Abstract
Aim Regenerative endodontics is a field dedicated to the repair and restoration of damaged dental tissues, in hopes of conserving and maintaining natural function of the affected tooth. Finding the suitable growth factors and stem cells are key factors that contribute to successful tissue engineering. Human stem cells from apical papilla (SCAPs) are known as potent cell sources for dental tissue regeneration, while the bone morphogenic protein-4 (BMP-4) is a growth factor that is vital in the growth and development of human teeth. This study was designed to investigate the impacts BMP-4 may have on the differentiation potential and matrix turnover SCAPs, by analyzing the expression of differentiation related markers, including osterix (Osx), N-cadherin, alkaline phosphatase (ALP), type I collagen (COL1A1), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). The signaling pathways underlying these effects were also explored. Materials and Methods Primary-cultured SCAPs were treated with different concentrations of BMP-4 (0, 10, 25, 50, 100, 200 ng/ml) for 24 hours. The expressions of Osx, N-cadherin, ALP, COL1A1, uPA, PAI-1 were evaluated through RT-PCR and western blot. In addition, pre-treatment of LDN193189 (a p-Smad1/5/8 inhibitor) and SB431542 (a p-Smad2/3 inhibitor) were used to investigate the Smad-dependent pathways induced by BMP-4 in SCAPs. Results SCAPs exposed to BMP-4 for 24 hours showed an increase in the mRNA and protein expressions of Osx, N-cadherin, ALP, COL1A1, and PAI-1, whereas the expression levels of uPA were decreased. Pre-treatment with inhibitors, LDN193189 and SB431542, showed inhibitory effects on the BMP-4-stimulated expressions. Moreover, the addition of LDN193189 significantly reduced the phosphorylation of Smad1/5/8 and Smad2/3. Similarly, when SB431542 was added, the phosphorylation level of Smad1/5/8 was diminished. Conclusion Our results demonstrate that SCAPs have the potential to undergo multi-lineage differentiation, including osteogenesis, odontogenesis, and extracellular matrix turnover. In addition, pre-treatment with LDN193189 significantly decreased the phosphorylation levels of Smad1/5/8 and Smad2/3, while SB431542 also reduced phosphorylation of Smad1/5/8, suggesting these two pathways may have some interaction or crosstalk with each other, and that the TGF-β signaling pathway may possibly play a role in the mechanism of BMP-4 signaling pathways. It is visible through our research that BMP-4 plays an important role in the differentiation and extracellular matrix turnover of SCAPs by regulation through the Smad-dependent signaling pathways. Thus, the knowledge obtained from this study on the combined use of SCAPs and BMP-4 may be a beneficial approach for future clinical applications in dental tissue repair and engineering.
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