric acid cycle after relatively simple breakdown (aerobic/anaerobic) by tannase enzymes, while condensed tannins, such as the mimosa extract, require different reductive breakdowns, which render them even more recalcitrant ,33. Howe tannins . For the tannins ,35,36,37 tannins .The hindering effect occurs when the presence of tannin inhibits the role of other enzymes of the microorganism. There are several studies that report this observation for many enzymes such as α-amylase, α-glucosidase, glucoamylase, cholinesterase and tyrosinase ,39,40,41Bacillaceae, indicating the ability to use the tannin–furanic polymer as a carbon source. Mimosa tannin does not significantly promote the growth of the bacteria without glucose, but as soon as glucose is added it can contribute to further enhance the carrying capacity supposing the occurrence of a synergic effect in the metabolisms, which is generally known to possibly occur in bacterial growth exposed to multiple carbon sources [The carrying capacity of media was increased by the TFP without glucose for  sources ,45.T. versicolor and A. aegerita grew well when exposed to a limited concentration of MT, confirming the finding of the literature that certain limited doses even enhance fungal growth in certain species of white- and soft-rot fungi [ot fungi . When thot fungi ,48,49.T. versicolor must be emphasized, suggesting that the polymerized tannin has a lower inhibitory effect against this strain than the mimosa tannin. Conversely, A. aegerita does not grow for high concentration of TFP, which means that, for this fungus, the inhibitory effect of tannin also remains relatively unaltered after polymerization.Within the low doses, no significant difference could be found for MT and TFP, but for 10 and 20% TFP, the higher tolerance of The good tolerance or even growth-enhancing properties at low concentrations of MT and TFP for the fungi and bacteria tested increases further interest in the suggested use as an ammonia mitigator in agriculture since studies showed their effectiveness already in very low amounts, where 1 w% of the polymer in 250 mL of 1:1 diluted cattle slurry led to a reduction in gaseous emissions by 60% .Bacillaceae and (iii) similar behavior in Pseudomonaceae and Agrocybe aegerita.The results of this study can be summarized as follows. In comparison to MT, the TFP registers (i) a similar feeding and slightly milder inhibition effect towards tested fungi, (ii) higher bacterial growth in Bacillaceae, which showed enhanced growth towards TFP. Findings in various literature suggest that such types of fungi and bacteria may even metabolize condensed tannins and, thus, TFP, by breaking them down through different oxygenases instead of tannase enzymes, before being reinserted into the citric acid cycle (feeding effect) [Staphylococcus aureus, Escherichia coli.) according to ISO 22196:2011 would, therefore, be particularly interesting. Although the aim of the study was not to evaluate biodegradability, it is worth highlighting the growth of Bacillaceae with TFP, which recorded comparable growth values as the glucose reference sample.At low concentrations, fungi can tolerate tannins (MT) and TFP or even benefit from their presence to grow faster, whereas bacteria were already inhibited without additional glucose, except  effect) . However effect) ,46. In cBacillaceae and Pseudomonaceae and two white-rot fungi (Trametes versicolor and Agrocybe aegerita). Tested bacteria and fungi were selected for the ability to tolerate or eventually degrade condensed tannins. The impact of the tannin content was evaluated for the fungal experiments while the selected concentration (1%) for bacteria gave complementary information to this study. In bacteria, the mimosa tannin, known for its antimicrobial properties, led to higher bacteria growth when it was combined with glucose compared to with glucose alone but did not show any growth enhancement without additional glucose. Aside from the synergistic effect occurring on the bacterial growth through the combination of glucose with MT/TFP, the bacteria of the family Bacillaceae even seems to be able to solely use TFP as a carbon source, as it shows a similar increase in growth as when glucose alone serves as the food source. In the white-rot fungi, MT and TFP enhanced mycelia growth for low concentrations up to 1%, whereas doses of 10% and higher clearly inhibited fungi growth. The influence of TFP on fungi and bacteria was very similar to that of MT, however showing slightly less inhibitory effect on the tested fungi Trametes versicolor and bacteria Bacillaceae. These results suggest that the tannin–furanic polymer loses some of its inhibitory effect on microorganisms compared to the natural mimosa tannin extract, maintaining similar antimicrobial effects on microorganisms as tannin. These results might be particularly interesting in the design of antimicrobial tannin-based bio-plastics or for supporting the use of these materials as ammonia mitigator in agriculture.This research work evaluated the influence of a tannin–furanic polymer compared to mimosa tannin on the in vitro growth of two strains of bacteria of the family


These include shear thinning for cell-friendly extrusion, post-printing structural stability as well as physiologically relevant elastic moduli needed for optimal cell attachment, proliferation, differentiation and tissue maturation. This work introduces a cost-efficient gelatin-methylcellulose based hydrogel whose rheological properties can be independently optimized for optimal printability and tissue engineering. Hydrogel viscosities were designed to present three different temperature regimes: low viscosity for eased cell suspension and printing with minimal shear stress, form fidelity directly after printing and long term structural stability during incubation. Enzymatically crosslinked hydrogel scaffolds with stiffnesses ranging from 5 to 50 kPa were produced, enabling the hydrogel to biomimic cell environments for different types of tissues. The bioink showed high intrinsic cytocompatibility and tissues fabricated by embedding and bioprinting NIH 3T3 fibroblasts showed satisfactory viability. This novel hydrogel uses robust and inexpensive technology, which can be adjusted for implementation in tissue regeneration, e.g., in myocardial or neural tissue engineering. It has the potential to mimic biological tissue closely and replicate natural tissue properties, thereby serving as a model for pharmacological assays ,2. The kTM or fibrin. In this work, we present a bioink based on affordable materials which are available in large quantities, and which feature all the properties needed for successful bioprinting of cell-laden scaffolds. The work is based on a combination of methylcellulose (MC) and gelatin similar to that used in food packaging [Researchers have used a plethora of natural and synthetic polymers and formulations in the attempt to meet the aforementioned criteria of bioinks ready for 3D-bioprinting . Howeverackaging . Since backaging . Conversackaging . CombiniIn addition, gentle and cell-friendly enzymatic crosslinking of gelatin after printing, which is characterized by different stiffnesses, may result in long term shape fidelity  and makeIn this work we study the combination of these two hydrogels to achieve favorable rheological characteristics for extrusion-based printing, with possible versatile implementation in regenerative tissue engineering, e.g., cardiac tissue reconstruction. Furthermore, printability of the developed bioink is characterized, elastic modulus alterations of the printed 3D constructs are examined and the ink is loaded with cells for 3D-bioprinting and long-term cell experiments.w/v gelatin solution and an 8% w/w MC solution mixed in a 2:1 ratio. Additional 6% w/v and 10% w/v gelatin solutions were produced during the optimization process. For the gelatin solutions porcine skin gelatin  was slowly added to prewarmed v/v phenol red  to help adjust the pH later. By adding 1 M NaOH  as well as 5 M NaCl the pH was adjusted to physiological conditions. The final solution was sterilized in a tabletop microwave autoclave , cooled to room temperature (RT) and stored at w/w MC solution. This freshly prepared MC solution was mixed while still at The process of bioink preparation was optimized for later bioprinting and consisted of two main components: a 15% ®-GS, 0.22 μm, Burlington, MA, USA) and produced freshly each time before use. To initiate crosslinking [To stabilize the printed construct mechanically a transglutaminase solution (TG) was prepared. TG solutions with varying concentrations were studied. Therefore, TG  was dissolved in DPBS to yield solutions with concentrations between 60–120 mg/mL, which were sterile filtered . The temperature-dependent complex viscosity and phase shift were assessed by oscillatory measurements at a frequency of 1 Hz. The temperature was varied in steps of The elasticity of inks was measured by nanoindentation  with probes having a spring constant of 0.025 N/m and a tip radius of 10 μm . 1 mL ofv/v fetal bovine serum  and 1% v/v penicillin–streptomycin . The cell medium was changed every second day and cells were used after reaching confluency higher than 80%. The cells were collected by adding 1X Trypsin  to the culture flask and incubated for 1 min. Cells were centrifuged for 7 min at 1000 rpm, the supernatant was discarded and cells were counted after resuspension a in fresh medium. Thereafter, the cells were ready to be used for 3D-bioprinting and cell viability assessment experiments.NIH 3T3 fibroblasts  were cultivated in an incubator at The aforementioned bioink was printed with a RegenHU 3D-printer  placed in a sterile environment. The designed print structure was a one or two layered 2 × 2 cm square meander with 1 mm line spacing. The design was chosen to be ready for printing into a 6-well plate . The optimized print parameters were: (1) printhead/bioink temperature: 3D-bioprinting was carried out following the same steps as for the ink without cells. NIH 3T3 cells were mixed into the ink before it was loaded into the cartridge to yield a 3 million cell per mL final concentration in the bioink. Enough cells were harvested in appropriate medium and centrifuged at 1000 rpm for 7 min. The supernatant was discarded, and the cells were mixed with prewarmed MCG. The cell-laden ink was immediately filled into a cartridge, which was manually turned horizontally and vertically to prevent the cells from sedimenting until the ink reached the printing temperature of Even though printability is a widely used term in BAM, currently there is no general agreement on when a bioink is considered ’ready for printing’ . Hence, lity (I) ,20. In ttographs  and ImagIn order to verify the biocompatibility of our produced bioink, cell viability was checked at different timepoints using a live/dead assay consisting of Hoechst 33342  and propidium iodide (PI) staining  . PrintedLenses of 4× and 10× with two fluorescence channels (Hoechst and PI) and a brightfield channel were used to capture the fluorescence microscopy images. Image stacking (channels and z-stack) was conducted by Fluoview software . Using ImageJ software , cell viability was further evaluated by converting the images to 8 bit gray value format, adjusting brightness and contrast values and threshold values . Cell viNext, the average cell viability of each timepoint was calculated by analyzing five randomly selected fluorescence images at each timepoint.v/v was chosen.The rheological properties of bioinks are crucial for successful extrusion as well as for achieving structural stability of the printed constructs. To reduce shear stress exerted on cells during extrusion, bioinks will typically show shear thinning and liquify during the printing process. Biomaterials with temperature-dependent viscosity can be used to render the ink liquid and further reduce the stress on cells. Gelatin is often used in that context, although it has a sharp viscosity transition between solid and liquid see a,b. The The rheological properties of the methylcellulose-gelatin (MCG) ink are shown in  at 28°C d indicatMechanical properties of the bioink are important for cell function and cell fate since human tissues are characterized by different stiffnesses . Hence, Besides mechanical stability of gelatin and its mixture by TG crosslinking, thermal stability is also reported to be influenced. According to previous work by Liu et al. , crossliBased on the rheological data, 3D-bioprinting with MCG15 was carried out. The best printing results were achieved at possible . Extrudepossible c,d confiPrintability of the developed bioink was analyzed by determining uniformity (U), pore factor  yielded 96% (day 1), 98% (day 3) and 96% (day 5) viability, indicating the high cyto-compatibility of the MCG ink. Meanwhile, another control of solely NIH 3T3 cells seeded on tissue culture plastic (TCP) yielded a 91% (day 1), 95% (day 3) and 89% (day 5) viability.The hydrogel preparation method applied produced MCG bioinks, which showed stable and repeatable rheological behavior immediately after preparation. Repeated shear stress assessments at constant temperatures produced replicable results, independent of the number of repetitions. In contrast, repeated temperature ramps induced a change in rheological behavior, indicating that the phase-separation of gelatin and MC varies with temperature cycles. Such temperature variations may be intentionally applied or can arise when moving the ink from Preliminary results (not shown here) indicate that the preparation protocol strongly influences the spatial phase distribution of gelatin and MC, which ultimately also influences the porosity of the crosslinked gelatin scaffold once the MC is removed. In our work we chose a 2:1 ratio of gelatin: MC in order to achieve an interconnected and structurally stable gelatin network after solidification. To optimize porosity, more investigations of mixing ratio as well as other parameters have to be performed. This is of importance for the precise engineering of the diffusion distance and transfer rate of nutrients to the embedded cells as well as the macro stiffness of the scaffold. Further investigation of these aspects are beyond the scope of this paper, however.Scaffold stiffness is a central parameter in tissue engineering since it influences the differentiation and maturation of cells, e.g., cardiomyocytes differentiate and mature best when embedded in a matrix with E-modulus of around 11 kPa , while nWith the rheological data at hand, optimal printing parameters could be determined: Bigger filament widths and heights compared to the used nozzle can be explained with a certain filament collapse during printing. Further investigations into better filament stabilization needs to be carried out to achieve higher print accuracy. To a certain extent, more precise structures could have been printed (lower line spacing) with the same printing parameters, but, for the existing cell experiments, no higher precision was necessary.As shown in Long-term cell viability analysis showed a high value for early timepoint (1 d) of around 86%, whereas this value dropped after three days to 68% and five days post-printing to 61% or 33%. We suggest that the major impact on viability is the lack of proper nutritional exchange since, as shown in The present study demonstrates the ability to 3D-(bio)print tissue mimicking constructs with widely available and cost-effective components, such as gelatin and MC. The MCG15 bioink displays favorable printing behavior, as demonstrated in the rheological assessment. The gelatin & MC combination widens the printability window by increasing the yield point to more physiological temperatures, while solidifying fast at slightly lower temperatures. Therefore, shear stress as well as temperature stress on the printed cells can be limited, shown by one day post-print cell viability assessment. Printability characterization showed uniform strands, satisfactory structural integrity and square pores. Furthermore, since scaffold stiffness is of paramount importance in cell differentiation and maturation ,29, it w


According to current guidelines, initial burn resuscitation should be performed with fluids alone. The aims of the study were to review the frequency of use of vasoactive and/or inotropic drugs in initial burn resuscitation, and assess the benefits and harms of adding such drugs to fluids.A systematic literature search was conducted in PubMed, Embase, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, UpToDate, and SveMed+ through 3 December 2021. The search included studies on critically ill burn patients receiving vasoactive and/or inotropic drugs in addition to fluids within 48 h after burn injury.The literature search identified 1058 unique publications that were screened for inclusion. After assessing 115 publications in full text, only two retrospective cohort studies were included. One study found that 16 out of 52 (31%) patients received vasopressor(s). Factors associated with vasopressor use were increasing age, burn depth, and % total body surface area (TBSA) burnt. Another study observed that 20 out of 111 (18%) patients received vasopressor(s). Vasopressor use was associated with increasing age, Baux score, and %TBSA burnt in addition to more frequent dialysis treatment and increased mortality. Study quality assessed by the Newcastle‐Ottawa quality assessment scale was considered good in one study, but uncertain due to limited description of methods in the other.This systematic review revealed that there is a lack of evidence regarding the benefits and harms of using vasoactive and/or inotropic drugs in addition to fluids during early resuscitation of patients with major burns. The quantity and quality of studies on the resuscitation of burn‐injured patients are low. Given the concerns about fluid overload and adverse skin effects from vasoactive drugs, clinical trials are urgently warranted.1Burn wounds are dynamic, and both depth and surface area may progress after the initial injury.Initial fluid resuscitation with crystalloids is generally recommended in major burns covering more than 20% total body surface area (TBSA).18Strategies to limit the amount of fluid by the use of colloids, sub‐target resuscitation, or the use of vasoactive and/or inotropic drugs have been proposed.The aims of the study were to review the frequency of use of vasoactive and/or inotropic drugs in initial burn resuscitation, and to assess benefits and harms of adding such drugs to fluids.22.1The purpose of the present study was to perform a systematic review and meta‐analysis of initial resuscitation of burn patients admitted to the intensive care unit (ICU), to identify, evaluate, and summarize the findings of all relevant individual studies on use of vasoactive and/or inotropic drugs.2.2The aims were to review how often vasoactive and/or inotropic drugs  were added to intravenous fluids during initial resuscitation of patients with major burns, and to assess benefits and harms of adding vasoactive and/or inotropic drugs. A PICO  diagram is presented in Table 2.3This systematic review was registered in the Prospero database 20 February 2019 (CRD42019120317).2.4A systematic literature search was performed through 3 December 2021 in the following electronic databases to identify relevant studies on burns and the vasoactive/inotropic drugs dobutamine, dopamine, epinephrine, and norepinephrine: PubMed, Embase, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, UpToDate and SveMed+. The search was conducted by an experienced librarian in collaboration with an intensive care clinician SB) and was not limited by year of publication or type of publication. Studies published in English, Swedish, Danish, and Norwegian languages were considered. A detailed search strategy is attached in Appendix  and was 2.5Two collaborators (KK and NGA) independently screened studies for eligibility according to predefined study selection criteria Appendix . Inclusi2.6Two independent collaborators (KK and NGA) extracted available data in duplicate according to a predefined data extraction form Appendix . This in2.7Two authors (KK and NGA) independently assessed the risk of bias of included studies using the Newcastle‐Ottawa quality assessment scale, which is a well‐established tool for assessing the quality of nonrandomized studies in meta‐analyses.2.8According to our protocol, we planned to perform a meta‐analysis using random effect models if the number of included studies was three or more, and to perform subgroup analyses covering the following topics: Burn injury population, burn injury mechanism, severity of burn injury and/or severity of organ failures.33.1The literature search identified 1058 unique publications that were screened for inclusion. After assessing 115 publications in full text, two retrospective cohort studies were included  and one receiving only fluids (NoPRESSOR group) (n = 36) during the first 48 h following burn injury.The Adibfar study included 52 patients in one group given vasopressor and fluids (PRESSOR group) (n = 20) versus only fluid (VP−) (n = 91).The study by Pape et al comprised 111 patients divided into patients receiving fluid and vasopressor (VP+) , registered 20 February 2019.Appendix S1 Supporting Information.Click here for additional data file.


Cryogels are a unique macroporous material for tissue engineering. In this work, we study the effect of hyaluronic acid on the physicochemical properties of cryogel as well as on the proliferation of a 3D model of mesenchymal stem cells. The functional groups of the synthesized cryogels were identified using Fourier transform infrared spectroscopy. With an increase in the content of hyaluronic acid in the composition of the cryogel, an increase in porosity, gel content and swelling behavior was observed. As the hyaluronic acid content increased, the average pore size increased and more open pores were formed. Degradation studies have shown that all cryogels were resistant to PBS solution for 8 weeks. Cytotoxicity assays demonstrated no toxic effect on viability of rat adipose-derived mesenchymal stem cells (ADMSCs) cultured on cryogels. ADMSC spheroids were proliferated on scaffolds and showed the ability of the cryogels to orient cell differentiation into chondrogenic lineage even in the absence of inductive agents. Thus, our results demonstrate an effective resemblance to extracellular matrix structures specific to cartilage-like microenvironments by cryogels and their further perspective application as potential biomaterials. Scaffolds play an important role in tissue engineering as they are often used as a basis for cell proliferation. Such polymers must be biodegradable and biocompatible, have a 3D structure, mechanical strength, etc. . To dateWhen comparing hydrogels and cryogels, hydrogels are crosslinked 3D networks with a high water content ,10. HydrMany cryogels were prepared from natural polymer-based materials and molecules because of their nontoxicity, biodegradability, biocompatibility and non-immunogenicity. Among others, glycosaminoglycans (GAG)-based cryogels are of a particular interest ,23. GAGsOne of the most widely used natural polymer is gelatin (Gel)—a biopolymer derived from animal collagen, the major protein component of cartilage, but with relatively low antigenicity and a lower cost than collagen, which is widely used in clinical applications ,19,27. GN-acetylgalactosamine, the latter being sulfated at O6 [Another good example of a natural polymer is chondroitin sulfate (CS). CS is a sulfated form of GAG, composed of a chain of alternating units of glucuronic acid and ed at O6 . CS attaed at O6 ,35.Mesenchymal stem cells (MSC) have found use in a wide range of fields, predominantly in regenerative medicine, e.g., bone and nerve regeneration ,37,38. TN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) [To date, similar works are known on the creation of crosslinked cryogels based on Gels, HA and CS using the crosslinking agent de (EDC) ,46,47. Kde (EDC) . Later, de (EDC) .The mainde (EDC) . In thatGelHACS cryogels were synthesized by cryopolymerization method at −12 °C as in our previous study . The rat–1, which corresponds to the stretching vibrations of the O–H and N–H functional groups (amide A) involved in the intramolecular hydrogen bond between HA and Gel molecules. Vibrations in the region of 2880 cm–1 refer to stretching vibrations of CH- groups. Peaks of 1631, 1541 and 1400 cm–1 are responsible to amide I for the C–O stretching vibration, amide II for the NH- bending vibration and the symmetric stretching vibration bands of the carboxyl groups of HA. The absorption band at 1338 cm–1 refers to bending vibrations of the amide group (amide III). The absorption peaks at 1233 and 1033 cm–1 are assigned to the asymmetric and symmetric stretching vibration bands of the OSO3—groups of CS. EDC is used to form covalent bonds between the carboxyl and amine groups of polymers to form amide bonds or ester bonds, resulting in bonds between Gel, HA and CS. No new peaks were observed upon crosslinking with EDC due to peak overlap, which was in a good agreement with the data published previously [The presence of the functional groups in the GelHACS cryogels is confirmed by FTIR measurements . The FTIDetermination of the gel fraction of the synthesized cryogels showed that with an increase in the content of HA in the cryogel, the content of the gel fraction in the polymer increases from 78.5 ± 1.3 to 88.5 ± 1.2% . ApparenAdditionally, with an increase in HA in cryogels, the pore volume (PV) of cryogels increases. Pore volume is an important factor for polymers used in tissue engineering. It is known that polymers with a pore volume above 80% can be used for cell proliferation . StudiesPolymers used in tissue engineering must have a porous structure in order to stimulate cell growth to the full extent . For exaChanges in the content of HA also affected their osmotic characteristics (swelling extent) of cryogels. The swelling results are shown in The degradation of biomaterials is critical for both drug delivery and tissue engineering, as implanted biomaterial must avoid chronic inflammation, fibrous encapsulation and implant rejection during implantation . During Thermal properties of cryogels were studied using DSC/dTG/TG curves A–C. The The cytotoxic effect of scaffolds towards ADMSC viability was measured by Alamar Blue assay. Rat ADMSCs were incubated at the presence of differing extracts concentrations of scaffolds for 24 h. The GelHACS-25 ranked second, showing 81% cell viability at the 10 mg/mL extract’s concentration and 91% at the lowest rate of the dose. Ranked third, GelHACS-10 had the strongest inhibiting effect on cell viability compared to control and other evaluated scaffolds. For instance, 77% of viable cells was detected at the highest dose of GelHACS-10, while the 81.7% cells were detected as viable after 24 h of incubation with 0.078 mg/mL scaffold extract. Taking in account obtained data, all three scaffolds showed lack of cytotoxicity even at the highest concentrations and could be further used as cell scaffolds for regenerative medicine.In order to examine the ability of the GelHACS scaffolds to mimic microenvironments specific for hyaline cartilage and induce chondrogenic cell differentiation even in absence of chondrogenic induction agents in vitro, the chondrogenic differentiation assay was performed. As a control groups, the scaffolds with ADMSC spheroids cultivated in culture medium containing chondrogenenic induction agents and TGF-β1 were used. Experimental groups were represented by scaffolds with ADMSC spheroids grown in culture medium without chondrogenenic induction agents and TGF-β1. At the end of the experiment the scaffolds with ADMSC spheroids were immunostained with specific antibodies to aggrecan and collagen type II. Aggrecan and collagen type II were chosen as markers of chondrogenic differentiation due to high abundance of collagen II and aggrecan in cartilage extracellular matrix. It is known that the structure, content, pore size and mechanical properties of scaffold have a crucial impact on cell fate and viability as well as on cell proliferation. The sponge structure, which was provided by cryogels, enhances cell–matrix interactions and oxygen and nutrient infiltration. As previously was demonstrated by Zhang and colleagues, human umbilical cord MSCs were able to undergo chondrogenic differentiation after their seeding on decellularized articular cartilage scaffold in the absence of inductive components .Sawatjui et al. showed that gene expression of main chondrogenic markers, such as aggrecan and collagen type II, were significantly increased in human MSCs after their cultivation in constructs comprised of Gel, CS and HA . The anaWe have studied the effect of HA on the properties of cryogels based on biopolymers of Gel and CS synthesized by cryopolymerization. It has been established that with an increase in the concentration of HA in the cryogels, the gel content, pore volume, swelling ratio and pore size increase. The MTT assay showed that cryogels do not have toxic properties and can be used as a biological material for cells. All three scaffolds promoted adhesion, growth and chondrogenic phenotype of ADMSC spheroids. The expression of aggrecan and collagen type II in ADMSC spheroids confirm the ability of scaffolds composed of Gel, HA and CS to sufficiently induce chondrogenesis in ADMSC spheroids without any addition of chondrogenic inductive agents as well as accumulate a cartilage-like matrix. Our work demonstrates the potential use of the synthesized cryogels in cartilage tissue engineering and regenerative medicine.N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), Dulbecco’s modified Eagle medium (DMEM) and phosphate buffered saline (PBS). Chondroitin sulfate A sodium salt was purchased from Glentham Life Sciences Ltd. . All aqueous solutions were prepared using MilliQ water (18.2 MΩ cm).All of the ingredients were obtained from Sigma-Aldrich and used as received, including gelatin (Type A), hyaluronic acid , A modified technique described previously was adapted for synthesis of cryogels in this work . Using a−1.FT-IR spectra of the samples were recorded using Thermo Scientific Nicolet iS10 FT-IR  spectrometer. The wavelength range is 4000–400 cmThe surfaces of dried cryogels were examined using scanning electron microscope . The coating of the samples was performed using 10 nm gold. Using ImageJ software, and the average diameter of the pores was calculated.i and Ww are the masses of the specimens before and after extraction with water.Thermogravimetry (TG), differential thermogravimetry (DTG) and differential scanning calorimetry (DSC) were used to analyze the thermal behavior of the cryogels using a Simultaneous Thermal Analyzer STA 6000  in a nitrogen atmosphere at a heating rate of 10 °C/min in the range of +30 to 800 °C. Gel fraction, swelling, degradation and pore volume of the cryogels were determined using a gravimetric method . The gelD in absolute ethanol for one day and then recording their final mass WS. Pore volume (PV) was calculated by Equation (2):The ethanol uptake into the pores was used to estimate the pore volume of the cryogels. The measurements were carried out by immersing dry cryogel specimens containing a mass of Ws) in 0.1 M PBS (pH 7.4) and the dry sample (Wd) over time (Equation (3)):The swelling ratio (SR) of the cryogels was calculated by comparing the masses of the swollen sample (W1) and transferred to 50 mL tubes filled with sterile 0.1 M PBS (pH 7.4). The tubes were incubated at 37 °C for 4 weeks, with the solution being refreshed twice a week. Cryogel samples were removed from the solution and washed with deionized water at predetermined intervals. After overnight freeze-drying and weighing (W2). The degree of degradation (DD) was calculated using the following Equation (4):Cryogel degradation was measured as follows: cryogels were weighed . The isolation of adipose tissue was performed using Wistar rats  as previously was described . BrieflySpheroids were established using the “hanging drops” technique as previously described . On day ® OCT compound  embedding and freezing at −70 °C. The frozen sections (7 µm thick) were cut using an MNT cryostat . After rehydration, scaffolds were washed in TBS containing NaCl , Tris  and pH 7.4 supplemented with 0.1% Tween-20 . Next, blocking buffer containing 1.5% goat serum  was added in order to prevent unspecific staining followed by incubation at room temperature for 30 min. Intensive washing procedures were performed and slides were covered with primary antibody dissolved in blocking solution. For detection of chondrogenic differentiation primary antibodies against aggrecan  and collagen type II  were added in 1:200 dilution and incubated overnight at 4 °C, samples with PBS instead of primary antibody were considered as a negative control. After staining with primary antibodies slides were extensively washed and covered with secondary antibodies Alexa Fluor 488 goat anti-mouse  and Alexa Fluor 594 goat anti-rabbit  for 1 h at room temperature followed by washing in TBS. To prevent evaporation, dye fading and to distinguish cell nucleus slides were covered with ProLong™ Gold Antifade Mountant with DAPI . The fluorescence images were taken by Axio Observer A1 . All incubations were performed in humidified chamber.After 14-day cultivation, scaffolds with spheroids were washed twice with PBS and fixed in 4% paraformaldehyde  at 4 °C at room temperature for 30 min followed by washing with PBS, cryopreservation, Tissue-Tek t test, and the difference between multiple groups was established using the one-way ANOVA (Tukey’s post hoc test). Statistically significant difference was defined as a p value 0.05. Data are presented as mean SD.Graphpad Prism  and Origin 8.1  statistical software was used to analyze the obtained data. The significant difference between two groups was found using the Student’s


To measure the impact of maternal body mass index (BMI) on the morphokinetics of embryo development as monitored by a time-lapse system.A retrospective chart review of in vitro fertilization (IVF) cycles from September 2016 to January 2019.Academic IVF practice.Patients <age 38 years undergoing IVF with their own gametes.Not applicable.The primary outcome was to compare embryo division timings between morbidly obese, obese, overweight, and normal-weight patients. A multilevel mixed effects model was performed to investigate the relationships between BMI categories and embryo division timings. Log or square transformation were used to improve fit.A total of 366 patients met inclusion criteria, yielding 4,475 embryos: 1,948 embryos from 162 normal-weight women (BMI 18.5–24.9), 1,242 embryos from 96 overweight women (BMI 25.0–29.9), 1,119 embryos from 91 obese women (BMI 30.0–39.9), and 166 embryos from 17 morbidly obese women (BMI ≥40). There were no differences in age, Antimüllerian hormone, or IVF cycle outcomes among the different BMI categories. When comparing embryo division timings based on BMI, controlling for covariates, embryos from obese patients had a shorter time to division to 2 cell embryo (T2) than normal-weight patients. When analyzing BMI as a continuous variable, there was no significant relationship between BMI and embryo division timing.Early embryo divisions were accelerated in only certain categories of obesity. This suggests a more complex mechanism for the effect of obesity on embryo development that may not be perceptible through the assessment of cell division timing events. Obesity is an epidemic with rates increasing in the last few decades; at least 50% of reproductive-age women are overweight or obese . ObesityDespite these demonstrated effects in clinical outcomes, results from traditional methods of evaluation of embryo quality, including morphological grade, do not consistently differ based on BMI, suggesting that morphology alone may not reflect the developmental potential of embryos conceived with oocytes from obese women undergoing IVF , 16, 17.Given the availability of time-lapse monitoring systems that track the specific division patterns and kinetics of embryos, it is now possible to observe and study the patterned and dynamic progression of embryos as they develop. Careful observation of these coordinated series of timed cell divisions may provide further insight into the effects of obesity on the observed decrease in fertility , 19. The2, they could not conclude an association between obesity and embryokinetics. This limits applicability to a population primarily comprised of overweight and obese reproductively aged women presenting for assisted reproductive technologies at clinics that provide treatment to women with higher BMI.Bellver et al.  comparedMore compelling evidence for an association between obesity and embryo division comes from Finger et al.  who, usiTo better elucidate the relationship between BMI and embryo morphokinetics, we aim to compare embryo morphokinetics in women undergoing their first cycle of IVF among patients of different BMI categories while controlling for several factors, including age, infertility diagnosis, Antimüllerian hormone (AMH) level, and smoking status. We hypothesize that embryo division timings, specifically T2 (time from one to two-cell embryo), will vary in a dose-dependent manner based on increasing maternal BMI.This project was approved by the institutional review board of the Medical College of Wisconsin under the project “Optimizing Embryo Selection—Clinical Utility of the Embryoscope,” project Number # PRO00029347 with Amendment ID: AME00019854. Using the Froedtert Hospital Reproductive Medicine Center IVF patient database, we reviewed Vitrolife EmbryoScope Time-Lapse System data and patient medical records from women undergoing their first cycle of IVF from September 2016 to January 2019. We collected data on BMI at the time of egg retrieval as our main dependent variable. We also obtained patient demographics and covariates related to the patient's IVF cycle, such as age, gravity, parity, infertility diagnosis, chronic medical conditions, antagonist/agonist protocol, ampules of gonadotropins, days of stimulation, peak estradiol, number of oocytes retrieved/fertilized, the timing of division of embryo to 2 cell stage (T2), 3 cell stage (T3), four-cell stage (T4) and 5 cell stage (T5), formation of a blastocyst, morphologic grade of an embryo, day of the embryo at transfer, beta-human chorionic gonadotropin (hCG) or chemical pregnancy, fetal cardiac activity, and pregnancy outcome.We attempted to minimize other factors that could negatively affect embryo development. Based on data from Liu et al, who reported that Embryoscope time-lapse imaging of polar body 2 extrusion was significantly delayed for women aged >38 years compared with those aged<35 years and between 35 and 38 years old, only patients aged <38 years were included in our study .2, which is the upper BMI limit per our clinic policy with anesthesia for office-based sedation.Exclusion criteria also encompassed those who had engaged in cigarette smoking within 6 months of the IVF cycle and who had a diagnosis of diminished ovarian reserve, based on AMH <1ng/mL, cycle day 2/3 serum Follicle Stimulating Hormone (FSH) >10 miu/mL, cycle day 2/3 serum estradiol >70 pg/mL) , 31. TheWe also excluded patients with diminished ovarian reserve as there is data that AMH and antral follicle count are comparable predictors of oocytes retrieved and of the number of good-quality embryos available for transfer and freezing . Some inRegimens used during the study included either a “short” or a “long” protocol of down-regulation with a GnRH agonist followed by stimulation via gonadotropins or stimulation with gonadotropins followed by pituitary suppression with a GnRH antagonist.Stimulation proceeded until at least 2 leading follicles reached 17–18 mm in size with appropriate estradiol levels.Ovulation was triggered via hCG and/or leuprolide acetate based on clinician judgment. For GnRH antagonist protocols in which patients were deemed at high risk of ovarian hyperstimulation syndrome, ovulation was triggered by 2 mg leuprolide acetate administered with a low dose  hCG or leuprolide acetate alone. Under conscious sedation, oocyte retrieval was performed 34–36 hours after hCG administration via transvaginal ultrasound-guided needle aspiration. The oocytes were then fertilized using intra-cytoplasmic semen injection (ICSI) or micro-insemination depending on patient preference, physician discretion, and/or male factor. Per protocol in our clinic, all cycles have at least a proportion of oocytes fertilized with ICSI. For luteal support, patients started either daily intramuscular progesterone (at 50 ng/mL) or vaginal gel the day after oocyte retrieval. In cases where leuprolide acetate and low-dose hCG trigger were used, the luteal phase was supported with 2 mg oral estradiol twice daily and one of the progesterone preparations. In cases at high risk for ovarian hyperstimulation syndrome, as deemed by clinic protocols , no luteal support was given and all embryos that were cryopreserved . During 2), 5% oxygen (O2), and the remaining balance of nitrogen, were used for the MINC incubators. Irvine Scientific (Fujifilm) Continuous Complete Culture NX (CSC-NX) was used for the full culture period. After fertilization was confirmed, the embryos were placed in the Embryoscope for continued culture until day 5/6 of development. Any blastocysts of adequate quality (minimum inner cell mass/trophectoderm grade of BB) according to a previously described grading system that were not transferred were cryopreserved on day 5 or 6 of culture using the Cryolock vitrification carrier system  oocytes were fertilized using ICSI or conventional insemination. Oocytes were placed in group microdrop culture until fertilization check, which occurred 15–18 hours post ICSI. The COOK and MINC incubators were used post-ICSI culture until fertilization check. Mixed gas tanks with 6% carbon dioxide . Cleavage time was considered the moment when cell division was completed, and the 2 originating cells were completely segregated and invested by their respective cytoplasmic membranes. The definitions of the time points assessed in the study were as follows: T2, the time at which the embryo presented two separate and distinct cells; T3, the time at which a 3 blastomeres embryo was achieved; T4, the time at which a 4 blastomeres embryo was achieved; and T5, time at which a 5 blastomeres embryo was achieved.The primary outcome was embryo morphokinetics, for which parameters were the time points listed above. Embryo quality score, ongoing pregnancy, and the number of available blastocysts to transfer or cryopreserve were secondary outcomes. To generate the embryo quality score, the main parameters of assessment on day 3 were the degree of fragmentation, the number of blastomeres, and symmetry among blastomeres, whereas on day 5, the main parameters were degree of expansion, hatching status, inner cell mass, and trophectoderm quality . ClinicaP<.05). Log transformation, or square transformation are used to improve fit. Logistic regression analysis was performed to test the effect of T2 on pregnancy results . A value of P <.05 was considered statistically significant. Statistical software SAS V9.4  was used for analysis.Median and interquartile range (IQR) were used to summarize the data. The chi-square test or Fisher’s exact test was used to examine the association between categorical variables, whereas the Kruskal-Wallis test was used for comparisons of continuous variables among BMI categories. A multilevel mixed effects model was employed to investigate the relationships between BMI categories or potential confounding factors  and the outcomes. A backward elimination method was used for model selection. The final model included BMI categories and any other significant factors  with a significance level of 0.05.2), 1,242 embryos from 96 overweight women (median = 27.2), 1,119 embryos from 91 obese women (median = 34.0), and 166 embryos from 17 morbidly obese women (median = 43.5). There were no differences between BMI categories in age, AMH level, number of oocytes retrieved, or number of blastocysts available , overweight  and morbidly obese  patients (P<.05).When comparing embryo division timings based on BMI, there were significant differences at the T2 time point. Embryos from obese patients had the shortest mean division timings (25.63 hours) compared with patients with normal weight . Similarly, the number of fertilized embryos was negatively associated with T2, T3, and T4 (P<.05). Interestingly, patient age, AMH level, and the total number of oocytes retrieved were not found to be significantly associated with the outcomes. Multivariable analysis using the backward elimination method showed that the number of blastocysts available and BMI categories were significant in the final model. The effect persisted where embryos from obese patients had the most rapid division timings compared with embryos from normal weight (P<.01), overweight (P<.05), and morbidly obese patients (P<.05) (P<.05) (P<.05).Univariate analysis was performed to evaluate the effects of possible covariates, which revealed that patients whose embryos had faster division timings were more likely to have more blastocysts ( (P<.05) . Again,  (P<.05) . The ratP=.35). Timing to T2 was not affected by precycle IVF laboratory results, including day 3 estradiol, day 3 FSH, AMH levels, and antral follicle counts (P>.05).However, when the data were analyzed using BMI as a continuous variable, there was no significant association between BMI and embryo division timing (P=.9). Distribution of infertility diagnosis was also analyzed between BMI categories, revealing a greater proportion of obese and morbidly obese patients carrying the diagnosis of anovulation . The BMI was found to differ significantly based on infertility diagnosis. Patients carrying a diagnosis of anovulation had a higher BMI than those with other infertility diagnoses (29.5 vs. 25.5 kg/m2) (P<.05).Overall, the timing of T2 was found to be unrelated to infertility diagnosis when comparing anovulation, idiopathic, or another infertility diagnosis (P>.05).Because of a possible confounding effect of anovulation, given the preponderance of this diagnosis among patients with higher BMI, data were analyzed excluding patients with a diagnosis of anovulation. Of the 366 patients, 272 did not carry a diagnosis of anovulation and were included for analysis. Within this patient group, 3,251 embryos were included in a multivariable analysis controlling for a covariate of the number of blastocysts available, which revealed no significant differences in embryo division timing based on maternal BMI  or anovulation, yet the average BMI did not differ between these diagnosis groups. To control for a possible confounding effect of infertility diagnosis, we analyzed the data from patients without a diagnosis of PCOS or anovulation, which revealed no differences in division timings between the BMI groups.Since we embarked on our study, more recent publications have examined the effect of obesity on embryo morphokinetics. Bartolacci et al.  studied Our results and the results of these other studies suggest that obesity may have a complex interaction with embryo development that cannot be measured by embryo morphokinetics alone. Similarly to Finger et al. , AndreasThese findings suggest a role of lipotoxicity, which is often described as a detrimental effect of obesity. Broughton and Moley describe a more severe infertility phenotype with combined obesity and PCOS, even when ovulatory, whether spontaneously or with assisted reproductive technology . PossiblOur study did not seek to investigate the relationship between paternal or combined parental obesity and embryodynamics, which may also contribute to our null results. We did not consistently collect data on the BMI of the sperm source and were not able to analyze this as a separate dependent variable, as was studied by Finger et al. . While wOne limitation of our study was that data were collected from a single site, although we met the requirements of our power calculation and had a BMI distribution that corresponded with the general population. Our strict exclusion criteria may also limit generalizability to the population of women and couples seeking evaluation for infertility. A final limitation of this study was that it was not designed to elucidate the effect of BMI on pregnancy outcomes, a result that may be more relevant to patients in the clinical setting.The strengths of our study include a larger sample size by comparison, especially of obese and morbidly obese women, to previous human studies analyzing time-lapse observation of embryo divisions , 27, 45.Previous studies examining embryodynamics in obese patients also included patients with lower mean BMI levels: 28.37 in Leary et al; 33.6 in Bellver et al. ; 32 in BOur statistical analysis attempted to control for the physiologic similarity within an individual patient by accounting for the variability of division timings of embryos that would be expected within a single patient embryo cohort. Although previous studies analyzed embryos as individual entities, it is physiologically and statistically true that multiple embryos from a single patient would be expected to behave more similarly than embryos compared from entirely different individuals.Having collected additional data on covariates related to patient IVF cycles, we could better analyze and control for possible confounders affecting embryo division timings. We acknowledge the strict exclusion criteria, which may have skewed our results. However, the purpose of our exclusion criteria was to attempt to limit as many parameters that could potentially also impact as many factors that could affect embryo division timings.The basis for the age cut-off, <38 years old, was based on data from Liu et al, who reported that Embryoscope time-lapse imaging of polar body 2 extrusion was significantly delayed for women >38 years than for those <35 years and between 35-38 years old . AdditioAlso, we excluded other factors that can contribute to abnormal embryo development. Specifically, we excluded patients with diminished ovarian reserve as there is data that AMH and antral follicle count are comparable predictors of oocytes retrieved and of the number of good-quality embryos available for transfer and freezing . Some inAlong those lines of factors that can predispose to abnormal embryo development, a retrospective cohort study of IVF cycles in the United States between 2009 and 2013 showed that cigarette smoking was associated with higher adjusted odds of cycle cancellation with no embryo transfer  . AdditioIn light of our findings, contrary to our hypothesis that division times would be consistently longer in patients with higher BMI categories, it is clear that further studies are warranted to evaluate other potential factors influenced by obesity. Our findings demonstrating a lack of BMI-dependent alteration in embryo division timings, together with our pattern of more rapid T2 corresponding to improved IVF outcomes, suggest a more complex, alternative mechanism by which BMI alters embryo development.