Tissue mimetic hyaluronan bioink containing collagen fibers with controlled orientation modulating cell migration and alignment.

Biofabrication is providing scientists and clinicians the ability to produce engineered tissues with desired shapes and gradients of composition and biological cues. Typical resolutions achieved with extrusion-based bioprinting are at the macroscopic level. However, for capturing the fibrillar nature of the extracellular matrix (ECM), it is necessary to arrange ECM components at smaller scales, down to the micron and the molecular level. Herein, we introduce a bioink containing the tyramine derivative of hyaluronan (HA; henceforth known as THA) and collagen (Col) type 1. In this bioink, similar to connective tissues, Col is present in the fibrillar form, and HA functions as a viscoelastic space filler. THA was enzymatically cross-linked under mild conditions allowing simultaneous Col fibrillogenesis, thus achieving a homogeneous distribution of Col fibrils within the viscoelastic HA-based matrix. The THA-Col composite displayed synergistic properties in terms of storage modulus and shear thinning, translating into good printability. Shear-induced alignment of the Col fibrils along the printing direction was achieved and quantified via immunofluorescence and second-harmonic generation. Cell-free and cell-laden constructs were printed and characterized, analyzing the influence of the controlled microscopic anisotropy on human bone marrow-derived mesenchymal stromal cell (hMSC) migration. Anisotropic HA-Col showed cell-instructive properties modulating hMSC adhesion, morphology, and migration from micropellets stimulated by the presence and the orientation of Col fibers. Actin filament staining showed that hMSCs embedded in aligned constructs displayed increased cytoskeleton alignment along the fibril direction. Based on gene expression of cartilage/bone markers and ECM production, hMSCs embedded in the isotropic bioink displayed chondrogenic differentiation comparable with standard pellet culture by means of proteoglycan production (safranin O staining and proteoglycan quantification). The possibility of printing matrix components with control over microscopic alignment brings biofabrication one step closer to capturing the complexity of native tissues.

Hyaluronic acid as a bioink for extrusion-based 3D printing.

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.

Thermoreversible hyaluronan-hydrogel and autologous nucleus pulposus cell delivery regenerates human intervertebral discs in an ex vivo, physiological organ culture model.

Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1ρ magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1ρ values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture set-up, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.

3D bioprinting of a hyaluronan bioink through enzymatic-and visible light-crosslinking.

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.

Effects of locally applied adipose tissue-derived microvascular fragments by thermoresponsive hydrogel on bone healing.

Insufficient vascularization is a major cause for the development of non-unions. To overcome this problem, adipose tissue-derived microvascular fragments (MVF) may serve as vascularization units. However, their application into bone defects needs a carrier system. Herein, we analyzed whether this is achieved by a thermoresponsive hydrogel (TRH). MVF were isolated from CD-1 mice and cultivated after incorporation into TRH, while non-incorporated MVF served as controls. Viability of MVF was assessed immunohistochemically over a 7-day period. Moreover, osteotomies were induced in femurs of CD-1 mice. The osteotomy gaps were filled with MVF-loaded TRH (TRH + MVF), unloaded TRH (TRH) or no material (control). Bone healing was evaluated 14 and 35 days postoperatively. MVF incorporated into TRH exhibited less apoptotic cells and showed a stable vessel morphology compared to controls. Micro-computed tomography revealed a reduced bone volume in TRH + MVF femurs. Histomorphometry showed less bone and more fibrous tissue after 35 days in TRH + MVF femurs compared to controls. Accordingly, TRH + MVF femurs exhibited a lower osseous bridging score and a reduced bending stiffness. Histology and Western blot analysis revealed an increased vascularization and CD31 expression, whereas vascular endothelial growth factor (VEGF) expression was reduced in TRH + MVF femurs. Furthermore, the callus of TRH + MVF femurs showed increased receptor activator of NF-κB ligand expression and higher numbers of osteoclasts. These findings indicate that TRH is an appropriate carrier system for MVF. Application of TRH + MVF increases the vascularization of bone defects. However, this impairs bone healing, most likely due to lower VEGF expression during the early course of bone healing. STATEMENT OF SIGNIFICANCE: In the present study we analyzed for the first time the in vivo performance of a thermoresponsive hydrogel (TRH) as a delivery system for bioactive microvascular fragments (MVF). We found that TRH represents an appropriate carrier for MVF as vascularization units and maintains their viability. Application of MVF-loaded TRH impaired bone formation in an established murine model of bone healing, although vascularization was improved. This unexpected outcome was most likely due to a reduced VEGF expression in the early phase bone healing.

Hydrogels in calcium phosphate moldable and injectable bone substitutes: Sticky excipients or advanced 3-D carriers?

The combination of hydrogels and calcium phosphate particles is emerging as a well-established trend for bone substitutes. Besides acting as binders for the inorganic phase, hydrogels within these hybrid materials can modulate cell colonization physically and biologically. The influence of hydrogels on the healing process can also be exploited through their capability to deliver drugs and cells for tissue engineering approaches. The aim of this review is to collect some recent progress in this field, with an emphasis on design aspects and possible future directions.