The aim of the study was the development of three-dimensional (3D) printed gene-activated implants based on octacalcium phosphate (OCP) and plasmid DNA encoding VEGFA. The first objective of the present work involved design and fabrication of gene-activated bone substitutes based on the OCP and plasmid DNA with VEGFА gene using 3D printing approach of ceramic constructs, providing the control of its architectonics compliance to the initial digital models. X-ray diffraction, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and compressive strength analyses were applied to investigate the chemical composition, microstructure, and mechanical properties of the experimental samples. The biodegradation rate and the efficacy of plasmid DNA delivery in vivo were assessed during standard tests with subcutaneous implantation to rodents in the next stage. The final part of the study involved substitution of segmental tibia and mandibular defects in adult pigs with 3D printed gene-activated implants. Biodegradation, osteointegration, and effectiveness of a reparative osteogenesis were evaluated with computerized tomography, SEM, and a histological examination. The combination of gene therapy and 3D printed implants manifested the significant clinical potential for effective bone regeneration in large/critical size defect cases.

Natural and synthetic hydrogel scaffolds containing bioactive components are increasingly used in solving various tissue engineering problems. The encapsulation of DNA-encoding osteogenic growth factors with transfecting agents (e.g., polyplexes) into such scaffold structures is one of the promising approaches to delivering the corresponding genes to the area of the bone defect to be replaced, providing the prolonged expression of the required proteins. Herein, a comparative assessment of both in vitro and in vivo osteogenic properties of 3D printed sodium alginate (SA) hydrogel scaffolds impregnated with model EGFP and therapeutic BMP-2 plasmids was demonstrated for the first time. The expression levels of mesenchymal stem cell (MSC) osteogenic differentiation markers Runx2, Alpl, and Bglap were evaluated by real-time PCR. Osteogenesis in vivo was studied on a model of a critical-sized cranial defect in Wistar rats using micro-CT and histomorphology. The incorporation of polyplexes comprising pEGFP and pBMP-2 plasmids into the SA solution followed by 3D cryoprinting does not affect their transfecting ability compared to the initial compounds. Histomorphometry and micro-CT analysis 8 weeks after scaffold implantation manifested a significant (up to 46%) increase in new bone volume formation for the SA/pBMP-2 scaffolds compared to the SA/pEGFP ones.

A new method for fabrication of highly porous bioresorbable polymer structures on the basis of various aliphatic polyesters for tissue engineering has been successfully designed and worked out. It has been shown that injection of polymer compositions plasticized in sub- or supercritical carbon dioxide into press forms at temperatures from 20 to 40°C through a nozzle of a certain diameter under atmospheric or elevated (up to 6 MPa) CO₂ pressure allows obtaining polymer matrices with a desired structure and morphology and mean porosity of up to 96 vol % with high reproducibility and avoiding the use of toxic organic solvents. The effect of chemical composition and molecular mass of starting polymers, as well as temperature and CO₂ pressure in the reaction cell and the receiver, on the morphology and internal structure of fabricated samples was studied using the method of scanning-electron microscopy.

Gene therapy is one of the most promising approaches in regenerative medicine to restore damaged tissues of various types. However, the ability to control the dose of bioactive molecules in the injection site can be challenging. The combination of genetic constructs, bioresorbable material, and the 3D printing technique can help to overcome these difficulties and not only serve as a microenvironment for cell infiltration but also provide localized gene release in a more sustainable way to induce effective cell differentiation. Herein, the cell transfection with plasmid DNA directly incorporated into sodium alginate prior to 3D printing was investigated both in vitro and in vivo. The 3D cryoprinting ensures pDNA structure integrity and safety. 3D printed gene-activated scaffolds (GAS) mediated HEK293 transfection in vitro and effective synthesis of model EGFP protein in vivo, thereby allowing the implementation of the developed GAS in future tissue engineering applications.

This paper concerns a detailed study of the phase separation and structure formation processes that occur in solutions of highly hydrophobic polylactic-co-glycolic acid (PLGA) in highly hydrophilic tetraglycol (TG) upon their contact with aqueous media. In the present work, cloud point methodology, high-speed video recording, differential scanning calorimetry, and both optical and scanning electron microscopy were used to analyze the behavior of PLGA/TG mixtures differing in composition when they are immersed in water (the so-called “harsh” antisolvent) or in a nonsolvent consisting of equal amounts of water and TG (a “soft” antisolvent). The phase diagram of the ternary PLGA/TG/water system was designed and constructed for the first time. The PLGA/TG mixture composition with which the polymer undergoes glass transition at room temperature was determined. Our data enabled us to analyze in detail the structure evolution process taking place in various mixtures upon their immersion in “harsh” and “soft” antisolvent baths and gain an insight into the peculiarities of the structure formation mechanism active in the course of antisolvent-induced phase separation in PLGA/TG/water mixtures. This provides intriguing opportunities for the controlled fabrication of a wide variety of bioresorbable structures—from polyester microparticles, fibers, and membranes to scaffolds for tissue engineering.

A device has been developed to study the physicochemical processes of the formation of a structure that occurs during temperature-induced aggregate transitions of liquid and gel compositions during three-dimensional printing under conditions of significant temperature gradients. The device is based on a three-dimensional coordinate system with a heated pneumatic dispenser of viscous fluid compositions and a substrate cooled to −190°C. Liquid nitrogen, solid carbon dioxide, or an integrated Peltier cooling system are used to cool the substrate. The device is equipped for contact temperature control and has a digital video camera.