6100 MOLECULAR MEDICINE REPORTS 17: 6100-6108, 2018 Effect of recombinant vascular endothelial growth factor and translationally controlled tumor protein on 2‑hydroxyethyl methacrylate‑treated pulp cells CHUNYANUT WONGKHUM1, WILAIWAN CHOTIGEAT1 and UREPORN KEDJARUNE-LEGGAT2 Departments of 1Molecular Biotechnology and Bioinformatics, Faculty of Science and 2Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Received September 13, 2017; Accepted February 6, 2018 DOI: 10.3892/mmr.2018.8593 Abstract. Vascular endothelial growth factor (VEGF)-A is promoted pulp cell growth and the survival of HEMA-treated a potential signaling protein that may promote angiogenesis. cells without synergistic effects. TCTP was required in lower VEGF also helps cells survive in stressfull or hazardous concentrations for these effects. VEGF and TCTP enhanced conditions. The present study aimed to compare the effect of cell differentiation and mineralization. VEGF with translationally controlled tumor protein (TCTP), an anti-apoptotic protein in human dental pulp cells (HDPCs), Introduction following exposure to 2-hydroxyethyl methacrylate (HEMA), which is a major residual monomer from resin restorative Growth factor typically acts as signaling molecules between dental materials. Cell viability, alkaline phosphatase (ALP) cells and is an important component required for tissue engi- activity, mineralization and gene expressions for odontogenic neering. There have been many attempts at developing dental and osteogenic differentiation markers of HDPCs were inves- biomaterial or scaffold supplemented with a single or combina- tigated, following exposure to HEMA and in combination with tion of active molecules to induce dentine or pulp regeneration. TCTP and VEGF. The results revealed that TCTP at 1 ng/ml One growth factor that was widely selected for this purpose is and VEGF at 10 ng/ml significantly promoted the proliferation vascular endothelial growth factor (VEGF). of HDPCs (P<0.05). TCTP (1 ng/ml) and VEGF (10 ng/ml) VEGF-A is one of the four subtypes of VEGF maintained the cell viability of 4 mM HEMA-treated cells (-A, -B, -C, and -D) that are expressed in dental pulp with at the same percentage as the control. However, cells treated autocrine and paracrine effects in blood vessels and immune with HEMA+TCTP+VEGF had a lower cell viability than cells (1). It is a potential signaling protein that can promote the groups treated with HEMA and TCTP or VEGF alone. angiogenesis and activate dentin regeneration (2). VEGF TCTP and VEGF promoted cell proliferation, ALP activity has been used in growth factor delivery-based tissue engi- and mineralization, and upregulated of DSPP, DMP-1, BMP-2, neering (3), including pulp tissue regeneration for regenerative and ALP mRNA expression compared with the control. endodontics (4). Moreover, VEGF has been shown to induce Furthermore, the HEMA+TCTP and HEMA+VEGF groups proliferation and differentiation of human pulp cells into had significantly higher percentages of calcium deposition odontoblasts (5). Another interesting property of VEGF is than HEMA-treated cells (P<0.001). HEMA was cytotoxic helping cells survive from stress or hazardous conditions. to HDPCs, reduced ALP activity and caused the significant There have been some studies reporting that VEGF played a downregulation of odontogenic and osteogenic gene expres- role in survival of serum-starved endothelial cells (6,7), as well sions (P<0.05). It was concluded that VEGF and TCTP as survival of these cells in hypoxic conditions (8). A previous study has also revealed that VEGF expression was upregu- lated in mouse odontoblast-like cells (MDPC-23) exposed to 2-hydroxyethyl methacrylate (HEMA) (9). HEMA is a major monomer released from incomplete Correspondence to: Dr Ureporn Kedjarune-Leggat, Department of polymerization of resin-based dental restorative materials. It is Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla cytotoxic to cells and can damage DNA (10), leading to prolif- University, 15 Karnjanavanich Road, Hat Yai, Songkhla 90112, Thailand eration impairment (11) and delayed cell differentiation (12). E-mail: [email protected] Translationally controlled tumor protein (TCTP) is a conserved protein found in many eukaryotic cells ranging Key words: vascular endothelial growth factor, translationally from plant to animal kingdoms (13). TCTP has many controlled tumor protein, 2-hydroxyethyl methacrylate, pulp cells, functions, including proliferation, maturation, and antiapop- cytotoxicity tosis (14). TCTP from Penaeus merguiensis (Pmer-TCTP) has been shown to have an antiapoptotic property against HEMA-treated dental pulp cells (15). A new formula of resin WONGKHUM et al: VEGF AND TCTP ON HEMA-TREATED PULP CELLS 6101 modified glass ionomer cement supplemented with TCTP can solution (5 mg/ml in PBS) were added to each well and incu- reduce cell death from residual HEMA release (16). The aim of bated in the dark for 4 h at 37˚C. The medium and MTT were this study was to compare cell proliferation, anticytotoxicity, then removed and 200 µl of DMSO and 25 µl of Sorensen's differentiation and mineralization of VEGF-A with TCTP on glycine buffer (0.1 M glycine plus 0.1 M NaCl equilibrated to HEMA-treated human dental pulp cells (HDPCs). This will pH 10.5 with 0.1 M NaOH) were added. The optical density assist the further development of a restorative material that (OD) of formazan production was measured at 570 nm. The may have less toxicity and be able to regenerate pulpal dentine OD values adjusted for a blank (medium only) of the experi- complex. mental groups were divided by the control (cell cultured in normal medium without TCTP and VEGF) and expressed as Materials and methods percentages of the control, which represented the percentages of viable cells. Expression and purification of recombinant TCTP and recombinant VEGF. Expression of Pmer‑TCTP gene was Cytotoxicity of HEMA. MTT assay was used to evaluate the performed in the bacteria Escherichia coli (E. coli) strain cytotoxicity of HEMA at two concentrations, which were BL21. The protein was purified according to methods previ- 4 mM and 6 mM. HDPCs were seeded at 8x103 cells/well in a ously described (14). Briefly, E. coli strain BL21 harboring 96‑well plate at a humidified atmosphere of 5% CO2 at 37˚C. pGEX-Pmer-TCTP was inoculated and induced by 1 mM After 24 h, the media was replaced with HEMA mixed in IPTG (isopropyl β-D-thiogalactopyronositol) for stimulating culture medium and left for 24 h. Then the media was refreshed protein expression. After induction for 3 h, the bacterial with normal media and incubated for 48 h before MTT assay cells were harvested by centrifugation and the soluble gluta- was performed as described previously (n=5 in each group). thione S-transferase (GST)-TCTP protein was purified by The results from HEMA cytotoxicity testing led to the using Glutathione Sepharose 4 Fast Flow (GE Healthcare selection of 4 mM HEMA as a cytotoxic reagent. In addition, Bio-Science, Piscataway, NJ, USA) and thrombin was used the result from effective concentrations of TCTP and VEGF for splitting of GST‑tagged protein. The purified Pmer-TCTP suggested the use of TCTP at 1 and 10 ng/ml of VEGF for protein with molecular mass about 19.2 kDa (168 amino further investigation. acid residues) was determined by SDS-PAGE and protein concentration was analyzed by a BCA protein assay kit The recovery effect of TCTP and VEGF on HEMA‑treated (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). HDPCs. There were five groups of HDPCs according to Recombinant human VEGF was purchased from Gibco® different medium conditions: HEMA, HEMA+TCTP, (Gibco; Thermo Fisher Scientific, Inc.).This protein has about HEMA+VEGF, HEMA+TCTP+VEGF, and control group 40 kDa (homodimer) with 165 amino acid residues/subunit. (culture medium only). HDPCs were seeded at 8x103 cells/well on a 96‑well plate at a humidified atmosphere of 5% CO2 at Cell culture. HDPCs were cultured from pulp tissue of sound 37˚C and fed with normal medium. After 24 h, the culture third molar teeth of adults aged 18-25 years at the Dental medium was replaced with media supplemented with Hospital, Faculty of Dentistry, Prince of Songkla University, substances as described above for 24 h. After that, the fresh with consent forms approved by the Research Ethics media was replaced and incubated for 48 h. The viable cells Committee (Number EC5703-09-P-LR), Faculty of Dentistry, were determined by the MTT assay (n=5 in each group). Prince of Songkla University. Primary culture of pulp cells was performed using an enzymatic method. Briefly, the pulp Alkaline phosphatase (ALP) activity assay. ALP activity assay tissue was cut into small pieces before digested in 3 mg/ml of was used to determine odontoblast-like differentiation. The collagenase type I and 4 mg/ml of dispase for 1 h at 37˚C. After experiment was divided to six groups according to substances centrifugation, cells were cultured in α-MEM, supplemented added to the medium: HEMA, HEMA+TCTP, HEMA+VEGF, with 10% fetal calf serum (FCS), 100 µM L-ascorbic acid TCTP, VEGF and cells cultured in normal medium as a control 2-phosphate, 100 µM L-glutamate, 100 U/ml penicillin, and group. HDPCs were seeded at 1x104 cells/well on a 24-well 100 µg/ml streptomycin and incubated at 37˚C with 5% CO2. plate and fed with normal culture medium using 15% FCS at a Pulp cell passages between three and five were used for this humidified atmosphere of 5% CO2 at 37˚C (n=3 in each group). study. The medium was changed every 2 days and the ALP activity of cells was determined after being cultured for 3, 7 and 14 days. Finding effective concentrations of TCTP and VEGF The ALP activity was measured by using p-nitrophenol phos- on HDPCs.