Engineering vascularized bone: osteogenic and pro-angiogenic potential of
murine periosteal cells
Nick van Gastel,1,4 Scott J. Roberts,2,4 Sophie Torrekens,1 Jan Schrooten,3,4 Frank P. Luyten,2,4
Geert Carmeliet,1,4
1
Laboratory of Experimental Medicine and Endocrinology, 2Laboratory for Skeletal Development and Joint Disorders, 3Department of
Metallurgy and Materials Engineering and 4Prometheus, Division of Skeletal Tissue Engineering, Katholieke Universiteit Leuven,
Leuven, Belgium
One of the key challenges in bone tissue engineering is the timely formation of blood vessels
that promote the survival of the implanted cells in the construct. The addition of angiogenic
factors or the presence of cells with pro-angiogenic properties could thus be crucial for the
successful outcome of a tissue engineered construct. Since fracture healing is enhanced when
the periosteum is present, periosteum-derived cells (PDC) may prove to contribute not only to
osteogenesis but also to angiogenesis. In this study, we therefore characterized the ability of
murine PDC (mPDC) to support the formation of vascular networks, in addition to their
osteogenic potential.
We first established a protocol to specifically isolate periosteal cells from long bones of adult
mice and verified the absence of perichondrial cells, articular chondrocytes, tenocytes or muscle
cells. Mesenchymal cells were abundantly present as more than 50 percent of the mPDC
expressed the mesenchymal markers CD105, CD73 and Sca-1, whereas the hematopoietic and
endothelial markers CD45, CD11b, CD31 and CD34 were not detected. The mPDC furthermore
possessed trilineage differentiation potential in vitro (chondrogenic, osteogenic, adipogenic),
confirming the presence of mesenchymal progenitor cells in the mPDC population and their
osteogenic capacity.
When seeded on a calcium phosphate-collagen carrier and implanted ectopically in mice for 8
weeks, mPDC formed mature bone, illustrated by the presence of osteocytes and osteocalcinpositive osteoblasts. When compared to carriers implanted without cells, mPDC-seeded
scaffolds attracted numerous blood vessels which were closely associated with the sites of bone
formation. Moreover, some of the blood vessels had developed into larger, sinusoid-like
structures and the formation of a bone marrow compartment could be observed.
To further elucidate the pro-angiogenic properties of mPDC, we used an in vitro culture system.
Culturing mPDC under low oxygen tension increased the secretion of vascular endothelial
growth factor, an angiogenic factor. In addition, hypoxia-conditioned medium from mPDC
enhanced the proliferation of human umbilical vein endothelial cells (HUVEC) and co-culture of
mPDC with HUVEC resulted in stabilization of the pseudo-vascular structures formed by
HUVEC, in which the mPDC were localized in a pericyte-like fashion. Consistent herewith,
mPDC express pericyte markers such as PDGFR-b and Thy-1.
In conclusion, we demonstrate that periosteal cells can contribute to fracture repair, not only
through their strong osteogenic potential, but also their pro-angiogenic features and thus may
provide an ideal cell source for bone regeneration.