Moisture adsorption by decellularized bovine pericardium collagen matrices studied by terahertz pulsed spectroscopy and solid immersion microscopy.

In this paper, terahertz (THz) pulsed spectroscopy and solid immersion microscopy were applied to study interactions between water vapor and tissue scaffolds-the decellularized bovine pericardium (DBP) collagen matrices, in intact form, cross-linked with the glutaraldehyde or treated by plasma. The water-absorbing properties of biomaterials are prognostic for future cell-mediated reactions of the recipient tissue with the scaffold. Complex dielectric permittivity of DBPs was measured in the 0.4-2.0 THz frequency range, while the samples were first dehydrated and then exposed to water vapor atmosphere with 80.0 ± 5.0% relative humidity. These THz dielectric measurements of DBPs and the results of their weighting allowed to estimate the adsorption time constants, an increase of tissue mass, as well as dispersion of these parameters. During the adsorption process, changes in the DBPs' dielectric permittivity feature an exponential character, with the typical time constant of =8-10 min, the transient process saturation at =30 min, and the tissue mass improvement by =1-3%. No statistically-relevant differences between the measured properties of the intact and treated DBPs were observed. Then, contact angles of wettability were measured for the considered DBPs using a recumbent drop method, while the observed results showed that treatments of DBP somewhat affects their surface energies, polarity, and hydrophilicity. Thus, our studies revealed that glutaraldehyde and plasma treatment overall impact the DBP-water interactions, but the resultant effects appear to be quite complex and comparable to the natural variability of the tissue properties. Such a variability was attributed to the natural heterogeneity of tissues, which was confirmed by the THz microscopy data. Our findings are important for further optimization of the scaffolds' preparation and treatment technologies. They pave the way for THz technology use as a non-invasive diagnosis tool in tissue engineering and regenerative medicine.

Chitosan-g-oligo(L,L-lactide) copolymer hydrogel for nervous tissue regeneration in glutamate excitotoxicity: in vitro feasibility evaluation.

Over the last decade, a number of hydrogels attracted great attention in the area of brain tissue engineering. The hydrogels are composed of hydrophilic polymers forming 3D network in water. Their function is promoting structural and functional restoration of damaged brain tissues by providing mechanical support and navigating cell fate. This paper reports on the neurocompatibility of chitosan-g-oligo(L,L-lactide) copolymer hydrogel with primary rat cortical neuron culture. The hydrogel was produced by a molding technique on the base of photocurable composition consisting of chitosan-g-oligo(L,L-lactide) copolymer, poly(ethylene glycol) diacrylate and photosensitizer Irgacure 2959. The influence of the hydrogel on cell viability, phenotype and calcium homeostasis, mitochondrial potential and oxygen consumption rate in glutamate excitotoxicity was analyzed using primary neuron cultures obtained from a neonatal rat cortex. This study revealed that the hydrogel is non-cytotoxic. Dissociated neonatal rat cortical cells were actively attaching to the hydrogel surface and exhibited the phenotype, calcium homeostasis and mitochondrial function in both standard conditions and glutamate excitotoxicity (100 µM) similar to the control cells cultured without the hydrogel. To conclude, in this study we assessed the feasibility of the application of chitosan-g-oligo(L,L-lactide) copolymer hydrogel for tissue engineering therapy of brain injury in an in vitro model. The results support that the hydrogel is able to sustain realization of the functional metabolic activity of neonatal rat cortical cells in response to glutamate excitotoxicity.

Tailoring the collagen film structural properties via direct laser crosslinking of star-shaped polylactide for robust scaffold formation.

Application of restructured collagen-based biomaterials is generally restricted by their poor mechanical properties, which ideally must be close to those of a tissue being repaired. Here, we present an approach to the formation of a robust biomaterial using laser-induced curing of a photosensitive star-shaped polylactide. The created collagen-based structures demonstrated an increase in the Young's modulus by more than an order of magnitude with introduction of reinforcing patterns (from 0.15 ±â€¯0.02 MPa for the untreated collagen to 51.2 ±â€¯5.6 MPa for the reinforced collagen). It was shown that the geometrical configuration of the created reinforcing pattern affected the scaffold's mechanical properties only in the case of a relatively high laser radiation power density, when the effect of accumulated thermomechanical stresses in the photocured regions was significant. Photo-crosslinking of polylactide did not compromise the scaffold's cytotoxicity and provided fluorescent regions in the collagen matrix, that create a potential for noninvasive monitoring of such materials' biodegradation kinetics in vivo.

2D/3D buccal epithelial cell self-assembling as a tool for cell phenotype maintenance and fabrication of multilayered epithelial linings in vitro.

Maintaining the epithelial status of cells in vitro and fabrication of a multilayered epithelial lining is one of the key problems in the therapy using cell technologies. When cultured in a monolayer, epithelial cells change their phenotype from epithelial to epithelial-mesenchymal or mesenchymal that makes it difficult to obtain a sufficient number of cells in a 2D culture and to use them in tissue engineering. Here, using buccal epithelial cells from the oral mucosa, we developed a novel approach to recover and maintain the stable cell phenotype and form a multilayered epithelial lining in vitro via the 2D/3D cell self-assembling. Transitioning the cells from the monolayer to non-adhesive 3D culture conditions led to formation of self-assembling spheroids, with restoration of their epithelial characteristics after epithelial-mesenchymal transition. In 7 days, the cells within spheroids restored the apical-basal polarity, and the formation of both tight (ZO1) and adherent (E-cadherin) intercellular junctions was shown. Thus, culturing buccal epithelial cells in a 3D system allowed us to recover and durably maintain the morphological and functional characteristics of epithelial cells. The multilayered epithelial lining formation was achieved after placing spheroids for 7 days onto a hybrid matrix, which consisted of collagen layers and reinforcing poly (lactide-co-glycolide) fibers and was proven promising for replacement of the urothelium. Thus, we offer an effective technique of forming multilayered epithelial linings on carrier-matrices using cell spheroids that was not previously described elsewhere and can find a wide range of applications in tissue engineering, replacement surgery, and regenerative medicine.

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.

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.

Specific visualization of tumor cells using upconversion nanophosphors.

The development of targeted constructs on the basis of photoluminescent nanoparticles with a high photo- and chemical stability and absorption/emission spectra in the "transparency window" of biological tissues is an important focus area of present-day medical diagnostics. In this work, a targeted two-component construct on the basis of upconversion nanophosphors (UCNPs) and anti-tumor 4D5 scFv was developed for selective labeling of tumor cells overexpressing the HER2 tumor marker characteristic of a number of human malignant tumors. A high affinity barnase : barstar (Bn : Bs) protein pair, which exhibits high stability in a wide range of pH and temperatures, was exploited as a molecular adapter providing self-assembly of the two-component construct. High selectivity for the binding of the two-component 4D5 scFv-Bn : UCNP-Bs construct to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2 was demonstrated. This approach provides an opportunity to produce similar constructs for the visualization of different specific markers in pathogenic tissues, including malignant tumors.