INTRODUCTION The vascularization of engineered bone tissue constructs is currently regarded as a sine qua non condition for successful regenerative strategies. However, doubts persist on the ideal type of endothelial cells to be used. Umbilical cord blood (UCB) has been suggested as a source of endothelial progenitors, namely the CD34+ cells sub-population. Carrageenans (CRG) are natural sulphated polyssacharides that have been recently proposed for regenerative medicine purposes.1 In the present work, a co-culture system composed by osteoblasts and the mononuclear cell fraction of UCB, using CRG membranes as carriers, was defined to take advantage of the osteoblast capacity to generate angiogenic conditions. The in vivo performance of those systems was then evaluated after subcutaneous implantation in nude mice. EXPERIMENTAL METHODS Hybrid kappa/iota CRG extracted from Chondrus crispus was used to produce membranes of 1.1 cm diameter and 1mm thickness. Osteoblasts were differentiated from adipose-derived stem cells, seeded in the CRG membranes and cultured for 7 days until confluence. The mononuclear fraction of UCB was isolated by differential centrifugation and seeded on top of the osteoblasts monolayer. Cells were co-cultured for 7 and 21 days and screened for DiI-AcLDL uptake, and CD31 and vWF expression by immunocytochemistry. Osteopontin, osteocalcin, collagen I, vWF, CD31 and VE-cadherin transcripts were also quantified. After each timepoint, the constructs were subcutaneously implanted in nude mice and the implants retrieved after 7 and 21 days for characterization by H&E and immunohistochemistry for CD31. The number of blood vessel around the implants was also quantified. RESULTS AND DISCUSSION After 7 and 21 days of co-culture in the CRG membranes, cells positive for CD31 and vWF were found throughout the culture. Gene expression analysis showed over expression of CD31 and VE-cadherin in the co-cultures, especially after 7 and 21 days of culture, suggesting the presence of cell committed to the endothelial lineage. H&E in vivo results showed the presence of a persistent and an enhanced inflammatory infiltrate surrounding the implants up to 21 days of implantation. The presence of polymorphonuclear neutrophils, macrophages and foreign body giant cells was detected. Similar infiltrates were detected when membranes with monoculture of osteoblasts were implanted, suggesting that the CRG might be causing the observed inflammatory reaction. Nevertheless, human CD31 positive cells were found as part of blood vessels surrounding the co-culture implants but not the osteoblast monoculture, demonstrating the existence of endothelial progenitors among the cultured UCB cell population. Figure 1- H&E staining (A) and hCD31 (B) immunostaining of the retrieved implants after 21 days of co-culture followed by 21 days of implantation. Arrow indicates a blood vessel incorporating hCD31 positive cells. Blood vessel quantification around the implants showed higher number of vessel in the vicinity of the implants with the co-cultures demonstrating that the cells from the endothelial lineage present in the mononuclear fraction of UCB were able to contribute to new vessel formation. CONCLUSION These results showed that osteoblasts can sustain the survival of endothelial progenitor cells present in the mononuclear fraction of UCB after co-culture in CRG membranes. Furthermore, the cells from the endothelial lineage were capable to contribute to new blood vessel formation in vivo in an inflammatory setting. This indicates how the mononuclear fraction of UCB can be used without selecting a determined population to improve the in vivo vascularization of bone tissue engineered constructs. REFERENCES 1. Santo VE, Frias AM, Carida M, Cancedda R, Gomes ME, Mano JoF, Reis RL (2009) Biomacromolecules 10(6):1392-1401. ACKNOWLEDGMENTS PhD grant SFRH/BD/44893/2008 to R.P. Pirraco by the Portuguese Foundation for Science and Technology is acknowledged
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