Irina Alexandra Paun

Thin films of polymer blends for controlled drug delivery deposited by matrix-assisted pulsed laser evaporation

We demonstrate the use of matrix-assisted pulsed laser evaporation technique for incorporating drugs (indomethacin) within thin films of polymer blends, to be used as biodegradable implants that deliver drugs in a controlled manner. For irradiation at fluences up to 1 J/cm2, the films show excellent surface morphology and the chemical structure of all constituent polymers within the blend is well preserved. The optical characteristics of the films are assessed by spectroscopic ellipsometry. The presence of the drug within the polymeric films is confirmed by its specific absorption at 319 nm.

Automated imaging, identification, and counting of similar cells from digital hologram reconstructions

This paper presents our method, which simultaneously combines automatic imaging, identification, and counting with the acquisition of morphological information for at least 1000 blood cells from several three-dimensional images of the same sample. We started with seeking parameters to differentiate between red blood cells that are similar but different with respect to their development stage, i.e., mature or immature. We highlight that these cells have different diffractive patterns with complementary central intensity distribution in a given plane along the propagation axis. We use the Fresnel approximation to simulate propagation through cells modeled as spheroid-shaped phase objects and to find the cell property that has the dominant influence on this behavior. Starting with images obtained in the reconstruction step of the digital holographic microscopy technique, we developed a code for automated simultaneous individual cell image separation, identification, and counting, even when the cells are partially overlapped on a slide, and accurate measuring of their morphological features. To find the centroids of each cell, we propose a method based on analytical functions applied at threshold intervals. Our procedure separates the mature from the immature red blood cells and from the white blood cells through a decision based on gradient and radius values.

Laser micro-patterning of biodegradable polymer blends for tissue engineering

We propose a multistep all laser, maskless, and solvent free synthesis of micro-patterned substrates of biodegradable polymer blends, with applicability for guided cell adhesion and localized hyaluronic acid (HA) immobilization. The polymer blends comprised polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), and polylactide-polyethylene glycol-polylactide (PPP) in 1:1:1 blending ratios. Polymer patterning was performed by laser processing in two steps. First, the polymers were patterned with periodic micro-channels by direct femtosecond laser ablation, which provided flexibility in design and spatial accuracy for the patterns. As a second step, the micro-patterned polymers were coated with thin layers of polymer blends using matrix assisted pulsed laser evaporation (MAPLE). The resulted sandwich substrates were composed of a bottom, micro-patterned layer and thin, top layer which conserved the patterns from the underlying layer and preserved the polymers chemical composition. Depending on the bottom/top layers, the substrates were denominated PU/PU:PLGA:PPP and PU:PLGA:PPP/PU:PLGA:PPP, respectively. The laser generated micro-patterns were used for selective attachment of oral keratinocyte stem cells and for HA immobilization. The highest cellular density was found on the PU:PLGA:PPP/PU:PLGA:PPP substrate, where the spongy-like micro-channels provided multiple anchoring points for the cells. For both substrates, the micro-channels enabled localized immobilization of HA. The effectiveness of HA immobilization was tested against cell adhesion and protein adsorption.

Laser-assisted fabrication and non-invasive imaging of 3D cell-seeding constructs for bone tissue engineering

We report on laser-assisted fabrication and non-invasive imaging of porous 3D cell-seeding constructs (3D-CSCs) for bone tissue engineering. The 3D structures were built by two-photon polymerization-direct writing (2PP_DW) of IP-L780 photopolymer and consist in arrays of vertical microtubes arranged in triangular lattices. The microtubes were tightly, medium, and rarely packed, according to the constants of the triangular lattices of 8, 12, and 24 μm, respectively. The efficiency of the laser-generated 3D-CSCs for new bone formation was assessed in MG63 osteoblast-like cells cultures. High spatial resolution 3D images of the cell-seeded 3D-CSCs were obtained by digital holographic microscopy (DHM). The recorded holograms allowed the simultaneous evaluation of the 3D-CSCs and of the seeded cells, in terms of 3D shapes and dimensions, without intruding into the cells natural environment. The seeded cells, in particular the cells nuclei, conformed to the micro-architectures of the 3D-CSCs. Furthermore, the osteogenic potential of the 3D-CSCs was assessed in terms of cell morphology, viability, and level of mineralization. The microtubes packing density that allowed the seeded osteoblasts to reach the highest level of mineralization was established.

Electrically stimulated osteogenesis on Ti-PPy/PLGA constructs prepared by laser-assisted processes.

This work describes a versatile laser-based protocol for fabricating micro-patterned, electrically conductive titanium-polypyrrole/poly(lactic-co-glycolic)acid (Ti-PPy/PLGA) constructs for electrically stimulated (ES) osteogenesis. Ti supports were patterned using fs laser ablation in order to create high spatial resolution microstructures meant to provide mechanical resistance and physical cues for cell growth. Matrix Assisted Pulsed Laser Evaporation (MAPLE) was used to coat the patterned Ti supports with PPy/PLGA layers acting as biocompatible surfaces having chemical and electrical properties suitable for cell differentiation and mineralization. In vitro biological assays on osteoblast-like MG63 cells showed that the constructs maintained cell viability without cytotoxicity. At 24 h after cell seeding, electrical stimulation with currents of 200 μA was applied for 4 h. This treatment was shown to promote earlier onset of osteogenesis. More specifically, the alkaline phosphatase activity of the stimulated cultures reached the maximum before that of the non-stimulated ones, i.e. controls, indicating faster cell differentiation. Moreover, mineralization was found to occur at an earlier stage in the stimulated cultures, as compared to the controls, starting with Day 6 of cell culture. At later stages, calcium levels in the stimulated cultures were higher than those in control samples by about 70%, with Ca/P ratios similar to those of natural bone. In all, the laser-based protocol emerges as an efficient alternative to existing fabrication technologies.

MAPLE deposited polymeric blends coatings for controlled drug delivery

We report on the use of Matrix Assisted Pulsed Laser Evaporation (MAPLE) for producing coatings of polymer blends for controlled drug delivery. The coatings consisting of blends of polyethylene glycol: poly(lactide-co-glycolide) (PEG: PLGA blends) are compared with those consisting of individual polymers (PEG, PLGA) in terms of chemical composition, morphology, hydrophilicity and optical constants. The release kinetics of an anti-inflammatory drug (indomethacin) through the polymeric coatings is monitored and possible mechanisms of the drug release are discussed. Furthermore, the compatibility of the polymeric coatings with blood constituents is investigated. Finally, the perspectives for employing MAPLE for producing coatings of polymer blends to be used in implants that deliver drugs in a controlled manner, along with the routes to be followed for elucidating the mechanism of drug release, are revealed.

3D Biomimetic Magnetic Structures for Static Magnetic Field Stimulation of Osteogenesis

We designed, fabricated and optimized 3D biomimetic magnetic structures that stimulate the osteogenesis in static magnetic fields. The structures were fabricated by direct laser writing via two-photon polymerization of IP-L780 photopolymer and were based on ellipsoidal, hexagonal units organized in a multilayered architecture. The magnetic activity of the structures was assured by coating with a thin layer of collagen-chitosan-hydroxyapatite-magnetic nanoparticles composite. In vitro experiments using MG-63 osteoblast-like cells for 3D structures with gradients of pore size helped us to find an optimum pore size between 20–40 µm. Starting from optimized 3D structures, we evaluated both qualitatively and quantitatively the effects of static magnetic fields of up to 250 mT on cell proliferation and differentiation, by ALP (alkaline phosphatase) production, Alizarin Red and osteocalcin secretion measurements. We demonstrated that the synergic effect of 3D structure optimization and static magnetic stimulation enhances the bone regeneration by a factor greater than 2 as compared with the same structure in the absence of a magnetic field.

Laser Structuring of Soft Materials: Laser-Induced Forward Transfer and Two-Photon Polymerization

This chapter discusses recent progress in 2D and 3D printing technologies, in particular laser-induced forward transfer and two-photon polymerization (TPP). We explore their potential for applications in micro-electronics, protein microarrays, sensors and biosensors, and tissue engineering. An overview of the factors that affect patterning, miniaturization and functionality is presented. A special focus is placed on laser direct writing via TPP for the fabrication of 3D vertical microtubes acting as microreservoirs for an osteogenic drug.

Empirical quantitative characterization of holographic phase images of normal and abnormal cervical cells by fractal descriptors

AbstractThe paper proposes a way to examine whether cervical cells qualify as normal or abnormal on the basis of phase profiles, and to subsequently perform local fractal descriptions of their thre...

Focusing criterion in DHM image reconstruction

This study is presenting the theoretical approach and the practical results of a precise activity involved in the hologram reconstruction in order to find the optimally focused image of MG63 osteoblast-like cells cultivated on polymeric flat substrates. The morphology and dynamic of the cell is investigated by digital holographic microscopy (DHM) technique. The reconstruction is digitally performed using an algorithm based on the scalar theory of diffraction in the Fresnel approximation. The quality of the 3D images of the cells is crucially depending on the focusing capability of the reconstruction chain to fit the parameters of the optical recorder, particularly the focusing value. Our proposal to find the focused image is based on the images decomposition on gray levels and their histogram analysis. More precisely the focusing criterion is based on the evaluation of the form of this distribution.