Cell Tissue Res DOI 10.1007/s00441-014-1877-x

REGULAR ARTICLE

Expression and effects of epidermal growth factor on human periodontal ligament cells Yoko Teramatsu & Hidefumi Maeda & Hideki Sugii & Atsushi Tomokiyo & Sayuri Hamano & Naohisa Wada & Asuka Yuda & Naohide Yamamoto & Katsuaki Koori & Akifumi Akamine

Received: 2 October 2013 / Accepted: 16 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1β were found to be upregulated in a perilesional pattern. Interleukin-1β induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded

Y. Teramatsu : H. Sugii : A. Tomokiyo : S. Hamano : A. Yuda : N. Yamamoto : K. Koori : A. Akamine Department of Endodontology and Operative Dentistry, Faculty of Dental Science, Kyushu University, Fukuoka, Japan H. Maeda (*) : N. Wada : A. Akamine Department of Endodontology, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan e-mail: [email protected]

PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue. Keywords Angiogenesis . Chemotaxis . Epidermal growth factor . Periodontal ligament cells . Wound healing

Introduction The periodontium is composed of four major tissues: the periodontal ligament (PDL) tissue, alveolar bone, cementum (covering the tooth root), and gingiva. The PDL is central in the periodontium, and is a highly specialized connective tissue located between the cementum and alveolar bone (Beertsen et al. 1997). The main role of PDL tissue is to tightly fix the tooth root to the bone socket, but it also has additional nutritive and sensory functions. However, deep infrabony caries, severe periodontitis and trauma to PDL tissue cause irreversible damage resulting in tooth loss. Therefore, development of regenerative therapy for PDL tissue is required (Maeda et al. 2011, 2013b). The cells comprising PDL tissue are heterogeneous populations composed of fibroblasts, which are the primary cells in PDL tissue (Beertsen et al. 1997; Berkovitz and Shore 1995), PDL stem cells (Fujii et al. 2006; Maeda et al. 2013b; Seo et al. 2004), epithelial rests of Malassez cells, and endothelial cells, among others. Epidermal growth factor was first isolated from mouse salivary glands, and was reported to promote corneal wound healing (Cohen and Elliott 1963). Since then, this factor has been described to have diverse actions, such as cell migration, proliferation, motility (Iwabu et al. 2004), wound healing (Schultz et al. 1991; Vranckx et al. 2007) in various cells and tissues, and bone healing (Marquez et al. 2013). In particular, its angiogenic activity was elucidated (Gospodarowicz et al. 1979). A study by Tamama et al. (2010) revealed that EGF enhances the production of vascular endothelial growth

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factor (VEGF) in bone marrow mesenchymal stem cells (BMMSCs), which promotes angiogenesis (Tamama et al. 2010). EGF also induces the production of interleukin-8 (IL8), which is known as an angiogenic chemokine in lung cancer cells (Zhang et al. 2012). A current study suggested that enzyme from Porphyromonas gingivalis, which is widely recognized as a main pathological bacterium for periodontitis, contributes to PDL tissue damage through inactivating EGF (Pyrc et al. 2013). A previous study that examined the localization of EGF receptor (EGFR, HER1, or ErbB1) in PDL tissue demonstrated that PDL cells, preosteoblasts, and paravascular cells expressed it in great numbers, whereas precementoblasts, cementoblasts, and osteoblasts did not, suggesting some significant roles of EGFR in PDL tissue (Cho and Garant 1996). From these reports, we hypothesized that EGF might have therapeutic potential in PDL healing. In this study, we investigated the roles of EGF in PDL tissue under inflammatory conditions and examined its effects on the chemotaxis of human PDL cells (HPDLCs). We also studied the effects of EGF-treated HPDLCs on capillary-tube formation by human umbilical vein endothelial cells (HUVECs).

Materials and methods Cell culture Three HPDLC lines were isolated from the healthy premolars of a 27-year-old female, a 22-year-old female, and a 26-yearold male with informed consent, and were named as HPDLC3A, HPDLC-3D, and HPDLC-3M, respectively. In this study, HPDLCs were maintained in alpha minimum essential medium (α-MEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 50 μg/ml streptomycin, 50 U/ml penicillin (Gibco-BRL), and 10 % fetal bovine serum (FBS; Bio-West, Nuaillé, France) (10 % FBS/α-MEM) in a humidified atmosphere of 5 % CO2 and 95 % air at 37 °C. The cells from passages 5 through 8 were used in this study. All procedures were performed in compliance with the Research Ethics Committee, Faculty of Dentistry, Kyushu University. HUVECs (Cell Applications, San Diego, CA, USA) were cultured on Primaria Easy Grip tissue culture dishes (BD Biosciences, Bedford, MA, USA) in endothelial cell growth medium (Cell Applications). HUVECs were expanded in endothelial cell growth medium (Cell Applications) supplemented with 2 % FBS. Culture medium was changed every 48 h in all experiments. Experimental animal model Surgically wounded PDL tissues in rats were prepared according to our recent study (Yamamoto et al. 2012). Briefly, 5-

week-old Sprague-Dawley male rats were purchased from Kyudo (Saga, Japan). The animals were anesthetized by an intraperitoneal injection of 0.03 % chloral hydrate. A periodontal defect, 2 mm in diameter and 2 mm in depth, was created using dental round bur #6 and extended from the mesio-palatal submarginal portion to the palatal root of the first molar in the left maxilla. First molars on the right side were not injured and were used as controls. Animals were sacrificed by transcardial perfusion with 4 % paraformaldehyde (PFA; Merck, Darmstadt, Germany) 2 days after surgery. The maxillae were then removed and immersed in 4 % PFA for 12 h. The tissues were further decalcified in 10 % ethylenediaminetetraacetic acid (Wako Pure Chemical Industries, Osaka, Japan) solution at 4 °C for 1 month before dehydration and embedding in paraffin. The rats were allowed free access to food and water throughout the experimental period. All procedures were approved by the Animal Ethics Committee and conformed to the regulations of Kyushu University. Immunohistochemical and immunofluorescent analyses Immunohistochemical analysis was performed as previously described (Fujii et al. 2010; Monnouchi et al. 2011). The immunolocalization of EGF or EGFR in rat PDL tissue was examined on 5-μm paraffin sections using primary rabbit polyclonal antibodies against rat/human EGF (anti-EGF, 1:150; Biomedical Technologies, Stoughton, MA, USA), rat/ human EGFR (anti-EGFR, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rat IL-1β (anti-IL-1β, 1:100; Santa Cruz Biotechnology). Sections were then incubated with a biotinylated secondary antibody, followed by an avidin-peroxidase conjugate (Nichirei Biosciences, Tokyo, Japan). Positive reactions were visualized with diaminobenzidine (DAB; Nichirei Biosciences). HPDLCs were fixed with 4 % PFA and 0.5 % dimethyl sulfoxide in phosphate-buffered saline (PBS) for 20 min. After blocking with 2 % BSA in PBS for 1 h, the cells were incubated for 24 h with anti-EGF or anti-EGFR antibody as described above. Next, cells were incubated for 1 h with fluorescein-conjugated goat anti-rabbit IgG (Fl-1000, Vector Laboratories, Burlingame, CA, USA) and then sealed with VECTASHIELD mounting medium containing 4,6diamidino-2-phenylindole dihydrochloride (DAPI; Vector Laboratories). The cells were imaged using a Biozero digital microscope (Keyence, Osaka, Japan). Semi-quantitative reverse-transcription polymerase chain reaction For semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), total cellular RNA was extracted from each culture using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

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First-strand cDNA was synthesized with an ExScript RT Reagent kit (Takara Bio, Shiga, Japan). PCR was performed as previously described (Fujii et al. 2008; Tomokiyo et al. 2008) using a PCR Thermal Cycler Dice (Takara Bio). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used as internal standards. All PCR assays were performed within the exponential amplification range. PCR products were separated on 2 % agarose gels (Seakem ME; BioWhittaker Molecular Applications, Rockland, ME, USA) by electrophoresis and imaged under ultraviolet excitation following ethidium-bromide staining. Specific primer sequences, annealing temperatures, cycle numbers, and product sizes for each gene are listed in Table 1. Primer sequences were designed using the GenBank database (National Center for Biotechnology Information; NCBI), and a BLAST search of GenBank was performed on the primer sequences to ensure specificity.

Systems) as previously described (Kono et al. 2013). The absorbance was measured at 450 nm with an Immuno-mini NJ-2300 microplate reader (Microtec, Chiba, Japan). Experiments were performed in duplicate.

Quantitative RT-PCR

Chemotaxis assays were performed using cell culture inserts (pore size 8 μm; Becton Dickinson Labware) and 24-well plates (Becton Dickinson Labware) as described (Tomokiyo et al. 2012b). HPDLCs (3.1×103 cells) in 200 μl of 10 % FBS/αMEM were seeded on the cell culture inserts. The lower chambers were filled with 750 μl of 10 % FBS/α-MEM containing PBS (control) or EGF (1, 10 and 50 ng/ml). Following incubation at 37 °C in 5 % CO2 for 12 h, HPDLCs on the upper side of the insert were scraped off with cotton-tipped swabs. The cells that had passed through the insert membranes were fixed with methanol for 2 min and stained with 1 % toluidine blue for 2 min. Stained cells were photographed on an inverted microscope. Cells were manually counted in four randomly chosen fields from each condition. Measurements were made using Scion Image Software (Scion, Walkersville, MD, USA). All samples were measured in duplicate.

Quantitative RT-PCR was performed with a SYBR Green II RT-PCR kit (Takara Bio) using a Thermal Cycler Dice Real Time System (Takara Bio) as previously described (Maeda et al. 2013a; Tomokiyo et al. 2012a). Specific primer sequences, annealing temperatures, and product sizes for each gene are listed in Table 2. Human ACTB (β-actin) was used as an internal standard. Expression levels of the target genes were calculated from the ΔΔCt values. Primer sequences were designed using GenBank and checked via BLAST to ensure specificity. Enzyme-linked immunosorbent assay Recombinant human EGF (R&D Systems, Minneapolis, MN, USA) was administered at 10 ng/ml to HPDLC-3A cells that had been cultured for 12 h in ϕ3 cm dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA). After 12 or 24 h incubation with EGF, the medium was collected from each well. To detect secreted BMP-2 and vascular endothelial growth factor A (VEGFA) in the medium, an enzyme-linked immunosorbent assay (ELISA) was performed using Quantikine (R&D

Table 1 Primer sequence, product size, annealing temperature, and cycle numbers for semi-quantitative RT-PCR

Target gene (abbreviation)

In vitro capillary-like tube structure formation assay HUVECs were incubated in 24-well culture plates precoated with Engelbreth-Holm-Swarm gel matrix (EZcell matrix gel; Asahi Glass, Shizuoka, Japan) and then cultured for 24 h with supernatant from HPDLCs pretreated with 10 ng/ml EGF for 24 h or untreated HPDLCs. Capillary-like tube structure formation was observed under a Leica DC300F microscope (Leica Microsystems, Wetzlar, Germany). Experiments were performed in duplicate. Chemotaxis assay

Statistical analysis All values are expressed as mean ± SD. Statistical analysis was performed using a Student’s paired t test. P values of