A Randomized Controlled Crossover Trial for Reinforcement of Epithelial Barrier Function by Vitamin D Induction of the Antimicrobial Peptide Cathelicidin - A Novel Therapeutic Approach in Chronic Periodontitis

Introduction

Chronic periodontitis is a polymicrobial inflammatory disease of multifactorial origin. It is initiated by the damage of oral epithelium which is the first barrier for entry of oral pathogens existing in dental plaque. Untreated periodontitis progressively results in tooth mortality which has significant social and economic burden on the patients. 

Antimicrobials have been used as a consistent chemotherapeutic modality for treating periodontitis in the past. The host immuno-inflammatory response to the bacteria dictates the magnitude of tissue destruction seen in periodontitis. The expanding knowledge of the host-microbial interaction in pathogenesis of periodontal diseases has led to investigation of new therapeutic approaches for periodontitis by focusing on host response in addition to conventional approaches for the treatment of diseases of periodontium.

With shifting paradigms in healthcare facilities from cure to prevention, much emphasis has been laid upon prevention of disease process at primary level. Oral epithelium forms a physical, chemical and immunological barrier against the invading microorganisms. The chemical barrier mainly comprises of anti microbialpeptides (AMPs) which are endogenously produced antibiotics. Cathelicidins are the most potent AMPs detected in the cavity of mouth.¹ The link between upscaling of AMP function and vitamin D is well established.² Thus, an increase in vitamin D should significantly improve the chemical barrier function by increasing AMPs.

This study intended to explore an economically viable, easily available and a well-accepted molecule like vitamin D in primary prevention of chronic periodontitis by reinforcing the epithelial barrier through increased action of cathelicidin.

Aim of the study

To evaluate the consequences of vitamin D administration on cathelicidin LL-37 levels in gingival crevicular fluid (GCF) of chronic periodontitis patients.

Objectives of the study

1. To study the role of vitamin D on cathelicidin LL-37 levels in periodontal diseases.

2. To evaluate cathelicidin LL-37 levels in GCF in chronic periodontitis patients pre-and post-administration of vitamin D.

3. To establish an association between Vitamin D, oral epithelium (chemical barrier) and anti-microbial peptides in prevention of periodontal diseases.

Methodology

Source of Data

Chronic periodontitis patients satisfying the inclusion criteria of the study were screened from the OPD of Sri Rajiv Gandhi College of Dental Sciences (SRGCDS) and hospital, Bangalore. All the patients were subjected for the estimation of vitamin D levels in the serum. Only the patients who were found to be vitamin D insufficient according to vitamin D council and Indian Endocrine Society were recruited for the study. The study group consisted of subjects belonging to both the sexes. Written informed consent was obtained from all the patients. This randomized controlled crossover trial was registered in Clinical Trials Registry, India (CTRI/2017/08/009193). This study was carried out between September 2017 to November 2019.

Inclusion Criteria

1. Study subjects >25 years of age

2. Patients suffering from chronic periodontitis i.e.

• a) Bleeding on probing (BOP)
• b) Probing pocket depth (PPD) of ≥ 5 mm
• c) Clinical attachment loss (CAL) of ≥ 3 mm

3. Vitamin D insufficient patients (serum levels < 30 ng/mL) according to vitamin D council

4. Systemically healthy individuals with minimum 20 teeth in occlusion.

Exclusion Criteria

1. Patients with systemic diseases such as hepatitis, diabetes mellitus, HIV and immunological disorders

2. Patients on any systemic antibiotics, NSAIDS, steroid therapy and any other drugs which are known to affect the periodontal status

3. Former smokers and current smokers

4. Patients who had received periodontal treatment in the last year or under any medication

5. Pregnant women or nursing mothers.

Method of Data Collection

The clinical periodontal parameters evaluated were probing pocket depth (PPD), clinical attachment level (CAL) and bleeding on probing (BOP) at six sites per tooth, excluding third molars in all patients.

Periodontitis was diagnosed based on clinical criteria which was put forward by the International Workshop for Classification of Periodontal Diseases and Conditions,1999. The subjects selected for the study were randomized by coin toss method and were allocated to two groups - the test and the control.

Study Design

This was a single-center, randomized, two-way crossover study. The study consisted of two intervention periods of three months divided by a six week washout period (Figure 1). 

Forty vitamin D insufficient subjects with chronic periodontitis were split into two groups:

Group I - Test group: Twenty chronic periodontitis patients who were prescribed vitamin D orally as an adjunct to scaling and root planning (SRP).

Group II – Control group: Twenty chronic periodontitis patients with SRP only.

Patients were scrutinized throughout the study by the same assessor assigned to the group. PPD and CAL measurements were done with a Williams periodontal probe. All the assessments were done by a scaled assessor. The intra examiner reliability was high as disclosed by the intraclass correlation coefficients of 0.85 and 0.87 for CAL and PPD, respectively.

Intra examiner reliability

PPD and CAL measurements were carried out on six surfaces on all the teeth of same patient (not a participant of the study) using Williams periodontal probe. Two measurements were done in a gap of two hours and both the sets of measurements were in excellent concurrence.

Collection of GCF

Sample GCF was collected a day after the examination of the periodontium. The buccal aspects of interproximal sites of the teeth were chosen for collecting sample. Gingival crevicular fluid collection was done from sites having probing pocket depth >5 mm and clinical attachment loss >3 mm. GCF sampling was done after the removal of plaque from interproximal surfaces with a sterile curette, drying the surfaces gently by an air syringe and isolating the site by cotton rolls. 3 µL of crevicular fluid was acquired by extra crevicular method using micro-capillary pipettes. The sulcular fluid was obtained as a pooled sample from the deepest site and fetched into a microcentrifuge tube which was frozen and stored at -70 ^о C till analysis was carried out.

After GCF collection, all the patients were subjected to SRP. Test group patients were prescribed oral dose of 2000 IU vitamin D daily for a period of three months. It was followed by a six week wash out period (no intervention). Then the groups were subjected to crossover (test becomes control and vice versa) and the same treatment sequence was followed. Cathelicidin LL-37 levels were estimated using enzyme linked immunosorbent assay (ELISA) method. Values obtained from the test were recorded, computed and subjected to statistical analysis.

Results

Data were conveyed as means and standard deviation. Full mouth clinical attachment levels, pocket probing depths, bleeding on probing and plaque index were recorded to distinguish participants in the study. Moreover, the means (± SD) of cathelecidin levels were calculated for those sites from which samples of GCF were collected. The relevant data was put into SPSS 13.0 program and was analyzed using the paired t-test. p <0.05 with 95% confidence intervals (CI) was set as the level of significance.

Figure 2 and 3 depicts the distribution of the GCF cathelicidin LL-37 in the study groups. The differences among the study groups were found to be significant (p <0.05). GCF cathelicidin concentrations strongly correlated with serum vitamin D concentrations in subjects with insufficient vitamin D levels.

In group I (Table 1), cathelicidin levels in GCF were found to increase post administration of vitamin D, which was followed by a washout period of six weeks. Before the washout, in Group I, the change in mean Cathelicidin LL-37 value post treatment as compared to before appeared to be significant (p <0.01). After the washout, in Group II (Table 2), the change in mean cathelicidine LL-37 value from before to after treatment appeared to be statistically significant (p <0.001).

Discussion

The oral epithelial barrier i.e the outer gingival epithelium acts as a physical barricade to separate dental plaque from gingival tissues. It provides the first line of defense against microbial invasion in periodontal diseases. An inflammatory response is triggered by the entry of exogenous pathogens into the host tissues due to breakage of oral epithelial barrier which may lead to the establishment of chronic infection. Research has proven that chronic inflammation observed in periodontitis can be due to gingival epithelial barrier dysfunction caused by IL-31 produced by mast cells as a feedback to P. gingivalis infection.³

The recognition of antimicrobial peptides in the epidermis illustrates a mechanism by which growth of microorganisms can be managed in the early hours following injury to epithelium, during the process of wound healing, and in any ongoing inflammation. Thus, they yield a robust defense system that can both protect the skin from any infection and send signals to the cells of host to change their etiquettes in response to injury.

Antimicrobial peptides (AMPs) of innate immune system are contemplated to be the fastest in responding to oral pathogens. Along with their antimicrobial effects, they also exhibit immunomodulation by inducing cell proliferation, differentiation, migration and by regulating production of cytokines, improving angiogenesis, wound healing and assisting the barrier function of skin.^4,5 Research has revealed that AMPs, such as cathelicidinLL-37, human-defensins (hBD)- 1&3, protein S100A7 escalate the levels of TJ-related proteins and assist epidermal barrier function.^6-9

Through this study, we tried to bring out the interrelation between oral epithelial barrier dysfunction in chronic periodontitis patients and vitamin D derived antimicrobial peptides to consider recruiting these peptides for barrier repair as an auxiliary therapeutic approach for CP.

Approximately one billion people in the world have vitamin D deficiency or insufficiency.¹⁰ Though the role of supplementation of vitamin D and calcium on bone health have been instrumental,^11,12 their probable role in diseases of periodontium has not been fully established. Literature reveals^13-16 that intake of vitamin D and/ or calcium results in alveolar bone gain, decreased inflammation of gingiva and decreased attachment loss.

Vitamin D aids in absorption of calcium in the intestine, maintains calcium equilibrium in the body and enables normal bone growth and preservation by stimulating osteoblasts. The bio-active form of vitamin D, 1α,25- dihydroxy vitamin functions as an immunomodulator by inhibiting cytokine production by immune cells and stimulating monocytes and macrophages to secrete peptides with potent antibiotic activity.¹⁷

This role of vitamin D has been linked to susceptibility to bacterial-mediated infections, with decreased vitamin D levels being associated with surge in the risk of infectious diseases.¹⁸ Therefore, vitamin D may be beneficial for the treatment of periodontal disease.

Although most epidemiologic studies have found beneficial results after supplementing with vitamin D and calcium, the use of oral supplementation remains low and varies greatly. With the discovery that bio-active forms of vitamin D instigate the CAMP gene expression, it has been hypothecated that status of vitamin D may affect the levels of circulating hCAP18.^19,20

Bhan and colleagues discovered a positive correlation in healthy individuals (mean age 39 years) at 25(OH) D levels <32 ng/mL, but not when levels were higher.²¹

Research by Alvarez-Rodriguez et al., involving 71 healthy individuals with about two-thirds of participants below 32 ng/mL serum 25(OH)D, established an optimistic inter relationship between levels of serum 25(OH) D and LL-37.²² Contrary to this, no relationship was noted between maternal 25(OH) D serum levels and LL-37 in samples of cord-blood²³ or in patients suffering from active pulmonary tuberculosis.²⁴

Considering the existing relationship between serum 25(OH)D and hCAP18/LL-37 levels, it is plausible that supplementation of vitamin D or sunlight exposure to generate vitamin D might help to increase hCAP18/ LL-37 systemically, thus amplifying protection against infection or sepsis. In another study, in vivo supplementation of subjects with serum 25(OH)D levels <32 ng/mL resulted in an increase in hCAP18 levels in those subjects showing greatest rise in serum 25(OH)D.21 In another study, supplementation of normal and atopic dermatitis (AD) patients with 4,000 IU oral vitamin D3 for 21 days showed a significant rise in expression of cathelicidin in the AD lesions.²⁵

The present study illustrated that GCF LL-37 levels were highly elevated in chronic periodontitis patients who were administered vitamin D. The presence of cathelicidin LL-37 in GCF suggests a pivotal role for this antimicrobial peptide in the pathogenesis of chronic periodontitis. Few studies^26,27,28 have investigated measurement of cathelicidin LL-37 in the GCF or gingival tissues of chronic periodontitis patients. In this study, we tried to elicit the amount of GCF cathelicidin in chronic periodontitis patients after the administration of vitamin D supplements through a crossover study method.

The advantage of a crossover design is gain in precision as we can compare treatments within patients; so discrepancy between patients is eradicated. Disadvantage of crossover design is the susceptible to carry over effects (lasting effects of one treatment on outcome in the following period), which can invalidate the results.

Vitamin D decreases the infection risk through varied mechanisms, one of them being modulating production of AMPs and boosting innate immunity. Antibiotics are an expensive option and their misuse can lead to significant antibiotic resistance. Vitamin D is an inexpensive option prophylactically and possibly therapeutically, either by itself or as a synergistic agent to conventional antimicrobial agents.

Vitamin D and calcium are considered crucial in addition to standard treatments for preventing and treating diseases of periodontium. Vitamin D supplementation (1,000 IU/ day) had a modest positive effect on periodontal health.²⁹ In subjects under periodontal maintenance, there was a drift for better periodontal health with supplementation of vitamin D and calcium.³⁰

Outcome of our study showed that vitamin D administration increases cathelicidin levels in periodontal diseases. Cathelicidin levels in GCF increase after intake of vitamin D and periodontal diseases might be intercepted by reinforcing the epithelial barrier (chemical barrier) by vitamin D induced production of AMPs. It was not established if vitamin D augmentation improved immunologic results in these studies; therefore, future randomized trials are needed to determine an immune strengthening role for vitamin D supplementation and to illustrate its optimal circulating levels. Further work in this direction will determine the effectiveness of such postulations and establish the use of these peptides in various treatments.

Conclusion

Progressive research on cathelicidin molecule with emphasis on the functional properties and its role in host defense will help the investigators reveal its multifunctional nature that may mediate various host responses, and thus represents an essential component of the innate immune system in humans.

The gene for cathelicidin carries a response element of vitamin D and the vitamin D pathway could therefore be targeted for regulation of cathelicidin. As the development and trajectory of periodontal diseases might be governed by vitamin D signaling, these pathomechanisms could explain the growing evidence connecting vitamin D to periodontal diseases.

Factually vitamin D has been associated with musculoskeletal health regulation. Recent research has shown its various systemic effects through vitamin D receptor (VDR) activation. The Vit-D-VDR system plays a pivotal role in oral homeostasis and its malfunction leads to periodontal disease.

Therefore, vitamin D research should make prime contributions to our comprehension of periodontal diseases and may be advantageous in the therapy due to its direct effect on bone metabolism and its anti-inflammatory properties. This research project is a step towards realizing the potential of vitamin D as a therapeutic modality in the management of periodontal diseases.

Limitations of the study

• Shorter duration of the study

• Inability of some patients to turn up after treatment

• Keeping a check on utilization of vitamin D tablets provided to patients

Future Perspective

The future of vitamin D looks bright. Believing that most people have vitamin D levels that are insufficient and that nearly a billion people across the world are deficient, appropriately designed studies in humans will be crucial for establishing the advantages from increasing vitamin D serum levels on functions of immune system.

It will be enthralling to establish if sufficient vitamin D levels will help in treating patients with TB and HIV infections. These are classically deficient populations and augmenting with vitamin D could probably be a very economical option for these conditions and might be alluring for countries where these diseases are very rampant.

Vitamin D research could help in identifying new targets of VDR through innovative approaches involving models of animals, studies on humans and genomic approaches. Most of the epithelial tissues such as oral mucosa, skin, urinary tract, reproductive organs, intestinal tract are constantly exposed to the environment. Additional studies focusing on vitamin D–cathelicidin pathway should elicit the importance of vitamin D in barrier defense of the body.

Conflict of interest

None

Acknowledgement

This research was externally funded by Advanced Research Wing of Rajiv Gandhi University of Health Sciences (RGUHS), Karnataka. [Research ID 17D029Consort Diagram