Integrin binding and MAPK signal pathways in primary cell responses to surface chemistry of calcium silicate cements

Abstract

Cell attachment, proliferation and differentiation on different materials depend largely on the surface properties of the materials. This study sheds light on the mechanism by which the modulation of the chemical composition of calcium silicate cements with different Si/Ca molar ratios could produce different cell responses. Two primary cell types (human mesenchymal stem cells (hMSCs) and human dental pulp cells (hDPCs)) were used to elicit the changes in total DNA content, integrin subunit levels, phosphor-focal adhesion kinase (pFAK) levels, and mitogen-activated protein kinase (MAPK) signaling pathway activity at the cell attachment stage. The effect of small interfering RNA (siRNA) transfection targeting collagen type I (COL I) and fibronectin (FN) was also evaluated. The results indicated that increased total DNA content, pFAK and total integrin levels were observed upon an increase in cement Si content. Cements with different Si/Ca ratios did not cause the variations of interleukin 1β (IL-1β), epidermal growth factor (EGF) and tumor necrosis factor-α (TNF-α) ligands. The Si-rich cement facilitated COL I and α2β1 subintegrin expression, while Ca-rich cement promoted FN and αvβ3 subintegrin expression. Si component of the calcium silicates stimulated cell adhesion via activation of MAPK/extracellular signal-regulated kinase (ERK) and p38 signaling pathways more effectively than did by Ca component, but it did not affect c-Jun NH₂-terminal kinase (JNK) activity. Inhibition of MAPK/ERK and MAPK/p38 signaling pathways in hMSCs and hDPCs significantly attenuated adhesion, proliferation and differentiation as assessed according to total DNA content and alkaline phosphatase activity. hMSCs and hDPCs from the three different donors exhibited a similar preference for cell behaviors. The results of the current study suggest that calcium silicate cements with a higher Si content have the potential to serve as excellent supports for primary cells. Unraveling the mechanism by which primary cells responded to calcium silicate materials will be beneficial for materials design in their eventual clinical use.

Introduction

The surface characteristics of biomaterials, such as chemical composition and topographical structure, has been a subject of long-time interest because they can affect protein absorption and modulate cell adhesion, proliferation, differentiation and bony tissue formation and deposition [1], [2], [3], [4], [5]. The outer membrane of a typical cell is covered by a forest of at least six different receptor systems that can be activated by interactions with adjacent cells, ligands in the surrounding extracellular matrix (ECM), and secreted signaling molecules [6]. Among the receptor systems, integrins bind to specific ECM components, such as COL I and FN, and mediate cell attachment [7], [8]. Cell growth on specific substrates depends on integrin-mediated cytoskeletal and signal transduction molecules, such as FAK and MAPKs [9]. Signaling network is increasingly important for our understanding of cell proliferation. In mammalian cells, there are three major members of the MAPK family, consisting of ERK, JNK and p38 kinases. ERK is essential not only for the growth and differentiation of osteoblasts but also for osteoblast adhesion, spreading, migration, and integrin expression [10]. The JNK pathway is mainly activated by cellular stress and by cytokines, and the p38 MAPK pathway can be activated by environmental stress, such as osmotic stress [11].

Calcium silicate-based bone graft substitutes, such as bioactive glass [12], [13] and calcium silicate ceramic [14], [15] have been developed for hard tissue applications. Newly formed bone tissue grew into the porous calcium silicate-based materials, along with the deposition of a bone-like apatite layer at the tissue/material interface [16], [17]. The in vitro cell culture studies have also shown that the calcium silicate-based materials can support the human bone mesenchymal stem cell [4], [18], human pulp cell [19], [20], and osteoblast-like cell [21], [22] attachment, proliferation and differentiation. It has been documented that extracellular Ca is a potent regulator of cell behavior and has significant effects on the proliferation and differentiation of osteoblasts [23]. On the other hand, although Si plays important roles in the early stages of bone formation and the calcification process, the mechanism by which Si promotes osteoblast activity, including initial cell attachment, remains unclear. To understand the mechanism of Si-induced cell attachment and proliferation enhancement is important to expand the applications of silica-based materials.

The proportions of Si or Ca in the calcium silicate materials may play a dose-dependent role in increasing cell growth on this material [24], [25]. In vitro tests using established cell lines or primary culture systems have provided valuable information for predicting biological reactions to materials placed into or on tissues in the body. From the clinical practice perspective, human mesenchymal stem cells (hMSCs) and human dental pulp cells (hDPCs) would provide clear advantages over MG63s in that the latter are an osteoblast-like cell line. Different cell types express specific patterns of integrins, which are involved in specifying the complex processes of cell growth and differentiation. The responses of various cell types to a material may be different in different cell culture environments. Therefore, in this study, we conducted a side-by-side comparison of the signaling pathways of the primary cell types containing hMSCs and hDPCs in response to altered Si/Ca ratios. hDPCs can differentiate along the osteoblast lineage and contribute to the dentinal regeneration process [26], [27]. The most striking feature of hDPCs is their ability to regenerate a dentin-pulp-like complex that is composed of mineralized matrix with tubules lined with odontoblasts, and fibrous tissue containing blood vessels in an arrangement similar to the dentin-pulp complex found in normal human teeth [26]. hMSCs can be isolated from adult bone marrow and are induced to differentiate along the adipogenic, osteogenic, and chondrogenic lineages under appropriate culture conditions [28]. hMSCs have numerous potential advantages over terminally differentiated cells for use in bone tissue engineering applications. Both hMSCs and hDPCs are suitable for evaluating the bone/dental tissue responses to biomaterials. This article focused on studies surrounding the effects of calcium silicate cements with three different Si/Ca molar ratios (6:4, 5:5, and 4:6) during cell attachment on signaling mechanisms that may coordinate cellular and molecular events preceding bone mineralization. Additionally, siRNA transfection was employed to explore the levels of ECM, including fibronectin (FN) and collagen I (COL I), in hMSCs and hDPCs at cell adhesion stage.