During the last decade, research on 3D-bioprinting has been growing exponentially. With the unlocked ability to produce engineered tissues with desired shapes, gradients of composition and biological cues, scientists and clinicians have focused their efforts on reproducing human native tissues. The current availability of diverse biomaterials (synthetic and natural) allows a variety of combinations with cells to mimic corresponding human tissues. Despite significant advances in controlling construct resolution, composition and shape, few studies address the complexity of reproducing the extracellular matrix (ECM) composition. Moreover, control over the microscopic architecture within the biomaterial ink is often overlooked. This macromolecular organization is yet paramount, as it strongly influences cell’s shape, migration, proliferation and differentiation (in case of stem cells). Thereby, reproducing anisotropic tissues – non-uniform tissues that have properties and/or structure oriented in a specific direction, like e.g. bones, cartilage or intervertebral discs – remains today a challenge.
A group of researchers from the AO Research Institute Davos led by Dr. Matteo D’Este developed a bioink containing both collagen (Col) type 1 and tyramine derivative hyaluronan (THA) as a promising composite biomaterial for connective tissue engineering. Hyaluronan (HA) is indeed known to induce cell proliferation, chondrogenic differentiation (cell profile typically found in cartilage) and matrix synthesis, as well as being able to hold osmotically a large amount of water molecules, thus providing compressive strength through fluid retention. Col is the most abundant protein in the ECM, displays a hierarchical fibrillar structure and provides a specific amino acid sequence (RGD) that allows cell attachment. This group previously developed an expertise in the interaction of those two complementary biomaterials. In their study entitled “Tissue mimetic hyaluronan bioink containing collagen fibers with controlled orientation modulating cell migration and alignment” published in the journal Materials Today Bio, the group aimed to print a homogeneous 3D hydrogel with a controlled Col fibril orientation at the microscopic level and evaluated the impact of this anisotropic architecture on embedded human bone marrow-derived mesenchymal stromal cell (hMSC).
The authors prepared separately an acidic Col solution and a THA solution containing a suspension of hMSC. Using concentrations and ratios previously defined optimal for cartilaginous tissue engineering, both biopolymers and cells were mixed, before the induction of simultaneously two mild crosslinking pathways: (i) the fibrillogenesis and physical gelation of collagen triggered with the pH increase up to 7, and (ii) the enzymatic chemical crosslinking of THA at 37 °C (optimal functioning temperature for the enzyme). After an incubation of 30 min at 37 °C, the 3D-scaffold was printed thanks to the shear-thinning properties of the composite bioink. Shear-thinning behavior describes the characteristic of some non-Newtonian fluids that have a decreasing viscosity when submitted to increasing shear stresses. In this case, the concentrated crosslinked THA flows through the narrow needle, while the Col fibrils present the ability to align themselves along the printing direction. This innovative method allows to print an anisotropic hydrogel, where for hydrogel casted with a pipette, macromolecules randomly disperse themselves (see Figure 1). After the printing, the scaffold structure was stabilized with a subsequent photo-crosslinking using eosin Y as a photoinitiator, triggered at 515 nm at 4 mm/s.
The presence and alignment of Col inside the composite printed THA-Col hydrogels was investigated with SHG imaging, confocal microscopy and turbidimetry, as Col fibrils reflect light and are able to generate SHG light. The shear-thinning behavior of the bioink was assessed through viscosity measurements with a rheometer. All crosslinking conditions and mixing ratios of both biopolymers were selected to obtain optimal extrudability conditions. And hMSC were cultivated inside the printed hydrogels for 21 days. Finally, the architecture of the printed scaffold was compared with the native knee articular cartilage, set in this study as an anisotropic 3D-bioprinting challenge. In the human body, this connective tissue displays parallel Col fibrils near the surface, and fibrils orthogonal to these, in the depths. The authors successfully reproduced this pattern.
Overall, the mechanical properties of the final printed scaffold weren’t measured. However, the authors showed that the mild crosslinking conditions used before printing did not alter the ability of Col fibrils to align when extruded, by complying with the applied shearing stress. Despite a moderate printing resolution due to the decrease of the storage modulus during this shear-thinning phenomenon, THA-Col bioink still allowed the printing of the construct mimicking the fibrils orientation of articular cartilage. By preserving the fibrillar structure of Col in combination with HA biological properties, (i) the cellular adhesion was enhanced, (ii) the migration of hMSC along the unidirectional orientation of Col fibrils was promoted, and (iii) the chondrogenic differentiation of hMSC resulted in cartilage-like matrix deposition throughout the hydrogel.
The target tissue in this study was the knee articular cartilage. But the developed 3D-bioprinting technology and innovative approach is definitely of interest for all anisotropic tissue of the human body. The cellular response stimulation, actually paramount to regenerate native tissue on the long term, depends not only on the composition and mechanical properties of the hydrogel, but also on the multiscale architecture control, whether at the microscopic level with macromolecules arrangement, at the macroscopic level with homogeneity assured, or at the design of the whole 3D-scaffold itself. This 3D-bioprinting study is a new milestone towards the regeneration and the mimic of native human tissues.
 Schwab, A., Hélary, C., Richards, R. G., Alini, M., Eglin, D., & d’Este, M. (2020). Tissue mimetic hyaluronan bioink containing collagen fibers with controlled orientation modulating cell migration and alignment. Materials Today Bio, 7, 100058.
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