The etiology of Anterior Cruciate Ligament (ACL) injury in women football results from the interaction of several extrinsic and intrinsic risk factors. Extrinsic factors change dynamically, also due to fatigue. However, existing biomechanical findings concerning the impact of fatigue on the risk of ACL injuries remains inconsistent. We hypothesized that fatigue induced by acute workload in short and intense game periods, might in either of two ways: by pushing lower limbs mechanics toward a pattern close to injury mechanism, or alternatively by inducing opposed protective compensatory adjustments.In this study, we aimed at assessing the extent to which fatigue impact on joints kinematics and kinetics while performing repeated changes of direction (CoDs) in the light of the ACL risk factors.This was an observational, cross-sectional associative study. Twenty female players  performed a continuous shuttle run test (5-m) involving repeated 180°-CoDs until exhaustion. During the whole test, 3D kinematics and ground reaction forces were used to compute lower limb joints angles and internal moments. Measures of exercise internal load were: peak post-exercise blood lactate concentration, heart rate (HR) and perceived exertion. Continuous linear correlations between kinematics/kinetics waveforms (during the ground contact phase of the pivoting limb) and the number of consecutive CoD were computed during the exercise using a Statistical Parametric Mapping (SPM) approach.r = −0.517, p < 0.01), while neither%HRmax nor [La–]b nor RPE were correlated with test duration before exhaustion (p > 0.05). Alterations in lower limb kinematics were found in 100%, and in lower limb kinetics in 85% of the players. The most common kinematic pattern was a concurrent progressive reduction in hip and knee flexion angle at initial contact (10 players); 5 of them also showed a significantly more adducted hip. Knee extension moment decreased in 8, knee valgus moment increased in 5 players. A subset of participants showed a drift of pivoting limb kinematics that matches the known ACL injury mechanism; other players displayed less definite or even opposed behaviors.The test lasted 153 ± 72 s, with a rate of 14 ± 2 CoDs/min. Participants reached 95% of maximum HR and a peak lactate concentration of 11.2 ± 2.8 mmol/L. Exercise duration was inversely related to lactate concentration (Players exhibited different strategies to cope with repeated CoDs, ranging from protective to potentially dangerous behaviors. While the latter was not a univocal effect, it reinforces the importance of individual biomechanical assessment when coping with fatigue. Non-contact anterior cruciate ligament (ACL) lesions are the most devastating injuries in female footballers, with an incidence as high as 0.9–2.2 ACL ruptures per 1,000 match hours  and accounting for 43% of total time loss due to injuries , with 32The etiology of ACL injury in women athletes is multifactorial and results from the interaction of several risk factors , includiThe susceptibility to injury change dynamically , also duThe level of evidence supporting the impact of fatigue on the risk factors of ACL injury remains elusive . A wide This lack of homogeneity in the existing literature originates from different sources of uncertainty: (i) a minor portion of studies adopting general fatigue models which simulate sport-specific peripheral and central fatigue effects, i.e., current fatigue protocols did not reflect the complexity of physical fatigue that occurs during an actual game ; (ii) few studies have quantified fatigue in a meaningful way; (iii) few to no data described the effects of the development/onset of fatigue throughout the exercise; (iv) subject groups and athletic tasks analyzed were not homogeneous.per se that affects lower limbs mechanics and in turn the susceptibility to injury, rather the acute workload in relatively short and intense game periods  technique . ModificThis was an observational, cross-sectional associative study aiming at evaluating the modifications of lower limb mechanics during a physically demanding exercise protocol. It was approved by the Institutional Ethics Committee (protocol n. 06/2020) and met the current ethical standards in Sport and Exercise Science. Participants were recruited on a voluntary basis and took part in the laboratory sessions after signing a written informed consent providing detailed explanation of the risks and benefits of the study.Communication about the research was shared with professional and semi-professional players of the main clubs of the region during the seasons 2018–2019 and 2019–2020. Inclusion criteria were: playing for a women football team competing in the first or second Italian division (“Serie A” or “Serie B”); participating in at least four training sessions per week; no history of previous severe injuries (>28 days of absence from the game) in the 6 months preceding the test; not having sustained any previous ACL or other ligamentous knee injury; being judged by a medical doctor to have no restrictions to sports practice.Twenty players (age range: 20–31 years) were selected and took part in the laboratory tests , that weLaboratory sessions were performed in the morning within a motion capture laboratory equipped with artificial turf, with a controlled ambient temperature of 20–22°C. Participants wore fitting sport clothing and their own indoor soccer shoes. After anthropometrics measurements obtained with a digital stadiometer , the players underwent a 10-min warm-up session led by a licensed strength and conditioning coach involving stretching, jogging, mobilization, lateral hopping, and familiarization with the test.Immediately following the warm-up routine, athletes started the actual exercise protocol, that was extensively described elsewhere , 2019 anDuring the whole test, three-dimensional kinematics was collected at 100 Hz with an 8-cameras SMART-DX optoelectronic system ; ground reaction forces data were recorded with two 46.5 × 51.8 cm force platforms  sampling at 200 Hz. Thirty-seven spherical (diameter: 15 mm) reflective markers were positioned on the following landmarks: radius styloid processes, olecranons, acromia and seventh cervical vertebra; anterior-superior and posterior-superior iliac spines; medial and lateral femoral epicondyles; medial and lateral malleoli; on the shoes in correspondence with the calcaneus and the first and fifth metatarsal heads; four clusters (three-markers each) positioned on the thighs and on the shanks. A 10-s static recording was obtained before the test.–]b) was determined with a portable lactate analyzer  every 2 min after the test, until a decrease in the measure was observed. Heart rate (HR) during the exercise was also recorded . After the shuttle test, subjects provided a rating of their perceived exertion (RPE) on the 0–10 Borg scale.Resting (before exercise) and peak post-exercise blood lactate concentration , using a 24° of freedom pelvis and lower extremities cluster-based model ; inverse•pelvis rotation and lower limbs three-dimensional joint angles ,•centre of mass (CoM) velocity in the running direction ,•three-dimensional ground reaction forces,•three-dimensional internal moments of force  through inverse dynamicsJoint moments and GRF were both normalized to body mass. After excluding the first three CoDs (used by the athletes to adjust themselves to the running pace), kinematics and kinetics waveforms during the ground contact phase of the dominant side  pivoting limb were resampled to 101 time points.1 approach. SPM analysis enabled exploration of the relationships between continuous parameters and a discrete value without having to impose the temporal focus bias . Variations in dependent variables  throughout the test were tested using a SPMcus bias . This mecus bias . This anmax) and with a peak post-exercise lactate concentration of 11.2 (2.8) mmol/L. Reported exertion substantially varied among individuals (4–9).A total of 699 CoDs were analyzed: participants completed between 15 and 68 CoDs with the preferred limb before exhaustion , corresponding to 14 (SD: 2) CoDs per minute. Average ground contact time was 0.4 (0.2) s. The shuttle test was performed at an average speed of 2.6 (0.2) m/s and lasted on average 153 (72) s; reaching 95% of the maximum HR , that is: the lower the running speed, the higher the lactate concentration . Neither%HRmax nor [La–]b nor RPE were correlated with test duration before exhaustion (p > 0.05).Exercise duration was inversely related to lactate concentration  and 12  of the participants, respectively. These two angles increased during the test in only 1/20 players (Player 5). More specifically, the observed drop in hip flexion at initial contact varied from about 10° (Players 6 and 13) up to 25° (Player 20). The reduction in knee flexion at initial contact ranged from about 5° (Players 6 and 18) to over 20° (Player 11).All the players showed statistically significant modifications of lower limbs kinematics in at least two of the considered variables. r = 0.617 to 0.912, p < 0.01) of 5–10° at initial contact  and did not change in 11 players. On the frontal and transverse planes, knee and ankle angles were primarily unchanged during the test, and a clear trend could not be identified. In particular, knee adduction and rotation angles did not change in 70 and 75% of the players, respectively. Pelvis rotation toward the new running direction was reduced in 5/20 players  and unchanged in 14/20, while CoM speed at ground contact increased in 6 players from 0.7–0.8 to 1.2–2.1 m/s  and was unchanged in 11.Hip abduction decreased in 8 players  and was unchanged in 7; knee extension moment decreased in 8  and was unchanged in the other players. Knee valgus moment increased in 5 and reduced in 2 players, with changes between early and late turns ranging from −2.5 and 0.2 Nm/kg at about 10% of the ground contact phase.Four players showed significant concurrent changes in 75% or more variables . However, a distinct trend was not noticeable in most cases . Two excr = −0.703 to 0.801, unchanged in 6). Hip and ankle internal rotation moments decreased in 5/16 players. The other ankle moments were unchanged in 75% or more players.Hip extension moment also decreased in 6/16 players by around 1.5 Nm/kg at 10% of the ground contact phase (The repetition of turning actions (180°-CoDs) induced alterations in lower limb kinematics in all the tested players, and in lower limb kinetics in 85% of the players. The nature and extent to which lower limbs mechanics was altered throughout the test were highly variable among individuals. Biomechanical modifications mostly occurred in the sagittal plane, but adaptations in the coronal and transverse planes were also detected. A subset of participants showed a drift of pivoting limb kinematics that tightly matches with the known ACL injury mechanism; other players displayed less definite, minimal or even opposed behaviors.The current study introduced a focal change in the analysis of fatigue effects: moving away from the traditional “pre- vs. post-fatigue” paradigm, we assessed the biomechanical waveforms throughout the whole exercise, by exploiting the capabilities of SPM. This approach tackles one of the uncertainty sources of existing studies  and allowed to detect deteriorations in biomechanics due to sub-maximal repetitive knee loadings replicated over a short time span while performing changes of directions , or to iWhen discussing our findings, we will embrace an injury perspective recently proposed by The most represented kinematic pattern we found was a progressive reduction in hip and knee flexion angle at initial contact, observed concurrently in 10/20 players. In 5 of these 10, a reduction of hip abduction (a more adducted hip) was also observed. Three players showed a trend toward knee valgus, and six players showed a progressively externally rotated foot. In six players the approaching CoM speed increased toward the end of the test (at constant shuttle speed). Only three players did not show a reduction of either the knee or the hip flexion angle.The relevance of these results to injury risk evaluation is two-fold: first, kinematic changes confirm the trend observed in our previous study on male athletes  and in o–]b > 11 mmol/L) might have become progressively less effective in dissipating impact forces through eccentric action, thus transmitting load to passive structures  is often referred to as a potentially deleterious posture, as it could increases the shear force on the ACL . It coulructures .A concurrent macroscopic result of our study was that a univocal trend in kinematics and kinetic changes could not be found. In 11/14 kinematic variables we did not detect a significant correlation at initial contact. There were a fraction of players showing minimal changes or even protective adaptations, suggesting that regulatory attempts to prevent harms can arise in response to a perturbed (fatigued) state. For instance, a reduction of knee extension moment was found in 8/16 players. This might be due to fatigued knee extensors muscles that forced to adopt a different strategy to negotiate the CoD. In this sense, our findings did not show a mechanistic link between fatigued muscles and an increase in the moments around the knee joints, as hypothesized by Savage et al. for 45-degrees sidestepping and crossover cutting tasks . On the In brief, these findings suggest that some players could compensate the impairment induced by acute workload  with a sort of “safety strategy” to preserve the integrity of passive structures and safely dissipate energy during CoDs. The reduction of knee adduction moment for 5/16 players, the shift toward a more adducted knee observed in four players and the increase in hip and knee flexion angles observed in Player 5 seem to support this view.max, [La–]b far above the aerobic threshold, RPE) and external load  denoted an high-intensity exercise, with a substantial muscle load due to repeated decelerations and accelerations  internal load was substantially the same.Even with individualized exercise load (70% of players’ MAS), each player showed a distinct pattern of alterations. It is unlikely that the underlying reason would just lie in the differences of the number of performed CoDs. For instance, Players 1, 5, and 17 completed 67–68 shuttles till exhaustion and showed several (but diverse) alterations in kinematics and kinetics. However, there were also cases like Player 2 who performed as little as half shuttles and still showed large modifications during the test. At the same time, players who performed similar amounts of CoDs (Player 10 and 11) at different speed (2.9 vs. 2.4 m/s) showed similar biomechanical patterns when coping with the exercise, even though perceived (RPE = 7) and measured  and ACL injury . GloballWe would like to point out that this is an associative study: we found a link between acute fatigue development during exercise and, in some cases, behaviors that can be associated to ACL injuries. This does not directly imply that fatigue alone is responsible of an injury , but thaAs the mechanical and neuromuscular demands of changes of directions are angle-dependent , resultsParticipants were wearing their own pairs of indoor soccer shoes; while this was a subject-wise analysis, we are aware that different shoe-surface mechanical coupling due to specific shoe models might in principle impact on the adaptations to exercise and constitute an additional source of inter-individual variability.Only the final plant step of the change of directions has been analyzed; it would be interesting to address in future work whether fatigue influences the role of the penultimate foot contact within a change of direction .This study used multiple linear regression for continuous time series to assess the change of kinematics and kinetics waveforms during exercise . HoweverLastly, while being a strictly defined sample, the players recruited belonged to different teams. We cannot exclude that a more homogeneous (and possibly larger) cohort would have returned more consistent results due to even exposure to injury prevention and athletic training programs.In female footballers, fatigue induced by repeated turns altered lower limbs mechanics. The fatigue effects were evaluated by assessing the biomechanical waveforms throughout the exercise, overcoming the conventional “pre- vs. post-” design. Biomechanical changes mainly occurred in the sagittal plane, with only a subset of participants also showing modifications in the coronal and transverse planes. Accordingly, the tested players demonstrated different strategies to cope with the exercise, ranging from protective to potentially dangerous behaviors . While not being in itself an isolated risk factors, we concluded that in some of the tested players fatigue affected the musculoskeletal system in a way that may contribute to bring an athlete toward an unsafe condition.As the physiological, and in turn neuromuscular and biomechanical adaptations to fatigue were strongly subject-specific , these fThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Comitato Etico Politecnico di Milano n. 6/2020. The patients/participants provided their written informed consent to participate in this study.MZ: study conception and design, data collection and statistical analysis, and initial draft of the manuscript. CB, AG, and FS: experimental setup and laboratory sessions, data collection and preparation, and data analysis. SD and FB: data collection, discussion on results, and critical revision and writing of selected parts of the manuscript. MG and CS: supervision, data interpretation, and critical revision of the study methods and results. MT and CG: supervision and critical revision of the manuscript. All the authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The photochemically-induced thrombosis (photothrombosis) method can create focal cerebral infarcts anywhere in the relatively superficial layers of the cerebrum; it is easy to implement and minimally invasive. Taking advantage of this versatility, we aimed to establish a new rat model of urinary frequency with focal cerebral infarction, which was characterized by its simplicity, nonlethal nature, and high reproducibility. The prefrontal cortex and the anterior cingulate cortex, which are involved in lower urinary tract control, were targeted for focal cerebral infarction, and urinary parameters were measured by cystometrogram. Cystometric analysis indicated that micturition intervals significantly shortened in photothrombosis-treated rats compared with those in the sham operative group on Days 1 and 7 (P < 0.01), but prolonged after 14 days, with no difference between the two groups. Immunopathological evaluation showed an accumulation of activated microglia, followed by an increase in reactive astrocytes at the peri-infarct zone after photothrombotic stroke. Throughout this study, all postphotothrombosis rats showed cerebral infarction in the prefrontal cortex and anterior cingulate cortex; there were no cases of rats with fatal cerebral infarction. This model corresponded to the clinical presentation, in that the micturition status changed after stroke. In conclusion, this novel model combining nonlethality and high reproducibility may be a suitable model of urinary frequency after focal cerebral infarction. Cerebral infarction is associated with a high incidence of lower urinary tract symptoms (LUTS), particularly urinary bladder overactivity, which reduces a patient’s quality of life ,2. TheraMiddle cerebral artery occlusion (MCAO) rats have generally been used as a model for bladder overactivity caused by cerebral infarction ,6. HowevEstablishing a simple and reproducible animal model of LUTS caused by cerebral infarction is needed to solve these issues. Therefore, we focused on applying photochemically-induced thrombosis (photothrombosis) as a method for creating focal cerebral infarcts . The priThe periaqueductal gray (PAG) region receives sacral afferents related to bladder filling and transmits efferent signals to the sacral spinal cord or bladder via the pontine micturition center (PMC) . PreviouOur aim was to establish a new rat model of urinary frequency, with focal cerebral infarction in PFC and ACC induced by photothrombosis, which is easy to perform and minimally invasive, and to observe histopathological changes in the brain.All experimental protocols were approved by the Animal Care Committee of Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan (No. H30M-57). The study design is shown in We used a modified version of the photothrombosis technique described by Watson to induce cerebral infarction . The rat2] × thickness [2 mm]) according to previously described methods [It is known that 2,3,5-triphenyl tetrazolium chloride (TTC)  staining allows for rapid assessment of the infarcted area . This TT methods .The modified neurological severity score (mNSS) was used to evaluate neurobehavioral functions . The mNSAfter the neurological evaluation, all rats underwent cystometry according to methods described previously –28. With2O (5 min). Antigen retrieval was performed by heating the sections in 10 mM trisodium citrate dehydrate (pH 6) in Decloaking Chamber™ NxGen  at 95°C for 40 min before cooling at room temperature for 30 min. After blocking the endogenous peroxidase activity by methanol containing 3% H2O2, the brain slice tissues were incubated overnight at room temperature with antibody against NeuN. After washing in buffer, the sections were immunostained by the avidin–biotin peroxidase method using the Vectastain Elite Kit  with 3–3-diaminobenzidine and hydrogen peroxide as the chromogen. For immunofluorescence staining, the brain slice tissues were incubated with anti-Iba1 and anti-GFAP overnight at 4°C, and incubated with goat anti-rabbit or goat anti-mouse secondary antibodies  for 1 h at 25°C. For immunostaining and immunofluorescence analyses, BZ-X800  was used to acquire digital images which were analyzed with a BZ-X800 Analyzer.We used NeuN  as a neural marker to assess the distribution of mature neurons. The area from which mature neurons had disappeared indicated the infarcted area. Anti-ionized calcium binding adaptor molecule 1 (Iba1)  and anti-glial fibrillary acidic protein (GFAP)  were used to assess activated microglia and reactive astrocytes in ischemic regions, respectively. After cystometrogram, the rats were exsanguinated from their right auricle, following inhalant anesthesia using isoflurane, and transcardially perfused with 20 mL/body of 1% heparinized saline followed by 25 mL/body 10% formalin. The brains were removed, postfixed overnight for 24 additional h in 10% formalin at 4°C, and embedded in paraffin. All brain tissue were sectioned into 4-μm-thick sagittal cross-sections of PFC and ACC, which is located 0.4 mm lateral to the midline, as described above. The sections were deparaffinized in D-limonene and 100% ethanol (3 times for 10 min) before rehydration in graded ethanol , followed by MilliQ-HFor quantification of immunofluorescence in the ischemic region, which is within 300 μm beneath the infarcted area, as indicated by NeuN staining, five microscopic images per rat were randomly captured at 40× objective using the microscope, according to previous reports . In the ® . The results are expressed as mean ± standard deviation (SD). Student’s t-tests were used for comparison between the two groups. A two-way repeated measures analysis of variance (ANOVA) was used to perform multiple group comparisons, followed by Tukey’s test for individual comparisons. The results were considered statistically significant at P < 0.05.All statistical analyses were performed using the Statistical Package of Social Sciences 22 software3. No evidence of infarction was found in any of the sham rats.As shown in The body and bladder weights are shown in In the PT group, mature neurons were lost in the frontal lobe of the cerebral cortex and the ACC from Days 1 to 28, whereas they were present in the sham group and preoperatively in the PT group . ImmunofThe representative cystometric curves of each group are shown in In this study, we used a photothrombotic stroke method to induce focal infarcts in PFC and ACC, and we observed decreased ICIs on Days 1 and 7, according to the cystometric analysis. Furthermore, all rats survived after the operation. To the best of our knowledge, this is the first study in which photothrombosis has been applied to manipulate urinary function.The method of inducing a stroke in the cerebral cortex using photochemical reactions was developed by C. Watson in 1985 . PhotothThere was no difference in bladder weight or bladder to body weight ratio between the sham and PT groups, which is the same result as obtained in the MCAO model . FurtherOnce necrotic, mature neurons do not recover, but the urinary status of the rats changed after stroke in this study. It is possible that the surrounding tissue near the necrotic lesion, which is called the penumbra, compensated for the loss function. Microglia, the immune cells resident in the brain, are the first to respond to ischemic neurons and promote their survival . FollowiAlthough this study has a limitation in that it is not directly comparable to the MCAO model, to the best of our knowledge, this is the first study to evaluate changes in the urinary status of rats after photothrombosis-induced cerebral infarction. Future studies should include more detailed investigations of changes in bladder tissue and local factors. We believe that this novel animal model, which reflects changes in voiding function as with clinical symptoms, may significantly contribute to development of new treatments for lower urinary tract dysfunction.Our photothrombosis model was characterized by its simplicity, ability to create localized infarcts, nonlethal nature, and high reproducibility. Similar to the clinical course of a patient after stroke, this model observed changes in micturition status. In other words, this model would be suitable as a new model of urinary frequency caused by cerebral infarction.S1 FigA: Identification of the bregma, the craniometric point at the junction of the sagittal and coronal sutures at the top of the cranium, marked with a black dot indicated by a yellow arrow. B: A fiber optic cable delivering a light source was fixed 1 mm anterior to the bregma on the midline under anesthesia inhalation. After Rose Bengal was injected, the skull was illuminated.(TIF)Click here for additional data file.S2 Fig2-mm slices of all brains in all sham and photothrombosis rats in the pilot study.(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.


Despite the extensive utilization of polysaccharide hydrogels in regenerative medicine, current fabrication methods fail to produce mechanically stable scaffolds using only hydrogels. The recently developed hybrid extrusion-based bioprinting process promises to resolve these current issues by facilitating the simultaneous printing of stiff thermoplastic polymers and softer hydrogels at different temperatures. Using layer-by-layer deposition, mechanically advantageous scaffolds can be produced by integrating the softer hydrogel matrix into a stiffer synthetic framework. This work demonstrates the fabrication of hybrid hydrogel-thermoplastic polymer scaffolds with tunable structural and chemical properties for applications in tissue engineering and regenerative medicine. Through an alternating deposition of polycaprolactone and alginate/carboxymethylcellulose gel strands, scaffolds with the desired architecture , and rheological characteristics  were prepared. The hybrid fabrication approach allows the fine-tuning of wettability (approx. 50–75°), swelling (approx. 0–20× increased mass), degradability (approx. 2–30+ days), and mechanical strength (approx. 0.2–11 MPa) in the range between pure hydrogels and pure thermoplastic polymers, while providing a gradient of surface properties and good biocompatibility. The controlled degradability and permeability of the hydrogel component may also enable controlled drug delivery. Our work shows that the novel hybrid hydrogel-thermoplastic scaffolds with adjustable characteristics have immense potential for tissue engineering and can serve as templates for developing novel wound dressings. Dressings that accelerate wound healing should be biocompatible and antiseptic and they should retain moisture and enable oxygen and nutrient diffusion. They should also mimic the biochemical, mechanical, and topographical properties of the native extracellular matrix (ECM) , which s®) [Hydrogels are promising candidates for this purpose, as they (mostly) fulfill all the aforementioned requirements ,8 by res®) . Like AL®) .By combining both polymers in different ratios, augmented characteristics can be obtained in one hybrid hydrogel formulation. The addition of ALG to CMC enhances the gelation mechanism, leading to marginally improved mechanical properties, which help to develop clinically relevant scaffolds . The polDespite the extensive utilization of polysaccharide hydrogels in tissue engineering and regenerative medicine, most fabrication methods have failed to produce stable 3D constructs and scaffolds using exclusively hydrogels , which aAmong many additive manufacturing techniques, extrusion-based 3D bioprinting is especially advantageous for fabricating tissue-regenerating scaffolds using a broad range of biocompatible materials . This teWe hypothesized that by using a hybrid 3D extrusion bioprinting process, we could fabricate scaffolds with tunable mechanical and physicochemical properties in terms of porosity, stiffness, swelling, and degradation. Such scaffolds would be suitable for advanced tissue engineering applications and the design of novel wound dressings.2) for scaffold production were all purchased from Sigma-Aldrich, Germany. All materials and chemicals were used as purchased without any further modification. For the preparation of all solutions and experiments, ultra-pure water (18.2 MΩ·cm at 25 °C) was used, prepared using an ELGA Purelab water purification system .The sodium salt of alginic acid , carboxymethyl cellulose , polycaprolactone , and other chemicals . As an alternative ‘ink’ (P ink), pure PCL was used, which was melted at 80 °C, followed by fused deposition 3D printing at the mentioned temperature. PCL-based scaffolds solidified again at room temperature, retaining their printer form without further post-processing.To be suitable for further processing and analysis, the deposited material had to hold its own weight and allow the preparation of structures with 10 or more layers that retain their shape  throughout the fabrication process. The basic ‘ink’ formulation to fulfill the mentioned requirements was a hydrogel of 5 wt.% ALG, 5 wt.% CMC, further stabilized by adding 3 g of 5 wt.% CaCl2 for 15 min at room temperature), P scaffolds (printed using pure PCL), and ACCP (hybrid) scaffolds. ACCP scaffolds were produced with the simultaneous use of two extruders by depositing alternating layers of ACC (using one extruder) and PCL (using the second extruder). The finished scaffolds were cross-linked with 5 wt.% CaCl2. Structure design and slicing were performed using Autodesk Fusion 360  and Slic3r v1.2.9 software, with a rectilinear infill pattern. Cuboid and cylindrical scaffolds were prepared, along with additional geometries to comply with tensile testing (prepared according to ISO 527-2:2012 type 1BB). All scaffolds were prepared using the BioScaffolder 3.1  bioprinter and conical, blunt-end extrusion nozzles  with an inner diameter of 0.25 mm. All manufactured scaffolds retained their structural stability and preserved shape fidelity after deposition of 50 subsequent layers, as well as prolonged periods in cell culture conditions (soaked in Advanced Dulbecco’s Modified Eagle’s Medium (ADMEM) at 37 °C and 5 wt.% CO2).Four types of scaffolds were prepared for further testing and analysis: AC scaffolds, AC scaffolds with additional crosslinking  using magnifications of 2×, 4×, and 10× in bright field (BF) mode, respectively. To evaluate the printed filament thickness and the scaffold macropores’ sizes, freely available NIH image analysis software ImageJ 1.52 g   was used−1. All measurements were performed at room temperature. For all samples, images of 10 × 10, 5 × 5, and 1 × 1 μm2 were recorded with a resolution of 1024 × 1024 pixels [q) and arithmetical mean height of the surface (Sa) according to ISO 25178.The topographical and surface roughness analysis of all optimized formulations was performed with atomic force microscopy (AFM) in tapping mode . Samples for AFM measurements were prepared by pressing the formulations between two silicon wafers, which were then removed, and the samples were appropriately post-processed. The images were scanned using silicon cantilevers  with a resonance frequency of 210–490 kHz and a force constant of 12–110 Nm4 pixels . Pico ImThe surface morphology and porosity of all final 3D printed scaffolds were analyzed by scanning electron microscopy (SEM) . Before The prepared 3D printing inks’ wettability was determined using an OCA15+ goniometer system  with the sessile drop method . Flat sa0). The dry scaffolds were immersed in phosphate-buffered saline (PBS) (pH 7.4) at 37 °C. At predetermined time intervals, the sample was removed from the PBS, wiped dry with filter paper to remove excess liquid from the surface, and weighed. The swelling ratio at time t was calculated using Equation (1):0 and Wt are the weights of the initial dry scaffolds and swollen scaffolds at fixed time intervals t, respectively. All measurements were executed in triplicate, and the final result is reported and plotted as the average with the corresponding standard error.The swelling kinetics of the scaffolds was investigated by the gravimetric method . Firstly3) were dried for 3 days at room temperature and then weighed (W0). Subsequently, dry scaffolds were placed in plastic flasks with 10 mL PBS (pH 7.4) at 37 °C and, at predetermined time intervals, the specimens were removed from PBS, washed with deionized water three times, and dried again. Separate samples were prepared for measurements at each degradation time point, so that sample handling was minimized. The weight remaining of the scaffolds was calculated according to Equation (2):0 is the initial weight of the dry scaffold and Wt is the weight of the scaffold at fixed time intervals. Again, all experimental data were obtained from triplicate samples, and the result is reported and plotted as the average with the standard error.Before the start of the in vitro degradation test, according to , freshlyThe 3D printed scaffolds’ mechanical properties were defined using quasi-static uniaxial compression and tensile tests, using a Tinius Olsen H10KT compression/tension testing machine  . Test spv/v% Foetal Bovine Serum , and incubated for 24 h at 37 °C in an atmosphere containing 5 wt.% CO2. The HaCaT and skin fibroblast cells  were seeded into a 96-well microtiter plate with a final volume of 100 μL of ADMEM medium supplemented with 5 v/v% FBS. All cells were exposed to the same volume (100 μL) of the as-prepared respective sample solutions, and their dilutions of 1:2, 1:4, 1:8, and 1:16, followed by incubation for 24 h at 37 °C in an atmosphere containing 5 wt.% CO2. All experiments were performed in four parallels. Spectrophotometrical detection of the color change was measured using the Varioskan multiplate reader  [All cell tests were conducted using human skin-derived cells; namely, commercially available skin fibroblasts  and an aneuploidy immortalized keratinocyte cell line from adult human skin (HaCaT). The latter were kindly provided by Prof. Dr. Elsa Fabbretti . The influence of different 3D printed scaffolds on cell viability was evaluated via the reduction reaction of the tetrazolium salt MTT -2,5-diphenyltetrazolium bromide), purchased from Sigma Aldrich, Germany. This is a widely accepted and reliable method to examine cell viability through cellular metabolic activity . The MTTMA, USA) . Final rp-values < 0.05 were considered statistically significant. Statistical analysis was performed using SPPS Statistics 27 .All numerical values are reported as mean ± standard deviation (SD). The Shapiro–Wilk test confirmed the normal distribution of the experimental data. Levene’s test was used to assess the equality of variances. As all data sets were well-modeled by a normal distribution and homoscedastic, one-way analysis of variance (ANOVA), followed by the Bonferroni post-hoc test, was carried out accordingly. Obtained Reproducible 3D printing of biocompatible and structurally stable hydrogels still represents a challenge . Improvi2 was used as a chemical crosslinking agent. Its addition further improved the formulation’s viscoelastic properties. The printing of this formulation yielded the AC scaffolds. Other divalent cations  or solutions containing calcium  can be used for ionic crosslinking. However, CaCl2 yields the fastest gelation rates and displays the highest crosslinking efficiency [2+ might be useful in all stages of the wound healing process. When released into the wound, Ca2+ ions may improve some cellular aspects of wound healing  and aid the clotting mechanism during the first stage of the wound healing process [2 was only added into the formulation and was not used for post-processing), the AC scaffolds failed to maintain their structural integrity in the cell culture medium at 37 °C. In comparison, the completely crosslinked ACC scaffolds  proved stable throughout the incubation cycle (72 h), as did the P and ACCP scaffolds.Reproducible 3D printing of scaffolds requires homogeneous coextrusion of both the hydrogel and the synthetic component, without crossover or retraction movements, which could interrupt the flow. The macropore size (distance between filaments) was precisely modeled in the g-code by considering the extruded filament’s outer diameter and the distance between the gridlines. Both the scaffold computer-aided design (CAD) model and the set macropore size (0.7 × 0.7 mm) were identical across all scaffolds , regardless of the composition. Because of the extensive utilization of polysaccharide polymers as the main components in various formulations for wound treatment, the ALG and CMC polymer mixture was chosen as the hydrogel base material. Comparative studies showed that CMC possesses the ability to absorb harmful bacteria and, additionally, ALG likewise exhibits antimicrobial activity, although to a lesser extent than CMC . Generalficiency . Aside fGeometrical characterization was performed by means of visual assessment of the fabricated structures’ geometry using an optical microscope . The printed layers of all scaffolds  were evaluated regarding macropore size  and filament thickness, and the construct’s overall macroporosity was calculated. The obtained images of the printed scaffolds’ meshes are presented in A pore is defined as a void space within a scaffold. In contrast, porosity, a morphological property independent of the material, can be considered a collection of pores and represents the percentage of empty space within a solid. The pore size and degree of porosity are also crucial in facilitating cell migration, proliferation, and vascularization. Macropores (pore size > 50 µm) facilitate nutrient distribution, enhanced oxygen diffusion, and improved catabolite removal . Due to Since the geometrical parameters were constant across all scaffolds in this work, the influence of different base formulations and the significance of crosslinking on the meshes of the resulting printed scaffolds was undoubtedly evident. By comparing the obtained images  and measOn the contrary, the hydrogel formulation’s low viscosity limits the fabrication of stable constructs, as is evident from the poorly defined filaments and pores of the AC scaffolds. Expectedly, both AC and ACC scaffolds displayed a significant increase in filament thickness, a decrease in macropore size, and a more significant variation in pore size and shape compared to P scaffolds. Among all results, the AC scaffolds displayed the thickest filaments, smallest macropores, and consequently the lowest macroporosity (1.53%). In comparison to AC scaffolds, their ionically crosslinked counterparts displayed a significant decrease in filament thickness and an increase in pore size. Furthermore, this also resulted in a nearly 10-fold increase in macroporosity (9.73%) and a more uniform pore size and shape. We showed that the inclusion of this simple post-processing step significantly improves the hydrogel scaffold’s mechanical characteristics and preserves its 3D-printed architecture.Considering the “softer” nature of hydrogels and the synthetic PCL’s favorable mechanical attributes, intermediate morphological features were expected when combining all the polymers into one scaffold. The hybrid ACCP lattice indeed exhibited intermediate macropore size and filament thickness. It is important to note that the pore length and width of hybrid scaffolds differed significantly, resulting in a seemingly more variable filament thickness. This is a consequence of significantly thicker hydrogel filaments (in agreement with AC and ACC scaffolds) and substantially narrower PCL filaments (similar to P scaffolds) being combined in one construct. Despite the low viscosity of the used AC hydrogel, the alternating AC-P lattice structure remained stable. More importantly, the hydrogel filaments did not fuse and no macropore closing was observed, arguably due to the mechanical support provided by the PCL filaments. By serving as a framework, PCL helps retain the scaffold’s printed geometry and results in the higher macroporosity of the hybrid construct (15.72%) compared to the hydrogel scaffolds. ACCP scaffolds also enable ionic crosslinking of the hydrogel constituent through post-processing, which has no noticeable effects on the synthetic component. Modulating the ratio of the ACCP hybrid scaffolds’ components presents the opportunity to tailor the geometrical properties of scaffolds, which influence their mechanical behavior. This is examined below. The highly microporous hybrid 3D construct is more beneficial for supporting the healing process through all stages than the “pure” hydrogel structures .To maintain the scaffolds’ long-term structural integrity, some crucial parameters of the hybrid biofabrication process have been established. When dispensing the thermoplastic PCL and a hydrogel mixture , all materials’ rheological properties should be considered. According to the literature , the filThe material’s surface characteristics  and morphology are important factors affecting cell adhesion. Through the physicochemical interaction between the cells and the substrate, cytoskeletal re-arrangement in cells is prompted, influencing migration, proliferation, and differentiation . Howeverq and Sa for respective scaffolds at 10 μm, 5 μm, and 1 μm scales are presented in The surface characteristics, morphology, and roughness of all formulations and the influence of ionic crosslinking on hydrogels and different material combinations (hydrogel + PCL) on the mentioned features were evaluated by performing AFM measurements directly on the formulations. The surface morphology and corresponding surface roughness parameters S2 deposits on the scaffolds’ surface. During the drying procedure, CaCl2 left over on the surface forms crystals, which can be clearly observed on the AFM images. The second factor, contributing to an overall increase in scaffold roughness, is the effect of crosslinking on the hydrogel formulation’s molecular nature. As shown in the results of optical (bright-field and fluorescence) microscopy, scaffold filaments of the post-processed ACC scaffolds were significantly finer than the untreated AC filaments, suggesting a denser polymer network in ACC scaffolds, which was also confirmed by SEM (see below). Apart from a rougher topography, the AFM images of ACCP scaffolds also show a less homogenous surface compared to the P scaffolds. Especially at the smallest size scale (1 μm), ACCP scaffolds exhibited surface characteristics that could be best characterized as an intermediate between the apparent homogeneous surface of P scaffolds and the particle-like attributes of the AC scaffold surface. This suggests that we can tailor the scaffolds’ roughness by adjusting the hydrogel composition and through post-processing within a hybrid scaffold.For AC, P, and ACCP samples, easily observable surface features were visible regardless of the image size. Based on the AFM micrographs and the calculated roughness parameters, it was evident (and expected) that the PCL-only scaffolds (P) exhibited the overall flattest and smoothest surface morphology. On the other hand, the AC formulation building blocks (ALG/CMC) were larger. Thus, it was anticipated that hydrogel scaffolds would display significantly rougher topography than P scaffolds . This caAFM’s main limitation is that it only provides topological information on a relatively small part of the scaffold’s surface (even if the test is repeated at multiple discrete locations on the sample). Therefore, SEM was used as a complementary technique to analyze the scaffold’s surface morphology and observe the influence of different material combinations and ionic crosslinking on scaffolds’ micro-geometry. By cutting the lyophilized scaffolds longitudinally, the scaffolds’ internal microporosity was also assessed. 2 crystals are visible on the scaffolds’ surface. Combined with AFM results, this suggests that only a thin layer of CaCl2 is uniformly distributed on the scaffolds’ surface. By examining the cross-sectional images, the scaffolds’ interior microporosity was assessed. Sufficient scaffold microporosity is considered beneficial. However, high microporosity can negatively influence the scaffold’s structural and degradation properties [The results obtained from SEM imaging were consistent with observations made using optical microscopy in terms of pore size and filament thickness. As the printing parameters (except for the printing temperature of PCL) and pore size were set as a constant for the fabrication of all scaffolds, it was evident that the scaffolds’ micro-geometrical characteristics and surface morphologies primarily depended on the material selection. For AC and ACC scaffolds, which differed only in the post-processing regime, significant size differences in pores and filaments were noticeable. Soon after 3D printing, AC scaffolds lose their 3D printed architecture, do not retain shape fidelity, and the scaffold’s filaments are prone to fusing, causing macropores to lose their set size and shape. On the other hand, crosslinked AC (ACC) scaffolds are superior in retaining their 3D structure and predefined macropore size and geometry and display significantly thinner filaments that do not fuse. In the case of ACC scaffolds, it is also important to point out that no larger CaCloperties . The SEMIn contrast, P scaffolds display virtually no inner microporosity. Smaller indentations on the PCL scaffold’s surface and in the filament’s core  are probably a consequence of trapped air bubbles, which tend to form during 3D printing of PCL at higher temperatures. P scaffolds display a more uniform filament thickness, pore size, and pore geometry than hydrogel scaffolds. Even in the cross-sectional micrographs, the filaments’ shape and inner structure are uniform. The main advantage of P scaffolds is that they maintain their shape fidelity and 3D printed geometry after the fabrication procedure. This makes PCL a great framework for supporting a softer “ECM-mimicking” hydrogel component. Indeed, that is what we observed in the SEM images of hybrid ACCP scaffolds. Mechanically advantageous PCL supports hydrogel filaments and prevents filament collapse and fusion. Therefore, hybrid scaffolds can retain their 3D printed architecture . The construct’s overall microporosity can be improved by introducing a microporous hydrogel component into the impermeable PCL.2O), which is correlated with the material’s hydrophilicity. Generally, CA(H2O) values are inversely proportional to water retention. However, it is important to note that the results obtained on a two-dimensional (2D) surface cannot be fully extrapolated to a 3D construct straightforwardly. The scaffold’s 3D structure on all size scales is also a key parameter in regulating its overall hydrophilicity, especially in synthetic polymers and fibrous materials [2O) values for all our samples are shown in Wound dressings are generally evaluated in terms of their water uptake capacity, whereas the sample’s hydrophilicity presents the main measured characteristic. A common method for assessing the latter is measuring the water contact angle CA = 13.7 ± 1.6° for ACC and CA(H2O) = 50.0 ± 3.8° for AC. The obtained values can most likely be ascribed to differences in surface morphology and increased surface roughness compared to synthetic polymers, and the high content of –OH and –COOH groups present in both polymers (ALG and CMC). These increase AC and ACC scaffolds’ surface affinity to water and contribute to hydrogel’s hydrophilic properties. According to the literature, pure ALG’s hydrophilicity depends on the polymer form and can vary from 54.0° to 90.0° [2O) = 50.0 ± 3.8° for the AC scaffold is expected and consistent with the literature.As polysaccharide-based hydrogels generally show increased hydrophilicity compared to synthetic materials , it was expected that both hydrogel-only formulations (AC and ACC) would exhibit the most hydrophilic properties among all formulations. Indeed, the values obtained for these two formulations were CA observed in ACC scaffolds, as well as the presence of CaCl2 on their surface. If we take the AFM surface roughness parameters into account, the most likely explanation for this increased hydrophilicity is an overall increase in surface roughness. A nearly 10-fold increase in surface roughness parameters was observed by crosslinking the scaffolds, which indicates a rougher surface compared to AC scaffolds. It is well established that specific surface morphology  significantly impacts a surface’s wettability [2O) is likely to reflect the rougher and less homogenous surface of ACC scaffolds. This result is of great importance because it shows that we were able to easily modulate the hydrogel’s hydrophobic/hydrophilic properties by changing the post-processing regime or the crosslinking agent.The results show that the post-processing regime and the choice of crosslinking agent affect the hydrogel formulation’s hydrophilic nature. By additionally crosslinking the AC scaffolds with Catability . Therefo2O) = 75.9 ± 0.9°), which is known for its hydrophobic nature, the highest contact angle among all tested materials was also anticipated. The ALG/CMC hydrogel addition into the PCL matrix reduced the CA(H2O), pointing to an incremental increase in the hybrid formulation’s hydrophilicity. The mixture of all three base materials (ALG/CMC/PCL) displays an intermediate CA(H2O) = 66.9 ± 2.2°, which lies between the AC and P scaffolds. This value drop demonstrates that incorporating polymers with a hydrophilic character  into the synthetic PCL framework presents a practical strategy for tailoring hydrophilic/hydrophobic properties of 3D printed constructs. Most of them, especially modern wound dressings, are hydrophobic, which, coupled with their exudate absorption, is of great importance in the elimination of passive bacteria [For pure PCL ; however, they differed in the post-processing step, in which the ACC scaffolds were also fully crosslinked. Additional application of Ca2+ ions forms extra ionic bonds with two carboxyl groups simultaneously, forming connections between polymer chains. This increases the polymer network’s rigidity and density and decreases the hydrogel’s microporosity and consequently hinders water diffusion into the construct [2+ with other, monovalent cations (mostly Na+) is expected. This effect disrupts the connections between polymer chains and reduces the overall crosslinking density over time. Therefore, it is safe to hypothesize that the greater the starting overall crosslinking density, the lower the scaffolds’ microporosity and, thus, the slower the hydrogel scaffolds’ weight loss. This is also supported by the degradation kinetics of AC and ACC scaffolds alongside the SEM results. For ACC scaffolds, after 30 days, presumably almost all crosslinking connections were broken, which leads to immediate degradation of the scaffold, similar to the behavior observed in the AC scaffolds between 24 and 48 h.onstruct . The ovePoor solubility in water and a relatively high melting point are some of the main factors that slow PCL’s degradation rate. Soaking PCL-only scaffolds (P scaffolds) in PBS for up to 32 days had negligible effects on their degradation as they lost only 8% of their initial dry weight. The degradation of PCL, which is a semi-crystalline polyester, involves two stages. Firstly, water diffuses into the amorphous regions, causing a random hydrolytic scission of the ester groups, resulting in additional crystallization and an overall increase in crystallinity. After the amorphous region’s degradation has started, the “hydrolytic attack” shifts towards the crystalline domain center. A molecular weight reduction occurs during water uptake, although it is not accompanied by loss of mass . As therIn contrast, the hybrid ACCP scaffolds lost approximately 20% of their starting weight in the first 48 h, which decreased slightly over the next 29 days. The degradation of the hydrogel component likely contributed the main part of the degradation in the hybrid scaffolds. However, the observed weight loss over time was significantly lower compared to pure hydrogel scaffolds, which can be attributed to two main factors. First, the hybrid scaffolds contain a lower amount of hydrogel. Thus, less material can be degraded. Secondly, the PCL component mechanically stabilizes the scaffold and reduces solvent access to the hydrogel component at the hydrogel–PCL interfaces . DespiteIn addition to exhibiting appropriate wettability and water uptake capacity, wound dressings must possess suitable mechanical properties. Therefore, the 3D-printed scaffolds were characterized based on their compression and tensile behavior to assess the differences in mechanical properties between base scaffolds. The results of compression and tensile tests have been plotted on stress-strain graphs, which are presented in Both hydrogel-only scaffolds (AC and ACC) showed similar mechanical behavior in the compression test. The stress-strain curves transitioned at approximately 180 kPa, where the curve flattened slightly . Structural irregularities can thereby cause oscillations within the otherwise smooth curve. Afterward, the curve showed exponential behavior, which is commonly observed in polymers . The traTensile tests proved extremely difficult to perform due to the “high hyper-elasticity” of the materials and the low elastic modulus of the materials, since clamping jaws needed to be appropriately adjusted to prevent material slippage in the jaws and avert specimen damage. Nevertheless, after several initial tests, three quality measurements were obtained for each material except AC. The results were again plotted in stress-strain graphs. The tensile behavior of ACC, P, and ACCP scaffolds is presented in The results for ACC showed slight local stochastic behavior in the tension region. This behavior can most likely be explained by structural irregularities due to the 3D fabrication process. ACC specimens showed almost linear behavior with low stresses for the given strains. The plastification zone is narrow, which indicates that the material deformed elastically and then broke suddenly, with little plastification. On the contrary, PCL was much stiffer compared to ACC and showed a higher elastic modulus. The yield point is around 11 MPa, and afterward a broad plastification zone begins. The plastification occurs at almost the same stress, which indicates that the material was stiffening due to the mechanisms occurring during plastic deformation. The strain at the material’s failure point is above 3, which is five times higher than that of ACC. ACCP again showed a combination of both the AC and PCL component. The curve’s initial slope, indicating the PCL component’s elastic modulus, is similar to the PCL-only scaffold. The lower yield strength of the ACCP material at ~0.7 MPa can be attributed to the fact that the specimen was manufactured as a combination of PCL and ACC. Since the test specimen’s size was the same as that of the specimen of homogeneous PCL, the hybrid ACCP showsed a smaller cross-sectional area of PCL than that of solid PCL. Therefore, the PCL’s true stress was much higher, probably comparable to the PCL specimen test. The yield point was clearly visible with the visible strengthening of the specimen. After critical stress was reached, a filament strand inside the specimen broke. The specimen settled at a lower stress value, where the strengthening of the specimen with increasing strain began again. This effect can be attributed to the structure of the PCL strands, which were not interconnected. The behavior was repeated until all filaments of the scaffold broke. The strain value at which the specimen failed was much higher than that observed in ACC and was similar to that of the PCL specimen. Generally, PCL was the stiffest material, with a broad plastification zone, and ACCP was effectively softer for the given area and showed a characteristic plastification zone. ACC showed the lowest elastic modulus, with the lowest yield point and sudden breakage.Based on the compression and tensile experiments, it is evident that ionic crosslinking influences the mechanical behavior of scaffolds . More importantly, scaffolds’ stiffness and durability to mechanical forces are significantly improved when the soft hydrogel is incorporated into a solid PCL framework. This suggests that mechanical characteristics of wound dressings can be tailored merely by adjusting the mass ratios of the hydrogel and synthetic components in the scaffolds. Our results indicate that hybrid 3D printing may present a great approach for researchers to regulate and fine-tune the scaffold’s mechanical characteristics to mimic native tissues’ characteristics or develop more durable wound dressings.The skin is a continuously self-renewing organ that dynamically copes with the human body’s exterior–interior interactions and actively participates in the host defenses . Ideally2+. Similarly, extracts from AC scaffolds showed the largest impact on SF viability, reducing metabolic activity with increasing concentration. However, the results after 24 and 48 h remained comparable.As the MTT assay reflects the total metabolic activity of cells in a sample, variances in the initial cell concentrations per sample are to be expected, resulting in fluctuations between samples. Extrapolating from the obtained data thus requires very careful consideration. Due to their poor solubility, extracts from P scaffolds showed little effect on cell viability. However, despite showcasing great biocompatibility, PCL’s hydrophobic nature impeded cell attachment. Although providing a stable environment for cell growth, a pure PCL scaffold is therefore not the best material for fabricating wound dressings . All oth+ from growth media for Ca2+ from the scaffold). The outcome of this cation exchange is a complete dissolution of ALG and CMC, which leads to extensive material transformation and eventually to its degradation [6H7O6Na; Mw: 198.0 g/mol), in comparison with a single CMC monomer unit . CMC has 0.9 carboxyl groups per one anhydro-glucose unit. Additionally, a single CMC polymer chain’s molecular weight is approximately nine times larger (700 kDa vs. 80 kDa) than a single ALG polymer chain, which probably also influences its dissolution [Degradation is most likely the consequence of cation exchange in growth media  of ACC extracts instead of lower pH values due to scaffold degradation. Previous studies showed that cultured HaCaT cells can switch between differentiated and basal states upon alterations in the Ca2+ concentration in media. The addition of Ca2+ triggers HaCaT hyperproliferation. When exposed to extracellular Ca2+, HaCaTs most likely undergo a spontaneous transformation and become relatively insensitive to Ca2+, which normally triggers reduced proliferation through the differentiation step [2+ environment, which induces the proliferation of HaCaT cells, which, after the formation of a cell monolayer, is decreased in favor of rapid differentiation. Once their differentiation is initiated, HaCaTs begin to slowly undergo apoptosis [2+ concentration, as their viability did not improve with increasing extract dilutions, regardless of the scaffold’s composition.Therefore, it was also anticipated (and confirmed by MTT results) that the additionally crosslinked ACC scaffolds, which degrade significantly slower than their non-post-processed counterparts (AC scaffolds), influence cell viability significantly less. The ACC scaffolds lost approximately 40% of their weight in the first 48 h. Thus, it can be hypothesized that much lower amounts of ALG and CMC are released into the growth medium. Nevertheless, because the ACC scaffolds’ degradation is not as extensive and the medium’s pH values are not lowered as considerably, the significantly lower viability of HaCaTs when exposed to ACC scaffolds can be attributed to another key factor. Namely, lower viability can be partially related to the excess Caion step . Degradapoptosis , which i2+ concentration. This resulted in significantly higher HaCaT viability after 48 h, relative to ACC scaffolds. Cell viability further increased with larger extract dilutions and even surpasses the control cells. The hybrid ACCP results again support the statement that SF cells, on the other hand, seem to be much more sensitive to alterations in pH values or Ca2+ concentrations, as no increasing trend was observed.If we exclude the P scaffolds, the most biocompatible scaffold seems to be the hybrid ACCP scaffold. In the first 24 h of incubation, no significant differences for either cell line between the ACC and the ACCP scaffolds were observed; however, after 48 h, significantly higher cell viabilities  were noticed for ACCP scaffolds. This result is also in agreement with the degradation results. For hybrid ACCP scaffolds, the viability was not decreased during the first 24 h, as only a small amount of the additionally crosslinked ALG-CMC component degraded. The total amount of the degraded ALG and CMC was too small to change the medium’s pH to the degree that would influence SF and HaCaT cells’ viability. The slower degradation process of the crosslinked hydrogel component, and lower ALG-CMC content per scaffold than hydrogel-only scaffolds, means that the pH of the growth media was maintained closer to physiological values. This also dictates a lower Ca2+) into the growth medium. As seen in the results from different cell types, the crosslinking solution should be tailored according to the materials used and fine-tuned based on the cell types that are in contact with the scaffolds. Finally, it is important to stress that all the prepared formulations displayed no visible cytotoxic effects on cells. Considering all results, the hybrid ACCP formulation demonstrated desirable properties and appropriate in vitro biocompatibility, making it the best candidate for wound healing applications.All things considered, it appears that the scaffold’s composition and degradation properties, combined with the cell type’s susceptibility to environmental changes , greatly affect cell viability outcomes. Therefore, when designing potential scaffolds for wound dressings or skin substitutes, especially in the design of infrequently changed dressings, one must first consider the cell type whose growth the scaffold is supposed to promote. The scaffold’s formulation and fabrication should be fine-tuned accordingly. In the case of skin-derived cells, even though the P scaffold’s biocompatibility is the highest, due to PCL’s inherently unfavorable properties, the best scaffold choice for supporting their growth is the hybrid ACCP scaffold. This form of scaffold combines the highly biocompatible PCL, which is crucial in long-term cell culturing (as it retains its integrity), with hydrogels’ desirable physicochemical properties. AC scaffolds show that small concentrations of hydrogel base components (ALG and CMC) improve long-term cell viability. As evident from our evaluation of AC and ACC scaffolds, additional ionic crosslinking of the hydrogel component improves hydrogels’ stability in the culture medium. ACC and ACCP scaffolds serve as an indication that appropriate post-processing of scaffolds delays hydrogel dissolution and mitigates pH changes. However, when ionically crosslinking hydrogels, we must also consider the subsequent release of ions  was prepared and optimized for 3D printing. The optimized hydrogel served as a “wound dressing template”, which was integrated into the synthetic polycaprolactone framework.By optimizing the 3D printing procedure and crosslinking, hybrid scaffolds with improved mechanical and physicochemical properties were fabricated. The hydrogel formulation was simultaneously printed with polycaprolactone in an alternating sequence. Systematic material and scaffold characterization tests were conducted to examine the influence of ionic-crosslinking and integration of the hydrogel component in the stiff synthetic framework. The methods used included evaluating the resulting material’s structural properties and the scaffold’s stability, durability, and mechanical behavior. Using a state-of-the-art printing strategy and employing a multi-head deposition system, we proved that combining soft hydrogels with stiff polymers into a single construct presents a straightforward approach to producing macroporous, highly organized, mechanically superior hybrid scaffolds. We further demonstrated that fabrication by sequential dispensing of the thermoplastic PCL and the ALG/CMC hydrogel fulfills both biomaterials’ thermal requirements and facilitates integrating polymers with different requirements into a single construct. Compared to hydrogel-only scaffolds, the hybrid constructs displayed significantly improved mechanical properties (improved from 0.2 MPa to 11 MPa), degradation kinetics (improved from approx. 2 days to 30+ days), appropriate water uptake capacity, wettability, and good surface morphology characteristics. Additionally, by carefully optimizing and adjusting the printing parameters, the post-processing regime, and ratios of both the hydrogel and synthetic components in the constructs, the hybrid approach enables rather simple control over the scaffolds’ characteristics. The mentioned parameters can be adjusted to fine-tune them for specific applications. The prepared 3D printed wound dressings were proven to be safe for use by measuring skin-derived cells’ viability when exposed to scaffolds with the MTT assay. All scaffolds were proven to be biocompatible and exerted no toxic effects on skin-derived cells. Based on the results, this study lays the groundwork for developing biocompatible hybrid scaffolds that can serve as advanced wound dressings with improved properties. Moreover, the hybrid printing approach used in this study opens up the possibility of additionally tuning the properties of scaffolds, so that they can more closely mimic the cells’ native microenvironment, thus guiding the cell behavior to promote the wound-healing process.


As a major epigenetic modification, DNA methylation plays an important role in coordinating plant responses to environmental changes. Methylation-sensitive amplified polymorphism (MSAP) technology was used in this study to investigate the epigenetic diversity of fifty japonica rice samples from five regions in Heilongjiang Province, China. In addition, the phenotypic indicators of japonica rice samples and the environmental conditions of the sampling sites were investigated and analysed. Based on the MSAP analysis technique, using eight pairs of selective primers, we identified a total of 551 amplified loci, of which 267 (48.5%) were classified as methylation loci. The methylation status and levels of the japonica rice genome in different regions differed significantly (p < 0.05). The results of the analysis of molecular variance (AMOVA) revealed that most of the molecular variation (91%) came from within the groups (regions) and was caused by individual variation within the region. Furthermore, the results of principal coordinates analysis (PCoA), cluster analysis, and population structure analysis indicated that there was no obvious correlation between the epigenetic differences and geographical locations, which may have been due to the limited range of sampling sites. When environmental factors, phenotypic indicators, and epigenetic data analysis are combined, it is easy to conclude that japonica rice grown in the same latitudinal region has increased epigenetic and phenotypic similarities due to similar climatic conditions and production practices. As the world's leading rice producing region, Asia is home to many of the world's top rice exporting countries2. As one of the main production areas of high-quality japonica rice in China, Heilongjiang Province is the coldest rice cultivation area in the world3. The rice planting area here accounts for approximately 12.8% of the total rice planting area in China4, the commodity volume of rice accounts for 34.2% of that in the country, and japonica rice accounts for 68.6% of production in the country. Heilongjiang Province has a cold environment with long sunshine hours, a large day-night temperature difference, and fertile soils, with high organic matter contents5. These unique geographical advantages ensure the unique high quality of Heilongjiang japonica rice. Rice is rich in protein, fat, starch and other nutrients, and its quality is affected by many factors, such as variety, genetics, geographical environment, growth conditions and processing.Approximately half of the world's population eats rice as a staple food6. Many previous studies have focused on the environmental heterogeneity and wide distribution of varieties, exploring the impact of different geographical distances and environmental factors on genetic diversity9. Few studies, however, attempted to explain the influence of environmental and geographic factors on phenotypic polymorphism by epigenetic factors. Epigenetics is a branch of genetics that investigates heritable changes in gene expression do not arise from the nucleotide sequences of the genes. DNA methylation, genomic imprinting, maternal effects, gene silencing, nucleolar dominance, dormant transposons activation, RNA editing, and other epigenetic phenomena exist11. DNA methylation, unlike DNA mutations, is a key epigenetic modification that influences gene regulation in response to environmental signals and also has evolutionary importance for adaption to various environments across extended evolutionary time periods12. DNA methylation variation in rice can be caused by diseases13, droughts15, anaerobic conditions16, radiation17, high salinity18, heavy metals19, nutritional deficiency20 and other biotic and abiotic environmental stresses. According to growing evidence, epigenetic mechanisms play a vital role in coordinating the heritable and reversible changes in plant gene expression.The biological environment and geographical differences can affect genetic diversity. To adapt to different environmental and developmental conditions, environmental factors and developmental signals will induce certain adjustments in genes, leading to changes in the final phenotype21, has been widely employed to analyse genomic DNA methylation levels and patterns in many species due to its dependability, high sensitivity, and convenience. It is possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome without requiring a reference genome22. MSAP is dependent on two different DNA methylation sensitive restriction isoschizomers, Hpa II and Msp I, which recognize the same tetranucleotide restriction site (5’-CCGG-3’), but Hpa II is sensitive to full methylation and cleaves the hemimethylated external cytosine, while Msp I is sensitive to only external cytosine methylation of the restriction site23. Previous studies25have proven that the MSAP technique is efficient for verifying the relationship between environmental factors and epigenetic diversity. Therefore, with the MASP molecular marker, this study aims to systematically assess the epigenetic diversity within and between Japonica rice from the five main production regions of Heilongjiang Province; the diversity is represented by genome-wide DNA methylation levels, patterns, and population structures. Exploring the phenotypic characteristics of landraces in different regions as well as elucidating the epigenetic diversity of rice in various growth environments and geographical locations, is important for preserving locally adapted varieties, determining basic agronomic characteristics of landraces, and improving rice quality and yield.The methylation-sensitive amplified polymorphism (MSAP) technique, as a molecular technology that is a modification of amplified fragment length polymorphism (AFLP) techniqueshttp://www.cma.gov.cn). Monthly (line chart) and annual (column chart) average data for precipitation, relative humidity, sunshine hours, temperature, atmospheric pressure, and wind speed from five different rice production regions are presented in Figure The environmental data for each of the five regions of Heilongjiang Province were collated from China’s daily surface climate dataset of the China Meteorological Administration  to 84 (E83/M42), with amplicon lengths ranging from 37 to 495 bp  was used to calculate the percentage of polymorphic loci (PLP%), the number of effective alleles (Ne), Shannon's information index (I), and Nei's gene diversity (h). The percentages of polymorphic loci (PLP%) for the five groups ranged from 49.09% (CHY) to 52.63% (FZ), with an average of more than 50%, indicating that the five populations were highly polymorphic. Compared with other groups, all indices  related to polymorphism were highest in XS , and lowest in CHY . Using the final epigenetic data matrix obtained by transforming the four recognized methylation types of each multistate epilocus into independent binary subepiloci, AMOVA was performed to quantify epigenetic variation among five regions. The majority of epigenetic variance (91%) occurred within the groups, whereas the differentiation index (Φst) of 8.5% indicated moderate epigenetic differentiation between them  based on genetic distance among the 50 sampled individuals Fig. . AlthougThe NJ phylogenetic tree based on the genetic distance of four types of subepiloci binary matrix data showed that the 50 individuals from five different regions were divided into two clades Fig. . PopulatK criterion reached its nodal value when K = 2 , indicating that there were two distinct subpopulations at the uppermost level of the epigenetic structure. The results for K = 2 and K = 5 were presented to illustrate the formation of populations  was primarily present in the CHY, FZ, and JSJ populations, whereas most individuals in population WC were assigned to subpopulation II (green color), except for the population XS, which was assigned into both subpopulation I (0.56) and subpopulation II (0.43). When K = 5, subpopulation II was further subdivided into three clusters , while light green and yellow clusters were found in some individuals from population FZ.The analysis of epigenetic population structure using subepiloci binary matrix data transformed from the MSAP dataset of 50 samples revealed that the ∆27. Apart from genetic mechanisms, increasing evidence has shown that epigenetic processes have important evolutionary significance in plant adaptation to stressful environments28. Among these epigenetic mechanisms, cytosine DNA methylation has received the most attention and has provided the most examples of transgenerational stability29. For example, mangrove plants from different nearby habitats with similar genetic profiles have divergent epigenetic profiles, indicating that cytosine methylation variation can be more closely correlated with environmental variation than genetic variation30. The results of global DNA methylation patterns of grapevines from different regions revealed that epigenetic profiles differed significantly despite the low genetic differentiation between vineyards31. In recent years, MSAP, as a modified AFLP technique, has been widely used to detect the genomic methylation status and extent of diverse species32.Previous research on rice has generally studied the genetic diversity of varieties and accessions using molecular marker techniques such as SSR (simple sequence repeat) and SNPs (single nucleotide polymorphism), and the impact of geographical environment and varieties on population structure has been analysed and clarified33. Meteorological conditions such as temperature, precipitation, and sunshine undoubtedly cause epigenetic effects, which then cause phenotypic variation34, and will eventually affect the yield and quality of rice35. In this study, six environmental factors that significantly affect the growth and development of rice and five phenotypic indicators that reflect rice quality were selected to discuss the effect of environmental conditions on phenotypic and epigenetic diversity. The results of the environmental condition analysis of five geographically isolated regions showed that most environmental data had no significant difference, which may have been due to the selected regions being all located in Heilongjiang Province, so the climate conditions were relatively similar. Even so, similar environmental conditions still generate significant phenotypic diversities  but did vary across regions. The polymorphism index of rice in the Chahayang and Wuchang regions was relatively low, and that in the Fangzheng and Xiangshui regions was relatively high. Understanding the causes and consequences of natural epigenetic variation is difficult because it can be influenced by a variety of factors39. The difference in epigenetic diversity among different regions might be related to the niche variation hypothesis40; that is, if the habitat range of the population was wider and more diverse, there would be more variation than if the population had a narrow habitat range.Based on the results of AMOVA, it was observed that a wide range (91%) of within group(region) differences were caused by individual variation within regions. Such high levels of molecular differences between individuals in the same region could be explained by the random accumulation of variation with age, which can be genetic or epigenetic in character. The apparent genetic variation of a single individual is a prerequisite for microevolution of plantsAlthough some individuals were clustered in other regions, the majority of individuals from each region were clustered in the same clade, possibly indicating that individuals within the same region have epigenetic similarity due to a relatively consistent geographical environment, climatic conditions, soil composition, production practices, and other factors Fig. . The PCoInterestingly, when we combined the analysis results of environmental conditions, phenotypic diversity and epigenetic diversity, we found that the phenotypic diversity results were closely related to the epigenetic diversity results. Compared with the Jiansanjiang region, rice in the Wuchang and Xiangshui regions had lower methylation levels, closer genetic distances, similar phenotypic data, and similar latitudes, altitudes and climatic conditions. Therefore, under long-term natural environmental selection and directional selection, to adapt to different environmental and developmental conditions, environmental factors and developmental signals will induce certain adjustments in plant genes, resulting in changes in the final phenotype.In this research, the epigenetic diversity of fifty rice samples from five different regions of Heilongjiang Province was investigated using eight pairs of MSAP primers. The differences in global genome methylation patterns and levels in rice of different regions were explored. AMOVA revealed that most of the genetic variation came from within populations. At the same time, the phenotypic analysis results of rice in different regions were consistent with the epigenetic analysis results. Due to the limited geographical scope of sampling, most environmental conditions in different regions did not differ significantly, and no obvious correlation between the epigenetic differences and geographic location was found, but it is not hard to speculate that they may have higher epigenetic and phenotypic similarity at the same latitude.The purpose of sampling was to obtain representative samples to evaluate the composition or characteristics of the sample system. By investigating the geography, ecology, environment, accumulated temperature, climate, variety, production mode, and degree of mechanization in various regions of Heilongjiang Province, in this study, a total of 50 japonica rice varieties from five main farming regions in Heilongjiang Province were selected to investigate epigenetic diversity, including Fangzheng (FZ) and Wuchang (WC) in the central region, Chahayang (CHY) in the western region, Jiansanjiang (JSJ) in the eastern region, and Xiangshui (XS) in the southern region Fig. . All japhttp://www.cma.gov.cn). The differences between groups were analysed using one-way ANOVA with IBM SPSS software, and p < 0.05 indicated a significant difference.The site-specific environmental data were collected from publicly accessible websites based on the latitude and longitude coordinates of the sampling sites. The data was collated from China’s daily surface climate data of the China Meteorological Administration . The five evaluation standard indicators at mature stages were selected to analyse the phenotypic diversity of rice in different regions, since the samples were all from mature rice landraces. The phenotypes were collected from ten plants or seeds from each sample. For the 1000-grain weight, grains harvested from ten plants were mixed, 100 randomly selected grains were weighed in grams with three replicates, and the average was multiplied by ten. To assess the phenotypic diversity of rice in the five regions, researchers utilized ANOVA and Duncan's multiple comparison.The phenotypic data were analysed using the Standard Evaluation System for Rice (SES)42 The sample was evenly mixed with 500 µL of CTAB buffer solution and 20 µL of proteinase K (20 mg/mL) evenly. After incubation at 65 °C for 60 min, 20 µL RNase A was added before centrifugation (1600 g for 10 min). With an equal volume of phenol and chloroform (1:1), the supernatant was extracted twice. The upper phase was combined with two-thirds volume of isopropanol. The mixture was centrifuged for 10 min. The pellet was washed with ethanol  twice and centrifuged at room temperature for 10 min. The supernatant was discarded carefully, the washing was repeated once, and the pellet was dried. All genomic DNA samples were stored at − 20 °C for later use after being dissolved in sterile deionized water.Ten healthy japonica seeds of each sample were randomly selected and cultivated at a room temperature of 25°. The fresh young leaves of the 3-leaf stage seedlings were mixed together, and the genomic DNA was extracted from 100 mg of each sample by the improved CTAB method43.The concentration and purity of the extracted DNA were detected by a UV–VIS spectrophotometer  and 1.0% agarose gel electrophoresis. The OD 260/280 ratio was between 1.762 and 1.806, and the concentration was approximately 1000 ng/µL, indicating that the extracted DNA met the quality requirements for subsequent experimentsHpa II and Msp I) to cytosine methylation21. In brief, DNA was double digested by EcoR I plus Hpa II and EcoR I plus Msp I  LTD) in digestion reaction buffer. Digestion and ligation were performed simultaneously in 20 μL system which contained DNA (400 ng), T4 DNA ligase (5 units), EcoR I (5 units), Hpa II or Msp I (5 units), EcoR I adaptor  analysis is an improvement on the typical amplified fragment length polymorphism (AFLP) method, which is based on the differential sensitivity of a pair of isoschizomers  consisted of 10 mmol dNTPs (0.4 μL), 10 × buffer (2 μL), 5unit Taq polymerase (0.2 μL), and 10 μmol 44.A total of 5 samples were randomly selected from each region for the optimal primer selection test. The primers with a clear spectrum, strong signal and high polymorphism from the twenty different primer combinations  at a volume ratio of 100:1, the PCR products were diluted tenfold (1 µL) and mixed with the above mixture (9 µL) in each well of a 96-well sample plate. The PCR products were analysed on an ABI 3730XL auto DNA sequencer . The selection of MSAP fragments was restricted to allele sizes ranging from 75 to 500 bphttps://softgenetics.com/GeneMarker.php) by detecting signals of varied intensities and locations46. The binary matrix comprising information on epilocus presence (1) or absence (0) was generated by side-by-side comparisons of the banding patterns of each EcoR I/Hpa II and EcoR I/Msp I enzyme combination. Four DNA methylation types were artificially categorized as follows: type I (1/1), type II (0/1), type III (1/0) and type IV (0/0). The modified approach of "mixed-scoring" was used to study the epigenetic diversity of rice in different regions to test the specific influence of different methylation types. The final epigenetic data matrix was generated by transforming each multistate epilocus and four recognized methylation types into binary subepiloci48 to analyse significant differences in methylation levels in different regions of rice. The epigenetic data matrix was analysed with GENALEX software (Version 6.5)49 to calculate diversity parameters, including the percentage of polymorphic loci (PLP%), number of effective alleles (Ne), Shannon’s information index (I) and Nei’s gene diversity (h). The analysis of molecular variance (AMOVA) method was applied to estimate population epigenetic differentiation within and between the five regional populations. Next, we used principal coordinate analysis (PCoA) to estimate epigenetic structure based on the genetic distance matrix of the total subepiloci matrix.The original binary matrix data was used to estimate the level of DNA methylation. One-way analysis of variance (ANOVA) and Duncan's multiple comparison were employed in IBM SPSS statistics software 50 based on four types of subepiloci binary matrix data . By clustering individual genotypes into a certain number of populations (K) and reducing divergence from Hardy–Weinberg equilibrium, this model infers population structure. We varied K value from 1 to 13, assumed 100,000 initial interactions (burn-in period) and 1,000,000 Monte Carlo Markov chain (MCMC) interactions, and conducted 10 independent iterations for each K. K was calculated using the method based on the likelihood's second-order rate of change (DK)51. Structure Harvester 52 was used to evaluate the results and determine the most likely value of K. Repeated sampling analysis was conducted on the results of structure analysis by CLUMPP software 53, and the Q-matrix results for the best K value were obtained. A structure graph was drawn by DISTRUCT software (http://rosenberglab.bioinformatics.med.umich.edu/distruct.html) according to the results of repeated sampling analysis. The neighbor-joining cluster analysis method based on Nei's genetic distance data was performed using the NTSYS-pc software .We assessed the population structure using the Bayesian clustering model implemented in STRUCTURE software (version 2.3.4, All the rice materials were obtained in the local fields of each region with the help of the local Bureau of Agriculture and Rural Affairs. We have been granted permission to conduct these fields and materials, and we have followed national and local regulations. We ensured the collection of rice experimental materials has no negative consequences for the local environment.Supplementary Information.


Hepatitis E is an enteric and zoonotic disease caused by hepatitis E virus (HEV) that is mainly transmitted via the faecal-oral route through contaminated food or the environment. The virus is an emerging infectious agent causing acute human infection worldwide. A high seroprevalence of the disease was reported in pregnant women in Addis Ababa, Ethiopia, raising significant public health concern. The presence of HEV specific antibodies were also reported in dromedary camels in the country; however, the infectious virus and/or the viral genome have not been demonstrated to date in animal samples.Camelus dromedarius, 45 samples) in west Hararghe were screened for the presence of HEV genome using universal primers in a fully nested reverse transcription polymerase chain reaction (nRT-PCR). The protocol is capable of detecting HEV in faecal samples from both pigs and camels.To address this gap, a total of 95 faecal samples collected from both apparently healthy pigs of uncharacterised types (50 samples) in Burayu and Addis Ababa areas and camels (The nRT-PCR detected HEV genes in six (12%) pig faecal samples and one camel sample (2.2%). Therefore, the results indicate that HEV is circulating in both pigs and camels in Ethiopia and these animals and their products could serve as a potential source of infection for humans.The detection of HEV in both animals could raise another concern regarding its public health importance as both animals’ meat and camel milk are consumed in the country. Further studies to determine the prevalence and distribution of the virus in different animals and their products, water bodies, food chain, and vegetables are warranted, along with viral gene sequencing for detailed genetic characterisation of the isolates circulating in the country. This information is critically important to design and institute appropriate control and/or preventive measures. Orthohepevirus in the Hepeviridae family that has four species, Orthohepevirus A with eight genotypes in mammals, the Orthohepevirus B circulating in chicken, the Orthohepevirus C circulating in rats and ferrets, and the Orthohepevirus D circulating in bats [Hepeviridae family namely genotypes 1–4 and 7 are known to infect humans [Hepatitis E is a zoonotic, emerging enteric infectious disease caused by Hepatitis E virus (HEV). The disease is a major cause of water-borne hepatitis epidemic in tropical and subtropical countries with poor sanitary conditions including southeast and central Asia, the Middle East, and Africa . The HEV in bats . The gen in bats . Two maj in bats . In cont in bats –6. The z in bats . Genotyp in bats . In genet humans , 10.Hepatitis E in humans is characterized by large scale water-borne epidemics of jaundice in regions of the world with contaminated water supplies and low sanitary conditions. According to World Health Organisation (WHO) report , there aIn African countries, a number of HEV outbreaks were reported including in Ethiopia, Somalia, Uganda, Democratic Republic of Congo, Sudan and South Sudan in different periods , 17. The2. Burayu town is bounded by Finfinnee (Addis Ababa) city in the East, Walmera district in West, Sululta district in North and Sebata Hawas district in South [The study was conducted from November to April in 2020 in Burayu (Oromia) and in Kolfe Keraniyo, Addis Ababa. Burayu town is located in Oromiya National Regional State and in the western direction of Addis Ababa at a distance of 15 km from the capital city. The town is a highland area located at an altitude of 2580 m above sea level with an area of 66.5 kmin South . In addiin South , 23.Apparently healthy pigs and camels of all age groups and both sexes kept under farmers management conditions were used for the study. The pigs were found in Addis Ababa, Ashewa Meda, and Burayu towns. The exact number of pig farms and pig population in the Burayu district was not known as there is no record of pig farms and population kept by the office of livestock and fisheries of Burayu district. However, in Burayu district and in Kolfe Keraniyo (a sub city in Addis Ababa), there were three and one extensive pig farms, respectively, with herd sizes of 50–100.The dromedary camels were found in western Hararghe zone of Oromia regional state, Ethiopia that has approximately 91,948 camel populations . Based oA cross sectional study design was conducted to select the pigs and camels for faecal sample collection from their respective study areas that is in and around Addis Ababa and western Hararghe, respectively. Four pig farms were included for sample collection. A total of 50 pig faecal samples were collected, of which 41 were directly collected from the pig rectum while 9 samples were collected as freshly voided faeces from the barn. The rectal faeces were collected by inserting two fingers into the rectum when the pigs were in recumbent position and placed in sterile universal tubes. These samples were labeled and transported at 4 °C to the National Animal Health Disease Investigation Centre (NAHDIC) virology laboratory, Sebeta. They were stored at − 80 °C until molecular detection was conducted. A total of 45 camel faecal samples (26 samples were collected from Chiro town and 19 from Mieso town) were collected. In addition, the pig watering area and grazing areas were visited to assess the potential for disseminating the disease to other animals and humans in the area. The camel faecal samples which had been collected in 2019 from western Hararghe were already stored at − 80 °C until the molecular detection commenced. A nearby watering point or river was visited to evaluate the area for contamination with swine faeces. In pigs all sample were collected from females while in camels both female and male were sampled.Total RNA was extracted from the faecal samples using the QIAamp viral RNA extraction mini kit . Briefly, 10% suspension of faecal samples were first prepared by suspending it in 5 ml of phosphate-buffered saline (PBS) and then centrifuged at 8000 revolution per minute (rpm) for 5 min. The supernatant obtained was collected and used for extraction of viral RNA according to the QIAamp® Viral RNA Mini kit 2018 instructions.The extracted RNA was amplified with fully nested conventional reverse transcriptase polymerase chain reaction (nRT-PCR) in two consecutive amplification rounds . The RT-2 thermal cycler was used for genome amplification. The amplification product was separated by gel electrophoresis on a 2% agarose gel for 40 min at 100 voltages. SYBR Safe Dye  staining was used to reveal the amplification products documented in a gel documentation apparatus. The bands of positive results were aligned at 325 base pair (bp) with 100 bp PCR marker (ladder)  and the amplification was gel documented.The second amplification was performed with internal primers: (HEV-CSN) 5’ TGT TGC CCT GTT TGG CCC CTG GTT TAG 3’ and (HEV-CASN) 5’ CCA GGC TCA CCR GAR TGY TTC TTG CA 3’ in a 50µL reaction mixture volume as described by López-Santaella et al. . A FlexCAge, sex, and location of both pigs and dromedary camels were recorded. Laboratory results was recorded in Microsoft Excel spread sheet for data analysis using proportion method. The RT-PCR results where documented in gel electrophoresis gel documenting apparatus.The age distribution of studied pigs range from 3 months to 4 years. Of the 50 pigs sampled, 54% (27/50) faecal samples were collected from females while 28% (14/50) were collected from males. The remaining 18% (9/50) samples were collected from freshly voided faeces from the barns. Pigs feeding in dirty environment around river in Burayu area were found and such feeding habit could further contaminate the river.Of the total 50 pig samples detected by fully nested RT-PCR, 12% of the collected faecal samples (6/50) gave positive result for HEV viral RNA genome amplification by giving expected band size of 325 bp as visualized on agarose gel electrophoresis (Fig. In this study, the 12% molecular detection of HEV genome in faecal samples of domestic pigs and the 2.2% results, in dromedary camels’ (Fig. Previous reports indicated that HEV viruses in pigs are zoonotic and belong to genotypes 3–6 , 33, 34.The role of pigs as potential HEV reservoirs is well documented in many parts of the world. For example, the incidence of pig HEV was reported in United Kingdom, China, Japan, and Canada , 38, 39.The 32% serological evidence of HEV infection in pregnant women in a hospital in Addis Ababa  suggestsIn dromedary camels, the detection of HEV genome strengthens previous serological report of anti-HEV IgG antibody in more than 20% of camels found in Afar regional state, Ethiopia  which isUsually, large numbers of camels and other domestic animals from many different herds/flocks congregate at watering and fresh grazing sites that create a perfect condition for disease transmission and spread among susceptible animals. During milking, washing of hands, milking vessels, the udder and teats are not washed properly by many milkers’ prior to milking the camels. Besides, the milking area is generally full of dust and dung and without shade. These conditions not only affect the quality and safety of the produced milk , 44 but HEV RNA has been detected in diverse food products ranging from meat and seafood, to fruits and vegetables . AccordiIn this study, molecular detection of the HEV from faecal samples of pigs and from a camel sample using a nested RT-PCR is reported for the first time in Ethiopia. The observation of pigs grazing in the field and around watering points, suggest possible environmental contamination of the virus that could lead to infection of susceptible animal species including humans. The HEV virus is becoming the leading cause of enterically transmitted viral hepatitis in humans . These v