Bull sperm selection by attachment to hyaluronic acid semi-interpenetrated hydrogels.

We report the development of a hydrogel-based approach to select bull spermatozoa, a crucial step for successful assisted reproductive techniques (ARTs). Hyaluronic acid (HA) semi-interpenetrated N-isopropylacrylamide (PNIPAM) co-20% N-Tris (hydroxymethyl) methyl acrylamide (HMA) hydrogels were synthetized on glass surfaces and cultured in presence of frozen-thawed bull spermatozoa. A fraction of motile bull spermatozoa population strongly attached to hydrogels and was partially released by treatment with hyaluronidase. Fifty-nine (59 ± 7.24) per cent of sperm cells attached to PNIPAM-HMA-HA hydrogels and 31.16 ± 4.81% of them were released upon treatment with medium containing hyaluronidase. This attached-released sperm fraction has acceptable characteristics of progressive motility (50.0 ± 5.0%), vigour (4), high viability (58.7 ± 11.7%) and low percentage of acrosome reacted spermatozoa (23.36 ± 4.1%). Our findings indicate that PNIPAM-HMA-HA hydrogels are non-toxic and allow the selection of high-quality sperm cells for ART.

Toxicity assessment and DNA repair kinetics in HEK293 cells exposed to environmentally relevant concentrations of Glyphosate (Roundup® Control Max).

Glyphosate is a systemic, non-selective, pre and post-emergence wide range herbicide. In 2015, IARC classified Glyphosate as "a probable carcinogenic agent for humans". The aim of this study was to evaluate the cytotoxicity and genotoxicity of the commercial formulation of glyphosate (Roundup® Control Max) at environmentally relevant concentrations and measure the potential effect of this herbicide over the cell capacity to repair DNA damage. HEK293 cells were exposed to 5 concentrations of Roundup® Control Max equivalent to 0.7; 7; 70; 700 and 3,500 µg/L glyphosate acid, for 1, 4 and 24 h. Cytotoxicity was quantified by the Trypan Blue staining method and by the MTT assay, while genotoxicity and evaluation of DNA damage repair kinetics were analyzed through the alkaline comet assay. In all treatments, cell viability was higher than 80%. The three highest glyphosate concentrations-70 µg/L, 700 µg/L, and 3,500 µg/L-increased levels of DNA damage compared to the control at the three exposure times tested. Finally, concerning the kinetics of DNA damage repair, cells initially exposed to 3,500 µg/L of glyphosate for 24 h were unable to repair the breaks in DNA strands even after 4 h of incubation in culture medium. The present study demonstrated for the first time that Roundup® Control Max may induce genetic damage and cause alterations in the DNA repair system in human embryonic kidney cells even at concentrations found in blood and breast milk of people exposed through residues of the herbicide in food, which values have been poorly assessed or not studied yet according to the existent literature.

Biointerfacial behavior of stallion spermatozoa adhered to hydrogel surfaces: Impact of the hydrogel chemical composition and the culture medium.

Novel soft materials based on hydrogel are proposed to enhance the selection of high-quality stallion sperm based on their adhesion capacity. The hydrogel surfaces are derived from polyacrylamide (PAAm), which is copolymerized with neutral and ionic co-monomers to modify the interfacial properties. The hydrogels undergo characterization through FTIR spectroscopy, assessment of swelling capacity, and wettability under various experimental conditions. Sperm adhesion capacity on the hydrogels is examined through several parameters including the percentage of bound sperm (%Sp) to hydrogels, tail oscillation intensity and flagellar movement. The biointerfacial properties of sperm-hydrogel systems vary based on the chemical composition of hydrogel as well as the components present in the culture medium. High %Sp and excellent metabolic activity of the spermatozoa are observed on hydrogel surfaces that possess moderate hydrophilicity. Specifically, a cationic hydrogel in BGM3 culture medium and a neutral surface in BGM3 medium supplemented with BSA exhibit favorable outcomes. Scanning Electron Microscopy (SEM) reveals the normal morphology of the head and tail in spermatozoa adhered to the hydrogel. Therefore, these hydrogel surfaces are potential materials for selecting stallion sperm with high quality, and their application could be extended to the study of other mammalian reproductive cells.

Mechanical and physicochemical behavior of a 3D hydrogel scaffold during cell growth and proliferation.

Some of the essential properties for cellular scaffolding are the capability to maintain the three-dimensional (3D) structure, good adhesion, and adequate elastic modulus during cell growth, migration, and proliferation. Biocompatible synthetic hydrogels are commonly used as cellular scaffolds because they can mimic the natural extracellular matrices (ECMs). However, it is possible that the physicochemical and mechanical behavior of the scaffold changes during cell proliferation and loses the scaffold properties but this is rarely monitored. In this work, the physicochemical and mechanical properties of a macroporous soft material based on poly(N-isopropyl acrylamide) (PNIPAM) have been studied during a period of 75 days at culture condition while bovine fetal fibroblasts (BFF) were grown within the matrix. The interconnected macroporous hydrogel was obtained by cryogelation at -18 °C. The swelling capacity of the scaffold was not altered during cell proliferation but changes in the mechanical properties were observed, beginning with the high elastic modulus (280 kPa) that progressively decreased until mechanical stability (40 kPa) was achieved after 20 culture days. It was observed that the matrix-cell interactions together with collagen production favor normal cellular processes such as cell morphology, adhesion, migration, and proliferation. Therefore, the observed behavior of macroporous PNIPAM as a 3D scaffold during cell growth indicates that the soft matrix is cytocompatible for a long time and preserves the suitable properties that can be applied in tissue engineering and regenerative medicine.

Cytotoxicity and bioadhesive properties of poly-N-isopropylacrylamide hydrogel.

Several hydrogel surfaces present properties that simulate the mechanical and physicochemical features of extracellular matrix (ECM), providing a platform that mimic the native cellular milieus. Poly-N-isopropylacrylamide (PNIPAM) hydrogels are receiving attention in biomedical field due to their thermosensibility and soft texture. However, more extensive biocompatibility and cellular interactions studies with cell lines are needed. Therefore, the aim of this study is focus on evaluating the biocompatibility of PNIPAM through cytotoxicity, genotoxicity, and proliferation tests in murine preadipose cells (3T3-L1), human embryonic kidney cells (HEK293) and human carcinoma-derived cells (A549) in presence of hydrogel surfaces. Bioadhesive capacity above PNIPAM surfaces was also analyzed. MTT and neutral red uptake assays shown non-cytotoxic effect of PNIPAM in the studied cell lines. Genotoxicity was evaluated by the single-cell gel electrophoresis assay, where DNA damages were not detected. [3H]-thymidine staining allowed to corroborate that cell proliferation had progressed correctly. Adopted morphologies for each cell line over PNIPAM were similar to cell growing observed on polystyrene, indicating that the surfaces favor the cell attachment during 5 days' culture. The good biocompatibility of PNIPAM surfaces make it an interesting scaffold with clinical potential in tissue regeneration engineering, and a possible adipose and kidney tissue-engineered construct.

Physicochemical properties of ionic and non-ionic biocompatible hydrogels in water and cell culture conditions: Relation with type of morphologies of bovine fetal fibroblasts in contact with the surfaces.

Cationic, anionic and non-ionic hydrogels having acrylamide polymer backbones were synthesized via free radical polymerization with N,N-methylenebisacrylamide (BIS) as crosslinker. The chemical structures of the hydrogels were characterized by Fourier Transform Infrared Spectroscopy (FTIR). Physicochemical properties such as swelling kinetic, maximum swelling capacity, volume phase transition temperature (VPTT) and wettability (static water contact angle) of hydrogels swollen in aqueous and cell culture medium, at room and cell culture temperatures were studied. In order to correlate the surface properties of the hydrogels and cellular adhesivity of bovine fetal fibroblasts (BFFs), cellular behaviour was analyzed by inverted fluorescence optical microscopy and atomic force microscopy (AFM). MTT assay demonstrated that the number of viable cells in contact with hydrogels does not significantly change in comparison to a control surface. Flattened and spindle-shaped cells and cell spheroids were the adopted morphologies during first days of culture on different hydrogels. Cell spheroids were easily obtained during the first 5days of culture in contact with PNIPAM-co-20%HMA (poly (N-isopropylacrylamide-co-20%N-acryloyl-tris-(hydroxymethyl)aminomethane)) hydrogel surface. After 15days of culture all hydrogels showed high adhesion and visual proliferation. According to obtained results, non-ionic and hydrophilic surfaces with moderated wettability induce the formation of BFFs cell spheroids. These hydrogel surfaces could be used in clinical and biochemical treatments at laboratory level to cell growth and will allow generating the base for future biotechnologic platform.