Small extracellular vesicles - A host for advanced bioengineering and "Trojan Horse" of non-coding RNAs.

Small extracellular vesicles (sEVs) are a type of membranous vesicles that can be released by cells into the extracellular space. The relationship between sEVs and non-coding RNAs (ncRNAs) is highly intricate and interdependent. This symbiotic relationship plays a pivotal role in facilitating intercellular communication and holds profound implications for a myriad of biological processes. The concept of sEVs and their ncRNA cargo as a "Trojan Horse" highlights their remarkable capacity to traverse biological barriers and surreptitiously deliver their cargo to target cells, evading detection by the host-immune system. Accumulating evidence suggests that sEVs may be harnessed as carriers to ferry therapeutic ncRNAs capable of selectively silencing disease-driving genes, particularly in conditions such as cancer. This approach presents several advantages over conventional drug delivery methods, opening up new possibilities for targeted therapy and improved treatment outcomes. However, the utilization of sEVs and ncRNAs as therapeutic agents raises valid concerns regarding the possibility of unforeseen consequences and unintended impacts that may emerge from their application. It is important to consider the fundamental attributes of sEVs and ncRNAs, including by an in-depth analysis of the practical and clinical potentials of exosomes, serving as a representative model for sEVs encapsulating ncRNAs.

The Role of Exosomes in Human Carcinogenesis and Cancer Therapy-Recent Findings from Molecular and Clinical Research.

Exosomes are biological nanoscale spherical lipid bilayer vesicles, 40-160 nm in diameter, produced by most mammalian cells in both physiological and pathological conditions. Exosomes are formed via the endosomal sorting complex required for transport (ESCRT). The primary function of exosomes is mediating cell-to-cell communication. In terms of cancer, exosomes play important roles as mediators of intercellular communication, leading to tumor progression. Moreover, they can serve as biomarkers for cancer detection and progression. Therefore, their utilization in cancer therapies has been suggested, either as drug delivery carriers or as a diagnostic tool. However, exosomes were also reported to be involved in cancer drug resistance via transferring information of drug resistance to sensitive cells. It is important to consider the current knowledge regarding the role of exosomes in cancer, drug resistance, cancer therapies, and their clinical application in cancer therapies.

Individual and Combined Treatments with Methylated Resveratrol Analogue DMU-214 and Gefitinib Inhibit Tongue Cancer Cells Growth via Apoptosis Induction and EGFR Inhibition.

The methylated resveratrol analogue 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214) has been revealed to exert the anti-cancer activity by a block of the cell cycle at the G2/M phase, apoptosis induction, and metastasis inhibition. These biological events may be involved in crosstalk with the epidermal growth factor receptor (EGFR), which belongs to the ErbB family of receptor tyrosine kinases. Several cancer therapeutic approaches employ small molecules capable of inhibiting tyrosine kinases (e.g., gefitinib). According to more recent reports, combining gefitinib with chemotherapeutics, such as cisplatin, seems to be more effective than monotherapy. The present study aimed to assess the molecular mechanism of the potential anti-proliferative activity of individual and combined treatments with DMU-214 and gefitinib in SCC-25 and CAL-27 human tongue cancer cell lines. We showed for the first time the anti-cancer effects of DMU-214, gefitinib, and their combination in tongue cancer cells triggered via cell cycle arrest, apoptosis induction, and inhibition of the EGFR signaling pathway. The anti-proliferative effects of DMU-214 and gefitinib are also suggested to be related to the EGFR and EGFRP (phosphorylated epidermal growth factor receptor) expression status since we found significantly weaker cytotoxic activity of the compounds tested in SCC-25 cells, which overexpressed EGFR and EGFRP proteins.

A Clinical Trial of Photobiomodulation Effect on Orthodontic Microscrews Stability Using a 635 nm Red Laser Light.

Background: One of the procedures enhancing implants stability can be photobiomodulation. Objective: To assess the effect of a 635 nm wavelength on orthodontic microscrews stability, survival rate, and an individual patient's pain score. Materials and methods: The study was done with 15 subjects, 30 orthodontic microscrews with a length of 10 mm and diameter 1.4 mm. Implants inserted on the right and left side of the maxilla. The implants in the test group (G1) were irradiated with the laser at palatal and buccal part of peri-implant area (two points). The diode laser (SmartM PRO; Lasotronix, Warsaw, Poland) was used with the following settings: power: 100 mW, spot size: 0.5 cm2, mean power density (irradiance): 200 mW/cm2, in continuous wave (CW) mode, energy per point: 4 J, radiant exposure: 8 J/cm2, time per point: 40 sec, the total energy dose (radiant energy) per session: 8 J in contact mode, handpiece diameter: 8 mm. Laser sessions: day of surgery and 3, 6, 9, 12, 15, 30 days later. Cumulative radiant energy was 59 J. Periotest appliance was used to assess the microscrews stability at day of surgery and 3, 6, 9, 12, 15, 30, 60 days later. Each patient received a survey for individual pain assessment (NRS-11) promptly after the implantation. Results: The analysis after 60 days revealed significantly higher secondary stability for the laser-irradiated microscrews group in comparison with control implants (G2 group) (p = 0.0037). We observed significantly higher stability in the experimental group compared with the control after 30 days (p = 0.0218). Moreover, we noted significantly higher microimplants stability for the control group in comparison with the test group, 9 days after implantation (p = 0.0374). Laser application had no effect on pain reduction noted in the first day in comparison with the control sites (p = 0.6690). No microscrews were lost in the study. Conclusions: Application of a 635 nm laser on peri-implant soft tissue increased the microscrews stability after 30 and 60 days.

Stemness Potency of Human Gingival Cells-Application in Anticancer Therapies and Clinical Trials.

Gingivae, as the part of periodontium, are involved in tooth support and possess the ability to heal rapidly, without scar formation. Recently, dental tissues have been identified as a potential source of mesenchymal stem cells (MSCs) and several populations of MSCs were isolated from the orofacial region, including gingival mesenchymal stem cells (GMSCs). GMSCs exhibit robust immunomodulatory and differentiation potential and are easily obtainable, which make them promising candidates for cellular therapies. Apart from being tested for application in immunologic- and inflammatory-related disorders and various tissue regeneration, GMSCs promise to be a valuable tool in cancer treatment, especially in tongue squamous cell carcinoma (TSCC) with the use of targeted therapy, since GMSCs are able to selectively migrate towards the cancerous cells both in vitro and in vivo. In addition to their ability to uptake and release anti-neoplastic drugs, GMSCs may be transduced with apoptosis-inducing factors and used for cancer growth inhibition. Moreover, GMSCs, as most mammalian cells, secrete exosomes, which are a subset of extracellular vesicles with a diameter of 40-160 nm, containing DNA, RNA, lipids, metabolites, and proteins. Such GMSCs-derived exosomes may be useful therapeutic tool in cell-free therapy, as well as their culture medium. GMSCs exhibit molecular and stem-cell properties that make them well suited in preclinical and clinical studies.

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials.

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

Photobiomodulation-Underlying Mechanism and Clinical Applications.

The purpose of this study is to explore the possibilities for the application of laser therapy in medicine and dentistry by analyzing lasers' underlying mechanism of action on different cells, with a special focus on stem cells and mechanisms of repair. The interest in the application of laser therapy in medicine and dentistry has remarkably increased in the last decade. There are different types of lasers available and their usage is well defined by different parameters, such as: wavelength, energy density, power output, and duration of radiation. Laser irradiation can induce a photobiomodulatory (PBM) effect on cells and tissues, contributing to a directed modulation of cell behaviors, enhancing the processes of tissue repair. Photobiomodulation (PBM), also known as low-level laser therapy (LLLT), can induce cell proliferation and enhance stem cell differentiation. Laser therapy is a non-invasive method that contributes to pain relief and reduces inflammation, parallel to the enhanced healing and tissue repair processes. The application of these properties was employed and observed in the treatment of various diseases and conditions, such as diabetes, brain injury, spinal cord damage, dermatological conditions, oral irritation, and in different areas of dentistry.

Epigenetic Research in Stem Cell Bioengineering-Anti-Cancer Therapy, Regenerative and Reconstructive Medicine in Human Clinical Trials.

The epigenome denotes all the information related to gene expression that is not contained in the DNA sequence but rather results from chemical changes to histones and DNA. Epigenetic modifications act in a cooperative way towards the regulation of gene expression, working at the transcriptional or post-transcriptional level, and play a key role in the determination of phenotypic variations in cells containing the same genotype. Epigenetic modifications are important considerations in relation to anti-cancer therapy and regenerative/reconstructive medicine. Moreover, a range of clinical trials have been performed, exploiting the potential of epigenetics in stem cell engineering towards application in disease treatments and diagnostics. Epigenetic studies will most likely be the basis of future cancer therapies, as epigenetic modifications play major roles in tumour formation, malignancy and metastasis. In fact, a large number of currently designed or tested clinical approaches, based on compounds regulating epigenetic pathways in various types of tumours, employ these mechanisms in stem cell bioengineering.

Inclusion Biogenesis, Methods of Isolation and Clinical Application of Human Cellular Exosomes.

Exosomes are a heterogenous subpopulation of extracellular vesicles 30-150 nm in range and of endosome-derived origin. We explored the exosome formation through different systems, including the endosomal sorting complex required for transport (ESCRT) and ESCRT-independent system, looking at the mechanisms of release. Different isolation techniques and specificities of exosomes from different tissues and cells are also discussed. Despite more than 30 years of research that followed their definition and indicated their important role in cellular physiology, the exosome biology is still in its infancy with rapidly growing interest. The reasons for the rapid increase in interest with respect to exosome biology is because they provide means of intercellular communication and transmission of macromolecules between cells, with a potential role in the development of diseases. Moreover, they have been investigated as prognostic biomarkers, with a potential for further development as diagnostic tools for neurodegenerative diseases and cancer. The interest grows further with the fact that exosomes were reported as useful vectors for drugs.

Human Umbilical Vein Endothelial Cells (HUVECs) Co-Culture with Osteogenic Cells: From Molecular Communication to Engineering Prevascularised Bone Grafts.

The repair of bone defects caused by trauma, infection or tumor resection is a major clinical orthopedic challenge. The application of bone grafts in orthopedic procedures is associated with a problem of inadequate vascularization in the initial phase after implantation. Meanwhile, the survival of cells within the implanted graft and its integration with the host tissue is strongly dependent on nutrient and gaseous exchange, as well as waste product removal, which are effectuated by blood microcirculation. In the bone tissue, the vasculature also delivers the calcium and phosphate indispensable for the mineralization process. The critical role of vascularization for bone healing and function, led the researchers to the idea of generating a capillary-like network within the bone graft in vitro, which could allow increasing the cell survival and graft integration with a host tissue. New strategies for engineering pre-vascularized bone grafts, that apply the co-culture of endothelial and bone-forming cells, have recently gained interest. However, engineering of metabolically active graft, containing two types of cells requires deep understanding of the underlying mechanisms of interaction between these cells. The present review focuses on the best-characterized endothelial cells-human umbilical vein endothelial cells (HUVECs)-attempting to estimate whether the co-culture approach, using these cells, could bring us closer to development and possible clinical application of prevascularized bone grafts.

New Gene Markers for Metabolic Processes and Homeostasis in Porcine Buccal Pouch Mucosa during Cells Long Term-Cultivation-A Primary Culture Approach.

The oral mucosal tissue is a compound structure composed of morphologically and physiologically different cell types. The morphological modification involves genetically determined lifespan, which may be recognized as the balance between cell survival and apoptosis. Although the biochemical processes and pathways in oral mucosa, with special regards to drug transport, delivery, and metabolism, are well known, the cellular physiological homeostasis in this tissue requires further investigation. The porcine buccal pouch mucosal cells (BPMCs) collected from 20 pubertal crossbred Landrace gilts, were used in this study. Immediately after recovery, the oral mucosa was separated micro-surgically, and treated enzymatically. The dispersed cells were transferred into primary in vitro culture systems for a long-term cultivation of 30 days. After each step of in vitro culture (IVC), the cells were collected for isolation of total RNA at 24 h, 7, 15, and 30 days of IVC. While the expression was analyzed for days 7, 15, and 30, the 24th hour was used as a reference for outcome calibration. The gene expression profile was determined using Affymetrix microarray assays and necessary procedures. In results, we observed significant up-regulation of SCARB1, PTGS2, DUSP5, ITGB3, PLK2, CCL2, TGFB1, CCL8, RFC4, LYN, ETS1, REL, LIF, SPP1, and FGER1G genes, belonging to two ontological groups, namely "positive regulation of metabolic process", and "regulation of homeostatic process" at 7 day of IVC as compared to down-regulation at days 15 and 30. These findings suggest that the metabolic processes and homeostatic regulations are much more intense in porcine mucosal cells at day 7 of IVC. Moreover, the increased expression of marker genes, for both of these ontological groups, may suggest the existence of not only "morphological lifespan" during tissue keratinization, but also "physiological checkpoint" dedicated to metabolic processes in oral mucosa. This knowledge may be useful for preclinical experiments with drugs delivery and metabolism in both animals and humans.

Influence of Estradiol-17beta on Progesterone and Estrogen Receptor mRNA Expression in Porcine Follicular Granulosa Cells during Short-Term, In Vitro Real-Time Cell Proliferation.

Progesterone (P4) and estradiol (E2) play a significant role in mammalian reproduction. Our study demonstrated that separated porcine cumulus cells (CCs) and/or granulosa cells (GCs) might proliferate in vitro during short-term, real-time primary culture. The GCs were analyzed according to gene expression of the progesterone receptor (nuclear form) (pgr), progesterone receptor membrane component 1 (pgrmc1), and estrogen-related receptor beta 3 (esrrb3) in relation to two housekeeping genes: actb and pbgd. GCs were cultivated in medium with the E2. Both pgr/actb and pgr/pbgd revealed higher expression between 24 and 168 h of IVC of prolonged E2 treatment and at 48 h of IVC after acute E2 administration. The pgrmc1/actb and pgrmc1/pbgd displayed increased expression after prolonged E2 treatment between 24 and 120 h of IVC. The highest level of esrrb3/actb at 120 and 144 h, as well as esrrb3/pbgd at 120 h, in untreated controls as compared to the hormone-stimulated group, was observed. We suggest that E2 significantly influences the upregulation of pgr, pgrmc1, and esrrb3 expression in porcine GCs during real-time cell proliferation. Since esrrb3 expression is stimulated by E2 in both an acute and prolonged manner, estradiol may be recognized as a potential estrogen receptor agonist in GCs.

[Smoking addiction and stress in dental practice]. / Nalóg palenia tytoniu i stres w praktyce zawodowej lekarzy dentystów.

UNLABELLED: Dentists in their practice are constantly exposed to psychic overloads, and yet they are rarely investigated as a professional group in the context of occupational risk. The aim of this epidemiological study was to analyze the frequency of smoking habit among dentists and to evaluate stress inducing factors which are affecting dentists in their everyday practice. MATERIALS AND METHODS: The questionnaire consisting of 12 closed questions arranged in Lickert scale were used in this cross sectional study. First part of the survey involved basic anthroposocial data (age, gender, weight), second ergonomic part referred to psychological overload involved in dental practice and stress factors outside their job. The question concerning smoking was a part of second section. 150 questionnaires were filled in by female dentists coming from western regions of Poland. Most of participants in the study were older than 40 years, only 10% represented younger than 30 years old generation (mean age 47.5 sd 9.1). For statistical analysis we used Kruskal Wallis, Anova and Dunn tests. Statistical significance in this study was established at the p < 0.05 level. RESULTS: The obtained results revealed that 17% of female dentists smoke cigarettes. 81% of respondents declare job related psychological overload. All of the respondents found physical overload as a main negative factor, 82% pointed out lack of good communication with patients and 65% lack of communication with coworkers as an important negative factor, 74% of respondents indicated long working hours and time pressure during procedures as a significant stress inducing factors. CONCLUSIONS: 1. The study confirmed that dentists who have long working hours consider their job as more stressful. 2. The results acknowledge the need to enhance and promote health-oriented attitudes among dentists.

[Smoking and periodontal disease of adolescents--results of an epidemiological survey]. / Palenie a choroby przyzebia dorastajacej mlodziezy--wyniki badan epidemiologicznych.

A questionnaire concerning oral health designed for young people aged between 15 and 18 and based on one produced by Tokyo University entitled Medical Outcome Study (MOS) and Oral Health Score (OHS) 8020, was submitted to a group of Polish citizens. One of the 10 questions in the OHS part of the questionnaire concerned smoking habits. 296 persons (157 girls and 139 boys) living in west Poland, took part. For a clearer evaluation of the health habits in this group, a comparison was made between the youngest (15 y old) and oldest (18 y old) groups. The Oral health Score (with a possible maximum 20 points) for the examined group was 14.4 which is one of the lowest among evaluated European countries. Smoking was most frequent among the 18 year olds (20.1%) whereas among the respondents in the younger group it was this value (16.5%). Moreover, in age 15 group, boys (29%) were more often smokers than girls (5.5%); 22% of 18 year old girls and 19.8% of boys smoked. Comparing the answers relating to oral health with the frequency of smoking revealed a connection between this habit and the presence of periodontal disease. Smoking increases a risk of developing periodontal disease. According to the results there is a need to increase an oral health education and identification of risk factors such smoking among Polish adolescents.