Authors: Malyshkina S.V., Poshelok D.M. Nikolchenko O.A., Vyshniakova I.V., Samoilova K.M.
Introduction. The study of the effect of hypothermia (decreased body temperature) on bone remodeling is one of the current problems in biology and medicine. Bone marrow contains stromal cells that are able to differentiate into оsteogenic direction, so there the number, structural and functional conditions are important for bone remodeling and osteoreparation. It is known that bone marrow stromal cells are extremely sensitive to unfavorable external and internal factors. The number of these cells and their ability to form cell colonies decreases with age and development of destructive changes in the bones. In the literature we have not found works in which the effect of mild hypothermia on bone marrow stromal cells would have been studied.
The purpose: to evaluate investigate in vitro the colony-forming ability of bone marrow stromal cells in rats of different ages after the mild hypothermia induced by cold exposure.
Materials and Methods. We studied the cytological indicators of the ability of bone marrow stromal cells to form colonies during the cultivation – the number and area of cell colonies, the morphological characteristics of the cultured cells. Experimental animals (10 rats) at the age of 6 and 24 months were kept in separate sections of the cold chamber at temperature −20°С for 5 days to 5 hours per day. Animals of the control groups (10 rats) were kept in the separate boxes at a room temperature (18-22 °C). Bone marrow was isolated from the rat femurs and tibias at 7 and 28 days after induced mild hypothermia. Euthanasia of animals was performed by the intraperitoneal administration of sodium thiopental (90 mg / kg). Bone marrow cells were seeded into plastic flasks (Falcon) based 1.5×106 cells per 1 cm2, and then cultured in a nutrient medium containing DMEM (Sigma), 2 mM L-glutamine, 20 % fetal calf serum and 100 U/ml penicillin and 100 mg/ml streptomycin. The nutrient medium was changed every three days up to 12 days of culture, then it was removed, and the cell cultures washed with PBS, fixed with May-Grunwald, stained with azure-eosin by Romanovsky. The cytological indicators were studied using light microscope OlympusCX41RF. The obtained digital values were processed by the methods of variation statistics. The differences between paired samples were evaluated by using Student t-test and considered statistically significant at P<0.05. Plan of the experimental study and its carrying out in accordance to the Law of Ukraine № 3447-IV «On protection of animals from cruelty» and European Convention for the protection of vertebrate animals used for experimental and other scientific purposes were approved by the positive decision of the local Committee on Bioethics (protocols No. 88, 30.05.2011, No. 131, 16.06.2014).
Results. In the cultures of bone marrow cells isolated from the bones of the young rats at 7 day after cold exposure the number and area of colonies are less as compared with the control, respectively, by 34.5 and 40.1 %. The cells with destructive changes (vacuolization of the cytoplasm, pyknosis of the nucleus) are identified on the periphery of the colonies. The colonies of the cells are no formed in the cultures of bone marrow cells of experimental old rats (the cells are arranged individually or in small clusters). In the cell cultures of control old rats the number and area of colonies are less than in the cell cultures of control young rats by 53.6 and 44.2 %, respectively. At 28 day after cold exposure the differences in the number and area of cell colonies between the experimental and control cultures of bone marrow cells both young and old rats are decreased compared with ones at 7 day. This suggests that the regenerative processes occur in the bone marrow of rats after mild hypothermia. At that, these processes are less active in old animals, so as at 28 day after cold exposure all cytological indicators remain the lowest in the cell cultures of experimental old rats.
Conclusion. The study showed that mild hypothermia induced in rats by cold exposure inhibits the proliferative activity of bone marrow stromal cells and their ability to form cell colonies in vitro. Negative effects of hypothermia are more expressed in the bone marrow cell culture of old animals.
Keywords: cell culture, bone marrow, cell colonies, hypothermia, rats.