3D bioprinting of mouse pre-osteoblasts and human MSCs using bioinks consisting of gelatin and decellularized bone particles.

One of the key challenges in biofabrication applications is to obtain bioinks that provide a balance between printability, shape fidelity, cell viability, and tissue maturation. Decellularization methods allow the extraction of natural extracellular matrix, preserving tissue-specific matrix proteins. However, the critical challenge in bone decellularization is to preserve both organic (collagen, proteoglycans) and inorganic components (hydroxyapatite) to maintain the natural composition and functionality of bone. Besides, there is a need to investigate the effects of decellularized bone (DB) particles as a tissue-based additive in bioink formulation to develop functional bioinks. Here we evaluated the effect of incorporating DB particles of different sizes (≤45 and ≤100µm) and concentrations (1%, 5%, 10% (wt %)) into bioink formulations containing gelatin (GEL) and pre-osteoblasts (MC3T3-E1) or human mesenchymal stem cells (hTERT-MSCs). In addition, we propose a minimalistic bioink formulation using GEL, DB particles and cells with an easy preparation process resulting in a high cell viability. The printability properties of the inks were evaluated. Additionally, rheological properties were determined with shear thinning and thixotropy tests. The bioprinted constructs were cultured for 28 days. The viability, proliferation, and osteogenic differentiation capacity of cells were evaluated using biochemical assays and fluorescence microscopy. The incorporation of DB particles enhanced cell proliferation and osteogenic differentiation capacity which might be due to the natural collagen and hydroxyapatite content of DB particles. Alkaline phosphatase activity is increased significantly by using DB particles, notably, without an osteogenic induction of the cells. Moreover, fluorescence images display pronounced cell-material interaction and cell attachment inside the constructs. With these promising results, the present minimalistic bioink formulation is envisioned as a potential candidate for bone tissue engineering as a clinically translatable material with straightforward preparation and high cell activity.

Fish scale containing alginate dialdehyde-gelatin bioink for bone tissue engineering.

The development of biomaterial inks suitable for biofabrication and mimicking the physicochemical properties of the extracellular matrix is essential for the application of bioprinting technology in tissue engineering (TE). The use of animal-derived proteinous materials, such as jellyfish collagen, or fish scale (FS) gelatin (GEL), has become an important pillar in biomaterial ink design to increase the bioactivity of hydrogels. However, besides the extraction of proteinous structures, the use of structurally intact FS as an additive could increase biocompatibility and bioactivity of hydrogels due to its organic (collagen) and inorganic (hydroxyapatite) contents, while simultaneously enhancing mechanical strength in three-dimensional (3D) printing applications. To test this hypothesis, we present here a composite biomaterial ink composed of FS and alginate dialdehyde (ADA)-GEL for 3D bioprinting applications. We fabricate 3D cell-laden hydrogels using mouse pre-osteoblast MC3T3-E1 cells. We evaluate the physicochemical and mechanical properties of FS incorporated ADA-GEL biomaterial inks as well as the bioactivity and cytocompatibility of cell-laden hydrogels. Due to the distinctive collagen orientation of the FS, the compressive strength of the hydrogels significantly increased with increasing FS particle content. Addition of FS also provided a tool to tune hydrogel stiffness. FS particles were homogeneously incorporated into the hydrogels. Particle-matrix integration was confirmed via scanning electron microscopy. FS incorporation in the ADA-GEL matrix increased the osteogenic differentiation of MC3T3-E1 cells in comparison to pristine ADA-GEL, as FS incorporation led to increased ALP activity and osteocalcin secretion of MC3T3-E1 cells. Due to the significantly increased stiffness and supported osteoinductivity of the hydrogels, FS structure as a natural collagen and hydroxyapatite source contributed to the biomaterial ink properties for bone engineering applications. Our findings indicate that ADA-GEL/FS represents a new biomaterial ink formulation with great potential for 3D bioprinting, and FS is confirmed as a promising additive for bone TE applications.