| Abstract|| |
Context: Periodontitis is a potential risk factor for adverse pregnancy outcomes due to the presence of a subgingival load of pathogenic bacteria. Instrumentation of periodontal pockets during treatment may result in bacteremia and/or endotoxemia.
Aims: The aim of this pilot clinical trial was to determine the immediate post-scaling and root planing (SRP) level of circulating endotoxin in females presenting with chronic periodontitis.
Settings and Design: A before-and-after pilot clinical trial among rural women of low socioeconomic status attending the outpatient department (OPD) for periodontitis.
Materials and Methods: Twenty-four women aged 25-35 years, with at least five teeth having probing depth (PD) ≥5 mm, were selected for the study. The clinical trial was divided into three phases: phase 0 (screening), phase I (just before scaling), and phase II (15 min after scaling). Phase 0, one day prior to SRP, was used to assess the eligibility of the patients and record the periodontal status. Endotoxin levels were assessed by a semiquantitiative gel-clot assay, the limulus amoebocyte lysate (Lonza®), at phase I and phase II.
Statistical analysis used: The Chi-square test was used for statistical analysis.
Results: There was significant increase of endotoxins levels (P<.05) immediately after scaling (15 min).
Conclusions: SRP can lead to endotoxemia, possibly by release of endotoxins by bacteria of periodontal origin, into the systemic circulation.
Keywords: Endotoxin, periodontitis, pregnancy, scaling and root planing, serum
|How to cite this article:|
Jacob SP, Nath S, Zade R M. Effect of periodontal therapy on circulating levels of endotoxin in women with periodontitis: A pilot clinical trial. Indian J Dent Res 2012;23:714-8
There is growing evidence that oral bacteria contribute to systemic disease.  Recent studies have shown that periodontal disease is linked to an increased risk of various systemic diseases, including stroke,  atherosclerosis,  acute myocardial infarction,  and adverse pregnancy outcomes such as preterm low birth weight (PTLBW). ,,, Specific microorganisms within the microbial ecology associated with periodontitis release endotoxins (lipopolysaccharides) that evoke an inflammatory response. The ulcerated pocket epithelium becomes a significant portal of entry for oral bacteria and their products into the general circulation. The term 'ulcerated' implies a breakdown in integrity of the epithelium. In this manner, periodontal biofilm infection contributes not only to local inflammation, resulting in destruction of tooth-supporting structures, but to systemic inflammation as well. ,
|How to cite this URL:|
Jacob SP, Nath S, Zade R M. Effect of periodontal therapy on circulating levels of endotoxin in women with periodontitis: A pilot clinical trial. Indian J Dent Res [serial online] 2012 [cited 2020 Nov 28];23:714-8. Available from: https://www.ijdr.in/text.asp?2012/23/6/714/111244
Endotoxin is a lipopolysaccharide (LPS) comprising the outer membrane of gram-negative bacteria. LPS acts through LPS-binding protein and Toll-like receptors 2 and 4.  The interaction between LPS and host cells results in up-regulation of the transcription of numerous genes, leading to elevated production of an array of cytokine, chemokine, and lipid mediators that contribute to acute and chronic inflammatory responses. 
Scaling and root planing (SRP) is the initial standard treatment of periodontitis.  SRP causes disruption of the ulcerated epithelium,  leading to translocation of periodontal bacteria and endotoxins into the systemic circulation.  Circulating endotoxins increase release of cytokines and prostaglandin, , leading to preterm labor, preterm birth (PTB), and low birth weight (LBW).
To our knowledge, no studies have determined the immediate effect of nonsurgical therapy like SRP on endotoxin level in women with periodontitis.
The aim of the present pilot clinical trial was to determine the immediate effects of SRP on the circulating levels of endotoxins in systemically healthy female patients presenting with untreated chronic periodontitis.
| Materials and Methods|| |
This before-and-after pilot interventional study was designed to determine the immediate effect of SRP on circulating endotoxin levels. The study was conducted over a 3-month period, between April 2011 and June 2011. The study was conducted after obtaining approval from the institutional ethical committee (CDCEC ethics protocol number: CDCEC/ 2011 / 2 / ET) and written informed consent from all the participants. The clinical trial was registered in clinical trial registry, India (Ref No. CTRI/2011/05/001766). The full protocol can be accessed on www.ctri.nic.in. The study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000.
A total of 24 female subjects of good general health with age ranging from 25-35 years, belonging to the rural population of Rajnandgaon, and who were diagnosed with periodontitis, were recruited for this trial. All subjects had at least 20 natural teeth, had not received periodontal debridement or surgery in the last 6 months, and had not received any antimicrobial therapy in the last 3 months. Periodontitis was defined as present when patients had at least five teeth with a probing depth ≥5 mm. ,
Subjects were not included in the study if they had any chronic immunological condition such as arthritis, gastrointestinal disorder, or diabetes; were taking steroids, immunosuppressants, or an anticoagulant for the past 6 months; or if they were pregnant or lactating.
This before-and-after clinical trial was divided into three phases: phase 0 (screening), phase I (just before scaling), and phase II (15 min after scaling).
Phase 0: During the screening examination, the participants were evaluated for eligibility by a complete case history and periodontal examination. The following parameters were recorded with a UNC-15 periodontal probe (Hu Friedy®); plaque index (PI) (Loe and Silness), modified gingival index (MGI), probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP). The presence or absence of bleeding within 30 s on probing was recorded. The PD and CAL were measured on six sites of each tooth (mesial and middle and distal points on buccal and palatal/lingual aspects) and assessed to the nearest millimeter. In order to avoid the effect of periodontal probing on endotoxin level the patients were recalled the next day for SRP. ,
Phase I and phase II: Venous blood was collected under aseptic conditions for endotoxin estimation. This was followed by complete SRP. A second sample of venous blood was taken 15 min after scaling (phase II).
For blood collection, the same procedure was adopted for both phase I and phase II. Venous blood (2 ml) was drawn from the antecubital fossa under aseptic conditions and transferred in plain tubes for serum preparation. Serum was prepared within an hour of collection by centrifugation of the blood samples. The samples were then stored at −20° C till the endotoxin assessment.
The serum was analyzed using the limulus amoebocyte lysate (LAL) gel-clot assay (Lonza®). This assay is a semi-quantitative measure of endotoxin activity. The test was carried out according to manufacturer's instructions. LAL, as supplied, was reconstituted with LAL reagent water and then mixed in equal parts with the serum. Then the tubes were incubated at 37°C for 60 min. A positive reaction for endotoxemia was recorded for the tubes that contained a firm gel clot which remained in place when the test tube was inverted at 180°. Gelation takes place in the presence of endotoxin. The samples were compared against a control standard Escherichia More Details coli endotoxin of known potency 055:B5 (Lonza®). All tests and dilutions were performed in endotoxin-free glassware and test tubes (Borosil®) and LAL reagent water (Lonza®). A positive reaction indicated an endotoxin level of 0.125 EU/ml.
Mean and standard deviation were calculated for each variable. Presence of endotoxemia was compared between phases I and II using the Chi-square test. Statistical significance was inferred at P < 0.05.
| Results|| |
The periodontal characteristics of the population are summarized in [Table 1]. Two subjects tested positive for endotoxemia at phase I as compared to eight patients at phase II (chi-value: 4.547; P = 0.036) [Table 2]. There was a significant increase in the number of subjects with endotoxemia at 15 min after SRP.
|Table 2: Comparison of endotoxemia before and after scaling among 24 healthy women|
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| Discussion|| |
The oral cavity has been estimated to harbor over 700 species of microorganisms, and there is wide variation in the colonization pattern and the number of potential virulence factors produced by these bacterial species. ,, Certain oral species have been demonstrated to have an enhanced capacity to invade host epithelial and endothelial cells, , which can be expected to play a role in their capacity to translocate into the systemic circulation. Therefore, in a patient who harbors 75-100 different species of periodontal bacteria, it is a challenge to document the frequency and magnitude of systemic bacterial translocation over time. Various molecular genetic techniques have been employed to identify the bacteria. The use of species-specific primers is limited by the range of species that could be translocating in an individual subject, while the prokaryotic primers are not uniform in their effectiveness for the broad range of bacteria.  Because the principal bacteria that have been associated with pathogenic biofilms in periodontitis are gram-negative, the availability of a system for detection of endotoxin could provide a broad evaluation of microbial challenge to the systemic circulation. In this study we did not attempt to assess the presence of bacteria within the circulation but, instead, focused on one of the bacterial products, i.e., endotoxins.
This before-and-after clinical trial was conducted in women of child-bearing age to find out if endotoxemia could be an explanation for the link between maternal periodontitis and PTLBW. Women enrolled in this study were all healthy except for periodontal disease. All participants were from the below-poverty-line (BPL) population, in which the incidence of periodontitis and adverse pregnancy outcomes like PTB and LBW is high. , Till now, no study has been conducted in a similar population to find out the acute effects of periodontal instrumentation.
This trial was divided into three phases; screening (phase 0), just before scaling (phase I), and 15 min after scaling (phase II). Ide  et al. used a similar trial design and observed changes at 15 min after scaling. Lee  et al. observed changes in endotoxin level at 5 min and 120 min. Probing measurement was taken a day before phase I to avoid confounding due the bacteremia induced by the probing itself. ,
In this study endotoxemia was assessed by the limulus amoebocyte lysate assay. The method is both sensitive and reproducible. , A first blood sampling was done before scaling and the second 15 min after scaling, the rationale being that scaling induces bacteremia by disruption of the ulcerated epithelium. ,,, The gingival tissues have an extensive capillary network that not only delivers cells and biomolecules from the vascular system, but also enables the translocation of oral bacteria and/or their products into the systemic circulation. During scaling, the diseased periodontium is subjected to high pressure or depression, which creates a 'pumping effect,' favoring the passage of bacterial components through the ulcerated epithelium.
Our results show that there is increase in the endotoxin level after SRP. Out of 24 women with periodontitis, 2 patients had endotoxemia before scaling compared to 8 patients after scaling, which was statistically significant (P<.05). Ide  et al. studied the short-term fluctuations in endotoxin levels and found increased levels at 15 min after scaling. Lee  et al. found raised endotoxin levels in samples taken 5 min after SRP in patients with periodontitis. The effects of toxemia have been studied by Mogardini  et al. who reported that 50% of patients who underwent intensive subgingival instrumentation over a short period reported pyrexia. In several studies, bacteremia was seen in the systemic circulation after initial periodontal therapy. ,,,
The two patients in our sample who tested positive for endotoxemia before scaling had the severest periodontal disease (as assessed by probing depth) among the study group. But due to the small sample size the aspect that severe periodontitis can by itself cause endotoxemia could not be analyzed. Geerts  et al. showed that even gentle mastication was capable of causing systemic dissemination of endotoxin and that this was directly related with increased probing depth. Endotoxemia can even occur as a result of simple brushing. Clearly, the severity and extent of periodontitis accentuates the possibility of bacteria entering the blood stream.
The translocation of endotoxin into the systemic circulation may cause subacute bacterial endocarditis. , More recent studies have suggested that the infection in periodontitis can enable oral bacteria to colonize in vascular tissues  and in placental/amniotic tissues  due to bacterial products translocation into systemic circulation. Endotoxin produced by maternal oral bacteria has been known to induce preterm birth in experimental animals by causing an increase in the cytokine level , and prostaglandin synthesis, , which leads to early intrauterine contraction and preterm labor and thereby to PTB and LBW. 
A few randomized trials have shown that treatment of periodontal disease during pregnancy lowers the chances of PTB and LBW. ,,, Other studies have shown that SRP has no effect on pregnancy outcomes. , A meta-analysis by Polyzos  et al. also showed that SRP cannot be considered to be an efficient way of reducing the incidence of preterm birth. A recent follow-up study  of an interventional trial showed that the infants of mothers treated for periodontitis during pregnancy were similar to infants of nontreated mothers with regard to neurodevelopment scores. Ferguson  et al. also concluded that treatment of periodontal disease did not reduce the risk of adverse pregnancy outcome and that the procedure can be performed safely during gestation. At this time, the best available evidence is mixed as to whether the treatment of periodontal disease during pregnancy influences gestational age at delivery or birth weight.
The finding from our study suggests that SRP during pregnancy can induce endotoxemia and thus increase the chances of delivering PTLBW babies. The possibility that the trauma of treatment (SRP) is itself responsible for the adverse pregnancy outcomes cannot be excluded, yet it is not implausible that endotoxemia caused by treatment may initiate host responses and lead to systemic changes. However, the results from this study should be considered in light of studies wherein nonsurgical periodontal treatment did not cause any adverse pregnancy outcomes. A large multicentric randomized controlled trial by Michalowicz  et al. found that treatment of periodontitis during pregnancy was completely safe. In a follow-up study,  the same group concluded that periodontitis treatment during pregnancy does not affect the growth and development of the infants. Disparate results have been seen in a review of five clinical trials by Ferguson  et al. The review indicates that the treatment of periodontal disease in pregnant women during the second and third trimesters is safe and does not significantly alter the rates of preterm birth, low birth weight, or fetal growth restriction. It is difficult to compare the results of the various trials due to differences in methodology. Unlike other studies, our trial assessed the transient effects of nonsurgical periodontal therapy on systemic endotoxin levels.
Our clinical trial had a few methodological limitations. Firstly, the small sample size of the study may affect the reproducibility of our results. Secondly, healthy female patients with periodontitis were selected instead of pregnant patients. Inclusion of pregnant subjects would have raised ethical issues so a similar population of non-pregnant women was considered. Thirdly, the endotoxin assay used for this study was a semi-quantitative measure of endotoxin activity and therefore the results should be interpreted with caution. Additionally, in this study, only the immediate effects of periodontal therapy was observed and additional follow-up was not done. We recommend further clinical trials with larger sample sizes to evaluate the effects of SRP.
Within the limitations of this study, it appears that an episode of nonsurgical treatment of periodontitis in healthy females could lead to transient increase in the levels of circulating endotoxin. Therefore, any intervention to reduce infection and inflammation of oral origin should be performed before onset of pregnancy. In view of the results of this study we would suggest caution when advocating scaling to pregnant periodontitis patients, especially those who are at risk for delivering PTLBW infants.
| Conclusion|| |
SRP in female patients with periodontitis increases circulating endotoxin levels. Endotoxemia during pregnancy may affect the developing fetus. Future studies are necessary to validate our findings and to properly elucidate the benefits or harms of SRP in pregnant women with periodontitis.
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Shaju P Jacob
Department of Restorative Dentistry, School of Dentistry, International Medical University, Kuala Lumpur
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]