Laser Thermo-Photobiomodulation of Bone Marrow Mesenchymal Stem Cells.

We studied the effect of laser radiation of moderate intensity with a wavelength of 970 nm on the efficiency of colony formation of rat bone marrow mesenchymal stem cells (MSC) in vitro. In this case, photobimodulation and thermal heating of MSC occur simultaneously. This combined laser treatment allows increasing the number of colonies by 6 times in comparison with the control and by more than 3 times in comparison with thermal heating alone. The mechanism of such an increase is associated with combined thermal and light effects of laser radiation of moderate intensity, which stimulates cell proliferation. This phenomenon can be used as the basis for solving the most important task of cell transplantation, associated with the expansion of autologous stem cells and activation of their proliferative potential.

Changes in the Number of Mesenchymal Stem Cells in Bone Marrow at Different Periods after In Vivo Exposure of the Bone Marrow to Local Infrared Laser Radiation.

We determined optimal parameters of bone marrow (BM) irradiation in vivo for rapid increase in the number of mesenchymal stem cells (MSC) at the initial stages of the culturing without changing the karyotype, polyploidy, which are observed at higher passages. Such an increase is necessary to achieve the required number of cells at the initial passages for subsequent transplantation into the body. It was shown that after irradiation with λ=0.97 µm, the maximum and similar increase in the content of BM MSC in comparison with the control (by 2.4 times) was observed on day 2 in the irradiated and contralateral tibia. An insignificant difference in the number of BM MSC for the irradiated and contralateral tibia remained at all terms after irradiation, with a general decrease in the number of BM MSC up to 21 days. After laser irradiation with λ=1.56 µm, the maximum number of BM MSC was also observed on day 2. In this case, the concentration of these cells in the irradiated and contralateral limbs exceeded the control by 3.1 and 1.7 times, respectively. With increasing the time after exposure, the number of BM MSC in both limbs showed the same tendency to a decrease as after irradiation at λ=0.97 µm.

Low-Intensity Femtosecond Radiation Activates the Natural Defenses of Mice in vivo.

The possibility of induction of cytogenetic damage in the bone marrow, changes in the cellularity of lymphoid organs and blood composition in mice irradiated with low-intensity femtosecond laser radiation at a power flux density of 5.1, 10.4, and 52 mJ/cm2 (0.5 mW for 5, 10, and 50 s) in vivo was shown. Using the radiation adaptive response test (0.1 Gy + 1.5 Gy), it was found that, when mice were exposed to femtosecond laser radiation in high doses, the body's natural defenses were activated in the same narrow range of energy flux density (2-16 mJ/cm2) as in the case of X-ray irradiation in a dose of 0.1 Gy (4 mJ/cm2). The data obtained suggest a similar mechanism of activation of the body's natural defense upon exposure to low doses of both ionizing and non-ionizing radiation.

Digging deeper: structural background of PEGylated fibrin gels in cell migration and lumenogenesis.

Fibrin is a well-known tool in tissue engineering, but the structure of its modifications created to improve its properties remains undiscussed despite its importance, e.g. in designing biomaterials that ensure cell migration and lumenogenesis. We sought to uncover the structural aspects of PEGylated fibrin hydrogels shown to contribute to angiogenesis. The analysis of the small-angle X-ray scattering (SAXS) data and ab initio modeling revealed that the PEGylation of fibrinogen led to the formation of oligomeric species, which are larger at a higher PEG : fibrinogen molar ratio. The improvement of optical properties was provided by the decrease in aggregates' sizes and also by retaining the bound water. Compared to the native fibrin, the structure of the 5 : 1 PEGylated fibrin gel consisted of homogenously distributed flexible fibrils with a smaller space between them. Moreover, as arginylglycylaspartic acid (RGD) sites may be partly bound to PEG-NHS or masked because of the oligomerization, the number of adhesion sites may be slightly reduced that may provide the better cell migration and formation of continuous capillary-like structures.

Effect of Low-Level Laser Irradiation on Proliferative Activity of Wharton's Jelly Mesenchymal Stromal Cells.

We studied the effect of low-level laser irradiation on proliferative activity of cultured human Wharton's jelly mesenchymal stromal sells. Cells were irradiated with a solid-state laser emitting at 650 nm; irradiation doses were 0.04, 0.4, or 4 J/cm2. Laser irradiation was performed once at the start of the cell proliferation experiment or daily throughout the experiment. Cells were cultured for 7 days. The number of viable cells was assessed using the MTT test. An increase in cell proliferative activity was detected after daily laser irradiations; the maximum stimulating effect was achieved at a dose of 0.04 J/cm2. These results substantiate medical use of lasers for expansion of cells intended for transplantation.

[Hydrogen Sulfide Donor NaHS Protects Mesenchymal Stem and Melanoma Cells from the Negative Effects of Infrared Laser Irradiation].

We have established earlier that 835-nm infrared laser irradiation results in a dose-dependent growth inhibition of human mesenchymal stem and melanoma cells and is able to induce cell death. In this work we have demonstrated that hydrogen sulfide donor NaHS is able to protect both cell types from the negative action of laser irradiation and the magnitude of protection depends on NaHS concentration. The mechanism of cell protection by NaHS is primarily attributable to its effects on intracellular processes occurring after irradiation, since the protective effect does not depend on whether NaHS is added before or after irradiation. Moreover, NaHS is able to exert its protective effect even when added 6 hours post irradiation.

New microorganism isolation techniques with emphasis on laser printing.

The study of biodiversity, growth, development, and metabolism of cultivated microorganisms is an integral part of modern microbiological, biotechnological, and medical research. Such studies require the development of new methods of isolation, cultivation, manipulation, and study of individual bacterial cells and their consortia. To this end, in recent years, there has been an active development of different isolation and three-dimensional cell positioning methods. In this review, the optical tweezers, surface heterogeneous functionalization, multiphoton lithography, microfluidic techniques, and laser printing are reviewed. Laser printing is considered as one of the most promising techniques and is discussed in detail.

Laser printing of microbial systems: effect of absorbing metal film.

Recently, it was shown that laser-induced forward transfer (LIFT) technology and the laser engineering of microbial systems (LEMS) technique (based on LIFT method) are effective for isolation of micro-organisms from different complex substrates. These techniques frequently utilize Au as an absorbing layer material. The purpose of this study was to investigate the influence of absorbing film materials (Au, Ti and Cr) on the effectiveness of laser printing of micro-organisms to improve LEMS and LIFT techniques. It was shown that application of Ti and Cr absorbing layers activates bacterial growth after laser printing and is significantly more effective in comparison to Au films, which actually show a suppressing effect on bacterial cells. Results of this study can be applied for LEMS and LIFT protocols for improving bacterial isolation and microbial growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Laser-induced forward transfer technique (LIFT) is currently used for printing of micro-organisms and in biosensor techniques, for single-cell isolation, and for culturing of micro-organisms from complex substrates. We have studied the influence of absorbing film materials (Au, Ti and Cr) on the effectiveness laser printing of micro-organisms. It was shown that application of Ti and Cr absorbing layers activates bacterial growth and is more effective in LIFT compared to Au films, which actually have a suppressive effect on bacteria cells. The results can improve LIFT protocols for bacteria isolation and culturing of microbial systems.

[Cytotoxic Effect of Low-Intensity Infrared Laser Irradiation on Human Melanoma Cells].

Continuous low-intensity laser irradiation (LILI) affects the state of cells in culture, including their proliferation rate. Data collected with various cell models vary significantly, but most studies have reported positive effects of LILI on cell proliferation. The effects of continuous infrared LILI (835 nm) was studied using three independent different melanoma cell lines. The LILI effect was shown to strongly depend on the irradiation dose. Higher doses (230 kJ/m^(2)) significantly suppressed the cell growth. A further increase in LILI dose led to a significant cytotoxic effect, which increased disproportionately quickly with the increasing light intensity. Human mesenchymal stem cells (MSCs) were found to be significantly more resistant to the cytotoxic effect of higher-dose LILI. Importantly, the effects were not due to the difference in culture conditions. Control experiments showed that 15 non-melanoma tumor cell lines were more resistant to LILI than melanoma cells. Selective sensitivity of melanoma cells to LILI in vitro was assumed to provide a basis for LILI-based approaches to melanoma treatment.

Combined Effect of Low-Intensity Helium-Neon Laser and X-Ray Radiation on in Vivo Cellular Response of the Whole Blood and Lymphoid Organs in Mice.

We studied the effect of exposure to helium-neon laser (dose range 0.16-50 mJ/cm2) on activation of natural protection reserve in mice using the adaptive response test. DNA comets method revealed a protective response manifested in DNA damage level in whole blood leukocytes of mice and in lymphoid organs by the thymus and spleen weight index; preexposure to laser did not induce the adaptive response. ROS level in the whole blood was assessed by the level of zymosan-induced luminol chemiluminescence. In mice subjected to adaptive laser irradiation in doses of 0.16-5 mJ/cm2 followed by X-ray irradiation in a dose of 1.5 Gy, the activation index calculated as the ratio of induced to spontaneous area of luminescence was by 1.4 times lower than that in non-irradiated animals, which attested to reduced ROSgeneration reserve capacity of neutrophils.

Induction of the Adaptive Response in Mice Exposed to He-Ne Laser and X-Ray Radiation.

We studied the dose-dependent induction of in vivo adaptive response in the bone marrow and blood of mice exposed to low-intensity radiation of He-Ne laser (633 nm) and X-ray radiation by the severity of cytogenetic injury and intensity of ROS production, respectively. Induction of the adaptive response in mice preexposed to He-Ne laser and X-ray radiation depended on the adaptive dose and the interval between the adaptive and main doses and correlated with changes in ROS generation. The adaptive response after exposure to low-intensity ionizing and non-ionizing radiation was observed in the same dose range, which attests to similar mechanisms of its induction.

In Vitro Effect of Laser-Induced Hydrodynamics on Cancer Cells.

We studied the effect of laser-induced hydrodynamic on viability of Colo-26 murine colon carcinoma cells in vitro. Laser-induced hydrodynamics was generated by a laser (λ=1.56 µ, power 3 W, 5 min exposure); to this end, the fiber end was submersed into a buffer above the cell monolayer. It was found that laser-induced hydrodynamics destructed the monolayer at standoff distances of between the working end of the laser fiber to cell monolayer of 1 and 5 mm and triggers apoptotic and necrotic death in remaining cells at a distance of 4 mm from the emitter.

[STIMULATION OF HaCaT KERATINOCYTES AND RATS MESENCHYMAL STROMAL CELLS PROLIFERATION BY FEMTOSECOND LASER PULSES].

The influence of femtosecond laser pulses on the proliferative activity of HaCaT keratinocytes and mesenchymal stromal cells (MSC) rats was studied. The growth media was exposed by laser pulses with wavelength 590 nm and duration 30 fs. The dependence of proliferative activity of cells on the dose was showed in the range 6-4299 J/cm2. Proliferative activity was assessed by the number of cells after 1 day after exposure. For both cell cultures obtained similar dose dependence: an increase in cell proliferation (32-54% for HaCaT and 19% for MSK) occurs when using lower doses, while higher doses no changes the rate of proliferation of cells. Conducted physical and chemical analysis found no increase in the concentration of active forms of oxygen in the culture medium. The impact of femtosecond laser pulses has led to the generation in culture medium acoustic oscillations in the range of 0.5 to 6.0 kHz. It is assumed that the increase in proliferative activity of cells, can be caused by mechanical effects of acoustic waves generated in the environment of optical breakdown in the focus of the laser radiation.

Effects of acoustic and EHF impulses on multipotent stromal cells during formation of bone marrow containing heterotopic organs in tissue engineered constructions.

We studied the effects of physical factors (acoustic impulses of laser-induced hydrodynamics, AILIH, and EHF-radiation) on the formation of heterotopic bone marrow organs. Suspension of precipitated mouse bone marrow cells was exposed to AILIH and EHF or their combinations (AILIH+EHF, EHF+AILIH). The developed tissue engineering constructions (gelatin sponges containing 107 nucleated bone marrow cells exposed to physical factors) were transplanted under the renal capsule of syngeneic mice. Analysis of newly formed hemopoietic organs was performed after 3 and 5 months. The total amount of hemopoietic cells, number of multipotent stromal cells, efficiency of colony formation from these cells, and weight of bone capsule of the transplants were measured. Microscopic study showed that 5-month transplants were significantly larger than 3-month transplants and contained 3-fold more hemopoietic cells (20-fold in the AILIH+EHF group). The number of multipotent stromal cells was maximum in EHF+AILIH group (by 2.2 times higher than in the control) and minimum in AILIH+EHF group. Exposure to EHF+AILIH had most pronounced effect on the formation of the bone marrow transplants. The weight of bone capsules more rapidly increased in gelatin sponges of 3-month transplants of EHF+AILIH and AILIH groups. These data suggest that the studied physical factors can be used for acceleration of rehabilitation process.