Influence of Periodontal Therapy on Systemic Lipopolysaccharides in Children with Localized Aggressive Periodontitis

Abstract

A previous study has shown that children with localized aggressive periodontitis (LAP) demonstrate a lipopolysaccharide (LPS) hyper-responsiveness in addition to elevated levels of systemic LPS when compared to periodontally healthy children.

Purpose

The objective of this study was to evaluate whether periodontal therapy modulates systemic LPS levels, and whether LPS levels may influence clinical outcomes.

Methods

Peripheral blood samples and clinical parameters [probing depth (PD), clinical attachment levels (CAL), percent sites greater than four mm, bleeding on probing (BoP), and visible plaque (P)], were collected from 29 LAP patients prior to as well as three, six, and 12 months following scaling and root planning and systemic antibiotics. Serum LPS levels were quantified using a chromogenic assay.

Results

Twenty-five patients were compliant with the prescribed antibiotic treatment and demonstrated a significant reduction in LPS as well as overall PD, CAL, and plaque at all time points post-therapy. Additionally LPS reductions correlated with reductions in PD, CAL, and plaque.

Conclusions

LAP therapy with antibiotics plays an important role in reducing systemic LPS levels. Since LPS is a key mediator of the LAP hyper-inflammatory response, its systemic reduction is especially important for the successful management of these children.

INTRODUCTION

Aggressive Periodontitis (AgP) is a rare but highly destructive form of periodontal disease. The disease is most commonly found at an early age and is characterized by rapid development along with a familial predisposition¹. When localized, it is referred to as localized aggressive periodontal disease (LAP), which affects primarily first molars and incisors with a higher bone-destruction to plaque ratio than observed in chronic periodontitis^{1, 2}. It has been documented that African-Americans have a higher incidence of LAP^{3, 4}, and that siblings of affected individuals are more susceptible to this disease^{5, 6}. We have demonstrated that individuals with LAP, and to a lesser extent their siblings, react to periodontopathic bacteria with a unique hyper-active inflammatory response⁷.

The severity of periodontal diseases, including LAP, is determined in large part by the host immune response. As the disease process advances both local and systemic inflammation can be detected and perpetuate disease progression^7–11. In periodontitis, the composition of bacteria is predominantly gram-negative species, which carry a unique set of virulence factors. During onset and progression of disease, the systemic dissemination of bacteria and their byproducts including endotoxins such as lipopolysaccharides (LPS), can occur^9–11. As LPS concentrations increase systemically, the production of host inflammatory agents can be provoked, exacerbating the local inflammatory response and ultimately perpetuating the breakdown of the peridontium. Indeed, we have reported plasma LPS levels were found to be higher in individuals with LAP as compared to periodontally healthy individuals¹¹. In addition, the elevated plasma LPS levels correlate with increased levels of local cyto/chemokines as well as with clinical parameters of disease ¹¹.

While there is some indication that periodontal treatment can reduce plasma LPS levels in chronic periodontitis ¹⁰, it is still unknown whether the same reduction may be achieved in children with LAP. The goal of this study was to determine the influence of periodontal therapy in combination with systemic antibiotics on the systemic levels of LPS and its correlations with changes in clinical parameters of the disease using a cohort of African-American children diagnosed with a similar form of LAP and a hyper-inflammatory response to LPS⁶.

MATERIALS & METHODS

Participant Population

The present investigation was part of a larger study registered at clinicaltrials.gov (NCT01330719). Patients were recruited through the Leon County Health Department, Tallahassee, Florida (February 2007 – November 2009). Data collection was carried out in conjunction with an approved Institutional Review Board informed consent. The inclusion criteria designated subjects to be African-American; between the ages of five to 21 years old; diagnosed with LAP as defined by equal to or greater than two teeth [incisor or first molar^{1, 2}] displaying pocket depth equal to or greater than five mm with bleeding on probing, attachment loss equal to or greater than two mm, and radiographically visible bone loss. Exclusion criteria: systemic diseases or conditions that influence the progression and/or clinical characteristics of periodontal disease, such as diabetes or autoimmune disorders; any antibiotics within the past three months; allergy to penicillin; use of medications that could influence the characteristics of periodontal disease; smokers or pregnant/lactating women. Clinical data presented for the cohort evaluated in the current paper may have been presented in a previous study, as this was a sub-study of a larger trial (NCT01330719). The purpose of the present investigation was to assess effectiveness of periodontal treatment in reducing circulating LPS levels and its correlation with clinical parameters after therapy.

Clinical Measurements

A medical and dental history was taken on all participants. At baseline and three, six and 12 months after periodontal treatment a four-mL sample of peripheral blood was collected, along with the following periodontal clinical parameters: pocket depth (PD), bleeding on probing (BoP); gingival margin position (GM), clinical attachment level (CAL) (measured from cementum-enamel junction (CEJ) to bottom of the pocket); visible plaque (P). All measurements were performed by one calibrated examiner using a UNC-15 periodontal probe at six sites per tooth and recorded using Florida Probe software (Gainesville, FL, USA).

Periodontal and Antibiotic Treatment

All participants were treated by the same practitioner. After completion of baseline measurements, all participants received full mouth debridement with an ultrasonic device (Cavitron Jet Plus, Dentsply, York, PA, USA) and scaling and root planing (SRP) of deeper sites under anesthesia, as required. At the end of the first visit, participants were placed on a systemic antibiotic regimen of 500mg of amoxicillin and 250mg of metronidazole three times per day for seven days (dosage was adjusted according to weight for children less than 40kg). Participants and their parents were provided verbal and written instructions on medication administration and side effects and a medication log. Medication compliance was monitored verbally with parent/patient and by the return of remaining medication at next follow up visit. Non-compliance to medication was established as the patient’s failure to fully complete the prescribed dose regimen in its entirety. Non-compliant patients were excluded from this analysis. Oral hygiene instructions (OHI) and tools, including an electric toothbrush (Vitality, Oral B, Belmont, CA, USA), were also provided. The subjects were re-examined at three, six and 12 months post-treatment where they received additional mechanical therapy for pockets greater than four mm, as required. Professional plaque removal and reinforcement of home care procedures were performed at all timepoints.

Plasma LPS Levels

A blood sample was collected from all participants at each time point. Plasma was then separated from red blood cells by centrifugation (~300 x g for 15 minutes) and frozen at −80 degrees Celsius until analysis was performed. Plasma lipopolysaccharides (LPS) levels were detected and semi-quantified by a chromogenic assay (Endpoint Chromogenic LAL Assay, Lonza, Basel, Switzerland). Endotoxin units/mL were calculated using a standard curve and best-fit linear trend line.

Statistical Analysis

ANOVA (Kruskal-Wallis with Dunn’s multiple comparisons) was used to evaluate reductions in LPS and clinical parameters over time. Spearman correlations were used to examine the associations of LPS reduction and clinical parameters over time.

RESULTS

Our study initially began with a cohort of 29 patients. (18 females/11 males; mean age 13.38± 3.75 years). Patients compliant (n = 25) with medications completed the full dose regiment of antibiotics as prescribed; however some may have missed one or more scheduled appointments. Non-compliant (n = four) patients failed to fully complete their prescribed dose regimen, and were excluded from the study. Patient attrition of the compliant group included 25 patients at baseline, 16 patients at three months, 11 patients at six months and 14 patients at 12 months; demographics of these patients are presented in Table 1.

Table 1.

Demographics, Clinical Parameters, and LPS at baseline for compliant and non compliant patients.

	Age	M/F	Mean PD sites	Mean CAL	%PD>4mm	%BoP	%Plaque	LPS
LAP Compliant, (n=25)	13.45±3.75	9m/16f	4.89±0.51	3.33±1.45	13.25±9.39	17.79±12.80	39.16±25.74	0.44±0.28

Values are given by means ± Standard deviation. M=male; F=female; %PD>4mm=the percentage of all sites presenting with a pocket depth of 4mm or greater; CAL=clinical attachment level of affected sites; Mean PD sites= Mean pocket depth of sites with PD>4mm; BoP=bleeding on probing; PD site = Pocket depth from sampled site; LPS=Lipopolysaccharide

Plasma LPS Levels and Clinical Parameters Following Treatment

Plasma levels of LPS in participants compliant with their antibiotic regimen demonstrated a significant reduction at three and 12 months following periodontal treatment (Fig. 1).

Fig 1.

(A) Plasma lipopolysaccharides (LPS) levels over time, for compliant patients (n=25) at each time point after periodontal therapy. Patients demonstrated a significant reduction from baseline in systemic LPS to 3 and 12 months after treatment. Kruskal wallis with Dunn’s multiple comparisons (*p<0.05, ***p<0.001).

Periodontal treatment resulted in significant improvement of most clinical parameters (Fig. 2). There was a significant decrease in mean pocket depth and clinical attachment levels at all timepoints after treatment (Fig. 2a and 2b) and a significant reduction in percent pockets greater than four mm at 12 months after treatment (fig. 2c). There were no significant differences in the percentage of sites with BoP and plaque post-treatment (Fig 2d and 2e). Figure 3 shows significant bone loss on permanent dentition first molars on a participant diagnosed with LAP before treatment and radiographic bone fill after treatment on those teeth. Figure 4 shows primary dentition affected with LAP before treatment and figure 5 shows permanent dentition erupted and free of disease 18 months after treatment of a primary LAP dentition affected participant.

Fig 2.

Clinical Parameters for compliant patients (n=25) at baseline, 3 months, 6 months and 12 months through periodontal therapy. (a) Mean pocket depth (PD), significant decrease between Baseline and all timepoints. (b) Mean clinical attachment level (CAL), significant decrease between Baseline and all timepoints. (c) % sites with PD > 4mm, overall significant reduction between baseline and 12 months (d) %bleeding on probing (BoP), no significant decrease. (e) %visible plaque, no significant decrease. Bars represent significant decreases among timepoints by Kruskal-Wallis with Dunn’s multiple comparisons (*p<0.05, **p<0.01, ***p<0.0001).

Fig. 3.

14 year old African American male patient diagnosed with Localized Aggressive Periodontitis of #3M, #14M at baseline and 12 month appointments during periodontal therapy. a. Intra-oral photograph #3 8mm PD. b. BW Radiograph #3 at baseline. c. BW radiograph #3 at 6 months following periodontal therapy. d. extra-oral photograph #14M, 8mm PD. e. BW radiograph #14 at baseline. f. BW radiograph #14M at 6 months following periodontal therapy.

Fig. 4.

7 year old African-American female patient diagnosed with Localized Aggressive Periodontitis in primary dentition at baseline appointment. a. Intra-oral photograph teeth A, B, and C showing 4mm pocket on mesial of B. b. PA Radiograph of teeth 3, A, and B at baseline showing bone loss on A mesial and B mesial and distal. c. Intra-oral photograph of teeth G, H and I showing probing depth of 7mm on mesial of I. d. PA Radiograph of teeth I, J, and 14 at baseline in mixed dentition showing bone loss on mesial of I.

Fig. 5.

9 year old African-American male patient diagnosed with Localized Aggressive Periodontitis of tooth A, B and S and T at baseline and 18 month appointments after periodontal therapy. a. BW Radiograph at baseline, mixed dentition affetcted with bone loss. b. BW Radiograph at 18 months following periodontal therapy, permanent dentition, no bone loss noted.

Plasma LPS Levels Correlate with Clinical Parameters

The decrease in circulating LPS over time was found to significantly correlate to a decrease in mean PD (r = 0.363, p=0.009), mean CAL (r= 0.487, p<0.0002), and plaque index (r= 0.472, p=0.001) (Table 2).

Table 2.

Correlation of LPS reduction with clinical parameters through each time point of periodontal therapy for antibiotic compliant patients.

		mean PD	mean CAL	%PD>4	BoP	Plaque
LPS	R2	0.363	0.487	0.087	0.139	0.472
	p value	0.009	0.0002	0.550	0.342	0.001

Mean PD = mean pocket depth; Mean CAL = mean calculated attachment loss; %PD = percent of sites with a probing depth > 4mm; BoP = bleeding on probing. All values recorded to the nearest millimeter.

^*Indicates significance according to Spearman Correlation.

DISCUSSION

Aggressive periodontitis is a severe and rapidly progressing form of periodontitis of children, typically asymptomatic in early stages. The host’s hyper inflammatory response to bacterial endotoxins, such as LPS, has been suggested as a key contributing factor to the progression of LAP ⁷. Circulating LPS has been found to be at high levels in LAP patients compared to unaffected siblings and controls ¹¹. Thus LPS may play a significant role in the cycle of the host’s hyper inflammatory response in LAP. Moreover, high levels of systemic LPS and a hyper-inflammatory response may also play a role in other complications in these patients, such as cardiovascular diseases ^{12, 13}.

Here, we examined whether periodontal therapy over time can successfully lower the systemic concentration of LPS, and, whether the change in serum LPS can be positively correlated with the clinical outcome of periodontal parameters. Our previous study has shown that individuals with LAP have significantly higher levels of systemic LPS levels as compared to periodontally healthy individuals ¹¹. Our present study found that patients under periodontal therapy while compliant with antibiotic prescription demonstrated an overall decrease in systemic LPS. While a significant decrease in serum LPS was observed among each subsequent time point, initially there appeared to be a strong decrease in serum LPS at 3 three months, a slight rebound at 6 six months but then again a reduction and a stabilization of these levels up to 12 months, which correlate well with clinical parameters of the disease, including a decrease in PD and CAL. This finding is consistent with previous research that reports a positive correlation between LPS levels and clinical parameters of disease, as well as reduction of LPS after treatment in chronic periodontitis in adult patients ¹⁰.

A greater improvement of clinical parameters with the use of systemic antibiotics in aggressive periodontitis has been previously reported ^14–16, as well as the effects of patient antibiotic compliance in clinical outcomes in AgP ¹⁵. Our present data suggests that periodontal treatment, a local therapy, in addition to compliance with antibiotic treatment, a systemic therapy, seem to be effective for lowering systemic LPS levels. The correlation between LPS and clinical parameters demonstrates the role of successful periodontal therapy in the reduction of bacteremia, and possible systemic complications it may cause.

In addition, this study adds to prior research highlighting the significance of LPS as a contributing factor to the hyper-inflammatory response found in LAP ⁷. LPS has a role in triggering the host’s immune response both on a local and systemic level. LPS has been associated with an increase of activity among destructive proteinases by leukocytes ¹⁷. On the other hand, LPS is able to interact with the host’s systemic immune system due to its release into the body’s circulation through a diseased periodontal pocket ⁹. When present in high levels, LPS was found to play an important role in triggering the host’s inflammatory response through its activation of macrophages, resulting in added cytokine production and release ^{7, 10}. Therefore, the role of LPS in activating macrophages and perpetuating the body’s inflammatory response may explain not only why significant inflammation and tissue destruction is observed in LAP individuals, but also the influence of periodontal diseases and bacteremia in other systemic inflammatory complications, such as cardiovascular disease and pre-term-low birth weight babies ^{18, 19}. For instance, serum LPS has been found to be positively correlated not only with increased radiographic bone loss, but also with levels of triglyceride and troponin, while inversely correlated with high density lipoprotein levels^{12, 13}. Thus, considering LAP patients’ tendency for immune hyper-responsiveness to LPS stimulation, the reduction of systemic LPS seems to be particularly desirable in these individuals, not only for the stability of periodontal tissue health but also for the prevention of future systemic complications in these individuals.

Future studies may include a randomized, delayed/different timing of antibiotic treatment, controlled trial with a higher number of patients to test the complete absence of any antibiotic therapy in order to conclusively evaluate its role in LPS reduction. Additionally, evaluating local inflammatory mediators over time and possible correlations to LPS and clinical parameters may add further insight into LPS’ role at a systemic and local level influencing clinical outcomes.

In conclusion, our results show that effective periodontal treatment along with a systemic antibiotic regimen in LAP patients leads to a reduction in clinical parameters of disease as well as in systemic LPS levels, which is maintained up to 12 months post-treatment. The reduction of circulating levels of LPS may be particularly important in LAP individuals, as they may be pre-disposed to a hyper inflammatory response to LPS. Additionally, since previous studies also point to a potential role of LPS in other systemic complications, its reduction may be not only a critical factor for the resolution and maintenance of periodontal clinical parameters, but also for the prevention of possible secondary complications.

CONCLUSIONS

The results of this investigation support the following conclusions:

1. Periodontal therapy, including antibiotic treatment, is effective in reducing clinical parameters of disease as well as reducing the level of systemic lipopolysaccharides.

2. The reduction of lipopolysaccharide in children with LAP was shown to correlate with an improvement in clinical outcomes over time.