The Stiffness and Structure of Three-Dimensional Printed Hydrogels Direct the Differentiation of Mesenchymal Stromal Cells Toward Adipogenic and Osteogenic Lineages

Abstract

The mechanical and physicochemical effects of three-dimensional (3D) printable hydrogels on cell behavior are paramount features to consider before manufacturing functional tissues. We hypothesize that besides good printability and cytocompatibility of a supporting hydrogel for the manufacture of individual tissues, it is equally essential to consider beforehand the desired tissue (bone, cartilage, fat). In light of its application, the structure and stiffness of printable hydrogel matrices influence cell geometry, which in turn impacts the differentiation fate. Embedded human mesenchymal stromal cells in printable type I collagen- and chitosan-agarose blends were induced to differentiate toward osteoblasts and adipocytes. Hydrogels' printability in air versus submerged printing in perfluorocarbon was evaluated according to the height, diameter, uniformity, and stability of 3D printed vertical cylinders. Bipotent differentiation within hydrogels was assessed histologically (morphology, cellularity), by immunohistochemistry (vimentin, smooth muscle actin), two-photon microscopy (spatial distribution), and real-time polymerase chain reaction (ALP, BGLAP, OPN, RUNX2, COL 1, aP2, PPARγ-2). Agarose and agarose blends revealed the most valid printability properties by generating uniform cylinders with an average height of 4 mm. Osteogenic differentiation was preferably achieved in anisotropic soft collagen-rich substrates, whereas adipogenic differentiation mostly occurred in isotropic stiff agarose-rich matrices. The conjugation of type I collagen to agarose with varying ratios is possibly a suitable bioink for a broad range of 3D printed mesenchymal tissues.

Cell‐secreted extracellular matrix formation and differentiation of adipose‐derived stem cells in 3D alginate scaffolds with tunable properties

“…In a previous study using polyacrylamide hydrogels, stem cell fate was shown to be solely dependent on scaffold stiffness and independent of porosity . This is however not the case in our scaffolds, which suggests that the scaffold material itself plays an important role in influencing the stem cell fate . In fact, it is not the pore size alone that determines cell proliferation, but a combination of material stiffness, pore architecture properties and overall structure of the scaffold.…”

Corneal bioprinting utilizing collagen‐based bioinks and primary human keratocytes

“…This is an indicator for unsuccessful integration of CSK inside the bioinks, because CSK are naturally dendritic in native corneal stromal tissue, as shown in Figure a,b. We hypothesized that the reason for this difference in cell morphology is due to used solid concentrations of the bioink (Duarte Campos et al, , ). Lower solids concentrations of the polysaccharide component (0.5% agarose) in the blended bioink allowed for increased cell spreading compared to higher solids concentrations (3.0% sodium alginate), as employed in the study by Isaacson et al…”

“…Using DoD, we bioprinted cultivated CSK‐loaded Type I collagen‐based bioinks suitable for corneal stromal regeneration (Figure ). The bioinks were made of two essential components: (a) Type I collagen for improved functionality, and (b) agarose hydrogel for fast and precise printability (Duarte Campos et al, , ). Working with collagen faces some drawbacks, since collagen alone lacks the stability needed to support a corneal construct, which makes further stabilization steps such as cross‐linking necessary (Duan & Sheardown, ).…”

Mechanically Tunable Bioink for 3D Bioprinting of Human Cells

“…The hMSCs were isolated from femoral heads of three independent donors as described previously. [41] For determining cell viability, 500 µL of each of the NA or CA solution was mixed at 37 °C and resuspended with equal volumes of the cell suspension resulting in a final cell concentration of 10 6 cells mL volume of 100 µL were printed using the 300 µm microvalve (Fritz Gyger, Gwatt, Switzerland) at an air pressure of 50 kPa and a valve gating time of 450 ms. As a control three samples with a volume of 100 µL of each hydrogel type were pipetted into the well plate. Immediately after printing cell viability was assessed using a vital fluorescence staining assay.…”