Abstract
Introduction
P-15 (GTPGPQGIAGQRGVV), a fifteen residue synthetic peptide, is a structural analogue of the cell binding domain of Type 1 collagen and creates a biomimetic environment for bone repair when immobilized on anorganic bovine mineral (ABM) scaffolds. ABM-P-15 scaffolds have been shown to enhance bone marrow stromal cell growth and differentiation. This study aimed at evaluating the osteogenic potential of human dental pulp stromal cells (HDPSCs) compared to human bone marrow stromal cells (HBMSCs) in monolayer and on 3D ABM-P-15 scaffolds in vitro and in vivo.
Materials and Methods
HDPSCs and HBMSCs were cultured as monolayers in basal or osteogenic media for 3 weeks. Osteogenic differentiation was confirmed using alkaline phosphatase (ALP) staining and ALP specific activity (ALPSA). In addition, the presence and distribution of osteogenic markers including Type 1 collagen, bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OCN) was determined by immunohistochemisty. Gene expression for COL1, RUNX2 and OCN was determined using RT-PCR after 1, 3 and 5 weeks in basal culture. For 3D culture, HDPSCs were seeded on ABM scaffolds ± P-15 (CeraPedics LLC) and cultured in basal media for 6 weeks. Cell viability and growth were visualized by confocal and scanning electron microscopy. Osteogenic differentiation was confirmed by ALP staining and ALPSA. For in vivo studies, HDPSCs were injected and sealed in diffusion chambers containing ABM-P-15 or ABM alone which were then implanted intraperitoneally in nude mice for 8 weeks. The retrieved samples were then processed for histology.
Results
In monolayers, HDPSCs showed stronger ALP staining compared to HBMSCs in both culture conditions. Type I collagen, BSP and OPN were detected by immunohistochemistry for both HBMSCs and HDPSCs; however, OCN was not detected. RT-PCR indicated an up regulation of all osteogenic markers in both cell types at weeks 1 and 3. At week 5, there was a marked down regulation of COL1 and RUNX2 in HDPSCS compared to HBMSCs. Confocal microscopy and SEM showed ABM-P-15 promoted HDPSCs bridge formation between the scaffold particles. Histological staining and biochemical analysis confirmed that P-15 enhanced HDPSC ALP activity in vitro and fibrillar collagen formation in vivo compared to ABM alone.
Discussion and Conclusion
HDPSCs have higher osteogenic capacity compared to HBMSCs. ABM-P-15 enhanced HDPSC ALPSA and collagen formation, suggesting that a combination of ABM-P15 with HDPSCs could be used as an autologous cell based therapy for bone tissue engineering. Acknowledgement: Supported by a University of Leeds studentships and Cerapedics Inc.
