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.

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.

Reinforced Hybrid Collagen Sponges for Tissue Engineering.

We created an anisotropic material based on collagen sponge and reactive polylactide structured by laser photopolymerization. The combination of collagen with reactive polylactide improves the resistance of the formed matrices to biodegradation in comparison with collagen sponge, while the existence of sites with different mechanical characteristics and cell affinity on the matrix provides directed cell growth during their culturing. It was shown that reinforcement of the collagen sponges 7-fold increased the mean Young's modulus for the hybrid matrix without affecting its cytotoxicity. The developed matrix provides cell adhesion and proliferation along reinforcement lines and can be used for fabrication of tissue engineering constructs.

The role of laser power and pullback velocity in the endovenous laser ablation efficacy: an experimental study.

Endovenous laser ablation is an effective and minimally invasive alternative to surgical removal of incompetent veins. However, many controversies concerning optimal laser parameters usage in this procedure still remain. The purpose of this experimental study was to assess the adequate parameters required for vein wall destruction and to evaluate the role of fiber pullback velocity on vessel wall degradation. Varicose vein segments were treated with 1470-nm diode laser 3 to 9.5 W in power. The fiber moved through the vein at a velocity of 0.7 or 1.5 mm/s; the applied linear endovenous energy density (LEED) was 40-95 J/cm. The temperature of the vein surface in the course of laser irradiation was controlled by IR thermography. The intact collagen in treated vein specimens was studied by differential scanning calorimetry. The increase in the surface temperature with applied energy was found to be about three times slower for the pullback velocity of 0.7 mm/s than that of 1.5 mm/s. The collagen in the tissue was totally denatured in the case of the surface temperature of about 91 °C. The critical values of LEED ensured complete degradation of vein wall were of 53 and 71.5 J/cm for velocities of 1.5 and 0.7 mm/s, respectively. Our experimental study supports the conception that it is laser power and pullback velocity rather than LEED value that determine the temperature as well the collagen framework degradation and therefore the thermal response of procedure.

Reconstruction of Ligament and Tendon Defects Using Cell Technologies.

We studied the possibility of restoring the integrity of the Achilles tendon in rabbits using autologous multipotent stromal cells. Collagen or gelatin sponges populated with cells were placed in a resorbable Vicryl mesh tube and this tissue-engineered construct was introduced into a defect of the middle part of the Achilles tendon. In 4 months, histological analysis showed complete regeneration of the tendon with the formation of parallel collagen fibers, spindle-shaped tenocytes, and newly formed vessels.

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.

Comparative Analysis of Proliferation and Viability of Multipotent Mesenchymal Stromal Cells in 3D Scaffolds with Different Architectonics.

3D biodegradable materials (scaffolds) containing bioactive hydroxyapatite molecules fabricated by foaming in supercritical carbon dioxide and by selective laser sintering were used for culturing of mesenchymal stromal cells from the human adipose tissue. Experiments showed that stromal cells from the human adipose tissue adhered and proliferated on all studied types of structures. Addition of hyproxyapatite to the scaffold stimulated proliferation of stromal adipose tissue cells.

Formation of Neural Networks in 3D Scaffolds Fabricated by Means of Laser Microstereolithography.

We developed and tested new 3D scaffolds for neurotransplantation. Scaffolds of predetermined architectonic were prepared using microstereolithography technique. Scaffolds were highly biocompatible with the nervous tissue cells. In vitro studies showed that the material of fabricated scaffolds is not toxic for dissociated brain cells and promotes the formation of functional neural networks in the matrix. These results demonstrate the possibility of fabrication of tissue-engineering constructs for neurotransplantation based on created scaffolds.

[Compatibility of cells of the nervous system with structured biodegradable chitosan-based hydrogel matrices].

Hydrogel matrices for cell cultivation have been generated by two-photon laser polymerization of unsaturated chitosan derivatives and methacrylated hyaluronic acid. The adhesive and toxic properties of the matrices have been assessed, and the matrices have been shown to have a good compatibility with primary hippocampal cell cultures. The formation of morphologically normal neural networks by cells of the nervous system cultured on the surface of hydrogel matrices has been observed. The metabolic status of dissociated hippocampal cells cultured on the matrices was similar to that of the control cultures, as shown by the results of MTT reductase activity assay. Thus, matrices based on unsaturated polysaccharide derivatives crosslinked by laser irradiation showed good compatibility with differentiated cells of the nervous system and considerable potential for use in neurotransplantation.

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.

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.

Design of tissue engineering implants for bone tissue regeneration of the basis of new generation polylactoglycolide scaffolds and multipotent mesenchymal stem cells from human exfoliated deciduous teeth (SHED cells).

Cultures of multipotent mesenchymal stromal cells from the pulp of human deciduous teeth (SHED cells) were characterized. The cells were used for population of 3D biodegradable polylactoglycolide scaffolds; their osteogenic potential was preserved under these conditions. Implantation of the scaffolds to mice induced no negative reactions in the recipients. These results suggest that the use of polylactoglycolide scaffolds populated with SHED cells is a promising approach for creation of implants for bone defect replacement.

Biocompatibility of tissue engineering constructions from porous polylactide carriers obtained by the method of selective laser sintering and bone marrow-derived multipotent stromal cells.

We studied the biocompatibility of porous polylactide carrier matrices obtained by means of surface selective laser sintering. Carrier matrices had no cytotoxic activity, but maintained adhesion and proliferation of cells. Subcutaneous transplantation of tissue engineering constructions from these carriers and bone marrow-derived multipotent stromal cells did not cause the inflammatory response and pathological changes in rats. The conditions for organotypic regeneration were provided at the site of transplantation (high degree of blood supply and considerable amount of immature precursor cells).

[Decreased collagen stability as a response to the loss of structural integrity of thyroid cartilage].

The thermal behavior, birefringence properties, and the biochemical composition of thyroid cartilage tissues have been studied. The hyaline cartilage, which was visualized as a quasi-isotropic medium, was composed of type II collagen, which did not denature at temperatures up to 100 degrees C. However, in hyaline cartilage digested by trypsin, the denaturation of collagen occured at 60 degrees C. Collagen fibers in the perichondrium were composed of type I and II collagen and formed a highly organized anisotropic structure (birefringence about 4.75 x 10(-3)) with a melting temperature of about 65 degrees C. The temperature of collagen denaturation in perichondrium in the whole system perichondrium-hyaline cartilage increased up to 75 degrees C, indicating the immobilization of perichondrium collagen by the extracellular matrix of the hyaline constituent.

Osteoinducing scaffolds with multi-layered biointerface.

This study was aimed to design and characterise hybrid tissue-engineered constructs composed of osteoinducing polylactide-based scaffolds with multi-layered cellular biointerface for bone tissue reconstruction. Three-dimensional scaffolds with improved hydrophilic and osteoinducing properties were produced using the surface-selective laser sintering (SSLS) method. The designed scaffold pattern had dimensions of 8 × 8 × 2.5 mm and ladder-like pores (∼700 µm in width). Hyaluronic acid-coated polylactide microparticles (∼100 µm in diameter) were used as building blocks and water was used as the photosensitizer for SSLS followed by photocross-linking with Irgacure 2959 photoinitiator. Resulting scaffolds provided successful adhesion and expansion of human bone marrow mesenchymal stromal cells from a single-cell suspension. Induced calcium deposition by the cells associated with osteogenic differentiation was detected in 7-21 days of culturing in basal medium. The values were up to 60% higher on scaffolds produced at a higher prototyping speed under the experimental conditions. Innovative approach to graft the scaffolds with multi-layered cell sheets was proposed aiming to facilitate host tissue-implant integration. The sheets of murine MS-5 stromal cell line exhibited contiguous morphology and high viability in a modelled construct. Thus, the SSLS method proved to be effective in designing osteoinducing scaffolds suitable for the delivery of cell sheets.

Surface micromorphology of cross-linked tetrafunctional polylactide scaffolds inducing vessel growth and bone formation.

In the presented study, we have developed a synthetic strategy allowing a gradual variation of a polylactide arms' length, which later influences the micromorphology of the scaffold surface, formed by a two-photon polymerization technique. It has been demonstrated that the highest number of cells is present on the scaffolds with the roughest surface made of the polylactide with longer arms (PLA760), and osteogenic differentiation of mesenchymal stem cells is most pronounced on such scaffolds. According to the results of biological testing, the PLA760 scaffolds were implanted into a created cranial defect in a mouse for an in vivo assessment of the bone tissue formation. The in vivo experiments have shown that, by week 10, deposition of calcium phosphate particles occurs in the scaffold at the defect site, as well as, the formation of a new bone and ingrowth of blood vessels from the surrounding tissues. These results demonstrate that the cross-linked microstructured tetrafunctional polylactide scaffolds are promising microstructures for bone regeneration in tissue engineering.

Atomic force microscopic study of the surface morphology of apatite films deposited by pulsed laser ablation.

Atomic force microscopy (AFM) has been used to study the surface morphology of apatite films deposited on metallic and polyethylene substrates by laser ablation using KrF and transversely excited atmospheric CO2 lasers. The films are found to consist of a smooth apatite coating with macroparticles scattered on the surface. A wide variety of macroparticles, differing in size, shape and roughness, were found and analysed employing the high spatial resolution of AFM (< 1 nm). We have investigated the correlation between the apatite film morphology and the deposition conditions. Of particular importance are laser fluence, gas pressure, the nature of the target and the substrate temperature. We have explained these dependencies on the basis of a theoretical model which includes evaporation and a cluster-type laser ablation mechanism.

Osteoblast growth on titanium foils coated with hydroxyapatite by pulsed laser ablation.

Pulsed laser ablation is a new method for deposition of thin layers of hydroxyapatite (HA) on to biomaterial surfaces. In this paper, we report activity and morphology of osteoblasts grown on HA surfaces fabricated using different laser conditions. Two sets of films were deposited from dense HA targets, at three different laser fluences: 3, 6 and 9 Jcm(-2). One set of the surfaces was annealed at 575 degrees C to increase the crystallinity of the deposited films. Primary human osteoblasts were seeded onto the material surfaces and cytoskeletal actin organisation was examined using confocal laser scanning microscopy. The annealed surfaces supported greater cell attachment and more defined cytoskeletal actin organisation. Cell activity, measured using the alamar Blue assay, was also found to be significantly higher on the annealed samples. In addition, our results show distinct trends that correlate with the laser fluence used for deposition. The cell activity increases with increasing fluence. This pattern was repeated for alkaline phosphatase production by the cells. Differences in cell spreading were apparent which were correlated with the fluence used to deposit the HA. The optimum surface for initial attachment and spreading of osteoblasts was one of the HA films deposited using 9 J cm(-2) laser fluence and subsequently annealed at 575 degrees C.

Biocompatibility of Laser-deposited Hydroxyapatite Coatings on Titanium and Polymer Implant Materials.

We have investigated the biocompatibility of calcium phosphate coatings deposited by pulsed laser ablation from hydroxyapatite (HA) targets onto polyethylene and Teflon substrates. It was found that the cell density, attachment, and morphology of primary rat calvaria osteoblasts were influenced by both the original polymer and by the nature of the apatite coatings. HA coatings on Teflon were found to have higher biocompatibility in terms of cell adhesion and spreading. In vivo studies of bone response to coatings deposited by KrF excimer and CO2 lasers on commercial Ti6A14V alloy implants show that both deposition techniques suppress fibrous tissue formation and promote osteogenesis. © 1998 Society of Photo-Optical Instrumentation Engineers.