Fabrication of Conductive Tissue Engineering Nanocomposite Films Based on Chitosan and Surfactant-Stabilized Graphene Dispersions.

Chitosan (CS)/graphene nanocomposite films with tunable biomechanics, electroconductivity and biocompatibility using polyvinylpyrrolidone (PVP) and Pluronic F108 (Plu) as emulsion stabilizers for the purpose of conductive tissue engineering were successfully obtained. In order to obtain a composite solution, aqueous dispersions of multilayered graphene stabilized with Plu/PVP were supplied with CS at a ratio of CS to stabilizers of 2:1, respectively. Electroconductive films were obtained by the solution casting method. The electrical conductivity, mechanical properties and in vitro and in vivo biocompatibility of the resulting films were assessed in relation to the graphene concentration and stabilizer type and they were close to that of smooth muscle tissue. According to the results of the in vitro cytotoxicity analysis, the films did not release soluble cytotoxic components into the cell culture medium. The high adhesion of murine fibroblasts to the films indicated the absence of contact cytotoxicity. In subcutaneous implantation in Wistar rats, we found that stabilizers reduced the brittleness of the chitosan films and the inflammatory response.

Photocurable Methacrylate Derivatives of Polylactide: A Two-Stage Synthesis in Supercritical Carbon Dioxide and 3D Laser Structuring.

A two-stage polylactide modification was performed in the supercritical carbon dioxide medium using the urethane formation reaction. The modification resulted in the synthesis of polymerizable methacrylate derivatives of polylactide for application in the spatial 3D structuring by laser stereolithography. The use of the supercritical carbon dioxide medium allowed us to obtain for the first time polymerizable oligomer-polymer systems in the form of dry powders convenient for further application in the preparation of polymer compositions for photocuring. The photocuring of the modified polymers was performed by laser stereolithography and two-photon crosslinking. Using nanoindentation, we found that Young's modulus of the cured compositions corresponded to the standard characteristics of implants applied in regenerative medicine. As shown by thermogravimetric analysis, the degree of crosslinking and, hence, the local stiffness of scaffolds were determined by the amount of the crosslinking agent and the photocuring regime. No cytotoxicity was observed for the structures.

Mechanical properties of anterior lens capsule assessed with AFM and nanoindenter in relation to human aging, pseudoexfoliation syndrome, and trypan blue staining.

The purpose of this study is the mechanical characterization of the mid-to- old-age human anterior lens capsules (ALCs) obtained by capsulorhexis using Atomic Force Microscopy (AFM) and a nanoindenter at different spatial scales. The dependencies on the human age, presence or absence of pseudoexfoliation syndrome (PEX), and application of trypan blue staining during the surgery were analyzed. The measurements on both the anterior (AS) and epithelial (ES) sides of the ALC were conducted and the effect of cells present on the epithelial side was carefully accounted for. The ES of the ALC had a homogenous distribution of the Young's modulus over the surface as shown by the macroscale mapping with the nanoindenter and local AFM indentations, while the AS was more heterogeneous. Age-related changes were assessed in groups ranging from the mid-age (from 48 years) to old-age (up to 93 years). We found that the ES was always stiffer than the AS, and this difference decreased with age due to a gradual decrease in the Young's modulus of the ES and an increase in the modulus of the AS. No significant changes were found in the mechanical properties of ALCs of PEX patients versus the PEX-free group, as well as in the properties of the ALC with and without trypan blue staining.

Cell spheroid fusion: beyond liquid drops model.

Biological self-assembly is crucial in the processes of development, tissue regeneration, and maturation of bioprinted tissue-engineered constructions. The cell aggregates-spheroids-have become widely used model objects in the study of this phenomenon. Existing approaches describe the fusion of cell aggregates by analogy with the coalescence of liquid droplets and ignore the complex structural properties of spheroids. Here, we analyzed the fusion process in connection with structure and mechanical properties of the spheroids from human somatic cells of different phenotypes: mesenchymal stem cells from the limbal eye stroma and epithelial cells from retinal pigment epithelium. A nanoindentation protocol was applied for the mechanical measurements. We found a discrepancy with the liquid drop fusion model: the fusion was faster for spheroids from epithelial cells with lower apparent surface tension than for mesenchymal spheroids with higher surface tension. This discrepancy might be caused by biophysical processes such as extracellular matrix remodeling in the case of mesenchymal spheroids and different modes of cell migration. The obtained results will contribute to the development of more realistic models for spheroid fusion that would further provide a helpful tool for constructing cell aggregates with required properties both for fundamental studies and tissue reparation.

Chitosan-g-oligo(L,L-lactide) Copolymer Hydrogel Potential for Neural Stem Cell Differentiation.

We evaluated the applicability of chitosan-g-oligo(L,L-lactide) copolymer (CLC) hydrogel for central nervous system tissue engineering. The biomechanical properties of the CLC hydrogel were characterized and its biocompatibility was assessed with neural progenitor cells obtained from two different sources: H9-derived neural stem cells (H9D-NSCs) and directly reprogrammed neural precursor cells (drNPCs). Our study found that the optically transparent CLC hydrogel possessed biomechanical characteristics suitable for culturing human neural stem/precursor cells and was noncytotoxic. When seeded on films prepared from CLC copolymer hydrogel, both H9D-NSC and drNPC adhered well, expanded and exhibited signs of spontaneous differentiation. While H9D-NSC mainly preserved multipotency as shown by a high proportion of Nestin+ and Sox2+ cells and a comparatively lower expression of the neuronal markers ßIII-tubulin and MAP2, drNPCs, obtained by direct reprogramming, differentiated more extensively along the neuronal lineage. Our study indicates that the CLC hydrogel may be considered as a substrate for tissue-engineered constructs, applicable for therapy of neurodegenerative diseases. Impact statement We synthetized a chitosan-g-oligo(L,L-lactide) hydrogel that sustained multipotency of embryonic-derived neural stem cells (NSCs) and supported differentiation of directly reprogrammed NSC predominantly along the neuronal lineage. The hydrogel exhibited no cytotoxicity in vitro, both in extraction and contact cytotoxicity tests. When seeded on the hydrogel, both types of NSCs adhered well, expanded, and exhibited signs of spontaneous differentiation. The biomechanical properties of the hydrogel were similar to that of human spinal cord with incised pia mater. These data pave the way for further investigations of the hydrogel toward its applicability in central nervous system tissue engineering.

Chemical cross-linking of xenopericardial biomeshes: A bottom-up study of structural and functional correlations.

Decellularized bovine pericardium (DBP)-based biomeshes are the gold standard in reconstructive surgery. In order to prolong their stability after the transplantation, various chemical cross-linking strategies are employed. However, structural and functional properties of the biomeshes differ in dependence on the cross-linker used. Here, we performed a bottom-up study of structural and functional alterations of DBP-based biomeshes following cross-linking with hexamethylene diisocyanate (HMDC), ethylene glycol diglycidyl ether (EGDE), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and genipin. The in vitro cytotoxicity tests supported their clinical applicability. Their structural differences (eg roughness, fibre thickness, pore morphology) were evaluated using the two-photon confocal laser scanning, atomic force, scanning electron and polarized light microscopies. HMDC and EDC samples appeared to be the roughest. Complex mechanical trials indicated the tendency to reduced Young's Modulus and mechanical anisotropy values of DBP upon cross-linking. The lowest mechanical anisotropy was found in EDC and genipin sample groups. In vitro collagenase susceptibility was the highest for EDC samples and the lowest for EGDE samples. The comparative analysis of the results allowed us to recognize the strengths and weaknesses of each cross-linker in relation to a particular clinical application.