The technology of cell 3D scaffolds laser fabrication is developed. 3D scaffolds are designed to repair osteochondral defects, which are poorly restored during the organism’s life. The technology involves the use of an installation, the laser beam of which moves along a liquid nanomaterial and evaporates it layer by layer. Liquid nanomaterial consists of the water-protein (collagen, albumin) suspension with carbon nanoparticles (single-walled carbon nanotubes). During laser irradiation, the temperature in the region of nanotubes defects increases and nanotubes are combined into the scaffold. The main component of installation is a continuous laser operating at wavelengh of 810 nm. The laser beam moves along 3 coordinates, which makes it possible to obtain samples of the required geometric shape. The internal and surface structure of the samples at the micro- and nanoscale levels were studied using the X-ray microtomography and scanning electron microscopy. In vitro studies of cell growth during 48 and 72 hours demonstrated the ability of cell 3D scaffolds to support the proliferation of osteoblasts and chondroblasts. Using fluorescence and atomic force microscopy, it was found that the growth and development of cells on a sample with a larger concentration of nanotubes occurred faster compared to samples with a smaller concentration of nanotubes.
A reliable connection of dissected biological tissues is a popular problem in modern surgery. In the last decade, two methods of biological tissues connection using laser radiation have been actively developed: laser-assisted vascular repair (LAVR) and anastomosis (LAVA). These methods make it possible to obtain a weld impenetrable for blood and other biological fluids immediately after the welding. A solder is applied to a welding area. The main characteristic of the weld at LAVA is the tensile strength. A weld should be flexible enough to withstand repeated cycles of alternation of diastolic and systolic pressures. Single-walled and multi-walled carbon nanotubes (SWCNTs and MWCNTs respectively) are used to increase the weld tensile strength. They form a spatial bovine serum albumin (BSA)- nanocarbon scaffold under an action of laser radiation. It in real time varies the power of laser radiation (in the range 0.2- 5 W), which is necessary to stabilize the welding temperature (~57 ºC). In the present work new compositions of laser solder are proposed and the scheme of the installation for LAVR are offered. The proposed solder is based on an aqueous dispersion of BSA, MWCNTs and SWCNTs, ICG and collagen. The using of the chromophore ICG is due to its absorption maximum corresponding to the wavelength of the diode laser used (~ 810 nm). The tensile strength was 0.8±0.3 MPa.
The conductivity of layers (thickness ~ 0.5-20 μm) of composite nanomaterials consisting of bovine serum albumin (BSA) with single-walled carbon nanotubes (SWCNTs) has been studied. The BSA/SWCNT composite nanomaterial was prepared according to a route map, some steps of which are: the preparation of an aqueous dispersion based on BSA and SWCNT; preparation of substrates; deposition of BSA/SWCNT dispersion on substrates; application of water paste from SWCNT on substrates; irradiation of layers by lasers when they were in a liquid state; drying of samples; carrying out electrical and temperature measurements. Half of the layer was covered with a light-tight hollow box and the other half of the layer was laser irradiated. The laser irradiation of the layer was carried out for about 20 sec, at which time the layers completely became dry, while the other half of the layer remained in liquid. Conductivity was increased (70 ÷ 650) % by laser irradiation of the layers when they were in the liquid state. Maximum values of specific conductivity for BSA/SWCNT-1 S/m layers, and for layers SWCNT - 70 kS/m. The investigated electrically conductive layers of 99 wt.% BSA/0.3 wt.% SWCNT are promising for medical practice.
Laser radiation limiters can be made on the basis of working substances, which have strong nonlinear effects after reaching a certain critical value (threshold limiting). Thus, it becomes possible to obtain a high transmission for a safe beam and a sharply reduced transmission for a hazardous beam. To determine the nonlinear and linear optical properties of these materials there were carried out comprehensive spectroscopic studies, experiments by Z-scan methods with an open aperture and a fixed location of the limiter. Working substances was developed which is suspension of conjugates J-type phthalocyanine dimers Zn or Mg with single-walled carbon nanotubes (SWCNTs) in water. Created conjugates can be used not only for protecting eyes and light-sensitivity elements, but for forming three-dimensional tissueengineered structures. Using conjugates J-type phthalocyanine dimers Zn and Mg with SWCNTs will increase the optical absorption in the wavelength range of laser processing by reducing the thermal effect on other substances in the composition of this structure. The Nd:YAG laser was used as the laser radiation source for generating pulses of 16 ns duration at a wavelength of 532 nm with the linearly polarized laser beam in the horizontal plane and a shape of Gaussian type. The threshold of limiting, linear and nonlinear absorption coefficients were determined by output characteristic, that was obtained by fixed location of the limiter. Created working substances have values of the following order: linear absorption coefficient ~ 3 cm-1 for layer of 0.2 cm thickness, low limiting threshold ~ 1 MW·cm-2 and high value of the nonlinear absorption coefficient ~ 550 cm GW-1 . Knowing the nonlinear optical parameters, Z-scan data with an open aperture can be calculated for comparison with experimental data.
The study of structural properties of nanocomposites, based on different types of single walled carbon nanotubes (SWCNTs) and proteins (albumin, collagen), was carried out. The binding of protein molecules to the carbon component was described by Raman spectroscopy. Complex analysis of the structure and microporosity of nanocomposites was performed by the X-ray microtomography. The nanoporosity study was carried out using the low-temperature nitrogen porosimetry method. Samples based on SWCNTs with smaller size had the most homogeneity. With an increase in the concentration from 0.01 to 0.1 %, the mean micropore size increased from 45 to 93 μm, porosity in general increased from 16 to 28 %. The percentage of open pores was the same for all samples and was 0.02. As it was shown by Raman spectroscopy the protein component in nanocomposites has undergone irreversible denaturation and can act as a biocompatible binder and serve as a source of amino acids for biological tissues. These nanocomposites are bioresorbable and can be used to repair cartilage and bone tissue. This is especially important in the treatment of diseases of hyaline cartilage and subchondral bone.
Creation of limiters for intensive laser radiation requires the development of effective methods for testing materials to determine the nonlinear optical parameters characterizing their properties. The limiting threshold, linear and nonlinear absorption coefficients can be determined not only from data of Z-scan with open aperture, but also with the help of a fixed location of the limiter. The use of this method makes it possible to determine the output characteristic of the studied material from which nonlinear optical parameters can be calculated. Characteristics of carbon nanotubes and graphene oxide in water were obtained with the fixed location of the limiter. The experiments were performed using an Nd:YAG laser that generates pulses of 16 ns duration at a wavelength of 532 nm with the linearly polarized laser beam in the horizontal plane and a shape closed to Gaussian type. Theoretical curves for method of fixed location of the sample according to threshold model was calculated and compared with the experimental data. Normalized weakening coefficients, limiting threshold, linear and nonlinear absorption coefficients were found for studied dispersions and calculation of Z-scan with open aperture was made. The value of normalized weakening coefficient was higher in dispersed medium of SWСNTs with water (Knorm≈20) in comparison with oxide graphene in water (Knorm≈14). The dependences of normalized weakening coefficient bias input energy were approximately linear in both cases.
Increase the weld strength is main directions of development of laser welding technology. Laser solders are used to increase tensile strength of welds and reduce of tissue temperature necrosis. Soldering components interaction effect the solder tensile strength characteristics of laser welds. Tensile strengths for welds obtained using of solder various concentration BSA and SWCNT was measured. Dimensions laser solder aggregates were measured. The dependence between the dimensions of the aggregates of laser solder and the tensile strength of the weld has been revealed.
The conductivity of layers (thickness 0.5 ÷ 50 μm) of composite nanomaterials consisting of bovine serum albumin (BSA) with single-walled carbon nanotubes (SWCNTs) has been studied. The aqueous dispersion of BSA / SWCNT was deposited on different substrates using the silk screening method. Conductivity was increased (30 ÷ 700) % by laser irradiation of the layers when they were in the liquid state. The investigated layers are promising for use in medical practice.
The results of experimental creation of nanocomposites using femtosecond laser are presented. We have theoretically proved the formation of a carbon nanotube frame in a protein matrix during laser structuring of single-walled carbon nanotubes. We have selected the technological parameters of synthesis of nanocomposites, which provide the proliferation of living cells.
This paper presents the composite biostructures created by laser structuring of the single-walled carbon nanotubes (SWCNTs) in an albumin matrix. Under the exposure of femtosecond laser radiation, the heating of the albumin aqueous solution causes liquid water to evaporate. As a result, we obtained a solid-state composite in the bulk or film form. Using the molecular dynamic method, we showed the formation of a framework from SWCNTs by the example of splicing of the open end of one nanotube with the defect region of another nanotube under the action of the laser heating. Laser heating of SWCNTs up to a temperature of 80°C to 100°C causes the C─C bond formation. Raman spectra measured for the composite biostructures allowed us to describe the binding of oxygen atoms of amino acid residues of the albumin with the carbon atoms of the SWCNTs. It is found that the interaction energy of the nanotube atoms and albumin atoms amounts up to 580 kJ/mol. We used atomic force microscopy to investigate the surface of the composite biostructures. The pore size is in the range of 30 to 120 nm. It is proved that the proliferation of the fibroblasts occurred on the surface of the composite biostructures during 72 h of incubation.
3-D bioconstructions were created using the evaporation method of the water-albumin solution with carbon nanotubes (CNTs) by the continuous and pulsed femtosecond laser radiation. It is determined that the volume structure of the samples created by the femtosecond radiation has more cavities than the one created by the continuous radiation. The average diameter for multi-walled carbon nanotubes (MWCNTs) samples was almost two times higher (35-40 nm) than for single-walled carbon nanotubes (SWCNTs) samples (20-30 nm). The most homogenous 3-D bioconstruction was formed from MWCNTs by the continuous laser radiation. The hardness of such samples totaled up to 370 MPa at the nanoscale. High strength properties and the resistance of the 3-D bioconstructions produced by the laser irradiation depend on the volume nanotubes scaffold forming inside them. The scaffold was formed by the electric field of the directed laser irradiation. The covalent bond energy between the nanotube carbon molecule and the oxygen of the bovine serum albumin aminoacid residue amounts 580 kJ/mol. The 3-D bioconstructions based on MWCNTs and SWCNTs becomes overgrown with the cells (fibroblasts) over the course of 72 hours. The samples based on the both types of CNTs are not toxic for the cells and don't change its normal composition and structure. Thus the 3-D bioconstructions that are nanostructured by the pulsed and continuous laser radiation can be applied as implant materials for the recovery of the connecting tissues of the living body.
Laser welding device for biological tissue has been developed. The main device parts are the radiation system and adaptive thermal stabilization system of welding area. Adaptive thermal stabilization system provided the relation between the laser radiation intensity and the weld temperature. Using atomic force microscopy the structure of composite which is formed by the radiation of laser solder based on aqua- albuminous dispersion of multi-walled carbon nanotubes was investigated. AFM topograms nanocomposite solder are mainly defined by the presence of pores in the samples. In generally, the surface structure of composite is influenced by the time, laser radiation power and MWCNT concentration. Average size of backbone nanoelements not exceeded 500 nm. Bulk density of nanoelements was in the range 106-108 sm-3. The data of welding temperature maintained during the laser welding process and the corresponding tensile strength values were obtained. Maximum tensile strength of the suture was reached in the range 50-55°C. This temperature and the pointwise laser welding technology (point area ~ 2.5mm) allows avoiding thermal necrosis of healthy section of biological tissue and provided reliable bonding construction of weld join. In despite of the fact that tensile strength values of the samples are in the range of 15% in comparison with unbroken strips of pigskin leather. This situation corresponds to the initial stage of the dissected tissue connection with a view to further increasing of the joint strength of tissues with the recovery of tissue structure; thereby achieved ratio is enough for a medical practice in certain cases.
A new method for the formation of composite nanomaterials based on multi-walled and single-walled carbon nanotubes (CNT) on a silicon substrate has been developed. Formation is carried out by ultrasound coating of a silicon substrate by homogenous dispersion of CNTs in the albumin matrix and further irradiation with the continuous laser beam with a wavelength of 810 nm and power of 5.5 watts. The high electrical conductivity of CNTs provides its structuring under the influence of the laser radiation electric field. The result is a scaffold that provides high mechanical strength of nanocomposite material (250 MPa). For in vitro studies of materials biocompatibility a method of cell growth microscopic analysis was developed. Human embryonic fibroblasts (EPP) were used as biological cells. Investigation of the interaction between nanocomposite material and cells was carried out by optical and atomic force microscopy depending on the time of cells incubation. The study showed that after 3 hours incubation EPP were fixed on the substrate surface, avoiding the surface of the composite material. However, after 24 hours of incubation EPP fix on the sample surface and then begin to grow and divide. After 72 hours of incubation, the cells completely fill the sample surface of nanocomposite material. Thus, a nanocomposite material based on CNTs in albumin matrix does not inhibit cell growth on its surface, and favours their growth. The nanocomposite material can be used for creating soft tissue implants
Dispersive and composite nanomaterials based on multi-walled and single-walled carbon nanotubes and its conjugates with dye of zinc phthalocyanine were produced. The composition and the structure of dispersive and composite materials were investigated using analytical methods of spectroscopy and microscopy. Nonlinear characteristic of nanomaterials of limiters by direct nonlinear scanning and Z-scan method were investigated. Studies suggest the possibility of using such nanomaterials in laser intensity limiters. Proposed threshold model characterizing limiters of powerful laser radiation takes into account the threshold nature of nonlinear interaction of irradiation with the nonlinear material. Threshold effect of nonlinear interaction of laser irradiation with several nonlinear material based on multi-walled and single-walled carbon nanotubes was experimentally found. It was shown that threshold model fit better with experimental data of Z-scan.
The important problem of modern laser medicine is the decrease of an exposure of biological tissues outside of an operational field and can be solved by optical radiation limiting. Organic dyes with reversibly darkening can be placed onto surfaces of irradiated tissues or can be introduced in solder for laser welding of vessels.
The limiting properties of a set of nontoxic organic compounds were investigated. Nonlinear optical properties of dyes having reverse saturable absorption (pyran styryl derivatives, cyanine and porphyrine compounds) were studied under XeCl and YAG:Nd (II harmonics) lasers excitation. The effect of attenuation of a visible laser radiation is obtained for ethanol solutions of cyanines: radiation attenuation coefficient ( AC) = 25-35 at N/S = 100-250 MW/cm2. In water solutions of such compounds in UV spectrum range AC ≈ 10. The spectral characteristics of compounds appeared expedient enough to operational use in laser limiters (broad passband in visible range of a spectrum). Under the data of Z-scanning (the scheme F/10) value AC ≈ 70 was reached. The limiting of power laser radiation in visible (λ = 532 nm) and UV- (λ = 308 nm) spectral region and nanosecond pulse duration (7 -13 ns) across porphyrine solutions and their complexes with some metals (13 compounds) was investigated too. The comparative study of optical limiting dependence on intensity of laser radiation, solvent type and concentration of solutions was carried out for selecte wavelength. There was shown a possible use of pyran styryl derivatives DCM as limiters of visual laser radiation.
To understand a mechanism of laser radiation limitation the light induced processes were experimentally and theoretically studied in organic molecules. The quantum-chemical investigation of one cyanine compound was carried out.
There were noted the perspectives of laser radiation limiting by application of inverted schemes of traditional laser shutters. Usage of phenomena of light -induced opalescence in one-component liquids and spinodal decay in stratifying liquid solutions is proposed.
Liquids used for cooling, filtration and transformation of radiation in laser pump systems, projection TV sets and IR searchlights were described. The most effective liquid types were shown. Specific features of physical and physico- chemical processes at resonant and non-resonant interaction of broadband radiation with liquid were point out. It is demonstrated that, by an appropriate choice of the composition of liquid cooling agents and conditions of cooling, one can increase the efficiency and service time of the liquid-cooling devices.
The selection of ballistic photons from a radiation, passed through a biological medium, is necessary for obtaining qualitative images in optical tomography. Within the framework of a non-stationary two-flux model of radiation transport in high scattering medium there is found the fraction of ballistic photons as function on macroscopic characteristics of a medium. With the purpose of separation of photons the application of bisphthalocianine dyes of rare-earth and transitional elements is considered. The experiments on deriving contrasted images in margarine and water solution of milk are carried out.
The criteria were determined for simultaneous observation of scattered and unscattered (ballistic) peaks in temporal distribution of a narrow pulsed laser beam passed through a strongly scattering medium. Conditions were found for such an observation on the basis of non-stationary two-flux model for radiation transport. Calculation results permitted to define a compromise between parameters of the initial laser pulse and properties of a scattering medium. This has provided the separate observation of various types of photons. The theoretical calculations were confirmed by experiments on recording pulse profiles of passed radiation of fs-laser in a water-milk solution.
The dependence of the optical density of a model strongly scattering medium-aqueous milk solution on its layer thickness was investigated. IR lasers generating pulses of various short (from nanosecond to femtosecond) duration were used as the radiation sources. There were determined the dependences of the attenuation coefficients of such pulses on the solution concentration in the areas of low and more high optical densities of the solution layer for different values of radiation detector angular aperture. A modification of the two-flux Kubelka-Munk model was used to derive an expression describing the dependence of the transmission of a solution layer on its parameters when radiation detectors with a finite angular aperture are used. The absorption and scattering coefficients of the medium were obtained. A comparison of the calculations and experiments revealed a forward scattering anisotropy of short-duration laser radiation in an aqueous milk solution characteristic for the Mie's scattering.
We carried out the synthesis of the new compounds -- sulfonated mono- and diphthalocyanines of d- and f-elements (sPcM, sPc2M). The unusual geometry of these compounds leads to the appearance of new properties -- they are highly water soluble, non- toxic and in contrast to porphyrines they strongly absorb clinically useful light. Suitable methods for synthesis of unknown before water soluble Ga, In and Ti phthalocyanines have been developed. Our investigations show that these compounds are prospects for plastic surgery.
There are some difficulties in the use of a well-known X-ray computer tomography technique due to the strong light scattering in biological tissues. We propose to analyze the whole light scattering picture for the development of novel processing algorithms in tomographical imaging. Time-resolved experiments on the forward scattering of IR-light from a model of biological media illuminated by a picosecond YAG-laser with 10 ps pulse duration have been performed. The scattering radiation was recorded by a streak camera having 1.5 ps time resolution. From these results and theoretical considerations the analytic expression for intensity against time distribution of the laser radiation passed through the media has been obtained. The expression can be used as the basis for tomographical reconstruction.
Time profiles of picosecond Nd:YAG-laser radiation transmitted through homogeneous and inhomogeneous scattering models of biological objects contrasted by new dyphtalocyanine dyes have been investigated. These dyes have specially synthesized as the contrasting substances for biological tissues in order to increase signal-to-noise ratio in optical image processing. By using this dye contrasting technique the ratio improvement has been demonstrated in time-resolved experiments on determination of inner details for some model biological objects.
For successful application of laser tomography methods for earlier medical diagnostics the signal-to-noise ratio (contrast) must be increased. For this purpose it is possible to use the absorbing dyes. We have theoretically investigated optical imaging conditions in high scattering medium on a model object. In our experiments a YAG:Nd laser generating picosecond pulses was employed. Output radiation has been recorded by a high speed streak camera with 1.5 ps temporal resolution. The high stability of the laser and of measurement scheme characteristics was provided. We looked for the contrasting substances having tropism with pathologically changed tissue of the tumor. For this purpose some dyphthalocyanines were synthesized. The experiments with laboratory animals have demonstrated that saturated dye concentrations were noticeably lower than toxicologic dangerous concentration values. We have demonstrated a possibility of the contrasting for a model object. The experimental temporal profile of scattered radiation can be explained by the nonstationary two-flow theory.
The introduction of sulpho groups into molecule of mono- and diphthalocyanines of hafnium and zirconium gave rise to the formation of water soluble compounds. This allows us to extend the field of their applications. The dyes of this class have been found to be efficient photosensitizers for PDT of cancer.
Suitable methods for synthesis of water soluble diphthalocyanines of rare earth elements have been developed. The synthesis was carried out by direct sulphonation of corresponding unsubstituted analogues. The sulphonation degree depends on the nature of a sulphonated agent, reaction time and molecular ratio of reagents. This allows us to vary the number of sulpho groups from one to the values corresponding to full sulphonation.
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