title: Prevascularization of a gas-foaming macroporous calcium phosphate cement scaffold via coculture of human umbilical vein endothelial cells and osteoblasts.
abstract: The lack of a vasculature in tissue-engineered constructs is currently a major challenge in tissue regeneration. There has been no report of prevascularization of macroporous calcium phosphate cement (CPC) via coculture of endothelial cells and osteoblasts. The objectives of this study were to (1) investigate coculture of human umbilical vein endothelial cells (HUVEC) and human osteoblasts (HOB) on macroporous CPC for the first time; and (2) develop a new microvasculature-CPC construct with angiogenic and osteogenic potential. A gas-foaming method was used to create macropores in CPC. HUVEC and HOB were seeded with a ratio of HUVEC:HOB=4:1, at 1.5×10(5) cells/scaffold. The constructs were cultured for up to 42 days. CPC with a porosity of 83% had a flexural strength (mean±SD; n=6) of 2.6±0.2 MPa, and an elastic modulus of 340±30 MPa, approaching the reported values for cancellous bone. Reverse transcription-polymerase chain reaction showed that HUVEC+HOB coculture on CPC had much higher vascular endothelial growth factor (VEGF) and collagen I expressions than monoculture (p<0.05). Osteogenic markers alkaline phosphatase, osteocalcin (OC), and runt-related transcription factor 2 (Runx2) were also highly elevated. Immunostaining of PECAM1 (CD31) showed abundant microcapillary-like structures on CPC in coculture at 42 days, as HUVEC self-assembled into extensive branches and net-like structures. However, no microcapillary was found on CPC in monoculture. In immunohistochemical staining, the neo-vessels were strongly positive for PECAM1, the von Willebrand factor, and collagen I. Scanning electron microscopy revealed microcapillary-like structures mingling with mineral nodules on CPC. Cell-synthesized minerals increased by an order of magnitude from 4 to 42 days. In conclusion, gas-foaming macroporous CPC was fabricated and HUVEC+HOB coculture was performed for prevascularization, yielding microcapillary-like structures on CPC for the first time. The novel macroporous CPC-microvasculature construct is promising for a wide range of orthopedic applications with enhanced angiogenic and osteogenic capabilities.
authors.affiliation: Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA.
authors.forename: WahWah
authors.initials: W
authors.lastname: Thein-Han
authors.forename: Hockin H K
authors.initials: HH
authors.lastname: Xu
mesh_terms: D002130:Calcium Phosphates / Q000737:chemistry*; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis / Q000502:physiology; D016062:Porosity; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D002130:Calcium Phosphates; C020243:calcium phosphate