The application of prostacyclin in promoting angiogenesis of dental pulp tissue in a three dimensional organ culture system

Abstract

Background: Dental pulp vitality can be threatened by carious or iatrogenic exposure that can lead to pulp tissue loss. The formation of a vascular network is crucial to provide a suitable environment for tissue regeneration. Thus, promoting angiogenesis is essential for successful pulpal repair in regenerative endodontics therapy. Iloprost, a prostacyclin analog, promotes vascularization in several organs such as, heart, lung, and corneal. Previously, it has been demonstrated that, iloprost induced pulpal blood flow and enhanced dentin formation in a rat mechanical pulp exposure model. However, the angiogenic effect of iloprost on the human dental pulp vasculature remains unknown. Objective: The present study investigated the effect of iloprost on promoting dental pulp angiogenesis using the tooth-slice organ culture system. Materials and methods: Tooth-slices with intact dental pulp tissues were prepared from extracted human third molars. Dental pulp tissue viability was analyzed by live/dead cell confocal microscopy. The tooth-slices were cultured without iloprost (control group) or with iloprost at a dose of 10-6 M. The microvessel density and vascular endothelial growth factor (VEGF) expression in the cultured tooth-slices were determined by immunohistochemical staining. The collagen deposition was determined by Masson’s Trichrome and immunofluorescent staining. A PKA-inhibitor was used to identify the molecular mechanisms regulated by the cAMP-dependent protein kinase A (PKA) that govern iloprost in promoting angiogenesis. Results: The dental pulp tissue architecture in the human tooth-slices model were maintained and remained viable when cultured in serum-free media. Iloprost increased the microvessel density as shown by a higher number of von Willibrand Factor-positive blood vessels when compare with the control tooth slice. A significant increased in the VEGF expression and the collange 1 (COL1) production were also observed in the tooth-slices cultured with iloprost. The effect of iloprost on angiogenesis and collagen synthesis were abolished following inhibition of protein kinase A activity. Conclusion: Human tooth-slices provide a valuable and easy-to-obtain model to investigate the effect of bioactive molecules used in dental pulp regeneration. This study, for the first time, showed that tooth slices could be kept viable under serum-free conditions for up to 3 days. Iloprost promoted angiogenesis, increased new vessel formation, and induced collagen deposition. This study proposes the clinical value of iloprost as a drug for inducing angiogenesis that can increase the success of pulp regeneration.