Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

STUDY QUESTION: Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? SUMMARY ANSWER: The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. WHAT IS KNOWN ALREADY: To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. STUDY DESIGN, SIZE, DURATION: The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. MAIN RESULTS AND THE ROLE OF CHANCE: In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). LIMITATION, REASONS FOR CAUTION: The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. STUDY FUNDING/COMPETING INTEREST(S): The study is funded by the Iran University of Medical Sciences and no competing interest exists. TRIAL REGISTRATION NUMBER: N/A.

Simple Method for On-Demand Droplet Trapping in a Microfluidic Device Based on the Concept of Hydrodynamic Resistance.

We demonstrate an innovative method to catch the desired droplets from a train of droplets and immobilize them in traps located in an integrated microfluidic device. To this end, water-in-oil droplets are generated in a flow-focusing junction and then guided to a channel connected to chambers designated for on-demand droplet trapping. Each chamber is connected to a side channel through a batch of microposts. The side channels are also connected to the flexible poly(vinyl chloride) tubes, which can be closed by attaching binder clips. The hydrodynamic resistance of the routes in the device can be changed by opening and closing the binder clips. In this way, droplets are easily guided into individual traps based on the user's demand. A set of numerical simulations was also conducted to investigate the authenticity of the employed idea and to find the optimal geometry for implementing our strategy. This simple method can be easily employed for on-demand droplet trapping without using on-chip valves or complex off-chip actuators proposed in previous studies.

Investigating the correlation between the protein adhesion simulation and the biocompatibility of polymeric substrate for skin-tissue-engineering applications.

Investigating the protein adhesion properties of polymeric scaffolds through computational simulations can predict the biocompatibility of scaffolds before an experimental assay is carried out. This prediction can be highly beneficial since it can cut costs and the time it takes for experimental assays. The current study aims to test the hypothesis that there is a correlation between the biocompatibility of a composite scaffold and the molecular dynamics simulations of protein adhesion. To this end, chitosan and gelatin were selected for fabricating a composite skin-tissue wound scaffold with five different polymer ratios. This polymeric blend has not been simulated for protein adhesion. The cell proliferation and viability of the samples were quantified via MTT assay using fibroblast cells. Then a series of molecular dynamics simulations were performed to measure the adhesion energy of two prominent extracellular matrix proteins - fibronectin, and collagen type I. Besides, a higher gelatin percentage in the scaffold leads to a decrease in the porosity. The results demonstrated a strong correlation between the experimental data and molecular dynamics simulations. The sample with equal amounts of chitosan and gelatin had the highest cell viability and the strongest adhesion energy, of 239 kcal mol-1 for collagen type I, and 149 kcal mol-1 for fibronectin. This correlation was also evident in other samples: samples with gelatin-to-chitosan ratios of 3 : 1 and 1 : 3 had the lowest cell viability and the weakest adhesion energy, respectively.

Label-free electrochemical microfluidic biosensors: futuristic point-of-care analytical devices for monitoring diseases.

The integration of microfluidics with electrochemical analysis has resulted in the development of single miniaturized detection systems, which allows the precise control of sample volume with multianalyte detection capability in a cost- and time-effective manner. Microfluidic electrochemical sensing devices (MESDs) can potentially serve as precise sensing and monitoring systems for the detection of molecular markers in various detrimental diseases. MESDs offer several advantages, including (i) automated sample preparation and detection, (ii) low sample and reagent requirement, (iii) detection of multianalyte in a single run, (iv) multiplex analysis in a single integrated device, and (v) portability with simplicity in application and disposability. Label-free MESDs can serve an affordable real-time detection with a simple analysis in a short processing time, providing point-of-care diagnosis/detection possibilities in precision medicine, and environmental analysis. In the current review, we elaborate on label-free microfluidic biosensors, provide comprehensive insights into electrochemical detection techniques, and discuss the principles of label-free microfluidic-based sensing approaches.

Effect of Material and Population on the Delivery of Nanoparticles to an Atherosclerotic Plaque: A Patient-specific In Silico Study.

Coronary artery disease (CAD) is the prevalent reason of mortality all around the world. Targeting CAD, specifically atherosclerosis, with controlled delivery of micro and nanoparticles, as drug carriers, is a very proficient approach. In this work, a patient-specific and realistic model of an atherosclerotic plaque in the left anterior descending (LAD) artery was created by image-processing of CT-scan images and implementing a finite-element mesh. Next, a fluid-solid interaction simulation considering the physiological boundary conditions was conducted. By considering the simulated force fields and particle-particle interactions, the correlation between injected particles at each cardiac cycle and the surface density of adhered particles over the atherosclerotic plaque (SDP) were examined. For large particles (800 and 1000 nm) the amount of SDP on the plaque increased significantly when the number of the injected particles became higher. However, by increasing the number of the injected particles, for the larger particles (800 and 1000 nm) the increase in SDP was about 50% greater than that of the smaller ones (400 and 600 nm). Furthermore, for constant number of particles, depending on their size, different trends in SDP were observed. Subsequently, the distribution and adhesion of metal-based nanoparticles including SiO2, Fe3O4, NiO2, silver and gold with different properties were simulated. The injection of metal particles with medium density among the considered particles resulted in the highest SDP. Remarkably, the affinity, the geometrical features, and the biophysical factors involved in the adhesion outweighed the effect of difference in the density of particles on the SDP. Finally, the consideration of the lift force in the simulations significantly reduced the SDP and consistently decreased the particle residence time in the studied domain.

Secondary Flows, Mixing, and Chemical Reaction Analysis of Droplet-Based Flow inside Serpentine Microchannels with Different Cross Sections.

Chemical bioreactions are an important aspect of many recent microfluidic devices, and their applications in biomedical science have been growing worldwide. Droplet-based microreactors are among the attractive types of unit operations, which utilize droplets for enhancement in both mixing and chemical reactions. In the present study, a finite-volume-method (FVM) numerical investigation is conducted based on the volume-of-fluid (VOF) applying for the droplet-based flows. This multiphase computational modeling is used for the study of the chemical reaction and mixing phenomenon inside a serpentine microchannel and explores the effects of the aspect ratio (i.e., AR = height/width) of rectangular cross-sectional geometries as well as three other cross-sectional geometries including trapezoidal, triangular, and circular, on consumption and production rates of chemical species. It is found that in these droplet bioreactors, the reaction begins from the forward section of the droplet. We investigate the secondary flows and chemical reactions inside the droplets in a serpentine microchannel with different cross-sectional geometries. Different transient Dean vortices and secondary flows in the presence and absence of the droplets are studied and explained based on the position of the droplets. It is found that as the droplets pass through the microchannel turns, the patterns and magnitude of the secondary flows change, depending on the cross-sectional geometry. Eventually, the results demonstrate that the AR = 2 rectangular cross-section is the most helpful geometry, whereas the trapezoidal cross-section takes into account the least efficient one between all geometries.

Evaluating the Multifunctionality of a New Modulator of Zinc-Induced Aß Aggregation Using a Novel Computational Approach.

The high concentration of zinc metal ions in Aß aggregations is one of the most cited hallmarks of Alzheimer's disease (AD), and several substantial pieces of evidence emphasize the key role of zinc metal ions in the pathogenesis of AD. In this study, while designing a multifunctional peptide for simultaneous targeting Aß aggregation and chelating the zinc metal ion, a novel and comprehensive approach is introduced for evaluating the multifunctionality of a multifunctional drugs based on computational methods. The multifunctional peptide consists of inhibitor and chelator domains, which are included in the C-terminal hydrophobic region of Aß, and the first four amino acids of human albumin. The ability of the multifunctional peptide in zinc ion chelation has been investigated using molecular dynamics (MD) simulations of the peptide-zinc interaction for 300 ns, and Bennett's acceptance ratio (BAR) method has been used to accurately calculate the chelation free energy. Data analysis demonstrates that the peptide chelating domain can be stably linked to the zinc ion. Besides, the introduced method used for evaluating chelation and calculating the free energy of peptide binding to zinc ions was successfully validated by comparison with previous experimental and theoretical published data. The results indicate that the multifunctional peptide, coordinating with the zinc metal ion, can be effective in Aß inhibition by preserving the native helical structure of the Aß42 monomer as well as disrupting the ß-sheet structure of Aß42 aggregates. Detailed assessments of the Aß42-peptide interactions elucidate that the inhibition of Aß is achieved by considerable hydrophobic interactions and hydrogen bonding between the multifunctional peptide and the hydrophobic Aß regions, along with interfering in stable bridges formed inside the Aß aggregate.

Microfluidic-Based Approaches in Targeted Cell/Particle Separation Based on Physical Properties: Fundamentals and Applications.

Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high-efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label-free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor-intensive steps of labeling molecular signatures of cells. In general, microfluidic-based cell sorting approaches can separate cells using "intrinsic" (e.g., fluid dynamic forces) versus "extrinsic" external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label-free microfluidic-based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic-based cell separation methods are discussed.

Design of peptide-based inhibitor agent against amyloid-ß aggregation: Molecular docking, synthesis and in vitro evaluation.

Formation of the amyloid beta (Aß) peptide aggregations represents an indispensable role in appearing and progression of Alzheimer disease. ß-sheet breaker peptides can be designed and modified with different amino acids in order to improve biological properties and binding affinity to the amyloid beta peptide. In the present study, three peptide sequences were designed based on the hopeful results of LIAIMA peptide and molecular docking studies were carried out onto the monomer and fibril structure of amyloid beta peptide using AutoDock Vina software. According to the obtained interactions and binding energy from docking, the best-designed peptide (d-GABA-FPLIAIMA) was chosen and synthesized in great yield (%96) via the Fmoc solid-phase peptide synthesis. The synthesis and purity of the resulting peptide were estimated and evaluated by Mass spectroscopy and Reversed-phase high-performance liquid chromatography (RP-HPLC) methods, respectively. Stability studies in plasma and Thioflavin T (ThT) assay were performed in order to measure the binding affinity and in vitro aggregation inhibition of Aß peptide. The d-GABA-FPLIAIMA peptide showed good binding energy and affinity to Aß fibrils, high stability (more than 90%) in human serum, and a reduction of 20% in inhibition of the Aß aggregation growth. Finally, the favorable characteristics of our newly designed peptide make it a promising candidate ß-sheet breaker agent for further in vivo studies.

Fluid-Structure Interaction Simulation of Blood Flow and Cerebral Aneurysm: Effect of Partly Blocked Vessel.

In this study, using fluid-structure interaction (FSI), 3-dimensional blood flow in an aneurysm in the circle of Willis - which is located in the middle cerebral artery (MCA) - has been simulated. The purpose of this study is to evaluate the effect of a partly blocked vessel on an aneurysm. To achieve this purpose, two cases have been investigated using the FSI method: in the first case, an ideal geometry of aneurysm in the MCA has been simulated; in the second case, modeling is performed for an ideal geometry of the aneurysm in the MCA with a partly blocked vessel. All boundary conditions, properties and modeling methods were considered the same for both cases. The only difference between the two cases was that part of the MCA parent artery was blocked in the second case. In order to consider the hyperelastic property of the wall and the non-Newtonian properties of the blood, the Mooney-Rivlin model and the Carreau model have been used, respectively. In the second case, the Von Mises stress in the peak systole is 26% higher than in the first case. With regard to the high amount of Von Mises stress, the risk of rupture of the aneurysm is higher in this case. In the second case, the maximum wall shear stress (WSS) is 12% higher than in the first case. And maximum displacement in the second case is also higher than in the first. So, the risk of growth of the aneurysm is higher in cases with a partly blocked vessel.

Designing a new multifunctional peptide for metal chelation and Aß inhibition.

According to the Amyloid hypothesis, as the foremost scientific explanation for Alzheimer Disease (AD), the neuropathology of AD is related to toxic fragments of amyloid beta (Aß) protein. Based on this hypothesis, an attractive therapeutic approach was demonstrated to identify multifunctional peptides able to modulate Aß pathologies as the source of AD. On this premise, a bifunctional polypeptide based on the iAß5p lead compound, was designed to inhibit Aß aggregation and free metal ions. Herein, the efficacy of this novel drug in Zn2+ and Cd2+ ion chelation was examined through an integrated technique comprising combined Docking, QM, and MD simulations. MD relaxation of a set of probable binding modes that were predicted by Molecular Docking, revealed six druggable hosts having considerable affinities. Further, free energy analysis indicated that the formation of the revealed polypeptide-ion complexes is more spontaneous than the presented Aß-ion+ ones. These findings certified the new ability of the modified lead compound to hamper Aß pathologies and provide helpful information in atomic details for further preclinical studies against AD.

Mechanical differences between ATP and ADP actin states: A molecular dynamics study.

This paper aims to give a comprehensive atomistic modeling of the nanomechanical behavior of actin monomer. Actin is a ubiquitous and essential component of cytoskeleton which forms many different cellular structures. Despite for several years great effort has been devoted to the investigation of mechanical properties of the actin filament, studies on the nanomechanical behavior of actin monomer are still lacking. These scales are, however, important for a complete understanding of the role of actin as an important component in the cytoskeleton structure. Based on the accuracy of atomistic modeling methods such as molecular dynamics simulations, steered molecular dynamics method is performed to assess tension of monomeric G-actin molecule under different types of mechanical loading including axial and lateral. As a result, stress-strain curves are obtained in aqueous solution, with either ATP or ADP bound in the nucleotide binding pocket. The obtained results yield evaluation of the tensile stiffness of a single actin monomer in lateral and normal direction. In order to compare the behavior of ATP and ADP G-actins, the number of hydrogen bonds and nonbonded interactions between the nucleotide and the protein are analyzed. Moreover, The effect of virtual spring of steered molecular dynamics on the mechanical behavior of actin monomer is investigated. The results reveal increasing the virtual spring constant leads to convergence of the stiffness. Moreover, in this paper, a generalized model is proposed to extend the obtained results for the monomeric G-actin scale to the actin filament. Our modeling estimated a persistence length of actin filament 15.41 µm, close to experimental measurements. Moreover, In this paper, the breaking force actin-actin bond is evaluated using steered molecular dynamics simulation. By applying a tensile force, actin-actin bond ruptured at 4197.5 pN.

Numerical analysis of a dielectrophoresis field-flow fractionation device for the separation of multiple cell types.

In this study, a dielectrophoresis field-flow fractionation device was analyzed using a numerical simulation method and the behaviors of a set of different cells were investigated. By reducing the alternating current frequency of the electrodes from the value used in the original setup configuration and increasing the number of exit channels, total discrimination in cell trajectories and subsequent separation of four cell types were achieved. Cells were differentiated based on their size and dielectric response that are represented in their real part of Clausius-Mossotti factor at different frequencies. A number of novel designs were also proposed based on the original setup configuration. It was seen that by reducing the length of the main channel and the number of electrodes at low frequencies and not changing the inlet flow velocities, cell separation was still achieved successfully, although with a slightly larger electrode voltage. The shorter main channel decreased the residence time for the cells on the chip and also reduced the overall size of the device-these were improvements over the original design. The obtained results can be used to analyze other cell types by knowing their size and dielectric properties to design geometries that can ensure separation.

Antimicrobial Wound Dressing Containing Silver Sulfadiazine With High Biocompatibility: In Vitro Study.

Many patients all over the world suffer from acute wounds caused by traumas or burns. In most crucial cases, skin regeneration cannot be promoted spontaneously, and skin grafts are applied as the main treatment. However, this therapy has some drawbacks which motivate researchers to develop wound dressings. In this study, electrospun mats consisting of polycaprolactone (PCL) and polyvinyl alcohol (PVA) incorporated with silver sulfadiazine (SSD) are proposed to be used as antimicrobial wound dressings with the capability of cell seeding. Various amounts of SSD were loaded into PVA nanofibers, and the effects of SSD particles on the morphological characteristics of nanofibers, mechanical behaviors, and physical properties of the mats were studied for the first time. The cellular viability, antimicrobial properties of the scaffolds, and release behavior of silver were also examined. Finally, the best concentration of SSD was determined based on the quality of nanofibers, antibacterial features, and the ability of cellular attachment and proliferation. Fibronectin was also coated to enhance the biocompatibility of the selective scaffold. It was shown that the mats have appropriate mechanical properties with good handling ability in wet environment and also have a hydrophilic surface to adhere to the wound bed. Results indicate that SSD particles increase the fiber diameter and hydrophilic properties, while they weaken the mechanical characteristics of the mats. Furthermore, 5 wt% SSD/PVA was determined as the best concentration of SSD as it results in a desirable fiber quality for the mats with enough antimicrobial properties and acceptable cell proliferation on the surface. Coating fibronectin was also introduced as an effective method to increase the biocompatibility of the scaffolds incorporated with SSD particles.

A model for cell density effect on stress fiber alignment and collective directional migration.

In this study, numerical simulation of collective cell migration is presented in order to mimic the group migration of endothelial cells subjected to the concentration gradients of a biochemical factor. The developed 2D model incorporates basic elements of the cell, including both the cell membrane and the cell cytoskeleton, based on a viscoelastic cell mechanic model. Various cell processes--including cell random walk, cell-cell interactions, cell chemotaxis, and cellular cytoskeleton rearrangements--are considered and analyzed in our developed model. After validating the model by using available experimental data, the model is used to investigate various important parameters during collective cell chemotaxis, such as cell density, cytoskeleton organization, stress fiber reorientations, and intracellular forces. The results suggest that increasing the cell density causes the cell-cell interactions to affect the orientation of stress fibers throughout the cytoskeleton and makes the stress fibers more aligned in the direction of the imposed concentration gradient. This improved alignment of the stress fibers correlates with the intensification of the intracellular forces transferred in the gradient direction; this improves the cell group migration. Comparison of the obtained results with available experimental observations of collective chemotaxis of endothelial cells shows an interesting agreement.

Toward Epileptic Brain Region Detection Based on Magnetic Nanoparticle Patterning.

Resection of the epilepsy foci is the best treatment for more than 15% of epileptic patients or 50% of patients who are refractory to all forms of medical treatment. Accurate mapping of the locations of epileptic neuronal networks can result in the complete resection of epileptic foci. Even though currently electroencephalography is the best technique for mapping the epileptic focus, it cannot define the boundary of epilepsy that accurately. Herein we put forward a new accurate brain mapping technique using superparamagnetic nanoparticles (SPMNs). The main hypothesis in this new approach is the creation of super-paramagnetic aggregates in the epileptic foci due to high electrical and magnetic activities. These aggregates may improve tissue contrast of magnetic resonance imaging (MRI) that results in improving the resection of epileptic foci. In this paper, we present the mathematical models before discussing the simulation results. Furthermore, we mimic the aggregation of SPMNs in a weak magnetic field using a low-cost microfabricated device. Based on these results, the SPMNs may play a crucial role in diagnostic epilepsy and the subsequent treatment of this disease.

In vitro and in vivo investigation of chitosan/silk fibroin injectable interpenetrating network hydrogel with microspheres for cartilage regeneration.

Articular cartilage is an avascular and almost acellular tissue with limited self-regenerating capabilities. Although injectable hydrogels have garnered a lot of attention as a promising treatment, a biocompatible hydrogel with adequate mechanical properties is yet to be created. In this study, an interpenetrating network hydrogel comprised of chitosan and silk fibroin was created through electrostatic and hydrophobic bonds, respectively. The polymeric network of the scaffold combined an effective microenvironment for cell activity with enhanced mechanical properties to address the current issues in cartilage scaffolds. Furthermore, microspheres (MS) were utilized for a controlled release of methylprednisolone acetate (MPA), around ~75 % after 35 days. The proposed scaffolds demonstrated great mechanical stability with ~0.047 MPa compressive moduli and ~145 kPa compressive strength. Moreover, the degradation rate of the samples (~45 % after 35 days) was optimized to match neo-cartilage formation. Furthermore, the use of natural biomaterials yielded good biocompatibility with ~76 % chondrocyte viability after 7 days. According to gross observation after 12 weeks the defect site of the treated groups was filled with minimally discernible boundary. These results were confirmed by histopathology assays were the treated groups showed higher chondrocyte count and collagen type II expression.

Fabrication of gelatin-based antibacterial bilayer wound dressing using direct writing and electrospinning methods.

Fabricating a fibrous well-ordered wound dressing for accelerating full-thickness wounds is a desirable treatment vector. Here, through modifications in the material extrusion device and adding a pneumatic-based injection, a material extrusion method for gelatin was introduced with the ability to fabricate 3D structure with repeat layers to support cell activity for the under layer. Furthermore, in the upper layer, the co-electrospinning of PU with gelatin was designed to simultaneously exploit the oxygen permeability and mechanical stability of PU with regenerative properties and collagen-like structure of gelatin. Moreover, zinc oxide nanoparticles (ZnO) was added into the 3D-printed under layer to synergistically benefit from the antibacterial properties of ZnO and the excellent biocompatibility of gelatin. The controllable porosity of the under layer, enabled through the additive manufacturing method, was adjusted to mimic the extracellular matrix of natural tissue with around (127.28 ± 20.70) µm pore size after swelling with smooth fibers. S. aureus, E. coli, Bacillus subtilis, and Pseudomonas with inhibition zone diameters at âˆ¼ 2.14 cm and âˆ¼ 1.96 cm, ∼ 4.01 cm, and âˆ¼ 2.24 cm, respectively. Moreover, the scaffold showed great biocompatibility toward fibroblast cells after 7 days of cell culture with âˆ¼ 89 % cell viability.

Design and evaluation of loop-mediated isothermal amplification for rapid detection of Enterocytozoon bieneusi.

Enterocytozoon bieneusi is one of the most prevalent microsporidia species, responsible for more than 90% of human and animal microsporidiosis. Microsporidia species, particularly E. bieneusi, are frequently reported from waterborne and foodborne outbreaks. Therefore, early detection is crucial in clinics and outbreak investigations. This study aimed to design a loop-mediated isothermal amplification (LAMP) for rapid detection of E. bieneusi. Total DNA was extracted from 30 E. bieneusi -positive samples, which had been confirmed with nested PCR. LAMP primers were designed based on the identical fragment of small subunit ribosomal RNA (SSU rRNA) gene. LAMP reactions were performed at 63 °C for 60 min. The sensitivity and specificity of the assay were analyzed and the results of amplification were compared to real-time PCR. Our results showed that the LAMP assay successfully amplified 25/30 (83.3%) samples. The specificity results indicated no false positive with other microorganisms. Furthermore, the LAMP method exhibited a sensitivity (limit of detection, LoD) as low as 34 ag/µL of total DNA. Compared to the LAMP assay, real-time PCR was able to detect all 30 nested PCR-positive samples. Our findings showed that the LAMP assay was able to detect 83.3% of E. bieneusi-positive samples. Although the current assay was not able to detect all nested PCR-positive samples, the lack of need for specific instruments, rapid processes, and high specificity makes LAMP assay a suitable tool for screening.

Engineering biomimetic scaffolds for bone regeneration: Chitosan/alginate/polyvinyl alcohol-based double-network hydrogels with carbon nanomaterials.

In this study, new types of hybrid double-network (DN) hydrogels composed of polyvinyl alcohol (PVA), chitosan (CH), and sodium alginate (SA) are introduced, with the hypothesis that this combination and incorporating multi-walled carbon nanotubes (MWCNTs) and graphene nanoplatelets (GNPs) will enhance osteogenetic differentiation and the structural and mechanical properties of scaffolds for bone tissue engineering applications. Initially, the impact of varying mass ratios of the PVA/CH/SA mixture on mechanical properties, swelling ratio, and degradability was examined. Based on this investigation, a mass ratio of 4:6:6 was determined to be optimal. At this ratio, the hydrogel demonstrated a Young's modulus of 47.5 ± 5 kPa, a swelling ratio of 680 ± 6 % after 3 h, and a degradation rate of 46.5 ± 5 % after 40 days. In the next phase, following the determination of the optimal mass ratio, CNTs and GNPs were incorporated into the 4:6:6 composite resulting in a significant enhancement in the electrical conductivity and stiffness of the scaffolds. The introduction of CNTs led to a notable increase of 36 % in the viability of MG63 osteoblast cells. Additionally, the inhibition zone test revealed that GNPs and CNTs increased the diameter of the inhibition zone by 49.6 % and 52.6 %, respectively.