Ján Vojtaššák

Expression of proliferation and apoptotic markers in human placenta during pregnancy

Trophoblast has unique properties in relation to its wide range of metabolic, endocrine and angiogenic functions. Trophoblastic cells invade endometrium and adjacent myometrium in a way that is imitated by malignant tumours. The aim of the present study was to analyse the expression of markers of proliferation and apoptosis in trophoblastic cells in normal human placenta during pregnancy. A total of 22 placentas, 12 of which were obtained from curettage and induced legal abortion and 10 placentas obtained from normal deliveries or caesarean sections were included in this study. Proliferation markers were strongly expressed in cytotrophoblast in early stages of gestation. In late term placentas, a distinct decrease in expression of these markers was observed. Syncytiotrophoblast was negative for proliferation markers in all placentas. Positive immunostaining for bcl-2, an anti-apoptotic marker, was observed only in syncytiotrophoblastic cells in first-trimester but also in third-trimester placentas. Cytotrophoblast and stromal mesenchymal cells of chorionic villi were negative for bcl-2. Expression of bcl-2 protein in syncytiotrophoblast may be one of the major factors preventing these structures from early cell death, which is indispensable for the maintenance of physiological pregnancy.

Biological and morphological characterization of in vitro expanded human muscle-derived stem cells.

Stem cells are generally characterised as clonogenic and undifferentiated cells with the capacity of self-renewal and plasticity. Over the past few years, the adult stem cells have been derived from various types of tissues including the skeletal muscle. The main goal of the present study was the isolation, in vitro expansion and characterisation of muscle-derived stem cells (MDSCs). Thereby obtained results showed that MDSCs have a fibroblast-like shape with a large nucleus having one to four nucleoli. The cytoplasm was transparent without any signs of vacuolisation. TEM analysis showed an ultrastructure of cells with high proteosynthetic activity. MDSCs had a large and irregular nucleus with variable number of nucleoli. The cytoplasm contained a richly developed and rough endoplasmic reticulum, prominent Golgi apparatus cisterns as well as transport vesicles containing glycogen granules and variable microvilli and filopodia. They expressed alpha-actin and desmin. Results of the phenotypic characterisation showed that the analyzed cells were positive for CD29, CD34, CD44, CD90, CD105 and HLA Class I. They did not express CD14, CD45, CD235a, HLA Class II and human fibroblast surface protein. According to these results it should be emphasised that MDSCs after performing the detailed studies focused on their immunological properties and differentiation potential may be used in the cell therapy of many degenerative diseases.

Morphological characterization of in vitro expanded human dental pulp-derived stem cells

Stem cells play pivotal role in the development of tissues and organs, as well as they maintain homeostasis and integrity of multicellular organisms. Human dental pulp-derived stem cells are capable of differentiation into osteoblasts, odontoblasts, adipocytes and neuronal-like cells and can have potential use in tissue regeneration. The aim of this study was a detailed description of human dental pulp-derived stem cells (HDPSCs) cultivated in vitro ultrastructural morphology. HDPSCs were isolated from the dental pulp of impacted third molars from healthy donors. Transmission electron microscopic analysis showed typical ultrastructural morphology similar to mesenchymal stem cells from other organs. The morphology of HDPSCs cells reflects their proteosynthetic and metabolic activity. Each cell contained spherical or irregularly-shaped large pale nucleus with a large amount of euchromatine. Nuclei had noticeable nucleolus (or two nucleoli) located nearby the nuclear envelope. Abundant cisterns of rough endoplasmic reticulum and numerous coated matrix vesicles as well as granules of glycogen were present in their cytoplasm. Nearby the nucleus small, elongated mitochondria were placed. Most of HDPSCs created and secreted vesicles; in plasmalemma bounded amorphous electron-lucide granules and also few glycogen granules. These secretory vesicles had around 0.5 μm in diameter. We assume that it can be a special type of communication between cells, probably paracrine type of cell signalling, but its real function is still unknown.

In vitro cytotoxicity testing of coladerm membrane

Atpresent, biodegradable and biocompatible membranes based on collagen andglycosaminoglycans play an important role in substitutive medicine. Modernbiomaterials use a chemically modified collagen-based matrix for implants withprogrammable biodegradability as a substitute of buccal mucosa, skin,cartilage,etc. Besides the requirements for biocompatibility and biodegradability, themembranes must be also non-toxic. Therefore, cytotoxicity testing of thesematerials in vitro is an integral part of introducingnewlydeveloped types of membranes into clinical practice. As a biological model forthe tested COLADERM membrane, cell cultures from human embryonic fibroblasts(B-HEF-2) were used for both cytotoxicity testing as well as in tests to assessthe ability of cells to proliferate on this membrane. Along with the ability ofcells to grow on the surface and inside the membrane, immunohistochemicalexamination and scanning electron microscopy (SEM) were performed as well. Theobtained results have shown that the COLADERM membrane is non-toxic withsuitable structural and biological properties for clinical application as asubstitute of buccal mucosa following surgical ablation of malignant tissuesfrom the oral cavity.

Mouth cavity base defect treated with biosynthetic graft

The use of in vitro prepared biosynthetic grafts can considerably improve the patient’s quality of life. This work reports on the use of an autologous graft prepared from a patient’s preputial cells cultivated on biodegradable polymeric membrane. Coladerm membrane is based on the chemically modified polyelectrolyte complex of atelocollagen and hyaluronan. The graft was used to cover a defect in the mouth cavity base and tongue after reconstruction surgery performed at this site in the past. The presented clinical case showed that the autologous biosynthetic graft prepared from foreskin cells can be successfully used for covering of medium-size defects in mouth cavity base resulting in the regeneration of target mouth structures with significant improvement of patient’s quality of life.

Chlamydia and mycoplasma infections during pregnancy and their relationships to orofacial cleft

Serum antibodies to Mycoplasma pneumoniae and Chlamydia trachomatis have been studied in a group of newborns with orofacial cleft (OC) and their mothers (n = 59) as compared to a control group of healthy newborns and their mothers (n = 40) assayed by ELISA and Western blot analysis. In the first group, IgG antibodies to M. pneumoniae were found by ELISA in 12 newborns with OC and 22 mothers, while IgA antibodies were detected only in 5 and 11 cases, respectively. IgM antibodies indicating an acute infection were found in 2 mothers only. IgG antibodies to C. trachomatis were found in 2 newborns with OC and 4 mothers. In the control group, IgG antibodies to M. pneumoniae were found in 3 newborns and 7 mothers. IgG antibodies to C trachomatis were observed in 1 newborn and 1 mother, while IgM antibodies to C trachomatis were present in 1 mother only. Immunoblot analysis revealed in newborns with OC and their mothers C. trachomatis-specific bands associated with MOMP 1, 29 kDa, 45 kDa, and heat shock proteins (HSP) 60 and 70. Based on these results we suggest that the risk associated with the exposure to M. pneumoniae and/or C. trachomatis is so far unknown and further study is needed for its elucidation.

Adult stem cells derived from skeletal muscle — biology and potential

Skeletal muscle contains at least two distinct populations of adult stem cells — satellite cells and multipotent muscle-derived stem cells. Monopotential satellite cells are located under the basal lamina of muscle fibers. They are capable of giving rise only to cells of myogenic lineage, which play an important role in the processes of muscle regeneration. Multipotent muscle-derived stem cells are considered to be predecessors of the satellite cells. Under proper conditions, both in vitro and in vivo, they undergo myogenic, cardiogenic, chondrogenic, osteogenic and adipogenic differentiation. The main purpose of the present article is to summarize current information about adult stem cells derived from skeletal muscle, and to discuss their isolation and in vitro expansion techniques, biological properties, as well as their potential for regenerative medicine.

SATB2 haploinsufficiency in patients with cleft palate

De novo translocation interrupting the transcription unit of SATB2 gene has been associated with cleft palate only (CPO). We tested for the presence of the copy number of SATB2 gene in a sample of 92 patients with CPO using a quantitative real-time PCR approach. In one patient (1%, 95% CI = 0.2%–6%), a 19 Mb de novo deletion encompassing the SATB2 gene was detected. These results suggest that SATB2 gene deletions do not play an important role in the etiology of cleft palate.