Abstract
Stromal cells derived from human dental pulp (HDPSCs) are of current interest for applications in skeletal tissue engineering. Angiogenesis and revascularization of bone grafts or bone constructs in vivo are of paramount importance for bone tissue regeneration and/or fracture healing. The aim of this study was to investigate the angiogenic and osteogenic potential of HDPSCs in combination with Bioglass¯ scaffolds in vitro and in vivo.
HDPSCs, isolated by collagenase digestion, were either maintained as monolayers or dynamically seeded on 3D Bioglass¯ scaffolds and cultured under either basal or osteogenic conditions for 2 and 4 weeks. Expression of osteogenic (COL1A1, ALP, RUNX2 and OC) and angiogenic markers (VEGFR2, CD34 and PECAM1) was determined using qRT-PCR. Alternatively, constructs were either cultured in vitro under basal/osteogenic conditions for 6 weeks or sealed in diffusion chambers which were then implanted intraperitoneally in immunosuppressed mice for 8 weeks. Retrieved constructs were fixed and embedded for histology and immunohistochemistry using antibodies against COL1, RUNX2, OC, VEGFR2, CD34 and PECAM1. qRT-PCR showed no significant differences in gene expression of osteogenic markers between basal and osteogenic media for both 3D construct and monolayers.
However when comparing 3D constructs to monolayers: COL1A1 showed a significantly lower expression (p< 0.05) in 3D compared to 2D at 2 weeks in both culture conditions, and this pattern was reversed after 4 weeks. ALPL was significantly lower in 3D constructs at 2 weeks under both conditions (p<0.01), and was significantly higher in basal conditions at 4 weeks (p<0.05). RUNX2 showed higher expression in 3D constructs at all time points and under both conditions while OC showed lower expression in 3D constructs at 2 weeks and higher expression at 4 weeks under both conditions. For the angiogenic markers, 3D constructs under osteogenic conditions showed an increase of expression in VEGFR2 and PECAM1 at 2 weeks followed by a decrease at week 4, while CD34 expression was undetected in 3D constructs at all times and under both sets of culture conditions. The expression of VEGFR2 and PECAM 1 under both conditions and at both time points was greater in 3D constructs compared to monolayers. After 8 weeks, the in vivo retrieved constructs showed no signs of inflammatory reactions. Immunohistology confirmed positive staining of osteogenic and angiogenic markers in 3D constructs from both in vitro and in vivo experiments with a greater staining intensity seen in the in vivo constructs. Furthermore, the in vivo constructs showed more intense sirius red staining and higher intensity of immunostaining using antibodies to type 1 collagen, with higher calcification as indicated by alizarin red staining.
In conclusion, this study indicated that a combination of HDPSCs and Bioglass¯ scaffolds has potential to provide a suitable microenvironment for angiogenic and osteogenic differentiation of HDPSCs which is essential for bone regeneration in preclinical and/or clinical applications.
