Influence of 2-hydroxyethyl methacrylate (HEMA) exposure on angiogenic differentiation of dental pulp stem cells (DPSCs)

Abstract

Objective

The angiogenic differentiation of dental pulp stem cells (DPSCs) is important for tissue homeostasis and wound healing. In this study the influence of 2-hydroxyethyl methacrylate (HEMA) on angiogenic differentiation was investigated.

Methods

To evaluate HEMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of non-toxic HEMA concentrations (0.1 mM and 0.5 mM). Subsequently, angiogenic differentiation was analyzed on the molecular level by qRT-PCR and protein profiler analyzes of angiogenic markers and flow cytometry of PECAM1. The influence of HEMA on angiogenic phenotypes was analyzed by cell migration and sprouting assays.

Results

Treatment with 0.5 mM HEMA during differentiation can lead to a slight reduction of angiogenic markers on mRNA level. HEMA also seems to slightly reduce the quantity of angiogenic cytokines (not significant). However, these HEMA concentrations have no detectable influence on cell migration, the abundance of PECAM1 and the formation of capillaries. Higher concentrations caused primary cytotoxic effects in angiogenic differentiation experiments conducted for longer periods than 72 h.

Significance

Non-cytotoxic HEMA concentrations seem to have a minor impact on the expression of angiogenic markers, essentially on the mRNA level, without affecting the angiogenic differentiation process itself on a detectable level.

Introduction

The pulp is the origin of dental pulp stroma/stem cells (DPSCs). These cells have mesenchymal stem cell properties. They are able to self-renewal and have the capability to differentiate in multiple lineages, like muscle, bone, neuronal and endothelial cells [1,2]. DPSCs also have a high proliferation rate and can produce colony-forming units [3]. They play an important role in tissue homeostasis and wound healing [4]. One important process for wound healing is angiogenesis [5]. Angiogenesis means the formation of new blood vessels from existing blood vessels and is primarily carried out by endothelial cells. Dental stem cells support or initiate this process by secreting angiogenic factors [6] or differentiating into endothelial cells [7]. Dental composite materials could interfere with differentiation and thus wound healing. The monomer 2-hydroxyethyl methacrylate (HEMA) is widely used in dental composite materials and accounts for 35–50% of most adhesion promoters [8] and is the most eluted monomer of primers and adhesives [9]. It reduces the viscosity and increases the bond strength of bonding agents [10,11]. Approximately 33–75% of the methacrylate monomers are not incorporated into the polymer network [12,13]. Enzymes in the oral cavity also degrade the polymer [14]. Due to its hydrophilicity and low molecular weight, free HEMA can diffuse into the pulp and cause adverse effects [15,16]. Local HEMA concentrations in the millimolar range may occur in the environment of dental resins (determined by in vitro studies) [17]. A study from Kaga et al. showed that 1.5–2% of co-monomers elute from polymerized adhesives [9]. Çetingüç et al., however, assumes that no toxic HEMA concentrations occur when used as dentin bonding agent [18]. However, HEMA can be metabolized to more toxic epoxides, which can multiply the toxicity of HEMA [19]. While HEMA-caused cytotoxicity has already been extensively investigated, studies on effects on angiogenic differentiation pathways, which could also occur at sub-toxic concentrations, are lacking. it has been shown that HEMA has an inhibitory effect on osteogenic differentiation of DPSCs [20] and that the composite monomer TEGDMA can interfere with angiogenic differentiation of dental pulp stem cells [7].

One of the most important angiogenic signaling molecules is vascular endothelial growth factor (VEGF). VEGF increases the formation of new capillaries and its expression is linked to factors such as hypoxia and shear stress. Another important angiogenesis marker is platelet endothelial cell adhesion molecule (PECAM1), which is located on endothelial cells and important for the cell junction integrity [21]. It was shown that blocking of PECAM1 by anti-PECAM1 antibodies completely inhibits in vitro tube formation [22]. In vitro tube formation is a very specific test for angiogenesis. All endothelial cells are able to form tubules in vitro [23].

The aim of this study was to investigate the effect of sub-toxic HEMA concentrations on the angiogenic differentiation of DPSCs in vitro. For this purpose, the relative mRNA levels of VEGF, the corresponding receptors VEGFR-1 and -2, and PECAM1 (CD31) were determined by qRT-PCR. Various angiogenic marker proteins were analyzed using protein antibody arrays and PECAM1 via flow cytometry. Additionally, the influence of HEMA on the cell migration and tube formation of DPSCs was analyzed by microscopy. MTT-assays were carried out initially to define non-cytotoxic concentrations of HEMA for DPSCs.