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Alkaline phosphatase activity in dental pulp of orthodontically treated teeth
ORIGINAL ARTICLE
Alkaline phosphatase activity in dental pulp of orthodontically treated teeth Giuseppe Perinetti,a Giuseppe Varvara,b Luisa Salini,c and Stefano Tetèd Chieti, Italy Introduction: The aim of this study was to examine alkaline phosphatase (ALP) activity in the dental pulp of orthodontically treated teeth. Methods: Sixteen healthy subjects (mean age 17.0 ⫾1.6 years) who required extraction of 4 first premolars for orthodontic reasons participated. One maxillary first premolar subjected to orthodontic force was the test tooth. The contralateral first premolar, bracketed but not subjected to mechanical stress, was the control tooth. After a week of treatment, the first premolars were extracted and the dental pulp removed from the teeth. ALP activity was determined spectrophotometrically and the results expressed as units/liter per milligram of pulp tissue [U/(L x mg)]. Results: ALP activity was 89 ⫾ 26 U/(L x mg) in the test teeth and 142 ⫾ 33 U/(L x mg) in the control teeth. The difference between the groups was statistically significant (P ⬍ .01). Conclusions: Orthodontic treatment can lead to significant early-phase reduction in ALP activity in human dental pulp tissue. (Am J Orthod Dentofacial Orthop 2005;128:492-6)
T
he response of dental pulp tissue to orthodontic force has been evaluated previously,1 and controversial results have been reported. Although a deleterious effect on the vitality of the teeth has been found as a result of orthodontic force,2-4 other investigations have shown that orthodontic force appears to have no significant effect on dental pulp.5-7 However, depression of tissue respiration rate,3,8 apoptosis,9 increased capillary numbers,10-12 and disturbances in blood circulation13 have all been described in dental pulp tissue from orthodontic force application to the teeth. Alkaline phosphatase (ALP) is an enzyme involved in tissue mineralization,14 and, in periodontal tissues, ALP activity has been correlated with inflammation.15 Several dental pulp cells, such as fibroblasts16 and odontoblasts,17 can synthesize and release ALP. Moreover, ALP activity in human dental pulp tissue has been shown to be up to 8 times higher in reversible pulpitis compared with healthy controls and irreversible pulpi-
tis.18 These results have demonstrated that ALP could have a role in the metabolic changes in dental pulp and other body tissues.19 To date, no studies have evaluated ALP activity in the dental pulp of teeth subjected to orthodontic force. Many previous studies have focused on only 1 dental movement— eg, intrusion20— but the orthodontist is faced with clinical conditions that involve the complex 3-dimensional movements of teeth. For this reason, Derringer et al11 and Derringer and Linden12 reported an increase in angiogenesis in dental pulp tissue using orthodontic forces exerted by a flexible archwire as a model that was responsive to 3-dimensional forces applied to the teeth. Similarly, Perinetti et al21 reported an increase in aspartate aminotransferase activity in orthodontically treated teeth compared with untreated controls. Our study was designed as a cross-sectional assessment to determine whether significant changes in ALP activity occur in dental pulp during the early phases of orthodontic treatment with fixed appliances in healthy young subjects.
a
Research fellow, Department of Oral Sciences, University G. D’Annunzio, Chieti, Italy, and Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy. b Research fellow, Department of Oral Sciences, University G. D’Annunzio, Chieti, Italy. c Clinical practice, Distretto Sanitario di Base, Unit of Dental Care, Francavilla al Mare, Chieti, Italy. d Professor, Department of Oral Sciences, University G. D’Annunzio, Chieti, Italy. Reprint requests to: Giuseppe Perinetti, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale 8/A, 66030 Santa Maria Imbaro, Chieti, Italy; e-mail, [email protected]. Submitted, February 2004; revised and accepted, July 2004. 0889-5406/$30.00 Copyright © 2005 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2004.07.042
492
MATERIAL AND METHODS
Sixteen orthodontic patients, 10 female and 6 male (age range, 15.0-19.6 years; mean, 17.0 ⫾ 1.6 years), who required extraction of the maxillary first premolars because of dental crowding participated in the study. The inclusion criteria were (1) need for fixed appliance therapy, (2) good general and periodontal health, (3) no use of anti-inflammatory drugs in the month before the study, (4) periodontal probing depth values not exceeding 3 mm in the whole dentition, and (5) no evidence of marginal periodontal bone loss after a periapical radio-
Perinetti et al 493
American Journal of Orthodontics and Dentofacial Orthopedics Volume 128, Number 4
Fig 1. Orthodontic appliance on TT.
graphic examination. During the study, the subjects were not allowed to take anti-inflammatory drugs that could affect the results. Informed consent was obtained from the patients and from the parents of those under 18 years of age before the study. The protocol was reviewed and approved by the Ethical Committee of the G. D’Annunzio University Medical Faculty. In each patient wearing the orthodontic appliance, the maxillary first premolars were the experimental teeth. A first premolar undergoing orthodontic force was used as the test tooth (TT), and its contralateral first premolar was the control tooth (CT). Both the TT and the CT were unrestored and asymptomatic, with no radiological evidence of caries or periapical radiolucency. Orthodontic brackets (MBT, 3M Unitek, Monrovia, Calif) were placed on the buccal surfaces of the incisors, canines, and premolars in the maxillary arch; the bands were bonded on the first molars. A unilateral circular orthodontic archwire, 0.018 inch (MBT, 3M Unitek), was then mounted in the same quadrant as the TT to activate the orthodontic appliance (Fig 1). The bracket on each TT was placed so that, when the archwire was placed into its slot, it exerted active forces on the TT. These forces (about 30-90 g) were measured as previously described by Grieve et al.22 The entire orthodontic appliance was placed in a single clinical session. After 7 days of treatment, the experimental teeth were extracted under local anesthesia, and the dental pulp samples were obtained. The teeth were immediately longitudinally grooved under water irrigation on the buccal and lingual surfaces with a diamond disc so as not to penetrate the root canals and then split in half with cutting pliers. Care was taken in extirpating dental pulp samples from the teeth. The samples were placed in plastic vials and immediately washed 2 or 3 times in ice-cold, heparinized, sterile saline solution to remove blood, which normally shows ALP activity.19 The samples were stored at ⫺80°C. Immediately before biochemical analysis, the spec-
imens were weighed and homogenized in 1 mL of 10 mmol/L potassium phosphate buffer, pH 7.0, containing 0.1% sodium cholate. This homogenate was centrifuged at 100,000xg for 60 minutes at 4°C, and the supernatant was recovered, diluted to a volume of 2 mL with the phosphate buffer, and used for the enzymatic activity determination. One milliliter of supernatant was taken for ALP activity determination; to it was added the substrate, with the final concentrations of 20 mmol/L p-nitrophenol phosphate, 5 mmol/L magnesium chloride, 200 mmol/L mannitol, 0.05% sodium azide, and carbonate buffer (pH 10.2 ⫾ 0.1) in a total volume of 2 mL. ALP hydrolyzes p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate. The rate of increase in absorbance was read with an ultraviolet-visible spectrophotometer at 405 nm for 4 minutes, by using the kinetic method to record the absorbance variation per minute as the p-nitrophenol formed. For each analysis, a control was used that consisted of the reagent and the phosphate buffer without the sample, and the value of absorbance variation per minute in this control was subtracted from the experimental values. By using 18.45 as the p-nitrophenol millimolar absorptivity, the absorbance was converted into enzyme activity units (1 unit ⫽ 1 micromole of p-nitrophenol released per minute at 30°C). ALP activity was expressed as units/liter per milligram of pulp tissue [U/(L x mg)]. The Statistical Package for Social Sciences (SPSS, Chicago, Ill) was used for data analysis. Each data set was tested for normality with the Shapiro-Wilks test and Q-Q normality plots; equality of variance was also tested with the Levene test and Q-Q normality plots of the residuals. Because each data set met the required criteria for parametric analyses, a paired t test was used to assess the significance of the differences in ALP activity between the experimental teeth. In addition, in each experimental group, the strength of the straightline relationship between age and enzymatic activity was tested with the Pearson r correlation coefficient. A P value less than .05 was used to reject the null hypothesis. RESULTS
ALP activity was 89 ⫾ 26 U/(L x mg) (range, 54-145 U/[L x mg]) in the TTs; enzymatic activity was 142 ⫾ 33 U/(L x mg) ([range, 78-198 U/[L x mg]) in the CTs (Fig 2). The paired t test showed that the 2 groups differed significantly in ALP activity (t15 ⫽ 5.3; P ⬍ .01). The 95% confidence interval of the differences of the mean values of ALP activities of the groups was 31-73 U/(L x mg). The Pearson r correlation coefficient demonstrated a nonsignificant straight-
494 Perinetti et al
Fig 2. ALP activity in test and control groups. ALP activities in units/liter per milligram of pulp tissue [U/(L x mg)] are presented as mean ⫾ SD (n ⫽ 15). Paired t test, P ⬍ .01.
line relationship between age and ALP activity in both TTs and CTs (P ⬎ .5). DISCUSSION
This cross-sectional study examined ALP activity in the dental pulp tissue of orthodontically treated teeth, compared with untreated teeth. The results demonstrate that a significant decrease in ALP activity (that does not correlate significantly with patient age) can be detected in the dental pulp tissue of teeth undergoing orthodontic force (Fig 2). The teeth were bonded, and a flexible archwire was mounted in the brackets (Fig 1) to maintain parity with the normal clinical situation, giving a continuous but reducing active force as tooth alignment occurred. Thus, although the forces were more difficult to standardize, this study design could accurately reproduce the clinical effects of 1 week of initial 3-dimensional archwire forces that are of direct clinical relevance, as reported in other studies.11,12,21 In several studies, dental pulp response to orthodontic force was evaluated through analysis of the mineralized tissue of the tooth, but conflicting results have been reported. An increase in the predentin width coincident with the peak of tooth movement has been described3,10; a reduction in the predentin zone with degeneration of the odontoblastic layer has also been reported.2 Subay et al7 found microscopic pulp stones in only 17% of teeth undergoing extrusive movement after 10 or 40 days of treatment. Considering that ALP has a key role in the physiological17,16 and pathological23 mineralization processes of pulp tissues, our results for the TTs are consistent with the observations that, in the early phases of orthodontic treatment, there is neither an increase in the predentin width2 nor a significant formation of pulp stones.7 However, the different experimental designs in these studies might explain the inconsistencies of the data.
American Journal of Orthodontics and Dentofacial Orthopedics October 2005
The decrease in ALP activity in the TTs might be explained by damage of the pulp cells responsive to the synthesis of this enzyme, as a consequence of applying orthodontic tools to the teeth. Apoptosis in rat dental pulp tissue of teeth under orthodontic load for 3 days to 2 weeks was evaluated by Rana et al,9 who observed that maximum apoptosis occurred after 3 days. Previous in-vivo studies4 reported a significant odontoblastic degeneration in the pulp of orthodontically treated teeth. Moreover, in an in-vivo study on human subjects, Perinetti et al21 showed a significant increase in the aspartate aminotransferase activity (an index of cell necrosis) in the pulp after 7 days of orthodontic treatment. These results are also supported by other histological evaluations2,24,25 that have correlated dental-pulp alterations with strangulation and stasis of blood flow after orthodontic force has been applied to the teeth. Such a reduction in dental pulp blood flow has been implicated as causing a decrease in oxygen availability.3,8 Because dental pulp fibroblasts16 and odontoblasts17 can produce ALP, it can also be hypothesized that the reduction in enzyme activity in the TTs should be mainly due to an alteration in these cells. However, our results do not appear to agree with other in-vitro studies, which have reported an increase in ALP activity from animal pulp cells under pathological conditions.23 An association between ALP activity and vesicular structures around degenerate and necrotic cells has been seen.23 Similarly, congested blood vessels and large vacuoles were reported by Mostafa et al4 in human dental pulp tissue after 1 week of orthodontic treatment. Conceivably, the apparent inconsistencies between our study and others might be because, under in-vivo conditions, several adjunctive processes are involved, compared with in-vitro conditions. The lack of collateral circulation in the pulp makes it one of the most sensitive tissues of the body, and orthodontic tooth movement has been described as causing a reduction in the level of oxygen and other degenerative damage. These conditions have been reported to be similar to wound healing and ischemic/hypoxic processes,12 in which angiogenic growth factors have been identified.26,27 Although no previous studies have evaluated angiogenic growth factor levels in the dental pulp of teeth undergoing mechanical stress, Derringer et al11 demonstrated an increase in the total number of pulp microvessels of orthodontically treated teeth after 5 and 10 days of treatment, and similar results have been reported in other investigations.10,12 Hence, these authors hypothesized an increase in angiogenic activity as a response to an increased level of growth factors. In this regard, it is of interest that a decrease in ALP activity has been shown to be consequent to the release
American Journal of Orthodontics and Dentofacial Orthopedics Volume 128, Number 4
of angiogenic growth factors.28-30 Among these, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor- and tumor necrosis factor-␣ have been mainly implicated in the reduction in dental pulp cell ALP activity. In particular, an inhibition of ALP activity up to 65% was shown in proliferating and stationary cell cultures when several growth factors were added.28 Hence, a combination of a degeneration of pulp cells, occurring in the earliest phases of orthodontic treatment, and an increase in angiogenic growth factors levels, which is likely to occur during the same treatment, appears to be the basis for the decrease in ALP activity in the test teeth. However, further studies that include both biochemical and histochemical/immunocytochemical analyses need to be carried out to confirm this possibility. Different pulpal reactions to orthodontic forces have been correlated with age and size of the apical foramen.5,31 These results are consistent with a relationship between the biological effects of orthodontic forces and the maturity of the tooth.32 Positive correlations between age and rate of tissue respiratory depression3 or other pulp tissue alterations5,31,32 during orthodontic treatment have been reported. We did not find a significant correlation between ALP activity and patient age in either the test or control teeth. This result, however, was not unexpected because the age range of our subjects was 15.0-19.6 years, which was more restricted than that in other studies.3 Thus, the lack of correlation between ALP activity changes in the dental pulp of orthodontically treated teeth and patient age requires further investigation. CONCLUSIONS
This cross-sectional study showed a decrease in ALP activity in dental pulp tissue during the earliest orthodontic stages. Further investigations are needed to fully understand the long-term effects of orthodontic tooth movement on ALP activity of the pulp and its possible clinical implications. We thank Dr Christopher P. Berrie for his critical appraisal of the text.
REFERENCES 1. Vandevska-Radunovic V. Neural modulation of inflammatory reactions in dental tissues incident to orthodontic tooth movement. A review of the literature. Eur J Orthod 1999;21:231-47. 2. Stenvik A, Mjör IA. Pulp and dentine reactions to experimental tooth intrusion: a histologic study of the initial changes. Am J Orthod 1970;57:370-85.
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3. Hamersky P, Weimar A, Taintor J. The effect of orthodontic force application on the pulpal tissue respiration rate in the human premolar. Am J Orthod 1980;77:368-78. 4. Mostafa YA, Iskander KG, El-Mangoury NH. Iatrogenic pulpal reactions to orthodontic extrusion. Am J Orthod Dentofacial Orthop 1991;99:30-4. 5. Butcher EO, Taylor AC. The vascularity of the incisor pulp of the monkey and its alteration by tooth retraction. J Dent Res 1952;31:239-47. 6. Huettner RJ, Whitman CL. Tissue changes occurring in the Macaque rhesus monkey during orthodontic movement. Am J Orthod 1958;44:328-45. 7. Subay RK, Kaya H, Tarim B, Subay A, Cox CF. Response of human pulpal tissue to orthodontic extrusive applications. J Endod 2001;27:508-11. 8. Unsterseher RE, Nieberg LG, Weimer AD, Dyer JK. The response of human pulpal tissue after orthodontic force application. Am J Orthod Dentofacial Orthop 1987;92:220-4. 9. Rana MW, Pothisiri V, Killiany DM, Xu XM. Detection of apoptosis during orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop 2001;119:516-21. 10. Nixon CE, Saviano JA, King GJ, Keeling SD. Histomorphometric study of dental pulp during orthodontic tooth movement. J Endod 1993;19:13-6. 11. Derringer KA, Jaggers DC, Linden RW. Angiogenesis in human dental pulp following orthodontic tooth movement. J Dent Res 1996;75:1761-6. 12. Derringer KA, Linden RW. Enhanced angiogenesis induced by diffusible angiogenic growth factors released from human dental pulp explants of orthodontically moved teeth. Eur J Orthod 1998;20:357-67. 13. Vandevska-Radunovic V, Kristiansen AB, Heyeraas KJ, Kvinnsland S. Changes in blood circulation in teeth and supporting tissues incident to experimental tooth movement. Eur J Orthod 1994;16:361-9. 14. Robinson R. The possible significance of hexosephosphoric esters in ossification. Biochem J 1923;17:286-93. 15. Lamster IB. The host response in gingival crevicular fluid: potential applications in periodontitis clinical trials. J Periodontol 1992;63(Supp 12):1117-23. 16. Tsukamoto Y, Fukutani S, Shin-Ike T, Kubota T, Sato S, Suzuki Y, et al. Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts. Arch Oral Biol 1992;37:1045-55. 17. Lindhe A. Structure and calcification of dentin. In: Bonucci E, editor. Calcification in biological systems. Boca Baton, Fla: CRC Press; 1992. p.269-311. 18. Spoto G, Fioroni M, Rubini C, Tripodi D, Di Stilio M, Piattelli A. Alkaline phosphatase activity in normal and inflamed dental pulps. J Endod 2001;27:180-2. 19. Delmas PD. Clinical use of biochemical markers of bone remodeling in osteoporosis. Bone 1992;13:17-21. 20. Ikawa M, Fujiwara M, Horiuchi H, Shimauchi H. The effect of short-term tooth intrusion on human pulpal blood flow measured by laser Doppler flowmetry. Arch Oral Biol 2001;46:781-7. 21. Perinetti G, Varvara G, Festa F, Esposito P. Aspartate aminotransferase activity in human dental pulps of orthodontically treated teeth. Am J Orthod Dentofacial Orthop 2004;125:88-92. 22. Grieve WG III, Johnson GK, Moore RN, Reinhardt RA, DuBois LM. Prostaglandin E (PGE) and interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid during human orthodontic tooth movement. Am J Orthod Dentofacial Orthop 1994;105:369-74.
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23. Hayashi Y, Imai M, Goto Y, Murakami N. Pathological mineralization in a serially passaged cell line from rat pulp. J Oral Pathol Med 1993;22:175-9. 24. Anstendig HS, Kronman JH. A histologic study of pulpal reaction to orthodontic tooth movement in dogs. Angle Orthod 1972;42:50-5. 25. Guevara MJ, McClugage SG Jr. Effects of intrusive forces upon the microvasculature of the dental pulp. Angle Orthod 1980;50: 129-34. 26. Knighton DR, Hunt TK, Scheuenstuhl H, Halliday BJ, Werb Z, Banda MJ. Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 1983;221:1283-5. 27. Schultz GS, Grant MB. Neurovascular growth factors. Eye 1991;5:170-80. 28. Nakashima M. The effects of growth factors on DNA synthesis, proteoglycan synthesis and alkaline phosphatase activity in bovine dental pulp cells. Arch Oral Biol 1992;37:231-6.
American Journal of Orthodontics and Dentofacial Orthopedics October 2005
29. Shiba H, Fujita T, Doi N, Nakamura S, Nakanishi K, Takemoto T, et al. Differential effects of various growth factors and cytokines on the syntheses of DNA, type I collagen, laminin, fibronectin, osteonectin/secreted protein, acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Phys 1998;174:194-205. 30. Shiba H, Nakamura S, Shirakawa M, Nakanishi K, Okamoto H, Satakeda H, et al. Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin (SPARC) and alkaline phosphatase, and calcification in cultures of human pulp cells. Dev Biol 1995;170:457-66. 31. Butcher EO, Taylor AC. The effects of denervation and ischemia upon the teeth of the monkey. J Dent Res 1951;30:265-75. 32. Scheinin A, Pohto M, Luostarinen V. Defense reactions of the pulp with special reference to circulation–an experimental study in rats. Int Dent J 1967;17:461-75.
Editors of the International Journal of Orthodontia (1915-1918), International Journal of Orthodontia & Oral Surgery (1919-1921), International Journal of Orthodontia, Oral Surgery and Radiography (1922-1932), International Journal of Orthodontia and Dentistry of Children (1933-1935), International Journal of Orthodontics and Oral Surgery (1936-1937), American Journal of Orthodontics and Oral Surgery (1938-1947), American Journal of Orthodontics (1948-1986), and American Journal of Orthodontics and Dentofacial Orthopedics (1986-present) 1915 1931 1968 1978 1985 2000
to to to to to to
1932 Martin Dewey 1968 H. C. Pollock 1978 B. F. Dewel 1985 Wayne G. Watson 2000 Thomas M. Graber present David L. Turpin
Alkaline phosphatase activity in dental pulp of orthodontically treated teeth Giuseppe Perinetti,a Giuseppe Varvara,b Luisa Salini,c and Stefano Tetèd Chieti, Italy Introduction: The aim of this study was to examine alkaline phosphatase (ALP) activity in the dental pulp of orthodontically treated teeth. Methods: Sixteen healthy subjects (mean age 17.0 ⫾1.6 years) who required extraction of 4 first premolars for orthodontic reasons participated. One maxillary first premolar subjected to orthodontic force was the test tooth. The contralateral first premolar, bracketed but not subjected to mechanical stress, was the control tooth. After a week of treatment, the first premolars were extracted and the dental pulp removed from the teeth. ALP activity was determined spectrophotometrically and the results expressed as units/liter per milligram of pulp tissue [U/(L x mg)]. Results: ALP activity was 89 ⫾ 26 U/(L x mg) in the test teeth and 142 ⫾ 33 U/(L x mg) in the control teeth. The difference between the groups was statistically significant (P ⬍ .01). Conclusions: Orthodontic treatment can lead to significant early-phase reduction in ALP activity in human dental pulp tissue. (Am J Orthod Dentofacial Orthop 2005;128:492-6)
T
he response of dental pulp tissue to orthodontic force has been evaluated previously,1 and controversial results have been reported. Although a deleterious effect on the vitality of the teeth has been found as a result of orthodontic force,2-4 other investigations have shown that orthodontic force appears to have no significant effect on dental pulp.5-7 However, depression of tissue respiration rate,3,8 apoptosis,9 increased capillary numbers,10-12 and disturbances in blood circulation13 have all been described in dental pulp tissue from orthodontic force application to the teeth. Alkaline phosphatase (ALP) is an enzyme involved in tissue mineralization,14 and, in periodontal tissues, ALP activity has been correlated with inflammation.15 Several dental pulp cells, such as fibroblasts16 and odontoblasts,17 can synthesize and release ALP. Moreover, ALP activity in human dental pulp tissue has been shown to be up to 8 times higher in reversible pulpitis compared with healthy controls and irreversible pulpi-
tis.18 These results have demonstrated that ALP could have a role in the metabolic changes in dental pulp and other body tissues.19 To date, no studies have evaluated ALP activity in the dental pulp of teeth subjected to orthodontic force. Many previous studies have focused on only 1 dental movement— eg, intrusion20— but the orthodontist is faced with clinical conditions that involve the complex 3-dimensional movements of teeth. For this reason, Derringer et al11 and Derringer and Linden12 reported an increase in angiogenesis in dental pulp tissue using orthodontic forces exerted by a flexible archwire as a model that was responsive to 3-dimensional forces applied to the teeth. Similarly, Perinetti et al21 reported an increase in aspartate aminotransferase activity in orthodontically treated teeth compared with untreated controls. Our study was designed as a cross-sectional assessment to determine whether significant changes in ALP activity occur in dental pulp during the early phases of orthodontic treatment with fixed appliances in healthy young subjects.
a
Research fellow, Department of Oral Sciences, University G. D’Annunzio, Chieti, Italy, and Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy. b Research fellow, Department of Oral Sciences, University G. D’Annunzio, Chieti, Italy. c Clinical practice, Distretto Sanitario di Base, Unit of Dental Care, Francavilla al Mare, Chieti, Italy. d Professor, Department of Oral Sciences, University G. D’Annunzio, Chieti, Italy. Reprint requests to: Giuseppe Perinetti, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale 8/A, 66030 Santa Maria Imbaro, Chieti, Italy; e-mail, [email protected]. Submitted, February 2004; revised and accepted, July 2004. 0889-5406/$30.00 Copyright © 2005 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2004.07.042
492
MATERIAL AND METHODS
Sixteen orthodontic patients, 10 female and 6 male (age range, 15.0-19.6 years; mean, 17.0 ⫾ 1.6 years), who required extraction of the maxillary first premolars because of dental crowding participated in the study. The inclusion criteria were (1) need for fixed appliance therapy, (2) good general and periodontal health, (3) no use of anti-inflammatory drugs in the month before the study, (4) periodontal probing depth values not exceeding 3 mm in the whole dentition, and (5) no evidence of marginal periodontal bone loss after a periapical radio-
Perinetti et al 493
American Journal of Orthodontics and Dentofacial Orthopedics Volume 128, Number 4
Fig 1. Orthodontic appliance on TT.
graphic examination. During the study, the subjects were not allowed to take anti-inflammatory drugs that could affect the results. Informed consent was obtained from the patients and from the parents of those under 18 years of age before the study. The protocol was reviewed and approved by the Ethical Committee of the G. D’Annunzio University Medical Faculty. In each patient wearing the orthodontic appliance, the maxillary first premolars were the experimental teeth. A first premolar undergoing orthodontic force was used as the test tooth (TT), and its contralateral first premolar was the control tooth (CT). Both the TT and the CT were unrestored and asymptomatic, with no radiological evidence of caries or periapical radiolucency. Orthodontic brackets (MBT, 3M Unitek, Monrovia, Calif) were placed on the buccal surfaces of the incisors, canines, and premolars in the maxillary arch; the bands were bonded on the first molars. A unilateral circular orthodontic archwire, 0.018 inch (MBT, 3M Unitek), was then mounted in the same quadrant as the TT to activate the orthodontic appliance (Fig 1). The bracket on each TT was placed so that, when the archwire was placed into its slot, it exerted active forces on the TT. These forces (about 30-90 g) were measured as previously described by Grieve et al.22 The entire orthodontic appliance was placed in a single clinical session. After 7 days of treatment, the experimental teeth were extracted under local anesthesia, and the dental pulp samples were obtained. The teeth were immediately longitudinally grooved under water irrigation on the buccal and lingual surfaces with a diamond disc so as not to penetrate the root canals and then split in half with cutting pliers. Care was taken in extirpating dental pulp samples from the teeth. The samples were placed in plastic vials and immediately washed 2 or 3 times in ice-cold, heparinized, sterile saline solution to remove blood, which normally shows ALP activity.19 The samples were stored at ⫺80°C. Immediately before biochemical analysis, the spec-
imens were weighed and homogenized in 1 mL of 10 mmol/L potassium phosphate buffer, pH 7.0, containing 0.1% sodium cholate. This homogenate was centrifuged at 100,000xg for 60 minutes at 4°C, and the supernatant was recovered, diluted to a volume of 2 mL with the phosphate buffer, and used for the enzymatic activity determination. One milliliter of supernatant was taken for ALP activity determination; to it was added the substrate, with the final concentrations of 20 mmol/L p-nitrophenol phosphate, 5 mmol/L magnesium chloride, 200 mmol/L mannitol, 0.05% sodium azide, and carbonate buffer (pH 10.2 ⫾ 0.1) in a total volume of 2 mL. ALP hydrolyzes p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate. The rate of increase in absorbance was read with an ultraviolet-visible spectrophotometer at 405 nm for 4 minutes, by using the kinetic method to record the absorbance variation per minute as the p-nitrophenol formed. For each analysis, a control was used that consisted of the reagent and the phosphate buffer without the sample, and the value of absorbance variation per minute in this control was subtracted from the experimental values. By using 18.45 as the p-nitrophenol millimolar absorptivity, the absorbance was converted into enzyme activity units (1 unit ⫽ 1 micromole of p-nitrophenol released per minute at 30°C). ALP activity was expressed as units/liter per milligram of pulp tissue [U/(L x mg)]. The Statistical Package for Social Sciences (SPSS, Chicago, Ill) was used for data analysis. Each data set was tested for normality with the Shapiro-Wilks test and Q-Q normality plots; equality of variance was also tested with the Levene test and Q-Q normality plots of the residuals. Because each data set met the required criteria for parametric analyses, a paired t test was used to assess the significance of the differences in ALP activity between the experimental teeth. In addition, in each experimental group, the strength of the straightline relationship between age and enzymatic activity was tested with the Pearson r correlation coefficient. A P value less than .05 was used to reject the null hypothesis. RESULTS
ALP activity was 89 ⫾ 26 U/(L x mg) (range, 54-145 U/[L x mg]) in the TTs; enzymatic activity was 142 ⫾ 33 U/(L x mg) ([range, 78-198 U/[L x mg]) in the CTs (Fig 2). The paired t test showed that the 2 groups differed significantly in ALP activity (t15 ⫽ 5.3; P ⬍ .01). The 95% confidence interval of the differences of the mean values of ALP activities of the groups was 31-73 U/(L x mg). The Pearson r correlation coefficient demonstrated a nonsignificant straight-
494 Perinetti et al
Fig 2. ALP activity in test and control groups. ALP activities in units/liter per milligram of pulp tissue [U/(L x mg)] are presented as mean ⫾ SD (n ⫽ 15). Paired t test, P ⬍ .01.
line relationship between age and ALP activity in both TTs and CTs (P ⬎ .5). DISCUSSION
This cross-sectional study examined ALP activity in the dental pulp tissue of orthodontically treated teeth, compared with untreated teeth. The results demonstrate that a significant decrease in ALP activity (that does not correlate significantly with patient age) can be detected in the dental pulp tissue of teeth undergoing orthodontic force (Fig 2). The teeth were bonded, and a flexible archwire was mounted in the brackets (Fig 1) to maintain parity with the normal clinical situation, giving a continuous but reducing active force as tooth alignment occurred. Thus, although the forces were more difficult to standardize, this study design could accurately reproduce the clinical effects of 1 week of initial 3-dimensional archwire forces that are of direct clinical relevance, as reported in other studies.11,12,21 In several studies, dental pulp response to orthodontic force was evaluated through analysis of the mineralized tissue of the tooth, but conflicting results have been reported. An increase in the predentin width coincident with the peak of tooth movement has been described3,10; a reduction in the predentin zone with degeneration of the odontoblastic layer has also been reported.2 Subay et al7 found microscopic pulp stones in only 17% of teeth undergoing extrusive movement after 10 or 40 days of treatment. Considering that ALP has a key role in the physiological17,16 and pathological23 mineralization processes of pulp tissues, our results for the TTs are consistent with the observations that, in the early phases of orthodontic treatment, there is neither an increase in the predentin width2 nor a significant formation of pulp stones.7 However, the different experimental designs in these studies might explain the inconsistencies of the data.
American Journal of Orthodontics and Dentofacial Orthopedics October 2005
The decrease in ALP activity in the TTs might be explained by damage of the pulp cells responsive to the synthesis of this enzyme, as a consequence of applying orthodontic tools to the teeth. Apoptosis in rat dental pulp tissue of teeth under orthodontic load for 3 days to 2 weeks was evaluated by Rana et al,9 who observed that maximum apoptosis occurred after 3 days. Previous in-vivo studies4 reported a significant odontoblastic degeneration in the pulp of orthodontically treated teeth. Moreover, in an in-vivo study on human subjects, Perinetti et al21 showed a significant increase in the aspartate aminotransferase activity (an index of cell necrosis) in the pulp after 7 days of orthodontic treatment. These results are also supported by other histological evaluations2,24,25 that have correlated dental-pulp alterations with strangulation and stasis of blood flow after orthodontic force has been applied to the teeth. Such a reduction in dental pulp blood flow has been implicated as causing a decrease in oxygen availability.3,8 Because dental pulp fibroblasts16 and odontoblasts17 can produce ALP, it can also be hypothesized that the reduction in enzyme activity in the TTs should be mainly due to an alteration in these cells. However, our results do not appear to agree with other in-vitro studies, which have reported an increase in ALP activity from animal pulp cells under pathological conditions.23 An association between ALP activity and vesicular structures around degenerate and necrotic cells has been seen.23 Similarly, congested blood vessels and large vacuoles were reported by Mostafa et al4 in human dental pulp tissue after 1 week of orthodontic treatment. Conceivably, the apparent inconsistencies between our study and others might be because, under in-vivo conditions, several adjunctive processes are involved, compared with in-vitro conditions. The lack of collateral circulation in the pulp makes it one of the most sensitive tissues of the body, and orthodontic tooth movement has been described as causing a reduction in the level of oxygen and other degenerative damage. These conditions have been reported to be similar to wound healing and ischemic/hypoxic processes,12 in which angiogenic growth factors have been identified.26,27 Although no previous studies have evaluated angiogenic growth factor levels in the dental pulp of teeth undergoing mechanical stress, Derringer et al11 demonstrated an increase in the total number of pulp microvessels of orthodontically treated teeth after 5 and 10 days of treatment, and similar results have been reported in other investigations.10,12 Hence, these authors hypothesized an increase in angiogenic activity as a response to an increased level of growth factors. In this regard, it is of interest that a decrease in ALP activity has been shown to be consequent to the release
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of angiogenic growth factors.28-30 Among these, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor- and tumor necrosis factor-␣ have been mainly implicated in the reduction in dental pulp cell ALP activity. In particular, an inhibition of ALP activity up to 65% was shown in proliferating and stationary cell cultures when several growth factors were added.28 Hence, a combination of a degeneration of pulp cells, occurring in the earliest phases of orthodontic treatment, and an increase in angiogenic growth factors levels, which is likely to occur during the same treatment, appears to be the basis for the decrease in ALP activity in the test teeth. However, further studies that include both biochemical and histochemical/immunocytochemical analyses need to be carried out to confirm this possibility. Different pulpal reactions to orthodontic forces have been correlated with age and size of the apical foramen.5,31 These results are consistent with a relationship between the biological effects of orthodontic forces and the maturity of the tooth.32 Positive correlations between age and rate of tissue respiratory depression3 or other pulp tissue alterations5,31,32 during orthodontic treatment have been reported. We did not find a significant correlation between ALP activity and patient age in either the test or control teeth. This result, however, was not unexpected because the age range of our subjects was 15.0-19.6 years, which was more restricted than that in other studies.3 Thus, the lack of correlation between ALP activity changes in the dental pulp of orthodontically treated teeth and patient age requires further investigation. CONCLUSIONS
This cross-sectional study showed a decrease in ALP activity in dental pulp tissue during the earliest orthodontic stages. Further investigations are needed to fully understand the long-term effects of orthodontic tooth movement on ALP activity of the pulp and its possible clinical implications. We thank Dr Christopher P. Berrie for his critical appraisal of the text.
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29. Shiba H, Fujita T, Doi N, Nakamura S, Nakanishi K, Takemoto T, et al. Differential effects of various growth factors and cytokines on the syntheses of DNA, type I collagen, laminin, fibronectin, osteonectin/secreted protein, acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Phys 1998;174:194-205. 30. Shiba H, Nakamura S, Shirakawa M, Nakanishi K, Okamoto H, Satakeda H, et al. Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin (SPARC) and alkaline phosphatase, and calcification in cultures of human pulp cells. Dev Biol 1995;170:457-66. 31. Butcher EO, Taylor AC. The effects of denervation and ischemia upon the teeth of the monkey. J Dent Res 1951;30:265-75. 32. Scheinin A, Pohto M, Luostarinen V. Defense reactions of the pulp with special reference to circulation–an experimental study in rats. Int Dent J 1967;17:461-75.
Editors of the International Journal of Orthodontia (1915-1918), International Journal of Orthodontia & Oral Surgery (1919-1921), International Journal of Orthodontia, Oral Surgery and Radiography (1922-1932), International Journal of Orthodontia and Dentistry of Children (1933-1935), International Journal of Orthodontics and Oral Surgery (1936-1937), American Journal of Orthodontics and Oral Surgery (1938-1947), American Journal of Orthodontics (1948-1986), and American Journal of Orthodontics and Dentofacial Orthopedics (1986-present) 1915 1931 1968 1978 1985 2000
to to to to to to
1932 Martin Dewey 1968 H. C. Pollock 1978 B. F. Dewel 1985 Wayne G. Watson 2000 Thomas M. Graber present David L. Turpin