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
1Laboratory 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 cell-based bone tissue engineering is the timelyformation of blood vessels that promote the survival of the cells in the implant. 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 alsoto angiogenesis.In this study, we therefore characterized theability of murine PDC (mPDC) to supportangiogenesis, in addition to their osteogenic potential.
We first established a protocol to specifically isolate periosteal cells from long bones of adult mice and characterized the obtained cell population. Mesenchymal stem cells (MSC) were abundantly present as more than 50% of the mPDC expressed the MSC markers CD105, CD90, CD73 and SCA-1,whereas hematopoietic and endothelial markers werenot detected. ThemPDC furthermorepossessed trilineage differentiation potentialin vitro(osteogenic, chondrogenic, adipogenic), confirming the presence of MSC 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 osteocalcin-positive osteoblasts. When compared to carriers implanted without cells, mPDC-seeded scaffolds attractednumerous blood vessels which were closely associated with the sites of bone formation.Moreover,some of the blood vessels had developed intolarger, sinusoid-like structuresand the formation ofa bone marrowcompartmentwas observed.
To further elucidate the pro-angiogenic properties of mPDC, we used an in vitroculture system. CulturingmPDCunder low oxygen tension increasedHIF-1α and VEGF protein levels andhypoxia-conditioned medium from mPDC enhancedthe proliferation of human umbilical vein endothelial cells (HUVEC). In addition, co-culture of mPDC with HUVEC resulted in stabilization of the pseudo-vascular structures formed by HUVEC, in which themPDCwere localized in a pericyte-like fashion.Consistent herewith, mPDC highly express pericyte markers such as PDGFR-β and α-SMA.
In conclusion, we demonstrate that periosteal cells can contribute to fracture repair, not only throughtheir strong osteogenic potential, but also their pro-angiogenic features and thus may provide an ideal cell source for bone regeneration.