Brazilian National Network of Alternative Methods (RENAMA) 10th Anniversary: Meeting of the Associated Laboratories, May 2022.

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

Cytocompatible and osteoinductive cotton cellulose nanofiber/chitosan nanobiocomposite scaffold for bone tissue engineering.

Natural polymeric nanobiocomposites hold promise in repairing damaged bone tissue in tissue engineering. These materials create an extracellular matrix (ECM)-like microenvironment that induces stem cell differentiation. In this study, we investigated a new cytocompatible nanobiocomposite made from cotton cellulose nanofibers (CNFs) combined with chitosan polymer to induce osteogenic stem cell differentiation. First, we characterized the chemical composition, nanotopography, swelling properties, and mechanical properties of the cotton CNF/chitosan nanobiocomposite scaffold. Then, we examined the biological characteristics of the nanocomposites to evaluate their cytocompatibility and osteogenic differentiation potential using human mesenchymal stem cells derived from exfoliated deciduous teeth. The results showed that the nanobiocomposite exhibited favorable cytocompatibility and promoted osteogenic differentiation of cells without the need for chemical inducers, as demonstrated by the increase in alkaline phosphatase activity and ECM mineralization. Therefore, the cotton CNF/chitosan nanobiocomposite scaffold holds great promise for bone tissue engineering applications.

Nanoencapsulated Lippia rotundifolia antimicrobial peptide: synthesis, characterization, antimicrobial activity, and cytotoxicity evaluations.

Antimicrobial peptides (AMP) are promising novel antibiotics but exhibit low stability and can be toxic. The AMP encapsulation can be used to protect the drug and control its release rates. The Lr-AMP1f encapsulated into chitosan nanoparticle (NP) by ionic gelation method reached 90% efficiency. The results indicated that the hydrodynamic particle size of NPs increased from 196.1 ± 3.14 nm (free NP) to 228.1 ± 12.22 nm (nanoencapsulated Lr-AMP1f), while the atomic force microscope showed the spherical shape. The Zeta potential of the nanoencapsulated Lr-AMP1f was high (+ 35 mV). These AMP-loaded NPs exhibited stability for up to 21 days of storage. The minimum inhibitory concentration (MIC) of free Lr-AMP1f was 8 µg/mL for E. coli and S. epidermidis. However, the nanoencapsulated Lr-AMP1f produced a bacteriostatic effect against both bacteria at 8 µg/mL. The MIC of nanoencapsulated Lr-AMP1f was 16 µg/mL for E. coli and 32 for S. epidermidis. Nanoencapsulated Lr-AMP1f was nontoxic to HEK293 cells. Promisingly, chitosan NP can be used as a vehicle for the antibacterial application of new AMP (Lr-AMP1f).

Challenges in the use of nanostructures as carriers of nucleic acids in clinical practice.

The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

Cytocompatibility and osteogenic differentiation of stem cells from human exfoliated deciduous teeth with cotton cellulose nanofibers for tissue engineering and regenerative medicine.

Cellulose nanofibers (CNFs) are natural polymers with physical-chemical properties that make them very attractive for modulating stem cell differentiation, a crucial step in tissue engineering and regenerative medicine. Although cellulose is cytocompatible, when materials are in nanoscale, they become more reactive, needing to evaluate its potential toxic effect to ensure safe application. This study aimed to investigate the cytocompatibility of cotton CNF and its differentiation capacity induction on stem cells from human exfoliated deciduous teeth. First, the cotton CNF was characterized. Then, the cytocompatibility and the osteogenic differentiation induced by cotton CNF were examined. The results revealed that cotton CNFs have about 6-18 nm diameters, and the zeta potential was -10 mV. Despite gene expression alteration, the cotton CNF shows good cytocompatibility. The cotton CNF induced an increase in phosphatase alkaline activity and extracellular matrix mineralization. The results indicate that cotton CNF has good cytocompatibility and can promote cell differentiation without using chemical inducers, showing great potential as a new differentiation inductor for tissue engineering and regenerative medicine applications.

Challenges in the use of nanostructures as carriers of nucleic acids in clinical practice

ABSTRACT The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

Cytocompatibility of carboxylated multi-wall carbon nanotubes in stem cells from human exfoliated deciduous teeth.

Carboxylated multi-wall carbon nanotube (MWCNT-COOH) presents unique properties due to nanoscale dimensions and permits a broad range of applications in different fields, such as bone tissue engineering and regenerative medicine. However, the cytocompatibility of MWCNT-COOH with human stem cells is poorly understood. Thus, studies elucidating how MWCNT-COOH affects human stem cell viability are essential to a safer application of nanotechnologies. Using stem cells from the human exfoliated deciduous teeth model, we have evaluated the effects of MWCNT-COOH on cell viability, oxidative cell stress, and DNA integrity. Results demonstrated that despite the decreased metabolism of mitochondria, MWCNT-COOH had no toxicity against stem cells. Cells maintained viability after MWCNT-COOH exposure. MWCNT-COOH did not alter the superoxide dismutase activity and did not cause genotoxic effects. The present findings are relevant to the potential application of MWCNT-COOH in the tissue engineering and regenerative medicine fields.

The distinct effect of titanium dioxide nanoparticles in primary and immortalized cell lines.

The titanium dioxide nanoparticles (NPs) have been applied to biomedical, pharmaceutical, and food additive fields. However, the effect on health and the environment are conflicting; thus, it has been reviewing several times. In this context, establishing standard robust protocols for detecting cytotoxicity and genotoxicity of nanomaterials became essential for nanotechnology development. The cell type and the intrinsic characteristics of titanium dioxide NPs can influence nanotoxicity. In this work, the cyto- and genotoxicity effects of standard reference material titanium dioxide NPs in primary bovine fibroblasts and immortalized Chinese hamster ovary epithelial (CHO) cells were determined and compared for the first time. Titanium dioxide NPs exposure revealed no cytotoxicity for primary bovine fibroblasts, while only higher concentrations tested (10 µg/ml) induce genotoxic effects in this cell model. In contrast, the lower concentrations of the titanium dioxide NPs cause the cyto- and genotoxic effects in CHO cells. Therefore, our finding indicates that the CHO line was more sensitive toward the effects of titanium dioxide NPs than the primary bovine fibroblast, which should be valuable for their environmental risk assessment.

Corrigendum to: The distinct effect of titanium dioxide nanoparticles in primary and immortalized cell lines.

[This corrects the article DOI: 10.1093/toxres/tfab040.].

Heat shock during in vitro maturation of bovine oocytes disturbs bta-miR-19b and DROSHA transcripts abundance after in vitro fertilization.

While microRNAs (miRNAs) are a class of non-coding RNAs important for embryo development, the relationship between them and heat stress during oocyte maturation has not yet been established. This study investigated the effect of heat shock during in vitro maturation (IVM) on the abundance of bta-miR-20a, -27b, -103, -21-5p, -19b, -1246 miRNAs and DROSHA and DICER1 mRNAs, previously reported for being involved in oocyte maturation, response to heat stress and miRNA biogenesis. Oocytes were exposed for 12h to heat shock during IVM, fertilized in vitro and the presumptive zygotes cultured for eight days. The relative quantification of miRNAs and mRNAs was performed by real-time PCR in vitro-matured oocytes and 8-cell stage embryos. Progression of meiosis, embryonic development and apoptotic indices was also evaluated. Heat shock compromised (p < .05) oocyte nuclear maturation, cleavage and embryo development, with a higher (p < .05) embryonic apoptotic index than the control group. The abundance of bta-miR-19b increased (p < .05) whereas the abundance of DROSHA transcripts decreased (p < .05) in embryos derived from heat-shocked oocytes. In conclusion, heat shock during IVM influences the abundance of bta-miR-19b and DROSHA in pre-implantation embryos, indicating a persistent effect of heat shock that can be associated with impaired embryo development.

Induction of osteogenic differentiation by demineralized and decellularized bovine extracellular matrix derived hydrogels associated with barium titanate.

Bone tissue-derive biomaterials have become of great interest to treat diseases of the skeletal system. Biological scaffolds of demineralized and decellularized extracellular matrices (ECM) have been developed and one of these options are ECM hydrogels derived from bovine bone. Nanomaterials may be able to regulate stem cell differentiation due to their unique physical-chemical properties. The present work aimed to evaluate the osteoinductive effects of ECM hydrogels associated with barium titanate nanoparticles (BTNP) on dental pulp cells derived from exfoliated teeth. The addition of BTNP in the ECM derived hydrogel did not affect cell proliferation and the formation of bone nodules. Furthermore, it increased the expression of bone alkaline phosphatase. The results demonstrated that the nanobiocomposites were able to promote the osteogenic differentiation, even in the absence of chemical inducing factors for osteogenic differentiation. In conclusion, bovine bone ECM hydrogel combined with BTNP presented and increased expression of markers of osteogenic differentiation in the absence of chemical inducing factors.

Preparation, Characterization and In Vivo Biocompatibility Studies of Cotton Cellulose Nanofibers.

Cellulose nanofibers have mechanical properties that make them very attractive in a myriad of fields such as biomedicine, tissue engineering, biosensors, cosmetics and food packet products. To evaluate the potential health risks of airborne cellulose nanofibers, the cellulose nanofiber was prepared and characterized and then its pulmonary potential toxicity to a mouse model was studied. Cellulose nanofiber has been prepared by acid hydrolysis of cotton cellulose and characterized by transmission electron microscopy, zeta potential and X-ray diffraction analysis. Then, using a short-term inhalation test, the pulmonary biocompatibility of cotton cellulose nanofibers at different concentrations (0.5 mg/mL, 1 mg/mL and 2 mg/mL) were evaluated. Transmission electron images showed needle-shaped particle with a diameter of about 6-18 nm and a length of 85-225 µm. Zeta potential was -25.3±7.80 mV and the X-ray diffraction patterns indicate that cotton cellulose nanofiber has pure structural characteristics. The In Vivo results revealed that the exposure to cotton cellulose nanofiber did not alter the number of inflammatory cells or cytokine secretion by lung cells (p > 0.05). The results demonstrate that the cotton cellulose nanofiber is biocompatible and it is an environment-friendly nanomaterial with promise in various industrial sectors.

Contrasting effects of heat shock during in vitro maturation on development of in vitro-fertilized and parthenogenetic bovine embryos.

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus-oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro-fertilized or activated with ionomycin and cultured in vitro for 192 hr post-in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat-shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down-regulated the expression of AQP3 (p < .01) and up-regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down-regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat-shocked oocytes.

Cotton cellulose nanofiber/chitosan nanocomposite: characterization and evaluation of cytocompatibility.

Cellulose is a renewable polymer quite abundant on the Earth and very attractive for applications in the construction of eco-friendly biomedical products. The aim of this study was to investigate the chemical-physical characteristics of cotton cellulose nanofiber (CCN)/chitosan nanocomposite and its cytocompatibility with human embryonic kidney cells. First, the chemical composition, swelling ratio and surface topography of the nanocomposite were evaluated. Cytocompatibility was then assessed through spreading, proliferation and viability of cells. The experimental results showed that the CCN was an effective nanomaterial agent for increasing the roughness surface of chitosan film. Cell proliferation and changes in cell morphology indicated that the nanocomposite led to improved cell spreading and growth. Cell viability did not decrease after 24 h. However, the cell survival on the nanocomposite was affected at 72 h. The results indicate that CCN/chitosan nanocomposite could be a promising biocompatible biomaterial for biomedical applications.

In vitro evaluation of barium titanate nanoparticle/alginate 3D scaffold for osteogenic human stem cell differentiation.

Nanomaterials can mimic properties of extracellular matrix molecules, promising great potential for scaffold composition in tissue engineering. In the present study, we investigated whether barium titanate nanoparticles (BT NP) combined with alginate polymer would provide a new cytocompatible three-dimensional (3D) scaffold to induce osteogenic stem cell differentiation. In vitro cytocompatibility and osteogenic differentiation potential were investigated using human mesenchymal stem cells (MSC). Firstly, we studied the cell viability and oxidative stress by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) thiazolyl blue tetrazolium bromide (MTT) and superoxide dismutase (SOD) assays. Overall, neither pure BT NP or BT NP/alginate 3D scaffold induced cytotoxicity. The scanning electron and atomic force microscopy revealed that BT NP/alginate 3D scaffold produced exhibited highly interconnected pores and surface nanotopography that were favorable for MSC differentiation. Von Kossa staining showed mineralization nodules and MSCs morphology changed from spindle to cuboid shape after 21 d. Finally, BMP-2 and ALP mRNA were significantly upregulated on cells grown into the BT NP/alginate 3D scaffold. Thus, the BT NP/alginate 3D scaffold showed an osteogenic differentiation induction potential, without the addition of osteogenic supplements. These results indicate that the BT NP/alginate 3D scaffold provides a cytocompatible and bioactive microenvironment for osteogenic human MSC differentiation.

Differential gene expression between in vivo and in vitro maturation: a comparative study with bovine oocytes derived from the same donor pool.

OBJECTIVE: In vitro maturation has been shown to influence gene expression in oocytes, but a common shortcoming in reports on the matter has been the use of different donors in each experimental group thus disregarding donor effects. This study aimed to investigate the abundance of mRNA in oocytes matured in vivo and in vitro obtained from the same group of donors. METHODS: A bovine model was used to assess the relative abundance of specific transcripts in in vitro-matured (IN VITRO-OPU) and in vivo-matured (IN VIVO-OPU) oocytes collected from the same donors by transvaginal ovum pick-up (OPU). Transcript abundance in oocytes from the IN VIVO-OPU group and oocytes matured in vitro but retrieved from different cows slaughtered at a commercial abattoir (IN VITRO-Abattoir group) was also compared. Total RNA was extracted from denuded oocytes and cDNA was produced via reverse transcription using an oligo(dT) primer for relative quantification of eight target transcripts by real-time PCR. RESULTS: Oocytes in the IN VITRO-OPU group had lower (p<0.05) abundance of peroxiredoxin 1 (Prdx1), heat shock protein 70.1 (Hsp70.1), growth and differentiation factor 9 (Gdf9), and maternal antigen that embryo requires (Mater) transcripts than the oocytes in the IN VIVO-OPU group, all obtained from the same pool of donor cows. Similar results were seen in the comparisons involving the IN VIVO-OPU and IN VITRO-Abattoir groups (p<0.05). CONCLUSION: In vitro maturation affected the abundance of polyadenylated transcripts in the oocyte cytoplasm when compared to in vivo maturation induced by exogenous hormones in oocytes collected from the same donor pool.

Effect of Multi-walled Carbon Nanotubes on Metabolism and Morphology of Filamentous Green Microalgae.

Multi-walled carbon nanotubes (MWCNTs) have potential applications in the industrial, agricultural, pharmaceutical, medical, and environmental remediation fields. However, many uncertainties exist regarding the environmental implications of engineered nanomaterials. This study examined the effect of the MWCNTs on metabolic status and morphology of filamentous green microalgae Klebsormidium flaccidum. Appropriate concentrations of MWCNT (1, 50, and 100 µg mL-1) were added to a microalgal culture in the exponential growth phase and incubated for 24, 48, 72, and 96 h. Exposure to MWCNT led to reductions in algal growth after 48 h and decreased on cell viability for all experimental endpoints except for 1 µg mL-1 at 24 h and 100 µg mL-1 after 72 h. At 100 µg mL-1, MWCNTs induced reactive oxygen species (ROS) production and had an effect on intracellular adenosine triphosphate (ATP) content depending on concentration and time. No photosynthetic activity variation was observed. Observations by scanning transmission electron microscopy showed cell damage. In conclusion, we have demonstrated that exposure to MWCNTs affects cell metabolism and microalgal cell morphology. To our best knowledge, this is the first case in which MWCNTs exhibit adverse effects on filamentous green microalgae K. flaccidum. These results contribute to elucidate the mechanism of MWCNT nanotoxicity in the bioindicator organism of terrestrial and freshwater habitats.

Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes.

The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos.

Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes.

Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62). Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P<0.05), when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01). BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01). Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01). Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes.

Biocompatibility assessment of fibrous nanomaterials in mammalian embryos.

Currently there is a growing interest in the use of nanotechnology in reproductive medicine and reproductive biology. However, their toxic effects on mammalian embryos remain poorly understood. In this work, we evaluate the biocompatibility of two fibrous nanomaterials (NMs): cotton cellulose nanofibers (CNF) and carboxylated multiwalled carbon nanotubes (MWCNT-COOH), by performing an investigation of the embryonic development, gene expression (biomarkers focused on cell stress, apoptosis and totipotency) and in situ apoptosis in bovine embryos. Exposure to NMs did not interfere in preimplantation development or in the incidence of apoptosis in the bovine embryo, but they did affect the gene expression. The results presented are important for an understanding of the toxicity of cotton CNF and MWCNT-COOH on mammalian embryos. To our knowledge, we report the first evaluation of biocompatibility between these NMs on preimplantation embryos, which may open a new window for reproductive biomedical applications.