In this research we compared the properties of three types of the dense materials suitable to be cells carriers at the creation of temporary artificial dermal equivalents. It has been established that shortcomings of modified polyacrylamide hydrogel matrixes are, at first, a decrease of transparency of those hydrogels owing to the introduction of inorganic nanoparticles into their structure, and secondly, a certain degree of fragility of the humidified flat hydrogel fragments. It has been found that main shortcoming of a symbiotic organism Medusomyces gisevi is the fact that living cells are not capable to be spread and survived normally on this matrix what raises doubts in their normal metabolic activity in such conditions. Finally it has been established that the commercial collagenic film "Belkozin" is the most suitable matrix from three checked options for creation of temporary artificial dermal equivalents. It has been supported by our results that application of the temporary dermal equivalent with structure "belkozin + gelatin + living cells" on burn wounds of model animals stimulates regeneration of a dermal layer of skin and epithelium restoration.
We have shown alsothat the hydrogel of carbopol (carbomer) prepared with using DMEM-HG culture medium can serve as the volume carrier of living cells and support their life within several hours. However, we consider that for better maintaining the cells viability it is necessary to carry out a combination of hydrogel and the cellular component ex tempore, just before its application. It is important that the presence of antibiotics in the carbopol hydrogel prepared with using DMEM-HG culture medium (which, in fact, contains antibiotics) gives some bactericidal, bacteriostatic and fungistatic properties for the finished bioconstructions, what might prevent wound infection and thus reveal a positive effect on the healing process.
We compared the wound-healing properties of such components of plastic carbopol hydrogel bioconstructions, as cells of three established lines (4BL, E8, A102) and cell-free conditioned mediums (CFCM), which have been produced by cultivating cells of these three lines (CFCM-4BL, CFCM-E8, CFCM-A102). The most effective for wound-healing in animals among all of six studied biostructures were ones which include either 4BL cells or CFCM-4BL.
It has been shown by us that the treatment of burn wounds using carbopol hydrogel biostructures loaded with the cells of 4BL line or with the corresponding cell-free medium conditioned by cells of 4BL line (CFCM-4BL), are accompanied, at first, by stimulation of capillary formation in the wound bed, and secondly, by a statistically significant decrease of regulatory proteins MMP2 and HIF-1α levels in blood, almost to the level inherent in healthy animals, which may indicate an inhibition of the inflammatory process and earlier completion of the first phase of wound-healing. It should be noted that the stimulating effect of wound-healing induced by the cell-free conditioned medium was much stronger than by the cells.
It has been found that the degree of 4BL cells confluence during their cultivation and production of the cell-free conditioned mediums is not significant for the expression of their regenerative wound-healing properties. The prepared conditioned medium should be stored in a frozen state, and deep freezing and storage of the obtained CFCM at –80°C is optimal, at the same time storage in a standard freezer at –20ºC is less good but acceptable.
Thus, the main results of this work are: a) creation of new biotechnological products, namely the dermal equivalents with the inclusion of human cells or their derivatives (cell-free conditioned mediums); b) preclinical studies of new dermal equivalents to determine their therapeutic efficacy and safety in vivo with using model animals; c) a method of obtaining these biotechnological products was patented. It has been shown that the new wound dressings promote burn wounds healing in experimental animals when applied to the wound surface.
The most important result from the point of view of both science and practice, is the establishment of the fact that cell-free conditioned media, produced by stem cells in vitro, can be used instead of living cells in the manufacture of dermal equivalents. For the first time it has been shown that CFCM samples conditioned by 4BL cells have a positive effect on the organism, and are stimulating regenerative processes at the most important early stage of burn wound-healing in model animals (mice, rats). The use of CFCM greatly simplifies and makes more economical the biotechnology for obtaining and using new dermal equivalents.
In the prospects, new bioconstructions developed by us might be the subject of preclinical studies at special accredited laboratory, and on the next step they might be admitted to clinical trials in burn departments of health care institutions of Ukraine to treat burns and other skin lesions.
