USH2A mutation was associated with high TMB and poor clinical prognosis. According to Gene Set Enrichment Analysis (GSEA) and the CIBERSORT algorithm, we determined that the USH2A mutation upregulates signaling pathways involved in the immune system and the antitumor immune response. In cases with a USH2A mutation, the immune score and MSI score of TCGA samples increased, the expression of immune checkpoint genes decreased significantly, and the TIDE score decreased significantly. Dependent on the presence or absence of a USH2A mutation, TCGA COAD samples were analyzed for differentially expressed genes, 522 of which were identified. Using a univariate Cox analysis and LASSO COX analysis of these differential genes, a prediction model was established, which established significant differences in the infiltration of immune cells, immune checkpoint gene expression, immune score, MSI score, TMB, and TIDE in patients in high- and low-risk groups. In conclusion, mutation of USH2A is frequent in COAD and is related to an increase in TMB and the antitumor immunity. The differential genes screened by USH2A mutation allowed the construction of a risk model for predicting the survival and prognosis of cancer patients, in addition to providing new ideas for COAD immunotherapy.Colon adenocarcinoma (COAD) is one of the diseases with the highest morbidity and mortality in the world. At present, immunotherapy has become a valuable method for the treatment of COAD. Tumor mutational burden (TMB) is considered to be the most common biomarker for predicting immunotherapy. According to reports, the mutation rate of COAD ranks third. However, whether these gene mutations are related to TMB and immune response is still unknown. Here, COAD somatic mutation data were downloaded from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases. Bioinformatics methods were used to study the relationships among gene mutations, COAD survival prognosis, and tumor immune response. A total of 22 of the top 40 mutations in TCGA and ICGC databases were the same. Among them, the  Colon cancer is the third leading cause of cancer deaths, with more than one million new cases diagnosed each year . COAD isPDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) pathways, has become a treatment strategy for various types of cancer , which targets programmed cell death ligand 1  and the International Cancer Genome Consortium (ICGC) databases to identify somatic mutations in COAD patients in the United States and China. Then, we identified common mutant genes in both cohorts, which were found to be related to TMB and prognosis, thus confirming that gene mutations are related to immune response and ICB treatment response. On the basis of their differential expression caused by mutations, we constructed a prognostic model composed of two genes with a predictive effect on tumor prognosis and ICB treatment response. These findings reveal that a gene mutation can be used as a biomarker for predicting immune response and for evaluating the response to ICB treatment in patients with COAD.n = 444), clinical data (n = 336), and somatic gene mutation data (n = 398) from TCGA database  (data updated on 29 October 2020). For clinical data, patients with COAD were included only when their clinical information was complete, and patients without survival time, survival status, age, gender, grade, or TNM classification data were not included. Next, the somatic gene mutation data of Chinese COAD patients (n = 305) was downloaded from the ICGC database (http://dcc.icgc.org/releases/current/Projects) (data updated on 27 November 2019), and the COAD dataset GSE39582 was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/).We used a method similar to that of Gongmin p-value < 0.05 was considered statistically significant. Software GSEA (version 4.1.0) was used for gene enrichment analysis. According to the gene mutations, TCGA expression data were divided into two groups: mutation and wild-type. The arrangement was set to 1,000, and the standardized enrichment score (NES) was applied using an FDR q-value < 0.05 as the significance threshold for enrichment  difference >2 . The enrichment analysis tool DAVID . R software was used to perform KM survival analysis and univariate Cox analysis of the differential genes using a p-value < 0.05 as the filter value. R software package glmnet was used to perform LASSO COX regression analysis and construct a prognosis-related risk model. To evaluate the risk model, R software package survival ROC was used to analyze the prediction accuracy of the model, and univariate and multivariate Cox analyses were used to evaluate whether the risk score of the tumor patient model could be used as an independent prognostic factor.PERL software (version 5.32) was used to extract and sort TCGA transcription data, somatic mutation data, clinical data, ICGC mutation data, and GEO transcription data for subsequent analysis. R software (version 4.0.3) package GenVisR was used to analyze and visualize the MAF file of Varscn, the somatic mutation data of colon cancer in the TCGA database. R software package GenVisR was used to analyze and visualize the colon cancer somatic mutation data TSV file of the ICGC database based on the hg19 genome reference information. The venn package in R software was used to take the intersection of the top 40 genes in the TCGA and ICGC datasets with mutation evaluation rates, followed by obtaining the intersection genes with the top mutation frequencies in both databases. Next, R software package ggpubr was used to analyze the relationship between gene mutations and TMB. The Kaplan–Meier (KM) method was used to analyze the relationship between gene mutation and survival prognosis. Univariate and multivariate Cox methods were used to analyze the relationships among patient clinical information , TMB, gene mutations, and tumor survival prognosis. In all comparisons, a richment . The edgol DAVID  was usedol DAVID functionsol DAVID  pathwaysTumor mutation burden (TMB) refers to the total number of gene coding errors, base substitutions, and gene insertion or deletion errors per megabit (Mb) of tumor tissue. All base substitutions and insertions in the coding region of the target gene are counted, whereas silent mutations that cannot cause amino-acid changes are not counted. The total number of mutations counted was divided by the exome size  to calculate the TMB score for each sample . A microp-value < 0.05 was used as the basis for the successful calculation of the sample.CIBERSORT is a deconvolution algorithm that can evaluate the proportion of 22 tumor-infiltrating lymphocyte subsets in a large number of tumor samples . This alR package estimate was used to calculate the immune score of the tumor sample. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm is a calculation method that uses gene expression profiles to predict immune checkpoint blockade (ICB) responses in non-small-cell lung cancer and melanoma . Accordip-value < 0.05 was considered statistically significant.R software (version 4.0.3) was used for statistical analysis and graphing. The logrank test was used for KM survival analysis, and the Mann–Whitney U test was used for analysis of the relationship between gene mutation and TMB. In all comparisons, a APC, TP53, TTN, KRAS, and SYNE1  and MUC4 mutation  were associated with a poor prognosis of COAD; thus, they could be considered independent risk factors. We further studied the relationship between the location of the USH2A mutation site in the COAD sample of the TCGA database and the survival of COAD. We searched the UCSC database  and found that the mutation sites provided are distributed in the exon region of the USH2A gene   , the expncreased . We compncreased . TIDE usncreased . We comp reduced . These rUSH2A mutation, we divided TCGA COAD samples into a USH2A mutant group and a wild-type group, and we used the edgeR package to analyze the differential expression of genes. A total of 522 differentially expressed genes (DEGs) were obtained, among which 440 DEGs were upregulated and 82 DEGs were downregulated  × TNNT1 + (0.2032 × ERFE)  . The ROC × ERFE) . We used × ERFE)  and draw × ERFE) . In the  × ERFE) , indicat × ERFE) . Univari × ERFE)  and GSE3 × ERFE) . Compari × ERFE) . We also × ERFE) . Our ana × ERFE) . These rUSH2A is frequently mutated in both cohorts, and its mutation is associated with high TMB and poor clinical prognosis. We also found that the USH2A mutation is positively related to the signaling pathway of the immune system. The results of tumor-infiltrating immune cell analysis showed an enrichment of activated NK cells, TFH cells, and γδT cells in the USH2A mutation samples, which is consistent with the results of previous studies , we found that NK cells and γδT cells are involved in the IL-17 signaling pathway, which confirms that the pathway enrichment of DEGs after USH2A mutation is correlated with tumor immune cell infiltration. After USH2A mutation, analysis showed that immune checkpoint gene expression and TIDE score decreased significantly, whereas immune score and MSI score increased significantly, thus indicating that USH2A mutation affects the antitumor immunity and is conducive to ICB treatment. By performing univariate Cox analysis and LASSO COX analysis of DEGs, we established a prognostic risk model based on the expression of genes TNNT1 and ERFE. Cox analysis showed that risk score is an independent risk factor for tumor survival and prognosis. The verification of the ROC curve using the GSE39582 dataset showed that the model has the ability to predict risk. Comparing the immune checkpoint gene expression, immune score, MSI score, TMB, and TIDE of patients in the high- and low-risk groups, significant differences were found, whereby CD8 T cells, TFH cells, and activated NK cells were all significantly increased in the high-risk group. These results indicate that the risk model can predict the survival prognosis of COAD patients and assess whether the patients will have a good ICB treatment response.In summary, by analyzing the somatic mutation characteristics of 398 USA COAD samples in TCGA database and 305 Chinese COAD samples in the ICGC database, we found that  studies . Dependeo the GO  and KEGGo the GO  enrichmeUSH2A  gene encodes a protein. The protein exists in the basement membrane and may play an important role in the development and homeostasis of the inner ear and retina  and cytotoxic T lymphocyte antigen 4 (CTLA4) pathways, has become a treatment strategy for various types of cancer . High numbers of TFH cells and high levels of CXCL13 are associated with increased survival of colon cancer patients  was used to group patients. The 5 years recurrence rate of patients in the high-immune-score group was only 4.8% (γδ T cells may be associated with a better colon cancer prognosis (USH2A mutations was poor; however, in the USH2A mutant tumor samples, there was an enrichment of activated NK cells, TFH cells, and γδ T cells, indicating a change in the recognition of immune surveillance, as well as an antitumor effect. Therefore, we found that, in COAD, the USH2A mutation can induce changes in infiltrating immune cells, thereby enhancing antitumor immunity.NK cells play a key role in innate and adaptive immune response and tumor immune surveillance by recognizing and killing tumor cells . Althougsynapses . TFH celsynapses . Currentsynapses . TFH celpatients . γδ T ce program . Evidencer cells . The knoes IL-17 . In brearognosis . There hnly 4.8% . Considerognosis . In thisTNNT1 gene encodes a protein of the troponin subunit, which is a regulatory complex located on the sarcomere filaments (TNNT1 is significantly upregulated in colon cancer samples and cell lines. The upregulation of TNNT1 is also related to a variety of clinicopathological characteristics, and its high expression is related to the poor prognosis of patients. Inhibition of TNNT1 can significantly inhibit cell proliferation, migration, and invasion, while promoting cell apoptosis (TNNT1 may promote the progress of COAD and mediate the EMT process (FAM132B, which is produced upon the stimulation of red blood cells by erythropoietin in the bone marrow and spleen (TNNTI gene expression in our research model was associated with poor tumor prognosis, which is consistent with previous studies (The increased migration and invasion potential of colon cancer cells leads to a significant decrease in the 5 years survival rate of colon cancer patients. Therefore, an accurate prediction of prognosis is essential for individualized treatment of these patients. Today, gene expression profiling has become an adjunct to cancer treatment; for example, Gene-expression prediction models were built using transcriptome to predicte colorectal cancer risk , the expilaments . Studiespoptosis . TNNT1 m process . On the d spleen . It has d spleen , whereas studies . Patient studies . In our USH2A mutations can affect the antitumor immunity of COAD and the responsiveness to ICB therapy. Furthermore, we constructed a prognostic model consisting of two DEGs, which could predict 1, 3, and 5 years survival rates in TCGA dataset and GEO validation dataset GSE39582 with a relatively high AUC. The main limitation of this study is that the ICGC database lacks corresponding clinical data on Chinese COAD; thus, we could not verify the significance of the USH2A mutation in the prognosis of Chinese COAD patients and whether it can cause the same immune response. Even though USH2A was frequently mutated in Chinese COAD samples, its impact may be somewhat heterogeneous among different races. Therefore, the relationship between USH2A mutation and prognosis, including the analysis of infiltrating immune cells and signaling pathways, needs to be further verified in Chinese colon samples. In addition, the differential expression of the two genes used to construct the risk model was identified from TCGA data; although TCGA data are of high quality, further experimental verification of the role of these two differential genes in colon cancer is needed in vitro and in vivo.The novelty of this study lies in the discovery that USH2A is frequently mutated in COAD, which is associated with a high TMB and poor prognosis. In addition, the USH2A mutation upregulates immune signaling pathways and promotes an antitumor immune response. On the basis of two DEGs associated with the USH2A mutation, we constructed a model with a predictive effect on the prognosis of tumor survival. These findings reveal a new gene whose mutation can be used as a biomarker for predicting the response to antitumor immunity and ICB treatment.In summary, this study showed that


The PF sows received unique daily blends of two isocaloric diets , whereas CON sows received a static blend throughout gestation to achieve 0.56% SID Lys. After weaning, sows were re-bred and entered the same feeding program as in the previous pregnancy for two subsequent pregnancy cycles . Sows on the PF program received 97%, 105%, and 118% (average over three pregnancy cycles) of dietary energy and 67%, 79%, and 106% of SID Lys intakes compared to CON between days 5 and 37, 38 and 72, and 73 and 108 of gestation, respectively. Estimated N (26.1%) retention did not differ between gestation feeding programs in any pregnancy, but excess N excretion was less  for PF vs. CON sows. Regardless of pregnancy cycle, sows that received the PF program had greater ADG between days 38 and 72  and between days 73 and 108  of gestation, and greater loin depth gain between days 63 and 110 of gestation , but BW (235.1 kg) and backfat (17.8 mm) and loin (70.5 mm) depths on day 110 of gestation did not differ. The number of piglets born alive, stillborn, and mummified, and litter birth weight (16.5 kg) did not differ in any pregnancy cycle, nor did piglet ADG during lactation (250 g/d) and piglet BW (6.7 kg) at weaning. Sows that received the PF program during gestation had lower ADFI during lactation . Therefore, using feeding programs that precisely match estimated daily energy and Lys requirements for gestating sows provides the opportunity to reduce N losses to the environment and reduce lactation feed usage, without negatively affecting sow reproductive and lactation performance.The objective of the current study was to determine the effects of precisely meeting estimated daily energy and Lys requirements for gestating sows over three consecutive pregnancies on sow reproductive and lactation performance. A total of 105 sows  were randomly assigned to a precision (PF;  The nutrient and energy requirements for gestating sows are not constant throughout the gestation period or across parities. For example, the estimated Lys requirement for a sow in the first reproductive cycle increases by 200% between days 0 and 114 of gestation, whereas the estimated energy requirement increases by 45% within the same timeframe . FurtherThe sow is adept at buffering moderate nutrient and energy deficiencies and excesses in the diet e.g., . HoweverTherefore, the aim of the current study was to determine the long-term effects of precisely meeting estimated daily Lys and energy requirements for individual sows on each day of gestation across three consecutive pregnancies on reproductive and lactation performance.The experimental protocol was approved by the University of Guelph Animal Care Committee and followed Canadian Council on Animal Care guidelines . Sow genetics were distributed equally between treatments and sows were randomly bred with either Yorkshire, Landrace, or Duroc semen. The ESF [modified according to One hundred five sows  were placed into one of four pens equipped with ESF 5 ± 3 d after breeding over five breeding batches (blocks). Sows were randomly assigned to one of two gestation feeding programs in the first gestation cycle: precision feeding (PF) or standard feeding  on days 5 ± 3, 62 ± 3, and 110 ± 1 of gestation and 3 d after weaning by a trained technician using a portable ultrasound machine with a 140 mm linear probe .g at room temperature for 15 min. Serum was aliquoted into microcentrifuge tubes and stored at −20 °C until further analysis. Serum from a subset of 20 sows (n = 10 per treatment) from each of three subsequent gestation cycles were analyzed at the University of Guelph Animal Health Laboratories  using a Cobas 6000 c501 biochemistry analyzer  to determine serum concentrations of glucose, urea, beta-hydroxybutyric acid (BHBA), and non-esterified fatty acids (NEFA).On days 5 ± 3 and 108 ± 1 of gestation, 10 mL of blood were collected from each sow via sub-orbital sinus puncture into serum tubes . Blood samples were centrifuged at 3000 × Immediately after birth and before first suckle, up to two piglets per sow were sacrificed for determination of physical and chemical body composition. Body weight and individual organ weights  were collected. The organs and carcass were combined per pig in a plastic bag and frozen at −20 °C until further analysis .Representative feed subsamples for the HP and LP diets were collected monthly (to include each batch of feed) and compiled into 3.5-mo intervals for analysis. Samples were ground and sent to SGS  and analyzed for dry matter (method 930.15), crude protein (method 990.03), and calcium and phosphorus  with individual sow as the experimental unit. The model included the fixed effects of gestation feeding program treatment , pregnancy number (PREG), and their interaction; initial reproductive cycle (INTPARITY) was used as a covariate and the repeated measures option was employed. The random effects of block, treatment nested within block, and sow nested within treatment were included. Piglet performance during the lactation period (after cross-fostering) was analyzed with the additional covariate of post-fostering litter size. Sow serum concentrations for BHBA, glucose, and NEFA were log transformed prior to analysis and back transformed for presentation of results. Pre-planned contrasts were constructed to compare treatments within pregnancy cycle. A The analyzed and calculated nutrient contents were comparable for both the high and low protein gestation diets, as well as the commercial lactation diet . The onlP-values are presented in the tables for the reader’s information. Initial and final sow body weights and ADG between gestation days 5 and 37 were not influenced by gestation feeding program for any of the three pregnancies (P < 0.05) where sows that received the PF program had greater ADG in pregnancies 2 and 3 than sows that received the CON program , whereas gestation feeding program did not affect ADG in pregnancy 1. Between gestation days 73 and 108, sows that received the PF program tended (P = 0.063) to have greater ADG than those that received the CON program; in pregnancy 1 only, sows fed the PF program had greater ADG than those fed the CON program . Generally, backfat and loin depth values, along with changes in backfat and loin depths over gestation, were not influenced by gestation feeding program in any of the pregnancies. However, the loin depth gain between days 63 and 110 of gestation was greater for sows that received the PF versus the CON program (P < 0.05); in pregnancy 3, sows fed the PF program had greater loin depth gain than those fed the CON program . The costs for the HP and LP diet usage per sow were less and greater, respectively, for sows that received the PF versus the CON program (P < 0.001 and P < 0.001). In pregnancies 1, 2, and 3, sows fed the PF program had lower feed cost associated with feeding the HP diet  and greater feed cost associated with feeding the LP diet . Cumulative feed cost (HP + LP) over the gestation period was greater for sows that received the PF vs. the CON program (P < 0.05). In pregnancies 1 and 2, cumulative feed cost tended to be  and was , respectively, greater for sows fed the PF vs. CON feeding program, but in pregnancy 3, cumulative gestation feed cost was not different between the feeding programs.The main effect of pregnancy is not mentioned in the results section since some variation in sows from different pregnancies was expected; the main effect gnancies . BetweenP = 0.071). Serum glucose tended to be influenced by the interactive effect of gestation feeding program and pregnancy (P = 0.059) where PF sows had greater serum glucose concentration on day 108 of gestation in pregnancy 2 (P < 0.05), whereas no differences in sercum glucose between gestation feeding programs were observed for pregnancies 1 or 3.The serum concentrations for BHBA and urea were not influenced by gestation feeding program in any pregnancy on either day 5  or day 108  of gestation . The serP = 0.092). Piglet BW, liver, and large intestine mass, and relative gastrointestinal tract weight (% of BW) at birth were not influenced by gestation feeding program in any pregnancy (P < 0.01), such that piglets from sows that received the PF program had greater stomach mass in pregnancy 3  but stomach masses were not different between feeding programs in pregnancies 1 or 2. Piglets from sows that received the PF program tended to have greater small intestine mass than piglets from sows that received the CON program in pregnancy 2 only . The protein content in piglet carcasses was less for sows that received the PF vs. the CON program (P < 0.05), which was especially evident in pregnancy 3 . The piglet ash and fat contents were not influenced by gestation feeding program in any pregnancy but the water content was greater for piglets from sows that received the PF vs. the CON program (P < 0.05).The number of piglets born alive, piglet birth weight, the variation in piglet birth weights before and after cross-fostering, litter birth weight, and the number of stillbirths were not influenced by gestation feeding program in any pregnancy . The numregnancy . Piglet P < 0.01). In pregnancy 1, the litter size for sows that received the PF program tended to be smaller than the litter size for CON-fed sows , whereas litter size was greater in pregnancies 2 and 3 for sows that received the PF program , such that the variation was greater for piglets from sows that received the PF program in pregnancy 1 , was less in pregnancy 2 , and not different in pregnancy 3 vs. piglets from sows that received the CON program. There was an interactive effect of feeding program and pregnancy on sow BW change over lactation (P < 0.05). In pregnancy 2, sows that received the PF program tended to lose less BW than sows that received the CON feeding program , but no differences in sow BW loss between feeding programs were observed in pregnancies 1 and 3. Changes in backfat and loin depths over lactation and re-breeding interval were not influenced by gestation feeding program in any pregnancy. Average daily feed intake and cumulative feed cost during lactation were less for sows that received the PF versus CON program (P < 0.01), which was especially apparent in pregnancy 2 .There was an interactive effect of sow feeding program and pregnancy on the number of piglets per litter after cross-fostering (P = 0.050), such that sows that received the PF program had lower excretion of excess N in pregnancies 2 and 3 vs. sows that received the CON program , whereas there was no difference in excess N in pregnancy 1. Overall, sows that received the PF program had lower amounts of excess N compared to sows that received the CON program (P < 0.01). Sows that received the PF program had greater excretion of excess P compared to sows that received the CON program (P < 0.01), particularly in pregnancies 2 and 3 .Estimated N and P retentions were not influenced by gestation feeding program in any pregnancy . There wThe purpose of the current study was to examine the long-term effects of closely meeting the dynamic estimated Lys and energy requirements for individual sows during gestation across three consecutive pregnancies on sow reproductive and lactation performance. Using the PF program, sows gained the same overall amount of BW during gestation, but with greater BW gain occurring after day 38 of gestation versus sows that received a static quantity and composition of gestation diet (CON). This, combined with positive and greater loin depth gains after day 63 of gestation and 7 and 2× (average over three pregnancies) lower day 108 fasted serum BHBA and NEFA concentrations, respectively, indicates that the dynamic and increasing SID Lys:NE ratio provided throughout gestation for the PF program, supplied adequate amounts of both Lys and energy to support maternal protein deposition and minimize lipid mobilization. However, since there were no differences in piglet birth weights, piglet ADG (indicator of sow milk production), re-breeding intervals, and sow retention, it appeared there were no negative long-term implications for CON-fed sows. In modeled scenarios, it has been demonstrated that a static SID Lys supply of 11 g/d throughout gestation resulted in maternal protein and lipid mobilization in late gestation, while providing 13.5 g/d SID Lys was above estimated requirements for both gilts and sows . FurtherSows that received the PF program during gestation consumed less lactation diet in the subsequent lactation period vs. those that received the CON program. Since overall sow BW change and piglet ADG during lactation were not different between sows from the two gestation feeding programs, sows that received the PF program during gestation were presumably more efficient at utilizing dietary nutrients and energy for milk production. This occurred despite PF sows unintentionally tending to have larger litter sizes than CON sows after cross-fostering. During lactation, when feed intake is no longer restricted, sows attempt to compensate for nutrient and energy deficiencies experienced in gestation by increasing feed intake . Thus, sIn the current study, there were minimal differences in litter characteristics and piglet physical and chemical composition at birth from sows that received the PF and CON gestation feeding programs. Piglets from sows that received the PF program in gestation, however, had greater variation in BW within litters at weaning in pregnancy cycle 1, less variation in pregnancy cycle 2, and no difference in variation in pregnancy cycle 3, despite no differences in BW variation within litters at birth or after cross-fostering. Variation in BW at weaning can be linked to poor lactation output ; howeverOverall, gestation feeding program had no obvious effect on sow retention throughout the study, although the number of sows enrolled was relatively small. Nearly equal numbers of sows were removed from the study for: lameness , reproductive failure , illness , savaging piglets , death with unknown cause , and incompatibility with the ESF system . It is interesting to note that 30% more CON sows were removed from the study due to reproductive failure compared to PF sows, with the most common reproductive issues being failure to enter estrus after weaning, failure to conceive after insemination, and miscarriage. The reproductive issue of failing to return to estrus is typically due to low BW at the time of breeding, which is caused by BW loss during lactation and poor adipose and protein reserves at weaning . In the The cumulative feed cost during gestation was greater for sows that received the PF vs. CON feeding program, which was largely driven by greater feed allowance  to meet estimated daily energy requirements. Considering the reduction in feed intake (and cost) during lactation for sows that received the PF program, the relative difference in feed cost per entire reproductive cycle was small (i.e. ~ $0.25 per sow in pregnancies 1 and 2). Conversely, in pregnancy 3, the sum of gestation and lactation feed costs per sow were $1.24 less for sows on the PF gestation feeding program. As sows mature, the estimated daily energy requirements increase slightly to account for greater maintenance needs, but AA requirements decrease as maternal protein retention approaches zero (once losses from the previous lactation are recuperated; Using feeding programs that precisely match estimated daily energy and Lys requirements for gestating sows provides the opportunity to reduce nitrogen losses to the environment and reduce lactation feed usage, without negatively affecting sow reproductive and lactation performance. Based on the conditions of the current study with the genetics used across three consecutive gestation cycles, there appeared to be no ill effects for sows that received a static amount of energy and Lys throughout gestation and across parities. Although there were no notable differences in piglet characteristics from the time of birth to weaning, it is not known if precisely meeting estimated energy and Lys requirements throughout gestation will influence offspring post-weaning growth performance. Depending on commodity prices and individual farm factors, implementing a precision feeding program in gestation could be a means to reduce gestation and lactation feed costs. Future studies should examine the impact of precision feeding gestating sows in commercial scenarios using modern, hyper-prolific sow genetics.


Polyvinyl alcohol (PVA) hydrogels have been widely studied for cartilage replacement due to their biocompatibility, chemical stability, and ability to be modified such that they approximate natural tissue behavior. Additionally, they may also be used with advantages as local drug delivery systems. However, their properties are not yet the most adequate for such applications. This work aimed to develop new PVA-based hydrogels for this purpose, displaying improved tribomechanical properties with the ability to control the release of diclofenac (DFN). Four types of PVA-based hydrogels were prepared via freeze-thawing: PVA, PVA/PAA (by polyacrylic acid (PAA) addition), PVA/PAA+PEG (by polyethylene glycol (PEG) immersion), and PVA/PAA+PEG+A . Their morphology, water uptake, mechanical and rheological properties, wettability, friction coefficient, and drug release behavior were accessed. The irritability of the best-performing material was investigated. The results showed that the PAA addition increased the swelling and drug release amount. PEG immersion led to a more compact structure and significantly improved the material’s tribomechanical performance. The annealing treatment led to the material with the most suitable properties: besides presenting a low friction coefficient, it further enhanced the mechanical properties and ensured a controlled DFN release for at least 3 days. Moreover, it did not reveal irritability potential for biological tissues. Osteoarthritis (OA) is the most common chronic joint condition, affecting more than 300 million people worldwide . Its preVarious polymeric biomaterials have been proposed to act as cellular scaffolds and support the growth of newly formed tissue ,7,8 or tThe high swelling capacity of hydrogels makes them ideal systems to encapsulate bioactive molecules or hydrophilic drugs that can be locally released into synovial joints, reducing the risks of drug degradation commonly associated with oral and intravenous administration, side effects, and required doses ,24. SeveThe main objective of this work was to develop a cartilage replacement material based on PVA and produced using FT with an adequate mechanical and tribological performance and the ability to control the release of DFN in the immediate postoperative period. Poly(acrylic) acid (PAA) was added to PVA to enhance the drug release profile . PAA is SEM images of the samples’ surfaces and cross-cut sections are shown in The SC and EWC of gels and porcine cartilage are shown in The results of the wettability measurements of the materials’ surfaces are given in Representative compressive stress–strain curves of all tested materials are provided in The angular frequency curves obtained for the different hydrogels are shown in The total amount of DFN loaded by the samples is depicted in p = 0.676).PVA loaded the lowest amount of drug, while PVA/PAA loaded the highest. The immersion in PEG led to an intermediate value. Annealing barely affected the loading capacity .The observation of CAM of hen’s eggs after 5 min of exposure to annealed samples loaded with DFN did not show signs of hemorrhage, lysis, or coagulation occurring during the test, similar to what happened with the negative control . In contIn this work, a set of PVA-based hydrogels were studied with the purpose of developing materials with adequate properties to be used as cartilage substitutes and simultaneously release an anti-inflammatory drug, namely, DFN. PVA and a blend of PVA/PAA were prepared. The latter was subsequently immersed in PEG (PVA/PAA+PEG) and thereafter submitted to an annealing treatment (PVA/PAA+PEG+A).The morphological analysis of the materials  showed tThe study of the swelling capacity of the hydrogels a showed Concerning wettability, the studied materials did not present significant differences between them. As expected, all materials showed hydrophilic behavior (water contact angles between 42 and 47°) since the polymers used in their preparation are hydrophilic ,51,52. WThe mechanical properties of the studied hydrogels were accessed using compressive tests. The stress–strain curves show the typical behavior observed for the studied materials. Hydrogels are biphasic materials consisting of a fluid phase and a solid matrix. At the beginning of the compressive test, when the load is applied, the resistance to deformation is mainly due to the fluid pressurization. As the fluid exits the hydrogel due to compression, the load is transferred to the solid phase, and the slope of the curve increases significantly. PVA/PAA exhibited the lowest compressive modulus, which was associated with the higher amount of water present in the sample and the high porosity of the hydrogel, which allowed for the easy release of water. In addition, PAA reduced the PVA chains’ intermolecular interactions decreasing the material’s stiffness. PVA/PAA+PEG and PVA/PAA+PEG+A gave rise to hydrogels with the highest compressive modulus, most likely due to their lower porosity and water content, as well as stronger interactions between the molecular chains and/or formation of microcrystallites.The rheological measurements showed that all the hydrogels displayed viscoelastic behavior, showing viscous and elastic characteristics simultaneously. The materials presented predominantly elastic behavior since the storage modulus was dominant over the loss modulus (G’ > G’’) for all analyzed frequencies. This means that the hydrogels are stable during storage . As bothThe study of the friction coefficient (CoF) of cartilage replacement materials is crucial, as it evaluates the resistance to movement of the joint interface. The friction of hydrogels depends on many factors, such as the materials’ ability to suffer deformation, EWC, hydrophilicity, roughness, and adhesion to opposing surfaces. The results showed that the lower the compressive modulus, the higher the CoF due to the higher deformability of the material. This increased the contact area, which enhanced the adhesive component of the tangential force and resulted in a higher counter body penetration depth into the hydrogel that impaired the sliding, explaining the higher value of CoF observed for PVA/PAA. The higher water amount in this hydrogel should have contributed to a decrease in the CoF relative to PVA. However, its higher deformation capacity and porosity played a more relevant role, increasing the friction. The remaining hydrogels presented CoF values that were similar to each other. As expected, increasing the applied load resulted in higher CoF values, which was also due to the increase in penetration depth and contact area. It should be stressed that all CoF values were in the range of those found in the literature for articular cartilage tested against stainless steel ,60.To evaluate the potential of the developed materials to be used as drug delivery platforms, loading and release studies were performed with DFN. Significant differences in the loading capacity of the samples were observed. PVA loaded the lowest amount of DFN (14.6 ± 0.9 μg DFN/mg dry sample) and released about 58% of that drug in less than 6 h. This hydrogel has a porous structure with free spaces that can contribute to the fast loading and release of the drug. Other authors  reached tM and ∞M are the masses released at time t and at infinite time (obtained via extrapolation using TableCurve software), respectively; n is the diffusional exponent; and K is the pseudo-kinetic constant. The fitting led to n = 0.4 and K = 0.03, with a correlation coefficient of R2 = 0.96, demonstrating that the model adequately described the drug release behavior. When n < 0.5, a pseudo-Fickian behavior with a diffusion-controlled mechanism occurs [R is the gas constant, T is the absolute temperature, AN is Avogadro’s number, and G the shear modulus, which can be calculated usingIn order to infer the drug release mechanism for the hydrogel that led to the best release behavior (PVA/PAA+PEG+A), the Korsmeyer–Peppas model  was fittm occurs , leadingm occurs . Howeverm occurs ,67,68:PAA was added to the formulation of hydrogels to obtain PVA/PAA samples;(2)PVA/PAA was further immersed in PEG to obtain PVA/PAA+PEG samples;(3)PVA/PAA+PEG was annealed at 120 °C, giving rise to PVA/PAA+PEG+A samples.In this work, PVA-based hydrogels were produced to be used as cartilage replacement materials. Aiming to improve their tribomechanical behavior and to provide them with the ability to act as drug release platforms of the anti-inflammatory drug DFN, several approaches were followed:The addition of PAA resulted in a porous material with slightly larger pores than PVA, greater swelling capacity, lower mechanical properties, and a higher CoF. Furthermore, it led to a significant increase in the amount of drug released, but as with PVA, the release was completed in a few hours. The lower mechanical resistance and the higher water absorption capacity played a determinant role in the frictional and drug release behavior, respectively.The immersion of PVA/PAA in PEG led to dehydration of the samples and established interactions with the PVA chains, which were responsible for the compaction of the structure and improvements in the mechanical properties. In turn, these last two factors explained the decrease in CoF relative to PVA/PAA. Concerning the DFN release, the lower porosity and swelling capacity contributed to the decrease in the amount of drug released compared to the hydrogel before PEG immersion. However, release the kinetics remained similar.After the annealing treatment, the hydrogels kept a non-porous structure, with similar swelling capacity and CoF compared to PVA/PAA+PEG. However, the higher crosslinking due to the increased degree of crystallinity led to a significant improvement in the mechanical properties and a more sustained release of DFN, which lasted for at least 3 days. The material proved to be a non-irritant to biological tissues.Overall, the final hydrogel (PVA/PAA+PEG+A), obtained through a set of sequential steps, revealed a superior tribomechanical behavior that approximated that of natural cartilage. Moreover, the material showed a high potential to ensure a sustained release of the anti-inflammatory DFN at the local level in the post-surgical period without inducing harmful effects to biological tissues. Further studies on the wear/fatigue behavior of this material, as well as on its ability to prevent an inflammatory response, would be needed to prove its efficacy for cartilage substitution.Polyvinyl alcohol (PVA) Poval™ 28–99  with a degree of hydrolysis of 99–99.8 mol% and average molecular weight (MW) of 145,000 g/mol and polyacrylic acid  from Sigma-Aldrich  were used as raw materials for the preparation of the hydrogel samples. Polyethylene glycol  was also purchased from Sigma-Aldrich .For the drug release experiments, diclofenac sodium salt (≥98%) was obtained from Sigma-Aldrich .As for the hen’s egg chorioallantoic membrane test, sodium chloride (NaCl ≥ 99%) was bought from PanReac AppliChem , and sodium hydroxide (NaOH ≥ 99%) was acquired from Merck . Fertilized chicken eggs (≈60 g/egg) were provided by Sociedade Agrícola da Quinta da Freiria S.A. .® Integral 3 water purification system . Phosphate buffered saline (PBS) tablets (pH = 7.4 at 25 °C) intended for the preparation of buffered solutions were supplied by Sigma-Aldrich .Working solutions were prepared in distilled and deionized (DD) water (resistivity ≥ 18 MΩ∙cm) obtained using a Milli-QArticular cartilage samples from the femoral condyles were obtained from a single knee joint of an adult porcine from a local slaughterhouse .w/w) at 95 °C for 20 h, with periodic stirring to ensure the formation of a clear and homogeneous solution. A similar procedure was followed to obtain PVA/PAA gels, but PAA was added to the solution in a ratio of 3:10 (w/w) relative to PVA. In both cases, the solutions were carefully poured into glass Petri dishes (preheated at 95 °C) and cooled to room temperature for 8 h. The polymeric solutions were submitted to 6 cycles of an FT process. Freezing occurred at −20 °C for 16 h and thawing at room temperature for 8 h.PVA hydrogels were prepared by dissolving the polymer powder in DD water  treatment at 120 °C for 1 h, giving rise to PVA/PAA+PEG+A hydrogels. The methodology followed for the materials’ preparation is summarized in the schematic diagram shown in After production, the hydrogels were washed in DD water for 48 h (exchanged at least three times per day) and then stored hydrated till being used. All of them were opaque white and presented an average thickness of 4.8 ± 1.2 mm in the swollen state. Before each characterization test, samples were cut with the proper dimensions, rinsed with DD water, carefully cleaned with absorbent paper, and soaked in fresh DD water for at least 24 h.The collection of the porcine knee joint was accomplished within 24 h post mortem to ensure the freshness of the tissues. Following joint dissection, full-thickness osteochondral plugs were harvested from the medial/lateral femoral condyles using a brazed hole cutter with an internal diameter of 6 mm. The underlying bone and vascularized tissue were removed from the deep zone of the cartilage plugs using a sharp scalpel, leaving the articular surface of the femoral condyles intact. Then, samples were washed with DD water and kept hydrated for 2 h at room temperature before being tested.The surface morphology of the materials was accessed using scanning electron microscopy (SEM). The images were acquired at 15 kV using an S-2400 microscope . Before being observed, the hydrated discs (diameter 10 mm) were frozen via immersion in liquid nitrogen, broken, and then lyophilized for 24 h in an LBFD-A21 freeze dryer . Subsequently, samples were coated with a conductive layer of gold/palladium (Au:Pd = 1:4) using a Q150T ES sputter coater . At least three images of the surface microstructure and cross-cut section of each material were taken with a magnification of 3000×.SC) was estimated through the following equation [EWC) was determined usingCylindrical samples (diameter 8 mm), fully hydrated in DD water (1 mL per sample), were dried in an oven at 37 °C for seven days. An OHAUS semi-micro analytical balance  was used to weigh the discs in their hydrated and dry states. The swelling capacity SCEach type of hydrogel was tested in triplicate. Measurements were also carried out in porcine cartilage samples (diameter 6 mm) following the same procedure.Surface wettability was assessed using the captive bubble method. The hydrated hydrogels were cut into rectangular strips (≈20 mm × 10 mm), fixed to a metal support, and immersed downward inside a quartz cell filled with DD water. Air bubbles (4.5 ± 1.5 μL) were formed beneath the material’s surface with a micrometric syringe with a J-shaped needle. Each bubble was monitored for 10 min, where it was recorded via several images at predefined times using a JAI CV-A50 video camera  mounted on an M3Z microscope  and connected to a DT3155 frame grabber . Image analysis was carried out with Axisymmetric Drop Shape Analysis Profile (ADSA-P) software. Three independent strips were used for each type of sample, where a total of 8–11 bubbles were analyzed. Measurements were also carried out in porcine cartilage samples following the same procedure.The mechanical properties were evaluated through compressive tests performed in a TA.XT Express Texture Analyzer  with a load cell of 50 N. Exponent Lite Express software (version 6) was used to acquire the stress–strain curves and process the results. The tests were performed with hydrated cylindrical samples (diameter 6 mm) in unconfined mode at room temperature. The discs were compressed at 0.1 mm/s to the maximum capacity of the load cell. Both loading and unloading responses were recorded. For comparison purposes, specimens (diameter 6 mm and thickness 3–4 mm) of the porcine cartilage were analyzed under the same testing conditions. Each type of sample was tested at least in quintuplicate.Rheological measurements were performed to evaluate the viscoelastic properties of the hydrated hydrogels. An MCR 92 rheometer in oscillatory mode with a parallel plate system (25 mm in diameter) was used. The gap was optimized for a 2% deformation of the hydrogels. Amplitude sweeps (γ = 0.01–100%) were carried out to determine the linear viscoelastic region (LVER) at a constant angular frequency of 10 rad/s. A strain value of γ = 0.1%, found to be within the LVER, was set during the dynamic frequency sweep tests (range: 0.1–100 rad/s) to determine the storage modulus (G’) and the loss modulus (G’’). All tests were done in quadruplicate for each hydrogel at 37 °C.The friction coefficient (CoF) of hydrogels against stainless steel was determined in reciprocal oscillating mode using a ball-on-disc geometry setup in a TRM 1000 tribometer . AISI 316L stainless steel spheres , purchased from Luis Aparicio SL , were used in the experiments. Before testing, the hydrogel discs (diameter 65 mm) were pre-equilibrated for 24 h in the lubricating media (PBS solution). The experiments were carried out in PBS at room temperature over a stroke length of 10 mm with a tangential sliding velocity of 42 mm/s and a sliding distance of 15 m. At least three replicates were performed for each hydrogel and testing condition.For the drug loading/release experiments, hydrogel discs (diameter 8 mm) were loaded via direct soaking of the dry samples  in 3 mL of a diclofenac (DFN) solution at 0.2% w/v, prepared in PBS. Once loaded, the discs were removed from the DFN solution, washed with DD water, superficially cleaned with absorbent paper, and placed in 3 mL of a PBS solution to perform the drug release assays. The experiments were done in sink conditions at 37 °C and 180 rpm using an Incubating Mini Shaker . At pre-established times, aliquots of 300 μL were taken and replaced with the same volume of fresh PBS solution. The absorbance of the collected solutions was measured at 276 nm with a Multiskan™ GO Microplate spectrophotometer  to estimate the amount of drug released. DFN solutions with different concentrations were prepared and their absorbance was measured at the referred wavelength to build a calibration curve. The release curves were obtained in triplicate. The methanol extraction method was applied to determine the total amount of DFN loaded into the samples . The druThe hen’s egg test (HET) on the chorioallantoic membrane (CAM) was accomplished with fertile white leghorn chicken eggs according to the ICCVAM recommendations  to predi® 3000 rotary tool . Then, the inner membrane was moistened with approximately 2 mL of NaCl aqueous solution (0.9% w/v). After 30 min of incubation, the saline solution was withdrawn and the inner membrane was carefully extracted with a surgical tweezer to expose the CAM.The eggs were placed in a PT-56S intelligent incubator  for 9 days at 37 °C and 60% relative humidity, and were automatically rotated every two hours. On the 9th day of incubation, the eggs were laid with the widest end facing upwards and a circular cut (diameter ≈ 18 mm) was made in the area of the air chamber to take out the eggshell using a Dremelw/v) or NaOH (0.1 M), respectively, into the eggs’ CAM. The membranes were monitored for the appearance of any signs of hemorrhage, vascular lysis, or coagulation, and the times it took for such injuries to occur  were recorded. The irritation score (IS) was calculated as follows [Hydrated hydrogel samples (diameter 8 mm) were directly placed on the eggs’ CAM and left for 5 min. Negative and positive controls were prepared, pouring 300 μL of aqueous solutions of NaCl (0.9%  follows :(6)IS= was used to perform the statistical analysis. Quantitative measurements are expressed in terms of mean ± standard deviation. Normality was verified using Shapiro–Wilk test and the homogeneity of variance using Levene’s test. When both were verified, one-way ANOVA was applied, followed by Tukey’s HSD post hoc test for multiple comparisons. Data that showed a normal distribution with unequal variances were analyzed using Welch’s ANOVA and Dunnett’s T3 post hoc test to determine significant differences between groups. An independent samples t-test was also used to compare means between two groups of data with a normal distribution when appropriate. A p-value lower than 0.05 was considered significant. All statistical differences between the data sets are indicated by asterisks . Unless stated otherwise, the asterisks are placed graphically on top of each group, referring to the comparison with the pair of the corresponding color.IBM


In the current study, rigid polyurethane foam (PUR) was modified with 10–30 wt.% sunflower press cake (SFP) filler, and its effect on performance characteristics—i.e., rheology, characteristic foaming times, apparent density, thermal conductivity, compressive strength parallel and perpendicular to the foaming directions, tensile strength, and short-term water absorption by partial immersion—was evaluated. Microstructural and statistical analyses were implemented as well. During the study, it was determined that 10–20 wt.% SFP filler showed the greatest positive impact. For instance, the thermal conductivity value improved by 9% and 17%, respectively, while mechanical performance, i.e., compressive strength, increased by 11% and 28% in the perpendicular direction and by 43% and 67% in the parallel direction. Moreover, tensile strength showed 49% and 61% increments, respectively, at 10 wt.% and 20 wt.% SFP filler. Most importantly, SFP filler-modified PUR foams were characterised by two times lower water absorption values and improved microstructures with a reduced average cell size and increased content in closed cells. It is estimated that within 10 years, global energy consumption will increase by ~50% [Rigid polyurethane foams are characterised by the lowest thermal conductivity, which varies from 0.018 W/(m·K) to 0.025 W/(m·K) when the traditional blowing agents are used, and from 0.033 W/(m·K) to 0.040 W/(m·K) when water is used as a chemical blowing agent [Polyurethane foams can be produced from mass polymerisation, which is the easiest technique for polyurethane foam synthesis and solely uses monomers such as polyols, isocyanates and physical or chemical blowing agents . CurrentCurrently, there are several studies on the use of natural fillers in polyurethane foam composites. For instance, the authors of  investigThe aim of this study is to investigate and evaluate the impact of high amounts of crushed SFP on rigid polyurethane (PUR) foam obtained from rapeseed oil- and sucrose-based polyols and water as a blowing agent. Moreover, rheological and structural characterisation, foaming process evaluation, and testing of thermal insulation, water resistance and mechanical performance were conducted, and the statistical interpretation of the results was implemented.Polyol BioPolyol RD was obtained from rapeseed oil  and Petol PZ 400-4G from sucrose . They were used as the main components for the production of PUR foam and SFP filler-modified PUR foams. Polyol from rapeseed oil has the hydroxyl value of 350 mg KOH/g, while the sucrose-based oil has a value of 421 mg KOH/g. Water contents of each polyol were <0.2% and <0.1%, respectively. As a second main component, polymeric 4,4′ -diphenylmethane diisocyanate Lupranate M20S  was selected. All PUR foams, including SFP filler-modified PUR foams, were blown using distilled water. To catalyse blowing and gelling reactions, Polycat 9  was incorporated. To stabilise foams and form cellular structures, silicone surfactant ST-52  was added. The SFP filler was supplied by a local company .The selected amounts of raw materials used for the preparation of PUR foam and SFP filler-modified PUR foams are presented in The system of two polyols was mixed with water, catalyst, and surfactant, and thoroughly mixed at a speed of 1800 rpm for 1 min. Before the addition of SFP filler, it was crushed and milled with a laboratory shredder for 1 min and thermally treated at a temperature of 110 °C for 24 h to remove the excess of moisture. After that, 10 wt.%, 20 wt.%, and 30 wt.% of SFP filler were added into the prepared mixture. The obtained premix was thoroughly mixed with isocyanate for 10 s at 1800 rpm. The obtained viscous mass was immediately poured into moulds and left to cure at a temperature of (23 ± 2) °C.The moisture content of particles was determined after drying three samples at (105 ± 2) °C until the change in mass between three weighings made 24 h apart was less than 0.1%, in accordance with the requirements outlined by . The structural studies of SFP filler, neat PUR foam and SFP filler-modified PUR foams were conducted using scanning electron microscopy (SEM) JEOL SM–7600F . Before the analysis of the samples, the filler and foams were coated with a gold layer under vacuum with the QUORUM Q150R ES  apparatus. The obtained images were used to further analyse structural parameters such as average cell size and the size of filler particles with ImageJ software.The test of dynamic viscosity was made using an SV-10 viscometer , which has an accuracy of 0.01 mPa·s and a measuring range of up to 1200 mPa·s.The characteristic foaming times and reaction profile were determined according to , Annex EThe linear dimensions of the samples were determined based on the requirements of , while tThermal conductivity measurements were conducted for 300 × 300 × 50 mm-sized samples, according to , with a The determination of the volumetric percentage of closed cells was carried out based on , method To determine the impact of water on the neat PUR foam and SFP filler-modified PUR foam, short-term water absorption by partial immersion was chosen according to ) with determination coefficient Thermal conductivity is one of the most important parameters for thermal insulating materials and is mainly determined by morphological features and apparent density . TherefoThe advantage of SFP incorporation can be noticed at 10 wt.% and 20 wt.% contents which reduce thermal conductivity values by 9% and 17%, respectively. Such a result may be explained by the increasing content of closed cells and reduced average cell size . Even thHowever, 30 wt.% SFP filler started to increase the thermal conductivity value and it became similar to the one at 10 wt.% SFP filler content. Even though the average cell size at such a filler content was reduced by 52% compared to the neat PUR foam, the microstructure d became Furthermore, it is evident that the results of thermal conductivity of modified PUR foams is dependent on the content of SFP filler. Therefore, it is of great importance to evaluate the strength of their impact on the final PUR foams. Test results indicate that the thermal conductivity of SFP filler-modified PUR foams may be approximated by the regression equation (Equation (2)) presented below.The resulting data show that the determination coefficients for the presented equation are The obtained determination coefficients indicate the suitability of the regression model for the proper description of thermal conductivity values of SFP filler-modified PUR foams.The mechanical performance of polymeric porous materials is influenced by a few factors such as closed cell content, apparent density of the final product, and filler topology, i.e., its ability to effectively adhere to the polymer matrix. Compared to neat PUR foam, SFP filler-modified PUR foams showed a noticeable improvement in compressive strength in the perpendicular and parallel directions . When itThe tensile strength test of the SFP filled PUR foams was conducted to investigate the PUR-SFP interaction. The interface between filler and matrix plays an important role in the mechanical properties of all composite materials because the load transfer to the dispersed phase occurs at the interface . The resAs a result, the improvements are attributed to three fundamental reasons concluded in the study conducted by . The firWater absorption is a vital property for materials which will have direct or indirect exposure to water. Short-term water absorption by partial immersion was chosen according to the requirements of the harmonised factory-made PUR products standard  and the 2).As the results show, SFP filler considerably improves water absorption of the resulting PUR foams. Interestingly, the reduction is two times higher compared to the neat PUR foam. Firstly, as sunflower press cake is a concentrated fodder which is obtained by pressing sunflower oil, the remaining oil (1.1–5.9 wt.% ) in SFP 3, while short-term water absorption by partial immersion results showed that the parameter was twice reduced at 20 wt.% SFP. Mechanical performance was significantly improved; compressive strength perpendicular to the foaming direction increased by 28%, compressive strength parallel to foaming direction increased by 67%, and tensile strength increased by 61%. However, among other properties, significant improvement in mechanical performance was achieved for PUR foams with 30 wt.%; the compressive strength increased by 114% and 71% in the perpendicular and parallel directions, respectively, and tensile strength by 89%.Polyurethane foam was modified with 10–30 wt.% sunflower press cake filler. The impact of sunflower press cake filler on selected properties of PUR foams, such as rheology , thermal insulating properties , mechanical properties  and water resistance  were investigated. It was determined that the greatest improvement was achieved for PUR foams with 20 wt.% SFP. The thermal conductivity was reduced by 17% at an average apparent density value of 65 kg/mIn summarising the obtained results for SFP filler-modified PUR foams, it is worth noting that such building materials are promising for future application as thermal insulating layers in building envelopes. In addition, the replacement of petroleum-based polyols with plant-based polyols, traditional blowing agents with water, and synthetic fillers with natural or plant-based fillers will contribute to the achievement of sustainability goals and the reduction of carbon dioxide emissions coming from the building industry. However, further studies on plant-based PUR foams are needed in order to reduce thermal conductivity values.


Scylla, a highly valued delicacy. The aim of this review paper is to summarise the status and information on the current nursery culture stage of the mud crab. Various aspects of mud crab hatchery described in this paper are expected to facilitate practitioners and lay people to easily understand a mud crab nursery. This review also provides guidelines for researchers in conducting future research and development on a mud crab nursery in order to increase the production of mud crab crablets for the farming industry.Nursery culture knowledge is important for successful commercial seed production especially for mud crab, genus Scylla, proceed from the megalopa stage to crablet instar stages. We review the definition and several of the key stages in mud crab nursery activities. The practice of the direct stocking of megalopa into ponds is not recommended due to their sensitivity. Instead, nursery rearing is needed to grow-out mud crabs of a larger size before pond stocking. Individual nursery rearing results in a higher survival rate at the expense of growth and a more complicated maintenance process compared with communal rearing. The nursery of mud crabs can be done both indoors or outdoors with adequate shelter and feed required to obtain a good survival percentage and growth performance. Artemia nauplii are still irreplaceable as nursery feed, particularly at the megalopa stage, while the survival rate may be improved if live feed is combined with artificial feed such as microbound diet formulations. Water quality parameters, identical to those proposed in tiger shrimp cultures, can be implemented in mud crab rearing. The transportation of crablets between different locations can be done with or without water. The provision of monosex seeds from mud crab hatcheries is expected to become commonplace, increasing seed price and thus improving the income of farmers. Numerous aspects of a mud crab nursery including nutrition; feeding strategies; understanding their behaviour, i.e., cannibalism; control of environmental factors and practical rearing techniques still need further improvement.The nursery stages of mud crab, genus  Scylla are a seafood product that sees high demand in both local and international markets [Scylla: S. olivacea, S. paramamosain, S. serrata and S. tranquebarica. All species are reported to have been commercially farmed [Scylla increases annually [Mud crabs of the genus  markets ,2. Currey farmed . The demannually , which hannually ,5. The iannually ,7. In adannually ,9,10, cuannually ,12 or inannually  and growannually ,15—couldannually . Diseaseannually .Portunus pelagicus, where a high survival has been observed when stocking into ponds at the megalopae phase, the critical transition step of M to Crablet 1 (C1) in mud crabs has produced inconsistent and unreliable results, with high mortality often reported during the metamorphosis from M to the C1 stage [Scylla, including the principal technical practices of a mud crab nursery from stocking to transportation. Several issues during this phase are identified and potential solutions are discussed.Mud crab hatcheries typically involve the maintenance of larvae to crablet size (>5 mm) before being marketed to farmers ,18. The C1 stage ,22. TherC1 stage ,23,24. TScylla, includes one megalopa stage and subsequent crab instar stages. The transition from M into the C1 stage is around 6–7 days [The nursery stage of mud crab, genus 6–7 days ,22 where6–7 days  (Table 16–7 days  and (ii)6–7 days ,28,29. T6–7 days ,31. Gene6–7 days ,28,29. TThe nursery area may be located indoors or outdoors. M are nursed in concrete, fiberglass or plastic tanks or net cages within brackish water ponds  22,23,2,31,32. TThe nursery of mud crabs in indoor conditions still needs to be further investigated in order to optimise the hatchery production of crablets. Among the methods that can be applied to indoor mud crab protocols is biofloc technology. Further research is needed to determine whether the biofloc system used in the shrimp farming industry can be used for future M maintenance and whether feed in the form of a biofloc meal can be a food substitution for M and crablets.Another important consideration is whether to rear individually or communally. The nursery rearing of M individually (indoor condition) in good water quality conditions could raise the survival up to 90–100% ,33. AlthIn the communal rearing technique, cannibalism is a major problem. To reduce cannibalism, black nylon, bunched netting or seaweed may be placed at the bottom of the rearing tanks as shelters while a few may also be allowed to float in the water column ,31,34,352) due to the fact that the M stage naturally stays at the bottom just before it moults to C1. Further studies related to the optimal density of the M per unit area need to be carried out in the future. The stocking of M with densities of 10, 20 and 30 ind/m2 in net cages within earthen ponds was shown to attain an average 50.5% survival rate [2 had a survival rate between 11.7–31.7% on different dietary treatments [The stocking density of 3 to 5 days old M in nursery tanks is typically around 1000–2000/ton of water ,23. The val rate  while theatments . Beside 2 for 15 days, survival rates of 71%, 62% and 58% were reported, respectively [2 had a survival rate of 52–66% while a further extended nursery to 60 days with a density of 30 ind/m2 attained a survival rate of 64–67% [2. Hence, further research is needed to identify the factors controlling the survival rate in relation to the stocking density.When C1 was cultured at high densities of 110, 175 and 230 crabs/mectively . A longef 64–67% . A lowerThe age of M at moulting is likely to affect the moulting success of M to C1. Syafaat  reportedDuring transportation, the avoidance of a stationary transport condition or continuous agitation has been indicated to reduce the probability of M to grasp each other . The traArtemia is often used as the main feed during the maintenance of M to crablet stages with additional dried shrimp, dried mud worms, prawn/shrimp meat, fish meat or artificial feed (shrimp larvae feed) being added as high as a 1–5 mg/L concentration or greater [Artemia. Apart from a suitable feed size, feed nutritional quality is another important component supporting the development of M into the C1 stage. The use of enriched Artemia (instar 2) resulted in a better survival than the use of only Artemia nauplii [Acetes is a potential live food in a mud crab nursery from megalopa to the crablet stage. The rearing of megalopa on Acetes alone or combined with minced shrimp meat, locally formulated feed or commercial feed showed a better survival than treatments fed with minced shrimp meat, locally formulated feed or commercial feed alone [ greater ,19,45,48 greater  clarifie nauplii . Acetes ed alone . It is red alone  while thed alone ,52.Artemia showed a higher survival rate when compared with the treatment being fed Artemia alone in rearing from the Z5 and M stages [Artemia nauplii, produced more C1 than the treatment being fed only artificial feed juice (without spirulina powder and digezym) and Artemia nauplii [Artemia suggested that an MBD can be formulated to contain beneficial nutrients insufficient in Artemia [Supplementation with artificial feed (shrimp post-larvae feed) and M stages ,45. The  nauplii . Shorter Artemia , which h Artemia ,54,55 an Artemia .Scylla has always been heavily dependent on live prey, particularly Artemia [Artemia compromised the vitality of larvae [Artemia nauplii could affect both the larval development and the survival of mud crab larvae [Artemia [Artemia nauplii [The nursery culture of mud crab  Artemia ,44. Nonef larvae ,56,57. Ab larvae . MBD fee[Artemia ,41,42. F nauplii  , a temperature of 27–32 °C, a pH of 7.5–8.5, a DO >4 ppm and ammonia <0.01 ppm . AlkalinTemperature and salinity are two key parameters to be considered both in the larvae rearing and the nursery phases of mud crabs ,62,63,64Vibrio spp. on the culture media [Vibrio spp. is still needed. However, the rearing of portunid crab larvae with probiotics has been proven to improve the production of mud crab crablets [Vibrio spp. populations [The use of artificial feed during mud crab larvae rearing may trigger the growth of re media . Hence, crablets  and theyulations ,69 whereThe harvesting process for the nursery phase (M to crablets or from early crablets (C1–C2) to a larger crablet phase) occurs between two and four weeks after stocking. Long nursery periods in small areas  will lead to a high mortality due to cannibalism. Two methods, the dry (moist) method ,70 and tS. paramamosain, the difference of the abdominal flap can be seen clearly at the C5 stage (CW of ±1 cm) using a microscope (magnification of 8–20×) whereas in the ±C9 stage (CW of ±2 cm), the difference can be seen with the naked eye [S. serrata, the different shape of the male and female abdomen enables the sexing of crabs above a 3 cm carapace width with a casual inspection while below this size, the sex can be determined at 30× by means of a binocular microscope [The morphological sex differentiation of mud crabs is mainly based on the shape of the abdominal flap and body size dimensions ,73. In taked eye . In mud croscope . Femalescroscope   compared with all females  and a cuP. pelagicus. Although operating a mud crab nursery for crablets individually exhibits a higher survival when compared with communal rearing, the individual nursery involves a higher complexity and resourcing so that it is necessary to consider rearing technology using automatic machinery especially for the feeding process. A mud crab nursery can be carried out both indoors  and outdoors  within net cages. In all applications, the rearing needs to be accompanied by the use of adequate shelter, particularly for a communal nursery.A mud crab nursery in the hatchery beginning from the M stage is important for production because the stocking of M directly to ponds has not shown good results when compared with species such as Artemia is still the most common main feed in the M phase but Artemia combined with additional feed  provides a better survival compared with Artemia alone. An MBD of the right composition is a feed candidate to replace or substitute for Artemia in the M phase.The availability of adequate food mainly contributes to suppressing cannibalism. Feed in the form of live Although mud crabs are believed to show a good resilience in nature and survive in extreme conditions , the water quality parameters for a shrimp culture can be used as a guide in mud crab farming activities. Further studies related to the transportation of crablets need to be carried out to produce practical, inexpensive methods and to determine the extent of crablet resistance during transportation without water. In addition, an improved knowledge of the sex differentiation of mud crabs during the early crablet stage would be expected to provide added value to mud crab hatcheries.Scylla, and implementing them in nursery operations. It is clear that current hatchery practices have room for development and the farming industry could improve in several aspects with future research on exploring the feasibility of individual rearing and biofloc application in controlling water quality, establishing monosex seed production as well as improving methods of transportation during critical mud crab stages. This paper is also expected to provide guidelines to researchers in conducting future research and development on a mud crab nursery, subsequently transferring the fundamental knowledge to the farming community.Various aspects of a mud crab hatchery described in this paper are expected to facilitate practitioners in understanding the natural conditions of mud crabs, genus


Traditional methods to assess hMSCs differentiation typically require long-term culture until cells show marked expression of histological markers such as lipid accumulation inside the cytoplasm or mineral deposition onto the surrounding matrix. In parallel, stem cell differentiation has been shown to involve the reorganization of the cell’s cytoskeleton shortly after differentiation induced by soluble factors. Given the cytoskeleton’s role in determining the mechanical properties of adherent cells, the mechanical characterization of stem cells could thus be a potential tool to assess cellular commitment at much earlier time points. In this study, we measured the mechanical properties of hMSCs cultured on soft gelatin-based hydrogels at multiple time points after differentiation induction toward adipogenic or osteogenic lineages. Our results show that the mechanical properties of cells (stiffness and viscosity) and the organization of the actin cytoskeleton are highly correlated with lineage commitment. Most importantly, we also found that the mechanical properties and the topography of the gelatin substrate in the vicinity of the cells are also altered as differentiation progresses toward the osteogenic lineage, but not on the adipogenic case. Together, these results confirm the biophysical changes associated with stem cell differentiation and suggest a mechanical interplay between the differentiating stem cells and their surrounding extracellular matrix. Mesenchymal stem cells (MSCs) lineage specification is tightly regulated by their microenvironment through a wide variety of chemical and mechanical cues . Since tThe mechanical properties of a cell can reflect cellular state and are closely related to diseases and pathological conditions . The cytWe have previously shown that the cell’s mechanical properties and the underlying organization of the cell’s cytoskeleton are strongly correlated and that they can be used to better understand at the cellular level physiological processes such as aging or cancer . In partUsing this approach, we have tracked at the single cell level the mechanical and morphological changes associated with hMSCs differentiation. In particular, the strongest trends found were a persistent increase in cytoskeletal stiffness for cells differentiating toward osteogenic linages and a consistent decrease in cytoskeletal stiffness for cells differentiating toward adipogenic lineages. Further, we have confirmed that the observed mechanical changes are associated with a marked reorganization of the underlying actin cytoskeleton. Finally, we have found that the differentiation process toward osteogenic lineages is also accompanied by mechanical changes in the neighboring gelatin scaffold. Our results thus point toward a mechanical interplay between differentiating cells and their ECM, and highlight the advantage of using biomimetic cell-modifiable soft substrates as scaffolds for regenerative medicine.Gelatin hydrogels (Gel) were made of gelatin powder  dissolved in either 10mM/20mM/40mM genipin  solution at a concentration of 3%/6%/9% (w/v). The mixture was kept at 40°C under moderate stirring until crosslinking was started, as indicated by the solution turning into a blue color. The polymer solution was cast in the mold and the obtained samples were left at room temperature for 24 h until polymerization was complete. The formed hydrogels were immersed in 70% ethanol solution to remove the excess genipin and sterilization. Then, the sterilized hydrogels were washed with PBS for 3 times and cultured in cell culture medium for 24 h in cell incubator before cell seeding.2. Growth media was removed and replenished every 3 days.Primary bone marrow MSCs were sourced from Promocell, Heidelberg, Germany. Expanded MSCs were used at passage 4. The basic media that was used to maintain cells during experiments and for proliferation was DMEM  supplemented with 10% FBS (Sigma), 0.1% FGF-basic1 (PeproTech) and 1% penicillin-streptomycin (Sigma). Cells were seeded at a concentration of 4000 cells/cmFor osteogenic induction media (ODM), DMEM (X-Pan) was supplemented with 50 μM ascorbic acid (Sigma), 100 nM dexamethasone (Sigma) and 10 mM β-Glycerophosphate. For adipogenic induction media (ADM), DMEM was supplemented with 500 μM IBMX(Sigma), 1 μM dexamethasone (Sigma), 100μM indomethacin, and 10μg/mL insulin (Sigma). In both cases, induction media was removed and replenished every 3 days.Oil Red O stock solution was prepared by adding 300 mg of Oil Red O (Sigma O0625) to 100ml of 99% isopropanol (0.3% w/v). To begin the histochemistry protocol, 6 mL of Oil Red O stock solution were mixed with 4 mL DI water. Cell samples were thrice rinsed in sterile PBS and fixed with 4% paraformaldehyde for 10 min. After being washed twice with DI water, 60% isopropanol was added and left to incubate for 5 min at RT. Subsequently, the Oil Red O working solution was added and incubated for 5 min at RT. Finally, the cell samples were rinsed repetitively with DI water until the solution became clear and left for storage. Imaging was carried out using phase contrast mode with an inverted microscope (Leica DMI4000B) with a 10× 0.5 NA objective lens and a CCD camera (Leica DFC300FX).Samples were thrice rinsed in sterile PBS and fixed with 4% paraformaldehyde for 10 min. Following, the samples were rinsed with distilled water, and stained with 0.5 mL of Alizarin Red S solution  for 10 min. Excess dye was rinsed with distilled water, and microscopy images were acquired by an inverted microscope (Leica DMI4000B) with a 10× 0.5 NA objective lens and a CCD camera (Leica DFC300FX).TM 488 Conjugate (Santa Cruz) at 1:500 dilution and 1:1000 DAPI (Santa Cruz) for 30 min at room temperature. All chemicals were purchased from Sigma Aldrich. Then samples were washed by PBS for 3 times and transferred with the seeded surfaces facing down into a cover glass. Zeiss LSM710 Confocal Microscope (Germany) was used to image cytoskeleton at 20× magnification and a cooled CCD camera.Mesenchymal stem cells cultured on gelatin hydrogels were fixed in 4% PFA (Sigma) and then permeabilized with 0.1% Triton X-100 (Sigma). Samples were incubated with Phalloidin CruzFluorThe cytoskeleton organization parameters were analyzed according to our previous studies using a dedicated pipeline (CSKMorphometrics) written in MATLAB .The mechanical properties of MSCs cultured on gelatin gels were assessed via AFM using QI mode . To probe the samples, a pyramidal silicon nitride tip, with a cantilever spring constant of ∼0.03 N/m  was used. Measurements were carried out by performing force-indentation ramps, using 100 × 100 μm range at a 2 μm/s ramping speed. A grid of 32 × 32 pixels was generated by performing a force-indentation curve for each pixel. The grid was defined broad enough to include also the surface of the gel neighboring the probed cell. Therefore, for each obtained grid, approximately half of the probed pixels corresponded to the cell and the other half to the surface of the gelatin gel. A minimum of 10 cells were probed for each timepoint and condition.During the data post-processing steps, the pixels on each grid were classified and split as belonging to the cell or the gelatin substrate based on their measured height, using Otsu’s method for image thresholding . SubsequCells were seeded in 35mm-diameter petri dish with seeding density of 30000 cells per dish and cultured for up to 14 days as described above. RNA was extracted using the RNeasy Plus Mini Kit  under manufacturer’s instructions at day 1, 7, and 14, and first-strand cDNA synthesized using QuantiTect Rev.Transcription Kit . Quantitative PCR was performed using the Taqman single-gene expression assay (Thermo Fisher Scientific) in a QuantStudio 7 Flex System (Thermo Fisher Scientific). The following oligonucleotide primers were used: PPARA (Hs00231882_m1) for adipogenesis and RUNX2 (Hs00231692_m1) for osteogenesis. In addition, GAPDH (Hs02758991_g1) was used for normalization. The comparative Ct method was applied to give relative gene expression values. All the values were compared with that gene expression at day 1.P-value of < 0.05 was considered significant.Statistical analysis was performed in GraphPad Prism. Each experiment was performed independently in triplicate. Data are presented as mean ± standard deviation (SD) values or mean ± standard errors (SE) according to different situations. For the majority of analysis, nested 1-way ANOVA followed by multiple comparisons against “day 1” data using Dunnett’s test was used. A In order to find the composition of gelatin-genepin gels for a given optimal stiffness (10 kPa), we produced a panel of 3%/6%/9% (w/v) gelatin hydrogels crosslinked in 10mM/20mM/40mM genipin solution. Subsequently, we measured their stiffness (reported throughout the manuscript as Young’s modulus) at 37°C using AFM. As expected, we observed a linear increase in hydrogel stiffness with increased gelatin powder concentration. For the 9% gelatin hydrogels, their stiffness grew linearly with increasing genipin solution. Conversely, for the 3% and 6% gelatin hydrogels, their stiffness did not significantly change until the concentration of genipin solution reached 40mM .Following AFM measurements, the hydrogels were sterilized and immersed in basic medium, and subsequently kept inside a cell culture incubator for 28 days. This procedure was carried out to confirm the stability of their mechanical properties when kept for a long time in temperature and bathing conditions that mimicked those of cell culture. After 28 days of incubation, the stiffness of all genipin-crosslinked gelatin hydrogels displayed no significant differences, thus confirming their mechanical stability in cell-less physiological conditions . SimultaFor all subsequent experiments using genepin-gelatin hydrogels, we chose to use only those composed of 6% gelatin in 10mM genipin solution. The reasoning was that the obtained stiffness (∼10 kPa) falls between those reported for adipogenic tissue (at around 4 kPa) and the bone tissue (at around 30 kPa) , while rhMSCs committed to different cell specifications on gelatin hydrogel, as confirmed by histological staining methods at Day 14. As expected, the presence of lipid droplets was observed in hMSCs cultured in ADM and phosphate deposits were found in hMSCs cultured in ODM . SimilarSingle cell topography and morphology were carried out by AFM at different time points. For both groups, cell shape and height were similar until Day 7 (not shown). As a qualitative observation, we found that hMSCs in ADM increased in height, likely as lipids accumulated in their cytosol, while cells in ODM started to remodel their neighboring ECM, which became rougher in its topography.We measured the mechanical properties of hMSCs at days 1, 4, 7, 10, and 14. At day 1, the mechanical properties of hMSCs cultured in either ADM or ODM were still very similar, and in both cases the stiffness of the cortical actin layer (around 0.5 kPa) was much lower than the stiffness of the cytoskeletal fiber’s underneath (around 2 kPa). For hMSCs cultured in ADM, the cortical stiffness had quadrupled to around 2 kPa by day 4 but remained constant for all later time points. This observation suggests that the re-organization of cortical actin takes place at an early stage of differentiation, with no further changes happening after that. Conversely, the cytoskeletal stiffness of ADM cells fell slightly between days 1 to 7 and then dropped markedly to around 0.5 kPa at day 14. Remarkably, the cytoskeletal stiffness observed at day 14 displays a similar stiffness to those reported for fat tissues at around 0.3 kPa . In addiFor hMSCs cultured in ODM, both the stiffness of the cortical actin layer and the underlying cytoskeleton increased monotonically with time, reaching at day 14 values that were fourfold and twofold those of day 1, respectively. In addition, and similar to the observations in ADM cells, the principal changes in cortical actin stiffness took place at the earliest time points, followed by a subsequent stabilization of their values. Conversely, the values of cytoskeletal stiffness displayed a rate of increase that was smaller but prevalent in time.The values for cellular viscosity and adhesive force were also obtained through the acquisition of AFM force-indentation curves. For both ADM and ODM, the values of viscosity increased significantly with time, even though the trends were less marked than those displayed for elasticity . SimilarTo obtain quantitative data cell gross morphology and F-actin organization, fluorescence images using phalloidin staining were obtained and subsequently processed using our image quantification pipeline. For both types of differentiation media used, cell area and F-actin assembly increased with time, even though hMSCs in ODM spread further since day 7 . Both thIn the present study, we were also able to monitor the changes in mechanical properties of the soft hydrogels in the vicinity of cultured cells. We found that the interaction between cell and gelatin scaffold could lead to matrix remodeling and measurable changes in the gels’ topography, stiffness, and viscosity. Matrix remodeling was already visible when assessing optical images obtained at low magnification using phase contrast imaging. For gels cultured with hMSCs in ODM, their surface became grainy, with small dot-like objects distributed randomly. In the particular case of a gel where ODM cells were kept for up to 9 weeks in culture, we even observed fibers over the gel’s surface . ConversThrough our force-indentation measurements, we were able to assess whether the changes in the gel’s topography in the case of ODM were also associated to changes in the mechanical properties. Indeed, we found that gel’s mechanics values showed a marked decrease with increasing time in ODM cell culture, dropping to 50% and 41% of their initial values by day 14 for Young’s modulus and viscosity, respectively . ConversIn this study, we have tracked at the single cell level the mechanical changes associated with hMSCs differentiation toward osteogenic and adipogenic lineages. While cortical actin stiffness displayed an abrupt and significant increase at the earliest time points of differentiation for both media, cytoskeletal stiffness showed significant and opposite monotonic changes between ODM and ADM for the duration of the experiments. The organization of the actin cytoskeleton was similarly significantly altered during differentiation, but the observed trends in ODM and ADM were largely parallel. Finally, we also found that ODM differentiation gave rise to mechanical softening and topography remodeling in the neighboring gelatin scaffold.A number of previous studies have tracked the mechanical changes of stem cells upon differentiation induction using AFM . NeverthThe organization of the actin cytoskeleton has also been considered a reliable biomarker of stem cell differentiation , even thIt should be stressed that, even though it is single cell and multiparametric in nature, our methodological approach doesn’t allow us to track the same individual cells for days along their differentiation path, nor measure the exact same cells with all techniques presented here . Because of that, we can’t rank our measured mechanical or CSK morphological parameters based on their positive (or negative) spearman correlation with day of differentiation of gene expression of lineage markers. Consequently, it is beyond the scope of this study to formally identify the most suitable biophysical biomarker of stem cell differentiation. Nevertheless, based only on the significances obtained in our statistical tests for all parameters measured, mechanical parameters appear to provide a more promising avenue than CSK morphological parameters, and as already discussed above, the cytoskeletal stiffness is the mechanical parameter that is more strongly related to differentiation lineage and maturity.de novo deposition and synthesis. In any case, our results highlight that the use of degradable biomimetic scaffolds as soft substrates for stem cell differentiation should allow a broader characterization of the interplay between differentiating cells and their ECM, including a potentially dynamic and finely tuned mechanical interplay between cells and scaffold. Accordingly, we anticipate that the use of biomaterials that can be readily remodeled and mechanically altered by their harbored cells will promote and enhance the use of bioscaffold-based regenerative therapies.While the role of the cell’s substrate stiffness in directing stem cell differentiation has been known for more than a decade, fewer studies have focused on assessing the mechanical changes of stem cells as they differentiate when cultured on soft scaffolds. Here the consensus is that ODM differentiation leads again to cell stiffening and actin reorganization, and that the choice of substrate affects the magnitude and rate at which these changes take place . In thisThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.HM and NG designed the studies. HM conducted all the experiments. HM, NG, and TC contributed to analyzing and interpreting the data, drafted the manuscript, and edited the final submission. All authors have read and agreed to the published version of the manuscript.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.