Bioprinting aims to provide new avenues for regenerating damaged human tissues through the controlled printing of live cells and biocompatible materials that can function therapeutically. Polymeric hydrogels are commonly investigated ink materials for 3D and 4D bioprinting applications, as they can contain intrinsic properties relative to those of the native tissue extracellular matrix and can be printed to produce scaffolds of hierarchical organization. The incorporation of nanoscale material additives, such as nanoparticles, to the bulk of inks, has allowed for significant tunability of the mechanical, biological, structural, and physicochemical material properties during and after printing. The modulatory and biological effects of nanoparticles as bioink additives can derive from their shape, size, surface chemistry, concentration, and/or material source, making many configurations of nanoparticle additives of high interest to be thoroughly investigated for the improved design of bioactive tissue engineering constructs. This paper aims to review the incorporation of nanoparticles, as well as other nanoscale additive materials, to printable bioinks for tissue engineering applications, specifically bone, cartilage, dental, and cardiovascular tissues. An overview of the various bioinks and their classifications will be discussed with emphasis on cellular and mechanical material interactions, as well the various bioink formulation methodologies for 3D and 4D bioprinting techniques. The current advances and limitations within the field will be highlighted.

This study aims to investigate the impact of kappa-carrageenan on dental pulp stem cells (DPSCs) behavior in terms of biocompatibility and odontogenic differentiation potential when it is utilized as a component for the production of 3D sponge-like scaffolds. For this purpose, we prepared three types of scaffolds by freeze-drying (i) kappa-carrageenan/chitosan/gelatin enriched with KCl (KCG-KCl) as a physical crosslinker for the sulfate groups of kappa-carrageenan, (ii) kappa-carrageenan/chitosan/gelatin (KCG) and (iii) chitosan/gelatin (CG) scaffolds as a control. The mechanical analysis illustrated a significantly higher elastic modulus of the cell-laden scaffolds compared to the cell-free ones after 14 and 28 days with values ranging from 25 to 40 kPa, showing an increase of 27–36%, with the KCG-KCl scaffolds indicating the highest and CG the lowest values. Cell viability data showed a significant increase from days 3 to 7 and up to day 14 for all scaffold compositions. Significantly increasing alkaline phosphatase (ALP) activity has been observed over time in all three scaffold compositions, while the KCG-KCl scaffolds indicated significantly higher calcium production after 21 and 28 days compared to the CG control. The gene expression analysis of the odontogenic markers DSPP, ALP and RunX2 revealed a two-fold higher upregulation of DSPP in KCG-KCl scaffolds at day 14 compared to the other two compositions. A significant increase of the RunX2 expression between days 7 and 14 was observed for all scaffolds, with a significantly higher increase of at least twelve-fold for the kappa-carrageenan containing scaffolds, which exhibited an earlier ALP gene expression compared to the CG. Our results demonstrate that the integration of kappa-carrageenan in scaffolds significantly enhanced the odontogenic potential of DPSCs and supports dentin-pulp regeneration.

Chronic or improperly healed wounds, either as a result of extended trauma or prolonged inflammatory response, affect a significant percentage of the world population. Hence, there is a growing interest in the development of biomimetic scaffolds that expedite wound closure at the early stages. Curcumin (Cur) is a plant-derived polyphenol with antimicrobial activity, and it accelerates the wound contraction rate. Recently, electrospraying has emerged for the precise deposition of bioactive molecules into scaffolds to improve therapeutic outcomes. In this study, we produced membranes for wound healing and endowed them with antibacterial properties to promote the healing of impaired wounds. Unlike previous studies that incorporated curcumin directly into electrospun fibers, we employed electrospraying to coat curcumin onto PVA/KC membranes. This approach improves the curcumin bioavailability and release kinetics, ensuring sustained therapeutic action. Toward this end, we fabricated four types of membranes, poly(vinyl alcohol) PVA and PVA/kappa carrageenan (KC), using electrospinning, and PVA/KC/Cur5 and PVA/KC/Cur20, in which the PVA/KC membranes were coated with two different concentrations of Cur by electrospraying. All membranes showed low cytotoxicity, good cell adhesion, the capability of enabling cells to produce collagen, and an adequate degradation rate for wound-healing applications. Antibacterial evaluation showed that both Cur-loaded membranes increased the antibacterial efficacy against both Escherichia coli and Staphylococcus aureus compared with PVA and PVA/KC membranes. These findings highlight the potential of electrosprayed curcumin as an effective strategy for bioactive wound dressings.

Main chain polyesters have been extensively used in the biomedical field. Despite their many advantages, including biocompatibility, biodegradability, and others, these materials are rather inert and lack specific functionalities which will endow them with additional biological and responsive properties. In this work, novel pH-responsive main chain polyesters have been prepared by a conventional condensation polymerization of a vinyl functionalized diol with a diacid chloride, followed by a photo-induced thiol-ene click reaction to attach functional carboxylic acid side-groups along the polymer chains. Two different mercaptocarboxylic acids were employed, allowing to vary the alkyl chain length of the polymer pendant groups. Moreover, the degree of modification, and as a result, the carboxylic acid content of the polymers, was easily tuned by varying the irradiation time during the click reaction. Both these parameters, were shown to strongly influence the responsive behavior of the polyesters, which presented adjustable pKα values and water solubilities. Finally, the difunctional polyesters bearing the alkene and carboxylic acid functionalities enabled the preparation of cross-linked polyester films by chemically linking the pendant vinyl bonds on the polymer side groups. The biocompatibility of the cross-linked polymers films was assessed in L929 fibroblast cultures and showed that the cell viability, proliferation, and attachment were greatly promoted on the polyester surface, bearing the shorter alkyl chain length side groups and the higher fraction of carboxylic acid functionalities.

In this work, a sodium alginate-based copolymer grafted by thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) chains was used as gelator (Alg-g-PNIPAM) in combination with methylcellulose (MC). It was found that the mechanical properties of the resulting gel could be enhanced by the addition of MC and calcium ions (Ca²⁺). The proposed network is formed via a dual crosslinking mechanism including ionic interactions among Ca²⁺ and carboxyl groups and secondary hydrophobic associations of PNIPAM chains. MC was found to further reinforce the dynamic moduli of the resulting gels (i.e., a storage modulus of ca. 1500 Pa at physiological body and post-printing temperature), rendering them suitable for 3D printing in biomedical applications. The polymer networks were stable and retained their printed fidelity with minimum erosion as low as 6% for up to seven days. Furthermore, adhered pre-osteoblastic cells on Alg-g-PNIPAM/MC printed scaffolds presented 80% viability compared to tissue culture polystyrene control, and more importantly, they promoted the osteogenic potential, as indicated by the increased alkaline phosphatase activity, calcium, and collagen production relative to the Alg-g-PNIPAM control scaffolds. Specifically, ALP activity and collagen secreted by cells were significantly enhanced in Alg-g-PNIPAM/MC scaffolds compared to the Alg-g-PNIPAM counterparts, demonstrating their potential in bone tissue engineering.

The application of mechanical stimulation on bone tissue engineering constructs aims to mimic the native dynamic nature of bone. Although many attempts have been made to evaluate the effect of applied mechanical stimuli on osteogenic differentiation, the conditions that govern this process have not yet been fully explored. In this study, pre-osteoblastic cells were seeded on PLLA/PCL/PHBV (90/5/5 wt.%) polymeric blend scaffolds. The constructs were subjected every day to cyclic uniaxial compression for 40 min at a displacement of 400 μm, using three frequency values, 0.5, 1, and 1.5 Hz, for up to 21 days, and their osteogenic response was compared to that of static cultures. Finite element simulation was performed to validate the scaffold design and the loading direction, and to assure that cells inside the scaffolds would be subjected to significant levels of strain during stimulation. None of the applied loading conditions negatively affected the cell viability. The alkaline phosphatase activity data indicated significantly higher values at all dynamic conditions compared to the static ones at day 7, with the highest response being observed at 0.5 Hz. Collagen and calcium production were significantly increased compared to static controls. These results indicate that all of the examined frequencies substantially promoted the osteogenic capacity.