Recombinant Human Plasminogen Activator Inhibitor-1 accelerates Odontoblastic Differentiation of Human Stem Cells from Apical Papilla

Abstract

Objectives Human dental mesenchymal stem cells (hDMSCs) are initially obtained from various types of dental tissue, such as periodontal ligament, pulp, periapical follicle, and apical papilla all of which have multi-lineage differentiation abilities. Apical papilla is apical to the epithelial diaphragm and there is an apical cell rich zone lying between apical papilla and pulp. Stem cells from apical papilla (SCAP) are a different population of DMSCS isolated from soft tissues residing in the apical papilla of incompletely developed teeth. Human SCAP are multi-potent and possess the ability to undergo various types of cell lineage differentiation, including odontogenic, osteogenic, chondrogenic, and adipogenic. In spite of the crucial functions of SCAP in dental tissue regeneration processes, the studies for searching a key molecule and its essential mechanism for differentiation are still ongoing.

Plasminogen activator inhibitor-1 (PAI-1) also known as serpin E1 is a member of the serine protease inhibitor (serpin) family. The functions of PAI-1 are the principal inhibitor of tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). Moreover, it has been reported that PAI-1 may play important roles in PRF-induced bone formation. However, the potential role of PAI-1 in tooth root formation has remained unclear.

Methods We first evaluated the PAI-1 effect on human stem cells from apical papilla (hSCAP) in vitro, which were derived from the human developing third molars. In addition, to evaluate the effects of PAI-1 in proliferation and differentiation of hSCAP performed MTT assay, ALP staining, Alizarin red S staining, real-time PCR, western-blot and so on. In order to observe the roles of PAI-1 during dentin-like structure formation, we first used that hSCAP were mixed with hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramic powder and the dentin matrix as scaffold, either with or without rhPAI-1, in a fibrin gel. The suspended cells and scaffold were then transplanted subcutaneously into immunocompromised mice for 12 week. We used to examine rhPAI-1 induced odontoblastic differentiation and dentin formation with hSCAP in mice immunohistochemical analysis and scanning electron microscopy.

Results We observed that rhPAI-1 promoted the proliferation and migration in hSCAP at a certain concentration and the activation of JNK pathway was essential for PAI-1-dependent hSCAP proliferation. Furthermore, PAI-1 accelerated mineral nodule formation in vitro and increased odontoblast-associated gene expression, such as type 1 collagen (COL1), alkaline phosphatase (ALP), bone sialoprotein (BSP), , osteopontin (OPN), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1). In addition, we also observed that PAI-1 significantly increased the expression of Smad4, and nuclear factor I-C (NFI-C), RUNX2, and osterix (OSX) during odontogenic differentiation. Moreover, the odontoblast-specific marker DSP was strongly expressed in the rhPAI-1 treated group compared with the control group in vivo. In addition, we used SEM to examine the newly-formed dentin-like tissue. These results suggest that rhPAI-1 not only provokes the proliferation and migration of hSCAP, but also promotes odontogenic differentiation of hSCAP in vitro to differentiate into odontoblast-like cells and to generate dentin like tissue.

Conclusions In this study, we demonstrated that PAI-1 accelerates proliferation and migration of hSCAP, but also promotes odontoblast differentiation of hSCAP and has the ability to differentiate into odontoblast-like cells with to generate dentin like tissue in vivo. The PAI-1 molecule that can be used for dentin formation during bio-tooth regeneration is considered to be clinically significant.