In-vitro polymicrobial oral biofilm model represents clinical microbial profile and disease progression during implant-related infections.

AIM: Clinically relevant in-vitro biofilm models are essential and valuable tools for mechanistically dissecting the etiopathogenesis of infectious diseases and test new antimicrobial therapies. Thus, the aim of this study was to develop and test a clinically relevant in-vitro oral polymicrobial biofilm model that mimics implant-related infections in terms of microbial profile. METHODS AND RESULTS: For this purpose, 24-well plate system was used to model oral biofilms, using three different microbial inoculums to grow in-vitro biofilms: (1) human saliva from periodontally healthy patients; (2) saliva as in inoculum 1 + Porphyromonas gingivalis strain; and (3) supra and subgingival biofilm collected from peri-implant sites of patients diagnosed with peri-implantitis. Biofilms were grown to represent the dynamic transition from an aerobic to anaerobic community profile. Subsequently, biofilms were collected after each phase and evaluated for microbiological composition, microbial counts, biofilm biomass, structure, and susceptibility to chlorhexidine (CHX). Results showed higher live cell count (P < .05) for biofilms developed from patients' biofilm inoculum, but biomass volume, dry weight, and microbiological composition were similar among groups (P > .05). Interestingly, according to the checkerboard DNA-DNA hybridization results, the biofilm developed from stimulated human saliva exhibited a microbial composition more similar to the clinical subgingival biofilm of patients with peri-implantitis, with proportions of the main pathogens closer to those found in the disease. In addition, biofilm developed using saliva as inoculum was shown to be susceptible to CHX with significant reduction in bacteria compared with biofilms without exposure to CHX (P < .05). CONCLUSION: The findings suggested that the in-vitro polymicrobial biofilm developed from human saliva as inoculum is a suitable model and clinically relevant tool for mimicking the microbial composition of implant-related infections.

Exploring Toxoplasma gondii´s Biology within the Intestinal Epithelium: intestinal-derived models to unravel sexual differentiation.

A variety of intestinal-derived culture systems have been developed to mimic in vivo cell behavior and organization, incorporating different tissue and microenvironmental elements. Great insight into the biology of the causative agent of toxoplasmosis, Toxoplasma gondii, has been attained by using diverse in vitro cellular models. Nonetheless, there are still processes key to its transmission and persistence which remain to be elucidated, such as the mechanisms underlying its systemic dissemination and sexual differentiation both of which occur at the intestinal level. Because this event occurs in a complex and specific cellular environment (the intestine upon ingestion of infective forms, and the feline intestine, respectively), traditional reductionist in vitro cellular models fail to recreate conditions resembling in vivo physiology. The development of new biomaterials and the advances in cell culture knowledge have opened the door to a next generation of more physiologically relevant cellular models. Among them, organoids have become a valuable tool for unmasking the underlying mechanism involved in T. gondii sexual differentiation. Murine-derived intestinal organoids mimicking the biochemistry of the feline intestine have allowed the generation of pre-sexual and sexual stages of T. gondii for the first time in vitro, opening a window of opportunity to tackling these stages by "felinizing" a wide variety of animal cell cultures. Here, we reviewed intestinal in vitro and ex vivo models and discussed their strengths and limitations in the context of a quest for faithful models to in vitro emulate the biology of the enteric stages of T. gondii.

Modeling the human placental barrier to understand Toxoplasma gondii´s vertical transmission.

Toxoplasma gondii is a ubiquitous apicomplexan parasite that can infect virtually any warm-blooded animal. Acquired infection during pregnancy and the placental breach, is at the core of the most devastating consequences of toxoplasmosis. T. gondii can severely impact the pregnancy's outcome causing miscarriages, stillbirths, premature births, babies with hydrocephalus, microcephaly or intellectual disability, and other later onset neurological, ophthalmological or auditory diseases. To tackle T. gondii's vertical transmission, it is important to understand the mechanisms underlying host-parasite interactions at the maternal-fetal interface. Nonetheless, the complexity of the human placenta and the ethical concerns associated with its study, have narrowed the modeling of parasite vertical transmission to animal models, encompassing several unavoidable experimental limitations. Some of these difficulties have been overcome by the development of different human cell lines and a variety of primary cultures obtained from human placentas. These cellular models, though extremely valuable, have limited ability to recreate what happens in vivo. During the last decades, the development of new biomaterials and the increase in stem cell knowledge have led to the generation of more physiologically relevant in vitro models. These cell cultures incorporate new dimensions and cellular diversity, emerging as promising tools for unraveling the poorly understood T. gondii´s infection mechanisms during pregnancy. Herein, we review the state of the art of 2D and 3D cultures to approach the biology of T. gondii pertaining to vertical transmission, highlighting the challenges and experimental opportunities of these up-and-coming experimental platforms.

Efectos de las diluciones homeopáticas altas sobre los modelos in vitro: revisión de la literatura / Effects of high homeopathic dilutions on in vitro models: a review of the literature

Los efectos de las diluciones homeopáticas altas (HD, por sus siglas en inglés) son controvertidos debido a que superan el número de Avogadro. Objetivo: Realizar una revisión de la literatura sobre los efectos de las HD sobre los modelos in vitro. Métodos: Se realizó una búsqueda sistemática en la base de datos PubMed de estudios que evaluaran las HD simples sobre modelos in vitro, publicados de 2007 en adelante. Resultados: 28 publicaciones cumplieron con los criterios de inclusión/exclusión. 26 estudios mostraron efectos evidentes de las HD simples sobre modelos in vitro. La mayor parte de tales estudios fueron realizados en países en donde la homeopatía ha alcanzado un alto nivel de institucionalización. Conclusiones: Los modelos in vitro evidencian de forma clara la actividad biológica de las HD superiores al número de Avogadro y dan cuenta de los efectos encontrados en la práctica clínica. La mayor parte de los estudios fueron realizados en países en donde la homeopatía es reconocida oficialmente, lo cual facilita el acceso a recursos para el desarrollo de la investigación.

the effects of homeopathic high dilutions (HDs) are controversial because they exceed Avogadro's number. Aim: to perform a literature review on the effects of HDs on in vitro models. Methods: a systematic search was performed in database PubMed for studies assessing simple HDs on in vitro models published from 2007 onward. Results: 28 publications met the inclusion/exclusion criteria; 26 studies demonstrated patent effects of simple HDs on in vitro models; most such studies were conducted in countries where homeopathy attained a high level of institutionalization. Conclusions: in vitro models patently evidence biological activity of HDs above Avogadro's number and account for effects found in clinical practice. Most studies were conducted in countries where homeopathy is officially recognized, which facilitates access to resources for the development of research.

The Role of Propolis as a Natural Product with Potential Gastric Cancer Treatment Properties: A Systematic Review.

Gastric cancer is one of the most common, aggressive, and invasive types of malignant neoplasia. It ranks fifth for incidence and fourth for prevalence worldwide. Products of natural origin, such as propolis, have been assessed for use as new complementary therapies to combat cancer. Propolis is a bee product with antiproliferative and anticancer properties. The concentrations and types of secondary metabolites contained in propolis mainly vary according to the geographical region, the season of the year, and the species of bees that make it. The present study is a systematic review of the main articles related to the effects of propolis against gastric cancer published between 2011 and 2021 in the PubMed and Science Direct databases. Of 1305 articles published, only eight studies were selected; among their principal characteristics was the use of in vitro analysis with cell lines from gastric adenocarcinoma and in vivo murine models of the application of propolis treatments. These studies suggest that propolis arrests the cell cycle and inhibits proliferation, prevents the release of oxidizing agents, and promotes apoptosis. In vivo assays showed that propolis decreased the number of tumors by regulating the cell cycle and the expression of proteins related to apoptosis.

In Vitro and In Vivo Modeling of Normal and Leukemic Bone Marrow Niches: Cellular Senescence Contribution to Leukemia Induction and Progression.

Cellular senescence is recognized as a dynamic process in which cells evolve and adapt in a context dependent manner; consequently, senescent cells can exert both beneficial and deleterious effects on their surroundings. Specifically, senescent mesenchymal stromal cells (MSC) in the bone marrow (BM) have been linked to the generation of a supporting microenvironment that enhances malignant cell survival. However, the study of MSC's senescence role in leukemia development has been straitened not only by the availability of suitable models that faithfully reflect the structural complexity and biological diversity of the events triggered in the BM, but also by the lack of a universal, standardized method to measure senescence. Despite these constraints, two- and three dimensional in vitro models have been continuously improved in terms of cell culture techniques, support materials and analysis methods; in addition, research on animal models tends to focus on the development of techniques that allow tracking leukemic and senescent cells in the living organism, as well as to modify the available mice strains to generate individuals that mimic human BM characteristics. Here, we present the main advances in leukemic niche modeling, discussing advantages and limitations of the different systems, focusing on the contribution of senescent MSC to leukemia progression.

Natural Antioxidant Evaluation: A Review of Detection Methods.

Antioxidants have drawn the attention of the scientific community due to being related to the prevention of various degenerative diseases. The antioxidant capacity has been extensively studied in vitro, and different methods have been used to assess its activity. However, the main issues related to studying natural antioxidants are evaluating whether these antioxidants demonstrate a key role in the biological system and assessing their bioavailability in the organism. The majority of outcomes in the literature are controversial due to a lack of method standardization and their proper application. Therefore, this study aims to compile the main issues concerning the natural antioxidant field of study, comparing the most common in vitro methods to evaluate the antioxidant activity of natural compounds, demonstrating the antioxidant activity in biological systems and the role of the main antioxidant enzymes of redox cellular signaling and explaining how the bioavailability of bioactive compounds is evaluated in animal models and human clinical trials.

Salivary gland cancer in the setting of tumor microenvironment: Translational routes for therapy.

Salivary gland carcinomas (SGC) are aggressive cancers that arise in minor and major salivary glands. Given the complexity and the multiple subtypes of this class of tumors, diagnosis and, treatment may be challenging for clinicians. Recently the tumor microenvironment, composed mainly of immune and stromal cells are been a target for treatment. Accumulating evidence indicates that cancer immunotherapies have made a significant impact on oncologic patients, however immunotherapeutic attempts in SGC have been shown limited improvement. Advances in the models that best translate aggressive SGC are needed for the development of clinical protocols grouping immunotherapies and other classes of drugs that will promote better responses in patients with advanced SGC stages. In this review, we introduced different experimental models for SGC with a focus on tumor microenvironment highlighting potential therapy applications for each model.

Resistivity Technique for the Evaluation of the Integrity of Buccal and Esophageal Epithelium Mucosa for In Vitro Permeation Studies: Swine Buccal and Esophageal Mucosa Barrier Models.

Permeation assays are important for the development of topical formulations applied on buccal mucosa. Swine buccal and esophageal epithelia are usually used as barriers for these assays, while frozen epithelia have been used to optimize the experimental setup. However, there is no consensus on these methods. In transdermal studies, barrier integrity has been evaluated by measuring electrical resistance (ER) across the skin, which has been demonstrated to be a simple, fast, safe, and cost-effective method. Therefore, the aims here were to investigate whether ER might also be an effective method to evaluate buccal and esophageal epithelium mucosa integrity for in vitro permeation studies, and to establish a cut-off ER value for each epithelium mucosa model. We further investigated whether buccal epithelium could be substituted by esophageal epithelium in transbuccal permeation studies, and whether their permeability and integrity were affected by freezing at -20 °C for 3 weeks. Fresh and frozen swine buccal and esophageal epithelia were mounted in Franz diffusion cells and were then submitted to ER measurement. Permeation assays were performed using lidocaine hydrochloride as a hydrophilic drug model. ER was shown to be a reliable method for evaluating esophageal and buccal epithelia. The esophageal epithelium presented higher permeability compared to the buccal epithelium. For both epithelia, freezing and storage led to decreased electrical resistivity and increased permeability. We conclude that ER may be safely used to confirm tissue integrity when it is equal to or above 3 kΩ for fresh esophageal mucosa, but not for buccal epithelium mucosa. However, the use of esophageal epithelium in in vitro transmucosal studies could overestimate the absorption of hydrophilic drugs. In addition, fresh samples are recommended for these experiments, especially when hydrophilic drugs are involved.

In Vitro and In Vivo Models for Studying SARS-CoV-2, the Etiological Agent Responsible for COVID-19 Pandemic.

The emergence and rapid worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted the scientific community to rapidly develop in vitro and in vivo models that could be applied in COVID-19 research. In vitro models include two-dimensional (2D) cultures of immortalized cell lines or primary cells and three-dimensional (3D) cultures derived from lung, alveoli, bronchi, and other organs. Although cell-based systems are economic and allow strict control of experimental variables, they do not always resemble physiological conditions. Thus, several in vivo models are being developed, including different strains of mice, hamsters, ferrets, dogs, cats, and non-human primates. In this review, we summarize the main models of SARS-CoV-2 infection developed so far and discuss their advantages, drawbacks and main uses.

Cell spheroids as a versatile research platform: formation mechanisms, high throughput production, characterization and applications.

Three-dimensional (3D) cell culture has tremendous advantages to closely mimic thein vivoarchitecture and microenvironment of healthy tissue and organs, as well as of solid tumors. Spheroids are currently the most attractive 3D model to produce uniform reproducible cell structures as well as a potential basis for engineering large tissues and complex organs. In this review we discuss, from an engineering perspective, processes to obtain uniform 3D cell spheroids, comparing dynamic and static cultures and considering aspects such as mass transfer and shear stress. In addition, computational and mathematical modeling of complex cell spheroid systems are discussed. The non-cell-adhesive hydrogel-based method and dynamic cell culture in bioreactors are focused in detail and the myriad of developed spheroid characterization techniques is presented. The main bottlenecks and weaknesses are discussed, especially regarding the analysis of morphological parameters, cell quantification and viability, gene expression profiles, metabolic behavior and high-content analysis. Finally, a vast set of applications of spheroids as tools forin vitrostudy model systems is examined, including drug screening, tissue formation, pathologies development, tissue engineering and biofabrication, 3D bioprinting and microfluidics, together with their use in high-throughput platforms.

Spheroids and organoids as humanized 3D scaffold-free engineered tissues for SARS-CoV-2 viral infection and drug screening.

The new coronavirus (2019-nCoV) or the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was officially declared by the World Health Organization (WHO) as a pandemic in March 2020. To date, there are no specific antiviral drugs proven to be effective in treating SARS-CoV-2, requiring joint efforts from different research fronts to discover the best route of treatment. The first decisions in drug discovery are based on 2D cell culture using high-throughput screening. In this context, spheroids and organoids emerge as a reliable alternative. Both are scaffold-free 3D engineered constructs that recapitulate key cellular and molecular events of tissue physiology. Different studies have already shown their advantages as a model for different infectious diseases, including SARS-CoV-2 and for drug screening. The use of these 3D engineered tissues as an in vitro model can fill the gap between 2D cell culture and in vivo preclinical assays (animal models) as they could recapitulate the entire viral life cycle. The main objective of this review is to understand spheroid and organoid biology, highlighting their advantages and disadvantages, and how these scaffold-free engineered tissues can contribute to a better comprehension of viral infection by SARS-CoV-2 and to the development of in vitro high-throughput models for drug screening.

Insights and controversies on sunscreen safety.

Although sunlight provides several benefits, ultraviolet (UV) radiation plays an important role in the development of various skin damages such as erythema, photoaging, and photocarcinogenesis. Despite cells having endogenous defense systems, damaged DNA may not be efficiently repaired at chronic exposure. In this sense, it is necessary to use artificial defense strategies such as sunscreen formulations. UV filters should scatter, reflect, or absorb solar UV radiation in order to prevent direct or indirect DNA lesions. However, the safety of UV filters is a matter of concern due to several controversies reported in literature, such as endocrine alterations, allergies, increased oxidative stress, phototoxic events, among others. Despite these controversies, the way in which sunscreens are tested is essential to ensure safety. Sunscreen regulation includes mandatory test for phototoxicity, but photogenotoxicity testing is not recommended as a part of the standard photosafety testing program. Although available photobiological tests are still the first approach to assess photosafety, they are limited. Some existing tests do not always provide reliable results, mainly due to limitations regarding the nature of the assessed phototoxic effect, cell UV sensitivity, and the irradiation protocols. These aspects bring queries regarding the safety of sunscreen wide use and suggest the demand for the development of robust and efficient in vitro screening tests to overcome the existing limitations. In this way, Saccharomyces cerevisiae has stood out as a promising model to fill the gaps in photobiology and to complete the mandatory tests enabling a more extensive and robust photosafety assessment.

An overview of neuroblastoma cell lineage phenotypes and in vitro models.

This review was conducted to present the main neuroblastoma (NB) clinical characteristics and the most common genetic alterations present in these pediatric tumors, highlighting their impact in tumor cell aggressiveness behavior, including metastatic development and treatment resistance, and patients' prognosis. The distinct three NB cell lineage phenotypes, S-type, N-type, and I-type, which are characterized by unique cell surface markers and gene expression patterns, are also reviewed. Finally, an overview of the most used NB cell lines currently available for in vitro studies and their unique cellular and molecular characteristics, which should be taken into account for the selection of the most appropriate model for NB pre-clinical studies, is presented. These valuable models can be complemented by the generation of NB reprogrammed tumor cells or organoids, derived directly from patients' tumor specimens, in the direction toward personalized medicine.

Preclinical toxicity of innovative molecules: In vitro, in vivo and metabolism prediction.

The lack of predictivity of animal's models has increased the failure rate of drug candidates. Thus, the reversion of this scenario using preliminary in vitro assays and metabolism prediction can reduce the unnecessary use of animals, as well as predict toxic effects at preclinical and clinical stages. The present study aimed to evaluate safety of four biologically active molecules (RN104, RI78, ICH, PCH) with potential therapeutic applications synthesized in our laboratory. Initially, we used MTT cytotoxicity against A549, H9C2, HepG2, LLC-PK1 and NEURO-2 cell lines. RN104 showed the lowest cytotoxicity and further studies were conducted with it. The neutral red (NR) test was performed according to OECD-129 and then acute toxicity test (OECD-423). According to NR results we administered at 300 mg/kg on animals; however, no toxic effect was observed, while 2,000 mg/kg resulted in the death of one animal per group. After, metabolism prediction studies, performed using both ligand-based and structure-based, suggests three potential metabolites. In silico results suggested that potential metabolites could be fast eliminated and, then, this could be an explanation for lower observed toxicity in in vivo experiments. The results showed limitations of the NR as a predictor of the initial dose for the acute toxicity study, which may be related to metabolism. Therefore, the combination of theoretical and experimental studies is relevant to a general understanding of new molecule's toxicity.

Full-Thickness Intraoral Mucosa Barrier Models for In Vitro Drug-Permeation Studies Using Microneedles.

The use of permeation enhancers such as microneedles (MNs) to increase drug penetration across intraoral mucosa has increased in recent years. Permeation studies, commonly performed using vertical diffusion cells, are a well-established way to preview formulations and enhance their performance during the development stage. However, to our knowledge, the existing intraoral mucosa barrier models do not permit permeation using MN-pretreated mucosa due to their insufficient thickness. Therefore, the objective of this study was to develop a barrier model using thick palate tissues to perform in vitro permeation studies, with physical enhancement of the permeability of intraoral mucosa by pretreatment with MNs. The adapted Franz-type cells used in the permeation experiments were validated (cell dimensions and volume, sealing effectiveness, stirring and dissolution efficiency, temperature control, and establishment of uniaxial flux). Commercially available MNs were used in the palatal mucosa. Optical images of the mucosa were acquired to analyze the microperforations created. In vitro permeation studies were conducted with the MN-pretreated mucosa. This work presents a new in vitro method for the evaluation of MNs as permeation enhancers, with the aim of improving the absorption of drug formulations topically applied within the oral cavity.

Pharmacological Effects of Carvacrol in In vitro Studies: A Review.

Carvacrol has a high therapeutic potential, with in vitro studies showing promising results in different cellular models using a variety of methodological designs. Therefore, the objective of this study was to conduct a systematic review to analyze the pharmacological effects of carvacrol in in vitro studies. A comprehensive search of the literature was made using four databases: Science Direct, Scopus, MEDLINE-PubMed, and Web of Science using different combinations of the following keywords: carvacrol, drug therapy, therapeutic uses, in vitro study. The search of the databases was for studies conducted in the period up to and including September 2016. A total of 3,269 studies were initially identified, with only 31 meeting the inclusion criteria. The included studies contained a variety of in vitro models able to determine the properties of Carvacrol. The following properties of Carvacrol were identified: antimicrobial (7 studies), bactericidal (4), bactericidal and antifungal (1), antiinflammatory (4), anticancer (4), mutagenic (4), antioxidant (3), antifungal (3), antidepressant (1), as a modulator of nerve impulses (1) and an immunological modulator (1). The In vitro studies with Carvacrol included in this review showed a diversity of models and confirmed the therapeutic potential of this product in relation to several diseases.

Microemulsion Formulations for the Transdermal Delivery of Lapachol.

This project was carried out to investigate the feasibility of using microemulsions for transdermal delivery of lapachol. From the screening of surfactants and oils, a range of microemulsions were developed using oleic acid, a mixture of Cremophor EL and Tween 20 and water. The solubility of lapachol was determined in these ingredients and in the formulated microemulsions. The microemulsions were characterised using cross-polarising light microscopy, their electrical conductivity, pH, zeta potential and rheology were analysed, and they were also investigated using small-angle X-ray scattering and differential scanning calorimetry. Ex vivo studies were performed using porcine ear skin and Franz diffusion cells to investigate the permeation and retention of lapachol. Systems containing different concentrations of Cremophor EL (8.4-41.6%), Tween 20 (5.4-41.6%) and oleic acid (12-31.9%) are able to form microemulsions. Lapachol was delivered more effectively through the skin from all of the microemulsions tested than by the control (oleic acid). These studies indicated that microemulsions incorporating lapachol were formed successfully and that these enhanced drug delivery and retention in the skin. Microemulsion systems may, therefore, provide promising vehicles for percutaneous delivery of lapachol.