Endothelial cells enhance the in vivo bone-forming ability of osteogenic cell sheets

Addressing the problem of vascularization is of vital importance when engineering three-dimensional (3D) tissues. Endothelial cells are increasingly used in tissue-engineered constructs to obtain prevascularization and to enhance in vivo neovascularization. Rat bone marrow stromal cells were cultured in thermoresponsive dishes under osteogenic conditions with human umbilical vein endothelial cells (HUVECs) to obtain homotypic or heterotypic cell sheets (CSs). Cells were retrieved as sheets from the dishes after incubation at 20 °C. Monoculture osteogenic CSs were stacked on top of homotypic or heterotypic CSs, and subcutaneously implanted in the dorsal flap of nude mice for 7 days. The implants showed mineralized tissue formation under both conditions. Transplanted osteogenic cells were found at the new tissue site, demonstrating CS bone-inductive effect. Perfused vessels, positive for human CD31, confirmed the contribution of HUVECs for the neovascularization of coculture CS constructs. Furthermore, calcium quantification and expression of osteocalcin and osterix genes were higher for the CS constructs, with HUVECs demonstrating the more robust osteogenic potential of these constructs. This work demonstrates the potential of using endothelial cells, combined with osteogenic CSs, to increase the in vivo vascularization of CS-based 3D constructs for bone tissue engineering purposes.We would like to acknowledge Mariana T Cerqueira for the illustration in Figure 1. This study was supported by Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology 'Cell Sheet Tissue Engineering Center (CSTEC)' and the Global CUE program, the Multidisciplinary Education and Research Center for Regenerative Medicine (MERCREM), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Financial support to RP Pirraco by the Portuguese Foundation for Science and Technology (FCT) through the PhD Grant SFRH/BD/44893/2008 is also acknowledged

Ectopic bone formation in cell-seeded poly(ethylene oxide)/poly(butylene terephthalate) copolymer scaffolds of varying porosity

Scaffolds from poly(ethylene oxide) and poly(butylene terephthalate), PEOT/PBT, with a PEO molecular weight of 1,000 and a PEOT content of 70 weight% (1000PEOT70PBT30) were prepared by leaching salt particles (425–500 μm). Scaffolds of 73.5, 80.6 and 85.0% porosity were treated with a CO2 gas plasma and seeded with rat bone marrow stromal cells (BMSCs). After in vitro culture for 7 days (d) in an osteogenic medium the scaffolds were subcutaneously implanted for 4 weeks in nude mice. Poly(d, l-lactide) (PDLLA) and biphasic calcium phosphate (BCP) scaffolds were included as references. After 4 weeks (wks) all scaffolds showed ectopic formation of bone and bone marrow. For the scaffolds of different porosities, no significant differences were observed in the relative amounts of bone (7–9%) and bone marrow (6–11%) formed, even though micro computed tomography (μ-CT) data showed considerable differences in accessible pore volume and surface area. 1000PEOT70PBT30 scaffolds with a porosity of 85% could not maintain their original shape in vivo. Surprisingly, 1000PEOT70PBT30 scaffolds with a porosity of 73.5% showed cartilage formation. This cartilage formation is most likely due to poorly accessible pores in the scaffolds, as was observed in histological sections. μ-CT data showed a considerably smaller accessible pore volume (as a fraction of the total volume) than in 1000PEOT70PBT30 scaffolds of 80.6 and 85.0% porosity. BMSC seeded PDLLA (83.5% porosity) and BCP scaffolds (29% porosity) always showed considerably more bone and bone marrow formation (bone marrow formation is approximately 40%) and less fibrous tissue ingrowth than the 1000PEOT70PBT30 scaffolds. The scaffold material itself can be of great influence. In more hydrophobic and rigid scaffolds like the PDLLA or BCP scaffolds, the accessibility of the pore structure is more likely to be preserved under the prevailing physiological conditions than in the case of hydrophilic 1000PEOT70PBT30 scaffolds. Scaffolds prepared from other PEOT/PBT polymer compositions, might prove to be more suited