Recapitulating cranial osteogenesis with neural crest cells in 3-D microenvironments

The experimental systems that recapitulate the complexity of native tissues and enable precise control over the microenvironment are becoming essential for the pre-clinical tests of therapeutics and tissue engineering. Here, we described a strategy to develop an in vitro platform to study the developmental biology of craniofacial osteogenesis. In this study, we directly osteo-differentiated cranial neural crest cells (CNCCs) in a 3-D in vitro bioengineered microenvironment. Cells were encapsulated in the gelatin-based photo-crosslinkable hydrogel and cultured up to three weeks. We demonstrated that this platform allows efficient differentiation of p75 positive CNCCs to cells expressing osteogenic markers corresponding to the sequential developmental phases of intramembranous ossification. During the course of culture, we observed a decrease in the expression of early osteogenic marker Runx2, while the other mature osteoblast and osteocyte markers such as Osterix, Osteocalcin, Osteopontin and Bone sialoprotein increased. We analyzed the ossification of the secreted matrix with alkaline phosphatase and quantified the newly secreted hydroxyapatite. The Field Emission Scanning Electron Microscope (FESEM) images of the bioengineered hydrogel constructs revealed the native-like osteocytes, mature osteoblasts, and cranial bone tissue morphologies with canaliculus-like intercellular connections. This platform provides a broadly applicable model system to potentially study diseases involving primarily embryonic craniofacial bone disorders, where direct diagnosis and adequate animal disease models are limited.