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