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Neutrophil elastase activity and concentrations of interleukin1-β in crevicular fluid after immediate replacement and immediate loading of implants
Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery 48 (2010) 228–231
Short communication
Neutrophil elastase activity and concentrations of interleukin1- in crevicular fluid after immediate replacement and immediate loading of implants Reinhard Gruber a,∗ , Jihan Nadir b , Robert Haas b a b
Department of Oral Surgery, Medical University of Vienna, Währingerstrasse 25a, 1090 Vienna, Austria Academy of Oral Implantology, Lazarettgasse 19, 1090 Wien, Austria
Accepted 15 October 2009 Available online 12 November 2009
Abstract Our objective was to calculate the amounts of neutrophil elastase and interleukin-1 (IL-1) in the crevicular fluid of dental implants that were placed and restored immediately after extraction of teeth. The crevicular fluid of 11 patients was obtained before the teeth were extracted and during the early (days 1–10) and late (day 10 onwards) follow-up periods. Samples were analysed by kinetic assay for neutrophil elastase and by immunoassay of IL-1. The absolute values remained unchanged during the early (p = 0.15; p = 0.2) and late (p = 0.4; p = 0.5) follow-up periods. Paired analysis showed that the absolute values in the periodontal crevicular fluid were similar compared with the corresponding samples obtained during the early (p = 0.5; p = 0.3) and the late (p = 0.6; p = 0.2) follow-up periods. These findings suggest that placement of implants according to the immediate loading protocol, although it is an invasive procedure, does not provoke an inflammatory reaction. © 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Immediate implantation; Dental implants; Crevicular fluid; Neutrophil elastase; Interleukin-1
Introduction Dental implants can be placed and restored immediately after the teeth have been extracted. Cellular events during the early phase of loading are only beginning to be discovered.1 During the inflammatory phase, neutrophil granulocytes immigrate into the blood clot, followed by macrophages. It is not until granulation tissue has developed that bone starts to form.2 Transient inflammation is necessary for bony regeneration3 whereas chronic inflammation causes destruction of periodontal and periimplant tissue.4 Analysis of the presence of inflammatory mediators in the crevicular fluid can therefore provide
∗
Corresponding author. Tel.: +43 1 4277 67011; fax: +43 1 4277 67019. E-mail address: [email protected] (R. Gruber).
insights into the cellular reaction after placement of implants. Activated neutrophils release the catabolic enzyme neutrophil elastase, the measurement of which in the crevicular fluid can help to distinguish healthy from inflamed periimplant and periodontal tissue.5,6 Monocytes are among the main sources of the inflammatory cytokine IL-1.7 Periimplantitis is associated with increased concentrations of IL-1.8 It was only recently that early peri-implant wound healing was followed for one week by measuring IL-1.9 Neutrophil elastase and IL-1 have not to our knowledge been analysed during the later stages of osseointegration. The aim of the present study was to assess possible changes of activity of neutrophil elastase and concentrations of IL1 in the crevicular fluid that happen as a consequence of teeth being replaced by dental implants that are subjected to loading.
0266-4356/$ – see front matter © 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2009.10.014
R. Gruber et al. / British Journal of Oral and Maxillofacial Surgery 48 (2010) 228–231
Patients and methods Patients were treated by surgery and prosthetic restoration. The ethics committee approval was number 195/2005. Implants (NobelreplaceTM Tapered Groovy, Nobel Biocare, Gothenburg, Sweden) were placed immediately after the teeth had been extracted without raising a soft tissue flap. Teeth were removed with a periotome technique (PeriotomeTM , Nobel Biocare, Gothenburg, Sweden). The teeth were extracted because of periodontal or endodontic failure. Once an insertion torque of more than 35 N/cm was obtained, the tooth was restored with a resin abutment and crown within an hour. The resin abutment was copied to receive a ceramic abutment of the same shape as the emergency profile of the extracted tooth. Ceramic abutments were tightened with 20 N/cm 2 days after operation. Provisional
229
crowns allowed the patient to eat normally. None of the patients were given antibiotics. A total of 204 samples were obtained from 11 patients (6 men, 5 women); 64 samples before extraction and 140 samples during recall visits: the first (day 1–10) and the second (day 10 onwards). Peri-implant and periodontal crevicular fluid were collected by the intracrevicular method.10 The surfaces of implants were air-dried and isolated with cotton rolls. Paper strips (Periopaper, ProFlow, Amittyville, NY) were inserted into the base of the pocket of the implant for 30 s. Samples were collected from the lingual and the buccal aspects of each implant and tooth before extraction. The adsorbed volume was calculated by impedance measurement based on a calibration curve (Periotron 8000, Oralflow Inc., Plainview, NY). Samples with a calculated volume of below 0.15 l were considered zero in the analysis. Filter
Fig. 1. Time response of matched-paired samples of crevicular fluid before extraction and after early and later type-I loading of implants. Box and whisker plots of matched-pair samples of crevicular fluid taken before extraction and during the recall visits. Absolute amounts of IL-1 and NPE (amount/filter strip), concentrations of IL-1, and activity of neutrophil elastase (amount/filter strip normalised to the adsorbed volume of crevicular fluid) were recorded. There was no significant difference between absolute amounts, activity of neutrophil elastase, and concentration of IL-1 in crevicular fluid obtained before extraction, during the early period after insertion of implants, or the later period.
230
R. Gruber et al. / British Journal of Oral and Maxillofacial Surgery 48 (2010) 228–231
strips were placed in 1.5 ml plastic tubes containing 400 l of phosphate-buffered saline and stored frozen. After they had been thawed the concentration of IL-1 was measured with an immunoassay (#DLB50; R&D Systems, Inc., Minneapolis, MN). The detection limit was typically below IL-1, 1 pg/ml. Neutrophil elastase activity was measured with the synthetic substrate Suc-Ala-Ala-Pro-Val p-nitroaniline (Sigma, St. Louis, MO). The amount of released p-nitroaniline was measured with a microplate reader, which reflected the neutrophil elastase activity (mmol of the released synthetic substrate/minute). Absolute amounts of IL-1/neutrophil elastase eluted/filter strips and the concentration or activity (amount IL-1/neutrophil elastase eluted/filter strip and normalised to the adsorbed volume of crevicular fluid) were recorded. Statistical analysis is based on the means from single teeth and the significance of differences on the results of the matched-pairs t test calculated using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL). Data are expressed as mean (SD).
Results There was no significant difference between absolute activities of neutrophil elastase and concentrations of IL-1 in crevicular fluid obtained before teeth were extracted during the early period after insertion of the implant or during the later period (Fig. 1). Crevicular fluid taken from patients before extraction had similar absolute amounts and activities of neutrophil elastase [114.5 (157.3) U; 256.7 (302.7) U/l] than those retrieved within 10 days [63.2 (61.7 U), p = 0.6; 112.3 (140.1) U/l, p = 0.2] and > 10 days [100.0 (117.5) U, p = 0.4; 201.6 (242.5) U/l, p = 0.5] after insertion of the implant, respectively. The same was true when the absolute amounts and concentrations of IL-1 were compared [4.8 (6.1) U; 9.9 (13.5) U/l] with measures obtained within 10 days [2.5 (2.4) U, p = 0.5; 5.4 (6.5) U/l, p = 0.3] and within >10 days of insertion of the implant [3.2 (2.8) U, p = 0.6; 8.4 (9.6) U/l, p = 0.2].
Discussion Inflammation has both physiological and pathological functions. Activated neutrophils and macrophages participate in the early stages of osseointegration, which is their physiological function,1 and both cell types are mediators of tissue destruction in chronic inflammation (such as periodontal disease and peri-implantitis), which is their pathological function.4 We have shown here that the activity of neutrophil elastase and the concentration of IL-1 in the crevicular fluid did not change significantly between teeth before extraction, after replacement, and after immediate loading of the implants. These findings are in line with a recent study that measured concentrations of IL-1 within one week of insertion of implants in patients given antibiotics.9 Interestingly,
IL-1 was reported to increase in patients given amoxicillin, suggesting that prophylactic antibiotics do not suppress inflammation during the early stages of osseointegration.9 Together, these data suggest that there is an ongoing inflammatory reaction in periodontal and peri-implant tissue. However, they do not tell us to what extent neutrophils and macrophages contribute to tissue destruction or tissue regeneration. There were large variations in the sizes of the samples, the activity of neutrophil elastase, and the concentration of IL-1 among individual patients and between the buccal and the lingual sites. A few samples even failed to reach the least detectable volume of crevicular fluid and the least detectable amounts of neutrophil elastase and IL-1, and these findings may reflect the heterogeneity of our study group. Nevertheless, the range of the mean measurements of inflammatory markers was much greater around teeth before extraction than after implantation, which indicates the different stages of periodontal inflammation before extraction. In theory, once implants have been inserted the pathological inflammation turns into a physiological inflammation during osseointegration. We should make clear that the clinical relevance of this study is limited because we studied only 11 patients. These preliminary findings may however provide a basis for longitudinal studies to follow inflammation until a stead-state is reached. Clearly, this is a speculation but an inspiring hypothesis to further reveal the “good and bad” of inflammation in implant research.
Acknowledgement The IL-1 immunoassay was sponsored by Nobel Biocare. We thank Barbara Kandler for assaying IL-1 and neutrophil elastase.
References 1. Berglundh T, Abrahamsson I, Lang NP, Lindhe J. De novo alveolar bone formation adjacent to endosseous implants. Clin Oral Implants Res 2003;14:251–62. 2. Schenk RK, Buser D. Osseointegration: a reality. Periodontol 2000 1998;17:22–35. 3. Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O’Keefe RJ. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 2002;109:1405–15. 4. Heitz-Mayfield LJ. Peri-implant diseases: diagnosis and risk indicators. J Clin Periodontol 2008;35(8 suppl):292–304. 5. Fartash B, Hultin M, Gustafsson A, Asman B, Arvidson K. Markers of inflammation in crevicular fluid from peri-implant mucosa surrounding single crystal sapphire implants. Clin Oral Implants Res 1997;8:32–8. 6. Boutros SM, Michalowicz BS, Smith QT, Aeppli DM. Crevicular fluid enzymes from endosseous dental implants and natural teeth. Int J Oral Maxillofac Implants 1996;11:322–30. 7. Kawai T, Matsuyama T, Hosokawa Y, et al. B and T lymphocytes are the primary sources of RANKL in the bone resorptive lesion of periodontal disease. Am J Pathol 2006;169:987–98.
R. Gruber et al. / British Journal of Oral and Maxillofacial Surgery 48 (2010) 228–231 8. Kao RT, Curtis DA, Richards DW, Preble J. Increased interleukin-1 beta in the crevicular fluid of diseased implants. Int J Oral Maxillofac Implants 1995;10:696–701. 9. Khoury SB, Thomas L, Walters JD, Sheridan JF, Leblebicioglu B. Early wound healing following one-stage dental implant placement
231
with and without antibiotic prophylaxis: a pilot study. J Periodontol 2008;79:1904–12. 10. Griffiths GS. Formation, collection and significance of gingival crevice fluid. Periodontol 2000 2003;31:32–42.
British Journal of Oral and Maxillofacial Surgery 48 (2010) 228–231
Short communication
Neutrophil elastase activity and concentrations of interleukin1- in crevicular fluid after immediate replacement and immediate loading of implants Reinhard Gruber a,∗ , Jihan Nadir b , Robert Haas b a b
Department of Oral Surgery, Medical University of Vienna, Währingerstrasse 25a, 1090 Vienna, Austria Academy of Oral Implantology, Lazarettgasse 19, 1090 Wien, Austria
Accepted 15 October 2009 Available online 12 November 2009
Abstract Our objective was to calculate the amounts of neutrophil elastase and interleukin-1 (IL-1) in the crevicular fluid of dental implants that were placed and restored immediately after extraction of teeth. The crevicular fluid of 11 patients was obtained before the teeth were extracted and during the early (days 1–10) and late (day 10 onwards) follow-up periods. Samples were analysed by kinetic assay for neutrophil elastase and by immunoassay of IL-1. The absolute values remained unchanged during the early (p = 0.15; p = 0.2) and late (p = 0.4; p = 0.5) follow-up periods. Paired analysis showed that the absolute values in the periodontal crevicular fluid were similar compared with the corresponding samples obtained during the early (p = 0.5; p = 0.3) and the late (p = 0.6; p = 0.2) follow-up periods. These findings suggest that placement of implants according to the immediate loading protocol, although it is an invasive procedure, does not provoke an inflammatory reaction. © 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Immediate implantation; Dental implants; Crevicular fluid; Neutrophil elastase; Interleukin-1
Introduction Dental implants can be placed and restored immediately after the teeth have been extracted. Cellular events during the early phase of loading are only beginning to be discovered.1 During the inflammatory phase, neutrophil granulocytes immigrate into the blood clot, followed by macrophages. It is not until granulation tissue has developed that bone starts to form.2 Transient inflammation is necessary for bony regeneration3 whereas chronic inflammation causes destruction of periodontal and periimplant tissue.4 Analysis of the presence of inflammatory mediators in the crevicular fluid can therefore provide
∗
Corresponding author. Tel.: +43 1 4277 67011; fax: +43 1 4277 67019. E-mail address: [email protected] (R. Gruber).
insights into the cellular reaction after placement of implants. Activated neutrophils release the catabolic enzyme neutrophil elastase, the measurement of which in the crevicular fluid can help to distinguish healthy from inflamed periimplant and periodontal tissue.5,6 Monocytes are among the main sources of the inflammatory cytokine IL-1.7 Periimplantitis is associated with increased concentrations of IL-1.8 It was only recently that early peri-implant wound healing was followed for one week by measuring IL-1.9 Neutrophil elastase and IL-1 have not to our knowledge been analysed during the later stages of osseointegration. The aim of the present study was to assess possible changes of activity of neutrophil elastase and concentrations of IL1 in the crevicular fluid that happen as a consequence of teeth being replaced by dental implants that are subjected to loading.
0266-4356/$ – see front matter © 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2009.10.014
R. Gruber et al. / British Journal of Oral and Maxillofacial Surgery 48 (2010) 228–231
Patients and methods Patients were treated by surgery and prosthetic restoration. The ethics committee approval was number 195/2005. Implants (NobelreplaceTM Tapered Groovy, Nobel Biocare, Gothenburg, Sweden) were placed immediately after the teeth had been extracted without raising a soft tissue flap. Teeth were removed with a periotome technique (PeriotomeTM , Nobel Biocare, Gothenburg, Sweden). The teeth were extracted because of periodontal or endodontic failure. Once an insertion torque of more than 35 N/cm was obtained, the tooth was restored with a resin abutment and crown within an hour. The resin abutment was copied to receive a ceramic abutment of the same shape as the emergency profile of the extracted tooth. Ceramic abutments were tightened with 20 N/cm 2 days after operation. Provisional
229
crowns allowed the patient to eat normally. None of the patients were given antibiotics. A total of 204 samples were obtained from 11 patients (6 men, 5 women); 64 samples before extraction and 140 samples during recall visits: the first (day 1–10) and the second (day 10 onwards). Peri-implant and periodontal crevicular fluid were collected by the intracrevicular method.10 The surfaces of implants were air-dried and isolated with cotton rolls. Paper strips (Periopaper, ProFlow, Amittyville, NY) were inserted into the base of the pocket of the implant for 30 s. Samples were collected from the lingual and the buccal aspects of each implant and tooth before extraction. The adsorbed volume was calculated by impedance measurement based on a calibration curve (Periotron 8000, Oralflow Inc., Plainview, NY). Samples with a calculated volume of below 0.15 l were considered zero in the analysis. Filter
Fig. 1. Time response of matched-paired samples of crevicular fluid before extraction and after early and later type-I loading of implants. Box and whisker plots of matched-pair samples of crevicular fluid taken before extraction and during the recall visits. Absolute amounts of IL-1 and NPE (amount/filter strip), concentrations of IL-1, and activity of neutrophil elastase (amount/filter strip normalised to the adsorbed volume of crevicular fluid) were recorded. There was no significant difference between absolute amounts, activity of neutrophil elastase, and concentration of IL-1 in crevicular fluid obtained before extraction, during the early period after insertion of implants, or the later period.
230
R. Gruber et al. / British Journal of Oral and Maxillofacial Surgery 48 (2010) 228–231
strips were placed in 1.5 ml plastic tubes containing 400 l of phosphate-buffered saline and stored frozen. After they had been thawed the concentration of IL-1 was measured with an immunoassay (#DLB50; R&D Systems, Inc., Minneapolis, MN). The detection limit was typically below IL-1, 1 pg/ml. Neutrophil elastase activity was measured with the synthetic substrate Suc-Ala-Ala-Pro-Val p-nitroaniline (Sigma, St. Louis, MO). The amount of released p-nitroaniline was measured with a microplate reader, which reflected the neutrophil elastase activity (mmol of the released synthetic substrate/minute). Absolute amounts of IL-1/neutrophil elastase eluted/filter strips and the concentration or activity (amount IL-1/neutrophil elastase eluted/filter strip and normalised to the adsorbed volume of crevicular fluid) were recorded. Statistical analysis is based on the means from single teeth and the significance of differences on the results of the matched-pairs t test calculated using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL). Data are expressed as mean (SD).
Results There was no significant difference between absolute activities of neutrophil elastase and concentrations of IL-1 in crevicular fluid obtained before teeth were extracted during the early period after insertion of the implant or during the later period (Fig. 1). Crevicular fluid taken from patients before extraction had similar absolute amounts and activities of neutrophil elastase [114.5 (157.3) U; 256.7 (302.7) U/l] than those retrieved within 10 days [63.2 (61.7 U), p = 0.6; 112.3 (140.1) U/l, p = 0.2] and > 10 days [100.0 (117.5) U, p = 0.4; 201.6 (242.5) U/l, p = 0.5] after insertion of the implant, respectively. The same was true when the absolute amounts and concentrations of IL-1 were compared [4.8 (6.1) U; 9.9 (13.5) U/l] with measures obtained within 10 days [2.5 (2.4) U, p = 0.5; 5.4 (6.5) U/l, p = 0.3] and within >10 days of insertion of the implant [3.2 (2.8) U, p = 0.6; 8.4 (9.6) U/l, p = 0.2].
Discussion Inflammation has both physiological and pathological functions. Activated neutrophils and macrophages participate in the early stages of osseointegration, which is their physiological function,1 and both cell types are mediators of tissue destruction in chronic inflammation (such as periodontal disease and peri-implantitis), which is their pathological function.4 We have shown here that the activity of neutrophil elastase and the concentration of IL-1 in the crevicular fluid did not change significantly between teeth before extraction, after replacement, and after immediate loading of the implants. These findings are in line with a recent study that measured concentrations of IL-1 within one week of insertion of implants in patients given antibiotics.9 Interestingly,
IL-1 was reported to increase in patients given amoxicillin, suggesting that prophylactic antibiotics do not suppress inflammation during the early stages of osseointegration.9 Together, these data suggest that there is an ongoing inflammatory reaction in periodontal and peri-implant tissue. However, they do not tell us to what extent neutrophils and macrophages contribute to tissue destruction or tissue regeneration. There were large variations in the sizes of the samples, the activity of neutrophil elastase, and the concentration of IL-1 among individual patients and between the buccal and the lingual sites. A few samples even failed to reach the least detectable volume of crevicular fluid and the least detectable amounts of neutrophil elastase and IL-1, and these findings may reflect the heterogeneity of our study group. Nevertheless, the range of the mean measurements of inflammatory markers was much greater around teeth before extraction than after implantation, which indicates the different stages of periodontal inflammation before extraction. In theory, once implants have been inserted the pathological inflammation turns into a physiological inflammation during osseointegration. We should make clear that the clinical relevance of this study is limited because we studied only 11 patients. These preliminary findings may however provide a basis for longitudinal studies to follow inflammation until a stead-state is reached. Clearly, this is a speculation but an inspiring hypothesis to further reveal the “good and bad” of inflammation in implant research.
Acknowledgement The IL-1 immunoassay was sponsored by Nobel Biocare. We thank Barbara Kandler for assaying IL-1 and neutrophil elastase.
References 1. Berglundh T, Abrahamsson I, Lang NP, Lindhe J. De novo alveolar bone formation adjacent to endosseous implants. Clin Oral Implants Res 2003;14:251–62. 2. Schenk RK, Buser D. Osseointegration: a reality. Periodontol 2000 1998;17:22–35. 3. Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O’Keefe RJ. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 2002;109:1405–15. 4. Heitz-Mayfield LJ. Peri-implant diseases: diagnosis and risk indicators. J Clin Periodontol 2008;35(8 suppl):292–304. 5. Fartash B, Hultin M, Gustafsson A, Asman B, Arvidson K. Markers of inflammation in crevicular fluid from peri-implant mucosa surrounding single crystal sapphire implants. Clin Oral Implants Res 1997;8:32–8. 6. Boutros SM, Michalowicz BS, Smith QT, Aeppli DM. Crevicular fluid enzymes from endosseous dental implants and natural teeth. Int J Oral Maxillofac Implants 1996;11:322–30. 7. Kawai T, Matsuyama T, Hosokawa Y, et al. B and T lymphocytes are the primary sources of RANKL in the bone resorptive lesion of periodontal disease. Am J Pathol 2006;169:987–98.
R. Gruber et al. / British Journal of Oral and Maxillofacial Surgery 48 (2010) 228–231 8. Kao RT, Curtis DA, Richards DW, Preble J. Increased interleukin-1 beta in the crevicular fluid of diseased implants. Int J Oral Maxillofac Implants 1995;10:696–701. 9. Khoury SB, Thomas L, Walters JD, Sheridan JF, Leblebicioglu B. Early wound healing following one-stage dental implant placement
231
with and without antibiotic prophylaxis: a pilot study. J Periodontol 2008;79:1904–12. 10. Griffiths GS. Formation, collection and significance of gingival crevice fluid. Periodontol 2000 2003;31:32–42.