Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) three-dimensional (3D) bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold, which underwent osteogenic differentiation for 2 weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during 2 more weeks of culture. We tested three experimental conditions: condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM): endothelial medium (EM); condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just 4 weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.

Gelatin is a natural biopolymer extensively used for tissue engineering applications due to its similarities to the native extracellular matrix. However, the rheological properties of gelatin formulations are not ideal for extrusion-based bioprinting. In this work, we present an approach to improve gelatin bioprinting performances by using pectin as a rheology modifier of gelatin and (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as a gelatin–pectin crosslinking agent. The preparation of gelatin–pectin formulations is initially optimized to obtain homogenous gelatin–pectin gels. Since the use of GPTMS requires a drying step to induce the completion of the crosslinking reaction, microporous gelatin–pectin–GPTMS sponges are produced through freeze-drying, and the intrinsic properties of gelatin–pectin–GPTMS networks (e.g., porosity, pore size, degree of swelling, compressive modulus, and cell adhesion) are investigated. Subsequently, rheological investigations together with bioprinting assessments demonstrate the key role of pectin in increasing the viscosity and the yield stress of low viscous gelatin solutions. Water stable, three-dimensional, and self-supporting gelatin–pectin–GPTMS scaffolds with interconnected micro- and macroporosity are successfully obtained by combining extrusion-based bioprinting and freeze-drying. The proposed biofabrication approach does not require any additional temperature controller to further modulate the rheological properties of gelatin solutions and it could furthermore be extended to improve the bioprintability of other biopolymers.

Developing biomaterial formulations with specific biochemical characteristics and physical properties suitable for bioprinting of 3D scaffolds is a pivotal challenge in tissue engineering. Therefore, the design of novel bioprintable formulations is a continuously evolving research field. In this work, the authors aim at expanding the library of biomaterial inks by blending two natural biopolymers: pectin and gelatin. Cytocompatible formulations are obtained by combining pectin and gelatin at different ratios and using (3‐glycidyloxypropyl)trimethoxysilane (GPTMS) as single crosslinking agent. It is shown that the developed formulations are all suitable for extrusion‐based 3D bioprinting. Self‐supporting scaffolds with a designed macroporosity and micropores in the bioprinted struts are successfully obtained by combining extrusion‐based bioprinting and freeze‐drying. The presence of gelatin in these formulations allows for the modulation of porosity, of water uptake and of scaffold stiffness in respect to pure pectin scaffolds. Results demonstrate that these new biomaterial formulations, processed with this specific approach, are promising candidates for the fabrication of tissue‐like scaffolds for tissue regeneration.