Comparative analysis of hypoxic response of human microvascular and umbilical vein endothelial cells in 2D and 3D cell culture systems.

In vitro cultivation conditions play a crucial role in cell physiology and the cellular response to external stimuli. Oxygen concentrations represent an essential microenvironmental factor influencing cell physiology and behaviour both in vivo and in vitro. Therefore, new approaches are urgently needed to monitor and control oxygen concentrations in 2D and 3D cultures, as well as cell reactions to these concentrations. In this work, we modified two types of human endothelial cells-human microvascular (huMECs) and umbilical vein endothelial cells (huVECs) with genetically encoded hypoxia biosensors and monitored cell reactions in 2D to different oxygen concentrations. Moreover, we fabricated 3D cell spheroids of different cell numbers and sizes to reveal the onset of hypoxia in huVECs and huMECs. We could demonstrate a quantitative sensor response of two cell types to reduced oxygen supply in 2D and reveal different thresholds for hypoxic response. In 3D cell spheroids we could estimate critical construct sizes for the appearance of a hypoxic core. This work for the first time directly demonstrates different hypoxic signatures for huVECs and huMECs in 2D and 3D cell culture systems.

Amphiphilic Polypeptides Obtained by the Post-Polymerization Modification of Poly(Glutamic Acid) and Their Evaluation as Delivery Systems for Hydrophobic Drugs.

Synthetic poly(amino acids) are a unique class of macromolecules imitating natural polypeptides and are widely considered as carriers for drug and gene delivery. In this work, we synthesized, characterized and studied the properties of amphiphilic copolymers obtained by the post-polymerization modification of poly(α,L-glutamic acid) with various hydrophobic and basic L-amino acids and D-glucosamine. The resulting glycopolypeptides were capable of forming nanoparticles that exhibited reduced macrophage uptake and were non-toxic to human lung epithelial cells (BEAS-2B). Moreover, the developed nanoparticles were suitable for loading hydrophobic cargo. In particular, paclitaxel nanoformulations had a size of 170-330 nm and demonstrated a high cytostatic efficacy against human lung adenocarcinoma (A549). In general, the obtained nanoparticles were comparable in terms of their characteristics and properties to those based on amphiphilic (glyco)polypeptides obtained by copolymerization methods.

Hybrid Nanoparticles and Composite Hydrogel Systems for Delivery of Peptide Antibiotics.

The growing number of drug-resistant pathogenic bacteria poses a global threat to human health. For this reason, the search for ways to enhance the antibacterial activity of existing antibiotics is now an urgent medical task. The aim of this study was to develop novel delivery systems for polymyxins to improve their antimicrobial properties against various infections. For this, hybrid core-shell nanoparticles, consisting of silver core and a poly(glutamic acid) shell capable of polymyxin binding, were developed and carefully investigated. Characterization of the hybrid nanoparticles revealed a hydrodynamic diameter of approximately 100 nm and a negative electrokinetic potential. The nanoparticles demonstrated a lack of cytotoxicity, a low uptake by macrophages, and their own antimicrobial activity. Drug loading and loading efficacy were determined for both polymyxin B and E, and the maximal loaded value with an appropriate size of the delivery systems was 450 µg/mg of nanoparticles. Composite materials based on agarose hydrogel were prepared, containing both the loaded hybrid systems and free antibiotics. The features of polymyxin release from the hybrid nanoparticles and the composite materials were studied, and the mechanisms of release were analyzed using different theoretical models. The antibacterial activity against Pseudomonas aeruginosa was evaluated for both the polymyxin hybrid and the composite delivery systems. All tested samples inhibited bacterial growth. The minimal inhibitory concentrations of the polymyxin B hybrid delivery system demonstrated a synergistic effect when compared with either the antibiotic or the silver nanoparticles alone.

Refolding, purification, and characterization of constitutive-active human-Smad8 produced as inclusion bodies in ClearColi® BL21 (DE3).

Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-ß (TGF-ß) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and ß-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

Self-Assembled Nanoparticles Based on Block-Copolymers of Poly(2-Deoxy-2-methacrylamido-d-glucose)/Poly(N-Vinyl Succinamic Acid) with Poly(O-Cholesteryl Methacrylate) for Delivery of Hydrophobic Drugs.

The self-assembly of amphiphilic block-copolymers is a convenient way to obtain soft nanomaterials of different morphology and scale. In turn, the use of a biomimetic approach makes it possible to synthesize polymers with fragments similar to natural macromolecules but more resistant to biodegradation. In this study, we synthesized the novel bio-inspired amphiphilic block-copolymers consisting of poly(N-methacrylamido-d-glucose) or poly(N-vinyl succinamic acid) as a hydrophilic fragment and poly(O-cholesteryl methacrylate) as a hydrophobic fragment. Block-copolymers were synthesized by radical addition-fragmentation chain-transfer (RAFT) polymerization using dithiobenzoate or trithiocarbonate chain-transfer agent depending on the first monomer, further forming the hydrophilic block. Both homopolymers and copolymers were characterized by 1H NMR and Fourier transform infrared spectroscopy, as well as thermogravimetric analysis. The obtained copolymers had low dispersity (1.05-1.37) and molecular weights in the range of ~13,000-32,000. The amphiphilic copolymers demonstrated enhanced thermal stability in comparison with hydrophilic precursors. According to dynamic light scattering and nanoparticle tracking analysis, the obtained amphiphilic copolymers were able to self-assemble in aqueous media into nanoparticles with a hydrodynamic diameter of approximately 200 nm. An investigation of nanoparticles by transmission electron microscopy revealed their spherical shape. The obtained nanoparticles did not demonstrate cytotoxicity against human embryonic kidney (HEK293) and bronchial epithelial (BEAS-2B) cells, and they were characterized by a low uptake by macrophages in vitro. Paclitaxel loaded into the developed polymer nanoparticles retained biological activity against lung adenocarcinoma epithelial cells (A549).

Functionalized Pt(II) and Ir(III) NIR Emitters and Their Covalent Conjugates with Polymer-Based Nanocarriers.

Two NIR-emitting platinum [Pt(N^N^C)(phosphine)] and iridium [Ir(N^C)2(N^N)]+ complexes containing reactive succinimide groups were synthesized and characterized with spectroscopic methods (N^N^C, 1-phenyl-3-(pyridin-2-yl)benzo[4,5]imidazo[1,2-a]pyrazine, N^C, 6-(2-benzothienyl)phenanthridine, phosphine-3-(diphenylphosphaneyl)propanoic acid N-hydroxysuccinimide ether, and N^N, 4-oxo-4-((1-(pyridin-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)butanoic acid N-hydroxysuccinimide ether). Their photophysics were carefully studied and analyzed using time-dependent density functional theory calculations. These complexes were used to prepare luminescent micro- and nanoparticles with the "core-shell" morphology, where the core consisted of biodegradable polymers of different hydrophobicity, namely, poly(d,l-lactic acid), poly(ε-caprolactone), and poly(ω-pentadecalactone), whereas the shell was formed by covalent conjugation with poly(l-lysine) covalently labeled with the platinum and iridium emitters. The surface of the species was further modified with heparin to reverse their charge from positive to negative values. The microparticles' size determined with dynamic laser scanning varies considerably from 720 to 1480 nm, but the nanoparticles' diameter falls in a rather narrow range, 210-230 nm. The species with a poly(l-lysine) shell display a high positive (>30 mV) zeta-potential that makes them essentially stable in aqueous media. Inversion of the surface charge to a negative value with the heparin cover did not deteriorate the species' stability. The iridium- and platinum-containing particles displayed emissions the spectral patterns of which were essentially similar to those of unconjugated complexes, which indicate retention of the chromophore nature upon binding to the polymer and further immobilization onto polyester micro- and nanoparticles for drug delivery. The obtained particles were tested to determine their ability to penetrate into different cells types: cancer cells, stem cells, and fibroblasts. It was found that all types of particles could effectively penetrate into all cells types under investigation. Nanoparticles were shown to penetrate into the cells more effectively than microparticles. However, positively charged nanoparticles covered with poly(l-lysine) seem to interact with negatively charged proteins in the medium and enter the inner part of the cells less effectively than nanoparticles covered with poly(l-lysine)/heparin. In the case of microparticles, the species with positive zeta-potentials were more readily up-taken by the cells than those with negative values.

Live reporting for hypoxia: Hypoxia sensor-modified mesenchymal stem cells as in vitro reporters.

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O2 ). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs.

Stronger Cytotoxicity for Cancer Cells Than for Fast Proliferating Human Stem Cells by Rationally Designed Dinuclear Complexes.

Cytostatic metallo-drugs mostly bind to the nucleobases of DNA. A new family of dinuclear transition metal complexes was rationally designed to selectively target the phosphate diesters of the DNA backbone by covalent bonding. The synthesis and characterization of the first dinuclear NiII2 complex of this family are presented, and its DNA binding and interference with DNA synthesis in polymerase chain reaction (PCR) are investigated and compared to those of the analogous CuII2 complex. The NiII2 complex also binds to DNA but forms fewer intermolecular DNA cross-links, while it interferes with DNA synthesis in PCR at lower concentrations than CuII2. To simulate possible competing phosphate-based ligands in vivo, these effects have been studied for both complexes with 100-200-fold excesses of phosphate and ATP, which provided no disturbance. The cytotoxicity of both complexes has been studied for human cancer cells and human stem cells with similar rates of proliferation. CuII2 shows the lowest IC50 values and a remarkable preference for killing the cancer cells. Three different assays show that the CuII2 complex induces apoptosis in cancer cells. These results are discussed to gain insight into the mechanisms of action and demonstrate the potential of this family of dinuclear complexes as anticancer drugs acting by a new binding target.

Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity.

Ligands used on the surface of colloidal nanoparticles (NPs) have a significant impact on physiochemical properties of NPs and their interaction in biological environments. In this study, we report a one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated methoxy polyethylene glycol (mPEG) molecules as a surface coordinating ligand. The resulting mPEG-Qdots were characterized by using ζ potential, FTIR, thermogravimetric (TG) analysis, and microscale thermophoresis (MST) studies. We investigated the effect of mPEG molecules and their grafting density on the Qdots photophysical properties, colloidal stability, protein binding affinity, and in vitro cellular toxicity. Moreover, cellular binding features of the resulting Qdots were examined by using three-dimensional (3D) tumor-like spheroids, and the results were discussed in detail. Promisingly, mPEG ligands were found to increase colloidal stability of Qdots, reduce adsorption of proteins to the Qdot surface, and mitigate Qdot-induced side effects to a great extent. Flow cytometry and confocal microscopy studies revealed that PEGylated Qdots exhibited distinctive cellular interactions with respect to their mPEG grafting density. As a result, mPEG molecules demonstrated a minimal effect on the ZnS shell deposition and the Qdot fluorescence efficiency at a low mPEG density, whereas they showed pronounced effect on Qdot colloidal stability, protein binding affinity, cytotoxicity, and nonspecific binding at a higher mPEG grafting amount.

Hydrogels for 3D mammalian cell culture: a starting guide for laboratory practice.

Hydrogels have become one of the most popular platforms for three-dimensional (3D) cultivation of mammalian cells. The enormous versatility of hydrogel materials makes it possible to design scaffolds with predefined mechanical properties, as well as with desired biofunctionality. 3D hydrogel constructs have been used for a variety of applications, including tissue engineering of microorgan systems, drug delivery, cytotoxicity testing, and drug screening. Moreover, 3D culture is applied for investigating cellular physiology, stem cell differentiation, and tumor models and for studying interaction mechanisms between the extracellular matrix and cells. In this paper, we review current examples of performance-based hydrogel design for 3D cell culture applications. A major emphasis is placed on a description of how standard analytical protocols and imaging techniques are being adapted to analysis of 3D cell culture in hydrogel systems.

Detection of Hypoxia in 2D and 3D Cell Culture Systems Using Genetically Encoded Fluorescent Hypoxia Sensors.

In vivo oxygen availability varies widely between cellular microenvironments, depending on the tissue of origin and its cellular niche. It has long been known that too high or too low oxygen concentrations can act as a biological stressor. Thus, the precise control of oxygen availability should be a consideration for cell culture optimization, especially in the field of three-dimensional (3D) cell culture. In this chapter, we describe a system for visualizing oxygen limitations at a cellular level using human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) that were genetically modified to express a fluorescent hypoxia sensor. This sensor can detect the activation of hypoxia-induced factors (HIF) transcription factors that lead to the expression of the oxygen-independent fluorescent protein, UnaG, at low oxygen concentrations. The response of these hypoxia reporter cells can be evaluated in two-dimensional (2D) and 3D cultivation platforms during exposure to hypoxia (1% O2) and normoxia (21% O2) using fluorescence microscopy and flow cytometry. We show that hypoxia reporter MSCs exhibit a hypoxia-induced fluorescence signal in both 2D and 3D cultivation platforms with fast decay kinetics after reoxygenation, rendering it a valuable tool for studying the cellular microenvironment and regenerative potential of hAD-MSCs in an in vivo-like setting.

Analysis of oxygen-dependent cytokine expression in human mesenchymal stem cells derived from umbilical cord.

Efficient cell expansion is a basic requirement for obtaining clinically relevant numbers of mesenchymal stem cells designed for cell-based therapies or tissue-engineering application. Previous studies have demonstrated that mesenchymal stem cells (MSC) cultivated under reduced atmospheric oxygen concentrations (2.5% O2) possess enhanced proliferation potential and can maintain their differentiation properties. We have analyzed the oxygen-dependent cytokine expression of human MSC derived from umbilical cord and attempted to link the results to the proliferation and differentiation capacities of these cells. By quantitative reverse transcription plus the polymerase chain reaction and by protein microarray, we measured the gene expression and intracellular protein concentration of several growth factors and growth factor receptors. Fibroblast growth factor-7, two growth factor receptors (vascular endothelial growth factor receptor 2 and stem cell factor receptor), and two growth-factor-binding proteins (insulin-like growth-factor-binding proteins 3 and 6) were over-expressed under hypoxic conditions, indicating that their signaling pathways participate in cell proliferation. On the other hand, typical differentiation factors such as bone morphogenetic protein-4, endothelial growth factor, and tissue growth factor-ß1 were absent in cells cultivated under hypoxic and normoxic conditions. The absolute concentration of some intracellular cytokines was also measured for the first time under hypoxia and normoxia. Our results in combination with previous findings indicate that enhanced proliferation potential and a maintained undifferentiated cell state can be ascribed to the oxygen-dependent expression of a set of cytokines. This knowledge might help in the understanding of MSC physiology and in the achievement of directed cell fate of MSC for clinical application.

Cultivated meat manufacturing: Technology, trends, and challenges.

The growing world population, public awareness of animal welfare, environmental impacts and changes in meat consumption leads to the search for novel approaches to food production. Novel foods include products with a new or specifically modified molecular structure, foods made from microorganisms, fungi, algae or insects, as well as from animal cell or tissue cultures. The latter approach is known by various names: "clean meat", "in vitro meat" and "cell-cultured" or "(cell-)cultivated meat". Here, cells isolated from agronomically important species are expanded ex vivo to produce cell biomass used in unstructured meat or to grow and differentiate cells on scaffolds to produce structured meat analogues. Despite the fast-growing field and high financial interest from investors and governments, cultivated meat production still faces challenges ranging from cell source choice, affordable expansion, use of cruelty-free and food-grade media, regulatory issues and consumer acceptance. This overview discusses the above challenges and possible solutions and strategies in the production of cultivated meat. The review integrates multifaceted historical, social, and technological insights of the field, and provides both an engaging comprehensive introduction for general interested and a robust perspective for experts.

Poly(lactic acid) and Nanocrystalline Cellulose Methacrylated Particles for Preparation of Cryogelated and 3D-Printed Scaffolds for Tissue Engineering.

Different parts of bones possess different properties, such as the capacity for remodeling cell content, porosity, and protein composition. For various traumatic or surgical tissue defects, the application of tissue-engineered constructs seems to be a promising strategy. Despite significant research efforts, such constructs are still rarely available in the clinic. One of the reasons is the lack of resorbable materials, whose properties can be adjusted according to the intended tissue or tissue contacts. Here, we present our first results on the development of a toolbox, by which the scaffolds with easily tunable mechanical and biological properties could be prepared. Biodegradable poly(lactic acid) and nanocrystalline cellulose methacrylated particles were obtained, characterized, and used for preparation of three-dimensional scaffolds via cryogelation and 3D printing approaches. The composition of particles-based ink for 3D printing was optimized in order to allow formation of stable materials. Both the modified-particle cytotoxicity and the matrix-supported cell adhesion were evaluated and visualized in order to confirm the perspectives of materials application.

Composites Based on Poly(ε-caprolactone) and Graphene Oxide Modified with Oligo/Poly(Glutamic Acid) as Biomaterials with Osteoconductive Properties.

The development of new biodegradable biomaterials with osteoconductive properties for bone tissue regeneration is one of the urgent tasks of modern medicine. In this study, we proposed the pathway for graphene oxide (GO) modification with oligo/poly(glutamic acid) (oligo/poly(Glu)) possessing osteoconductive properties. The modification was confirmed by a number of methods such as Fourier-transform infrared spectroscopy, quantitative amino acid HPLC analysis, thermogravimetric analysis, scanning electron microscopy, and dynamic and electrophoretic light scattering. Modified GO was used as a filler for poly(ε-caprolactone) (PCL) in the fabrication of composite films. The mechanical properties of the biocomposites were compared with those obtained for the PCL/GO composites. An 18-27% increase in elastic modulus was found for all composites containing modified GO. No significant cytotoxicity of the GO and its derivatives in human osteosarcoma cells (MG-63) was revealed. Moreover, the developed composites stimulated the proliferation of human mesenchymal stem cells (hMSCs) adhered to the surface of the films in comparison with unfilled PCL material. The osteoconductive properties of the PCL-based composites filled with GO modified with oligo/poly(Glu) were confirmed via alkaline phosphatase assay as well as calcein and alizarin red S staining after osteogenic differentiation of hMSC in vitro.

Physiologic isolation and expansion of human mesenchymal stem/stromal cells for manufacturing of cell-based therapy products.

The utilization of mesenchymal stem/stromal cells raises new hopes in treatment of diseases and pathological conditions, while at the same time bringing immense challenges for researchers, manufacturers and physicians. It is essential to consider all steps along the in vitro fabrication of cell-based products in order to reach efficient and reproducible treatment outcomes. Here, the optimal protocols for isolation, cultivation and differentiation of mesenchymal stem cells are required. In this review we discuss these aspects and their influence on the final cell-based product quality. We demonstrate that physiological in vitro cell cultivation conditions play a crucial role in therapeutic functionalities of cultivated cells. We show that three-dimensional cell culture, dynamic culture conditions and physiologically relevant in vitro oxygen concentrations during isolation and expansion make a decisive contribution towards the improvement of cell-based products in regenerative medicine.

Studies on oxygen availability and the creation of natural and artificial oxygen gradients in gelatin-methacryloyl hydrogel 3D cell culture.

Three-dimensional (3D) cultivation platforms allow the creation of cell models, which more closely resemble in vivo-like cell behavior. Therefore, 3D cell culture platforms have started to replace conventional two-dimensional (2D) cultivation techniques in many fields. Besides the advantages of 3D culture, there are also some challenges: cultivation in 3D often results in an inhomogeneous microenvironment and therefore unique cultivation conditions for each cell inside the construct. As a result, the analysis and precise control over the singular cell state is limited in 3D. In this work, we address these challenges by exploring ways to monitor oxygen concentrations in gelatin methacryloyl (GelMA) 3D hydrogel culture at the cellular level using hypoxia reporter cells and deep within the construct using a non-invasive optical oxygen sensing spot. We could show that the appearance of oxygen limitations is more prominent in softer GelMA-hydrogels, which enable better cell spreading. Beyond demonstrating novel or space-resolved techniques of visualizing oxygen availability in hydrogel constructs, we also describe a method to create a stable and controlled oxygen gradient throughout the construct using a 3D printed flow-through chamber.

Corrigendum: Hypoxia Onset in Mesenchymal Stem Cell Spheroids: Monitoring With Hypoxia Reporter Cells.

[This corrects the article DOI: 10.3389/fbioe.2021.611837.].

Biocompatible Nanoparticles Based on Amphiphilic Random Polypeptides and Glycopolymers as Drug Delivery Systems.

In this research, the development and investigation of novel nanoobjects based on biodegradable random polypeptides and synthetic non-degradable glycopolymer poly(2-deoxy-2-methacrylamido-d-glucose) were proposed as drug delivery systems. Two different approaches have been applied for preparation of such nanomaterials. The first one includes the synthesis of block-random copolymers consisting of polypeptide and glycopolymer and capable of self-assembly into polymer particles. The synthesis of copolymers was performed using sequential reversible addition-fragmentation chain transfer (RAFT) and ring-opening polymerization (ROP) techniques. Amphiphilic poly(2-deoxy-2-methacrylamido-d-glucose)-b-poly(l-lysine-co-l-phenylalanine) (PMAG-b-P(Lys-co-Phe)) copolymers were then used for preparation of self-assembled nanoparticles. Another approach for the formation of polypeptide-glycopolymer particles was based on the post-modification of preformed polypeptide particles with an oxidized glycopolymer. The conjugation of the polysaccharide on the surface of the particles was achieved by the interaction of the aldehyde groups of the oxidized glycopolymer with the amino groups of the polymer on particle surface, followed by the reduction of the formed Schiff base with sodium borohydride. A comparative study of polymer nanoparticles developed with its cationic analogues based on random P(Lys-co-d-Phe), as well as an anionic one-P(Lys-co-d-Phe) covered with heparin--was carried out. In vitro antitumor activity of novel paclitaxel-loaded PMAG-b-P(Lys-co-Phe)-based particles towards A549 (human lung carcinoma) and MCF-7 (human breast adenocarcinoma) cells was comparable to the commercially available Paclitaxel-LANS.

3D-Printed composite scaffolds based on poly(ε-caprolactone) filled with poly(glutamic acid)-modified cellulose nanocrystals for improved bone tissue regeneration.

The manufacturing of modern scaffolds with customized geometry and personalization has become possible due to the three-dimensional (3D) printing technique. A novel type of 3D-printed scaffolds for bone tissue regeneration based on poly(ε-caprolactone) (PCL) filled with nanocrystalline cellulose modified by poly(glutamic acid) (PGlu-NCC) has been proposed in this study. The 3D printing set-ups were optimized in order to obtain homogeneous porous scaffolds. Both polymer composites and manufactured 3D scaffolds have demonstrated mechanical properties suitable for a human trabecular bone. Compression moduli were in the range of 334-396 MPa for non-porous PCL and PCL-based composites, and 101-122 MPa for porous scaffolds made of the same materials. In vitro mineralization study with the use of human mesenchymal stem cells (hMSCs) revealed the larger Ca deposits on the surface of PCL/PGlu-NCC composite scaffolds. Implantation of the developed 3D scaffolds into femur of the rabbits was carried out to observe close and delayed effects. The histological analysis showed the lowest content of immune cells and thin fibrous capsule, revealing low toxicity of the PCL/PGlu-NCC scaffolds seeded with rabbit MSCs (rMSCs) to the surrounding tissues. The most pronounced result on the generation of new bone tissue after implantation of PCL/PGlu-NCC + rMSCs scaffolds was detected by both microcomputed tomography and histological analysis. Around 33% and 55% of bone coverage were detected for composite 3D scaffolds with adhered rMSCs after 1 and 3 months of implantation, respectively. This achievement can be a result of synergistic effect of PGlu, which attracts calcium ions, and stem cells with osteogenic potential.