Bioprinting has emerged as a novel technological approach with the potential to address unsolved questions in the field of tissue engineering. We have recently shown that Laser Assisted Bioprinting (LAB), due to its unprecedented cell printing resolution and precision, is an attractive tool for the in situ printing of a bone substitute. Here, we show that LAB can be used for the in situ printing of mesenchymal stromal cells, associated with collagen and nano-hydroxyapatite, in order to favor bone regeneration, in a calvaria defect model in mice. Also, by testing different cell printing geometries, we show that different cellular arrangements impact on bone tissue regeneration. This work opens new avenues on the development of novel strategies, using in situ bioprinting, for the building of tissues, from the ground up.

Vascularization plays a crucial role in bone formation and regeneration process. Development of a functional vasculature to improve survival and integration of tissue-engineered bone substitutes remains a major challenge. Biofabrication technologies, such as bioprinting, have been introduced as promising alternatives to overcome issues related to lack of prevascularization and poor organization of vascular networks within the bone substitutes. In this context, this study aimed at organizing endothelial cells in situ, in a mouse calvaria bone defect, to generate a prevascularization with a defined architecture, and promote in vivo bone regeneration. Laser-assisted bioprinting (LAB) was used to pattern Red Fluorescent Protein-labeled endothelial cells into a mouse calvaria bone defect of critical size, filled with collagen containing mesenchymal stem cells and vascular endothelial growth factor. LAB technology allowed safe and controlled in vivo printing of different cell patterns. In situ printing of endothelial cells gave rise to organized microvascular networks into bone defects. At two months, vascularization rate (vr) and bone regeneration rate (br) showed statistically significant differences between the ‘random seeding’ condition and both ‘disc’ pattern (vr = +203.6%; br = +294.1%) and ‘crossed circle’ pattern (vr = +355%; br = +602.1%). These results indicate that in vivo LAB is a valuable tool to introduce in situ prevascularization with a defined configuration and promote bone regeneration.