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.

Tissue engineering of large organs is currently limited by the lack of potent vascularizationin vitro. Tissue-engineered bone grafts can be prevascularizedin vitrousing endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors.

Described for the first time in 1971, Schimke immuno-osseous dysplasia (SIOD) is an autosomal-recessive multisystem disorder that is caused by bi-allelic mutations of SMARCAL1, which encodes a DNA annealing helicase. To define better the dental anomalies of SIOD, we reviewed the records from SIOD patients with identified bi-allelic SMARCAL1 mutations, and we found that 66.0% had microdontia, hypodontia, or malformed deciduous and permanent molars. Immunohistochemical analyses showed expression of SMARCAL1 in all developing teeth, raising the possibility that the malformations are cell-autonomous consequences of SMARCAL1 deficiency. We also found that stimulation of cultured skin fibroblasts from SIOD patients with the tooth morphogens WNT3A, BMP4, and TGFβ1 identified altered transcriptional responses, raising the hypothesis that the dental malformations arise in part from altered responses to developmental morphogens. To the best of our knowledge, this is the first systematic study of the dental anomalies associated with SIOD.

Grafts aside, current strategies employed to overcome bone loss still fail to reproduce native tissue physiology. Among the emerging bioprinting strategies, laser-assisted bioprinting (LAB) offers very high resolution, allowing designing micrometric patterns in a contactless manner, providing a reproducible tool to test ink formulation. To this date, no LAB associated ink succeeded to provide a reproducible ad integrum bone regeneration on a murine calvaria critical size defect model. Using the Conformité Européenne (CE) approved BioRoot RCS® as a mineral addition to a collagen-enriched ink compatible with LAB, the present study describes the process of the development of a solidifying tricalcium silicate-based ink as a new bone repair promoting substrates in a LAB model. This ink formulation was mechanically characterized by rheology to adjust it for LAB. Printed aside stromal cells from apical papilla (SCAPs), this ink demonstrated a great cytocompatibility, with significant in vitro positive impact upon cell motility, and an early osteogenic differentiation response in the absence of another stimulus. Results indicated that the in vivo application of this new ink formulation to regenerate critical size bone defect tends to promote the formation of bone volume fraction without affecting the vascularization of the neo-formed tissue. The use of LAB techniques with this ink failed to demonstrate a complete bone repair, whether SCAPs were printed or not of at its direct proximity. The relevance of the properties of this specific ink formulation would therefore rely on the quantity applied in situ as a defect filler rather than its cell modulation properties observed in vitro . For the first time, a tricalcium silicate-based printed ink, based on rheological analysis, was characterized in vitro and in vivo , giving valuable information to reach complete bone regeneration through formulation updates. This LAB-based process could be generalized to normalize the characterization of candidate ink for bone regeneration.