Purpose: Mesenchymal stem cells isolated from bone marrow (BM-MSC) and periodontal ligament (PLSC) are cells with high proliferative potential and ability to self-renewal. Characterization of these cells under genotoxic stress conditions contributes to the assessment of their prospective usage. The aim of our study was to evaluate changes in BM-MSC and PLSC caused by ionizing radiation. Methods: Human BM-MSC and PLSC were irradiated with the doses up to 20 Gy by Co⁶⁰ and observed 13 days; viability, proliferation, apoptosis and senescence induction, and changes in expression and phosphorylation status of related proteins were studied. Results: Irradiation with the doses up to 20 Gy significantly reduces proliferation, but has no significant effect on cell viability. The activation of tumor suppressor protein 53 (p53) and its phosphorylations on serines 15 and 392 were detected from the first day after irradiation by 20 Gy and remained elevated to day 13. Expression of cyclin-dependent kinases inhibitor 1A (p21^Cip1/Waf1) increased. The cell cycle was arrested in G2 phase. Instead of apoptosis we have detected hallmarks of stress-induced premature senescence: increase in cyclin-dependent kinases inhibitor 2A (p16^INK4a) and increased activity of senescence-associated β-galactosidase. Conclusion: Mesenchymal stem cells isolated from bone marrow and periodontal ligament respond to ionizing radiation by induction of stress-induced premature senescence without apparent differences in their radiation response.

A new and relatively simple method for purification of betulin from birch bark extract was developed in this study. Its five purification steps are based on the differential solubility of extract components in various solvents and their crystallization and/or precipitation, on their affinity for Ca(OH)₂ in ethanol, and on the affinity of some impurities for silica gel in chloroform. In addition, all used solvents can be simply recycled. Betulin of more than 99% purity can be prepared by this method with minimal costs. Various observations including crystallization of betulin, changes in crystals during heating, and attempt of localization of betulin in outer birch bark are also described in this work. The original extract, fraction without betulinic acid and lupeol, amorphous fraction of pure betulin, final crystalline fraction of pure betulin and commercial betulin as a standard were employed to determine the antiproliferative/cytotoxic effect. We used WST-1 tetrazolium-based assays with triple negative breast cancer cell line BT-549. The decrease in cell survival showed clear relationship with the purity of the samples, being most pronounced using our final product of pure crystalline betulin. WST-1 proliferation/cytotoxicity test using triple negative breast cancer cell line BT-549 clearly showed the importance of purity of betulin for biological experiments and, apparently, for its medicinal use.

Adult human dental pulp contains stem cells (DPSCs) that are capable of differentiation into osteoblasts, odontoblasts, adipocytes, and neuronal-like cells. Because these cells have potential use in tissue regeneration, herein we characterized the response of DPSC lines to ionizing radiation (IR). These DPSC lines have been developed from the extracted molars of healthy donors. DPSCs were cultivated in a unique media supplemented with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Since tissue homeostasis depends on a precise balance among cell proliferation, senescence, and cell death, we explored the effects of IR (2-20 Gy) on the proliferative activity of DPSCs and the molecular pathways involved. Even the highest dose used (20 Gy) did not induce DPSC apoptosis. After irradiation with doses of 6 and 20 Gy, DPSCs accumulated in the G2 phase of the cell cycle. DPSCs responded to IR (20 Gy) with senescence detected as SA-β-galactosidase positivity, beginning on the third day after irradiation. Twenty-four hours after irradiation, p53 and its serine 15 and 392 phosphorylated forms were detected. At this time, p21 (WAF1) was induced. Increases in protein p16 were observed from the third day following irradiation and continued till the end of the examination (Day 13). We conclude that DPSCs respond to IR-induced damage by permanent cell cycle arrest in the G2 phase and by stress-induced premature senescence.