The Expression of the Human Cathelicidin LL-37 in the Human Dental Pulp : An In vivo Study

Bernardo F. Sarmiento1, Anita Aminoshariae2*, Mohammed Bakkar2, Tracey Bonfield3, Santosh Ghosh4, Thomas A Montagnese5 and Andre K Mickel5 1Department of Graduate Endodontics, Case Western Reserve University School of Dental Medicine 2Department of Endodontics, Case School of Dental Medicine 3Division of Pediatric Pulmonology, Allergy & Immunology, Case School of Medicine 4Department of Biological Sciences, Case School of Dental Medicine 5Diplomate, American Board of Endodontics, Case School of Dental Medicine International Journal of Pharmacovigilance Open Access Research Article

To date, the antimicrobial peptide LL37 is the only cathelicidin found in humans [6].It is formed from the last 37 amino acid residues of the C-terminus of human Cationic Antimicrobial Peptide 18 (hCAP18) [7].LL-37 is amphipathic, helical peptide found throughout the body and being expressed in epithelial layers of the skin, saliva, and junctional epithelium of the gingiva [8] bone and bone marrow [9].LL37 has been shown to exhibit a broad spectrum of antimicrobial activity [10] against bacteria, fungi, and viruses [11], and inhibits lipopolysaccharide bioactivity [12].In addition to its antimicrobial properties LL37 induces the migration of monocytes, T cells, and keratinocytes [13]; inhibits the increase in cytokine production induced by lipopolysaccharide in dendritic cells [14]; and induces angiogenesis [15].Furthermore, LL37 has antimicrobial activity against cariogenic and periodontopathogenic bacteria [16].LL37 plays a central role in innate immune responses and inflammation [17].To date there has not been an in vivo study that has detected the expression of LL37 directly in the human dental pulp.
Thus, the purpose of this study was to identify the expression of LL37 in the normal and inflamed dental pulp diagnosed as symptomatic irreversible pulpitis with symptomatic apical periodontitis.

Introduction
The human dental pulp is exposed to a variety of biologic, chemical, and physical stimuli during caries development and dental therapy demonstrating a wide range of responses, including defense and repair processes [1].The dental pulp is equipped to mount an innate and adaptive response in order to defend itself [2].Although the mechanism of the host defense against microbial invasion in the human dental pulp is not fully

The Expression of the Human Cathelicidin LL-37 in the Human Dental Pulp: An In Vivo Study
Copyright: © 2016 Aminoshariae et al.
from Case Western Reserve University, Cleveland, Ohio-Graduate Endodontics program were randomly selected from August 2014 -October 2014 to participate in this study.The treatment plans from any of the patients remained without any modification during this investigation.Patients were given a written description of the study; approval and informed consent were obtained.The Institutional Review Board (IRB) approval was granted.Patients selected in this study were included with the following diagnosis: symptomatic irreversible pulpitis with symptomatic apical periodontitis, and for the control group, samples were processed from teeth with normal pulp with normal apical tissues from teeth requiring prophylactic endodontic treatment.The inclusion criteria are the following: American Society of Anesthesiologists (ASA) physical status 1, no history of known allergies, non-smokers, age between eighteen and sixty-five, healthy periodontal status and restorable teeth.Exclusion criteria: patient on antibiotics, patients with any known allergies, pregnancy, diabetes, immunocompromised patients, any periodontal probing depth greater than 5mm or teeth with a furcation or trifurcation involvement, teeth diagnosed with a necrotic pulp, previously initiated endodontic treatment and/or previously endodontically treated teeth.Each patient's pulpal and periradicular status was evaluated by the cold test (HYGIENIC ®; ENDO-ICE ®; Coltène/ Whaledent Inc., Cuyahoga Falls, OH, USA) and electric pulp test (EPT) (Vitality Scanner; SybronEndo, Orange, CA, USA) to assess pulp vitality.Percussion, palpation, mobility and periodontal examinations were performed.Digital periapical and bitewing radiographs of the tooth in question (Planmeca® ProSensor ™; PLANMECA USA, Inc., Roselle, IL, USA) were obtained.
The following type of patients was subsequently included: samples collected from 10 teeth of patients diagnosed with Symptomatic Irreversible Pulpitis (SIP) and Symptomatic Apical Periodontitis (SAP).As a negative control, samples were also taken from 6 teeth diagnosed as having normal pulp, which had no clinical or radiographic evidence of, pulpal and periapical pathosis but needed routine endodontic treatment for prosthodontic reasons.The definition of the SIP SAP is described in a previous study [18].Briefly, the clinical characteristics of the cases included subjective and objective findings that are described: Symptomatic Irreversible Pulpitis (SIP): Included sharp pain upon thermal stimulus, lingering pain (often 30 seconds or longer after stimulus removal).

Symptomatic Apical Periodontitis (SAP):
Pain to percussion and/or palpation.

Control
Normal pulp with normal apical tissues, where the teeth indicated absence of caries, symptoms, cracks and restorations.

Operative Procedure & Site Selection
The sampling procedure is as described by Martinho, et al. [19].After local anesthesia and absolute isolation with the rubber dam, an access opening was made into the pulp chamber using a sterile size #2 round carbide bur (DENTSPLY Maillefer, Tulsa, OK) friction grip in a dental high speed hand piece.A safe ended (Endo-Z bur) (DENTSPLY Maillefer, Tulsa, OK) was used to deroof the pulp chamber.Hemorrhage and tooth structure remnants were controlled, and removed using a saline irrigating solution.Standard size #30 sterile paper points (Lexicon®; DENTSPLY Tulsa Dental Specialties, John City, TN, USA) were introduced into the canal orifice remaining for 30 seconds for the blood sample absorption.Thereafter, the paper points were transferred into Eppendorf tubes (Eppendorf Tubes®, Lakewood, OH, USA) containing 400 µL of phosphate-buffered saline (PBS) (Gibco® PBS, Grand Island, NY, USA) at pH 7.4, centrifuged at 1500 xg at 4C° for 15 minutes and stored in a freezer compartment at -70°C until use.

Bicinchoninic acid assay (BCA)
Bicinchoninic acid assay was first introduced by Smith, et al. [20] and is a sensitive methodology for protein quantification [21].The BCA protein assay (Pierce, Rockford, IL, USA) measured the total concentration of protein in the samples according to manufactures instructions.

Measurement by Enzyme-linked Immunosorbent Assay (ELISA)
LL37 levels in the paper points were measured using ELISA assay (Hycult Biotech Inc.®; Plymouth Meeting, PA, USA).LL37 was detected at from a range of 0.1 ng / ml to 100 ng / ml.The working volume of 100 μl / well was the standard for the dilution.The human LL-37 ELISA a solid-phase enzymelinked Immunosorbent assay based on the sandwich principle was performed.Micro titer wells were coated with antibody to recognize the human LL-37, a total (100 µL) of the sample was added to each well and the plates were incubated for 60 min.at 20-25°C the samples were washed four times, thereafter a Biotinylated tracer antibody (100 μl) was added to bind and capture the peptide and incubated for 60 min.at 20-25°C the samples are washed four times.Streptavidin-peroxidase conjugate (100 μl) was added to bind to the tracer; the samples were again washed four times.A substrate was added Tetra Methylbenzidine (TM solution) (100μl) to react with the conjugate incubated for 30 min at 20-25°C.Finally, the enzyme reaction was stopped by the addition (100μl) of oxalic acid stop solution and immediate readings of the plates at 450 nm measurements of absorbance are done with the spectrophotometer.

Statistical Analyses
Mann-Whitney test was used to compare the protein expression levels (BCA) and LL-37 levels between normal pulp and symptomatic irreversible pulpitis.The level of significance was set at P-value = 0.05 with a post Dunn test.Data was analyzed by using Graphpad Prism and SPSS 22 software (IBM SPSS Statistics for Windows, Version 22.0.Armonk, NY).

Results
In this study there were a total of sixteen teeth (normal pulp

The Expression of the Human Cathelicidin LL-37 in the Human Dental Pulp: An In Vivo Study
Copyright: © 2016 Aminoshariae et al.

BCA Levels
The mean total protein concentration for the normal pulp was 884.4 ± 466.4 mg/ml and symptomatic irreversible pulpitis was 786.6 ± 463 mg/ml.There was no statistical significance difference in protein levels between normal pulp and symptomatic irreversible pulpitis ( p = 0.79) Figure 1.

LL37 Levels
LL37 were detected only in one sample out of 6 normal pulps, however in symptomatic irreversible pulpitis with symptomatic apical periodontitis (SIP SAP) samples, LL37 were detected in 8 out of 10 samples.The mean pulpal concentration of LL-37 in normal pulps 0.2 ± 0.6 ng/ml and symptomatic irreversible pulpitis 1.5 ± 1.2 ng /ml.The levels of LL37 with symptomatic irreversible pulpitis are significantly higher than normal pulp (p = 0.03) Figure 2.

Discussion
To the best of our knowledge this is the first study to report on the expression of LL-37 directly in the human dental pulp.A previous study by Kajiya, et al. [22] had hypothesized that LL37 could be expressed in the human dental pulp due to presence of neutrophil infiltration which produces LL37, and could be expressed in inflamed pulp tissues.Based on the results of the present study LL37 expression of the SIP SAP group was statistically significant compared to the group with normal pulp implicating that there are differences upon the upregulation of LL37 between the healthy and inflamed pulp tissue.
The importance of LL37 among its diverse biological effects is the contributable role it plays in maintaining micro biota-host homeostasis and promoting repair responses to inflammatory insults [9].The current trend in the endodontic literature is the focus on the ability of LL37 to induce human pulp cell migration which is an attributing factor in the regeneration of the pulpdentin complex for the formation of reparative dentin commonly known as Dentin Bridging [22].Aforementioned, among other characteristics of LL37 its angiogenic effect is being widely observed as potential novel therapeutic agent in the medical field [15].In the field of Endodontics the potential use of LL37 as a pulp-capping agent for the reversible healing of the inflamed dental pulp is being contemplated for a promising clinical use [23].
It has been reported that levels of total protein in the Gingival Crevicular Fluid (GCF) of diseased teeth with apical periodontitis were higher than contralateral healthy teeth in the same patient [24].However, in our study the total protein concentration levels were not statistically significant between normal and inflamed dental pulp, p = 0.79 Figure 1.Due to the nature of the study obtaining SIP and normal pulp from the same patient were inapplicable.
Enzyme-linked ImmunoSorbent Assay (ELISA) was used to evaluate human pulpal blood with respect to LL37 levels collected by paper points.Nakanishi, et al [25] used ELISA to analyze human pulpal blood samples collected by nylon pellets and found that IgG, IgA, IgM, elastase, and especially prostaglandin E2, but not IL-1, IL-6 or TNF-α, were elevated in inflamed compared to healthy teeth.Another study found significantly higher levels (P < 0.05) of IL-6, IL-8, IL-10, TNF-α and IFN-γ were detected in irreversible pulpitis as compared to normal pulp using ELISA and samples were collected by sterile cotton pellets [26].

The Expression of the Human Cathelicidin LL-37 in the Human Dental Pulp: An In Vivo Study
Copyright: © 2016 Aminoshariae et al.The limitation of our study was the sample size, as it was very difficult to obtain higher amount of samples and equal distribution among both groups especially in the control group, where obtaining normal pulp samples from teeth undergoing root canal therapy for prosthodontic considerations are very limited and/or impossible to acquire.Pulpal tissue vascularization provenience is difficult to determine in hyperemic dental pulp tissues as with the endodontic clinical testing we are only able to diagnose based on subjective and objective findings [27].This perhaps might dictate the result in the normal pulp group in which expression of LL37 was detected in one sample only consequently of a potential misdiagnosis; as an accurate diagnosis of different pulpal inflammation forms is only possible by using histopathologic or molecular biologic techniques [28].
In conclusion, based on the results of the present study it is acknowledged that inflammation induces the expression of LL37 [13] in the human dental pulp; these findings suggest that LL37 plays a role in the innate immunity [29].Further research is needed to better quantify expression levels of LL37 as well as detect the degree of inflammation which might up regulate LL37 in human dental pulps.

Figure 2 :
Figure 2: Enzyme-linked Immunosorbent Assay (ELISA) to determine the LL37 levels (ng/ml) in normal pulp with normal apical tissues and symptomatic irreversible pulpitis with symptomatic apical periodontitis.Statistically significant (p=0.03),Mann-Whitney test with post-Dunn test.
Table details the patient age and gender, and data distribution.

Table 1 :
Demographic characteristics and endodontic diagnosis of the healthy patients UD: undetected; N/A: not available data.