title: Recapitulating cranial osteogenesis with neural crest cells in 3-D microenvironments.
abstract: 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.
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Bumjin
authors.initials: B
authors.lastname: Namkoong
authors.affiliation: Demirci BAMM Labs, Canary Center at Stanford for Early Cancer Detection, Department of Radiology, Department of Electrical Engineering (By courtesy), Stanford School of Medicine, Palo Alto, CA 94304, USA; Izmir Biomedicine and Genome Center, Dokuz Eylul University Health Campus, Balcova, 35350 Izmir, Turkey.
authors.forename: Sinan
authors.initials: S
authors.lastname: Güven
authors.affiliation: Demirci BAMM Labs, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Shwathy
authors.initials: S
authors.lastname: Ramesan
authors.affiliation: Demirci BAMM Labs, Canary Center at Stanford for Early Cancer Detection, Department of Radiology, Department of Electrical Engineering (By courtesy), Stanford School of Medicine, Palo Alto, CA 94304, USA.
authors.forename: Volha
authors.initials: V
authors.lastname: Liaudanskaya
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA; Current address: Department of Life Sciences, Imperial College London, Silwood Park Campus Buckhurst Road, Ascot, Berkshire SL5 7PY, United Kingdom; Current address: Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom. Electronic address: a.abzhanov@imperial.ac.uk.
authors.forename: Arhat
authors.initials: A
authors.lastname: Abzhanov
authors.affiliation: Demirci BAMM Labs, Canary Center at Stanford for Early Cancer Detection, Department of Radiology, Department of Electrical Engineering (By courtesy), Stanford School of Medicine, Palo Alto, CA 94304, USA; Demirci BAMM Labs, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address: utkan@stanford.edu.
authors.forename: Utkan
authors.initials: U
authors.lastname: Demirci
mesh_terms: D000469:Alkaline Phosphatase / Q000502:physiology; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D050659:Core Binding Factor Alpha 1 Subunit / Q000502:physiology; D020285:Cryoelectron Microscopy; D003470:Culture Media; D005614:Freeze Fracturing; D005780:Gelatin / Q000737:chemistry; D020100:Hydrogels / Q000737:chemistry; D007150:Immunohistochemistry; D051379:Mice; D008855:Microscopy, Electron, Scanning; D009432:Neural Crest / Q000166:cytology*; D015675:Osteocalcin / Q000502:physiology; D010012:Osteogenesis / Q000502:physiology*; D053495:Osteopontin / Q000502:physiology; D012886:Skull / Q000196:embryology*; D000076182:Sp7 Transcription Factor; D023822:Tissue Engineering / Q000379:methods*; D014157:Transcription Factors / Q000502:physiology
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D003470:Culture Media; D020100:Hydrogels; C493716:Runx2 protein, mouse; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; C506896:Spp1 protein, mouse; D014157:Transcription Factors; D015675:Osteocalcin; D053495:Osteopontin; D005780:Gelatin; D000469:Alkaline Phosphatase
keywords: 3-D culture; Bone; Controlling 3-D tissue microenvironment; Ectodermal to mesodermal differentiation