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Human interleukin-1β and interleukin-1 receptor antagonist secretion and velocity of tooth movement
Archives of Oral Biology 46 (2001) 185 – 189 www.elsevier.com/locate/archoralbio
Short communication
Human interleukin-1b and interleukin-1 receptor antagonist secretion and velocity of tooth movement L.R. Iwasaki a,*, J.E. Haack b, J.C. Nickel a,c, R.A. Reinhardt c,d, T.M. Petro c a
Department of Growth and De6elopment, College of Dentistry, Uni6ersity of Nebraska Medical Center, P.O. Box 830740, Lincoln, NE 68583 -0755, USA b USAF, 3rd Dental Squadron, 5955 Zeamer A6enue, Elmendorf AFB, AK 99506, USA c Department of Oral Biology, College of Dentistry, Uni6ersity of Nebraska Medical Center, P.O. Box 830740, Lincoln, NE 68583 -0755, USA d Department of Surgical Specialities, College of Dentistry, Uni6ersity of Nebraska Medical Center, P.O. Box 830740, Lincoln, NE 68583 -0755, USA Accepted 1 July 2000
Abstract The cytokines interleukin-1b (IL-1b) and IL-1 receptor antagonist (IL-1RA) probably play a part in orthodontic tooth movement. Here, the force magnitudes and the area of force application in the compressed periodontal ligament (PDL) were controlled and the velocity of tooth movement correlated with concentrations of IL-1b and IL-1RA in the gingival crevicular fluid (GCF). Seven individuals undergoing orthodontic treatment involving maxillary first premolar extractions and distal movement (bodily retraction) of the maxillary canines participated in the 84-day study. For each participant, continuous retraction forces were applied so that they received equivalent PDL stresses of 13 kPa for one canine and 4 kPa for the other. GCF cytokine concentrations from experimental and control teeth were expressed relative to total protein in the GCF and compared using an ‘Activity Index’ (AI) = Experimental (IL-1b/IL-1RA)/Control (IL-1b/IL-1RA). The results showed that the velocity of tooth movement in an individual was related to their AI. The correlation between AI and tooth movement was stronger from the distal (Rd = 0.78) than from the mesial (Rm =0.65) of retracted teeth. The results demonstrate that equivalent force systems produce individual differences in cytokine production, which correlate with interindividual differences in the velocity of canine retraction. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cytokines; Tooth movement; IL-1b; IL-1RA; Gingival crevicular fluid
Biologically active substances, such as cytokines and enzymes, are expressed by cells within the periodontium in response to mechanical stress from orthodontic apAbbre6iations: IL, interleukin; RA, receptor antagonist. * Corresponding author. Tel.: + 1-402-4721303; fax: + 1402-4725290. E-mail address: [email protected] (L.R. Iwasaki).
pliances. The overall objective of many investigations has been to understand better the mechanisms for converting physical stress to the cellular responses resulting in tooth movement (Sandy et al., 1993; Hill, 1998). In order to monitor the expression of biologically active substances non-invasively in humans, changes in the composition of gingival crevicular fluid (GCF) dur-
0003-9969/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 0 0 ) 0 0 0 8 8 - 1
186
L.R. Iwasaki et al. / Archi6es of Oral Biology 46 (2001) 185–189
ing orthodontic (Last et al., 1988; Sorsa et al., 1992; Samuels et al., 1993; Grieve et al., 1994; Insoft et al., 1996; Uematsu et al., 1996a,b) and orthopedic tooth movement (Tzannetou et al., 1999) have been studied. It was assumed that substances involved in bone remodeling were produced by periodontal ligament (PDL) cells in sufficient quantities to diffuse into the crevicular fluid. The main limitations of in vivo studies of cytokine production during tooth movement have been the lack of stress-distribution control in the PDL, the short time over which samples of crevicular fluid were taken, and the lack of three-dimensional quantification of tooth movement. Our purpose now was to test the hypothesis that under equivalent stress conditions in the PDL, when bodily tooth movement occurs, the tooth movement velocity for an individual is related to the concentrations in their gingival crevicular fluid of IL-1b and IL-1RA. Interleukin-1 (IL-1) comes in two forms, IL1a and IL-1b. Interleukin-1a (IL-1a) is primarily cellassociated except in severe disease conditions, and is not expected to be found commonly in the circulation or body fluids (Lonnemann et al., 1989). Interleukin-1b (IL-1b) is more potent for bone resorption and the inhibition of bone formation (Stashenko et al., 1989), and its role in orthodontic tooth movement has been the focus of previous studies (Saito et al., 1991; Grieve et al., 1994; Shimizu et al., 1994; Uematsu et al., 1996a). In view of this, IL-1a was not measured. Seven healthy patients from the Graduate Orthodontic Clinic at the University of Nebraska Medical Center (UNMC) College of Dentistry volunteered to participate. The study group and methods used to produce and measure controlled tooth movement are described by Iwasaki et al. (2000). The participants’ rights were protected, and informed consent and assent were obtained according to the UNMC Institutional Review Board. The participants had good oral hygiene and treatment plans that included maxillary first premolar extractions for the distal retraction of maxillary canines. Each volunteer was scheduled for nine appointments, starting at days 0, 1, and 3, and then as close as possible to 14-day intervals until day 84, the last day of the study. One week before day 0, each individual was fitted with maxillary ‘anchorage’ appliances and started on twice-daily chlorhexidine mouth-rinses. At each appointment, beginning on day 0, fluid was sampled from the mesial and distal gingival crevice of each experimental maxillary canine, and the gingival crevice of a control site (at or near a mandibular canine), oral hygiene was evaluated with a modified Gingival Index (Lobene et al., 1986), and an oral prophylaxis was performed. Each experimental and control tooth was washed gently with water, isolated with cotton rolls, and then dried gently with air. Sterile paper strips
(Periopaper; Proflow Inc., Amityville, NY, USA) were inserted into each gingival crevice for 30 s and then removed. After 1 min, a second strip was placed at each of the same sites for another 30 s. The paper strips were sealed in polypropylene containers (ClickSeal tubes; National Scientific Supply Co., San Marcos, CA, USA) and stored at − 70°C. Crevicular fluid samples from each site, for the same individual at the same appointment, were thawed and combined to produce approximately 0.8 ml of fluid. The combined samples were then eluted into 400 ml of phosphate-buffered solution and agitated on an orbital shaker for 1 h. From the original dilution, specific amounts were pipetted and assayed in duplicate to determine concentrations of total protein, IL-1b and IL-1RA. The concentrations of IL-1b and IL-1RA in the fluid were expressed in terms of total protein concentration. To test the sensitivity of the method, total protein concentrations of two 40-ml samples of the original elution of crevicular fluid were quantified using standard absorbance spectrophotometric techniques. IL-1b and IL-1RA concentrations were quantified with commercially available enzyme-linked immunosorbent assay kits (Cayman Chemical, Ann Arbor, MI and R&D Systems, Inc., Minneapolis, MN, USA) according to manufacturers’ instructions. None of the participants had signs of gingival inflammation during the study; therefore, Gingival Index scores remained zero and equal to baseline measures. The mean concentration (and standard error) for total protein was 111 (13.7) mg/l, for IL-1b was 634 (35.1) ng/g protein, and for IL-1RA was 173 (24.2) mg/g protein. Cytokine concentrations were compared using a modified IL-1 Activity Index (AI; Ishihara et al., 1997) defined by the equation, AI = Experimental (IL1b/IL-1RA)/Control (IL-1b/IL-1RA). The strength of the correlation between velocity of distal canine movement and the AI was measured with a Pearson product – moment correlation coefficient. Because the potential for error was high due to the small number of experimental teeth, a method of filtering extreme values from small samples was applied (Dixon and Massey, 1969). Like other reports (Grieve et al., 1994; Uematsu et al., 1996a,b), our results showed that IL-1b concentration spiked relative to that at control sites within 3 days after loading, and then dropped to baseline by 14 – 28 days (Fig. 1A). The concentrations of IL-1b then showed a periodicity of approximately 28 days. Interleukin-1 receptor antagonist (IL-1RA) also showed peaked concentrations within 3 days after loading, but unlike IL-1b, no periodicity was evident (Fig. 1B). Ratios of experimental to control protein were mostly near unity (Fig. 1C). The exception was at day 70, when the average ratio of experimental to control
L.R. Iwasaki et al. / Archi6es of Oral Biology 46 (2001) 185–189
protein was 0.45. Overall means and standard errors are listed in Table 1. Details of the resultant tooth movement are presented elsewhere (Iwasaki et al., 2000). In general, the mechanics applied produced canine retraction with velocities averaging 1.27 and 0.87 mm per month for 13 and 4 kPa of stress, respectively, with minimal unwanted linear or angular tooth movement. A leastsquare regression indicated that there was a correlation between velocity and AI. The Pearson product–moment correlation coefficient was stronger for velocity and AI from distal sites (Rd =0.78) than from mesial sites (Rm =0.65).
187
It was expected that AI ratios in GCF from sites of compressive stresses (distal experimental sites; area of net bone resorption) would be correlated with tooth velocity. Correlation of the ratios in fluid from sites of tensile stresses (mesial experimental sites; area of net bone formation) may reflect the reported role of IL-1 in the coupling of bone resorption with formation during bone turnover (Linkhart and MacCharles, 1992; Williams et al., 1992). The least-square fit lines did not intersect the ordinate axis at an AI ratio of unity, suggesting further that other factors were involved in the process of bony remodeling for controlled tooth movement. Clearly, the AI ratio applied in this study is
Fig. 1. (A – C) Mean experimental/control concentration ratios for gingival crevicular fluid (GCF) from all seven participants for each of the four experimental sample sites: higher stress distal ( — — ) and mesial ( — — ), lower stress distal (--- ---) and mesial (------) over time (days) for (A) IL-1b; (B) IL-1RA and (C) protein. (D) Average tooth velocity (mm/day) over 84 days versus the Activity Index for GCF samples from the distal sites (") and the mesial sites (2) of each maxillary canine for each participant, after filtering values with an alpha coefficient of 0.05 (Dixon and Massey, 1969). The best-fit lines and correlation coefficients for the distal site data ( — , Rd) and mesial site data (---, Rm) are shown. Table 1 Mean IL-1b, IL-1RA, and protein concentrations and standard errors in gingival crevicular fluid (GCF) for the 84-day study Sites
IL-1b (ng/g protein) IL-1RA (mg/g protein) Protein (mg/l GCF) a
Significantly Significantly c Significantly d Significantly b
greater greater greater greater
than than than than
Distal
Mesial
Control
Higher stress (13 kPa)
Lower stress (4 kPa)
17139 178 8259 48b 559 3.8
1934 9 222a 7029 41 55 93.1
1423 9204 651 950 67 96.4
1970 9217a 752 9 43c 55 93.6d
1685 9187 775 9 48c 53 9 3.2
IL-1b control-site concentration (PB0.01). IL-1RA mesial and control-site concentrations (PB0.01). IL-1RA control-site concentration (PB0.01). protein control-site concentrations (PB0.05).
188
L.R. Iwasaki et al. / Archi6es of Oral Biology 46 (2001) 185–189
too simple, as the R 2 coefficients indicate that, at best, 61% of the tooth velocity is accounted for by the distal AI ratio. The results suggest the need for further modifications to the AI that will reflect the effects of concentrations of several other candidate factors involved in bony remodeling, rather than just IL-1b and IL-1RA (Tai et al., 1997; Hill, 1998; Lacey et al., 1998). We believe the current study is unique in that quantified stresses of low magnitude were used to investigate human gingival cytokine secretion in relation to tooth velocity. When well-controlled, continuous, and equivalent stress systems were applied to human canines, individual-dependent differences in IL-1b/IL1RA ratios in crevicular fluid were found. These cytokine differences correlated with interindividual differences in the amount of tooth translation during the 84-day study.
Acknowledgements This investigation was funded in part by a Biomedical Research Award from the American Association of Orthodontists Foundation and by the Seed Grant Program of the UNMC College of Dentistry. We gratefully acknowledge K. Theesen, Illustrator, Learning Resources, UNMC College of Dentistry, for his assistance with the figures.
References Dixon, W.J., Massey, F.J., 1969. Introduction to Statistical Analysis, third ed. McGraw-Hill, New York. Grieve, W.G., Johnson, G.K., Moore, R.N., Reinhardt, R.A., DuBois, L.M., 1994. Prostaglandin E (PGE) and interleukin-1b (IL-1b) levels in gingival crevicular fluid during human orthodontic tooth movement. Am. J. Orthodontics Dentofacial Orthop. 105, 369–374. Hill, P.A., 1998. Bone remodelling. Br. J. Orthodontics 25, 101 – 107. Insoft, M., King, G.J., Keeling, S.D., 1996. The measurement of acid and alkaline phosphatase in gingival crevicular fluid during orthodontic tooth movement. Am. J. Orthodontics Dentofacial Orthop. 109, 287–296. Ishihara, Y., Nishihara, T., Kuroyanagi, T., Shirozu, N., Yamagishi, E., Ohguchi, M., Koide, M., Ueda, N., Amano, K., Noguchi, T., 1997. Gingival crevicular interleukin-1 and interleukin 1 receptor antagonist levels in periodontally healthy and diseased sites. J. Periodontal Res. 32, 524 – 529. Iwasaki, L.R., Haack, J.E., Nickel, J.C., Morton, J., 2000. Human tooth movement in response to continuous stress of low magnitude. Am. J. Orthodontics Dentofacial Orthoped. 117, 175 – 183.
Lacey, D.L., Timms, E., Tan, H.L., Kelley, M.J., Dunstan, C.R., Burgess, T., Elliott, R., Colombero, A., Elliott, G., Scully, S., Hsu, H., Sullivan, J., Hawkins, N., Davy, E., Capparelli, C., Eli, A., Qian, Y.X., Kaufman, S., Sarosi, I., Shalhoub, V., Senaldi, G., Guo, J., Delaney, J., Boyle, W.J., 1998. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165 – 176. Last, K.S., Donkin, C., Emberg, G., 1988. Glycosaminoglycans in human gingival crevicular fluid during orthodontic movement. Arch. Oral Biol. 33, 907 – 912. Linkhart, T.A., MacCharles, D.C., 1992. Interleukin-1 stimulates release of insulin-like growth factor-I from neonatal mouse calvaria by a prostaglandin synthesis dependent mechanism. Endocrinology 131, 2297 – 2305. Lobene, R.R., Weatherford, T., Ross, N.M., Lamm, R.A., Menaker, L., 1986. A modified gingival index for use in clinical trials. Clin. Prev. Dentistry 8, 3 – 6. Lonnemann, G., Endres, S., Van der Meer, J.W., Cannon, J.G., Koch, K.M., Dinarello, C.A., 1989. Differences in the synthesis and kinetics of release of interleukin 1 a, interleukin 1 b and tumor necrosis factor from human mononuclear cells. Eur. J. Immunol. 19, 1531 – 1536. Saito, M., Saito, S., Ngan, P.W., Shanfeld, J., Davidovitch, Z., 1991. Interleukin-1b and prostaglandin E are involved in the response of periodontal cells to mechanical stress in vivo and in vitro. Am. J. Orthodontics Dentofacial Orthop. 99, 226 – 240. Samuels, R.H.A., Pender, N., Last, K.S., 1993. The effects of orthodontic tooth movement on the glycosaminaglycan components of gingival crevicular fluid. J. Clin. Periodontol. 20, 371 – 377. Sandy, J.R., Farndale, R.W., Meikle, M.C., 1993. Recent advances in understanding mechanically induced bone remodeling and their relevance to orthodontic theory and practice. Am. J. Orthodontics Dentofacial Orthop. 103, 212 – 222. Shimizu, N., Yamaguchi, M., Goseki, T., Ozawa, Y., Saito, K., Takiguchi, H., Iwasawa, T., Abiko, Y., 1994. Cyclictension force stimulates interleukin-1b production by human periodontal ligament cells. J. Periodontal Res. 29, 328 – 333. Sorsa, T., Ingman, T., Mikkonen, T., Suomalainen, K., Golub, L.M., Thesleft, I., 1992. Characterization of interstitial collagenase in gingival crevicular fluid during orthodontic tooth movement in man. In: Davidovitch, Z. (Ed.), The Biological Mechanisms of Tooth Movement and Craniofacial Adaptation. The Ohio State University College of Dentistry, Columbus, OH, pp. 47 – 51. Stashenko, P., Obernesser, M.S., Dewhirst, F.E., 1989. Effect of immune cytokines on bone. Immunol. Invest. 18, 239 – 249. Tai, H., Miyaura, C., Pilbeam, C.C., Tamura, T., Ohsugi, Y., Koishihara, Y., Kubodera, N., Kawaguchi, H., Raisz, L.G., Suda, T., 1997. Transcriptional induction of cyclooxygenase-2 in osteoblasts is involved in interleukin-
L.R. Iwasaki et al. / Archi6es of Oral Biology 46 (2001) 185–189 6-induced osteoclast formation. Endocrinology 138, 2372 – 2379. Tzannetou, S., Efstratiadis, S., Nicolay, O., Grbic, J., 1999. Interleukin-1b and b-glucuronidase in gingival crevicular fluid from molars during rapid palatal expansion. Am. J. Orthodontics Dentofacial Orthop. 115, 686– 696. Uematsu, S., Mogi, M., Deguchi, T., 1996a. Interleukin (IL)1b, IL-6, tumor necrosis factor-a, epidermal growth factor, and b2 -microglobulin levels are elevated in gingi-
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val crevicular fluid during human orthodontic tooth movement. J. Dental Res. 75, 562 – 567. Uematsu, S., Mogi, M., Deguchi, T., 1996b. Increase of transforming growth factor-b1 in gingival crevicular fluid during human orthodontic tooth movement. Arch. Oral Biol. 41, 1091 – 1095. Williams, S.R., Jiang, Y., Cochran, D., Dorsam, G., Graves, D.T., 1992. Regulated expression of monocyte chemoattractant protein-1 in normal human osteoblastic cells. Am. J. Physiol. 263, C194 – C199.
Short communication
Human interleukin-1b and interleukin-1 receptor antagonist secretion and velocity of tooth movement L.R. Iwasaki a,*, J.E. Haack b, J.C. Nickel a,c, R.A. Reinhardt c,d, T.M. Petro c a
Department of Growth and De6elopment, College of Dentistry, Uni6ersity of Nebraska Medical Center, P.O. Box 830740, Lincoln, NE 68583 -0755, USA b USAF, 3rd Dental Squadron, 5955 Zeamer A6enue, Elmendorf AFB, AK 99506, USA c Department of Oral Biology, College of Dentistry, Uni6ersity of Nebraska Medical Center, P.O. Box 830740, Lincoln, NE 68583 -0755, USA d Department of Surgical Specialities, College of Dentistry, Uni6ersity of Nebraska Medical Center, P.O. Box 830740, Lincoln, NE 68583 -0755, USA Accepted 1 July 2000
Abstract The cytokines interleukin-1b (IL-1b) and IL-1 receptor antagonist (IL-1RA) probably play a part in orthodontic tooth movement. Here, the force magnitudes and the area of force application in the compressed periodontal ligament (PDL) were controlled and the velocity of tooth movement correlated with concentrations of IL-1b and IL-1RA in the gingival crevicular fluid (GCF). Seven individuals undergoing orthodontic treatment involving maxillary first premolar extractions and distal movement (bodily retraction) of the maxillary canines participated in the 84-day study. For each participant, continuous retraction forces were applied so that they received equivalent PDL stresses of 13 kPa for one canine and 4 kPa for the other. GCF cytokine concentrations from experimental and control teeth were expressed relative to total protein in the GCF and compared using an ‘Activity Index’ (AI) = Experimental (IL-1b/IL-1RA)/Control (IL-1b/IL-1RA). The results showed that the velocity of tooth movement in an individual was related to their AI. The correlation between AI and tooth movement was stronger from the distal (Rd = 0.78) than from the mesial (Rm =0.65) of retracted teeth. The results demonstrate that equivalent force systems produce individual differences in cytokine production, which correlate with interindividual differences in the velocity of canine retraction. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cytokines; Tooth movement; IL-1b; IL-1RA; Gingival crevicular fluid
Biologically active substances, such as cytokines and enzymes, are expressed by cells within the periodontium in response to mechanical stress from orthodontic apAbbre6iations: IL, interleukin; RA, receptor antagonist. * Corresponding author. Tel.: + 1-402-4721303; fax: + 1402-4725290. E-mail address: [email protected] (L.R. Iwasaki).
pliances. The overall objective of many investigations has been to understand better the mechanisms for converting physical stress to the cellular responses resulting in tooth movement (Sandy et al., 1993; Hill, 1998). In order to monitor the expression of biologically active substances non-invasively in humans, changes in the composition of gingival crevicular fluid (GCF) dur-
0003-9969/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 0 0 ) 0 0 0 8 8 - 1
186
L.R. Iwasaki et al. / Archi6es of Oral Biology 46 (2001) 185–189
ing orthodontic (Last et al., 1988; Sorsa et al., 1992; Samuels et al., 1993; Grieve et al., 1994; Insoft et al., 1996; Uematsu et al., 1996a,b) and orthopedic tooth movement (Tzannetou et al., 1999) have been studied. It was assumed that substances involved in bone remodeling were produced by periodontal ligament (PDL) cells in sufficient quantities to diffuse into the crevicular fluid. The main limitations of in vivo studies of cytokine production during tooth movement have been the lack of stress-distribution control in the PDL, the short time over which samples of crevicular fluid were taken, and the lack of three-dimensional quantification of tooth movement. Our purpose now was to test the hypothesis that under equivalent stress conditions in the PDL, when bodily tooth movement occurs, the tooth movement velocity for an individual is related to the concentrations in their gingival crevicular fluid of IL-1b and IL-1RA. Interleukin-1 (IL-1) comes in two forms, IL1a and IL-1b. Interleukin-1a (IL-1a) is primarily cellassociated except in severe disease conditions, and is not expected to be found commonly in the circulation or body fluids (Lonnemann et al., 1989). Interleukin-1b (IL-1b) is more potent for bone resorption and the inhibition of bone formation (Stashenko et al., 1989), and its role in orthodontic tooth movement has been the focus of previous studies (Saito et al., 1991; Grieve et al., 1994; Shimizu et al., 1994; Uematsu et al., 1996a). In view of this, IL-1a was not measured. Seven healthy patients from the Graduate Orthodontic Clinic at the University of Nebraska Medical Center (UNMC) College of Dentistry volunteered to participate. The study group and methods used to produce and measure controlled tooth movement are described by Iwasaki et al. (2000). The participants’ rights were protected, and informed consent and assent were obtained according to the UNMC Institutional Review Board. The participants had good oral hygiene and treatment plans that included maxillary first premolar extractions for the distal retraction of maxillary canines. Each volunteer was scheduled for nine appointments, starting at days 0, 1, and 3, and then as close as possible to 14-day intervals until day 84, the last day of the study. One week before day 0, each individual was fitted with maxillary ‘anchorage’ appliances and started on twice-daily chlorhexidine mouth-rinses. At each appointment, beginning on day 0, fluid was sampled from the mesial and distal gingival crevice of each experimental maxillary canine, and the gingival crevice of a control site (at or near a mandibular canine), oral hygiene was evaluated with a modified Gingival Index (Lobene et al., 1986), and an oral prophylaxis was performed. Each experimental and control tooth was washed gently with water, isolated with cotton rolls, and then dried gently with air. Sterile paper strips
(Periopaper; Proflow Inc., Amityville, NY, USA) were inserted into each gingival crevice for 30 s and then removed. After 1 min, a second strip was placed at each of the same sites for another 30 s. The paper strips were sealed in polypropylene containers (ClickSeal tubes; National Scientific Supply Co., San Marcos, CA, USA) and stored at − 70°C. Crevicular fluid samples from each site, for the same individual at the same appointment, were thawed and combined to produce approximately 0.8 ml of fluid. The combined samples were then eluted into 400 ml of phosphate-buffered solution and agitated on an orbital shaker for 1 h. From the original dilution, specific amounts were pipetted and assayed in duplicate to determine concentrations of total protein, IL-1b and IL-1RA. The concentrations of IL-1b and IL-1RA in the fluid were expressed in terms of total protein concentration. To test the sensitivity of the method, total protein concentrations of two 40-ml samples of the original elution of crevicular fluid were quantified using standard absorbance spectrophotometric techniques. IL-1b and IL-1RA concentrations were quantified with commercially available enzyme-linked immunosorbent assay kits (Cayman Chemical, Ann Arbor, MI and R&D Systems, Inc., Minneapolis, MN, USA) according to manufacturers’ instructions. None of the participants had signs of gingival inflammation during the study; therefore, Gingival Index scores remained zero and equal to baseline measures. The mean concentration (and standard error) for total protein was 111 (13.7) mg/l, for IL-1b was 634 (35.1) ng/g protein, and for IL-1RA was 173 (24.2) mg/g protein. Cytokine concentrations were compared using a modified IL-1 Activity Index (AI; Ishihara et al., 1997) defined by the equation, AI = Experimental (IL1b/IL-1RA)/Control (IL-1b/IL-1RA). The strength of the correlation between velocity of distal canine movement and the AI was measured with a Pearson product – moment correlation coefficient. Because the potential for error was high due to the small number of experimental teeth, a method of filtering extreme values from small samples was applied (Dixon and Massey, 1969). Like other reports (Grieve et al., 1994; Uematsu et al., 1996a,b), our results showed that IL-1b concentration spiked relative to that at control sites within 3 days after loading, and then dropped to baseline by 14 – 28 days (Fig. 1A). The concentrations of IL-1b then showed a periodicity of approximately 28 days. Interleukin-1 receptor antagonist (IL-1RA) also showed peaked concentrations within 3 days after loading, but unlike IL-1b, no periodicity was evident (Fig. 1B). Ratios of experimental to control protein were mostly near unity (Fig. 1C). The exception was at day 70, when the average ratio of experimental to control
L.R. Iwasaki et al. / Archi6es of Oral Biology 46 (2001) 185–189
protein was 0.45. Overall means and standard errors are listed in Table 1. Details of the resultant tooth movement are presented elsewhere (Iwasaki et al., 2000). In general, the mechanics applied produced canine retraction with velocities averaging 1.27 and 0.87 mm per month for 13 and 4 kPa of stress, respectively, with minimal unwanted linear or angular tooth movement. A leastsquare regression indicated that there was a correlation between velocity and AI. The Pearson product–moment correlation coefficient was stronger for velocity and AI from distal sites (Rd =0.78) than from mesial sites (Rm =0.65).
187
It was expected that AI ratios in GCF from sites of compressive stresses (distal experimental sites; area of net bone resorption) would be correlated with tooth velocity. Correlation of the ratios in fluid from sites of tensile stresses (mesial experimental sites; area of net bone formation) may reflect the reported role of IL-1 in the coupling of bone resorption with formation during bone turnover (Linkhart and MacCharles, 1992; Williams et al., 1992). The least-square fit lines did not intersect the ordinate axis at an AI ratio of unity, suggesting further that other factors were involved in the process of bony remodeling for controlled tooth movement. Clearly, the AI ratio applied in this study is
Fig. 1. (A – C) Mean experimental/control concentration ratios for gingival crevicular fluid (GCF) from all seven participants for each of the four experimental sample sites: higher stress distal ( — — ) and mesial ( — — ), lower stress distal (--- ---) and mesial (------) over time (days) for (A) IL-1b; (B) IL-1RA and (C) protein. (D) Average tooth velocity (mm/day) over 84 days versus the Activity Index for GCF samples from the distal sites (") and the mesial sites (2) of each maxillary canine for each participant, after filtering values with an alpha coefficient of 0.05 (Dixon and Massey, 1969). The best-fit lines and correlation coefficients for the distal site data ( — , Rd) and mesial site data (---, Rm) are shown. Table 1 Mean IL-1b, IL-1RA, and protein concentrations and standard errors in gingival crevicular fluid (GCF) for the 84-day study Sites
IL-1b (ng/g protein) IL-1RA (mg/g protein) Protein (mg/l GCF) a
Significantly Significantly c Significantly d Significantly b
greater greater greater greater
than than than than
Distal
Mesial
Control
Higher stress (13 kPa)
Lower stress (4 kPa)
17139 178 8259 48b 559 3.8
1934 9 222a 7029 41 55 93.1
1423 9204 651 950 67 96.4
1970 9217a 752 9 43c 55 93.6d
1685 9187 775 9 48c 53 9 3.2
IL-1b control-site concentration (PB0.01). IL-1RA mesial and control-site concentrations (PB0.01). IL-1RA control-site concentration (PB0.01). protein control-site concentrations (PB0.05).
188
L.R. Iwasaki et al. / Archi6es of Oral Biology 46 (2001) 185–189
too simple, as the R 2 coefficients indicate that, at best, 61% of the tooth velocity is accounted for by the distal AI ratio. The results suggest the need for further modifications to the AI that will reflect the effects of concentrations of several other candidate factors involved in bony remodeling, rather than just IL-1b and IL-1RA (Tai et al., 1997; Hill, 1998; Lacey et al., 1998). We believe the current study is unique in that quantified stresses of low magnitude were used to investigate human gingival cytokine secretion in relation to tooth velocity. When well-controlled, continuous, and equivalent stress systems were applied to human canines, individual-dependent differences in IL-1b/IL1RA ratios in crevicular fluid were found. These cytokine differences correlated with interindividual differences in the amount of tooth translation during the 84-day study.
Acknowledgements This investigation was funded in part by a Biomedical Research Award from the American Association of Orthodontists Foundation and by the Seed Grant Program of the UNMC College of Dentistry. We gratefully acknowledge K. Theesen, Illustrator, Learning Resources, UNMC College of Dentistry, for his assistance with the figures.
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