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RESEARCH REPORT |
1 Department of Oral Biology and Maxillofacial Pathology,
2 Office of Biostatistics, and
3 Department of Orthodontics, Medical College of Georgia, School of Dentistry, Augusta, GA 30912-1129, USA;
*corresponding author, JBORKE{at}mail.mcg.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: mechanotransduction • orthodontic tooth movement • plasma membrane disruption • periodontal ligament
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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It has been shown that when mechanical stress is imposed on various tissues in vivo, transient, survivable disruptions of the plasma membrane are created. This form of cell injury has been termed "cell wounding". Cells of the gut, skin, aortic endothelium, and skeletal muscle frequently suffer survivable plasma membrane disruption in vivo under physiological and pathological conditions of mechanical stress (McNeil and Ito, 1989, 1990; McNeil and Khakee, 1992; Yu, 1992).
Two important molecules in bone remodeling—IL-1ß (Auron et al., 1984) and bFGF (Abraham et al., 1986)—lack the signal peptide sequence, a prerequisite for protein secretion via the classic exocytotic pathway. Because of this, it has been proposed that these two polypeptides are released from dead, damaged, or injured cells (Klagsbrun and Vlodavsky, 1988; Young et al., 1988). To date, there have been no studies linking the placement of orthodontic forces with plasma membrane disruption in cells of the PDL.
In the present study, we applied the plasma membrane disruption hypothesis to the cells of the periodontal ligament, a tissue frequently exposed to mechanical stress. We developed an in vivo rat model using an orthodontic device and endogenous albumin as a "wound marker". These studies provide insight into this novel cellular mechanism, previously undescribed in the PDL.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Tooth Movement
For this study, we used 15 Harlan retired breeder female rats with an average body weight of 300 ± 2 g (mean ± SD). Rats were provided with feed and water ad libitum. Our animal protocol was reviewed and approved by the Committee for Care and Use of Laboratory Animals at the Medical College of Georgia. The animals were divided into two groups, one group with moved teeth and one control group with unmoved teeth, each composed of 7 animals. One additional control rat was used for Western blot analysis.
A round stainless steel orthodontic wire (0.018 inch, Sybron Dental Specialties, Glendora, CA, USA) was bent into a rectangular form with one helical loop to construct 7 customized uniform springs. The schematic occlusal view of the orthodontic appliance is shown in Fig. 1A
, and a photograph of the spring placement is shown in Fig. 1B. We adjusted the initial expansion force for each rat by measuring the interproximal maxillary molar distance and adjusting each spring to exert a 50-g load upon activation. This supra-clinical force was used to accentuate differences seen between groups during the short duration of this study. Calibration of the individual springs was conducted by means of a Vitrodyne V100 Universal Tester (Chantillon, Greensboro, NC, USA). With the animals under anesthesia, a small circular depression was made in the enamel of the lingual side of the crown of the right and left upper first molars, by means of a dental handpiece operating at slow speed with a coarse round diamond bur. The springs were engaged in the depressions so that the appliance would not be dislodged during the five-minute experimental period and so that the force was delivered primarily in the lingual-to-buccal direction. Rats were not killed until 2 hrs after removal of the spring, allowing for either cell recovery or cell dissolution. The control group followed the same protocol, but no spring was placed across the maxilla.
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Immunohistochemistry
The tissue sections for localization of intracellular albumin were processed by means of a modified avidin-biotin-peroxidase technique (Hsu et al., 1981; Borke et al., 1987). Briefly, paraffin sections on poly-L-lysine-coated slides were deparaffinized in limonene and rehydrated in descending concentrations of ethanol. Endogenous peroxidase activity was blocked by five-minute incubation in 0.3% H2O2. Non-specific binding of antibodies to tissue sections was blocked by incubation for 1 hr in 10 mg/mL casein. Sheep anti-rat albumin (Accurate Antibodies, Westbury, NY, USA) was applied to the sections at a dilution of 1:2000 for 1 hr. Control sections were processed with normal sheep serum substituted for the anti-albumin antibody. After sections were washed in PBS, a 1:200 dilution of the secondary biotin-conjugated rabbit anti-sheep Ig antibody (Vector Laboratories, Burlingame, CA, USA) was applied for 30 min. After being further washed in PBS, the tissue sections were incubated for 30 min in the avidin-peroxidase complex reagent (ABC reagent, Vector). Following additional PBS washes, the peroxidase molecule in the immobilized avidin-peroxidase complex was used to reduce H2O2 in the presence of diaminobenzidine tetrahydrochloride (DAB), to produce a brown reaction product over the sites of antibody binding to albumin. After being stained, tissue sections were dehydrated in ascending concentrations of ethanol to xylene, and coverslipped with Permount. Some sections were also counter-stained with Mayerapos;s hematoxylin for histological identification.
Western Blot Analysis
We used Western blot analysis to determine if there was cross-reactivity of the anti-albumin antibody with other proteins of the rat maxilla. Samples from the soft tissues of the rat maxilla were homogenized in a solution containing 10 mM Tris-HCl (pH 7.4), 0.5 mM dithiothreitol, 1 mM EDTA, and 5 mM benzamidine. Rat plasma was also processed for Western blot analysis as a control. Each sample was assayed for total protein by means of the BCA protein assay (Pierce Inc., Rockford, IL, USA). Samples were standardized based on protein concentration and combined 1:1 with a 2X sample buffer containing 100 mM Tris-HCl (pH 7.4), 4% sodium dodecylsulfate (SDS), 10% ß-mercaptoethanol, 20% glycerol, and 0.2% bromophenol blue. Samples containing 20 µg of protein were applied to each lane of a 7.5% SDS-polyacrylamide gel (Laemmli, 1970). Marker proteins of known molecular weight were loaded onto the same gel for comparison with sample proteins. After approximately 45 min of electrophoresis, proteins were transferred to nitrocellulose membrane via a Trans-Blot apparatus (Bio-Rad, Richmond, CA, USA). Transferred proteins were processed for immunodetection of albumin by an avidin-biotin-peroxidase technique similar to that used for immunohistochemistry. Briefly, the nitrocellulose membrane was rinsed in PBS (pH 7.4) at room temperature before incubation for 5 min in 3% H2O2. The blot was then rinsed in PBS and incubated with 10 mg/mL casein in PBS for 1 hr with gentle shaking. This solution was removed, and a 1:5000 dilution of sheep anti-rat albumin antibody in PBS was applied to the blot for 1 hr. The blot was washed thoroughly with PBS-0.05% Tween-20 (PBS-Tween) before incubation in a 1:200 dilution of biotin-conjugated rabbit anti-sheep antibody for 30 min. The blot was washed in PBS-Tween and next incubated with the avidin-peroxidase complex solution for 30 min. After being washed in PBS-Tween, the blot was incubated with H2O2 in the presence of the DAB substrate solution for 2 min, and the reaction was terminated with de-ionized water.
Quantitative Analysis of Intracellular Albumin
Densitometric evaluation of immunostained PDL was performed by means of an Olympus BX40 microscope (Olympus Optical Co, Ltd., Tokyo, Japan). Microscope illumination intensity and magnification remained constant during the acquisition of images via a mounted video camera. The first root of each sample was captured, and the optical densities of immunoreactions at the tension, compression, mesial, and distal sides of the most mesial root of the maxillary first molar were measured by means of the Image Tool software (University of Texas Health Science Center, San Antonio, TX, USA). We obtained 12 measurements from each root (3 for tension, 3 for compression, 3 for mesial, and 3 for distal). Areas of identical size (64-pixel squares) were outlined in each image and digitized by the program to yield light intensity levels ranging from 0 (black) to 255 (white). The light intensity per outlined area was recorded and logged directly into the Excel program for further analysis.
Statistical Analysis
A nested repeated-measures factorial analysis of variance model was used to determine if differences in the mean density readings existed between areas of PDL surrounding the tooth roots and between control and treatment (moved teeth) groups. The following two hypotheses were tested. First, within each group (control and experimental), do differences exist between sides (lingual, distal, mesial, and buccal)? Second, within each side, do differences exist between groups? We used a Tukey multiple-comparison procedure to determine where differences within groups between sides or within each side between groups existed. The significance level was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Our blots showed a single band in both maxilla tissue homogenates and rat plasma corresponding to the known molecular weight of albumin (~ 66 kDa). No cross-reactivity was seen with proteins of either higher or lower molecular weight.
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). This indicates that the other reagents used in the immunohistochemical localization studies did not cross-react with tissue molecules.
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shows a representative section of rat maxilla processed after exposure of the first molar for 5 min to a heavy spring exerting an initial force of 50 g. Immunohistochemical localization with anti-rat albumin antibody shows intracellular albumin in cells of the PDL (Figs. 3D-3F). Areas of the PDL under tension (buccal, Fig. 3E) and compression (lingual, Fig. 3F) show a broad distribution of cells demonstrating immunoreactivity for albumin. No albumin is seen in the extracellular connective tissue. Figs. 3G-3I show a representative cross-section of rat first molar root from a control tooth that did not undergo orthodontic tooth movement. These Figs. represent sections that were processed identically to those of the moved teeth represented by Figs. 3D-3F. No localization of intracellular albumin is seen in these control sections, suggesting that plasma membrane disruption occurred as a result of the applied force and did not occur prior to loading.
A statistically significant difference was found between groups (p < 0.0001) and between sides within the experimental group (p = 0.0023). No statistically significant differences were found between sides of the control group (p = 0.4207). For the experimental group, although the immunohistochemistry demonstrates the uptake of albumin by cells on all sides of the moved teeth, the buccal (tension) side shows a significantly higher mean density reading than the lingual (compression, p = 0.0002), the distal (p = 0.0011), and the mesial (p = 0.0002) sides. Comparison of buccal (tension) sides from control and experimental groups shows that the control group has a significantly lower mean density reading than the experimental group (p = 0.0001) (Table).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate this hypothesis, we developed a rat model of orthodontic tooth movement. We used a spring design that was simple to construct and able to deliver a consistent force that was adjustable for calibration and to compensate for anatomical differences between rats in terms of the maxillary intermolar distance (Fig. 1). In our model, we considered the possibility of the applied force being diminished by transmission to the midline suture. However, this force was dispersed over a large area and is therefore negligible.
The forces used to produce orthodontic tooth movement in rats vary among studies and authors, even when the same method or device is applied. Heavy forces that produce tooth movement have been shown to range from 40 g to 60 g (Bridges et al., 1988; Gibson et al., 1992; Kyomen and Tanne, 1997; Shirazi et al., 1999). In our model, we used 50 g of force.
In our studies, we followed the uptake by rat PDL cells of the ~ 66-kDa rat serum albumin molecule. We used the animalapos;s native albumin as a probe by postulating that this molecule would be unable to permeate the phospholipid bilayer barrier in only 5 min unless a disruption of the plasma membrane had occurred. Intracellular albumin as a marker for cell injury has been used in different tissues, such as rat tricep, endothelial cells of the rat aorta, and cardiac myocyte of normal and overloaded rat heart (McNeil and Khakee, 1992; Yu and McNeil, 1992; Clark et al., 1995; Fischer et al., 1997). Although the intracellular albumin was observed on all sides of experimentally moved teeth, cytoplasmic staining with antibodies to endogenous albumin was significantly higher in cells from the tension side of the moved teeth when compared with the other sides within the same group and when compared with cells from the PDL of unmoved control teeth. This suggests that when high mechanical forces are imposed on the PDL, wounding of the plasma membrane occurs in numerous cells. Whether wounding also occurs with lesser force, permitting the passage of smaller molecules, seems likely but was not investigated in the present study.
The inclusion of 2 hrs of recovery time following the release of force ensured that if the cells had not resealed or had died, the intracellular albumin would have had sufficient time to diffuse from the cytoplasm. Further, the morphology of the PDL cells appeared essentially normal, also suggesting that the cells had survived plasma membrane disruption (Fig. 3).
The lack of intracellular albumin in our control group suggests that for membrane disruption to allow passage of a 66-kDa molecule may require a large mechanical force. The possibility remains open that normal occlusal forces may generate lesser disruptions.
In summary, the work described here provides the first in vivo evidence of a role for plasma membrane disruption in the mechanotransduction of orthodontic tooth movement. Studies in other tissues and species show that this cellular phenomenon occurs frequently in vivo. Our work suggests that this overlooked but important mechanism also occurs in cells of the PDL.
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ACKNOWLEDGMENTS |
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Received June 11, 2001; Last revision November 9, 2001; Accepted November 14, 2001
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REFERENCES |
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