Effect of collagen denaturation degree on mechanical properties and biological activity of nanofibrous scaffolds.

Further progress in regenerative medicine and bioengineering highly depends on the development of 3D polymeric scaffolds with active biological properties. The most attention is paid to natural extracellular matrix components, primarily collagen. Herein, nonwoven nanofiber materials with various degrees of collagen denaturation and fiber diameters 250-500 nm were produced by electrospinning, stabilized by genipin, and characterized in detail. Collagen denaturation has been confirmed using DSC and FTIR analysis. The comparative study of collagen and gelatin nonwoven materials (NWM) revealed only minor differences in their biocompatibility with skin fibroblasts and keratinocytes in vitro. In long-term subcutaneous implantation study, the inflammation was less evident on collagen than on gelatin NWM. Remarkably, the pronounced calcification was revealed in the collagen NWM only. The results obtained can be useful in terms of improving the electrospinning technology of collagen from aqueous solutions, as well as emphasize the importance of long-term study to ensure proper implementation of the material, taking into account the ability of collagen to provoke calcification.

Chitosan/hyaluronic acid polyanion bilayer applied from carbon acid as an advanced coating with intelligent antimicrobial properties for improved biological prosthetic heart valves.

The tightly bonded shielding coating on biomatrix significantly enhances the functionality of medical devices, bioprostheses in particular. In our work we have obtained a polyelectrolyte coating on a biomatrix by sequentially depositing chitosan and hyaluronic acid (HA) from solutions in carbonic acid under pressure. This approach makes it possible to obtain hybrid biomatrix with a firmly bonded polymer screen due to the electrostatic bonding of polyions. High-precision analysis using a tritium label shows a 3-fold increase in quantity of HA in carbonic acid under pressure compared to the conventional method. The presence of the chitosan layer increases the HA adsorption by 15-20 % due to electrostatic interaction of differently charged polymers. Antimicrobial results show the possibility of implementing an induced antimicrobial response, due to the lysis of the upper layer of the coating (HA) and the release of antimicrobial agents in the case of growth of pathogens on the bioprosthesis.

Influence of γ-Radiation on Mechanical Stability to Cyclic Loads Tubular Elastic Matrix of the Aorta.

A significant drawback of the rigid synthetic vascular prostheses used in the clinic is the mechanical mismatch between the implant and the prosthetic vessel. When placing prostheses with radial elasticity, in which this deficiency is compensated, the integration of the graft occurs more favorably, so that signs of cell differentiation appear in the prosthesis capsule, which contributes to the restoration of vascular tone and the possibility of vasomotor reactions. Aortic prostheses fabricated by electrospinning from a blend of copolymers of vinylidene fluoride with hexafluoropropylene (VDF/HFP) had a biomechanical behavior comparable to the native aorta. In the present study, to ensure mechanical stability in the conditions of a living organism, the fabricated blood vessel prostheses (BVP) were cross-linked with γ-radiation. An optimal absorbed dose of 0.3 MGy was determined. The obtained samples were implanted into the infrarenal aorta of laboratory animals-Landrace pigs. Histological studies have shown that the connective capsule that forms around the prosthesis has signs of high tissue organization. This is evidenced by the cells of the fibroblast series located in layers oriented along and across the prosthesis, similar to the orientation of cells in a biological arterial vessel.

Biomechanical behaviour of PEDOT:PSS-based hydrogels as an electrode for stent integrated enzyme biofuel cells.

The possibility of creating a biofuel cell based on a metal stent was shown in this study. Given the existing stent implantation approaches, the integration of a biofuel cell into a stent naturally entails capacity for biofuel cells to be installed into a human body. As a counter electrode, a hydrogel based on iota-carrageenan, polyvinyl alcohol, and PEDOT:PSS, with an immobilized glucose oxidase enzyme, was proposed. Tension tests demonstrated that the hydrogel mechanical behavior resembles that of a bovine's vein. To obtain an analytical description, the deformation curves were fitted using Gent and Ogden models, prompting the fitting parameters which can be useful in further investigations. During cyclic biaxial studies the samples strength was shown to decreases insignificantly in the first 50 cycles and, further, remains stable up to more than 100 cycles. The biofuel cell was designed with the PEDOT:PSS based material as an anode and a Co-Cr self-expanding stent as a cathode. The maximum biofuel cell power density with a glucose concentration of 5 mM was 7.87 × 10-5 W in phosphate buffer and 3.98 × 10-5 W in blood mimicking buffer. Thus, the biofuel cell integration in the self-expanding stent was demonstrated.

True Molecular Composites: Unusual Structure and Properties of PDMS-MQ Resin Blends.

Poly(dimethyl siloxane)-MQ rubber molecular composites are easy to prepare, as it does not require a heterophase mixing of ingredients. They are characterized by perfect homogeneity, so they are very promising as rubber materials with controllable functional characteristics. The manuscript reveals that MQ resin particles can significantly, more than by two orders of magnitude, enhance the mechanical properties of poly(dimethyl siloxane), and, as fillers, they are not inferior to aerosils. In the produced materials, MQ particles play a role of the molecular entanglements, so rubber molecular weight and MQ filler concentration are the parameters determining the structure and properties of such composites. Moreover, a need for a saturation of the reactive groups and minimization of the surface energy of MQ particles also determine the size and distribution of the filler at different filler rates. An unusual correlation of the concentration of MQ component and the interparticle spacing was revealed. Based on the extraordinary mechanical properties and structure features, a model of the structure poly(dimethyl siloxane)-rubber molecular composites and of its evolution in the process of stretching, was proposed.

New Principles of Polymer Composite Preparation. MQ Copolymers as an Active Molecular Filler for Polydimethylsiloxane Rubbers.

Colorless transparent vulcanizates of silicone elastomers were prepared by mixing the components in a common solvent followed by solvent removal. We studied the correlation between the mechanical behavior of polydimethylsiloxane (PDMS)-rubber compositions prepared using MQ (mono-(M) and tetra-(Q) functional siloxane) copolymers with different ratios of M and Q parts as a molecular filler. The composition and molecular structure of the original rubber, MQ copolymers, and carboxyl-containing PDMS oligomers were also investigated. The simplicity of the preparation of the compositions, high strength and elongation at break, and their variability within a wide range allows us to consider silicone elastomers as a promising alternative to silicone materials prepared by traditional methods.

The Effect of Mechanical Properties of Synthetic Prostheses Made by Electrospinning on the Results of Experimental Implantation in the Infrarenal Abdominal Aorta.

BACKGROUND: The research aims to study the effect of circumferential compliance of synthetic vascular prostheses on their healing during implantation in the infrarenal abdominal aorta of pigs. METHODS: In an experiment, 12 pigs were implanted with blood vessel prostheses in the infrarenal abdominal aorta. The prostheses possessed elastic characteristics obtained by a tensile testing machine, and differed in circumferential compliance: rigid (polycaprolactone [PCL]); less compliant than the native aorta (polyurethane [PU]); comparable in compliance to the native aorta (copolymer of vinylidene fluoride with hexafluoropropylene) before (FKM) and after radiation treatment (FKM-γ). The implanted prostheses compliance was measured by aortography during the first 3 days and 1 month after implantation, the condition of the prosthesis capsule was evaluated by macroscopic preparations and histologic examination. RESULTS: Pulsation on PCL prostheses was nonexistent immediately after implantation. On PU prostheses, slight pulsation was noted during the first 3 days and disappeared after 1 month. On FKM prostheses, although pulsation persisted after 1 month, a significant expansion of prostheses was also recorded as a result of fatigue plastic deformation. On FKM-γ prostheses, pulsation comparable in magnitude with aortic pulsation was present 1 month after implantation with no change in the size of the prosthesis. Macroscopic preparations reveal significant differences in the formed connective tissue capsule. The PCL prosthesis capsule is thick, narrowing the lumen of the vessel from the outside. The outer surface of PU prostheses is covered with a thinner uniform fibrous capsule. The inner surface of the FKM and FKM-γ prostheses is covered with a thin layer of smooth whitish tissue. The FKM prosthesis, unlike the FKM-γ prosthesis, is sharply expanded. In all cases, moderate aortic expansion was observed distal to the prosthesis. According to the histologic data, the outer and inner capsules of PCL prostheses are covered with a thick layer of fibrous tissue with signs of productive inflammation and foci of calcification. PU prostheses are surrounded by a thick connective tissue capsule partially endothelialized from the inside; the outer capsule is randomly populated with fibroblastic cells. FKM prostheses have a thin outer capsule where smooth muscle cells are visible, mainly oriented along and across the prosthesis axis; the inner capsule is thin and completely covered with a layer of endothelial cells from the side of the lumen. A layered structure is visible in the prosthesis capsule of FKM-γ, and the fibroblast cells in each layer of the capsule are oriented along or across the prosthesis axis, similar to the structure of a natural arterial vessel. The inner surface of the prosthesis is completely endothelialized. CONCLUSIONS: The healing and degree of inflammation in a capsule of blood vessel prostheses implanted in the infrarenal abdominal aorta of pigs depend on the degree of their circumferential compliance. Although maintaining pulsations, the cellular structure of the capsule is characterized by a greater degree of differentiation and approaches the structure of the native arterial wall.

Viscoll collagen solution as a novel bioink for direct 3D bioprinting.

Collagen is one of the most promising materials for 3D bioprinting because of its distinguished biocompatibility. Cell-laden constructs made of pure collagen with or without incorporated growth supplements support engineered constructs persistence in culture and are perfectly suitable for grafting. The limiting factor for direct 3D collagen printing was poor printability of collagen solutions, especially admixed with cells or tissue spheroids. In our study, we showed that concentrated solutions of native collagen branded Viscoll were effective as bioinks with high fidelity performance. Viscoll containing 20, 30, or 40 mg/ml collagen were used for direct extrusion 3D bioprinting to form scaffolds appropriate to support spatial arrangement of tissue spheroids into rigid patterns with resolution of 0.5 mm in details. Incorporated cells demonstrated sufficient viability. Associated rheological study showed that good printability of the collagen solutions correlates with their increased storage modulus value, notably exceeding the loss modulus value. The proper combination of these physical parameters could become technological criteria for manufacturing various collagen bioinks for 3D bioprinting.

Non-woven bilayered biodegradable chitosan-gelatin-polylactide scaffold for bioengineering of tracheal epithelium.

OBJECTIVES: The conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non-woven material in order to obtain a well-differentiated human airway epithelium. MATERIALS AND METHODS: The bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy. RESULTS: With the use of a copolymer of chitosan-gelatin-poly-l-lactide, a bilayered non-woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed. CONCLUSIONS: The structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well-differentiated equivalent of respiratory epithelium on non-woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co-culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.

Structural and mechanical characteristics of collagen tissue coated with chitosan in a liquid CO2/water system at different pressures.

Chitosan coatings of biological heart-valve prostheses enhance their biocompatibility, resistance to pathogenic microflora and lifetime. Collagen tissues can be coated with chitosan in aqueous solution acidified, to make chitosan soluble, with H2CO3 formed from a coexisting liquid CO2 phase under pressure. The advantage of H2CO3 is that it can be easily removed after the coating procedure. This study assessed the effects of 6-50 MPa CO2 pressure during the coating procedure on the structure and mechanical properties of the resulting biocomposite matrices. The dependence of chitosan adsorption on CO2 pressure was bell-shaped, reaching a maximum adsorption of 0.8 mass % at 40 MPa. Tissue surface became highly porous upon pressure treatment. At 50 MPa, the pores merged to form furrows with lengths of several hundred micrometers, accompanied by collagen fibril reorganisation. Chitosan coating did not affect tissue tensile strength in the axial direction, but increased it by 75% in the radial direction in the tissue coated at 50 MPa pressure. Strain at break, a measure of elasticity, increased in both directions by up to 100% upon coating with chitosan. CO2 pressure of 30-50 MPa seems thus optimal in terms of chitosan incorporation and tissue mechanical properties.

In vitro assessment of electrospun polyamide-6 scaffolds for esophageal tissue engineering.

Artificial tissue-engineered grafts offer a potential alternative to autologous tissue grafts for patients, which can be traumatic. After decellularizing Papio hamadryas esophagus and studying the morphology and physical properties of the extracellular matrix (ECM), we generated electrospun polyamide-6 based scaffolds to mimic it. The scaffolds supported a greater mechanical load than the native ECM and demonstrated similar 3D microstructure, with randomly aligned fibers, 90% porosity, 29 µm maximal pore size, and average fiber diameter of 2.87 ± 0.95 µm. Biocompatibility studies showed that human adipose- and bone marrow-derived mesenchymal stromal cells (AD-MSC and BMD-MSC) adhered to the scaffold surface and showed some proliferation: scaffold cell coverage was 25% after 72 h of incubation when seeded with 1000 cells/mm2 ; cells elongated processes along the polyamide-6, although they flattened 1.67-4 times less than on cell culture plastic. Human umbilical vein endothelial cells, however, showed poor adherence and proliferation. We thus provide in vitro evidence that polyamide-6 scaffolds approximating the esophageal biomechanics and 3D topography of nonhuman primates may provide a biocompatible substrate for both AD-MSC and BMD-MSCs, supporting their adhesion and survival to some degree. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 253-268, 2019.

Multi-hierarchical tissue-engineering ECM-like scaffolds based on cellulose acetate with collagen and chitosan fillers.

A novel high-tech composite biomimetic matrixes for a wide range of medical purposes were prepared. The structure of scaffolds was inspired by the architecture of native decellularized tissue: material consists of a sponge and fibrous components of different spatial geometry based on cellulose acetate with collagen or chitosan filler. The fibrous component was prepared by electrospinning, the sponge - freeze-drying technique. The influence of main technological parameters, such as freeze mode, polymer type and concentration, etc. on the fiber-sponge architecture and properties was examined. It was shown that scaffolds with different types of microstructure can be obtained employing this technique. The impregnation of chitosan or collagen filler in fiber matrix also significantly improves mechanical properties up to 40 MPa for strength and 600 MPa for Young's modulus.

Collagen tissue treated with chitosan solution in H2O/CO2 mixtures: Influence of clathrates hydrates on the structure and mechanical properties.

A mixture of water/carbon dioxide is a "green" perspective solvent from the viewpoint of biomedical applications. Clathrate hydrates are formed this solvent under certain conditions and a very interesting question is the impact of clathrates hydrates on the structure and properties of bovine pericardium, which is used in biomedicine, in particular as a main part of biological heart valve prostheses. The aim of the present work is to investigate the influence of clathrates on the structure and mechanical properties of the collagen tissue treated with chitosan in H2O/CO2 mixtures under pressure 3.0-3.5MPa and temperatures 2-4°C. It was first found that the clathrate hydrates in this media due to the strong fluctuations "bomb" collagen tissue of bovine pericardium, which is manifested in the appearance of numerous small gaps (pores) with mean size of 225±25nm and large pores with size of 1-3µ on the surface and within collagen matrices. High porosity leads to averaging characteristics of the organization structure in tissues with different orientation of the collagen fibers. As a result, the mechanical properties of the collagen tissue with a different orientation of the collagen fibrils become similar, which is quite different from their original properties. The structural changes caused by the influence of the environment clathrate hydrates led to a significant decrease of the tensile strength (30-47% in total, p<0.05) and initial elastic moduli (74-83%, p<0.05). However, the final elastic moduli and the maximum tensile virtually unchanged compared to the control. Nevertheless, it was found that the direct deposition of chitosan from the H2O/CO2 mixtures with clathrate improve the mechanical-strength properties of the porous matrices. We believe that these improved mechanical properties are achieved due to particularly deep and uniform impregnation of the collagen matrix with chitosan from its pressurized solutions in H2O/CO2 mixtures.

SEM and TEM for structure and properties characterization of bacterial cellulose/hydroxyapatite composites.

Preparation of composites with different properties and gradient of components is aimed at better performance of materials for bone substitution. Bacterial cellulose-hydroxyapatite (BC-HAP) composites with various mass ratio of the components (BC-25HAP, BC-4HAP, and BC-HAP) were prepared by a novel method of growing HAP nanocrystals (the linear size ≤30 nm) in water solutions in the presence of the BC gel-film micro-fragments. Varying the BC-HAP ratios leads to a gradual change of the physical properties of the materials. It was found that an increase in the BC content results in a decrease of the HAP crystal length and specific surface area, porosity, and pore volume while the values of density and Young's modulus values increase. SCANNING 38:757-765, 2016. © 2016 Wiley Periodicals, Inc.

Orthotopic transplantation of a tissue engineered diaphragm in rats.

The currently available surgical options to repair the diaphragm are associated with significant risks of defect recurrence, lack of growth potential and restored functionality. A tissue engineered diaphragm has the potential to improve surgical outcomes for patients with congenital or acquired disorders. Here we show that decellularized diaphragmatic tissue reseeded with bone marrow mesenchymal stromal cells (BM-MSCs) facilitates in situ regeneration of functional tissue. A novel bioreactor, using simultaneous perfusion and agitation, was used to rapidly decellularize rat diaphragms. The scaffolds retained architecture and mechanical properties and supported cell adhesion, proliferation and differentiation. Biocompatibility was further confirmed in vitro and in vivo. We replaced 80% of the left hemidiaphragm with reseeded diaphragmatic scaffolds. After three weeks, transplanted animals gained 32% weight, showed myography, spirometry parameters, and histological evaluations similar to native rats. In conclusion, our study suggested that reseeded decellularized diaphragmatic tissue appears to be a promising option for patients in need of diaphragmatic reconstruction.