Thousands of research studies concerning genome-wide association studies (GWAS) in Alzheimer’s disease (AD) have been published in the last decades. However, a comprehensive understanding of the current research status and future development trends of GWAS in AD have not been clearly shown. In this study, we tried to gain a systematic overview of GWAS in AD by bibliometric and visualization analysis.The literature search terms are:  AND (“Alzheimer’s Disease” or “Alzheimer Disease”). Relevant publications were extracted from the Web of Science Core Collection (WoSCC) database. Collected data were further analyzed using VOSviewer, CiteSpace and R package Bibliometrix. The countries, institutions, authors and scholar collaborations were investigated. The co-citation analysis of publications was visualized. In addition, research hotspots and fronts were examined.Neurobiology of Aging was the journal with the highest number of publications. Aβ, tau, immunity, microglia and DNA methylation were research hotspots. Disease and causal variants were research fronts.A total of 1,350 publications with 59,818 citations were identified. The number of publications and citations presented a significant rising trend since 2013. The United States was the leading country with an overwhelming number of publications (775) and citations . The University of Washington and Harvard University were the most prolific institutions with 101 publications each. Bennett DA was the most influential researcher with the highest local H-index. TREM2 as a potential immunotherapy target, and (3) DNA methylation. The research fronts are (1) looking for genetic similarities between AD and other neurological diseases and syndromes, and (2) searching for causal variants of AD. These hotspots suggest noteworthy directions for future studies on AD pathogenesis and genetics, in which basic research regarding immunity is promising for clinical conversion. The current under-researched directions are (1) GWAS in AD biomarkers based on large sample sizes, (2) studies of causal variants of AD, and (3) GWAS in AD based on non-European populations, which need to be strengthened in the future.The most frequently studied AD pathogenesis and research hotspots are (1) Aβ and tau, (2) immunity and microglia, with  Alzheimer’s disease (AD) is the most common cause of dementia, with tremendous economic and social burden. About 6.7 million Americans living with AD in 2023 are 65 years and older, and as the aging population increases, the prevalence of dementia is predicted to increase to 131.5 million in 2050 . HoweverAPP, PSEN1 and PSEN2 to cause early-onset AD, while early-onset form only accounts for less than 5% of all AD cases , immune response , endocytosis  have been proven to be associated with the risk of AD  in AD . By usinsk of AD . HoweverBibliometrics is a reliable means for literature analysis. It allows quantitative description for publications of a specific field using mathematical and statistical methods, and enables visualization of bibliometric statistics by CiteSpace, VOSviewer and R software. It helps researchers identify research trends, hotspots and critical cooperation networks that collectively guide academic decisions . In thisA comprehensive search was performed within the Web of Science Core Collection (WoSCC) database  using the following search strategy: TS =  AND (“Alzheimer’s Disease” or “Alzheimer Disease”), document type = (article or review), and language = English. The time frame was limited from January 1, 2007 to December 31, 2022. All data searches and retrievals were completed in a single day on April 3, 2023, to minimize the bias led by database updates .All bibliographical information of publications, including publication year, title, author’s names, affiliations, nationalities, abstracts, keywords, journal names and citation numbers, were recorded and then converted to text format. Subsequently, the data was imported into Microsoft Excel 2019 (Microsoft Corporation). Non-English publications and duplicate publications were removed. Then two researchers (Junyao Zhang and Yinuo Wang) independently examined and manually excluded the publications unrelated to the topic, 1,221 publications were agreed to meet the inclusion criteria. Then, the third researcher (Yingying Zhang) examined the parts where the other two researchers disagreed (234 publications) and made a final assessment to reach a consensus among researchers. In the end, 1,350 publications  were included in the final dataset .The final dataset was imported into bibliometric software. VOSviewer (version 1.6.18) was used for co-authorship analysis of countries, institutions and researchers, and also used for keyword co-occurrence analysis. CiteSpace (version 5.8.R3) was used for publication co-citation analysis, knowledge base analysis, knowledge flow analysis and burst keyword analysis. R package Bibliometrix (version 4.2.1) was used for the authors’ local H-index analysis. In addition, the Microsoft Excel 2019 (Microsoft Corporation) software was used to present global trends of annual publications and citations. SCImago Graphica (version beta 1.0.21) was used for geographic visualization in country co-authorship analysis.All the 1,350 publications  were cited 59,818 times from the first publication in 2007 to the end of 2022. The trend of annual publication and citation numbers was generally on the rise. The annual publication number increased rapidly after 2013 and peaked in 2021 with 187 publications. The average number of citations per year increased and maintained at a high level after 2013 .Seventy countries and regions participated in the publication of GWAS in AD. When ranked by the number of publications, the United States was the most productive country with 775 publications cited 42,237 times, followed by China with 309 publications cited 7,259 times, and the United Kingdom with 302 publications cited 25,311 times. The map indicating the contributions of countries or regions in this field is shown in A country co-authorship map is shown in Overall, a total of 1,819 institutions performed GWAS in AD. As presented in An institution co-authorship map is shown in According to the overlay map generated by VOSviewer, the Mayo Clinic, the University of Pittsburgh, the University of Toronto, the University of Miami and the University of California Los Angeles started working on GWAS in AD the earliest. Columbia University, Harvard University, Rush University, the University of Washington, the University of Cambridge and King’s College London published the most between 2016 and 2017. Research by Stanford University, the University of Oxford and Duke University were the institutions published the most after 2018 .Local H-index refers to h publications cited at least h times for each paper in a certain field. It is an important index to comprehensively measure the quantity and quality of a researcher’s output. To investigate the most influential researchers, the researchers with the highest H-index were shown. As demonstrated in As indicated by the researcher co-authorship map, Bennett DA, De Jager PL and Saykin AJ were critical researchers and cooperated extensively with others. Researchers from China were in the red cluster, with strong collaborative relationships established with each other, while little collaboration between Chinese researchers and researchers in other countries was observed. And Yu JT and Tan L were influential researchers in this cluster .According to the publication co-citation analysis in TREM2, #meta-analysis, #PICALM, #eQTLs, #brain expression, #snRNA-seq, #linkage analysis, #microglia and #Mendelian randomization.In the knowledge base map, articles co-cited by another article had a close academic relationship and were therefore grouped into the same cluster. Tags that represent the knowledge base of the cluster were extracted from the titles of the publications. As demonstrated in Neurobiology of Aging had 105 publications and 2,910 total citations, followed by J Alzheimer’s Disease with 95 publications cited 1,561 times, Plos One with 49 publications cited 2,277 times, and Alzheimer’s & Dementia with 47 publications cited 1,805 times. Although Nature Genetics only had 22 publications, it received an overwhelming 11,327 citations.As demonstrated in To display the evolutionary relationship between citing and cited journals, the knowledge flow overlay map was generated . The dotPICALM, #CLU, #CD33 and #ABCA7, were shown in the yellow cluster  belonged to the red cluster. #genetic association, #protein, #SNP and #population were assigned to the blue cluster. While the most often mentioned genes, including # cluster .APOE, #late-onset AD, #suseptibility gene, #identifies variant, #CLU, #PICALM, #common variant, #CD2AP, #EPHA1, #CD33 had burst time before 2016. The keywords #Mendelian randomization, #Aβ, #UK Biobank, #meta-analysis, #causal association, #causal effect, #disease, #microglia and #sensitivity analysis has become popular since 2020 till now, suggesting the present research fever of the field.To visualize the dynamics of frontier topics in the field, the burst analysis of keywords from 2007 to 2022 was performed. The whole segments stand for the time span of each keyword, while the red segments represent sudden increases in the frequency of the keyword occurrence during this period. The burst strength is a statistical value calculated from the keyword occurrence frequency over time. The top 20 keywords with the strongest bursts are shown in We investigated 1,350 publications, including original articles and reviews on GWAS in the AD field between 2007 and 2022. Since several small GWAS opened the new era of AD research in 2007, the number of annual publications of GWAS in AD has been generally on the rise, indicating it is an academic concern. There were not many citations in the first few years, while the average citations per year increased and maintained at a high level after 2013 , which sNeurobiology of Aging was the journal that published the most research in this field, and Nature Genetics was the authority of this field with the largest number of citations.Then, we got an overview of GWAS in AD by demonstrating the main contributors and their cooperation networks. The highest number of studies was generated by the United States (775), followed by China (309) and the United Kingdom (302). When ranked by total citations, the United States had an overwhelming 42,237 citations, and the United Kingdom ranked second with 25,311 citations. Although China had many publications, the total citations were not high  . The topTREM2, #meta-analysis, #PICALM, #eQTLs, #snRNA-seq, #linkage analysis, #microglia and #Mendelian randomization, which were important words or phrases that summarize the basic concepts underlying the field, including research objects and methods of GWAS in AD.As indicated in Knowledge flow analysis is an intuitive way to show the developmental trajectory of research. The cited literature was published earlier, and the citing literature was published later. As Our keyword analysis revealed that #Aβ and #tau were frequently mentioned keywords in this field. Extracellular plaques that consist of Aβ and intracellular neurofibrillary tangles (NFT) with over-phosphorylated tau protein are the basic pathological features of AD, and are recognized as major players in disease progression .APOEε4 and ABCA7 were identified to be most strongly associated with Aβ production  and endosomes (FERMT2 and SORL1) can also modulate APP metabolism and Aβ production  Aβ and tau levels has been conducted. Although in its initial stage, it has identified vital genes associated with Aβ and tau . Ramananβ burden . In the entified . At presAPOE, TREM2, CD33, CLU, CR1, ABCA7, EPHA1, MS4As, INPP5D, SPI1, PLCG2, ABI3, HLA-DR15, etc.) have been reported to be associated to AD  is a microglial receptor encoded by function . TREM2 R as 2013 , while mammation . In contβ uptake . Interesβ uptake .TREM2 in the spotlight for immunotherapy. A study by van Lengerich B et al. reported that a TREM2-activating antibody is capable of promoting microglial glucose metabolism in AD mice  silencing leads to reduced tau pathology  is a biomedical database containing half a million volunteers’ genetic and health information . UK BiobAs GWAS continues to evolve, some methodologies have also been developed. Mendelian randomization (2020–2022) is a statistical method based on GWAS, which is used to infer causal associations between exposures and disease outcomes using genetic variants. Mendelian randomization can effectively reduce confounding bias due to the advantage of alleles being randomly assigned to offspring. Meanwhile, sensitivity analysis (2021–2022) is a way to assess the reliability of conclusions obtained from Mendelian randomization studies and further enhance the studies’ credibility. In recent years, Mendelian randomization and sensitivity analysis have become cutting-edge methods frequently used in AD genetic studies.There are certain limitations to our study. Only references in the WoSCC database and publications written in English were included in our study owing to the nature of bibliometric software. The publications we included in this work were selected by database retrieval as well as manual screening, and therefore have an unavoidable subjective judgment bias. Although we covered a majority of publications, the study cannot fully describe the bibliometric information of the field.TREM2 as a potential immunotherapy target, and (3) DNA methylation. The important pathogenesis implied by genetic studies is valuable for future mechanism studies and clinical conversion. The research fronts were (1) looking for genetic similarities between AD and other neurological diseases and syndromes, and (2) searching for causal variants of AD. Current weaknesses of the discipline are (1) GWAS in AD biomarkers based on relatively large sample sizes are scarce, (2) studies of causal variants of AD are few, and (3) GWAS in AD based on non-European populations are inadequate. It is necessary to pay more attention to these under-researched directions in the future. Our study provides a comprehensive view of GWAS in AD. Researchers new to the field can easily obtain useful information from our research and better investigate the genetic etiology of AD.This bibliometric and visualization study demonstrates what GWAS in AD is like, what it tells us, and where it is headed. According to our bibliometric analysis, the most concerned AD pathogenesis and current research hotspots were (1) Aβ and tau, (2) immunity and microglia, with The original contributions presented in the study are included in the article/JZ: Conceptualization, Data curation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. YW: Data curation, Methodology, Writing – original draft. YZ: Data curation, Methodology, Writing – review & editing. JY: Conceptualization, Funding acquisition, Investigation, Writing – review & editing.


Several approaches have evolved to facilitate the exploration of hydrogel systems in biomedical research. In this sense, poly (PVA) has been widely used in hydrogel (HG) fabrication for several therapeutic applications. The biological properties of PVA hydrogels (PVA-HGs) are highly dependent on their interaction with protein receptors and extracellular matrix  deposition, for which there is not enough evidence from existing research yet. Thus, for the first time, the functional properties, like protein and mineral interactions, related to the proliferation of mesenchymal stem cells (MSCs) by silver nanoparticle (AgNP)-loaded PVA hydrogels (AgNPs-PVA-HGs) were investigated in the present study. The UV absorption spectrum and TEM microscopic results showed a maximum absorbance of synthesized AgNPs at 409 nm, with an average particle size of 14.5 ± 2.5 nm, respectively. The functional properties, such as the calcium-binding and the protein adsorption of PVA-HG, were accelerated by incorporating AgNPs; however, the swelling properties of the HGs were reduced by AgNPs, which might be due to the masking of the free functional groups (hydroxyl groups of PVA) by AgNPs. SEM images showed the presence of AgNPs with a more porous structure in the HGs. The proliferative effect of MSCs increased over culture time from day 1 to day 7, and the cell proliferative effect was upregulated by HGs with more pronounced AgNPs-PVA-HG. In addition, both HGs did not produce any significant cytotoxicity in the MSCs. The histological (bright light and H&E staining) and fluorescence microscopic images showed the presence of a cytoskeleton and the fibrillar structure of the MSCs, and the cells adhered more firmly to all HGs. More fibrillar bipolar and dense fibrillar structures were seen in the day 1 and day 7 cultures, respectively. Interestingly, the MSCs cultured on AgNPs-PVA-HG produced extracellular matrix deposition on day 7. Accordingly, the present results proved the biocompatibility of AgNPs-PVA-HG as a suitable system for culturing mammalian stem cells for regenerative tissue applications. Hydrogels (HGs) are formed by the hydrophilic interaction of water and polymers, which produce a three-dimensional network that facilitates the swelling ratio in a significant amount upon interaction with water ,2,3. TheAmong the different polymers, naturally derived biopolymer-based hydrogels had poor mechanical and chemical stability, which limits their potential application in tissue regeneration. In this sense, the use of PVA in hydrogel fabrication overcomes the limitation of natural hydrogels in practical application. As a hydrophilic polymer, PVA is considered a suitable biocompatible synthetic material, which has the major functional hydroxyl groups. The presence of a hydroxyl group makes PVA more readily available for crosslinking with several polymers to form hydrogels . Due to Numerous studies investigated the fabrication of composite PVA hydrogels associated with different natural biopolymers, such as alginate, cellulose, ovalbumin, dextran, heparin, gelatin, collagen, hyaluronic acid, chondroitin sulfate, chitosan, silk fibroin, starch, fibrin, gellan, and carrageenan and synthetic polymers, such as ultra-high molecular weight poly-ethylene (UHMWPE), poly (acrylic acid), polydimethyl siloxane, polyurethanes, poly(N-isopropyl acrylamide), poly (vinyl pyrrolidone), poly (glycolic acid), poly , bioceramics , and their potential in tissue engineering applications .The use of silver nanoparticles (AgNPs) is of great interest in the field of nanotechnology due to its optical, electrical, catalytic, and biofunctionality properties. In particular, both their physicochemical properties and biological functionality would dramatically depend on their morphology, size, and distribution, as well as on their electrochemical environment in the case of nanoparticles in a liquid medium. Indeed, AgNPs belong to the most commonly used nanomaterials in biomedical applications mainly due to their antimicrobial potential, but they are also very promising against nonliving organisms, including human immunodeficiency viruses that can induce several viral diseases . In this3  molecular weight 169.87 g/mol, NaBH4  molecular weight 37.83 g/mol and were acquired from Sigma-Aldrich . Liquid vegetable glycerin, pharmaceutical grade, was acquired from EQM chemical solution store .Polyvinyl alcohol (PVA) molecular weight 9000–10,000 g/mol (CAS: 9002-89-5), AgNO3 (silver nitrate) as the initial reagent and NaBH4 (sodium borohydride) as the reducing agent. The corresponding chemical reaction.Hydrogels were fabricated as follows. First of all, AgNPs were synthesized by the chemical reduction method ,24,25, u4 adsorption plays a crucial dual role as a reducing agent but also as a stabilizer during the growth of silver (Ag) nanoparticles, providing a surface charge on the particle. Thus, nanoparticles are created and, at the same time, stabilized in suspension through repulsive electrostatic forces between them due to adsorbed NaBH4. Besides, the amount of NaBH4 must be controlled. Indeed, it must be enough to stabilize the particles when the reaction occurs, but not too high; otherwise, the ionic strength will increase, promoting the aggregation of the particles. The stability of these solutions strongly depends on the proportion of reducing agents and varies between minutes and/or weeks. In fact, this route offers the fabrication of NPs with diameters ranging from ~10 nm to a few dozen of nm, with a Surface Plasmon Resonance (SPR) around ~400 nm and full width at half maximum around ~35–100 nm. If the amount of reductant is not adequate, the particles agglomerate, and the solution turns yellow, violet, to gray. The latter aggregation can be avoided by adding dispersing/stabilizing agents such as polyvinylpyrrolidone (PVP).During the AgNPs synthesis process, the NaBH3 and 2.0 mM NaBH4 were prepared in distilled water beforehand. Typically, 10 mL of the AgNO3 solution is added dropwise (~1 drop/s or using a burette instead) to a vial containing 16 mL of the NaBH4 solution; in this way, the ratio [NaBH4]/[AgNO3] was 1.6. The whole reaction was carried out in an ice bath with vigorous stirring. The solution turns to yellow color at the end of the AgNO3 addition, and the whole process takes about ~3 to 5 min after which stirring is turned off. Just before switching off the agitation, 1 mL of an aqueous solution of PVP at 0.3 wt.% is also added drop by drop  the former PVA solution (8 g) with the silver nanoparticle solution (8 g) and the crosslinker, vegetal glycerin (4 g) in a ratio of 40/40/20 . All the UV-vis spectra recorded within the wavelength range of 300 nm to 700 nm and systematically background-corrected were recorded by using a UV-1800 spectrophotometer  and using quartz cells with an optical path of d ≈ 10 mm. Spectra analysis was performed by using Peakfit v4.12 software. For TEM measurements, 20 µL of the AgNPs solution was deposited on a formvar/carbon-coated nickel-300 mesh grid. The TEM micrographs were obtained with an accelerating voltage of 120 kV by using a JEOL JEM 1011  Transmission Electron Microscope equipped with an Orius SC200 High Contrast Digital Camera . For statistical analysis of the AgNPs size distribution, 100 measurements were performed using the ImageJ software. Subsequently, the silver nanoparticle’s size distribution was obtained by performing a Gaussian analysis of the histogram plotted using GraphPad Prism 9.0 Software, LLC.Morphological characterizations of the PVA and PVA-AgNPs hydrogel were carried out by scanning electron microscopy ), and energy dispersive X-ray spectroscopy ). For SEM/EDX analysis, the samples were freeze-dried and then deposited on aluminum sample holders with double-sided carbon tape. In order to guarantee the adequate conductivity of the sample, one drop of carbon ink was also used to fix the sample on the carbon tape. Then, the samples were coated with a platinum ~5 nm coating thickness conductive layer (99.99% purity) using a sputtering evaporator (BIORAD-POLARON). Sample images were collected at a variable magnification from 300× to 2000× with an accelerating voltage of 5 kV.The water-holding property measured the ability of hydrogels to hold/retain water within the intramolecular spaces and was investigated by following our previous protocol ,26. BrieThe swelling measures the ability of hydrogels to expand their shape and size in an aqueous medium, and the percentage of the swelling ratio of hydrogel was calculated by following our previous method . BrieflyThe ability of hydrogel in mineral binding was evaluated by following our earlier methods ,27. BrieThe ability of hydrogel in protein binding was done as per our previous protocols . Briefly2 culture flasks and subcultured eight times on confluence. In each culture, some cells were stored for future use, and cells from subcultures 4 to 7 were used for the present study [In vitro cell culture work was done by using Human Mesenchymal Stem Cells from Bone Marrow (MSCs). The cells were procured in the cryopreserved form from PromoCell GmbH, Heidelberg, Germany . The cells were cultured as per the standard protocol using mesenchymal culture medium (PCS-500-030) with growth kit (PCS-500-041), 5% FBS (Gibco), 1% Penicillin-Streptomycin-Amphotericin B Solution (ATCC PCS-999-002). Initially, the cells were seeded in two T75 cmnt study .4 in 24 well culture plates. The cells cultured without hydrogel in the culture plate were considered as control. Both control cells and hydrogel groups were cultured with complete MSC culture medium in a CO2 incubator at 37 °C, 98% relative humidity, at 5% CO2 for 1, 3, and 7 days. On each period, the culture medium was removed, and the cells were treated with Alamar blue , Cat No: DAL1025) reagent (1:9 v/v) prepared in culture medium for 4 h at 37 °C. Then the cell viability was measured spectrophotometrically at 570 nm using SpectraMax iD3.For proliferation, the cells were seeded on hydrogel with a cell density of 1 × 102 incubator at 37 °C, 98% relative humidity, at 5% CO2. Then, the cells were washed with PBS to remove the existing culture medium, and a fresh culture medium containing 0.5 mg/mL 3--2,5-diphenyl tetrazolium bromide . The cells were incubated for 4 h in a CO2 incubator, the unbound yellow dye was removed gently by PBS wash, and the formed formazan dye was solubilized with DMSO, followed by measuring the OD at 570 nm using SpectraMax iD3.For cytotoxicity, the cells were seeded the same as above in 4 in 12-well culture plates using complete MSC culture medium for 1, 3, and 7 days. Each time, the cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at room temperature, followed by consecutive fixing with 2.5% glutaraldehyde for another 1 h at room temperature. The cells on the hydrogel were captured in white light using a fluorescence microscope coupled with Axiocam 305 mono  before histological staining. Then, the cells on hydrogels were stained with hematoxylin stain for 30 min, incubated in bluing reagent, followed by eosin stain for 30 min. The bright field light images and HE-stained images were captured using an Axio Vert A1 microscope.The morphology of the MSCs on hydrogel was visualized by using a histological staining method. In brief, the cells were cultured on hydrogel with a cell density of 5 × 10We further investigated the morphological structure of the MSCs cultured on the hydrogel via a fluorescence microscope. Briefly, the cells were seeded on hydrogels and cultured in a complete MSCs culture medium for 1, 3, and 7 days, as described in t-test, and a p-value less than 0.05 was considered statistically significant.In the present study, all the experiments were carried out in three independent set-ups and the results were summarized as the mean and standard deviation. The statistical analysis and graph diagram were carried out by using Prism GraphPad 9.5.1. The statistically significant differences between groups were analyzed using a The flexible and elastic properties of fabricated hydrogels are shown in The AgNPs’ spectrum absorbance can be used to indicate the average particle size. Indeed, the wavelength of the absorption maximum and its peak width at half maximum (PWHM) in each solvent for AgNPs provide useful information. The microstructure of the PVA and PVA-AgNPs hydrogels is presented in The ability of the hydrogel to hold water in intramolecular space was investigated using the weight difference before and after immersion in water, as seen in Though the water-holding capacity of both hydrogels did not change significantly, the pattern of swelling ratio differed between the PVA hydrogel and AgNPs-PVA-HG. As shown in The effect of hydrogels in support of extracellular matrix formation was investigated by treating the hydrogels with calcium, and the ability of the hydrogels to bind with minerals was investigated by the alizarin red-S staining method.p < 0.05)  B. The ra < 0.05) . In orde < 0.05) D.In order to investigate the potential compatible interaction of a hydrogel with cellular protein receptors, we have estimated the ability of the hydrogels in their protein-binding ability. To do this, the hydrogels were incubated with fetal bovine serum, and the hydrogel-bounded protein was quantified using the CBB G-250 method C,E. Simip < 0.05). On day 1, a similar proliferation rate for the MSCs was observed between PVA hydrogel and the control (without hydrogel); on the other hand, the proliferation rate of MSCs improved in AgNPs-PVA-HG. On day 3, the cells cultured on the PVA hydrogel (p < 0.05) and AgNPs-PVA-HG (p < 0.01) had more proliferative effects than the control cells. Among the hydrogels, AgNPs-PVA-HG had a higher proliferation rate regarding MSCs on day 3 and day 7.As the hydrogels showed excellent functional properties, the biocompatibility using MSCs was investigated for 1, 3, and 7 days of treatment with hydrogels. In general, cell proliferation increased as expected with culture time, i.e., the cells cultured for 7 days had a higher proliferation rate compared to the day 1 and day 3 cultures  microscopy. As shown in In the light microscopy, the cells stained with H&E stains clearly showed the presence of a cytoskeleton and the fibrillar structures of the cellular components. On day 1, the cells cultured without hydrogels were more fibrillar bipolar structures compared to day 3 and day 7. In contrast, the cells cultured without hydrogels for 3 and 7 days showed a more dense fibrillar structure, which was more pronounced on day 7. When compared to the control cells, the cells cultured on hydrogels showed different morphology and more cell clumps were observed on hydrogel-cultured cells. Among the hydrogel groups, the H&E staining showed more fibrillar cells in AgNPs-PVA-HG. Interestingly, the cells cultured on AgNPs-PVA-HG for 7 days potentially produced extracellular matrix deposition (yellow arrows) and more cell clumps compared to the PVA hydrogel. In addition, the light microscopic pictures clearly showed that the cells were firmly adherent to the hydrogels, and the cell’s morphology was different between the control (without hydrogels) and hydrogels groups, showing more fibrillar structures even on day 3 and day 7 in hydrogel groups; in contrast, the cells become more aggregated with less fibrilar structures for those cultured in the control group. The extracellular matrix formation of MSCs cultured on AgNPs-PVA-HG was clearly seen in the light microscopic picture.We further evaluated the proliferative and biocompatibility effect of hydrogels on MSCs through FITC and DAPI fluorescence microscopy. As observed in H&E staining and light microscopy, the fluorescent intensity of FITC and DAPI significantly increased in the control cells cultured for 7 days when compared to the cells cultured for 1 and 3 days . SimilarIn the present study, the content of PVA- and glycerine-modulated mechanical properties, such as elasticity, yield strength, young modulus, and concomitant physicochemical properties, were analyzed. Indeed, more glycerine will provide more flexibility and serve to modulate the intrinsic micro and macroporosity structure, which are of paramount importance in tissue engineering applications . The maxPreviously, Wang et al. tested the water-holding capacity of cellulose-based hydrogel copolymerized with polyacrylic acid (PAA)-grafted quaternized cellulose (QCE) and PVA at different pH , and also different QCE and PVA contents from 0% to 1.5%, and 0% to 8%, respectively. The authors concluded that the water adsorption behavior of the hydrogel was significantly affected by the pH range and the abundant carboxyl groups of PAA. The authors also stated that the water adsorption capacity of the hydrogel decreased as the PVA content increased . SimilarSimilar to the present study, the swelling ratio of cellulose nanocrystal (CNC)-reinforced PVA was tested by Tanpichai and Oksman, and they found that the swelling ratio of the PVA (4%) hydrogel was 577%, which was higher than the present study. The authors further reported that the swelling ratio of the hydrogel was reduced from 577 to 165% as the PVA concentration increased from 4 to 12%, respectively . SeveralIt has been reported that the swelling behavior of synthesized nanocomposites was significantly altered by the presence of nanosilver in the polymer network . SimilarThe ability of the mineral-binding rate of hydrogel was potentially improved by AgNPs, which ultimately shows the efficiency of the hydrogels in extracellular matrix deposition during bone formation. For instance, PVA hydrogels were fabricated with biomimetic mineral hydroxyapatite or biomimetic apatite for cartilage repair ,40. In oIn addition, the calcium biomineralization of a PVA hydrogel was sufficiently proved by several studies ,43,47. WThe microstructural properties revealed that the presence of these micropores could be attributed to the interactions between AgNPs and the rapid sublimation process occurring during freeze-drying, promoting reduced pressure surrounding the nanoparticles. It is worth noting that, at this scale, EDX was unable to provide a microanalysis to verify the presence of the silver nanoparticles, which demonstrated that AgNPs are homogeneously distributed through the 3D structure as an individual or in a small cluster of nanoparticles. The microstructure  of hydrogel had a great influence on tissue engineering applications because it allows for cell intrusion, adhesion, and proliferation. Additionally, high interconnectivity promotes uniform cell distributions as well as the diffusion of nutrients to and from the cell substrates. Thus, in the PVA-AgNPs hydrogel, the existence of a distribution of micropores would be favorable in terms of biocompatibility since it increased the interconnectivity and interface area of the porous structure.The applicability of PVA hydrogels in biological cell cultures (PVA-cell receptor interaction) was indirectly determined by the absorption of plasma protein by PVA hydrogel ,52,53. HIn addition to the PVA hydrogel potential in protein adsorption, the accelerated rate of protein adsorption of AgNPs could be explained by several reports. The interaction of nanoparticles with protein in the biological environment produces a nanoparticle–protein corona complex, which is the primary cause of in vivo biodistribution and pharmacokinetic profiles ,62. The In vitro studies, such as proliferation, cytotoxicity, histological staining, and fluorescence staining, showed that the growth of the cell was superiorly supported through culturing the cells on hydrogels than the control, and the effect was more pronounced in cells cultured on AgNPs-PVA HG. Similar to the present study, earlier studies proved the proliferative effect of PVA hydrogel in MSC cell growth ,69,70,71The hydrogel fabricated with AgNPs and the chitosan-PEG mixture showed excellent healing properties for treating diabetic chronic wounds ,76. AddiThe actual signaling mechanism of AgNPs in cell proliferation was reported by several authors. For instance, AgNPs potentially alter the gene expression that is involved in multiple cellular pathways, including cell proliferation and hormone signaling pathways, such as tumor necrosis factor (TNF), matrix metalloproteinases, and interleukin (IL)-12 and IL-1 .More detailed molecular signaling mechanisms of AgNPs in cell proliferation were reported by Li et al.  by treatIn the present study, the functional properties and in vitro biological response of AgNPs-PVA-HG were investigated. Our study clearly demonstrated that the functional properties, such as protein adsorption and the calcium-binding abilities of PVA hydrogel, increased due to AgNPs, and the swelling properties of PVA hydrogel decreased due to AgNPs incorporation. The prominent protein and mineral affinity of AgNPs directly proved the efficacy of the fabricated composite PVA hydrogel in regenerative tissue applications. Further, the biocompatibility of the composite hydrogels in culturing MSCs was investigated. In general, the proliferative rate of MSCs increased with AgNPs-PVA-HG treatment. Histological and fluorescence images proved that the MSCs adhered to both the PVA hydrogel and AgNPs-PVA-HG more efficiently, and the cells cultured for 7 days on AgNPs-PVA-HG deposited extracellular matrix. Based on the above findings, this study concludes that the fabricated AgNPs-PVA-HG could be an effective system for culturing mammalian cells, especially for stem cell differentiation. However, further studies are essential to prove the biological role of this system in regenerative tissue applications.


While no differences were found in self-reported health, there were differences in self-reported exposure to self-reported exercise and non-exercise physical activity, with the Viennese population being more inactive than their more western comparison group. In addition, the objective indicators for muscle strength, balance and flexibility of the lower extremities differed significantly in favour of the more western Austrian population. We recommend assessing the situation of older people in Austria regarding their physical activity and fitness on a regional basis, even if they live in cities of the same category. Future projects should therefore aim to consider specific regional needs during development and incorporate both subjective and objective indicators when monitoring the success of such programs.The aim of this study is to compare data on the health status, self-reported exercise and non-exercise physical activity as well as fitness parameters, such as grip strength, of people in retirement in two cities that are both considered urban centres according to the statistical office of the European Union (EUROSTAT), but differ by geographic location. Self-reported physical activity questionnaires and objective assessments of physical fitness indicators collected by sports scientists were used and examined for differences. A total of 210 people (66.3 years ± 2.3) in Salzburg ( An active lifestyle for people over 65 is more important than ever for two reasons: First, an inactive lifestyle can cause high personal burdens . As repoSecond, the over-65s are the population group that is the only one growing in Europe , which mPhysical activity (PA) is defined as any bodily movement generated by the skeletal muscles that results in raised energy expenditure ,17. SubsAny PA can be considered a health-enhancing physical activity (HEPA) if it is beneficial to the health and functioning of the organism without harming or endangering it . To be cIn 2019 about 45% of the Austrian population between 60 and 65 years of age stated that they comply with the WHO recommendation ,23. LessLooking more closely at the data regarding trends in life expectancy, it becomes clear that although life expectancy has increased since 2003, the ratio to years in health has deteriorated. For example, Austrian men live 2.1 years longer, but 1.3 of these years with limitations. The situation is similar for female citizens as they live 1.8 years longer but 1.1 years thereof with limitations. These numbers indicate that about 60% of the gained lifetime comes with limitations in everyday life . These limitations affect the quality of life of older people. Acknowledging that there are numerous definitions of QoL , we descTo counteract this, it is important to stay regularly physically active. However, according to recent findings, not all people are equally at risk of being inactive or insufficiently active, as residency and gender both seem to matter ,30,31 wiFurthermore, we lack region-specific data on objectively measured components of fitness. Since the results of self-assessed questionnaires can be strongly influenced by many factors such as a lack of comparison possibilities, biased feedback from others, personality traits and socio-economic status , it is cWith our work we wanted to investigate whether there is a difference in the older Austrian population of Vienna and Salzburg regarding subjective measures such as self-perceived health and objective measurements of indicators of physical fitness, namely muscular strength and flexibility. Moreover, we want to answer the question of whether there are different behaviours regarding the subsets of PA, namely exercise and NEPA, between these regions. In addition, we want to substantiate all self-reported data with objectively collected data on physical fitness. With our contribution, we aim to provide additional information that will help in the development of future physical-activity-promotion programs for older urban residents in Austria.To investigate the research questions, we analysed the data of a randomised controlled intervention study recently reported elsewhere . We chosn = 90, 43%), located in the western part of the country, or the city of Vienna  in the east, both cities including urban centres, according to EUROSTAT. As the retirement age in Austria differs, men (69.7 ± 1.5) were on average older than women (65.5 ± 1.6) in the total sample  = 15.785; p = 0.001). The gender distribution was identical, with 80% women in Salzburg (n = 72) and Vienna (n = 95), and 20% men in Salzburg (n = 18) and Vienna (n = 25). This study design leads to an overall homogenous group of participants clustered into two very similarly composed and thus well-comparable regional groups regarding age and gender in Salzburg (66.3 yrs ± 2.5) and Vienna (66.4 yrs ± 2.2). The selected study participants did not follow structured exercises but were interested in taking up such activities. The inclusion criteria comprised being free of physical and mental disorders, having no chronic disease and the medical allowance to engage in a regular exercise program. Moreover, the fact that only participants who did not receive a compensatory allowance or care allowance and who had to meet various technical requirements, such as a flat-screen TV and access to the Internet, were included ensured similar general conditions regarding illnesses and socioeconomic conditions. Another important inclusion criterion of the parent study was being retired for at least 2.5 years, as the acute retirement phase can affect PA with an initial increase followed by a subsequent decrease in the long run ; thus, tBoth items were collected via an online questionnaire. Self-perceived general health was assessed according to the EHIS  . AnswersThe amount of HEPA was assessed using the single-item questionnaire by Milton et al. ,41. It aWe relied on guided interviews to assess the exercise load and NEPA ,43. PartWe assessed the frequency, intensity and duration of a typical session to determine the load of exercise and NEPA. Modes of exercise included, for example, sports such as tennis, skiing, hiking and golf, while non-exercise physical activity included housework and gardening or, for example, shopping. Incidental physical activity, e.g., walking as a means of locomotion , was notThe three items  were used to create indices that allowed for the overall load of exercise and NEPA. Before these indices were calculated, the 10-part scale was reduced to a five-part scale by merging every two categories in each case. Intensity 1 and 2 of the 10-part scale thus became intensity 1 of the 5-part scale. Intensity 3 and 4 of the 10-part scale became intensity in the 5-part scale, and so on see . This way all three components of the respective index were weighted equally, to be then used in calculating the mean indices, ranging from 1 as the lowest score up to 5 as the highest score. Based on this calculative framework one can expect to score an index of 1 to 2 if no or little efforts are made, an index of 3 to 4 if the requirements for health-enhancing physical activities are met and even a 5 if the person is very active in the corresponding activity, namely, exercise or NEPA. We termed these indices the exercise index and NEPA index, respectively.The handgrip strength  was used as a surrogate marker for overall muscular strength . The sitTo gain more detailed insights, the isometric muscular strength  was assessed with a handheld dynamometer , as this method is reliable and valid in diverse settings ,55,56. TStatistical analysis was conducted using SPSS . The description of ordinal-scaled data was conducted using the mode and mean ranks. A Mann–Whitney U-test was conducted to detect group differences in non-parametric data of the questionnaire. t-test and took Cohen’s d as the effect size. As proposed in Cohen [The interval-scaled data such as indices and objectively measured indicators of physical fitness were described with mean and standard deviation. To evaluate any group difference, we calculated an independent-samples Welch’s in Cohen , we usedR²| = 0.02, a moderate goodness-of-fit at |R²| = 0.13 and a high goodness-of-fit at |R²| = 0.26.Multiple regressions were run to predict the indicators of fitness from the exercise index, NEPA index, sex and age. Linearity was assessed using partial regression plots and a plot of studentized residuals against the predicted values. The independence of residuals was assessed with Durbin–Watson statistics. There was homoscedasticity, as assessed by visual inspection of a plot of studentized residuals versus unstandardized predicted values. There was no evidence of multicollinearity, as assessed by tolerance values greater than 0.1. There were no studentized deleted residuals greater than ±3 standard deviations, no leverage values greater than 0.2 or values for Cook’s distance above 1. The assumption of normality was met, as assessed with a Q-Q Plot. As proposed in Cohen , we assuTo provide additional insights into the relationship between the different datasets, all ordinal and continuous data correlations were calculated using Spearman’s rank-order correlation, with results inspected via scatterplot and interpreted via Spearman correlation coefficient, rs (between 1 and −1). The correlations were calculated for the total sample as well as gender- and region-specific subsets and can be found as p < 0.05 in all analyses. Adjusted p values are presented. Missing values were checked for randomness and, if so, were not imputed. The level of significance was set to n = 134 of 209 (64%) reporting “good” health without any significant gender-specific  or region-specific  difference in the distribution of responses. The comparison of the reported HEPA resulted in significant differences  = 3.5; p = 0.001) between the regions, but not the genders  = 0.811, p = 0.418). While the participants living in Salzburg stated that they had at least 30 min of HEPA on an average of 3 (±1.7) days per week, the Viennese had only 2 (±1.7) such days per week. The participants attested to a good perceived state of health, resulting in p = 0.001). Looking at the distribution of NEPA indices, a similar picture emerges, with an average of 2 in Salzburg and 1 in Vienna  = 7.083; p = 0.001).Comparison of the exercise as well as the NEPA indices showed a significant difference in the distribution of indices between regions in both areas of PA. While the exercise index in Salzburg showed a mean of 3, Viennese participants scored 2 on average  = 7.921; The distribution of NEPA indices  shows thFor a more detailed overview of the factors leading to these index values, see The correlation analyses showed that self-reported health and HEPA in Vienna are strongly related to exercise load. Positive correlations with the frequency as well as intensity of exercise were also shown in Salzburg. In contrast, however, in Salzburg there were slightly negative correlations between self-reported health and NEPA intensity as well as between HEPA and NEPA frequency. For the detailed presentation of the correlation analyses carried out in the total sample and regional sub-samples, we refer to the The balance, isometric strength and ranges of motion of the Salzburg participants were significantly higher than those of the Viennese. However, only shoulder flexion mobility and the 30 s chair-rise test results for the lower body strength were higher in the Viennese participants, but with small effect sizes.A multiple-regression analysis resulted in the following findings see .p < 0.001, ShoulderF; F = 40.233, p < 0.001; HipExtF, F = 22.618, p < 0.001; HipAbdF, F = 17.622, p < 0.001; and BicF, F = 41.844, p < 0.001, all models with a high goodness-of-fit according to Cohen, 1988 [p < 0.001, and LegMob, F = 10.598, p < 0.001, both models indicating a moderate goodness-of-fit according to Cohen, 1988 [p < 0.001, but the model indicated a less-than-moderate goodness-of-fit according to Cohen, 1988 [Significant prediction was possible for grip strength, F = 78.082, en, 1988 . Signifien, 1988 . Signifien, 1988 . SignifiWe investigated whether there is a difference between senior citizens living in Vienna and those living in Salzburg regarding self-perceived health, self-reported physical activity and objectively collected physical fitness parameters. Our data show that the place of residence accounts for no difference in self-perceived health but makes a significant difference in self-reported physical activity and objectively collected physical fitness data. The participants mostly perceived their health as “good” without any region- or gender-specific difference. More so, the overall findings show consistently good fitness for all participants in terms of strength, balance and flexibility compared to normative values. Since the test group at the age of 65 in Austria can still expect an average of seven years without functional limitation, this result aligns with the self-reported values of the Austrian Health Survey 2019 . However, regional differences are reflected in the count of weekly days where the HEPA recommendations are reached and even more clearly in the subsets of PA, i.e., exercise and NEPA. While the regions differed strongly in the levels of exposure to exercise and NEPA load, the correlation analyses see  indicateOur results, which show that Viennese participants incorporate significantly less physical activity into their daily lives than Salzburg participants, confirm the PA behaviour east–west gradient reported in previous work  and suppNevertheless, both sit-to-stand tests and the handgrip strength test did not provide any region-specific difference; hence, they do not support the idea of an east–west gradient.The overall results of the 30 s chair-rise test are slightly better than the proposed normative scores within the age group of 65–69 years   and in lThe handgrip strength independent of region or gender was in line with normative data for this age group  and abovDespite being an evident screening tool for individuals at risk of mobility limitations, physical fitness tests based on movement patterns of daily life might have a lower discriminatory function in fit, older individuals living without functional limitations . As a reIn contrast, our other data point to an east–west gradient. The Salzburg population shows better physical fitness in terms of balance, isometric muscle strength and flexibility than the Viennese. Balance ability, measured via unipedal stance, showed a significant difference between regions and genders, whereas all groups were well above the proposed norm values  for ages 60–69 years  and far The better results in balance could be due to the higher isometric strength scores and flexibility in the lower limbs, as there is evidence that both strength and flexibility can positively affect balance . This isThe region-specific differences are visible in the comparison of the isometric strength in the hips, shoulder and biceps and hip flexibility. Strengthening activities positively affect hip flexion mobility, while in contrast, there is limited evidence for the same mechanisms in shoulder flexion ,84,85. TThese findings regarding differences in exposure to exercise, NEPA and indicators of fitness are further strengthened by the results of the multiple regression analysis, which show the exercise index to be highly predictive of most indicators of physical fitness. As suspected by the results of the correlation analysis, the multiple regression analysis confirms that NEPA has much less influence on fitness than exercise. Hence, structured exercise seems to be the key for the studied population to achieve HEPA as well as better fitness.We would like to point out the limitations of this study, which mean that our results cannot be considered generalizable. The sample size does not allow us to speak of a representative group of all people over 65 in the respective regions. Rather, we are dealing with comparable samples from different regions, which were used to determine whether regional differences in subjective and objective markers of PA could be found. Despite a similar gender ratio between the regional groups, men are underrepresented in this study when considering their share of the population studied. The lack of exercise modalities meant that MET could not be assigned, making comparability with other studies difficult. Due to the complex physical tests, the interviews were conducted in a very abbreviated manner, so that various co-variates could not be collected, which could have helped explain the differences in exercise and NEPA indices between regions. Hence, the potential root causes for regional disparities remain unclear and should be subject to further investigation.Our data show differences in HEPA, muscle strength, balance and hip flexibility between Vienna and Salzburg. This aligns with previous findings regarding the regionality of PA and fitness in Austria  despite


Gestational exposure of mice to valproic acid (VPA) is one currently used experimental model for the investigation of typical failure symptoms associated with autism spectrum disorder (ASD). In the present study we hypothesized that the reduction of dopaminergic source neurons of the VTA, followed by perturbed growth of the mesotelencephalic dopamine pathway (MT), should also modify pattern formation in the dopaminoceptive target regions (particularly its mesoaccumbens/mesolimbic portion). Here, we investigated VPA-evoked cellular morphological , as well as synaptic proteomic (western blotting) changes, in selected dopaminoceptive subpallial, as compared to pallial, regions of mice, born to mothers treated with 500 mg/kg VPA on day 13.5 of pregnancy. We observed a surge of apoptosis on VPA treatment in nearly all investigated subpallial and pallial regions; with a non-significant trend of similar increase the nucleus accumbens (NAc) at P7, the age at which the MT pathway reduction has been reported . Of the CaBPs, calretinin (CR) expression was decreased in pallial regions, most prominently in retrosplenial cortex, but not in the subpallium of P7 mice. Calbindin-D 28K (CB) was selectively reduced in the caudate-putamen (CPu) of VPA exposed animals at P7 but no longer at P60, pointing to a potency of repairment. The VPA-associated overall increase in apoptosis at P7 did not correlate with the abundance and distribution of CaBPs, except in CPu, in which the marked drop of CB was negatively correlated with increased apoptosis. Abundance of parvalbumin (PV) at P60 showed no significant response to VPA treatment in any of the observed regions we did not find colocalization of apoptotic (Casp3+) cells with CaBP-immunoreactive neurons. The proteomic findings suggest reduction of tyrosine hydroxylase in the crude synaptosome fraction of NAc, but not in the CPu, without simultaneous decrease of the synaptic protein, synaptophysin, indicating selective impairment of dopaminergic synapses. The morpho-functional changes found in forebrain regions of VPA-exposed mice may signify dendritic and synaptic reorganization in dopaminergic target regions, with potential translational value to similar impairments in the pathogenesis of human ASD. Autism Spectrum Disorder (ASD) is one of the most common neurodevelopmental disorders associated with altered social behavior. A cover term for a continuum of failure symptoms from high functioning to seriously defective cases, ASD has been extensively investigated in the recent years both in preclinical and clinical studies. With all its phenomenological variations, impairment of social adaptation and behavior are characteristic features of all forms of ASD, however, its etiology has remained largely elusive. Two main groups of pathogenetic/risk factors  are usually associated with the disease. While only a limited contingent of human autism cases could be traced backed to known genetic components , an everpost-partum  is one currently used experimental model for the investigation of typical failure symptoms associated with autism spectrum disorder (ASD). Administration of VPA (a.k.a. 2-propylpentanoic acid), a commonly used anticonvulsant, antiepileptic, and mood stabilizer , to pregt-partum . VPA-tret-partum . The valt-partum  or birdst-partum . The VPAt-partum .Teratogenic effects, including an elevated risk of ASD following administration of VPA  have lonin utero after VPA administration to the mother, must influence critically timed events of CNS development, and they must also affect those brain regions participating in, or linked to, the social brain network , the developmental dynamics of the ventrotegmental–accumbens (mesolimbic) pathway, including the chemorepellent or chemoattractant factors specifically involved in the segregation of distinct dopaminergic pathways/connections see .de novo missense mutation of the dopamine transporter gene , and the selective drop of DA in the NAc, observed in prenatally VPA-exposed animals at P7  into the occipital skin fold. Control animals were injected with vehicle, under identical conditions. After birth, pups remained with the dams until day 7 . Thereafter, the rest of the pups were separated from the dams (at the age of 4 weeks) and kept together with their littermates until 60 days of age .For VPA treatment, 6-week-old female C57BL mice with an established pregnancy of 7 days  were used. The animals were kept in pairs, with Valproic acid-treated and control mice of either sex , were terminally anaesthetized with an intraperitoneal injection of ketamine (50 mg/ml) and xylazine (20 mg/ml) . The animals were transcardially perfused with a solution of 4% paraformaldehyde in PBS, at 4°C. After removal, the brains were post-fixed in the same solution for at least 24 h.Immunocytochemistry in paraffin-embedded sections. The brains were paraffin-embedded and 10 μm sections were cut in the sagittal or coronal plane, serially mounted on glass slides, and immunostained as performed previously . The ima2O2 , washed again for 3 × 10 min, in PBS, and blocked for 30 min in normal goat serum (NGS), 5% in PBS. For those sections processed for DARPP-32, normal horse serum (NHS) was used instead of NGS throughout. Then the sections were incubated with the respective combination of primary antisera , overnight. The primary antibodies were as follows: a-calretinin 1:1,000 chicken (LOT 22024) Thermo Fisher Scientific, a-calbindin 1:1,000 chicken (LOT 22654) Thermo Fisher Scientific, a-caspase-3 1:1,000 rabbit (LOT CN89330), Bioworld Technology, a-parvalbumin 1:1,000 chicken (LOT 22579) Thermo Fisher Scientific, a-DARPP-32 1:1,000 goat (LOT XHG0320061), RD Systems. Following washing for 3 × 10 min, in PBS, the sections were incubated with the respective ‘ary antibodies,  1:1,000, Invitrogen; Donkey a-rabbit, Alexa Fluor 488 (LOT VC296619) 1:1,000, Invitrogen), for 1 h, followed by another washing for 3 × 10 min, in PBS. The sections were mounted on glass slides and coverslipped using a glycerol/PBS mixture (1:1).Immunocytochemistry in free-floating frozen sections. The brains were immersed in 30% sucrose  and cut into 30 μm coronal sections with a Leica SM2000R freezing microtome. The sections were stored in 0.01% of azide-containing PBS at 4°C until further processing. Altogether five series of sections were prepared from each brain. From P7 animals, three section series were doubly immunostained for calretinin (CR) and caspase-3 (Casp3), for calbindin (CB) and Casp3, as well as for DARPP-32 and Casp3, respectively. From P60 animals, two section series were singly stained for parvalbumin (PV) or CB. In each case, the technical procedure was similar, always ensuring that the sections from control and VPA-treated animals were handled simultaneously, using the same batch of reagents under the same conditions. The sections were washed for 3 × 10 min, in PBS, treated with H2. We performed manual counting throughout, including manual thresholding. The latter meant that the diameter of each labeled cell to be counted was measured using the feature of the image viewer program (Aperio ImageScope) of the fluorescent microscope. Only those cell bodies larger than 5 μm in longest diameter were counted. Counting and further analysis was done on photographic images off-line, the images were coded by one observer and counted blind by another colleague, who was not aware of the code until the end of the analysis.For cell counting studies, the specimens were viewed and photographed under a Nikon Eclipse E800 fluorescence microscope at 10x, 20x, or 40x magnifications. In the reference frames, marked out according to the coordinates of the atlas by 1 as defined consistently for each region of interest (ROI), at identical A/P levels in every brain. Both hemispheres were used for counting, but the values were not combined, since, in some cases, only one hemisphere remained intact. Rather than using regular quadrangles, the measuring frames for each ROI were marked out by hand, to better fit to the boundaries of nuclei, to avoid noise from artifacts and to exclude non-relevant areas, such as brain pathways . The relevant anatomical landmarks and stereotaxic coordinates defining the sectional level, as well as the boundaries for each ROI, are summarized by diagrammatic drawings depicting the location of the measuring frames for every brain region analyzed ,analyzed .Of the pallial regions, the anterior cingulate cortex (ACC), posterior cingulate cortex (PCC) and retrosplenial cortex (RSC) were analyzed. Regarding subpallial regions, we quantified the number of cells in lateral septum (LS), nucleus accumbens (NAc), bed nucleus of stria terminalis (BNST), and caudate-putamen (CPu).2 in every brain region. The linear mixed model included the data of the two hemispheres separately to control for within-subject variance but, since the ID of the individuals was included as a random effect, the two hemispheres’ data were NOT treated as statistically independent. Accordingly, the datapoints on the graphs represent the cell density values of the given brain region from unilateral hemispheres, rather than the mean value of those regions from bilateral hemispheres.Statistical analysis of results was based on individual hemispheric data , using the R Studio program. To determine the effects of treatment (VPA vs. control) and age (7 vs. 60 days) as fixed effects, we applied a generalized linear mixed model (GLMM) based on negative binomial distribution with a Log link functionAlthough the design of the experiment was not intended to specify sex differences, the sex of the individuals was also included in the model in order to control for it. In most cases, the gender of the animals neither had any significant effect, nor was it included in any significant interaction (expect for one specific case mentioned below).For high-resolution microscopic analysis of colocalization, one group of the mounted specimens were viewed and photographed under a LSM 780 confocal laser microscope , the images were stitched using the ZEN 2010 program. The degree of cellular overlapping was assessed on Z-stacks. For analysis of another contingent of specimens an Olympus Fluoview FV3000 confocal laser scanning microscope was used, applying the Olympus FV31S-SW image processing software.For quantification of cellular overlapping, confocal image stacks were analyzed using an Imaris software package  operating on a HP Z4 control system. The “Coloc” feature of Imaris implements the Costes and Lockett method , which aTwenty-two 7-day-old mouse pups of either sex was used. Immediately after decapitation of the animals, the brains were removed and dissected into coronal slabs with the help of a steel brain matrix for reproducible blocking. Further dissection of the slabs was carried out on top of a black anodized aluminum box containing ice, under a stereomicroscope. Tissue samples of 10–20 mg wet weight from the NAc and CPu were dissected bilaterally. The diagrams in g for 10 min followed. The supernatant was centrifuged again at 15,000 g for 20 min at 4°C. The sediment, containing the crude synaptosome fraction, was resuspended according to the wet weight, Then, all samples were diluted to an even protein concentration (1.25 μg/μl), as determined by the BCA method  were immediately placed into pre-weighed Eppendorf tubes and stored in dry ice until further processing. The samples were homogenized in ice-cold lysis buffer . To separate the cellular debris from total protein content, centrifugation at 4°C, 1,200 A method  with theIn agreement with the previously reported findings , a markeTo specify the nature of potential sequelae of impaired DAergic input, first, we analyzed neuronal degeneration (apoptosis) in the NAc (main target of the mesolimbic MT), parallel with other subpallial and pallial regions, by quantitatively measuring the areal density of the perikarya expressing Casp3, a common apoptosis marker enzyme , compariIn P7 mice, the overall level of apoptosis was expected to be high, since this period corresponds to the second  apoptotic surge in the CNS . In our Next, we analyzed the distribution of calcium binding proteins (CaBP) in the above subpallial and pallial regions in VPA-exposed and control P7 mice. Given the observed alterations in apoptosis, it was reasonable to expect changes also in the expression of these, potentially neuroprotective, proteins. The density of calretinin CR;  immunore2 were detected in the CPu (in both age groups), and the largest between-age differences were found in the LS and BNST (2.5-3-fold increase with age), whereas in the CPu and NAc the values tended to drop down at P60 by ca. 15%. Based on further statistical analysis, the age had a significant effect (p < 0.05) on the CB cell density in all of the brain regions observed. Furthermore, age and VPA treatment also had a significant interaction in the CPu (p = 0.01). Notably, the only significant effect of sex observed in the present dataset of cell density values was an interaction with the age in the case of CB in the LS (p = 0.013).Conversely, the abundance of calbindin-D 28K CB;  was not 2 in the cingulate and retrosplenial  is still absent in P7 mice and appears only later in development . At P60 splenial  cortices the NAc  and BNSTreatment .We did not find considerable overlap between Casp3 + and CR + structures in either the NAc  or the RFurther to cellular changes, we searched for alterations of synaptic development by comparing two dopaminoceptive regions of subpallium: CPu and NAc. Proteomic investigation (western blotting) of tissue samples harvested from 7-day-old mouse pups born to VPA-treated or vehicle-treated mothers was focused on simultaneous detection of synaptophysin and TH . We founThe findings point at a general increase of apoptosis on VPA treatment; although no significant increase was detected in the NAc, the most exposed target of the MT, the direction of the difference does not contradict the general trend. It is worth considering that, in previous studies , the greConcerning the density and distribution of CaBPs, the corollary from the findings is that at P7, the age at which the MT pathway reduction was observed  and enhaCalbindin is selectively reduced in the CPu of VPA exposed animals at P7 but no longer at P60. Thus, the VPA-associated overall increase in apoptosis at P7 does not seem to correlate with the abundance and distribution of CaBPs . The only exception is CPu, in which the marked drop of CB may be negatively correlated with increased apoptosis. This may well reflect a protective effect of CB, apparently getting compromised in the striatum by gestational VPA exposure, at least by the age of P7. It is of interest, however, that the observed drop in CB in CPu disappears by P60, which may reflect an increased resilience of the region. Thus, an early detrimental effect can, visibly, be restored. This is further supported by the apparent stability of PV, not responding to gestational VPA exposure at P60, whereas, at P25  a distinct drop of the number of PV + cells was reported in the striatum (but not in the cortex) of mice born to VPA exposed dams . It is wThe non-overlapping distribution of Casp3 + cells vs. CaBP-immunoreactive neurons, observed in the present study, is in apparent agreement with The proteomic findings suggest selective reduction of TH in NAc, in agreement with our previous histological and neurochemical data  without At variance with our findings . The appAccording to recent findings, elevation of Casp3 may not necessarily be followed by neuronal death. Such non-apoptotic Casp3 is thought to regulate dendritic regression and the pruning of dendritic spines . In a reThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Food Chain Safety and Animal Health Directorate of the Government Office for Pest County, Hungary (XIV-I-001-2269-4/2012).CF, RK, and SH carried out the experiments and histological processing of specimens. CF, RK, SH, and ÁÁ contributed to the microscopy and quantitative analysis. CF and RK performed the immunoblotting. IA and ÁÁ acted as Ph.D supervisors for the experiments by CF. GZ and DE participated in the design and evaluation of experiments, and in the writing of manuscript. AC conceptualized the study and wrote the first version of manuscript. All authors contributed to the article and approved the submitted version.


Background: This study evaluates the association between dietary acid load (DAL) and hyperuricemia in Chinese adults. Methods: The China Health and Nutrition Survey (CHNS) in 2009 was used in this cross-sectional study. Potential renal acid load (PRAL) and net endogenous acid production (NEAP) were applied to estimate DAL. A multiple logistic regression model was used to test the relationship between DAL and hyperuricemia risk. Results: A total of 7947 participants were included in this study, of whom 1172 had hyperuricemia. The PRAL score was positively related to the prevalence of hyperuricemia, even when potential covariates were taken into account. In comparison with Q1, the ORs were 1.12 , 1.20  and 1.42  in Q2, Q3 and Q4. However, there was no significant relationship between NEAP scores and hyperuricemia. Every 10 g increase in energy-adjusted fat, protein and animal protein intakes caused a 10%, 17% and 18% increase in hyperuricemia risk, respectively . An obvious linear correlation was also suggested by the restricted cubic spline. Conclusions: Hyperuricemia risk was associated with higher PRAL among Chinese adults. This means that a diet low in PRAL scores could be a very valuable uric acid-lowering dietary pattern. Hyperuricemia and gout have become serious worldwide public health issues owing to their high global prevalence, significant younger trend and huge impact on physical function, health-related quality of life and medical expenditures ,2,3. It The most important factor affecting serum uric acid (SUA) levels is renal excretion, which accounts for approximately 90% of all cases of hyperuricemia . The infTwo approaches are generally taken in assessing DAL, namely potential renal acid load (PRAL) and net endogenous acid production (NEAP) ,28. AccoExisting epidemiological evidence indicates that SUA values may be negatively affected by a high DAL, but prior studies have tended to focus on high-income countries. Several studies have demonstrated that the prevalence of hyperuricemia varies widely across dietary patterns, ethnic groups and geographic regions ,33,34, wThis study analyzed data from the CHNS , a longiA cross-sectional study was developed to collect the general demographics of Chinese inhabitants as well as their health and nutritional status. In 2009, 18,805 people took part in the survey. After excluding participants with incomplete dietary information (N = 7541), implausible energy intake (N = 31) , no serum uric acid data (N = 53) or missing data on covariates (N = 162), and those who were pregnant (N = 57) or younger than 18 years (N = 781), 7947 participants were eventually enrolled .Before taking part in the study, each individual provided informed consent. The Carolina Population Center at the University of North Carolina at Chapel Hill and the National Institute for Nutrition and Health at the Chinese Center for Disease Control and Prevention (CCDC) both gave their approval to the investigation (2015017).Personal dietary consumption information was obtained by asking participants to record their daily food varieties and intake over 24 h periods. The dietary records needed to be investigated for 2 weekdays and 1 day at the weekend. Meanwhile, three days’ worth of food weighing was used to obtain information about edible oil and condiments. The questionnaire used to collect food consumption in this study was the validated Food Frequency Questionnaire (FFQ) . Daily eWe used the following equation to calculate PRAL and NEAP scores PRAL  was classified into four categories: less than 18.5 was considered low weight,18.5–23.9 normal, 24 to 27.9 overweight and 28 or more obese. Medical histories included diabetes (yes/no) and hypertension (yes/no). Based on serum creatinine, we calculated the estimated glomerular filtration rate (eGFR) according to the chronic kidney disease–Epidemiology Collaboration (CKD-EPI) equation .t-test  and the Wilcoxon rank sum test  were employed to calculate the differences in the continuous variables, and the results were expressed as means  and medians . The final covariates included in the study were judged according to whether the basic characteristics of the two groups were different.In our descriptive analysis, categorical variables were calculated by chi-squared tests, and the results were expressed as counts (percentages). Student’s We adopted a multilevel logistic regression model to analyze whether hyperuricemia and DAL (PRAL and NEAP) were related. Four groups were identified based on PRAL and NEAP scores quartiles. The odds ratio (OR) was calculated based on the quartiles of the DAL score. The reference group was Q1, the lowest group. Considering that dietary fiber and water intake can affect the excretion of uric acid, we adjusted for them as covariates. Three models were developed: model 1 without covariate adjustment; model 2 adjusted for age, gender, residence area, smoking status, marital status, alcohol intake, education level, BMI, hypertension, diabetes and eGFR; and model 3 further adjusted for dietary fiber and water intake. Analysis of subgroups was conducted by gender and age (<60 and ≥60 years).In order to assess whether dietary fiber intake  and water intake , <1700 ; ≧1500 mL , ≧1700 ) could modify the association between DAL and hyperuricemia, we conducted interaction analyses.We examined linearity using multivariable-adjusted restricted cubic splines (model 3) and investigated the dose–response correlation between DAL and the prevalence of hyperuricemia.p < 0.05 was defined as statistically significant.SPSS Statistics 23 was used for all the analyses. The figures were produced in R version 4.2.2 and Graphpad Prism 9.4.1. This study included 7947 subjects. The participants’ mean age was 50.7 ± 15.0 years, of whom 4215 (53%) were females, 3732 (47%) were males, and 6701 (84.3%) were married. The rural population was 5520 (69.5%) and the urban population was 2427 (30.5%). The history of alcohol consumption and smoking was 1652 (20.8%) and 2468 (31.1%), respectively. In total, 2531 subjects had hypertension (31.8%) and 606 subjects had diabetes (7.6%).A total of 1172 subjects in our study had hyperuricemia, a prevalence of 14.7% (19% for men and 11% for women). The median values for PRAL and NEAP were 21.9 mEq/d and 75.2 mEq/d, respectively. The distribution of subject characteristics is summarized in The subjects’ total energy, water intake and energy-adjusted carbohydrate, protein, fat, cholesterol, dietary fiber and mineral intake are shown in In all three logistic regression models, an increased prevalence of hyperuricemia and elevated PRAL scores were related . In compp-values > 0.05), shown in As shown in models 1 and 2, the higher the NEAP scores, the greater the hyperuricemia risk. The ORs in model 2 were 1.25 , 1.30  and 1.40  in Q2, Q3 and Q4. However, confounding factors were further adjusted with model 3 (model 3), where NEAP scores and hyperuricemia risk did not appear to be correlated . When all the confounders were taken into account, Q3 and Q4 female subjects also demonstrated an increased risk of hyperuricemia versus Q1 female subjects . After controlling for all potentially confounding factors, the age-stratified results indicated a link between PRAL and hyperuricemia prevalence. In the age <60 years group, compared with Q1, the OR for Q4 was 1.44 . For the age ≥60 years subjects, in comparison with Q1, the ORs were 1.38 , 1.24  and 1.47  in Q2, Q3 and Q4. The results are shown in According to subgroup analysis, elevated NEAP was significantly associated with hyperuricemia among the age <60 years group. After adjusting for all confounders, compared with Q1, the OR for Q4 was 1.38 . Concerning the gender groups and age ≥60 years group, the NEAP and hyperuricemia risks did not appear to be related after all the confounding factors were taken into account, as shown in p for overall <0.005 and p for nonlinearity >0.05), as displayed in p for overall = 0.2137), shown in Multivariable-adjusted restricted cubic spline analyses showed a significant linear relationship between PRAL and hyperuricemia risk . For each 100 g of energy-adjusted phosphorus consumed, the hyperuricemia risk increased by 6% . Intake of energy-adjusted carbohydrate, plant-derived proteins and dietary fiber may reduce the risk of hyperuricemia . There was no correlation between energy-regulated potassium, cholesterol, calcium and magnesium intake and the risk of hyperuricemia .From This study is the first investigation in China to examine the relationship between DAL and hyperuricemia in a large and geographically representative sample size, to the best of the authors’ knowledge. Our analysis revealed that the PRAL score was linked to the prevalence of hyperuricemia, following a linear correlation. The NEAP score and the hyperuricemia risk were positively related in the age <60 years group, while no significant results were found in the overall participants, male, female and ≥60 years groups. Even when confounding factors were taken into account, this correlation still existed. Additionally, we identified that the intake of energy-adjusted protein, fat, animal protein and phosphorus was positively linked to the risk of hyperuricemia, while energy-adjusted dietary fiber, plant protein and carbohydrate were inversely correlated.In other countries, the association between DAL and hyperuricemia had been examined. An analysis of 6894 German adults showed that PRAL and SUA values had a positive correlation, and in the highest PRAL score group, the hyperuricemia risk was higher than that in the lowest group  , which iWe further explored the risk of hyperuricemia based on nutrient intakes, and found that a higher energy-adjusted fat and protein intake was linked to hyperuricemia prevalence in Chinese adults. The finding is consistent with previous results, which have suggested that Western dietary patterns with high protein and fat content can directly contribute to the modern hyperuricemia epidemic . We alsoOur study found that hyperuricemic subjects consumed less energy-adjusted dietary fiber than non-hyperuricemic subjects. In addition, the results of multiple logistic regressions indicated a reduced risk of hyperuricemia with high dietary fiber ingestion. This association persisted while adjusting for full confounding factors. The positive effect of dietary fiber ingestion on reducing hyperuricemia risk had been mentioned several times. A study from Korea comparing the nutritional intake and dietary quality of hyperuricemia and non-hyperuricemia groups demonstrated that subjects with hyperuricemia had a lower dietary fiber intake than the non-hyperuricemic subjects . A studyA correlation between the risk of hyperuricemia and energy-regulated calcium, potassium and magnesium intake was not observed. It is generally believed that calcium, potassium and magnesium have a high alkalinizing capacity in favor of lower PRAL . There aOur research has several strengths. First, a large and nationally representative sample was used to evaluate whether there was an existing relationship between DAL and the prevalence of hyperuricemia. Therefore, the results are reliable and typical. Second, when a broad range of confounding variables (dietary and non-dietary) were taken into account, the positive association of PRAL with hyperuricemia remained significant. Our findings not only accumulate epidemiological evidence for exploring the association between DAL and hyperuricemia in Chinese adults, but also demonstrate that a diet low in PRAL scores is likely to be an effective dietary management strategy to prevent elevated uric acid levels.Our study also had some limitations. Firstly, DAL and hyperuricemia were assessed cross-sectionally, which limits measuring their causal association. Future studies could be carried out prospectively and longitudinally to verify these findings. Secondly, the ascertainment of hyperuricemia was based on only one fasting serum uric acid value of the subjects; however, fasting serum uric acid levels need to be measured on two different days, according to the guidelines for diagnosing hyperuricemia. In addition, there was no medically documented information on the use of anti-gout medications, hyperuricemia and gout history in the CHNS questionnaire. Therefore, SUA levels may not reflect the actual situation of individuals. Thirdly, although we used validated measures (PRAL and NEAP) to evaluate the overall DAL, the calculation based on dietary intake is an indirect method rather than a direct measurement. Due to individual factors, nutrient absorption in the gastrointestinal tract may vary considerably, but this is not considered by the calculated equations . FinallyIn summary, elevated PRAL was correlated with hyperuricemia risk among Chinese adults. A significant linear correlation was also suggested by the multivariable-adjusted restricted cubic spline. This means that a low PRAL diet has the potential to lower uric acid and could be a very valuable uric acid-lowering dietary pattern. However, no significant results were seen for the relationship between NEAP scores and hyperuricemia. Further research to verify this association is warranted.


Healthcare professionals face an ongoing challenge in managing both acute and chronic wounds, given the potential impact on patients’ quality of life and the limited availability of expensive treatment options. Hydrogel wound dressings offer a promising solution for effective wound care due to their affordability, ease of use, and ability to incorporate bioactive substances that enhance the wound healing process. Our study aimed to develop and evaluate hybrid hydrogel membranes enriched with bioactive components such as collagen and hyaluronic acid. We utilized both natural and synthetic polymers and employed a scalable, non-toxic, and environmentally friendly production process. We conducted extensive testing, including an in vitro assessment of moisture content, moisture uptake, swelling rate, gel fraction, biodegradation, water vapor transmission rate, protein denaturation, and protein adsorption. We evaluated the biocompatibility of the hydrogel membranes through cellular assays and performed instrumental tests using scanning electron microscopy and rheological analysis. Our findings demonstrate that the biohybrid hydrogel membranes exhibit cumulative properties with a favorable swelling ratio, optimal permeation properties, and good biocompatibility, all achieved with minimal concentrations of bioactive agents.  Ochrophyta, class Phaeophyceae), is composed of repeating mannuronic acid (M) and glucuronic acid (G) units [With a growing population and increased life expectancy, it is crucial for the healthcare system to adapt to the complex medical needs of patients, while the industry must provide accessible and cost-effective solutions. Recent research conducted in 2021 confirmed the significant impact of chronic wounds on patients’ quality of life . In ordeG) units ,14,15. AG) units ,17,18,19G) units ,23, hydrG) units ,25,26, fG) units ,28,29, nG) units ,31,32, oG) units ,34,35. AG) units ,37. AlgiG) units ,39. DuriG) units , gelatinG) units , or polyG) units ,42. PolyG) units . ProductG) units , nanofibG) units  or hydroG) units . ChemicaG) units ,49,50,51G) units , biocompG) units  or microG) units  of the fG) units ,44,55,56G) units  obtaininG) units ,60,61,62G) units ,64,65,66G) units ,68,69. TG) units ,74,75,76G) units ,73. DuriG) units . An ideal wound dressing should incorporate a range of qualities, including the promotion of healing, biocompatibility, biodegradability, suitable mechanical properties, adaptability, moisture variations, semipermeability, conformity to body contours, ease of use, and affordability . Due to To ascertain the most performant formulation that could act as a matrix for integrating bioactive additives, we conducted an initial evaluation of seven formulations using alginate (Alg) and polyvinyl alcohol (PVA). We evaluated hydrogel membranes made of biopolymer (BP40 and BP50), synthetic polymer (SP25 and SP15), or a combination of both  macroscopically and via in vitro studies for their swelling index, degradation rate and gel fraction. As presented in The incorporation of different concentrations of collagen (COL) and hyaluronic acid (HA) had no observable effect on the appearance of the biohybrid hydrogel membranes . HoweverAs depicted in As presented in The swelling ratio, which indicates the water retention capacity of the hydrogels, was determined via a gravimetric assay. In order to mimic in vivo conditions, the samples were tested immediately following the completion of the crosslinking procedure and not fully dried prior to the analysis. The addition of COL and HA affected the swelling behavior differently depending on the PVA/Alg ratio, as depicted in 2O, pH 7.4) and simulated wound fluid . As expected, the addition of enzymes resulted in an elevated degradation rate of the samples, although the increase was not substantial but rather discrete. The results of the degradation study illustrated in The biodegradation of hydrogel membranes primarily occurs through the action of enzymes that recognize and target specific regions within the polymer network formed through crosslinking ,105,106.The number of crosslinked molecules forming an insoluble gel fraction is reflected by the gel content of a hydrogel, while the non-crosslinked portion of the hydrogel dissolves upon immersion in a solvent, leading to a reduction in the sample weight . In addi2 h and at 48 h between 106.99 ± 1.53–127.32 ± 2.65 g/m2 h. As it is shown in 2 h, while for wounded skin it can increase by a factor of ten or more, depending on the wound thickness [In this study, we evaluated the water vapor transmission rate (WVTR) at 24 and 48 h using the gravimetric method while filter paper served as a control. At 24 h, the WVTR for the biohybrid hydrogel membranes was in the range of 104.33 ± 3.06–125.56 ± 3.06 g/mhickness . In order to evaluate the protein adsorption of the biohybrid hydrogel membranes, we implemented a modified protocol inspired by one in the existing literature ,127. BovThe hydrogels were assessed for their anti-inflammatory properties using the protein denaturation inhibition assay, using BSA as a model protein and aspirin as a control. According to our results, the BH1.20 formulation exhibited the most significant inhibition of protein denaturation, with a value of 59.26 ± 0.86% in comparison to aspirin. We presume that this effect is primarily attributed to the presence of COL and HA in the formulation . AdditioThe assessment of in vitro cytotoxicity is crucial in determining the biocompatibility of biomaterials, and one commonly used method is the LDH assay (lactate dehydrogenase assay). LDH is a stable enzyme present in the cytosol of cells, and its release into the surrounding medium indicates cellular membrane permeabilization induced by chemicals or factors that affect cellular integrity ,139. In Several factors may account for these findings, including potential variations in the purity or molecular weight of the natural components employed in the study. Additionally, physical interactions due to the surface morphology and mechanical properties of the hydrogels could play a role, along with possible concentration-dependent effects. The specialized literature reported minimal cytotoxicity for the components used in the study. Charron et al. (2020) demonstrated that the number of freeze–thaw cycles (F-H cycles) did not have an influence on the cytotoxicity of gels incorporating polyvinyl alcohol (PVA) and gelatin . SchulzeThe scanning electron microscope (SEM) images  revealedMechanical characteristics are crucial in the design of new biomaterials, including wound dressings, as they must conform to body contours, provide mechanical protection, and maintain their inherent properties during their application period. Key parameters including Young’s modulus, elongation at break, and tensile strength are essential in assessing dressing performance and clinical indications. We evaluated the rheological characteristics of samples BH1.80 and H1, identified as potential optimal wound dressing formulations based on previous assays. Our focus was on rheological properties influenced by moisture content, aiming for a versatile material for both dry and exudative wounds. The addition of COL and HA was found to influence the behavior of the hydrogel membranes, aligning with findings from previous tests. Our analysis revealed that within a moisture content range of 30–40%, the Young’s modulus of sample BH1.80 exceeded that of H1 A. Above The objective of this study was to produce and to explore the influence of polymer ratio and the addition of bioactive agents on the properties of biohybrid hydrogel membranes that can be used as wound dressings. Through a comprehensive analysis using various in vitro and instrumental methods, the study revealed important insights into the performance of these membranes. The hybrid hydrogel membranes displayed cumulative properties, including improved stability and an enhanced swelling ratio. The PVA/Alg ratio had a notable influence especially on the swelling ratio and gel fraction.The addition of bioactive substances, specifically collagen (COL) and hyaluronic acid (HA), had a significant influence on the performance of the biohybrid hydrogel membranes compared to the hybrid hydrogel membranes. The presence of COL and HA had an impact on the crosslinking process and influenced various characteristics of the obtained membranes, including moisture content, gel fraction, swelling rate, morphological appearance, and mechanical properties.Interestingly, variations in hyaluronic acid concentrations did not significantly affect the overall properties of the biohybrid hydrogel membranes. The introduction of bioactive agents had varying effects on LDH release during cell studies. Cells treated with BH2 formulations (a higher PVA concentration) demonstrated lower LDH release, indicating a decrease in cytotoxicity. On the other hand, cells treated with BH1 formulations  showed an increase in LDH release. Further research is required to comprehensively understand the underlying mechanisms and fine-tune the concentrations of bioactive agents to enhance biocompatibility.2, and hyaluronidase (1000 U) were purchased from Sigma. Sodium alginate, glycerol , sodium hyaluronate (≥98.0%), and collagen (rat tail) were purchased from VWR Chemicals, Honeywell, MedChemExpress and Roche, respectively. Polyvinyl alcohol , bovine serum albumin , collagenase , CaCl2 1%) were dispersed in glycerol. Warm distilled water (68 °C) was added to the homogeneous mixture, and the ensuing solution was homogenized at 1000–1500 rpm for 15 min. The solution was poured onto a Petri dish and left at 28 °C for 48 h. The obtained hydrogel membranes were sterilized by UV light for 15 min and kept at room temperature until further investigations.The biopolymer hydrogel membranes (BP40 and BP50) were prepared via ionic crosslinking, using the solvent casting method. Briefly, the proper amounts  from stoThe synthetic polymer hydrogel membranes (SP25 and SP15) were prepared using the freezing–thawing (F–T) method reported by Peppas and Stauffer, 1991. The proper amounts of hot PVA solution (10%) and glycerol  were mixThe hybrid hydrogel membranes  were prepared by stirring mixtures (according to Stock solutions of collagen (0.35%) and hyaluronic acid (0.25%) were prepared in distilled water. The thickness variations were achieved using a digital micrometer  and were evaluated as a dependent variable to optimize the hydrogel formulation. We measured five different points (center and edges). For the weight variation test, the hydrogels were weighed individually. The pH of the hydrogel formulation was determined using a digital pH meter (Thermo Fisher Orion Star A211).To determine the moisture content, at the end of the crosslinking process, the samples were cut in the form of a circle with a diameter of 2 cm and weighed before (Wi) and after drying (Wd) . The samFor moisture uptake determination, the crosslinked hydrogel membranes were cut into circles with a diameter of 2 cm and left for 24–48 h in an oven at 55 °C, until their weight became constant (W1) . The samThe swelling ratio was evaluated in vitro via the gravimetric method ,170 usin2O (pH 7.4), SWF (pH 8.3), hyaluronidase solution , collagenase solution  and a mixed solution of HAasa and COLasa —using an adapted protocol previously described in the literature [The biodegradation of the hydrogel membranes was studied in vitro using different solutions—dHterature ,173. TheThe gel fraction of the hydrogel membranes was evaluated in vitro via the gravimetric method ,174 usinThe water vapor transmission rate of the biohybrid hydrogel membranes was assessed using a modified method described by Razzak et al. . To measw/v) in phosphate-buffered solutions . Batch experiments were carried out by adding 5 mL of the protein solutions to the hydrogel samples (which had a diameter of 1 cm and thickness of 1 mm). The samples were placed in an orbital shaker (200 rpm) at 37 °C, for 24 h. Aliquots of the solution were collected and initial and equilibrium protein concentrations were assayed spectrophotometrically in a UH5300 Hitachi spectrophotometer (Japan) using the Bradford reagent. The amount of protein adsorbed by the hydrogel membranes was calculated using the following equation: 0 and Ce (mg/L) are the initial and equilibrium concentrations of protein in the solution, respectively; V (L) is the solution volume; and m (g) is the hydrogel membrane mass. The evaluation of protein adsorption of the hydrogel membranes was assayed using BSA as the protein model. The evaluation protocol employed in this study was adapted from the existing literature ,127. TheTo assess the anti-inflammatory properties of the hydrogels, we evaluated their ability to inhibit protein denaturation. The methodology was adapted from the literature ,135,136.2 at 37 °C. Cytotoxicity was assessed via cellular LDH release . In total, 10,000 cells were seeded in triplicates overnight in 96-well plates and incubated the next day with triplicates of the gel formulations. Two controls were included—a negative control (cell culture medium only) and a positive control . Additional cell-free wells were incubated with tested gels, for background subtraction. For the LDH analysis, 50 µL of the supernatant was collected from each well and incubated with the assay reagent for 30 min, in the dark. After the addition of the stop solution, absorbance was read at 490 nm with the microplate reader Anthos Zenyth 3100. Cytotoxicity was assessed as a % of that of the lysis control using the described formula:For the LDH assay , human nThe morphological features of the biohybrid hydrogel membranes compared to those of the hybrid hydrogel membranes were acquired using Nova NanoSEM 630 Scanning Electron Microscope  with an accelerating voltage of 5 kx. For the SEM images, all samples were coat-sputtered with Au to ensure the conductivity of the sample (60 s).The samples were cut into strips (0.5 cm × 4 cm) to analyze the mechanical properties of obtained hydrogels using a protocol adapted from the literature ,178. The