Gene therapy is one of the most promising approaches in regenerative medicine. Gene-activated matrices provide stable gene expression and the production of osteogenic proteins in situ to stimulate osteogenesis and bone repair. In this study, we developed new gene-activated matrices based on polylactide granules (PLA) impregnated with BMP2 polyplexes and included in chitosan hydrogel or PRP-based fibrin hydrogel. The matrices showed high biocompatibility both in vitro with mesenchymal stem cells and in vivo when implanted intramuscularly in rats. The use of porous PLA granules allowed the inclusion of a high concentration of polyplexes, and the introduction of the granules into hydrogel provided the gradual release of the plasmid constructs. All gene-activated matrices showed transfecting ability and ensured long-term gene expression and the production of target proteins in vitro. At the same time, the achieved concentration of BMP-2 was sufficient to induce osteogenic differentiation of MSCs. When implanted into critical-size calvarial defects in rats, all matrices with BMP2 polyplexes led to new bone formation. The most significant effect on osteoinduction was observed for the PLA/PRP matrices. Thus, the developed gene-activated matrices were shown to be safe and effective osteoplastic materials. PLA granules and PRP-based fibrin hydrogel containing BMP2 polyplexes were shown to be the most promising for future applications in bone regeneration.

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.

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.

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 β-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.

A comparative study of gelation processes in aqueous solutions of sodium alginate initiated by their chemical crosslinking with divalent Ca²⁺ or Ba²⁺ ions is carried out. 3D cryoprinted hydrogel scaffolds with certain architectonics are formed from the studied compositions containing various concentrations of crosslinking agents. It is shown that increasing the concentration of the crosslinking agent from 2 up to 10 wt % leads to the improvement in the strength characteristics of alginate structures. Here, scaffolds crosslinked with 10% aqueous solutions of both CaCl₂ and BaCl₂ retain their structural stability during 21 days of incubation in a culture medium at 37°C. It is found during in vitro experiments on cultures of multipotent mesenchymal stromal cells derived from rat adipose tissue that samples crosslinked with Ba²⁺ ions demonstrate a moderate cytotoxic effect and manifest their inability to maintain cell adhesion as opposed to nontoxic scaffolds crosslinked in aqueous solutions of CaCl₂. In vivo analysis of experimental samples on an intramuscular implantation model for male Wistar rats also confirms moderate cytotoxicity of the alginate scaffolds formed using Ba²⁺ ions as crosslinking agents. The results obtained make it possible to assert that sodium alginate crosslinked with a 10% aqueous solution of CaCl₂ can be considered the most promising for various biomedical applications among all the materials studied by us.

The possibility of improving the biocompatibility of poly(lactic-co-glycolic acid) (PLGA) scaffolds for tissue engineering by their seeding with mesenchymal stem cells (MSCs) is studied. The cell seeding procedure can generally affect the physical and mechanical properties of these scaffolds, which are mainly determined by their architectonics. Three types of PLGA scaffolds were manufactured and studied: (1) monolithic blocks fabricated by injection molding, (2) porous scaffolds formed by supercritical fluid plasticization followed by foaming, and (3) fibrous nonwoven scaffolds fabricated by electrospinning. A quantitative XTT test showed no cytotoxicity for all types of studied scaffolds as well as the higher efficiency of the dynamic seeding method in comparison with the static method. Also, already after 48 h of incubation of the scaffolds with cells, their physical and mechanical properties changed both at the macro and at the micro level. It is assumed that these changes are determined by the processes of aqueous and enzymatic hydrolysis of poly(lactic-co-glycolic acid)s while adhered MSCs altered internal scaffold structure and surface morphology of scaffold surface. Similar transformations of some physicochemical and structural properties of the scaffolds based on other biodegradable polymers or their seeding can also occur as a result of their seeding with various cell cultures, which should indeed be taken into account when applying them for development of tissue engineering constructions.