Prostaglandin E₂ Induces Expression of Mineralization Genes by Undifferentiated Dental Pulp Cells

Abstract

Prostaglandin E₂ (PGE₂) is a lipid mediator usually released during inflammation. This study aimed to investigate the potential of soluble or microsphere-loaded PGE₂ on inducing differentiation of dental pulp stem cells. PGE₂-loaded microspheres (MS) were prepared using an oil-in-water emulsion solvent extraction-evaporation process and were characterized. Mouse dental pulp stem cells (OD-21) were stimulated with soluble or PGE2-loaded MS (0.01 and 0.1 µM). Cell viability was determined by MTT colorimetric assay. Ibsp, Bmp2 and Runx2 expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 3, 6, and 24 h. The results showed that the soluble PGE₂ reduced dental pulp stem cells viability after 24 h of stimulation whereas PGE₂-loaded MS did not. Soluble PGE₂ up-regulated Ibsp and Bmp2 at 3 h, differently from PGE₂-loaded MS. On the other hand, PGE₂-MS induced Bmp2 and Runx2 at 6 h and Ibsp at 24 h. In conclusion, our in vitro results show that PGE₂, soluble or loaded in MS are not cytotoxic and modulateIbsp,Bmp2, andRunx2gene expression in cultured OD-21 cells.

Key Words:
microspheres; prostaglandin E₂; dental pulp stem cell; differentiation

Resumo

A Prostaglandina E₂ (PGE₂) é um mediador lipídico comumente liberado durante a inflamação. Este estudo teve como objetivo investigar o potencial da PGE₂, solúvel ou na forma de microesferas, na diferenciação de células-tronco de polpa dentária. Microesferas de PGE₂ (MS) foram preparadas por meio do processo de extração/evaporação de solvente em emulsão óleo-em-água e foram caracterizadas. Células-tronco de linhagem derivadas da polpa dentária de camundongos (OD-21) foram estimuladas com PGE₂ solúvel ou na forma de MS (0,01 e 0,1 µM). A citotoxicidade foi determinada por ensaio colorimétrico MTT. A expressão gênica de Ibsp, Bmp2 e Runx2 foi avaliada por meio de reação em cadeia da polimerase em tempo real (qRT-PCR) após 3, 6 e 24 h. Os resultados mostraram que as MS contendo PGE₂ não foram citotóxicas para células-tronco da polpa dentária, enquanto MS vazias ou PGE₂ solúvel reduziram a viabilidade celular após 24 h de estimulação. PGE₂ solúvel aumentou a expressão de Ibsp e Bmp2 após 3 h, diferentemente da PGE₂ em MS. Por outro lado, PGE₂-MS induziram a expressão de Bmp2 e Runx2 após 6h de estímulo e Ibsp após 24h. Em conclusão, nossos resultados in vitro demonstram que a PGE₂, solúvel ou em microesferas não são citotóxicas e modulam a expressão gênica deIbsp,Bmp2 eRunx2em células OD-21.

Introduction

Dental pulp present high regeneration capacity because of the presence of progenitor stem cells within the tissue. Acidic dissolution of carious dentin release repair signals that regulate the synthesis of reparative dentin, angiogenesis, and innervation ¹. Thus, in response to caries, dental pulp responds dynamically by inflammation, expression of mineralization proteins and/or reparative dentin formation by odontoblast-like cells derived from dental pulp mesenchymal stem cells ².

Successful handling of regeneration in this scenario involves the regulation of a complex molecular signalling network that shares similarities to the immune response to infection. Within that response, eicosanoid signalling are crucial players ³. Eicosanoids are oxidation products of C20 polyunsaturated fatty acids (e.g., arachidonic acid [AA]) that include prostaglandins (PGs) and leukotrienes (LTs) ⁴. During pulp inflammation, AA metabolism generates prostaglandin E₂ (PGE₂) ⁵ and this lipid mediator produced by stem cells shows such a potent immunomodulator with anti-inflammatory effects through regulation of cell maturation and activation ⁶. PGE₂ have demonstrate a dual role in proliferation and cell differentiation, because inhibit osteogenic differentiation of mesenchymal stem cells ⁷, but also has anabolic effects on osteogenic and odontogenic markers in vitro, enhances tertiary dentin formation in vivo ⁸. These findings shed light on this mediator as a potential therapeutical agent to accelerate pulp repair.

One shortcoming is that PGE₂ shows a half-life relatively short. Therefore to preserve its biological activities and protect the mediator from degradation, loading of lipid mediators in microspheres has been used as a pharmacological strategy ^9,10. However, the role of lipid mediator, loaded in microspheres or are not, in dental pulp cell differentiation is not clear. For this reason, in this study we investigated the effects of the PGE₂ on the regulation of genes that encodes proteins as an indicator of odontoblastic cell differentiation. The null hypothesis of this study was that PGE₂ did not impact cell differentiation.

Material and Methods

Preparation of Microspheres

Microspheres (MS) were prepared as a pharmacological strategy using an oil-in-water emulsion solvent extraction-evaporation process ¹¹. Briefly, PGE₂ (CAYM-14010; Cayman Chemical Company, Ann Arbor, MI, USA) was dissolved in absolute ethanol (5 mg/mL). Then, 0.3 mL of the organic phase, equivalent to 7 x 10-3 M of the PGE₂ solution was added to 10 mL of methylene chloride supplemented with 30 mg of 50:50 poly (lactic-co-glycolic acid) (PLGA) (Boehringer Ingelheim, Germany). Next, 40 mL of 3% polyvinyl alcohol (3% w/v PVA) (Sigma-Aldrich Co., St. Louis, MO, USA) were added and the mixture was mechanically stirred at 600 rpm for 4 h (RW-20; Ika®-Werke GmbH & CO. KG, Staufen, Germany). Microspheres were washed (3x) with deionized water (Milli-Q®, Merck Millipore, Darmstadt, Germany), lyophilized, and stored at -20 °C until use.

Microspheres were tested for LPS contamination using the Limulus Amebocyte Lysate (LAL) QCL-1000™ kit (Lonza Walkersville, Inc., Olten, Switzerland) according to the manufacturer’s instructions. In addition, a sterility test was performed. Briefly, small microsphere aliquots were diluted in 500 µL of 1x PBS (phosphate buffered saline) and 100 µL of solution was spread on Brain Heart Infusion (BHI)-Agar medium and kept in an incubator at 37 °C for 24 h to detect microbial contamination.

Characterization of Microspheres

Size distribution of MS was determined using a LS 13 320 Laser Diffraction Particle Size Analyzer (Beckman Coulter, Inc., Atlanta, GA, USA). Samples (1 mg) of either unloaded-MS or PGE₂-loaded MS was dispersed in 0.4 mL of purified sterile water and then analyzed at 25 °C. Zeta potential of MS was determined using a Zetasizer Nano (Malvern Instruments, England). Each sample was prepared dispersing 1 mg of unloaded-MS or PGE₂-loaded MS in 0.4 mL of purified water containing 10 mM NaCl and then analyzed at 25 °C. Morphology of MS samples was assessed by scanning electron microscopy (SEM) using a FEI Inspect S 50 scanning microscope (FEI; Oregon, USA).

Efficiency of PGE2 Encapsulation in MS

For calculation of encapsulation efficiency, samples of PGE₂-loaded MS (2 mg) were dissolved in 1 mL of acetonitrile/ethanol (7:3 v/v), to disrupt the MS structure. The solvent was then evaporated off in a vacuum concentrator centrifuge for 4 h, and the residue was reconstituted in 100 mL of methanol. Then, the supernatants were transferred to appropriate vials for determination of the concentration of PGE₂ by high-performance liquid chromatography-tandem mass spectrometry using an Acquity UPLC-MS system coupled to a Xevo TQ-S mass spectrometer (Waters Corporation, Milford, MA, USA). Chromatographic separation was conducted using a Supelco Ascentis EXPRESS C₁₈ HPLC column (Sigma-Aldrich Co.) with dimensions of 100 mm×3.0 mm, 2.7 µm. A binary gradient system was employed in which phases A and B consisted of water/acetonitrile/acetic acid (70:30:0.02, v/v) and acetonitrile/isopropanol (70:30, v/v), respectively. Samples (10 µL) were eluted with a flow rate of 0.6 mL/min, with a linear gradient starting with 0% B, which was increased to 15% B at 2 min, 20% B at 5 min, 35% B at 8 min, 40% B at 11 min, 100% B at 15 min, 100% B at 18 min, and 0% B at 19 min and held there for 30 min. Analyses were performed using multiple reaction monitoring (MRM) scan mode employing negative ionization (transitions 351.2 ® 171 for PGE₂). Quantification was accomplished using calibration lines constructed with PGE₂ synthetic standards (Cayman Chemical Co.). Data were collected and analyzed using the software Mass Lynx 4.0 (Waters Corporation).

Cell Culture

Murine immortalized undifferentiated pulp cells line (OD-21) was used. The cells were kept in cryopreservation and stored in a freezing solution composed of 90% Bovine Fetal Serum (Gibbs, Carlsbad, CA, USA) and 10% Dimethylsulfoxide (DMSO) (Mallinckodt Chemicals , Mallinckrodt Baker Inc., Phillipsburg, USA). For cell culture, cells were transferred to a conical tube (Falcon ™, Corning Inc., NY, USA) containing 10 mL of DMEM with 10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin (Gibco, USA), supplemented with 10% FBS (DMEM-c) and centrifuged at 1200 rpm for 10 min at 10 ° C. The supernatant was discarded and cells resuspended in 30 mL of DMEM-c. After that, the cells were transferred to 150 cm² culture bottles (FalconTM, Corning Inc., NY, USA) at 37 °C in humidified incubator with 5% CO₂/95% air, according Ferreira et al. ¹² The medium was changed every 3 days, and the bottle was washed with 30 mL Phosphate Buffered Saline (PBS) in order to remove debris and dead cells present in the culture.

After the culture reached confluence, the culture medium was removed, and the bottle was washed with 30 mL of PBS. Then, 15 mL of DMEM-c were placed in the bottle, and with a sterile scraper (Cell Scraper, Corning Glass Workers, NY, USA) cells were detached. After that, the supernatant was transferred to a conical tube and centrifuged at 1200 rpm for 10 min at 10 °C. The supernatant was discarded and the pellet resuspended in 10 mL of DMEM-c. The viable cells number was counted in Newbauer Chamber (BOECO Germany, Hamburg, Germany) using 10 µL of the solution containing the cells were added to 90 µL of Trypan blue solution (Gibco, NY, USA) (1:10).

For the experiments, 1 × 10⁵ cells/well were plated into 48-well cell culture plates (Cell Wells; Corning Glass Workers, NY, USA) using DMEM without FBS (DMEMi) and cells were left to attach overnight.

Next, the culture medium was removed; wells were washed with 1x PBS and 300 µL PGE₂-loaded MS or soluble PGE₂ were added to each well. The experiments were done in duplicate for 3, 6, 24 or 36 h. Following incubation, the stimuli were removed and plates were either assayed or stored at -80 °C.

Cell Viability - MTT Colorimetric Assay

Cell viability was evaluated using MTT assay according manufacturer instructions. Briefly, 1 x 10⁵ OD-21 cells/well were plated into 96-well cell culture plates and stimulated with PGE₂-loaded MS or soluble PGE₂ (Cayman Chemical Co.) for 24 h.

The stimuli were removed and 10 µL of MTT (3-(4,5-dymethylthiazol-2-yl)-2,5-diphenyltetrazoluim bromide, Sigma-Aldrich Co., Catalog number M2128) supplemented with 150 µL RPMI (Roswell Park Memorial Institute) medium 1640 (Gibco) was added to the plates. After 3 h incubation, 40 µL of SDS (sodium dodecyl sulphate) buffer was added and cell viability was determined using a SpectraMax® Paradigm® spectrophotometer (Molecular Devices, LLC, Sunnyvale CA, USA). Cell viability was expressed as percentages, according to the formula: cell viability (%)=100× Experimental absorbance / Negative control absorbance (medium alone).

RNA Extraction, Reverse Transcription, and qRT-PCR

For evaluation of cell differentiation and mineralization signalling, Ibsp, Bmp2, Runx2, Alpl, Msx1, and Bglap mRNA levels were assayed by qRT-PCR. For that, total RNA was extracted using the RNeasy® Mini kit (Qiagen Inc., Valencia, USA) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, MA, USA). Next, cDNA synthesis was done from 800 ng of total RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) in a thermal cycler (Veriti® Thermal Cycler, Applied Biosystems). Quantitative reverse transcriptase-polymerase chain reactions (qRT-PCR) were done in duplicate using the TaqMan® system in a StepOne Plus® real-time PCR system (StepOne Plus® Real-Time PCR System, Applied Biosystems) using the following cycle program: 95 °C for 20 s, followed by 40 cycles at 95 °C for 1 s, and 60 °C for 20 s. All protocols were performed according to the manufacturers’ instructions. Primer-probe pairs were obtained commercially, and thus their sequences are not available. The quantifications were normalized using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and beta-actin (Actb) as reference genes. The results were analyzed based on cycle threshold (Ct) values. For each gene, relative expression was calculated by the ΔΔCt method.

Statistical Analysis

The groups were compared using analysis of variance (ANOVA) followed by the Tukey’s test (a=0.05).

Results

Endotoxin levels in PLGA MS were less than 0.1 EU / µg, as detected by the LAL assay and no bacterial growth was detected after 24 h of incubation in BHI-agar at 37 ^oC.

MS prepared with PLGA co-polymers presented monomodal population with similar diameter. For PGE₂- loaded MS average diameter was 5.332±3.480 µm and for unloaded-MS was 4.153±2.723 µm (p>0.05). Zeta potential of all MS was near the neutrality (-25±8.78 mV for PGE₂- loaded MS and -21.7±5.78 mV for unloaded-MS; p>0.05). Regarding morphology, MS prepared with PLGA co-polymers were nonporous, spherical and non-aggregated (Fig. 1).

Figure 1
PGE2-MS and unloaded-MS morphology assessed by scanning electronic microscopy (SEM)

The efficiency of encapsulation was determined using mass spectrometry to quantify PGE₂. The concentration of PGE₂ in 2 mg of PGE₂- loaded MS was calculated to be 853.9 ng/mL, which corresponds to an encapsulation efficiency of 85.4%.

Soluble PGE₂ at 0.01 and 0.1 µM reduced dental pulp cell viability (p<0.05) while PGE₂-loaded microspheres did not (p>0.05). Reduced cell viability was also detected in unloaded-MS (Fig. 2).

Figure 2
Effect of soluble or microsphere-loaded PGE2 on undifferentiated dental pulp cells (OD-21) viability by the MTT assay after 24 h. *p<0.05 compared to medium alone; #p<0.05 compared to unloaded-MS; §p<0.05 comparison between PGE2-loaded-MS and corresponding soluble PGE2 at the same concentration; &p<0.05 comparison between different concentration of soluble or PGE2-loaded-MS

Soluble PGE₂ up-regulated gene expression at 3 h, differently from PGE₂-loaded MS, that sustained delivery for longer periods. Ibsp expression was stimulated by soluble PGE₂ (0.01 µM) compared to control (DMEM) cells after 3 h, differently from PGE₂-loaded MS (0.01 µM) or soluble PGE₂ in a higher concentration (0.1 µM) (p<0.05). After 6 h, no difference was found between treatments (p>0.05) whereas Ibsp expression at 24 h was inhibited by soluble PGE₂ (0.01 and 0.1 µM) (p<0.05). Treatment with PGE₂-loaded MS (0.01 µM) induced Ibsp expression compared to unloaded-MS (p<0.05) (Fig. 3).

Figure 3
Ibsp expression after stimulation with soluble and PGE2-MS after 3 (A), 6 (B), and 24 h (C). *p<0.05 compared to medium alone; #p<0.05 compared to unloaded-MS; §p<0.05 comparison between PGE2-loaded-MS and corresponding soluble PGE2 at the same concentration; &p<0.05 comparison between different concentration of soluble or PGE2-loaded-MS

Bmp2 expression was stimulated by soluble PGE₂ (0.1 µM) compared to control (DMEM) cells after 3 h (p<0.05) whereas PGE₂-loaded MS exerted no effect (p>0.05). After 6 h, there was an invertion in this pattern, because PGE₂-loaded MS (0.1 µM) induced Bmp2 expression higher than PGE₂-loaded MS (0.01 µM) or controls (DMEM and unloaded-MS). At 24 h, PGE₂-loaded MS (0.01 or 0.1 µM) sustained Bmp2 expression, differently from soluble PGE₂ that inhibited gene expression (p<0.05; Fig. 4).

Figure 4
Bmp2 expression after stimulation with soluble and PGE2- MS after 3 (A), 6 (B), and 24 h (C). *p<0.05 compared to medium alone; #p<0.05 compared to unloaded-MS; §p < 0.05 comparison between PGE2-loaded-MS and corresponding soluble PGE2 at the same concentration; &p<0.05 comparison between different concentration of soluble or PGE2-loaded-MS.

Runx2 expression was not modulated after 3 h of treatment (p<0.05) but was upregulated by PGE₂-loaded MS (0.1 µM) after 6 h, differently from controls (DMEM and unloaded) or soluble PGE₂ (0.1 µM) (p<0.05). At 24 h, PGE₂-loaded MS (0.01 and 0.1 µM) exerted no effect on gene expression while soluble PGE₂ inhibited (0.1 µM) (p<0.05) (Fig. 5).

Figure 5
Runx2 expression after stimulation with soluble and PGE2- MS after 3 (A), 6 (B), and 24 (C) h. *p<0.05 compared to medium alone; #p<0.05 compared to unloaded-MS; §p<0.05 comparison between PGE2-loaded-MS and corresponding soluble PGE2 at the same concentration; &p<0.05 comparison between different concentration of soluble or PGE2-loaded-MS.

Bglap, Alpl, and Msx1 gene expression were not detected in dental pulp stem cells with or without PGE₂ stimulation at any concentration or time.

Discussion

In dental pulp cells, little was known about the mechanisms involved in cell differentiation induced by prostaglandins. In this study, we showed that PGE₂ is not cytotoxic to dental pulp cells, although when loaded in microspheres, it reduced cell viability. Nonetheless, the percentage of cell death is not considered cytotoxic according to International Organization for Standardization ¹³. Therefore, PGE₂-loading strategy is suitable for further dental pre-clinical investigation. It should be noted that PLGA degradation products are biocompatible and consist of lactic acid and glycolic acid ¹⁴, but these products are important cell metabolites ¹⁵. A study that used another lipid mediator (Leukotriene B4 - LTB4) loaded microspheres to stimulate murine bone marrow-derived macrophages showed that cells were activated by unloaded microspheres and produced significant amounts of cytokines, confirming that PLGA microspheres act as particulate adjuvants ^16,17. Conversely, in this study we showed that the polymer used had inhibitory on cell viability and that PGE₂- loaded MS is able to recover and even upregulate gene expression, demonstrating sustained delivery of the mediator overtime.

Bmp2 expression was upregulated by soluble PGE₂ (0.1 µM) after 3 h and by PGE₂- loaded MS (0.1 µM) after 6 h, indicating that this mediator is involved in early Bmp2 expression. Interestingly, PGE₂-loaded MS sustained gene expression up to 24 h, differently from soluble PGE₂. The human bone morphogenetic protein 2 gene is essential for odontogenic differentiation of dental pulp cells ¹⁸. PGE₂ stimulation of human tendon stem cells (hTSC) in culture induces BMP2 production and hTSC differentiation into osteoblast-like cells ¹⁹.

In addition to Ibsp and Bmp2, Runx2 (runt-related transcription factor 2) expression was also upregulated by PGE₂- loaded MS in the first 6 h of stimulation. Runx2 regulates stem cell differentiation and is the main gene controlling odontoblast differentiation. Runx2 is expressed in odontoblast-like cells and dental pulp stem cells at sites of dentin deposition, thus promoting the differentiation of dental pulp stem cells to form reparative dentin ^20,21. Because increased PGE₂ production has been shown in experimentally-induced inflamed pulp ⁵, it is important to determine if there is a correlation between Runx2 and PGE₂. PGE₂ plays a dual role by promoting both bone resorption and formation ²². Similarly to what we observed for undifferentiated dental pulp cells in the current study, PGE₂ upregulated Runx2 expression in human periodontal ligament cells (hPDLC) cultured in osteogenic medium at different molarities, indicating that PGE₂ modulates the osteogenic differentiation of hPDLC via regulation of Runx2 expression ²³.

However, differently to that observed in loaded microspheres, PGE₂ did not exert effects on OD-21 cells after 24 h or even inhibited gene expression. That might be explained by the fact that eicosanoid encapsulation preserves the biological activity of lipid mediators by preventing their degradation ⁹. In fact, PGE₂ have been effectively encapsulated in PLGA with no loss of biological activity, because lactic/glycolic acid polymers show high encapsulation efficiency and adequate stability ¹⁰. A similar method for loading of PGE₂ showed that peak levels were reached in the first 4 h, but it could also be released continuously over longer periods ¹¹. Additionally, release of encapsulated products has been observed after 21 days and their effects on cells remained unaltered ²⁴. Our findings shed light on a novel pharmacological strategy to delivery stimuli capable of inducing differentiation of dental pulp cells obtained from a mouse cell lineage. The encapsulation of bioactive molecules in microspheres has shown to be an interesting pharmacological strategy because besides modulate the immune response and protect biomolecules against fast degradation, this method prolong the delivery over longer periods of time and prevents toxic side effects ²⁵. It has been already demonstrated that biomaterials in microspheres have the ability to induce stem cells differentiation and results in odontogenic differentiation and mineralized tissue regeneration ²⁶.

Therefore, in accordance to satisfactory results in this study, PGE₂- loaded MS should be further investigated in direct contact with dental pulp tissue, aiming to promote the repair by mineralization gene expression regulation. Our in vitro results show that PGE₂, soluble or loaded in MS were not cytotoxic and modulateIbsp,Bmp2, andRunx2gene expression in cultured OD-21 cells. Based on these results we speculate that PGE₂may play an important role in dentin-pulp complex response to inflammatory stimuli and that delivery of PGE₂in microspheres might be an interesting approach to induce dental pulp cell differentiation. Further studies should be conducted in primary human dental pulp cells that represent a mixed cell culture in short and long terms to investigate the effects of PGE₂on both cell differentiation and mineralization.

Acknowledgements

We would like to thank Dr. Karina Fittipaldi Bombonato Prado at School of Dentistry of Ribeirão Preto at University of Sao Paulo (FORP/USP) for providing OD-21cells. This study was supported by Grants from The São Paulo Research Foundation (FAPESP) 2010/17611-4to FWGPS and CAPESFellowship to FLS.

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Publication Dates

• Publication in this collection
  03 June 2019
• Date of issue
  May-Jun 2019

History

• Received
  14 Dec 2018
• Accepted
  14 Jan 2019