Biofabrication is enriching the tissue engineering field with new ways of producing structurally organized complex tissues. Among the numerous bioinks under investigation, hyaluronic acid (HA) and its derivatives stand out for their biological relevance, cytocompatibility, shear-thinning properties, and potential to fine-tune the desired properties with chemical modification. In this paper, we review the recent advances on bioinks containing HA. The available literature is presented based on subjects including the rheological properties in connection with printability, the chemical strategies for endowing HA with the desired properties, the clinical application, the most advanced preclinical studies, the advantages and limitations in comparison with similar biopolymer-based bioinks, and future perspectives.

Extrusion-based three-dimensional bioprinting relies on bioinks engineered to combine viscoelastic properties for extrusion and shape retention, and biological properties for cytocompatibility and tissue regeneration. To satisfy these conflicting requirements, bioinks often utilize either complex mixtures or complex modifications of biopolymers. In this paper we introduce and characterize a bioink exploiting a dual crosslinking mechanism, where an enzymatic reaction forms a soft gel suitable for cell encapsulation and extrusion, while a visible light photo-crosslinking allows shape retention of the printed construct. The influence of cell density and cell type on the rheological and printability properties was assessed correlating the printing outcomes with the damping factor, a rheological characteristic independent of the printing system. Stem cells, chondrocytes and fibroblasts were encapsulated, and their viability was assessed up to 14 days with live/dead, alamar blue and trypan blue assays. Additionally, the impact of the printing parameters on cell viability was investigated. Owing to its straightforward preparation, low modification, presence of two independent crosslinking mechanisms for tuning shear-thinning independently of the final shape fixation, the use of visible green instead of UV light, the possibility of encapsulating and sustaining the viability of different cell types, the hyaluronan bioink here presented is a valid biofabrication tool for producing 3D printed tissue-engineered constructs.

Photo-crosslinked synthetic biodegradable polymer networks are highly interesting materials for utilization in biomedical applications such as drug delivery, cell encapsulation and tissue engineering scaffolds. Varying the architecture, chemistry, degree of functionalization and molecular weight of the macromer precursor molecules results in networks with a wide range of physical- and mechanical properties, crosslinking densities, degradation characteristics and thus in potential applications. Photo-crosslinked networks can easily be prepared and have the possibility to entrap a wide range of (biologically active) substances and cells. Additionally, spatial and temporal control over the crosslinking process when using additive manufacturing processes, allows for the preparation of network structures with complex shapes. Photo-crosslinked networks have been used to prepare drug delivery devices, as these networks allow for drug delivery in a controlled way over a prolonged period of time. Furthermore, additive manufacturing techniques such as extrusion-based additive manufacturing and stereolithography have been used to prepare photo-crosslinked tissue engineering scaffolds. This allows for the preparation of designed porous structures with precise control over the pore size and pore architecture and optimal mechanical properties. In particular for stereolithography, a wide variety of resins based on biodegradable photo-crosslinkable macromers has been developed.

The introduction of two-photon polymerization (2PP) to the field of tissue engineering and regenerative medicine (TERM) has led to great expectations for the production of scaffolds with an unprecedented degree of complexity and tailorable architecture. Unfortunately, resolution and size are usually mutually exclusive when using 2PP, resulting in a lack of highly-detailed scaffolds with a relevant size for clinical application. Through the combination of using a highly reactive photopolymer and optimizing key printing parameters, we propose for the first time a biodegradable and biocompatible poly(trimethylene-carbonate) (PTMC)-based scaffold of large size (18 × 18 × 0.9 mm) with a volume of 292 mm³ produced using 2PP. This increase in size results in a significant volumetric increase by almost an order of magnitude compared to previously available large-scale structures (Stichel 2010 J. Laser Micro./Nanoeng. 5 209–12). The structure’s detailed design resulted in a highly porous scaffold (96%) with excellent cytocompatibility, supporting the attachment, proliferation and differentiation of human adipose-derived mesenchymal stem cells towards their osteogenic and chondrogenic lineages. This work strongly attests that 2PP is becoming a highly suitable technique for producing large-sized scaffolds with a complex architecture. We show as a proof-of-concept that an arrayed design of repetitive units can be produced, but a further perspective will be to print scaffolds with anisotropic features that are more representative of human tissues.