Gene expression regulating lipopolysaccharide and lipid metabolic processes in porcine oral mucosal cells in long-term primary in vitro culture.

Lipids are an alternative energy source for cells and provide structural integrity in cell membrane and their metabolism is regulated with the use of different pathways, such as integrin signalling, oxidative stress, mechanical stress, and pH changes. All of those processes take place in the oral mucosa which is subject to different environmental impacts. In this study, porcine buccal pouch mucosal cells (pBPMCs) were used during long-term primary in vitro culture. The cultured cells were collected at 7, 15 and 30 days of IVC and subsequently transferred to RNA isolation. In the results of the following microarray analysis, we analyzed the genes detected, belonging to ontology groups, such as "cellular lipid metabolic process", "response to lipid" and "response to lipopolysaccharides. All of the genes involved in these ontological groups were expressed at higher levels at 7 days of IVC and substantially decreased in expression at days 15 and 30 of primary culture. We observed new genes, which may be recognized as markers in regulation of lipid metabolism in mucosal cells in vitro. The results suggested that the biochemical mechanism-involved lipids were accompanied by increased enzymatic activation and synthesis of crucial growth factors reaching high activity at day 7 of culture, which is also well documented as a stage of tissue regeneration period within oral mucosa. Therefore, this "biochemical fingerprint" may be an additional checkpoint of the integrity, resistance and easy adaptability of oral tissues, which are important conditions of success in tissue engineering and grafting for tissue reconstruction.

Overview of the different methods used in the primary culture of oral mucosa cells.

The culture of primary cells in vitro has enabled to gain knowledge in the field of cell biology, disease mechanisms and to offer great potential in drug testing. To date, two main techniques of isolating and culturing oral mucosal cells, the direct explant method and the enzymatic method, dominate the literature and practice. In the present study, both techniques are discussed in detail, comparing the advantages and disadvantages of the two approaches in setting up a primary culture of oral mucosal cell. The direct explant technique is well-established and has been commonly used for the past 20-30 years. Although the method of setting up the cultures did not show much variations in the methodology described by authors, the culturing conditions varied according to the aims of the projects.

The p75 neurotrophin receptor in cells of oral mucosal epithelium.

The p75 neurotrophin receptor (p75NTR) can play different roles in cells. This protein can on the one hand act in the regulation of cell growth and survival, while being an apoptosis inducing factor in different contexts. p75NTR regulates cell cycle not only in nerve cells but also in epithelial oral mucosal cells. In the former, neurotrophin-p75NTR signaling affects cell growth and survival. Recent studies showed that p75NTR is expressed in basal cells of oral mucosal epithelium and can be used as one of the markers of epithelial stem/progenitor cells. This role of p75NTR can be utilised in aspects such as tissue engineering and gene therapy. One of the examples of clinical use of cultivated oral mucosal cells is ocular surface reconstruction. p75NTR can be a significant marker of stem cells in studies of epithelial tissues, especially when the cells will exhibit other specific markers, such as CK13, CK14 and PCNA..

Genes involved in regulation of cellular metabolic processes, signaling and adhesion are the markers of porcine buccal pouch mucosal cells long-term primary cultured in vitro.

Extraordinary abilities for continuous proliferation and differentiation, associated with constant renewal triggered by stimulation from the mastication process, together with the relative lack of aesthetic complications associated with post-surgery healing, have highlighted buccal pouch mucosa as a potential source of explants that could be used in transplantation and tissue engineering. Additionally, this tissue plays a major role in the oral drug delivery process, which brings special interest to its molecular properties in the context of new drug development. There is therefore a need to analyse the exact mechanisms of oral mucosa functioning, especially when it comes to the processes that are associated with the potential clinical applications. In this study we analysed a complete transcriptome of long-term in vitro cultures of porcine buccal pouch oral mucosa cells. Using a microarray approach, we focused on genes associated with cellular metabolic processes, signalling and adhesion, from 4 gene ontology groups: "Positive regulation of cellular component movement", "Positive regulation of cellular process", "Positive regulation of intracellular signal transduction" and "Single organism cell adhesion". Nineteen genes (CCL8, CXCL2, PLK2, DUSP5, PTGS2, LIF, CCL2, ATP1B1, REL, ITGB3, SCARB1, UGCG, PDPN, LYN, ETS1, FCER1G, TGFB1, RFC4, LMO2) with fold changes higher than |2| and p value Extraordinary abilities for continuous proliferation and differentiation, associated with constant renewal triggered by stimulation from the mastication process, together with the relative lack of aesthetic complications associated with post-surgery healing, have highlighted buccal pouch mucosa as a potential source of explants that could be used in transplantation and tissue engineering. Additionally, this tissue plays a major role in the oral drug delivery process, which brings special interest to its molecular properties in the context of new drug development. There is therefore a need to analyse the exact mechanisms of oral mucosa functioning, especially when it comes to the processes that are associated with the potential clinical applications. In this study we analysed a complete transcriptome of long-term in vitro cultures of porcine buccal pouch oral mucosa cells. Using a microarray approach, we focused on genes associated with cellular metabolic processes, signalling and adhesion, from 4 gene ontology groups: "Positive regulation of cellular component movement", "Positive regulation of cellular process", "Positive regulation of intracellular signal transduction" and "Single organism cell adhesion". Nineteen genes (CCL8, CXCL2, PLK2, DUSP5, PTGS2, LIF, CCL2, ATP1B1, REL, ITGB3, SCARB1, UGCG, PDPN, LYN, ETS1, FCER1G, TGFB1, RFC4, LMO2) with fold changes higher than |2| and p value less than 0.05 were identified, described in context and analysed. While the study needs much further validation to become applicable in a clinical environment, it yields valuable information about the transcriptomic basis of oral mucosal cell functioning in vitro, that might serve as a reference for further research, aiming to apply this knowledge in clinical situations.0.05 were identified, described in context and analysed. While the study needs much further validation to become applicable in a clinical environment, it yields valuable information about the transcriptomic basis of oral mucosal cell functioning in vitro, that might serve as a reference for further research, aiming to apply this knowledge in clinical situations.

Gender and age-related variability of macrophage representation in the internal thoracic artery wall: does it matter?

Some recent reports suggested that elderly and female patients did not benefit from implantation of the second internal thoracic artery (ITA) during coronary artery bypass surgery (CABG). Macrophages, among other cells, were described to be involved in both atherosclerosis and aortocoronary grafts failure. The aim of the study was to examine the age and gender association with different distribution of CD68+ cells within the layers of ITA wall. This study involved 158 consecutive patients (95 male and 63 female), with the mean age of 64.5±9.5 years, who underwent elective CABG procedures. During surgery, the surplus distal segments of ITA were harvested for immunohistochemical analysis. The number and distribution of CD68+ cells was calculated and plotted against the age and gender of the study participants. CD68+ cells were present in all of the harvested ITA fragments (median 44), more in women (55) than in men (42) (p less than 0.001). However, this difference was of statistical significance exclusively in the tunica intima. Approximately 70% of macrophages were found in the tunica adventitia. The total number of CD68+ cells the in arterial wall as well as in the tunica intima and adventitia correlated positively with the age of patients (r=0.544, r=501 and r=0.462, respectively). The lack of significant advantages of the use of two thoracic arteries, in elderly patients and women, might have resulted from the larger population of CD68+ cells in their walls, especially the tunica intima. However, this result from immunohistochemical analysis needs validation in long-term clinical research on a larger cohort of patients.

Haematopoiesis: “living in the shadow” of stem cell differentiation.

Haematopoiesis is one of the most well understood stem-cell associated processes. It is a process in which pluripotent hematopoietic stem cells (HSCs) self-proliferate and differentiate into all types of blood cells. The process takes place in marrow of the flat bones in adults, however its location changes several times through embryonic and foetal development. Given the broad range of blood cells and the major differences in their build and function, together with the fact that their numbers need to be maintained within relatively narrow margins in order to maintain homeostasis despite changing environmental conditions, makes the whole process of haematopoiesis highly regulated and depending on a variety of growth factors. When influenced by those, HSCs undergo several irreversible steps, with every next one committing them to an even more specialised fate, ending with all the specific types of mostly short-lived blood cells, that are unable to proliferate on their own and need constant replenishment from the HSC pool. Because the process of haematopoiesis is the only source of all the members of the group of cells performing a range of highly important roles in functioning of the organism, significant damage to the underlying stem cells can cause a range of severe diseases. Many treatments are suggested for managing their symptoms or slowing progress, with bone marrow transplant being one of the only ones that offer possible permanent solution and, despite being a relatively risky procedure, is being widely performed, with the methods constantly improving in order to achieve progressively better results in both treatability and survivability of the patients.

Expression of genes responsible for cell morphogenesis involved in differentiation in porcine buccal pouch mucosal cells during long-term primary culture and real-time proliferation in vitro.

Recently, using experimental animal model, we demonstrated that porcine buccal pouch mucosal cells reflect increased proliferation capability during primary cultivation in vitro. Although the histological structure and morphogenesis in oral cavity is well recognized, the molecular mechanisms which regulate this process still need further investigation. This study was aimed to analyze the molecular marker expression profile involved in morphogenesis and differentiation capacity of porcine buccal pouch mucosal cells during their long-term primary cultivation in vitro. The experiment was performed on buccal pouch mucosal cells isolated from 80 pubertal crossbred Landrace gilts. After collection, the cells were treated enzymatically and transferred into a primary in vitro culture (IVC) system and cultured for 30 days. The cells were collected for RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using the RTCA system. We found an increased expression of FN1 and SOX9 genes when calculated against ACTB after 7, and 30 days of IVC, (P less than 0.01, P less than 0.001, respectively). The CXCL12 mRNA was down-regulated after 7, 15 and 30 days of IVC, but not statistically significant. Similar expression profile was observed when calculated against HPRT, however, DAB2 was found to be higher expressed at day 15 of IVC, (P less than 0.05). The cell index measured during real-time cell proliferation was substantially increased between 96 h and 147h of IVC and reached the log phase. Since FN1 and SOX9 revealed significant increase of expression after long-term culture in vitro, it is suggested that expression of these differentiation and stemness genes is accompanied by cell proliferation. Moreover, FN1 and SOX9 might be recognized as new markers of buccal pouch mucosal cell proliferation and differentiation in pigs in in vitro primary culture model.

Expression of connexins in porcine buccal pouch mucosa cells during real-time long-term cell proliferation in vitro: a primary culture approach.

In this study we investigated the expression of connexins Cx36, Cx37, Cx40, Cx43, and Cx45 mRNAs during real-time cellular proliferation in vitro. The oral mucosa cells were isolated from 80 pubertal crossbred Landrace gilts. The cells were transferred into primary in vitro culture (IVC) and cultured for 30 days. The cells were collected to RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using real-time cell analysis (RTCA). We found an increased expression of Cx43 mRNA after 30 days of IVC as compared to control (P<0.05). The expression level of Cx36 was significantly decreased after 30 days. The expression of Cx37, Cx40 and Cx45 mRNAs was not changed. The expression of Cx43 was statistically increased when compared to Cx40, Cx37, Cx45 and Cx36 (P<0.001, for all time periods, respectively). We confirmed the expression of selected connexins in porcine buccal mucosa cells during their long-term primary IVC, which suggests the existence of functional gap junction connections (GJCs) communication network between these cells. We also confirmed the observations of other authors that Cx43 plays a substantial role in GJC structure. However, the increased expression of Cx43 in buccal mucosa cells, accompanied with their proliferation during real-time primary culture, is presented, to our knowledge, for the first time.

Expression of cell mitotic progression proteins and keratinocyte markers in porcine buccal pouch mucosal cells during short-term, real-time primary culture.

The porcine model is often used in clinical trials. The pig has many fundamental anatomic, physiological and nutritional similarities to humans. Additionally, the European Medicines Agency (EMA) demands the use large animals in clinical studies. Oral mucosa has received special attention due to its regenerative properties. Oral tissue is composed of several types of cells including fibroblasts and keratinocytes. The porcine oral mucosa/buccal pouch mucosa has a cellular structure with defined proliferation and differentiated capability. In this study, we investigated the expression pattern of porcine buccal pouch mucosal cell proliferation and differentiation markers such as Ki-67, proliferating cell nuclear antigen (PCNA), and involucrin. We observed a clear monolayer culture of spindle-shaped, porcine buccal pouch mucosal cells during 168 h of real-time in vitro culture. The RTCA assays revealed parametric and progressive increases in proliferation after 72 h of IVC. We found an altered proliferation index (PI) in the replicated groups of experiments except through the 144-168 h proliferation period. The RT-qPCR results demonstrated a significant increase in Ki-67 and PCNA expression after 48, 120, and 168 h of IVC as compared to other culture periods (P<0.001). The involucrin mRNA displayed increased expression after 168 h of IVC as compared to other periods. We observed a lack of PCR product at 24 h in the case of Ki-67 and both before IVC (0h) and after 24 h of IVC for PCNA mRNA. When we analyzed the three transcripts together, we found the highest expression of involucrin during each of the culture periods. It has been suggested that Ki-67, PCNA, and involucrin may be successfully used as markers of porcine buccal pouch mucosal cell proliferation and differentiation capability in vitro.

The origin, in vitro differentiation, and stemness specificity of progenitor cells.

Since the successful collection of the first progenitor stem cells (SCs), there has been an increased interest in these cells as a model for undiscovered and unlimited potential of differentiation and development. Additionally, it was shown that SC populations display an ability to form pluripotent and/or totipotent cell populations. It was found that human ovarian granulosa cells (GCs) maintain a large capacity for differentiation into several other cell lineages, such as chondrogenic, osteogenic, neurogenic, and adipogenic, particularly during long-term, in vitro culture. In these cases, the specific media supplements that promote various pathways of differentiation, such as leukemia-inhibiting factor (LIF) and/or FSH, are well recognized. However, these are only some examples of the differentiation possibilities of human SCs in vitro and other pathways still require further investigation. Many SC populations, which are directed to differentiate into specific cell types, are also successfully used in several human disease therapies, e.g. leukemia. Moreover, SCs are used for tissue scaffold construction in patients with respiratory and cardiovascular diseases. In this review, the most recent knowledge about the in vitro growth and differentiation capacity of SCs is presented. Furthermore, we discuss the possible worldwide application of SCs in advanced cell and tissue bioengineering. In conclusion, it is suggested that, in the future, SCs will be a basic strategy in human therapy, and their use will open new gates in regenerative and reconstructive medicine in the 21st century.

The biomedical aspects of oral mucosal epithelial cell culture in mammals.

In recent years, there has been a growing interest in epithelial cell tissue culture, particularly oral mucosa and its application utilizing in vitro cell culture in medicine. This involves tests using animal models to better understand oral mucosa function, and the differences in its construction in various animal models. The use of buccal pouch mucosal cell culture provides insight into the processes of trans mucosal transport and regeneration of the oral epithelium. The processes associated with epithelium regeneration is the base for stem cell research and/or oral cancer investigation. These artificially cultured tissue equivalents are used in transplant surgery for the treatment of a variety of tissue dysfunctions, i.e. eye, esophagus, or urethra. In this review, the most recent results from studies carried out on in animal models, which may be applied in areas such as regenerative medicine and reconstructive surgery, were explored.

Differential expression and distribution of cytokeratins and vimentin in buccal pouch mucosal cells during real-time cell proliferation: research based on a porcine model.

In recent years, buccal pouch oral mucosa cells were used as a source of potential biological grafting material in advanced tissue engineering. However, there are several limitations in the process of graft fabrication: donor and recipient patient availability as well as an incomplete knowledge of in vitro procedures related to tissue surgical recovery, in vitro cell culture (IVC) and/or tissue processing in "human somatic cell therapy." Therefore, the animal model for oral mucosa grafting is still recognized as a source for xenografts and a useful model for biomedical research. In this study, the porcine buccal pouch oral mucosa cells were used in analysis of the stromalization/epithelialization process during short-term, in vitro real-time cell proliferation. We evaluated cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression as epithelial and stromal cell markers, respectively. The porcine buccal pouch oral mucosa cells were cultured in vitro for 168 h, and the protein expression/ distribution was analyzed every 24 h during real-time cell proliferation. In our analysis of protein expression using fluorescence intensity (FI), followed by confocal microscopic observations, we found the highest expression of CK18 occurred after 24 h of IVC, panCK after 72 h, and Vim after 48 h of IVC, as compared to other cultivation periods. We also found a substantial increase in Vim expression (3-4 fold) as compared to CK18 and panCK, and all of the investigated proteins were distributed in the cellular cytoplasm. The lag phase of cell proliferation occurred during the first 24 h of IVC, whereas the log phase was observed between 24 h-120 h of IVC. Throughout 7 days of IVC, statistically significant differences were found in Cell Index (CI) of the analyzed cells. Increased Vim expression in buccal pouch oral mucosa cells, as compared to CK18 and panCK, suggested that the stromal cells substantially predominated during in vitro cell cultivation. This may be a result of significant specificity of porcine oral mucosa cells isolated from the buccal pouch.

Association between expression of cumulus expansion markers and real-time proliferation of porcine follicular granulosa cells in a primary cell culture model.

Folliculogenesis is a compound process that involves both ovarian follicle growth and oocyte development, which is tightly attached to the follicular wall. During this process, cells that form the follicle structure undergo substantial morphological and molecular modifications that finally lead to differentiation and specialization of ovarian follicular cells. The differentiation of ovarian cells encompasses formation of follicle, which is composed of theca (TCs), mural granulosa (GCs), and cumulus cells (CCs). It was previously hypothesized that GCs and CCs represent undifferentiated and highly specialized follicular cells, respectively, which may have similar primordial cell origins. In this study, we investigated the expression pattern of cumulus expansion markers such as COX2, HAS2, PTX3, and TSG6 in porcine GCs during short-term, in vitro culture. We hypothesized that these genes may display an important function in GCs in relation to cellular real-time proliferation. The expression pattern of COX2, HAS2, PTX3, and TSG6 was evaluated after using RT-qPCR in relation to confocal microscopy observations of protein expression and distribution during real-time proliferation of porcine follicular GCs. The COX2 and HAS2 mRNAs were highly expressed after 120 h of in vitro culture (IVC), whereas PTX3 and TSG6 mRNAs were increased during the first 24-48 h of IVC (P less than 0.001, P less than 0.01). Conversely, all of the encoded proteins were highly expressed after 144-168 h of IVC as compared to other culture periods (P less than 0.001, P less than 0.01). When analyzing the realtime proliferation of GCs in vitro, we observed a logarithmic increase of cell proliferation between 0 h and 120 h of IVC. However, after 120-168 h of IVC, the cells reached the lag phase of proliferation. Since it is well accepted that porcine GCs undergo luteinization shortly after 24-48 h of IVC, the expression pattern of investigated genes indicated that Cox2 and Has2 are independent from the LH surge, but their increased levels may be upregulated by cell proliferation in vitro. Moreover, higher expression of PTX3 and TSG6 during first 24 h and/or 48 h of IVC suggested that their levels are accompanied by porcine GCs luteinization process.

The influence of amalgam fillings on the detection of approximal caries by cone beam CT: in vitro study.

OBJECTIVES: The aim of this CBCT investigation on the detection of caries was to assess the influence of artefacts produced by the presence of amalgam fillings located in the vicinity. METHODS: 102 non-cavitated pre-molar and molar teeth were placed in blocks of silicone with approximal contacts consisting of 3 sound or carious teeth and 1 mesial-occlusal-distal amalgam-filled tooth in-between. Radiographs of all the teeth were recorded using the CBCT system (NewTom™ 3G; QR Srl, Verona, Italy; field of view, 9 inches). Data from the CBCT unit were reconstructed and sectioned in the mesiodistal tooth plane. Images were evaluated twice by two observers, using a five-step confidence scale. After the CBCT examination, the teeth were individually sectioned in the mesiodistal direction with a diamond saw. Using a light microscope at ×40 magnification, the true morphological status of all approximal surfaces was established. RESULTS: Sensitivity of the CBCT for the detection of caries on surfaces located proximally and distally to an amalgam filing ranged from 0.27 to 0.30 for enamel and from 0.47 to 0.56 for dentin. Specificity values for enamel proximal and distal lesions were 0.48 and 0.53, respectively, for enamel and 0.33 to 0.38, respectively, for proximal and distal dentin cases. Intra-observer reliability was 0.84, and interobserver reliability was 0.49. CONCLUSIONS: Owing to its low specificity, scans from a CBCT examination should not be used to determine the presence of demineralization of the tooth surface when amalgam fillings are present in the region of interest.