Effects of hyperbaric oxygen on aggressive periodontitis and subgingival anaerobes in Chinese patients

Abstract

Objective:

To investigate the effects of hyperbaric oxygen (HBO₂) on aggressive periodontitis (AgP), and subgingival obligate anaerobes in Chinese patients.

Materials and Methods:

Sixty cases of Chinese patients with AgP were randomly divided into two groups –the HBO₂ group (30 cases) and the control group (30 cases). Study teeth were divided into four groups –: the HBO₂ therapy, the HBO₂ + scaling scaling group, the scaling group and the control group. Subgingival anaerobic organisms were measured with anaerobic culture, and number of obligate anaerobes and facultative anaerobes and Bacteroides melaninogenicus was counted. Comparisons of changes in the clinical indices, and subgingival anaerobes were made between the groups.

Results:

Highly significant differences in gingival index (GI), probing depth (PD), attachment loss (AL), and Plaque index (PLI), and tooth odontoseisis (TO) were seen in the HBO₂, the HBO₂ + scaling and the scaling groups when compared with the control group (P<0.01). The number of subgingival anaerobes as well as the types of obligate anaerobes and facultative anaerobes and the number of Bacteroides melaninogenicus were reduced markedly in these three treatment groups. Highly statistical differences in clinical indices, subgingival anaerobe number and types of obligate anaerobes and facultative anaerobes and Bacteroides melaninogenicus were found when comparisons were made between the HBO₂ + scaling and the HBO₂ groups, as well as between the HBO₂ + scaling and the scaling groups. Clinical follow-ups indicated that the GI, PD, AL, TO, PLI and subgingival anaerobes number of the three therapeutic groups were reduced more severely than the control group.

Conclusions:

HBO₂ had good therapeutic effects on Chinese patients with AgP. HBO₂ therapy combined with scaling and root planing was the most beneficial in the treatment of AgP. The therapeutic effect of HBO₂ on AgP is most likely through inhibition of the growth of subgingival anaerobes. Clinical follow-ups suggest that the effect could last more than 2 years.

INTRODUCTION

Microorganisms mainly include bacteria and virus and fungus. Subgingival bacteria are closely correlated with periodontitis, and these bacteria are mainly anaerobes. As we know, anaerobes are the most important factor on the onset of periodontitis. Anaerobes are divided into obligate anaerobes and facultative anaerobes, and most of the anaerobes belong to Bacteroides melaninogenicus, whose characteristic surface structures mediate subgingival bacteria growth and proliferation. Various virulent factors that cause destruction of gingival supporting alveolar bone and immune response of the body play an important role in the occurrence and development of various periodontal diseases.[1–3] Because of the prevalence and complex pathophysiology, there is at present no effective treatment for aggressive periodontitis (AgP). However, our experimental work indicates that hyperbaric oxygen (HBO₂) can inhibit the growth of anaerobes in the periodontal pockets of humans and animals[4–6] and improve gingival microcirculation and increase gingival blood flow.[7–9] Previous studies have also shown that HBO₂ had good therapeutic effects on human severe periodontitis[10] and inhibition of synthesis of prostaglandins.[11] In recent years, some clinical case reports of HBO₂ therapeutic effects on senile periodontitis and periodontitis after periodontal flap surgery have been reported in Chinese medical journals.[12,13] Pasquier et al. reported that HBO₂ can stimulate angiogenesis, fibroblast and osteoblast proliferation as well as collagen formation in irradiated tissues, and improve cellular levels of oxygen, and increase angiogenesis and cellular density.[14] But, to date, no research reports have been published on the treatment regimen or mechanism of HBO₂ therapy of AgP. In order to further explore the mechanism of HBO₂ in the treatment of AgP, outpatients with AgP were treated with HBO₂ in a controlled manner. We selected Chinese aggressive periodontitis patients and anaerobes and Bacteroides melaninogenicus as the experimental parameter to investigate the effects of HBO₂ on anaerobes and Bacteroides melaninogenicus in AgP of Chinese to provide the scientific basis for clinical application of HBO₂.

MATERIALS AND METHODS

Patient selection

Sixty patients (34 males, 26 females) with AgP among the Chinese (36 generalized AgP, 24 localized AgP) were instructed on oral hygiene and were referred to the Periodontal Department of Stomatological Research Center and chosen for our study. New International Classification standards of Periodontal Diseases (1999) were used as case selection standards. Their ages ranged from 14 to 30 years (mean age 23.5 ± 3.8 years). Patients were selected on the basis of severe AgP in Chinese, where pockets could be probed to 4 mm or more and where approximately one quarter or more of the original alveolar bone height had been lost. All patients had at least 20 remaining teeth and were willing to participate in the study. They had not received any irradiation and other periodontal therapy in the past 3 years, nor did they have any history of allergy, diabetes, blood dyscrasias or chronic liver or kidney disease. None of the patients were heavy smokers or alcohol drinkers. None of the women were pregnant.

It was made clear to the subjects that participation should be voluntary. Consent was obtained from all patients after careful explanation of the HBO₂ therapy used and the risks and benefits of the study. Ethical clearance was obtained for the study from the Ethical Committee, Hospital 411 of CPLA.

Study groups

The study groups were designated as follows: patients were randomly divided into two groups – HBO₂ therapy and non-HBO₂ therapy. For the HBO₂ therapy, patients were exposed to a pressure of 2.5 ATA (0.25 MPa) breathing pure oxygen once a day for 90 min with an air break for 10 min in the middle of the session (exposure schedule: pure oxygen 45 min, air 10 min and pure oxygen 45 min). The subjects received a total of 10 treatment sessions.[4] The left-sided teeth of all patients were treated with scaling and root planing with the same techniques, namely ultrasonic combined with manual mechanical scaling. The right-sided teeth remained untreated. The initial two groups of patients were each divided into two additional sub-groups based on different treatments. The HBO₂ group was divided into the HBO₂ group (G1) and the HBO₂ + scaling group (G2). The non-HBO₂ group was divided into the scaling group (G3) and the control group (G4).

Clinical assessment

For all patients, the following clinical parameters were recorded during the first clinical visit, immediately after the last HBO₂ and 2 years after HBO₂ therapy. Gingival Index (GI) was measured in accordance with the Loe Standard[15] and plaque deposits were recorded using the Plaque Index (PLI, Silness).[16] Probing depth (PD) and attachment loss (AL) were recorded in the nearest millimeter by a single investigator (Ramfjord 1959).[17] PD and AL were the average values of the selected teeth buccomesial, buccocentral, buccodistal and linguocentral.[10] Clinical data of six test teeth of each group were measured, i.e. three maxillary and three mandible teeth of each group were chosen at random to measure the clinical indices. Tooth odontoseisis (TO) measured with the routine technique. Two teeth of the six teeth in each group were selected for the assessment of the subgingival anaerobes of AgP, obligate anaerobes and facultative anaerobes and Bacteroides melaninogenicus. Measurement of anaerobes, subgingival bacteria and other clinical indices were made before HBO₂ therapy, immediately after the last HBO₂ therapy and 2 years after HBO₂ therapy, respectively.

Subgingival bacterial sampling obtained

Subgingival bacterial samples in each periodontal pocket base were obtained as follows: when no detectable supra-gingival microbial accumulations were present, a clean and standard curette was introduced through the pocket orifice as far as possible and the contents were removed. The process was repeated to obtain enough material. If the crown was covered with substantial accumulation of bacterial debris, the latter was first scraped off from the tooth surface corona to the pocket orifice prior to sampling. The plaque from the base of each periodontal pocket was placed in 1.0 mL of sterile thioglycolate salt solution and then made ultrasonication for 30 s. Afterwards, this was air-sealed with liquid paraffin and then immediately sent to the laboratory for anaerobic bacteria separation.

Assessment and observation of anaerobes and Bacteroides melaninogenicus

Bacterial suspensions of 10-fold dilution were prepared and 0.1 mL of the diluents were spread on 5% blood agar plates, one with and one without 1.0 mL of streptomycin and 300 ug of clindamycin phosphate per milliliter. All plates were cultured in the anaerobic glove incubator at 37°C for 4 days by using CDC anaerobic basal culture medium. Properly diluted plates (within 200 colonies) were selected for bacterial counting. Colonies of anaerobes were confirmed by Gram stain, colony morphology and biochemical tests. The number of anaerobes was calculated and the total sum of anaerobes in the diluted specimen and the types of obligate anaerobes and facultative anaerobes and Bacteroides melaninogenicus in the diluted specimen was assessed.[18,19]

Statistical analysis

Statistics was performed using the SPSS software. Differences between pre-HBO₂ therapy and after last HBO₂ therapy, and between after the last HBO₂ therapy and 2 years after HBO₂ therapy values, for each parameter were analyzed using the Student's t-test for paired observations. Differences between groups were analyzed by means of Student's t-test for unpaired observations. The level of significance was set at P<0.05.

RESULTS

Effects of HBO₂ on GI, PD, AL, PLI and TO

Before therapy, various indices between the groups were close to one another. Assessment of GI, PD, AL, PLI and TO after therapy indicated that highly statistically significant differences were seen in the HBO₂ group, the HBO₂ + scaling group and the scaling group when compared with the control group. Significant differences could also be seen by comparison of the HBO₂ + scaling group and the HBO₂ group, as well as between the HBO₂ + scaling group and the scaling group [P<0.01, Table 1]. Very significant differences could also be seen in certain clinical data, e.g., GI, PD, AL, PLI and TO between pre-and post-therapy in the HBO₂ group, the HBO₂ + scaling group and the scaling group (P<0.01). However, no significant differences were observed in the GI, PD, AL and TO between the HBO₂ group and the scaling group. There was a significant difference in the amplitude of changes in PLI, GI, PD, AL and TO when a comparison was made between the therapy group and the control group [P<0.01, Table 2].

Table 1.

Comparison of PLI, GI, PD, AL and TO between groups after HBO₂ therapy (x±s)^‡

Table 2.

Comparison of the amplitude of changes in PLI, GI, PD, AL and TO between groups after HBO₂ therapy (x±s)^‡

Effects of HBO₂ on subgingival anaerobes

The number of subgingival anaerobes from the Chinese patients with AgP included in the three treatment groups decreased significantly when compared with the baseline figures. Very significant differences in subgingival anaerobes were seen in the three treatment groups and the control group. More significant differences were also seen between the HBO₂ + scaling group and the HBO₂ group, and also between the HBO₂ + scaling group and the scaling group (P<0.01), whereas no significant difference in the number of anaerobic bacteria could be seen between groups pre-therapy [Table 3].

Table 3.

Comparisons of subgingival anaerobe number between groups (x±s, CFU/mL)^‡

Effects of HBO₂ on subgingival obligate anaerobes

Types of subgingival obligate anaerobes from the Chinese patients with AgP included in the three treatment groups decreased markedly when compared with pre-therapy. Very significant differences in subgingival obligate anaerobe types were seen in the three treatment groups and the control group. More significant differences were also seen between the HBO₂ + scaling group and the HBO₂ group, and also between the HBO₂ + scaling group and the scaling group [P<0.01, Table 4].

Table 4.

Comparisons of subgingival obligate anaerobe types between groups (x±s)^‡

Effects of HBO₂ on subgingival facultative anaerobes

Types of subgingival facultative anaerobes from the Chinese patients with AgP included in the three treatment groups decreased significantly when compared with pre-therapy. Very significant differences in subgingival facultative anaerobes types were seen in the three treatment groups and the control group. More significant differences were also seen between the HBO₂ + scaling group and the HBO₂ group, and also between the HBO₂ + scaling group and the scaling group. However, no significant differences were observed between the HBO₂ group and the scaling group [Table 5].

Table 5.

Comparisons of subgingival facultative anaerobe types between groups (x±s)^‡

Effects of HBO₂ on subgingival Bacteroides melaninogenicus anaerobe

The number and positive rate of subgingival Bacteroides melaninogenicus from the Chinese patients with AgP included in the three treatment groups decreased significantly when compared with the pre-therapy figures. Very significant differences in subgingival Bacteroides melaninogenicus anaerobe positive rate were seen in the three treatment groups and the control group. More significant differences were also seen between the HBO₂ + scaling group and the HBO₂ group, and also between the HBO₂ + scaling group and the scaling group. The positive rate of Bacteroides melaninogenicus for the HBO₂ group after therapy (34.5%) was significantly lower than that of the control group (96.5%). And, for the HBO₂ + scaling group, the positive rate of Bacteroides melaninogenicus post-therapy (27.6%) was significantly lower than that of the other three groups [Table 6].

Table 6.

Comparisons of the number and positive rate of subgingival Bacteroides melaninogenicus anaerobe between groups

Follow-up Data on the effects of HBO₂ on AgP

Assessment of GI, PD, AL, PLI, TO and subgingival anaerobe number 2 years after treatment indicated statistically significant differences in the HBO₂ group, the HBO₂ + scaling group and the scaling group compared with the control group. Identical results could be seen when comparisons were made between the HBO₂ + scaling group and the HBO₂ group, and also between the HBO₂ + scaling group and the scaling group. Significant differences were also observed in clinical data such as GI and subgingival anaerobe number measured after termination of HBO₂ therapy and 2 years after HBO₂ therapy in the HBO₂ group, the HBO₂ + scaling group and the scaling group. However, no significant differences were found in the PD, AL, PLI and TO assessed between termination of HBO₂ therapy and 2 years after therapy [Table 7].

Table 7.

Comparison of PLI, GI, PD, AL, TO and subgingival anaerobe number between groups 2 years after HBO₂ therapy (x±s)^‡

DISCUSSION

Our previous study provides good evidence that HBO₂ can inhibit the growth of subgingival bacteria and anaerobes, improve gingival microcirculation and increase gingival blood flow thus promoting healing of peridontium, which will be of help in the treatment of periodontitis. We also found that the mechanism of HBO₂ lies in the inhibition of growth and reproduction of subgingival plaque and spirochetes. HBO₂ therapy, combined with scaling and root planing , appears to be more effective for treating the human severe periodontitis. This treatment effect can last more than 1 year.[4–9,20] Gram negative Bacteroides melaninogenicus was considered to be the main pathogen of chronic periodontitis, which included Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Prevotella nigrescenes (Pn), etc. These bacteria and their products were of great significance to alveolar resorption and AL in chronic periodontitis. In our previous study, we also found that PLI, GI, PD and AL decreased significantly after patients with chronic periodontitis were treated with HBO₂, indicating that HBO₂ is very effective in periodontitis.[9–11] Costantino et al.[21] reported that HBO₂ therapy could reduce the incidence and severity of bone necrosis and inhibit alveolar resorption. Our research showed that TO for the HBO₂ group alleviated, with the extent of alleviation being greater for the HBO₂ group than that for the control group. At the same time, the alleviation of TO after HBO₂ therapy might not be just the result that dental plaque and inflammation were brought under good control, but that most probably HBO₂ promoted the formation of new bones and inhibited alveolar resorption. Anaerobes are culprits of periodontitis. HBO₂ kills or inhibits anaerobes, and can be applied in the treatment of periodontitis.[5,22] Our controlled study clearly showed that PLI, GI, PD and AL of all the three treatment groups decreased, and those of the HBO₂ and scaling groups decreased markedly, indicating that HBO₂ had significant effects on AgP and also indicating that HBO₂ combined with scaling and root planing is most beneficial for treating the patients with AgP. The treatment effects can last at least 2 years. It has been reported that bacteria can secrete different enzymes that can destroy collagen and growth factors. When the oxygen concentration in gingival tissue is low, the number of bacteria in the periodontal pockets is increased. HBO₂ can effectively decrease the number of bacteria and inhibit secretion of collagenase. A study by Rabkin showed that oxygen at 0.20 MPa could inhibit the growth of certain pathogens related to periodontitis.[23] The PLI and GI in the scaling group decreased to some extent because of good removal of dental calculus and plaque on the surface of teeth, decreasing the sedimentation of the dental plaque and controlling gingivitis.

Pg and Pi in Bacteroides melaninogenicus are highly correlated with the occurrence and development of periodontitis. The fimbriae, outer membrane proteins, lipopolysaccharide, agglutinin and vesicles, etc. on the surface of Pg and Pi are closely related with the pathogenesis of periodontitis. In addition, Pg secretes protease, hyaluronidase, alkaline phosphatase, etc.[24] The characteristic structure and secretion of these bacteria may cause edema, ischemia, hypoxia and inflammation of the periodontal tissue involved. The application of HBO₂ will increase oxygen tension in tissues and elevate oxygen concentration in periodontal pockets; thus, the growth of such Bacteroides melaninogenicus as Pg and Pi is inhibited, and metabolism is recovered. Experiments showed that oxygen at 0.2–0.3 MPa could obviously inhibit the growth of Diplococcus catarrhalis, Bacillus diphtheriae, Lactobacillus casei and such bacteria as Actinobacillus, Bacteroides and Streptococci,[23] which are closely correlated with periodontitis. Our research showed that the number of anaerobic bacteria decreased considerably, types of obligate anaerobes and facultative anaerobes and positive rate of Bacteroides melaninogenicus also decreased markedly for the HBO₂ group after therapy. For the HBO₂ group, Bacteroides melaninogenicus was detected after therapy, the positive rate being 34.5% of the original level, indicating that HBO₂ can decrease markedly the number of Bacteroides melaninogenicus in cases of AgP. And, for the HBO₂ + scaling group, the positive rate of Bacteroides melaninogenicus after therapy was significantly lower than that of the other three groups. Jiang (1995) reported that the oxygen pressure in periodontal pockets was lowered when periodontitis was active.[25] Yoblon[26] reported that rats with a fracture exposed to 0.3 MPa pressures two sessions a day, 1 h each, had abundant new subperiosteal bones formed at 40 days, indicating that HBO₂ could enhance new bone formation. Constantino et al. (1995) reported that radiotherapy coupled with HBO₂ treatment significantly reduced the occurrence of osteoradionecrosis and the amount of bone loss, indicating that HBO₂ therapy could effectively inhibit alveolar resorption.[21] As can be seen from our experiment and other literature, HBO₂ kills or inhibits actinomyces, Bacteroides and streptococcus, which contribute to the development of periodontitis. HBO₂ exposure can increase local oxygen delivery, which will raise oxygen concentration in the periodontal pocket base, on the one hand inhibiting the growth of anaerobes and on the other enabling ischemic tissues to get enough oxygen supply to facilitate early recovery of tissue metabolism.[27,28] our results showed that the number of anaerobes in the HBO₂ group obviously decreased. Scaling and root planing may decrease markedly the number of microorganisms in periodontal pockets; therefore, HBO₂ therapy combined with scaling and root planing will be most effective for treating periodontitis. According to a series of studies, our “study group” proved the effects of HBO₂ on aggressive periodontitis and pocket anaerobes in Chinese patients. The mechanism may be related to HBO₂ inhibiting the anaerobes’ reproduction and growth. The detailed mechanism still needs further investigation.

CONCLUSIONS

Our investigation provides good evidence that HBO₂ inhibits the growth of subgingival obligate anaerobes and facultative anaerobes and Bacteroides melaninogenicus thus promoting healing of peridontium, which will be of help in the treatment of AgP. HBO₂ therapy combined with scaling and root planing appears to be even better for synergistic treatment of AgP. The effects can last more than 2 years. The mechanism of HBO₂ therapy on AgP is not clear. The following might be involved in the mechanism of action of HBO₂: an inhibition of growth and reproduction of subgingival plaque and anaerobes, and in particular the growth of obligate and facultative anaerobes and Bacteroides melaninogenicus at the base of the pockets. Collectively, these effects indicate a therapeutic benefit of HBO₂ for the treatment of Chinese patients with AgP, and it can be used widely.