Laser nanoengineering of biocompatible scaffolds and microbial systems
Currently, the use of laser technology for three-dimensional bio-printing or manipulation of biological objects is one of the most important trends in modern science. Laser radiation allows highly effective dosed local impact with high performance on the required objects. The reliability of modern equipment allows to carry out the necessary process around the clock for a long time. This work is aimed at the development of this technology and methods for the subsequent implementation of the results in the field of regenerative medicine and pharmacy.
Part 1. Formation biocompatible scaffolds by two-photon femtosecond micro stereolithography.
Technology for creating biocompatible tissue-engineering structures using three-dimensional printing method is currently experiencing exponential growth. Early experiments in this direction have shown in practice possible to obtain fragments of different tissues and organs. The decisive step in the development of organ recovery technology is the ability to create a biocompatible porous matrix - "scaffolds". Scaffolds for tissue engineering be used as a temporary substrate for attachment and growth of cells and must correspond a lot of requirements, such as biocompatibility, bioresorbable, compliance with specified mechanical properties.
One of the most of perspective methods for solving this problems is two-photon femtosecond photopolymerization. This method allows to carry out with high efficiency process of radical polymerization of micron size area to filling a volume of scaffold for by three-dimensional model using the femtosecond laser. We used the photo sensitivities compositions based on chitosan and its copolymers with photoinitiator Irgacure 2959 (Ciba ®), and with the addition of various crosslinkers and photo sensitivity polylactides materials.
Fig.1 Photo and a schematic of the two-photon laser microstereolithography.
The complex of two-photon femtosecond laser micro-stereolithography (Fig. 1) has been created within the work. We used second harmonic laser «TeMa-100» (Avesta-Project, Russia) (Fig. 1a) as a femtosecond light source. Laser radiation was controlled by acousto-optical modulator (Fig. 1b) on frequencies up to 1 MHz. To adjust the output power used optical system (Fig. 1c), consisting of a half-wave plate, a polarizing beam-splitting cube and power meter. As a focusing element used microscopic planar lenses, mounted on galvoscanner (Fig. 1e), which provides high-speed movement of the focused laser beam in the focal plane of the microscope objective. Galvoscanner is mounted on the Z-precision of three-axis table motions ABL-1000 (Aerotech, USA), which provides a change of the laser focus in the required plane using a CCD camera with a objective (Fig. 1d). The submicron positioning accuracy of the system was provided by the movement of the table along the rails on an air bearing using linear brushless servomotor, feedback presence, controlling the position with an accuracy of nanometers and a massive granite base, mounted on viboisolated optical table. A photosensitive composition was placed on a glass slide, was limited to a silicone spacer and a cover glass (Fig. 1f), through which the laser light delivered by a given program in accordance with computer model.
In the center of the focused laser light is a region with the shape of ellipsoid prolate in the vertical axis. Inside this region by the influence of high-intensity femtosecond laser pulses took place two-photon absorption, causing the collapse of the photoinitiator into radicals that trigger the process of three-dimensional cross-linking (Fig. 1g). Depending on the used microscope lens height crosslinkable region was 1-8 µm, and the diameter of the cross section along the XY axes was 0.3-4µm. Area size also depends on the size of the laser power. Filling volume polymerized structure was made in layers-by-layers. With the help of galvoscanner laser beam filled area of each layer separate lines for a program at a high speed. Then, the focus shifted in the vertical plane through the axis Z-translator and start irradiated next layer. Thus, the whole volume of three-dimensional model was filled by creating unit volumes of the structures of the polymerized composition inside the starting material. Then, with the X-Y translator sample shifted to a new location and repeat the process a single structure polymerization. According to the described method create an array of contiguous structures polymerized unit predetermined configuration, providing quick filling of the entire volume scaffold with micron or submicron resolution and high productivity.
In the first stage, as unitary structures used model of a hollow cylinder with an outer diameter equal to the diameter of the microscopic field 20x lens - 250 mm (Fig. 2). Structure printed in a hexagonal order with little overlap with each other, forming a periodic porous scaffold.
Fig.2 Image of unit structure (left) and model of scaffold (right).
To increase the productivity of manufacturing scaffolds, as a single unit structures was decided to use a microscopic lens with a smaller increase, but with a larger diameter of the field (400mkm). It is possible to use more complex unit structure (Fig. 3), Through which matrixes formed for model experiments on prototypes tsitosovmestimost materials and scaffolds for the cultivation of neuronal cells.
Fig.3 Image of unit structures used for experiments on cytocompatibility scaffolds (left) and model used for the cultivation of neural scaffolds for preclinical testing (right).
Currently, work is underway to create scaffolds from other unit models with a complex structure that allows, controllable place different cell cultures in the internal volume the scaffold. Work is under the selection of the optimal parameters of scaffolds, for example for different cell cultures - mesenchymal stem cell or neuronal cells. The results of this work will be presented at the conference.
Part 2. Application of laser bioprintinga for manipulating microbiological objects.
Two years ago, a group of scientists (Ringeisen et al, 2014) demonstrated the possibility of cultivation of microorganisms in the soil microportions isolated using laser radiation. To do this, they used a method based on laser bioprinting (Laser-assisted bioprinting, LAB). In laser bioprintinge biomolecules and living cells are mixed with a hydrogel and applied on a glass plate with a laser radiation absorbing thin metal layer. This substrate is called the donor, inverted and subjected to laser irradiation. A laser pulse is absorbed in the metal layer, the metal evaporates and between the glass plate and hydrogel the steam bubble is formed. The expansion and subsequent collapse of the bubble leads to the formation of a hydrogel spray. Thus, each laser pulse results in the transfer of the printed droplets with a donor material to the receiving plate disposed in parallel or in a well of a standard microbiological plate. The above mentioned group of scientists made the transfer of microportions soil with diameter from 170 to 430 micrometers on the glass or in the culture medium, using ultraviolet laser radiation. Due to the nature of the experiment - the transport of dry soil, there was a strong sprinkling of soil, the amount of transferred particles was great, it is not possible to talk about the isolation of individual colonies of microorganisms, and it was not received definitive data on the survival of microorganisms.
In this work we propose a new method of laser transfer of soil particles in a liquid medium. It is applicable to any soil, provides vitality, allows you to transfer a given concentration of the soil particles. It was designed and engineered plant-based fiber laser infrared (NTO "IRE-Polus", Russia) and galvoscanner with F-theta lens with a working field of 100 * 100mm and motorized XY table for moving the receiving glass plate or a standard microbiological plate.
Fig.4 Photography of prototype system with mounted microbiological tablet (left) and scheme of transfer soil particles (right).
In this configuration, experimental system allowed to carry out high-speed transfer of small soil particles, operating a laser beam with a spot diameter at the focus ~ 30 µm with adjustable pulse duration (from 4 to 100 ns) and pulse energy (from 10-100 µJ).
Fig.5 Photo of Petri dishes with the nutrient medium after laser transfer array drops of soil mixture with water (left) and a mixture of water and soil hydrogel (right). Insets show pictures of the results of experiments with the transfer of the soil on a glass plate.
Decisive importance in the laser transfer process is the energy of the laser pulse. There is a threshold below which the soil transfer was not observed. With the laser pulse energy increases (Fig. 6), the increase in droplet size observed, as demonstrated through experiments on the receiving plates. Also, an increase leads to disruption of the droplet shape and appearance of the spray, which is associated with an increase in speed of the jet and the violation of its laminar.
Fig.6 Photo of drops soil mixture with hydrogel after transfer of laser with different energies of the laser radiation.
A result of experiments were identified modes of laser printing by soil particles with microorganisms in the liquid and solid culture media, optimized modes of laser system with finding the laser energy threshold for effective transfer. The results of analysis of direct sowing data on solid agar growth medium were obtained. Spend the processing parameters of the structural biodiversity accounted forms. The results of evaluation of the functional biodiversity of microbial objects received via MCT method, show significant differences in the structural and functional characterization of microbial communities chernozem soil due to the impact of laser transfer procedure.
Fig.7 Distribution of the groups of microorganisms in percentage using a laser transfer method (left) and the classical method of seeding (right).
According to an analysis of more than 600 colonies revealed significant differences on the basis of the analysis of phenotypic traits (by direct microscopy) and KOH reaction distributions Gram-positive and Gram-negative organisms to the classical method of planting and seeding and laser transfer of microorganisms (Fig. 7)
Fig.8 The three-dimensional model of the second prototype of the installation of the laser transfer (left) and install the photo in the process of creating, as of May 2016 (right).
At the current moment a new version of the installation, allowing to carry out the process of the transfer of laser to operate in a fully automatic mode. System adapted to work with the 1 or 2 standard microbiological tablets. Installation of the process allows for working at the same time with several substrates with different types of objects supported on different receiving plate or in specified wells microbiological plate. The possibility of of observation through a digital camera with a microobjective on the samples before, during and after exposure. It is possible to select the diameter of the laser beam spot is in the range of tens to hundreds of micrometers, the ability to fine tune the laser power. The design is maximally adapted to operate in a laminar flow hood. As of May 2016 is in the final assembly and adjustment.