The coculture of osteogenic and angiogenic cells and the resulting paracrine signaling via soluble factors are supposed to be crucial for successfully engineering vascularized bone tissue equivalents. In this study, a coculture system combining primary human adipose-derived stem cells (hASCs) and primary human dermal microvascular endothelial cells (HDMECs) within two types of hydrogels based on methacryloyl-modified gelatin (GM) as three-dimensional scaffolds was examined for its support of tissue specific cell functions. HDMECs, together with hASCs as supporting cells, were encapsulated in soft GM gels and were indirectly cocultured with hASCs encapsulated in stiffer GM hydrogels additionally containing methacrylate-modified hyaluronic acid and hydroxyapatite particles. After 14 days, the hASC in the stiffer gels (constituting the "bone gels") expressed matrix proteins like collagen type I and fibronectin, as well as bone-specific proteins osteopontin and alkaline phosphatase. After 14 days of coculture with HDMEC-laden hydrogels, the viscoelastic properties of the bone gels were significantly higher compared with the gels in monoculture. Within the soft vascularization gels, the formed capillary-like networks were significantly longer after 14 days of coculture than the structures in the control gels. In addition, the stability as well as the complexity of the vascular networks was significantly increased by coculture. We discussed and concluded that osteogenic and angiogenic signals from the culture media as well as from cocultured cell types, and tissue-specific hydrogel composition all contribute to stimulate the interplay between osteogenesis and angiogenesis in vitro and are a basis for engineering vascularized bone.

Bone tissue is one of the most frequently transplanted tissues. Since procedures like the transplantation of autologous bone bear risks, though, regenerative medicine and tissue engineering reach to face those problems by engineering bone substitutes by using suitable materials and living cells. For the fabrication of bone tissue equivalents, evolving manufacturing techniques like bioprinting can be used. We developed bioinks that can either support the osteogenic differentiation of human adipose-derived stem cells (hASCs) and formation of a bone matrix by further addition of hydroxyapatite, or the formation of vascular structures by or human microvascular endothelial cells (ECs). The bioinks were used to build up geometries like 3D grids, cylindrical structures and combination hydrogels of bone and vascularization hydrogels via a microextrusion-based printing system, which were afterwards cultured for up to four weeks under static or dynamic culture conditions in a bioreactor. Evaluation of the hydrogels by mechanical analysis and staining of bone specific proteins like collagen type I, alkaline phosphatase and osteopontin showed formation of a bone matrix. This was also observes after culture under control conditions without the addition of osteogenic supplements, indicating osteoinductive properties of the hydroxyapatite. Co-culture of ASCs and ECs in a suitable hydrogel environment resulted in improved formation of bone matrix and capillary structures compared to the respective monocultures. Additionally, the perfusion culture in a bioreactor allowed the build-up and successful culture of cell-laden hydrogel constructs with a volume of >1 cm 2 . In conclusion, we were able to develop bioinks and a printing process which allow the successful build-up of bone tissue equivalents whose bioreactor culture enables the setup of relevant geometries and sizes.