Leukotriene B 4 Loaded in Microspheres Regulate the Expression of Genes Related to Odontoblastic Differentiation and Biomineralization by Dental Pulp Stem Cells

Background: Leukotriene B 4 (LTB 4 ) is a potent lipid mediator that stimulate the immune response. Because dental pulp inammation and dentin repair are intrinsically related responses, the aim of this research was to investigate the potential of LTB 4 in inducing differentiation of dental pulp stem cells. Methods: Microspheres (MS) loaded with LTB 4 were prepared using an oil emulsion solvent extraction evaporation process and sterility, characterization, eciency of LTB 4 encapsulation and in vitro LTB 4 release assay were investigated. Mouse dental pulp stem cells (OD-21) were stimulated with soluble LTB 4 or MS loaded with LTB 4 (0.01 and 0.1 μM). Cytotoxicity and cell viability was determined by lactate dehydrogenase (LDH) and MTT (methylthiazol tetrazolium) assays . Gene expression were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 3, 6, 24, 48 and 72 h. Results: Mineralized nodule formation was assessed after 28 days of OD-21 cell stimulation with LTB 4. Groups were compared using the one-way ANOVA test followed by Dunnett's post-test (α = 0.05). Treatment with LTB 4 or MS loaded with LTB 4 (0.01 and 0.1 micrometer-μM) were not cytotoxic to OD-21 cells. Treatment with LTB 4 modulated the expression of the Ibsp (integrin binding sialoprotein) and Runx2 (runt-related transcription factor 2) genes differently depending on the experimental period analyzed. Interestingly LTB 4 loaded in microspheres (0.1 μM) allowed long term dental pulp cell differentiation and biomineralization. LTB 4 loaded in MS was not cytotoxic and induced an odontoblastic cell phenotype differentiation. Conclusion: These ndings shed light on a novel pharmacological strategy to induce dental pulp cell differentiation. For evaluation of cell differentiation biomineralization signaling, binding runt-related transcription factor 2 (Runx2),dentin sialophosphoprotein(Dspp) and dentin matrix protein-1 (Dmp1) mRNA levels were assayed by qRT-PCR. mRNA levels were measured by quantitative reverse transcriptase-polymerase chain reactions (qRT-PCR). To this end, total RNA was extracted using the RNeasy® Mini kit and quantied using NanoDrop 2000 spectrophotometer A total of 1 µg of RNA were used for cDNA synthesis with the High Capacity cDNA Reverse Transcription kit in a thermal cycler (Veriti® qRT-PCR reactions were performed in duplicate using the TaqMan® system in a StepOne Plus® real-time PCR system (StepOne Plus® Real-Time PCR System, Applied Biosystems) and the following cycle program: 95 °C for 20 s, 40 cycles at 95 °C for 1 s, and 60 °C for 20 s. Primer-probe pairs were obtained and thus their sequences are not available(TaqMan® Gene Expression Assay, Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as reference genes for normalization purposes. The results were analyzed based on cycle threshold (Ct) values. Relative expression was calculated by the ΔΔCt method.


Introduction
Pulp and dentin are closely related tissues, being assembled as a single unit, the dentin-pulp complex, which is a strategic and dynamic barrier in face of injuries suffered by teeth, being caries the most common cause of injury to this complex [1,2]. Odontoblasts, located around the pulp, are the rst to have contact with pathogens, producing dentine matrix in order to protect the pulp [3,4]. However, deep cavity preparations or dental pulp exposure can disrupt the integrity of the dentin-pulp complex and may cause odontoblast cell death [5]. Thus, the regeneration of these tissues occurs through stimulation and proliferation of mesenchymal progenitor cells, which are attracted to the injury site to differentiate into odontoblast-like cells and produce reparative dentin [6,7].
Response to infection that occurs in the dental pulp is a complex molecular reaction that aims to eliminate the foreign pathogen. Cells and tissues at the injury site express receptors that recognize pathogenic signals, such as lipopolysaccharides, lipoteichoic acids and bacterial DNA [8]. In response to that, several in ammatory mediators are produced locally to orchestrate the immune response. Among those are the eicosanoids, a class of lipid mediators that are synthesized from arachidonic acid through the action of cyclooxygenases or lipoxygenases to produce prostaglandins and thromboxanes or leukotrienes (LT) and lipoxins, respectively [9,10]. In the presence of FLAP (5-lipoxygenase activating protein), a nuclear protein associated with the membrane, the enzyme 5-LO is activated and oxidizes arachidonic acid, converting it to 5S-hydroxyperoeicosatetraenoic acid (5S-HpETE), which is further reduced by the enzyme peroxidase to 5S acid -hydroxyieicositetraenoic (5S-HETE) or is converted into LTA4, which, by the action of LTA4 hydrolase, results in LTB 4 production [11].
Leukotriene B 4 (LTB 4 ) is a potent in ammatory mediator that also stimulates the immune response, induces the recruitment of phagocytes and potentiates the ingestion and death of pathogens, being one of the most recognized neutrophil activators, modulating the release of cytokines and increasing vascular permeability [12][13][14]. LTB 4 binds either to high a nity receptor (BLT1), mainly in leukocytes, or to low a nity receptor (BLT2) [15]. However, soluble LTB 4 present a short half-life and is rapidly degraded [16].
As a therapeutical strategy, the use of microspheres could preserve the biological activity and stability of the mediator for prolonged periods [13,[17][18]. However, studies are lacking to investigate the role of these lipid mediators in dental pulp cell behavior, especially through the synthesis and deposition of dentinal matrix in undifferentiated cells. Therefore, the objective of this study was to investigate if LTB 4 loaded in microspheres would induce odontoblastic cell differentiation and biomineralization. The null hypothesis of this study was that LTB 4 did not impact odontoblast cell differentiation and function.

LPS contamination tests
For sterility test 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 o C for 24 h to detect microbial contamination.
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. To obtain the standard curve, the serial dilution regime was performed, starting from 1.0 EU / mL of E. coliendotoxin 0111: B4 (E50-640). Optical density was analyzed using a μQuantTM spectrophotometer at a wavelength of 405 ηm (BioTek® Instruments Inc., Winooski, USA), with KC4 TM Data Analysis Software (BioTek® Instruments Inc.), in order to determine the concentration of endotoxin units/ml of solution containing microspheres (EU / ml).

Characterization of microspheres
Size distribution of MS was determined using a LS 13 320 Laser Diffraction Particle Size Analyzer (Beckman Coulter, USA). Samples (1mg) of either unloaded-MS or LTB 4 -loaded MS was dispersed in 0.4mL of puri ed 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 1mg of unloaded-MS or LTB 4 -loaded MS in 0.4 mL of puri ed 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).

E ciency of LTB 4 encapsulation in MS
For calculation of encapsulation e ciency, samples of LTB 4 -loaded MS (4 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 μL of methanol. Then, the supernatants were transferred to appropriate vials for determination of the concentration of LTB 4 by a competition enzyme immunoassay, according to manufacturer's instructions (EIA, Amershan Biosciences, Piscataway, NJ, USA). Quanti cation in μM was accomplished using calibration curve containing LTB 4 synthetic standards (Cayman Chemical, Ann Arbor, MI, USA).

In vitro LTB 4 release assay
The release kinetics of LTB 4 from LTB 4 -MS were monitored in vitro. LTB 4 (4 mg) was suspended in 1 mL of PBS/ethanol (50:50, v/v), pH 7.4, and incubated at 37 °C on a rotating incubator. At each time point 6, 12, 18, 24, 30, 36, 42, 48 and 54 h of rotation, the suspension was centrifuged and the supernatant was collected for assay of LTB 4 concentration, then 1 mL of fresh PBS/ethanol was added to the ask containing the LTB 4 -MS and the experiment was continued.
The supernatants were transferred to appropriate vials for determination of the concentration of LTB 4 by a competition enzyme immunoassay, according to manufacturer's instructions (EIA, Amersham Biosciences, Piscataway, NJ, USA). Quanti cation was accomplished using calibration curve containing LTB 4 synthetic standards (Cayman Chemical, Ann Arbor, MI, USA).

OD-21 cell culture
Undifferentiated mice dental pulp stem cells (OD-21 cell line) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1% Penicilin/Streptomicin (Gibco) in an incubator at 37 °C and 5% CO 2 . For the experiments, 1 × 10 5 cells/well were plated into 48-well cell culture plates (Cell Wells, Corning Glass Workers, NY, USA) using DMEM without FBS and cells were left overnight for attachment.
Next, the culture medium was removed; wells were washed with phosphate buffered saline (PBS) and 300 µL LTB 4 -loaded MS or soluble LTB 4 were added to each well. The experiments were done in duplicate and the stimuli were maintained for 3, 6, 24, 48 and 72 h for short term experiments or 28 days for long term biomineralization assay.

Cytotoxicity -Lactate dehydrogenase (LDH) assay
For cytotoxicity assessment, cells were plated in serum-free medium, at a concentration of 1 × 10 5 cells per well, kept in an incubator at 37 o C and 5% CO 2 for 12 hours (overnight). After this period, cultures were

Biomineralization assay
Mineralized nodule formation was assessed by culturing con uent OD-21 cells in biomineralization media for 28 days with changes of media every third-day. Biomineralization media consisted of DMEM culture media supplemented with 10 mM β-glycerophosphate, 50 μg / ml ascorbic acid, and 1% FBS. OD-21 cells were treated with LTB 4 -MS or mineralizing media alone and with the combination of both.
Mineralized monolayer cell cultures were stained for matrix biomineralization as described previously [20]. Brie y, cultures were xed with 70% ethanol for 10 minutes and stained with 2% Alizarin Red solution (Sigma) for 5 minutes at room temperature. To quantify the degree of calcium accumulation in the mineralized extracellular matrix, Alizarin Red-stained cultures were incubated with 100 mM cetylpyridinium chloride (Sigma) for 1 hour to release calcium-bound dye into solution. The absorbance of the released dye was measured at 570 nm using a spectrophotometer, and normalized by the total protein concentration in the culture.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 6 software (GraphPad software Inc., La Jolla, USA). Groups were compared using the one-way ANOVA test followed by Dunnett's post-test (α = 0.05).

PLGA microspheres (loaded with LTB 4 or empty) exhibited no bacterial growth after 24h incubation in
BHI-agar at 37º C ( Figure 1A). Also, the endotoxin levels in all samples (encapsulated LTB 4 or in empty microspheres) were less than 0.1 EU/μg ( Figure 1B

Discussion
Here we found that LTB 4 induced an odontoblastic phenotype in dental pulp cells and production of mineralized nodules. LTB 4 is a proin ammatory mediator derivate from the enzymatic oxidation of arachidonic acid involved in dental pulp in ammatory reactions [10,14,[21][22][23], but none of them evaluated your effect in the osteogenic and odontogenic differentiation of dental pulp stem cells. Therefore, the null hypothesis was rejected once LTB 4 loaded in microspheres regulated the expression of genes related to odontoblastic differentiation and biomineralization in mouse dental pulp stem cells.
As LTB 4 shows a half-life relatively short, in this study the use of microspheres had the aim to preserve its biological activities a longer time and protect the mediator from degradation [24]. LTB 4 showed no cytotoxic to dental pulp cells, measured by the percentage of cell death of less than 30% and in accordance to the International Organization for Standardization guidelines [25]. Other studies that used the PLGA microspheres demonstrated that it is biocompatible and act as particulate adjuvants [17,24,[26][27][28][29]. All these studies showed that microspheres are a viable way to delivery mediators for prolonged time.
The expression ofRunx2 was upregulated by LTB 4 soluble after 3 h and after 6, 24, 48 and 72h by LTB 4loaded MS in different concentrations (0.01 and 0,1 μM), indicating the involvement of this mediator in Runx2expression (30).Runx2 is a transcription factor highly expressed in mesenchymal cells and dental papilla, which is essential for osteoblast and odontoblast differentiation and regulates these cell proliferations [31][32][33]. Hight doses of LTB 4 can stimulate the osteoblastic cell proliferation while low doses exhibited an inhibitory effect [34]. In this study, the use of microspheres prolongated the action of LTB 4 and it may have corroborated to this effect by increasing the expression of Runx2.
Integrin binding sialoprotein belongs to a family of proteins, exclusively located in mineralized tissues and crucial for the homeostasis of bone remodeling. The role of this protein involves the initiation of mineral deposition (hydroxyapatite) and increasing of osteoclastogenesis (bone resorption) [35]. In bacterial-induced apical periodontitis, the LTB 4 is involved in the signaling for osteclastogenesis by the action of leukotriene B4 type 1 receptor (BLT1) [10].
In this studyIbsppresented high relative expression after 3 hours of stimulation with LTB 4 soluble, however it decreases in the other times analyzed, 6, 24, 48 and 72 hours. While LTB 4 -loaded MS upregulated the expression of Ibsp at 48 and 72 hours. This upregulation can be associated to high expressions of Runx2 as some in-vitro studies demonstrated that the expression of bone matrix protein genes, as integrin binding sialoprotein (Ibsp) can be upregulated by Runx2 [33,36].
Two LTB 4 receptor have been cloned: BLT1 and BLT2. BLT1 is the high-a nity receptor predominantly expressed in leukocytes and acts as a potent chemotactic receptor for in ammatory cells [15,37]. LTB 4 can stimulate the osteoclast differentiation and bone resorption [38] by the activation of LTB 4 /BLT1 mechanism [39]. BLT2 is the low-a nity receptor and has been associated with reduction of pain and wound-healing acceleration by cell proliferation [40]. The prolongated effect of LTB 4 promoted by the microspheres could activate the LTB 4 /BLT2 mechanism and promote cell proliferation and differentiation. The increase in the relative expression of Runx2andIbsp might be related to that as BLT2 plays an important role in the wound-healing by cell proliferation [18].
A recent study demonstrated that LTB 4 needs an incubation time of 24 hours to assure an adequate ligation with the receptor and present the intended pharmacological effects, as accelerated woundhealing rate [40]. Therefore, the use of microspheres can be a strategy to preserve the biological activities of the mediator for prolonged times and activated this receptor. One should not expect a direct correlation between in vitroand in vivoconcentration of mediators released from microspheres, specially because the environment might in uence that, due to in ammation, edema, dilution, etc. In this preclinical in vitro study, cell differentiation under LTB 4 stimuli was investigated. Later on, in vivoinvestigation should be performed to optimize the deliver to in vivo preclinical and clinical studies.
There are several clinical procedures that the materials can be directly applied to dental pulp which includes direct pulp capping, partial pulpotomy or full pulpotomy. Our ndingsshed light on a novel pharmacological strategy to deliverystimuli capable of inducing differentiation of dental pulp cells. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.