Photoluminescent Scaffolds Based on Natural and Synthetic Biodegradable Polymers for Bioimaging and Tissue Engineering.

Non-invasive visualization and monitoring of tissue-engineered structures in a living organism is a challenge. One possible solution to this problem is to use upconversion nanoparticles (UCNPs) as photoluminescent nanomarkers in scaffolds. We synthesized and studied scaffolds based on natural (collagen-COL and hyaluronic acid-HA) and synthetic (polylactic-co-glycolic acids-PLGA) polymers loaded with ß-NaYF4:Yb3+, Er3+ nanocrystals (21 ± 6 nm). Histomorphological analysis of tissue response to subcutaneous implantation of the polymer scaffolds in BALB/c mice was performed. The inflammatory response of the surrounding tissues was found to be weak for scaffolds based on HA and PLGA and moderate for COL scaffolds. An epi-luminescent imaging system with 975 nm laser excitation was used for in vivo visualization and photoluminescent analysis of implanted scaffolds. We demonstrated that the UCNPs' photoluminescent signal monotonously decreased in all the examined scaffolds, indicating their gradual biodegradation followed by the release of photoluminescent nanoparticles into the surrounding tissues. In general, the data obtained from the photoluminescent analysis correlated satisfactorily with the histomorphological analysis.

Natural and Synthetic Polymer Scaffolds Comprising Upconversion Nanoparticles as a Bioimaging Platform for Tissue Engineering.

Modern biocompatible materials of both natural and synthetic origin, in combination with advanced techniques for their processing and functionalization, provide the basis for tissue engineering constructs (TECs) for the effective replacement of specific body defects and guided tissue regeneration. Here we describe TECs fabricated using electrospinning and 3D printing techniques on a base of synthetic (polylactic-co-glycolic acids, PLGA) and natural (collagen, COL, and hyaluronic acid, HA) polymers impregnated with core/shell ß-NaYF4:Yb3+,Er3+/NaYF4 upconversion nanoparticles (UCNPs) for in vitro control of the tissue/scaffold interaction. Polymeric structures impregnated with core/shell ß-NaYF4:Yb3+,Er3+/NaYF4 nanoparticles were visualized with high optical contrast using laser irradiation at 976 nm. We found that the photoluminescence spectra of impregnated scaffolds differ from the spectrum of free UCNPs that could be used to control the scaffold microenvironment, polymer biodegradation, and cargo release. We proved the absence of UCNP-impregnated scaffold cytotoxicity and demonstrated their high efficiency for cell attachment, proliferation, and colonization. We also modified the COL-based scaffold fabrication technology to increase their tensile strength and structural stability within the living body. The proposed approach is a technological platform for "smart scaffold" development and fabrication based on bioresorbable polymer structures impregnated with UCNPs, providing the desired photoluminescent, biochemical, and mechanical properties for intravital visualization and monitoring of their behavior and tissue/scaffold interaction in real time.

3D-printed hyaluronic acid hydrogel scaffolds impregnated with neurotrophic factors (BDNF, GDNF) for post-traumatic brain tissue reconstruction.

Brain tissue reconstruction posttraumatic injury remains a long-standing challenge in neurotransplantology, where a tissue-engineering construct (scaffold, SC) with specific biochemical properties is deemed the most essential building block. Such three-dimensional (3D) hydrogel scaffolds can be formed using brain-abundant endogenous hyaluronic acid modified with glycidyl methacrylate by employing our proprietary photopolymerisation technique. Herein, we produced 3D hyaluronic scaffolds impregnated with neurotrophic factors (BDNF, GDNF) possessing 600 kPa Young's moduli and 336% swelling ratios. Stringent in vitro testing of fabricated scaffolds using primary hippocampal cultures revealed lack of significant cytotoxicity: the number of viable cells in the SC+BDNF (91.67 ± 1.08%) and SC+GDNF (88.69 ± 1.2%) groups was comparable to the sham values (p > 0.05). Interestingly, BDNF-loaded scaffolds promoted the stimulation of neuronal process outgrowth during the first 3 days of cultures development (day 1: 23.34 ± 1.46 µm; day 3: 37.26 ± 1.98 µm, p < 0.05, vs. sham), whereas GDNF-loaded scaffolds increased the functional activity of neuron-glial networks of cultures at later stages of cultivation (day 14) manifested in a 1.3-fold decrease in the duration coupled with a 2.4-fold increase in the frequency of Ca2+ oscillations (p < 0.05, vs. sham). In vivo studies were carried out using C57BL/6 mice with induced traumatic brain injury, followed by surgery augmented with scaffold implantation. We found positive dynamics of the morphological changes in the treated nerve tissue in the post-traumatic period, where the GDNF-loaded scaffolds indicated more favorable regenerative potential. In comparison with controls, the physiological state of the treated mice was improved manifested by the absence of severe neurological deficit, significant changes in motor and orienting-exploratory activity, and preservation of the ability to learn and retain long-term memory. Our results suggest in favor of biocompatibility of GDNF-loaded scaffolds, which provide a platform for personalized brain implants stimulating effective morphological and functional recovery of nerve tissue after traumatic brain injury.

Fabrication of moldable chitosan gels via thermally induced phase separation in aqueous alcohol solutions.

Wide application of chitosan in modern technologies is limited by the lack of reliable and low-cost techniques to prepare size-tuned constructs with a complex surface morphology, improved optical and mechanical properties. We report a new simple method for preparation of transparent thermoreversible chitosan alcogels from chitosan/H2O/ethanol ternary systems. This method, termed "low temperature thermally induced phase separation under non-freezing conditions" (LT-TIPS-NF), fine tunes gelation by adjusting only temperature (from 5 to -25 °C) and varying the initial content of chitosan (from 0.5 to 2.0 wt%) and ethanol (from 28.5 to 47.5 vol%). Transparent non-swelling final constructs of complex shape are prepared by fixing the pre-formed alcogels with a base solution. The size of the gel constructs is limited only by the dimensions of the mold and the cooling chamber. The LT-TIPS-NF is applicable both in injection molding and 3D printing techniques. The in vitro and in vivo experiments show the absence of prominent cytotoxicity and well-defined cell adhesion on the obtained hydrogels. Thus, this facile and scalable technique provides the multifunctional chitosan gel preparation with easily controlled properties exploiting inexpensive, renewable, and environmentally friendly source polysaccharide. These materials have prospects for a variety of uses, especially for biomedical applications.

Facile Cell-Friendly Hollow-Core Fiber Diffusion-Limited Photofabrication.

Bioprinting emerges as a powerful flexible approach for tissue engineering with prospective capability to produce tissue on demand, including biomimetic hollow-core fiber structures. In spite of significance for tissue engineering, hollow-core structures proved difficult to fabricate, with the existing methods limited to multistage, time-consuming, and cumbersome procedures. Here, we report a versatile cell-friendly photopolymerization approach that enables single-step prototyping of hollow-core as well as solid-core hydrogel fibers initially loaded with living cells. This approach was implemented by extruding cell-laden hyaluronic acid glycidyl methacrylate hydrogel directly into aqueous solution containing free radicals generated by continuous blue light photoexcitation of the flavin mononucleotide/triethanolamine photoinitiator. Diffusion of free radicals from the solution to the extruded structure initiated cross-linking of the hydrogel, progressing from the structure surface inwards. Thus, the cross-linked wall is formed and its thickness is limited by penetration of free radicals in the hydrogel volume. After developing in water, the hollow-core fiber is formed with centimeter range of lengths. Amazingly, HaCaT cells embedded in the hydrogel successfully go through the fabrication procedure. The broad size ranges have been demonstrated: from solid core to 6% wall thickness of the outer diameter, which was variable from sub-millimeter to 6 mm, and Young's modulus ∼1.6 ± 0.4 MPa. This new proof-of-concept fibers photofabrication approach opens lucrative opportunities for facile three-dimensional fabrication of hollow-core biostructures with controllable geometry.

In Vitro and in Vivo Analysis of Adhesive, Anti-Inflammatory, and Proangiogenic Properties of Novel 3D Printed Hyaluronic Acid Glycidyl Methacrylate Hydrogel Scaffolds for Tissue Engineering.

In this study, we prepared hydrogel scaffolds for tissue engineering by computer-assisted extrusion three-dimensional (3D) printing with photocured (λ = 445 nm) hyaluronic acid glycidyl methacrylate (HAGM). The developed product was compared with the polylactic-co-glycolic acid (PLGA) scaffolds generated by means of the original antisolvent 3D printing methodology. The cytotoxicity and cytocompatibility of the scaffolds were analyzed in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests, flow cytometry, and scanning electron microscopy. Anti-inflammatory and proangiogenic properties of the scaffolds were evaluated in the dorsal skinfold chamber mouse model by means of intravital fluorescence microscopy, histology, and immunohistochemistry throughout an observation period of 14 days. In vitro, none of the scaffolds revealed cytotoxicity on days 1, 2, and 5 after seeding with umbilical cord-derived multipotent stromal cells, and the primary cell adhesion to the surface of HAGM scaffolds was low. In vivo, implanted HAGM scaffolds showed enhanced vascularization and host tissue ingrowth, and the inflammatory response to them was less pronounced compared with PLGA scaffolds. The results indicate excellent biocompatibility and vascularization capacity of the developed 3D printed HAGM scaffolds and position them as strong candidates for advanced tissue engineering applications.

Multichannel hydrogel based on a chitosan-poly(vinyl alcohol) composition for directed growth of animal cells.

In this study, a new method for production of hydrogels with oriented multichannel structure based on chitosan-poly(vinyl alcohol) compositions was developed. Microscopic and biological studies of the obtained hydrogels were conducted to determine the optimal composition, which would ensure that structure of the material mimics that of the epineurium and perineurium in a nerve. Structure of the hydrogels was adjusted by variation of the initial concentration of the precipitant, poly(vinyl alcohol), and acid in the chitosan compositions. A single cycle of freezing and thawing of the produced hydrogels resulted in lower structural heterogeneity, which is promising for the production of a scaffold that simulates the structure of the native peripheral nerve. in vitro cytotoxic assays showed biocompatibility of the manufactured hydrogels.