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.

A Hydrophobic Derivative of Ciprofloxacin as a New Photoinitiator of Two-Photon Polymerization: Synthesis and Usage for the Formation of Biocompatible Polylactide-Based 3D Scaffolds.

A hydrophobic derivative of ciprofloxacin, hexanoylated ciprofloxacin (CPF-hex), has been used as a photoinitiator (PI) for two-photon polymerization (2PP) for the first time. We present, here, the synthesis of CPF-hex and its application for 2PP of methacrylate-terminated star-shaped poly (D,L-lactide), as well a systematic study on the optical, physicochemical and mechanical properties of the photocurable resin and prepared three-dimensional scaffolds. CPF-hex exhibited good solubility in the photocurable resin, high absorption at the two-photon wavelength and a low fluorescence quantum yield = 0.079. Structuring tests showed a relatively broad processing window and revealed the efficiency of CPF-hex as a 2PP PI. The prepared three-dimensional scaffolds showed good thermal stability; thermal decomposition was observed only at 314 °C. In addition, they demonstrated an increase in Young's modulus after the UV post-curing (from 336 ± 79 MPa to 564 ± 183 MPa, which is close to those of a cancellous (trabecular) bone). Moreover, using CPF-hex as a 2PP PI did not compromise the scaffolds' low cytotoxicity, thus they are suitable for potential application in bone tissue regeneration.

Solute Diffusion into Polymer Swollen by Supercritical CO2 by High-Pressure Electron Paramagnetic Resonance Spectroscopy and Chromatography.

High-pressure electron paramagnetic resonance (EPR) was used to measure translational diffusion coefficients (Dtr) of a TEMPONE spin probe in poly(D,L-lactide) (PDLLA) and swollen in supercritical CO2. Dtr was measured on two scales: macroscopic scale (>1 µm), by measuring spin probe uptake by the sample; and microscopic scale (<10 nm), by using concentration-dependent spectrum broadening. Both methods yield similar translational diffusion coefficients (in the range 5-10 × 10-12 m2/s at 40-60 °C and 8-10 MPa). Swollen PDLLA was found to be homogeneous on the nanometer scale, although the TEMPONE spin probe in the polymer exhibited higher rotational mobility (τcorr = 6 × 10-11 s) than expected, based on its Dtr. To measure distribution coefficients of the solute between the swollen polymer and the supercritical medium, supercritical chromatography with sampling directly from the high-pressure vessel was used. A distinct difference between powder and bulk polymer samples was only observed at the start of the impregnation process.

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.

Photocurable Methacrylate Derivatives of Polylactide: A Two-Stage Synthesis in Supercritical Carbon Dioxide and 3D Laser Structuring.

A two-stage polylactide modification was performed in the supercritical carbon dioxide medium using the urethane formation reaction. The modification resulted in the synthesis of polymerizable methacrylate derivatives of polylactide for application in the spatial 3D structuring by laser stereolithography. The use of the supercritical carbon dioxide medium allowed us to obtain for the first time polymerizable oligomer-polymer systems in the form of dry powders convenient for further application in the preparation of polymer compositions for photocuring. The photocuring of the modified polymers was performed by laser stereolithography and two-photon crosslinking. Using nanoindentation, we found that Young's modulus of the cured compositions corresponded to the standard characteristics of implants applied in regenerative medicine. As shown by thermogravimetric analysis, the degree of crosslinking and, hence, the local stiffness of scaffolds were determined by the amount of the crosslinking agent and the photocuring regime. No cytotoxicity was observed for the structures.

Cell spheroid fusion: beyond liquid drops model.

Biological self-assembly is crucial in the processes of development, tissue regeneration, and maturation of bioprinted tissue-engineered constructions. The cell aggregates-spheroids-have become widely used model objects in the study of this phenomenon. Existing approaches describe the fusion of cell aggregates by analogy with the coalescence of liquid droplets and ignore the complex structural properties of spheroids. Here, we analyzed the fusion process in connection with structure and mechanical properties of the spheroids from human somatic cells of different phenotypes: mesenchymal stem cells from the limbal eye stroma and epithelial cells from retinal pigment epithelium. A nanoindentation protocol was applied for the mechanical measurements. We found a discrepancy with the liquid drop fusion model: the fusion was faster for spheroids from epithelial cells with lower apparent surface tension than for mesenchymal spheroids with higher surface tension. This discrepancy might be caused by biophysical processes such as extracellular matrix remodeling in the case of mesenchymal spheroids and different modes of cell migration. The obtained results will contribute to the development of more realistic models for spheroid fusion that would further provide a helpful tool for constructing cell aggregates with required properties both for fundamental studies and tissue reparation.

Tepidiforma bonchosmolovskayae gen. nov., sp. nov., a moderately thermophilic Chloroflexi bacterium from a Chukotka hot spring (Arctic, Russia), representing a novel class, Tepidiformia, which includes the previously uncultivated lineage OLB14.

A novel aerobic moderately thermophilic bacterium, strain 3753OT, was isolated from a Chukotka hot spring (Arctic, Russia) using the newly developed technology of laser engineering of microbial systems. Сells were regular short rods, 0.4×0.8-2.0 µm in size, with a monoderm-type envelope and a single flagellum. The temperature and pH ranges for growth were 42-60 °C and pH 6.5-8.5, the optima being 50-54 °C and pH 7.3. Strain 3753OT grew chemoorganoheterotrophically on a number of carbohydrates or peptidic substrates and volatile fatty acids, and chemolithoautotrophically with siderite (FeCO3) as the electron donor. The major cellular fatty acid was branched C19 : 0. Phosphatidylethanolamine, phosphatidylglycerol and two unidentified phospholipids as well as two yellow carotenoid-type pigments were detected in the polar lipid extract. Strain 3753OT was inhibited by chloramphenicol, polymyxin B, vancomycin, streptomycin, neomycin and kanamycin, but resistant to the action of novobiocin and ampicillin. The DNA G+C content was 69.9 mol%. The 16S rRNA gene as well as 51 conservative protein sequence-based phylogenetic analyses placed strain 3753OT within the previously uncultivated lineage OLB14 in the phylum Chloroflexi. Taking into account the phylogenetic position as well as phenotypic properties of the novel isolate, the novel genus and species Tepidiforma bonchosmolovskayae gen. nov., sp. nov., within the Tepidiformaceae fam. nov., the Tepidiformales ord. nov. and the Tepidiformia classis nov. are proposed. The type strain of Tepidiforma bonchosmolovskayae is 3753OT (=VKM B-3389T=KTCT 72284T).

Two-Photon-Induced Microstereolithography of Chitosan-g-Oligolactides as a Function of Their Stereochemical Composition.

Chitosan-g-oligolactide copolymers with relatively long oligolactide grafted chains of various stereochemical compositions have been synthetized via a solvent-free mechanochemical technique and tailored to fabricate three-dimensional hydrogels using two-photon induced microstereolithography. An effect of the characteristics of chitosan and oligolactide used for the synthesis on the grafting yield and copolymer's behavior were evaluated using fractional analysis, FTIR-spectroscopy, dynamic light scattering, and UV-spectrophotometry. The lowest copolymer yield was found for the system based on chitosan with higher molecular weight, while the samples consisting of low-molecular weight chitosan showed higher grafting degrees, which were comparable in both the cases of l,l- or l,d-oligolactide grafting. The copolymer processability in the course of two-photon stereolithography was evaluated as a function of the copolymer's characteristics and stereolithography conditions. The structure and mechanical properties of the model film samples and fabricated 3D hydrogels were studied using optical and scanning electron microscopy, as well as by using tensile and nanoindenter devices. The application of copolymer with oligo(l,d-lactide) side chains led to higher processability during two-photon stereolithography in terms of the response to the laser beam, reproduction of the digital model, and the mechanical properties of the fabricated hydrogels.