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Three-dimensional (3D) bioprinting is the use of computer-controlled transfer processes for assembling bioinks (cell clusters or materials loaded with cells) into structures of prescribed 3D organization. The correct bioprinting parameters ensure a fast and accurate bioink deposition without exposing the cells to harsh conditions. This study seeks to optimize pneumatic extrusion-based bioprinting based on hydrogel flow rate and extrusion speed measurements. We measured the rate of the hydrogel flow through a cylindrical nozzle and used non-Newtonian hydrodynamics to fit the results. From the videos of free-hanging hydrogel strands delivered from a stationary print head, we inferred the extrusion speed, defined as the speed of advancement of newly formed strands. Then, we relied on volume conservation to evaluate the extrudate swell ratio. The theoretical analysis enabled us to compute the extrusion speed for pressures not tested experimentally as well as the printing speed needed to deposit hydrogel filaments of a given diameter. Finally, the proposed methodology was tested experimentally by analyzing the morphology of triple-layered square-grid hydrogel constructs printed at various applied pressures while the printing speeds matched the corresponding extrusion speeds. Taken together, the results of this study suggest that preliminary measurements and theoretical analyses can simplify the search for the optimal bioprinting parameters.

Abstract Purpose The aim of this study was to evaluate the antioxidant potential, antimicrobial activity, the in vitro anticancer effect (tested on MCF-7 breast cancer cell line), as well as the antiangiogenic and immunomodulatory potential of Populus nigra L. bud (Pg) extract collected from the western part of Romania. Results Populus nigra L. bud extract presents an important antioxidant activity, due to the rich phytochemical composition. Regarding the biological activity, results have shown that poplar bud extract presents a significant inhibitory activity against Gram-positive bacteria and a dose-dependent decrease of MCF-7 tumor cell viability with an IC50 of 66.26 μg/mL, while not affecting healthy cells. Phenomena of early apoptotic events at the maximum concentration tested (150 μg/mL) were detected by Annexin V-PI double staining. The extract induced G0/G1 phase cell cycle arrest. In addition, Pg extract showed antiangiogenic potential on the chorioallantoic membrane. Also, at the highest concentration (150 μg/mL), good tolerability and no signs of toxicity upon vascular plexus were observed. Moreover, in low concentrations, the Pg extract had immunomodulatory activity on primary human dendritic cells by upregulating IL-12 and IL-23 subunits. Conclusion The study concludes that poplar bud extract elicited antioxidant activity, antitumor properties on the breast cancer cell line, followed by an antiangiogenic effect and an immunomodulatory potential on human primary dendritic cells. The biological activity of Populus nigra L. buds extract may open new directions of research on the topic addressed.

Few are known about the molecular mechanism controlling the early embryo development. The reduce dimension of the embryos, only a few µm, the small quantities of proteins synthesized and the artificial environment influence makes difficult to decode the mechanisms controlling early embryonic stages of development. Although, in the last few years many genes have been showed to be active in the early embryonic stages of development, only a few have been characterized and found to be implicated in the molecular mechanism responsible of preimplantational embryos development. Ped gene (Preimplantational embryo development) is considered to be involved in regulation of embryonic cleavage division and subsequent embryo survival. This review presents, based on a rich documentation, the main mechanisms involved in early embryo development.

The purpose of the ongoing research is to improve our current skills and knowledge in stem cell isolation, cultivation and differentiation from the utricular and saccular epithelia of young mice. We harvested utricles and sacculi from 7 days old NMRI mice. Utricles were trypsinized in order to isolate single cells. Obtained cells were cultivated in DMEM with F12 Nutrient mixture, B27, N2 supplement, IGF-1 and EGF. Sphere pluripotency was established with stem cell markers Nanog and Oct-4. We mechanically dissociated primary spheres and cultivated. Secondary spheres were placed on fibronectin coated tissue culture slide chambers in the absence of IGF-1 and EGF. Cells were characterized by immunofluorescence and immunohistochemistry for myosinVIIA (hair cell marker) and nestin (intermediate filament VI marker). Vestibular epithelia contain pluripotent stem cells able to form cell clusters (spheres). Pluripotency was demonstrated by the expression of nanog, oct 4 and nestin markers (cell progenitors). Also sphere dissociation and separate cultivation of the isolated cells lead to the formation of larger spheres and in higher amounts compared to the original spheres, proving that these cells are not only pluripotent but also capable of self-renewal. Single spheres harvested and cultivated on fibronectin produced through differentiation different cell types including neuron like-cells, positive for myosin VIIA and nestin.

Cardiac pathological hypertrophy is the major risk factor that usually progresses to heart failure. We hypothesized that extracellular vesicles (EVs), known to act as important mediators in regulating physiological and pathological functions, could have the potential to reduce the cardiac hypertrophy and the ensuing cardiovascular diseases. Herein, the effects of mesenchymal stem cell-derived extracellular vesicles (EV-MSCs) on cardiac hypertrophy were investigated. EVs were isolated from the secretome of human adipose tissue-derived stem cells (EV-ADSCs) or bone marrow-derived stem cells (EV-BMMSCs). Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were stimulated with AngII and TGF-β1, in absence or presence of EVs. The results showed that exposure of hiPSC-CMs to AngII and TGF-β1 generated in vitro model of hypertrophic cardiomyocytes characterized by increases in surface area, reactive oxygen species production, protein expression of cardiac-specific biomarkers atrial natriuretic factor, migration inhibitory factor, cTnI, COL1A1, Cx43, α-SMA and signalling molecules SMAD2 and NF-kBp50. The presence of EV-ADSCs or EV-BMMSCs in the hiPSC-CM culture along with hypertrophic stimuli reduced the protein expressions of hypertrophic specific markers (ANF, MIF, cTnI, COL1A1) and the gene expressions of IL-6 molecule involved in inflammatory process associated with cardiac hypertrophy and transcription factors SMAD2, SMAD3, cJUN, cFOS with role in cardiomyocyte hypertrophic response induced by AngII and TGF-β1. The EV-ADSCs were more effective in reducing the protein expressions of hypertrophic and inflammatory markers, while EV-BMMSCs in reducing the gene expressions of transcription factors. Notably, neither EV-ADSCs nor EV-BMMSCs induced significant changes in cardiac biomarkers Cx43, α-SMA and fibronectin. These different effects of stem cell-derived EVs could be attributed to their miRNA content: some miRNAs (miR-126-3p, miR-222-3p, miR-30e-5p, miR-181b-5p, miR-124-3p, miR-155-5p, miR-210-3p hsa-miR-221-3p) were expressed in both types of EVs and others only in EV-ADSCs (miR-181a-5p, miR-185-5p, miR-21-5p) or in EV-BMMSCs (miR-143-3p, miR-146a-5p, miR-93-5p), some of these attenuating the cardiac hypertrophy while others enhance it. In conclusion, in hiPSC-CMs the stem cell-derived EVs through their cargo reduced the expression of hypertrophic specific markers and molecules involved in inflammatory process associated with cardiac hypertrophy. The data suggest the EV potential to act as therapeutic mediators to reduce cardiac hypertrophy and possibly the subsequent cardiovascular events.

The tumor microenvironment (TME) influences cancer progression. Therefore, engineered TME models are being developed for fundamental research and anti-cancer drug screening. This paper reports the biofabrication of 3D-printed avascular structures that recapitulate several features of the TME. The tumor is represented by a hydrogel droplet uniformly loaded with breast cancer cells (106 cells/mL); it is embedded in the same type of hydrogel containing primary cells—tumor-associated fibroblasts isolated from the peritumoral environment and peripheral blood mononuclear cells. Hoechst staining of cryosectioned tissue constructs demonstrated that cells remodeled the hydrogel and remained viable for weeks. Histological sections revealed heterotypic aggregates of malignant and peritumoral cells; moreover, the constituent cells proliferated in vitro. To investigate the interactions responsible for the experimentally observed cellular rearrangements, we built lattice models of the bioprinted constructs and simulated their evolution using Metropolis Monte Carlo methods. Although unable to replicate the complexity of the TME, the approach presented here enables the self-assembly and co-culture of several cell types of the TME. Further studies will evaluate whether the bioprinted constructs can evolve in vivo in animal models. If they become connected to the host vasculature, they may turn into a fully organized TME.

Populus nigra L. (Salicaceae family) is one of the most popular trees that can be found in deciduous forests. Some particularities that characterize the Populus genus refer to the fact that it includes more than 40 species, being widespread especially in Europe and Asia. Many residues, parts of this tree can be used as a bioresource for different extracts as active ingredients in pharmaceuticals next to multiple benefits in many areas of medicine. The present review discusses the latest findings regarding the phytochemical composition and the therapeutic properties of Populus nigra L. buds. The vegetal product has been described mainly to contain phenolic compounds (phenols, phenolic acids and phenylpropanoids), terpenoids (mono and sesquiterpenoids), flavones (e.g., apigenol and crysin), flavanones (e.g., pinocembrin and pinostrombin), caffeic/ferulic acids and their derivates, and more than 48 phytocompounds in the essential oils. The resinous exudates present on the buds have been the major plant source used by bees to form propolis. Several studies depicted its antioxidant, anti-inflammatory, antibacterial, antifungal, antidiabetic, antitumor, hepatoprotective, hypouricemic properties and its effects on melanin production. All these lead to the conclusion that black poplar buds are a valuable and important source of bioactive compounds responsible for a wide range of therapeutic uses, being a promising candidate as a complementary and/or alternative source for a large number of health problems. The aim of the review is to gather the existing information and to bring an up to date regarding the phytochemical and therapeutic uses of Populus nigra L. buds.

Betulinic acid, a very promising anti-melanoma agent, has very low water solubility that causes low bioavailability. To overcome this inconvenience, a highly water-soluble cyclodextrin was used (octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonic acid)]-γ-cyclodextrin). The complex was physico-chemically analyzed using differential scanning calorimetry (DSC), X-ray and scanning electron microscopy (SEM) methods and then in vitro tested for its antiproliferative activity by the MTT assay and by cell cycle analysis. Finally, the complex was tested in vivo using an animal model of murine melanoma developed in C57BL/6J mice, where it caused a reduction in tumor volume and weight. The study revealed the beneficial influence of betulinic acid inclusion into the cyclodextrin in terms of antiproliferative activity and in vivo tumor development.

Apigenin (4′,5,7-trihydroxyflavone) (Api) is an important component of the human diet, being distributed in a wide number of fruits, vegetables and herbs with the most important sources being represented by chamomile, celery, celeriac and parsley. This study was designed for a comprehensive evaluation of Api as an antiproliferative, proapoptotic, antiangiogenic and immunomodulatory phytocompound. In the set experimental conditions, Api presents antiproliferative activity against the A375 human melanoma cell line, a G2/M arrest of the cell cycle and cytotoxic events as revealed by the lactate dehydrogenase release. Caspase 3 activity was inversely proportional to the Api tested doses, namely 30 μM and 60 μM. Phenomena of early apoptosis, late apoptosis and necrosis following incubation with Api were detected by Annexin V-PI double staining. The flavone interfered with the mitochondrial respiration by modulating both glycolytic and mitochondrial pathways for ATP production. The metabolic activity of human dendritic cells (DCs) under LPS-activation was clearly attenuated by stimulation with high concentrations of Api. Il-6 and IL-10 secretion was almost completely blocked while TNF alpha secretion was reduced by about 60%. Api elicited antiangiogenic properties in a dose-dependent manner. Both concentrations of Api influenced tumour cell growth and migration, inducing a limited tumour area inside the application ring, associated with a low number of capillaries.

Chamomile, parsley, and celery represent major botanical sources of apigenin, a well-known flavone with chemopreventive properties. The aim of this study was to assess the phytochemical composition, antioxidant, and anti-inflammatory potential of methanol extracts obtained from chamomile, parsley, and celery collected from Romania, as well as the biological activity against A375 human melanoma and human dendritic cells. Results have shown that all three extracts are rich in polyphenolic compounds and flavonoids, and they generate a radical scavenger capacity, iron chelation potential, as well as lipoxygenase inhibition capacity. Chamomile and celery extracts present weak antiproliferative and pro-apoptotic properties in the set experimental conditions, while parsley extract draws out significant pro-apoptotic potential against A375 human melanoma cells. Parsley and chamomile extracts affected the fibroblast-like morphology of the screened tumor cell line. On the other hand, chamomile and celery extracts abrogated the expansion of LPS-activated dendritic cells, while the metabolic activity was attenuated by stimulation with celery extract; chamomile and parsley extracts had no effect upon this parameter. Chamomile and parsley extracts incubation with naive dendritic cells did not trigger cytokine secretion (TNF-alpha, IL-6, IL-10), but celery extract stimulation significantly reduced the anti-inflammatory, cytokine IL-10.

Given the present high incidence of melanoma and skin cancer, interest in potential drugs of plant origin has increased significantly. Pentacyclic lupane-type triterpenes are widely distributed in plants, offering numerous pharmacological benefits. Betulin is an important compound in the bark of Betula pendula Roth and has important therapeutic properties, including antitumor activities. Its biological effect is limited by its poor water solubility, which can be improved by cyclodextrin complexation. The best results have been obtained by using a novel cyclodextrin derivative, octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonate)]-γ-CD. The complexes between betulin and the previously mentioned cyclodextrin were analyzed by scanning electron microscopy (SEM)and differential scanning calorimetry (DSC) and pharmacologically evaluated in vitro (MTT and immunocytochemistry tests) and in vivo in C57BL/6J mice. The solubility of betulin is improved by cyclodextrin complexation, which creates a stable complex that improves the in vitro and in vivo properties of the active compound.

Sequencing technology proposes new insights into neoantigens and mutation-targeted therapies derived from transcriptional patterns. Combined with classic diagnostic methods, immune profile and microenvironmental assessment, can provide in-depth information that can reframe our understanding of human cancer biology. In order to know tumor biology, it is necessary to obtain information about the genetic changes in the DNA of the tumor cells. We performed an extensive genomic characterization by next-generation sequencing (NGS) of three melanoma cell lines with the aim of analyzing the genetic changes occurring in melanoma and their clinical and therapeutic relevance. We identified known melanoma driver mutations, such as BRAF V600E, NRAS Q61R, and additionally found other mutated genes that appear in melanoma pathogenesis and some of them targeted by novel therapies. These cell lines are used to improve the genetic understanding of melanoma and to study the functional significance of individual mutational changes, but turning cancer genomic data into knowledge remains a challenge. For establishing the appropriate therapeutic approach in melanoma, the personalized treatment should be envisioned, based on both next generation sequencing, surface markers expression, as well as microenvironment assessment. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) are a type of cells that can be generated directly from adult cells and can be differentiated into many cell lines, including cardiomyocytes. The first reprogramming methods had low efficiency but lately safe lines of iPSCs without insertional mutations have been achieved. The human iPSCs technology offers a unique opportunity to obtain patient-specific hiPSC-derived cardiomyocytes. This platform allows iPSCs to be used for drug testing, disease modeling and cell therapy development. In this review, we discuss the progress and applications regarding human iPSCs, including their potential, limitations and future directions. [ABSTRACT FROM AUTHOR]

Bioprinting is an evolving technology that uses 3d printing in order to create complex macro and micro structures capable of replicating and enhancing the healing capacity of the native body. Through the printing process different materials and cells can be mixed in order to obtain artificial tissues and organs which in the future can represent an alternative to tissue transplant or implants. We have reviewed the main techniques of cell bioprinting options and presented the advantages and drawbacks of each method. [ABSTRACT FROM AUTHOR]

Ultraviolet B (UVB) or medium wave irradiation, with a wavelength in the range 315-280 nm and a energy per photon between 3.94-4.43 eV, regulates ultraviolet-responsive genes, including matrix metalloproteinases. Moreover, it causes connective tissue damage and the skin to become wrinkled and aged. The aim of this study was to investigate the evolution of the mouse skin papillomas initiated by 7,12-dimethylbenzanthracene (DMBA) and promoted by 12-O-decanoylphorbol-13-acetate (TPA), which progressed more rapidly to carcinoma on mice exposed to UVB. The biological effects of these two tumors (chemically and photochemically initiated) were evaluated for twelve weeks on the SKH1 hairless mice skin by using of non-invasive techniques (tewameter, skin-pH-meter, melanin and erythema measurements). It was found that UVB irradiation affects very much the parameters which were monitored and which characterizing the skin condition. [ABSTRACT FROM AUTHOR]

The aim of the present study is to investigate the probability of dog periosteum-derived cells and bone marrow-derived cells to differentiate along the osteogenic, chondrogenic and adipogenic lineages. Results of this experiment aim to highlight the similarities and differences between the cells obtained as a result of periosteum-derived culture and bone marrow-derived cells cultivated in three differentiation media. While the dog bone marrow derived-cells differentiate towards all three lineages, with variable intensity, thus confirming the results of other research studies in this field referring to other species, canine periosteum-derived cells obtained using culture of periosteal explants, differentiate mostly towards the osteoblastic and chondrogenic lineage, and to lesser extent towards the adipogenic lineage; the cells obtained using the digestion method of periosteal fragments positively differentiate towards all the three lineages: osteoblastic, chondrogenic and adipogenic. In addition, it was demonstrated that cryopreserved studied cells have osteogenic, chondrogenic and adipogenic differentiation capacity. [ABSTRACT FROM AUTHOR]

One priority in developping better immunotherapies in cancer is the use of natural killer cells to treat different types of cancer. The citotoxicity of natural killer cells to tumoral cells depends on the type of immunological synapse formed. By establishing a litic immunological synapse, the effector cells is activated and release enzymes to destroy the target cells. The termination of the interaction between two cells is as important as the engagement in immunological synapse, because the detachment of the target cell can influence the serial killing of natural killer cells. Here, we followed the behavior of NK-92 cells in interaction with 3 different types of tumoral cell lines. The diferences observed in the interaction of natural killer cells with tumoral cells gives insight into the factors invovled in citotoxicity. [ABSTRACT FROM AUTHOR]

This paper investigates the intricate interplay of fetal human leukocyte antigen (HLA) and maternal killer cell immunoglobulin-like receptor (KIR) interactions, focusing on their implications for pregnancy outcomes. The study explores the impact of HLA mismatches, particularly in the context of the HY immunity system, on the maternal immune response. The presence of HLA antibodies is assessed, with consideration given to the correlation with total immunoglobulin levels, aiming to discern associations with potential pregnancy complications. Complement activity is examined, adding a layer of understanding to the dynamics of fetal HLA and maternal KIR interactions. Furthermore, the paper delves into the delicate balance between Th1 and Th2 helper T cells, crucial components of the immune system, and their potential influence on maternal-fetal immune tolerance. Natural Killer Cell Cytotoxic Activity (NKa) is investigated, providing insights into the cytotoxicity of these immune cells and their role in the context of maternal-fetal interactions. The findings presented contribute to a nuanced comprehension of the immunological factors shaping pregnancy outcomes and may inform future research and clinical approaches aimed at optimizing maternal and fetal health. [ABSTRACT FROM AUTHOR]

Na+/K+ ATPase is a protein involved in the active transport of ions across the cellular membrane. Ouabain is a cardiotonic glycoside that, by inhibiting the Na+/K+ pump, interferes with cell processes mediated directly by the pump, but also indirectly influences other cellular processes such as cell cycle and proliferation, growth, cell differentiation, angiogenesis, migration, adhesion, and invasion. We used the SK-BR-3 breast cancer cell line, mesenchymal stem cells (MSCs), and tumor-associated fibroblasts (TAFs) in vitro to determine the effects of ouabain exposure on these cellular types. The results showed a multi-level effect of ouabain mainly on tumor cells, in a dose-dependent manner, while the TAFs and their normal counterparts were not significantly influenced. Following exposure to ouabain, the SK-BR-3 cells changed their morphologic appearance, decreased the expression of immunophenotypic markers (CD29, Her2, VEGF), the proliferation rate was significantly decreased (Ki67 index), the cells were blocked in the G0 phase of the cell cycle and suffered necrosis. These data were correlated with the variable expression of α and β Na+/K+ pump subunits in tumor cells, resulting in decreased ability to adhere to the VCAM-1 substrate in functional flow chamber studies. Being indicative of the pro-apoptotic and inhibitory effect of ouabain on tumor invasion and metastasis, the results support the addition of ouabain to the oncological therapeutic arsenal, trailing the “repurposing drugs” approach.

Pectus excavatum (PE) is the most frequent anterior chest deformity occurring in approximately 1 in 1000 live births. Despite the excellent achievements in the surgical treatment of the disease the etiology of PE is yet to be clarified. Nowadays the most accepted etiopathogenic theory is that the posterior displacement of the sternum is caused by the overgrowth of the disturbed costal cartilages. Insulin Growth Factor 1 (IGF1) is the main anabolic growth factor for human hyaline cartilages and plays a key role in cartilage homeostasis, promoting proteoglycans synthesis and cell division. In this article we present the results of our study regarding the involvement of IGF1 in the etiology and pathogenesis of PE in children. Samples of the deformed costal cartilages from 25 children with PE were obtained during the surgical intervention for the correction of the disease. Cartilage samples were obtained during autopsy from 17 children in whom the cause of death was unlikely to affect the cartilage. The samples were immunohistochemically stained with fluorescent antibody for phosphorylated IGF1 receptor (IGF1R). The majority of the samples were negative for phosphorylated IGF1R and there were no significant differences between the two groups (p> 0.05). In both PE patients and in general populations, the majority of the costal chondrocytes doesn't reveal activated IGF1R. PE in children is not produced by a defect of the IGF1 axis. [ABSTRACT FROM AUTHOR]

In vivo conditions, the innate immune arm signals shape the activation of naive T cells, however, it is unclear how T cells maintain their function in vitro, in experimental settings, where different culture conditions signals influence the effector T-cell function. In this study of two sets of healthy donors designated here group A (n=10) and group B (n=10), we assessed the cytokine profile of PBMCs, cluster of differentiation 3 (CD3+) cells and cluster of differentiation 8 (CD8+) T cells, which were cultured in different environmental conditions, including Interleukin-2 (IL-2) and/or CD3/CD28 T cell activator, supplemented culture media. The (IL-2), Interleukin-5 (IL-5) and Interferon- γ (IFN-γ) secretion at 24, 48, 72, and 96 hours was determined in a 4.5-hour solid-phase ELISA immunoassay method. The medium containing IL-2 and CD3/CD28 T cell activator was the one that induced increased secretion of IL-2, IL-5, and IFN-γ in all three cell populations, mainly in the CD3+ cells, irrespective of the time frame sampled. [ABSTRACT FROM AUTHOR]

Worldwide, cancers are one of the main leading causes of death and the conventional treatment has its own limits. Researches from immunotherapy field are focusing on discovering feasible altemative therapeutic approaches for treating this disorder. In cancers, immunotherapy has the role to enhance anti-tumor effect of the immune cells involved in the process. Depending on the type of targeted antigens, side effects of this therapy may vary from local and mild to systemic and more severe reactions. The principle of CART cells and Tlls immunotherapies is based on using T cells that are specific for a certain antigen and re-infusing back to the patient after those cells were expended ex vivo, in order to cause tumor destruction. Association of these therapies with a lymphodepleting regime and T cell growth factor IL-2 leads to a potent effect on patients. Within this article, we make an overview of the CART cell therapy and Tlls therapy, by describing the principles of this immunotherapies, their clinical applications, as well as their advantages, disadvantages and future perspectives. [ABSTRACT FROM AUTHOR]