|Year : 2012 | Volume
| Issue : 1 | Page : 9-14
Nitric oxide level around dental implant: Indicator of an inflammatory process
Farhan Durrani1, Urvashi Ojha1, VP Singh1, Royana Singh2
1 Department of Periodontics, Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Web Publication||24-May-2012|
Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The purpose of the present study is to determine the measure of inflammation of nitric oxide (NO) in healthy dental implants and diseased implants.
Materials and Methods: A total 102 subjects constituting of two groups: Tooth and dental implant sites were selected, constituting of healthy and inflamed subgroup. NO levels were spectrophotometrically determined. For comparison Chi square and paired t-test analysis was done. The correlation between NO levels and clinical inflammatory status were analyzed with Spearman's correlation coefficient.
Results: Gradual increase in the GCF and PISF volume was exhibited in the inflamed tooth as well implants. The total NO level was significant higher in the inflamed sites (P<0.05) than the non-inflamed sites in both tooth and the implant sites.
Conclusion: The PISF (peri-implant sulcus fluid) may have a diagnostic potential for reflecting the biological changes around load-bearing endosseous dental implants.
Keywords: Dental implants, inflammation, natural teeth, nitric oxide
|How to cite this article:|
Durrani F, Ojha U, Singh V P, Singh R. Nitric oxide level around dental implant: Indicator of an inflammatory process. J Dent Implant 2012;2:9-14
|How to cite this URL:|
Durrani F, Ojha U, Singh V P, Singh R. Nitric oxide level around dental implant: Indicator of an inflammatory process. J Dent Implant [serial online] 2012 [cited 2021 Oct 21];2:9-14. Available from: https://www.jdionline.org/text.asp?2012/2/1/9/96557
| Introduction|| |
Dental implants have been remarkably improved in the past half century. When good-quality dental implants and techniques are used, implants show similar cumulative success rates at 10 years after implantation. The 10-year cumulative success rates are about 88% for maxillary and 93% for mandibular implants  but some implants are lost as a result of primary failure or by loosening, a mode of failure resulting from implant movement or migration in the bone.  Unfortunately, these patients, who are mostly about 60 years old, are still quite young in terms of current human longevity.  As a result of failure or loosening of the implants and the longevity of the patients, some patients would later need to be re-implanted. But often the possibilities for revision operations are limited, due to general and local circumstances.  The high and increasing number of dental implantations performed  is evidence that loosening of dental implants is a common complication affecting thousands of patients annually worldwide.
More recently, criteria for implants were approved by The American Academy of Periodontology in 2000.  These include: "1) absence of persistent signs/symptoms such as pain, infection, neuropathies, paresthesias, and violation of vital structures; 2) implant immobility; 3) no continuous peri-implant radiolucency; 4) negligible progressive bone loss (less than 0.2 mm annually) after physiologic remodeling during the first year of function; 5) patient/dentist satisfaction with the implant-supported restoration".  Thus the control of etiologic agents that cause onset of peri-implant tissue inflammation plays a critical role in long-term success of endosseous dental implants. Periodontally compromised patients (PCP) present a potentially higher risk for implant failure. The reason for this is that period following such initial peri-implant inflammation, which generally resembles the reversible plaque-related tissue reactions around natural tooth, can induce irreversible destruction of peri-implant tissues. Dental implant studies have demonstrated that any loss of integrity of biological seal of peri-implant tissues is an important factor that may trigger peri-implant soft tissue inflammation and alveolar bone loss (peri-implantitis), which may result in implant failure.
Early recognition of any peri-implant pathology including peri-implant soft tissue inflammation is vital for long-term proper functioning of dental implants.The development of simple and reliable diagnostic tools for early detection of initial peri-implant inflammatory process and prevention of any irreversible host reactions, such as destructive peri-implant disease, may be of particular importance. There is a large body of evidence that NO is involved in several inflammatory disorders. Indeed, virtually every cell and many immunological parameters are modulated by NO. It has been shown that NO can be both pro-inflammatory (immune-stimulatory, anti-apoptotic) or anti-inflammatory (immunosuppressive, pro-apoptotic), host-protective or host damaging during infections. For these reasons NO has been described as "double-edge sword mediator" and this phenomenon is often referred to as the NO paradox.
Menaka, et al.,  revealed an increase in NO expression in periodontitis and demonstrated that enhanced formation of NO played a significant role in the pathogenesis of periodontitis. Also that the deactivation of NO by activated PMNs as one of the pathomechanisms of disorders in periodontitis. 
Thus, the present status of the role of NO in periodontal disease is not clearly defined and this study was therefore undertaken to reveal the involvement of NO in periodontal disease and to see the probability of use of NO estimation as reliable diagnostic tool for early detection of initial peri-implant inflammatory process and, henceforth, chances of implant failure.
| Materials and Methods|| |
Patients and samples
Patient participation in this study did not endanger their health. They underwent procedures deemed necessary for their oral health. Participation did not involve them in any extra dental procedures. Participation was voluntary. All patients gave their informed consent. The project was approved by the local ethics committee of the participating institutes.
Gingival crevicular fluid (GCF) sample is an accepted method for evaluation of clinical periodontal status of the natural dentition and for better understanding of pathogenesis of periodontal disease.
Inclusion criteria and selection of participants
The patients were required to be healthy with no allergies or metabolic bone diseases;
No history of antibiotic prior 3 months.
Patients with dental implants and natural teeth preferred in order to minimize the inter-patient variation. However, patients with only implants or teeth were not excluded;
All the patient who were included in the study in clinical periodontal health should have one site of each of the following:
- Healthy site
- Dental implant restoration need to be in function for at least six months and
- Patients attending faculty for periodontal care and implant maintenance
Determination of the clinical status of the soft Tissue
Clinical status of peri-implant soft tissues and clinical periodontal status of natural tooth sites were evaluated by assessing the probing depth (PD), Plaque Index (PI) score, Gingival Index (GI) score, and the Gingival Bleeding Time Index (GBTI). These measurements were used to assess the presence/extent of both periodontal and peri-implant inflammatory destruction. All measurements were performed at four sites around each implant and natural tooth (mesial, distal, buccal, and lingual) and were carried out to the nearest mm using a Michigan 'O' probe. To avoid any volumetric disturbance, all of the clinical measurements were recorded after PISF and GCF sampling.
PISF/GCF sampling was performed at the dental implant and natural tooth sites as guided by Rüdin et al., Chapple IL, Atici K et al., and Tözüm TF et al. PISF/GCF samples were obtained according to the method described by Rüdin et al. using standardized paper strips (Periopaper, no. 593525; Ora Flow, Amityville, NY). Briefly following the isolation of the sampling area with sterile cotton rolls, supragingival plaque was removed, and the site was gently air-dried to reduce any contamination with plaque and saliva. Care was taken to minimize the level of mechanical irritation. Paper strips were placed at the entrance of the peri-implant sulcus or natural tooth crevice and were inserted to a standardized depth of 1 mm at each site for 30 s. Samples with evidence of gingival bleeding were not included. To eliminate the risk of evaporation, paper strips were immediately transported for electronic volume determination. PISF/GCF was measured electronically. The PISF/GCF samples were then placed in sterile, wrapped Eppendorf tubes and stored at -20°C until the day of laboratory analysis.
Determination of nitrite Level of PISF/GCF
To each PISF/GCF sample in the Eppendorf tube, 300 μL extraction buffer (10 mmol/L phosphate buffer containing 0.5%,hexadecyltrimethylammonium bromide [HETAB], ph 6.0), was added, and the samples were vigorously mixed for the extraction of nitrite into the buffer. For the determination of nitrite levels, 150 μL of the extract was mixed with 150 μL of freshly prepared Greiss reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% hydrochloric acid) using a microplate. After 10 min of incubation at room temperature, the absorbance of each sample in microplate wells was determined at 540 nm. , A standard curve was prepared using sodium nitrite to calculate nitrite concentration in PISF/GCF.
Formula for measurement of nitrite level
Nitrite concentration in samples was calculated by relating the net optical density of the sample to nitrite concentration in standard curve in the following way:
| Results|| |
A total of 107 sites in 21 subjects were included in the present cross-sectional study. Of these, 21 patients (with a mean age of 44 years), 10 men and 11 women, had been treated with screw-type endosseous dental implants. Ten of 21 patients had both dental implants and natural teeth; 51 peri-implant and periodontal sites in these patients were measured. Eleven subjects had only either dental implants or natural teeth; 58 sites in these patients were measured. Of the sites, 42 were dental implant sites, while 67 were natural tooth sites with clinical health or some state of inflammation. The descriptive statistical analysis and actual P values are provided in [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6] and [Table 7]. The impact of severity of inflammation on clinical measures, together with descriptive statistical analysis and actual P values, is shown in [Table 8] and [Table 9].
|Table 1: Analysis of plaque index of natural teeth and dental implants grouped by state of inflammation|
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|Table 2: Analysis of probing depth of natural teeth and dental implants grouped by state of inflammation|
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|Table 3: Analysis of gingival bleeding time index of natural teeth and dental implants grouped by state of inflammation|
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|Table 4: Analysis of gingival crevicular fluid/peri-implant sulcus fluid index of natural teeth and dental implants respectively, grouped by state of inflammation|
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|Table 5: Analysis of nitrite level of GCF/PISF of natural teeth and dental implants grouped by state of inflammation|
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|Table 6: Analysis of total nitrite concentration of natural teeth and dental implants grouped by state of inflammation|
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|Table 7: Analysis of plaque index and probing index of natural teeth and dental implants grouped by state of inflammation|
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|Table 8: Analysis of plaque index of natural teeth and dental implants grouped by state of inflammation|
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|Table 9: Analysis of nitrite level and total nitrite concentration of natural teeth and dental implants grouped by state of inflammation|
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For both of the laboratory measures, total and concentration modes of data presentation did not match and presented different trends. While in GCF the total nitrite levels stayed quite stable in all clinical circumstances (periodontal health: 0.050 nmol; gingivitis: 0.053 nmol; periodontitis: 0.052 nmol; P>0.05), GCF nitrite concentration clearly presented a different pattern (periodontal health: 0.433 nmol/μL; gingivitis: 0.159 nmol/μL; periodontitis: 0.051 nmol/μL; P=0.0001). Where dental implant sites were concerned, the PISF total nitrite (P=0.001) was significantly higher. While no difference was observed in any of the laboratory parameters between inflamed GCF and PISF samples, total nitrite level demonstrated a pattern of increase in GCF from healthy natural tooth sites compared to PISF from non-inflamed implant sites. Despite a trend of increase in clinical parameters and the severity of inflammation at moderately/severely inflamed sites, GCF total nitrite levels stayed stable and were not influenced by the severity of clinical inflammation. At dental implant sites, higher PI and GBTI scores were observed at moderately/severely inflamed sites than both the clinically non-inflamed sites (P = 0.0001). Total nitrite levels presented a trend of increase in the inflamed sites compared with the clinically non-inflamed dental implant sites, while slightly inflamed sites demonstrated a significantly increased total nitrite level compared to non-inflamed peri-implant sites (P = 0.004). However, no significant difference was observed between slightly inflamed and moderately/severely inflamed sites (P>0.05). However, no correlation was found for total nitrite level and nitrite concentration at natural tooth and dental implant sites.
| Discussion|| |
In addition to the examination of clinical parameters, quantitative and qualitative analysis of GCF samples is an accepted method for the evaluation of early and destructive stages of inflammatory periodontal diseases around natural teeth, while PISF analysis may serve for further clarification of the biologic mechanisms around dental implants and the pathophysiology of peri-implant diseases ,,,,,,, There is the possibility that these biological fluids may share much in terms of their diagnostic potential and their involvement in the pathogenesis of periodontal or peri-implant disorders. ,,,,,,, The volumetric findings of the present study support the idea of such a similarity; both the GCF and PISF volumes exhibited clear increases at diseased sites compared to clinically healthy sites, in addition to the similarities between the sites with respect to clinical peri-implant or periodontal parameters. Further, despite the higher PISF volume at non-inflamed sites, no significant difference between PISF and GCF volume could be observed at inflamed sites. Thus, it seems that that PISF and GCF share similar volumetric features in their local response to existing clinical inflammation. It is clear that molecular changes around natural teeth and/or load-bearing dental implants are complex, and a close relationship has been demonstrated between inflammatory status and the molecular pathophysiology. ,,, This, in fact, seems to be the main reason for the great interest in GCF and PISF and their potential changes with inflammation. ,,,,,, The lack of significant correlations between certain laboratory measures and clinical parameters has been attributed to the lack of a correlation between time of GCF profiling and clinical status. ,, The relatively "early" nature of such laboratory measures (e.g., enzymatic changes in GCF profile), which precede clinically detectable changes, has also been suggested. , Thus, analysis of various laboratory-based parameters (e.g., biochemical measures), rather than clinical parameters, may serve for the development of reliable diagnostic tests for early detection of periodontal inflammation. ,, In addition to these, the significant limitations of most clinical parameters (e.g., having subjective elements in reflecting only past events, providing limited information on actual disease status, the potential for missing early signs of disease, lack of 100% specificity and sensitivity, having limited prognostic value) continue to give rise to laboratory-based studies that aim to overcome these limitations. ,
NO has been considered an important molecular signal in a wide variety of tissues and may play a significant role as a cytotoxic mediator of the nonspecific immune response, with beneficial and harmful effects. ,, Inducible NO synthase (inos) is expressed in response to inflammatory stimuli, resulting in higher amounts of NO production, and much of the share and the diagnostic potential of PISF, the results should be interpreted with caution due to the limited number of samples analyzed. Further studies to evaluate and compare the components of PISF and GCF, especially with respect to the inflammatory process and bone metabolism, are needed to increase our understanding of the role of each component and the diagnostic potential of PISF for peri-implant pathologies as a biological fluid.
Nitrite of body fluids is formed from oxidation of NO produced by inos , Increased numbers of inos positive cells have been demonstrated in periodontally diseased tissues. ,,, Because of the reactivity of NO and its short life, direct measurement of NO from body fluids has been thought hard to perform.  Thus, nitrite, a stable end-product of NO oxidation, has been measured instead. , In the present study, using Greiss reactions, increased NO metabolism with peri-implant inflammation, represented by higher PISF nitrite levels at inflamed dental implant sites, compared to healthy ones was observed in patients with edentulous mandibles rehabilitated with overdentures with ball attachments supported by implants.  The finding of an increase in PISF NO metabolism at inflamed sites is in line with the previously reported results of a comparative analysis of PISF nitrite levels at inflamed and non-inflamed peri-implant sites in subjects with implant-supported fixed prostheses  Based on the previously demonstrated discrepancy between "concentration" and "total activity" modes of data presentation for GCF , in the present study, it was not surprising to observe that these two modes of data presentation for GCF were not completely correlated. The same discrepancy was also observed for PISF samples. As concentration expression is affected by the available PISF or GCF volume in a given site, it may be suggested that GCF and PISF share similar volumetric features with respect to the appropriate mode of data presentation, ,, NO metabolism may be affected by force and loading. , Thus, besides the inflammatory process, PISF nitrite levels may also be affected by the loading of dental implants. It is possible that the design of the implant-supported prosthesis (e.g., a complete mandibular prosthesis supported by a ball attachment) may affect NO production at dental implant sites and the subsequent PISF nitrite levels. In the present study, two biological fluids, PISF and GCF, were comparatively analyzed. Although the results may shed light on the features of the two fluids, share and the diagnostic potential of PISF, the results should be interpreted with caution due to the limited number of samples analyzed. Further studies to evaluate and compare the components of PISF and GCF, especially with respect to the inflammatory process and bone metabolism, are needed to increase our understanding of the role of each component and the diagnostic potential of PISF for peri-implant pathologies as a biological fluid.
| Conclusions|| |
The findings of the present study support the contribution of NO metabolism to the inflammatory process around both natural teeth and dental implants. PISF appears to have diagnostic potential for the discrimination between peri-implant health/disease and for a better understanding of the peri-implant biological mechanisms on a molecular level.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]