Laser Bioprinting with Cell Spheroids: Accurate and Gentle.

Laser printing with cell spheroids can become a promising approach in tissue engineering and regenerative medicine. However, the use of standard laser bioprinters for this purpose is not optimal as they are optimized for transferring smaller objects, such as cells and microorganisms. The use of standard laser systems and protocols for the transfer of cell spheroids leads either to their destruction or to a significant deterioration in the quality of bioprinting. The possibilities of cell spheroids printing by laser-induced forward transfer in a gentle mode, which ensures good cell survival ~80% without damage and burns, were demonstrated. The proposed method showed a high spatial resolution of laser printing of cell spheroid geometric structures at the level of 62 ± 33 µm, which is significantly less than the size of the cell spheroid itself. The experiments were performed on a laboratory laser bioprinter with a sterile zone, which was supplemented with a new optical part based on the Pi-Shaper element, which allows for forming laser spots with different non-Gaussian intensity distributions. It is shown that laser spots with an intensity distribution profile of the "Two rings" type (close to Π-shaped) and a size comparable to a spheroid are optimal. To select the operating parameters of laser exposure, spheroid phantoms made of a photocurable resin and spheroids made from human umbilical cord mesenchymal stromal cells were used.

Controlled Structure of Polyester/Hydroxyapatite Microparticles Fabricated via Pickering Emulsion Approach.

Biodegradable polyester/hydroxyapatite microparticles are widely proposed as microcarriers for drug/cell delivery or scaffolds for bone tissue regeneration. The current research implements the surfactant-free approach for the fabrication of polyester-based microparticles filled with hydroxyapatite nanoparticles (nHA) via the oil/water Pickering emulsion solvent evaporation technique for the first time, to the best of our knowledge. The process of polyester microparticle fabrication using nHA for the oil/water interface stabilization was studied as a function of phase used for nHA addition, which allows the preparation of a range of microparticles either filled with nHA or having it as a shell over the polymeric core. The effect of processing conditions (polymer nature, polymer/nHA ratio, ultrasound treatment) on particles' total yield, size distribution, surface and volume morphology, and chemical structure was analyzed using SEM, EDX, Raman spectroscopy, and mapping. Addition of nHA either within the aqueous or oil phase allowed the effective stabilization of the oil/water interface without additional molecular surfactants, giving rise to hybrid microparticles in which total yield, size distribution, and surface morphology depended on all studied processing conditions. Preliminary ultrasound treatment of any phase before the emulsification process led to a complex effect but did not affect the homogeneity of nHA distribution within the polymeric core of the hybrid microparticles.

Theoretical and Experimental Assay of Shock Experienced by Yeast Cells during Laser Bioprinting.

Laser-induced forward transfer (LIFT) is a useful technique for bioprinting using gel-embedded cells. However, little is known about the stresses experienced by cells during LIFT. This paper theoretically and experimentally explores the levels of laser pulse irradiation and pulsed heating experienced by yeast cells during LIFT. It has been found that only 5% of the cells in the gel layer adjacent to the absorbing Ti film should be significantly heated for fractions of microseconds, which was confirmed by the fact that a corresponding population of cells died during LIFT. This was accompanied by the near-complete dimming of intracellular green fluorescent protein, also observed in response to heat shock. It is shown that microorganisms in the gel layer experience laser irradiation with an energy density of ~0.1-6 J/cm2. This level of irradiation had no effect on yeast on its own. We conclude that in a wide range of laser fluences, bioprinting kills only a minority of the cell population. Importantly, we detected a previously unobserved change in membrane permeability in viable cells. Our data provide a wider perspective on the effects of LIFT-based bioprinting on living organisms and might provide new uses for the procedure based on its effects on cell permeability.

Endovenous laser coagulation: asymmetrical heat transfer and coagulation (modeling in blood plasma).

The objective of this study was to describe the dynamics of blood plasma heating and coagulation processes carried out by continuous laser radiation with wavelengths 1.55 and 1.94 µm through bare-tip fibers and fibers with radial output (radial fibers) used for endovenous laser coagulation (EVLC). The study was performed in previously thawed frozen donor blood plasma using high-speed shooting of the heating process through the shadow optical method. It has been shown that in the case of highly water-absorbed laser radiations, convection, explosive, and small-bubble boiling play a major role in the process of heat transfer and coagulation. It has been shown that in the case of radiation with wavelength λ = 1.94 µm, effective heat transfer begins at significantly lower levels of power compared to radiations with λ = 1.55 µm. It has been established that heat transfer is sharply asymmetrical and is directed mainly upwards and forwards (bare-tip fiber) or upwards (radial fibers). For a wavelength of 1.94 µm, the effect of self-cleaning of the fiber surface from coagulated plasma fragments was found. Except for short-term acts of explosive boiling, the heat transfer is asymmetrical and directed mainly upwards. This effect should lead to uneven heating and thermal damage to the vein wall with the maximum at its upper part. For EVLC, the use of radiation with a wavelength of 1.94 µm is more efficient and safer.

Endovenous laser coagulation: asymmetrical heat transfer (modeling in water).

The objective of this study was to describe the dynamics of water heating carried out by continuous laser radiation with wavelengths 1.47, 1.55, and 1.94 µm with different types of fibers used for endovenous laser coagulation. The study was conducted in water using high-speed surveying of the heating process through the shadow optical method. It has been shown that in the case of highly water-absorbed laser radiations, convection and boiling play a major role in the process of heat transfer. It has been shown that in the case of radiation with λ = 1.94 µm that is heavily absorbed by water, effective heat transfer begins at significantly lower levels of power compared to the weaker-absorbed radiations with λ = 1.47 and 1.55 µm. Mathematical models based only on thermal conductivity inadequately describe the process of real heat transfer during endovenous laser coagulation. It has been established that heat transfer is sharply asymmetrical and is directed mainly up-and-forward (bare-tip fiber) or upward ("radial" and "two-ring" fibers). Heat transfer for laser light with wavelength 1.94 µm is most effective than for 1.47 and 1.55 µm.

Beyond 2D: effects of photobiomodulation in 3D tissue-like systems.

SIGNIFICANCE: Currently, various scaffolds with immobilized cells are widely used in tissue engineering and regenerative medicine. However, the physiological activity and cell viability in such constructs might be impaired due to a lack of oxygen and nutrients. Photobiomodulation (PBM) is a promising method of preconditioning cells to increase their metabolic activity and to activate proliferation or differentiation. AIM: Investigation of the potential of PBM for stimulation of cell activities in hydrogels. APPROACH: Mesenchymal stromal cells (MSCs) isolated from human gingival mucosa were encapsulated in modified fibrin hydrogels with different thicknesses and concentrations. Constructs with cells were subjected to a single-time exposure to red (630 nm) and near-infrared (IR) (840 nm) low-intensity irradiation. After 3 days of cultivation, the viability and physiological activity of the cells were analyzed using confocal microscopy and a set of classical tests for cytotoxicity. RESULTS: The cell viability in fibrin hydrogels depended both on the thickness of the hydrogels and the concentration of gel-forming proteins. The PBM was able to improve cell viability in hydrogels. The most pronounced effect was achieved with near-IR irradiation at the 840-nm wavelength. CONCLUSIONS: PBM using near-IR light can be applied for stimulation of MSCs metabolism and proliferation in hydrogel-based constructs with thicknesses up to 3 mm.

Effects of Photobiomodulation on Daphnia magna Straus and their Sensitivity to Toxicant.

This paper deals with the effect of photobiomodulation (PBM) on Daphnia magna S. and their sensitivity to cadmium sulfate, a known high toxic pollutant. In a first series of experiments, the effect of different He-Ne laser fluences irradiation (range 0.9-4300 mJ cm-2 ) on the fertility of both parent and filial generations (F1-F3) of the crustacean was studied. It was found that PBM in some cases significantly influenced the fertility of both irradiated crustaceans and their nonirradiated offspring. By selecting two fluences (9 ± 2 mJ cm-2 reducing fertility and 4.3 ± 0.9 J cm-2 increasing it), the effect of these on toxicity of cadmium sulfate was evaluated. These experiments have shown that prior irradiation with low-intensity light of a helium-neon laser with 632.8 nm wavelength can change the sensitivity of aquatic organisms to toxin cadmium sulfate. The degree and direction of changes depend on the toxicant concentration and the irradiation dose.

From Aggregates to Porous Three-Dimensional Scaffolds through a Mechanochemical Approach to Design Photosensitive Chitosan Derivatives.

The crustacean processing industry produces large quantities of waste by-products (up to 70%). Such wastes could be used as raw materials for producing chitosan, a polysaccharide with a unique set of biochemical properties. However, the preparation methods and the long-term stability of chitosan-based products limit their application in biomedicine. In this study, different scale structures, such as aggregates, photo-crosslinked films, and 3D scaffolds based on mechanochemically-modified chitosan derivatives, were successfully formed. Dynamic light scattering revealed that aggregation of chitosan derivatives becomes more pronounced with an increase in the number of hydrophobic substituents. Although the results of the mechanical testing revealed that the plasticity of photo-crosslinked films was 5⁻8% higher than that for the initial chitosan films, their tensile strength remained unchanged. Different types of polymer scaffolds, such as flexible and porous ones, were developed by laser stereolithography. In vivo studies of the formed structures showed no dystrophic and necrobiotic changes, which proves their biocompatibility. Moreover, the wavelet analysis was used to show that the areas of chitosan film degradation were periodic. Comparing the results of the wavelet analysis and X-ray diffraction data, we have concluded that degradation occurs within less ordered amorphous regions in the polymer bulk.

Photoluminescence of Drying Droplets with Silicon Nanoparticles.

This study is dedicated to the formation of structures during drying of droplets of sols of silicon nanoparticles (SiNPs) in dimethylsulfoxide (DMSO) with a diameter of 1-5 mm on the horizontal glass and mica surfaces. Drying of such droplets with pinning (sticking) of the droplet contact line causes gradual gathering of the SiNPs on its edge with the formation of a thin ring. It has been found that the integral photoluminescence intensity IPL greatly varies during the drying process. At the initial stage, IPL monotonically decreases by several orders of magnitude and then abruptly increases several times at the final stage of ring formation. It has been shown that the rate of IPL decrease is maximal at a very early stage and depends both on the aggregative state (solid film SiNPs/sols of the SiNPs) and volume of the SiNPs sols. It is minimal for the solid film SiNPs and gradually increases as the volume of SiNPs sol in DMSO decreases (optical cell → big droplet → small droplet). The obtained experimental dependencies between the luminescence decrease rate and aggregative state and volumes of the SiNPs sol in DMSO are attributed to the combination of three mechanisms of luminescence quenching: photobleaching, quenching with atmospheric oxygen, and Förster resonance energy transfer quenching. The appearing of the luminescence leap at the final stage of ring formation is associated with the emergence of cracks in the ring.