Abstract
Angiogenesis and the ability to provide appropriate vascular supply are crucial for skeletal tissue engineering. The aim of this study was to investigate the angiogenic potential of human dental pulp stromal cells (HDPSCs) and stro-1 positive populations as well as their role in tissue regeneration (the clinical reality).
HDPSC were isolated from the pulp tissues of human permanent teeth by collagenase digestion. STRO-1 positive cells were enriched using monoclonal anti- STRO-1 and anti- CD45 PE conjugated antibodies together with and fluorescence activated cell sorting (FACS). Cells isolated by FACS were grown to passage4 and cultured as monolayers or on 3D Matrigel scaffold in endothelial cell growth medium-2 (EGM-2) with/without 50ng/mL of vascular endothelial growth factor (VEGF). Cells cultured in alpha MEM supplemented with 10% FCS were used as controls. After 24, 48 and 72 hours angiogenic marker expression (CD31, CD34, vWF and VEGFR-2) was determined by qRT-PCR and immuno-histochemistry.
Using three different donors, 0.5-1.5% of total HDPSCs population was characterized as STRO-1+/CD45- cells At each time point cells cultured as monolayer in EGM-2 with VEGF showed up regulation of CD31 and VEGFR-2 expression compared to the control group while expression of CD34 and vWF remained unaffected. However on Matrigel, all four genes were up regulated to different extents. CD31 and VEGFR-2 were up regulated to a greater degree compared to CD34 and vWF. Changes in gene expression in both cell types were time dependent. Immuno-histochemical staining confirmed that the HDPSCs cultured in the test group showed positive staining for the four angiogenic markers (CD31, CD34 vWF and VEGFR-2) when grown in both monolayer and 3D Matrigel culture compared to control cultures. When cultured on Matrigel (but not Monolayer) for 7 days, HDPSC formed tube-like structures in the VEGF treated group.
This indicates the potential of use HDPSCs and their STRO-1 positive population for angiogenesis to enhance skeletal tissue repair and/or regeneration toward translational research for clinical benefit.
