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Is there an optimal force level for sutural expansion?
ORIGINAL ARTICLE
Is there an optimal force level for sutural expansion? Sean Shih-Yao Liu,a Lynne A. Opperman,b Hee-Moon Kyung,c and Peter H. Buschangd Indianapolis, Ind, Dallas, Tex, and Daegu, Korea
Introduction: The purpose of this study was to establish the causal relationships between expansion force magnitudes, sutural separation, and sutural bone formation. Methods: Thirty-seven 6-week-old rabbits were randomly assigned to 4 force groups (0, 50, 100, or 200 g). Constant forces were delivered for 42 days by nickel-titanium open-coil springs to miniscrew implants (MSIs) placed in the frontal bone on both sides of the midsagittal suture. Inter-MSI and bone marker widths were measured biweekly to quantify sutural separation and MSI movements. Sutural bone formation was quantified based on the incorporation of fluorescent bone labels administered at days 18, 28, and 38. Results: Nine of 74 MSIs failed between days 0 and 14, including 4 in the controls and 5 in the 50-g group. A decelerating curvilinear pattern of sutural separation was evident in the 50-g, 100-g, 200-g groups. Bone markers showed that sutural widths increased by 0.6, 3.2, 5.1, and 6.2 mm in the control, 50-g, 100-g, and 200-g groups, respectively. Except for the 200-g group, significantly greater amounts of bone formation were observed between days 18 and 28 than between days 28 and 38. Sutural bone formation also increased with increasing forces up to 100 g; there was no difference between the 100-g and the 200-g groups. Sutural separation explained 71% and 53% of the variations in bone formation between days 18 and 28 and days 28 and 38, respectively. Conclusions: Within the limits of this study, sutural bone formation is directly related to the amount of sutural separation, which is in turn related to the amount of force applied. The results suggest that there is a level of induced sutural separation that provides the greatest amount of bone formation. (Am J Orthod Dentofacial Orthop 2011;139:446-55)
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t has been well established that sutural expansion stimulates bone formation. Once expansion forces are applied, tissue trauma occurs with disruption of collagen fibers and blood vessels,1,2 together with cell death of fibroblasts.3 However, most of the collagen fibers and the periosteum remains intact, and the osteoblast layers close to the bone margins survive to form successive lamellae within 3 to 4 days.3 As the suture separates, new bone forms along the collagen fibers aligned perpendicularly across the suture.3 a Assistant Professor, Department of Orthodontics and Oral Facial Genetics, School of Dentistry, Indiana University, Indianapolis. b Professor, Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M Health Science Center, Dallas. c Professor, Department of Orthodontics, Dental School, Kyungpook National University, Daegu, Korea. d Professor, Department of Orthodontics, Baylor College of Dentistry, Texas A&M Health Science Center, Dallas. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Supported financially by Texas A&M Health Science Center, Baylor College of Dentistry’s Department of Orthodontics. Reprint requests to: Sean Shih-Yao Liu, Department of Orthodontics and Oral Facial Genetics, Indiana University School of Dentistry, 1121 W Michigan St, Indianapolis, IN 46202; e-mail, [email protected]. Submitted, January 2009; revised and accepted, March 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.03.056
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Although higher forces have been related to greater sutural separation, the exact nature of the relationship remains unclear. Histologic and biometric studies have shown that short-term sutural separation relates to the magnitude of the expansion forces.4-8 Morndal,5 who expanded rats’ interpremaxillary sutures for 27 hours, confirmed that the amount of sutural separation was linearly related to the magnitude of force. Over a longer time period (28 days), Yen et al8 showed that the springs activated 2 mm with heavier forces produced faster rates of expansion than those activated 2 mm with lighter forces, even though both ultimately produced similar amounts of sutural separation. Importantly, all experimental studies used helical springs. Because the forces of these springs decay as the suture expands, it is difficult to quantify the exact nature of the relationships between forces and sutural separation.9 The relationship between sutural bone formation and the amount of expansion force remains poorly understood. The increases in alkaline phosphatase-stained cells associated with heavy force levels indicate increased bone formation.10,11 It has also been shown that DNA synthesis of fibroblasts and osteoblasts increases with increasing forces, and eventually plateaus at higher force levels.5 DNA synthesis of fibroblasts and osteoblasts has also
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been shown to decrease at higher forces, possibly due to tissue damage.7,12 In the only study that directly evaluated the relationships between bone formation and sutural expansion, Parr et al13 reported no significant difference in bone formation rates between 1-N and 3-N expansion forces. However, their mineral apposition rates suggested a possible difference; because of small sample sizes, the study’s power might have been insufficient to detect the differences. Whether there is an optimal force level to produce the greatest sutural bone formation remains questionable. Our goal was to better understand how sutures respond to varying amounts of force application. Histomorphometric and biometric analyses were performed to test the hypotheses that (1) sutural separation increases as a function of increasing expansion forces, (2) bone formation increases as a function of expansion force magnitude, and (3) bone formation is related to sutural separation. Our clinical objective was to determine whether there is an optimal force that maximizes sutural bone formation. If there is such a force, it provides a potential means of making expansion therapies more effective and efficient. MATERIAL AND METHODS
The sample included 37 six-week-old male New Zealand white rabbits. The housing, care, and experimental protocol were in accordance with the guidelines of the Institutional Animal Care and Use Committee of Baylor College of Dentistry. The rabbits were randomly assigned to 4 groups: 50-g force (n 5 11), 100-g force (n 5 9), 200-g force (n 5 9), or 0-g force (control) (n 5 8). The expansion forces were delivered for 42 days; no forces were used in the control group. All animals were anesthetized with ketamine at 75 mg per kilogram intramuscularly and 0.5% acepromazine at 5 mg per kilogram intramuscularly (Marcaine, Abbott Laboratories, Abbott Park, Ill), with 1:200,000 epinephrine serving as the local anesthetic. All surgical procedures were performed under sterile conditions. After reflecting the skin and the periosteum midway between the anterior and posterior limits of the orbital rims, the midsagittal suture and the frontal bone were exposed (Fig 1, A). Pilot holes were drilled with a size-2 round burr with a low-speed hand piece (\600 rpm) and copious saline-solution irrigation. Two custom-made Dentos (Daegu, Korea) miniscrew implants (MSIs; 3.0 mm long 3 1.7 mm in diameter) were placed approximately 4 mm on either side of the midsagittal suture with a manual driver (Fig 2). Four 99.95% tantalum bone markers (1.5 mm long 3 0.8 mm in diameter) were tapped into the skull 2 to 3 mm anterior and 2 to 3 mm posterior to the MSIs by using a custom-made stainless steel appliance. The bone
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markers were used to radiographically quantify the sutural separation and implant movements. All animals were given penicillin (60000 IU/lb/im) immediately after surgery to prevent infection and buprenorphine (0.02 mg/kg/sc; prn) to minimize discomfort. A 20-mm long and 0.020-in diameter stainless steel interabutment guide wire was engaged in the holes in the MSI heads (Fig 1, D). Fifteen-millimeter long Sentalloy (GAC International, Bohemia, NY) nickel-titanium open-coil springs, delivering 50, 100, or 200 g of force, were telescoped over the wire between the 2 MSIs. Two stop loops were bent to prevent the spring and wire from becoming dislodged. The forces exerted were maintained because the springs remained compressed at lengths ranging from 8 to 12 mm, and the springs’ lengths were checked biweekly.14 When necessary, sliding tubes were added to the guide wire to maintain the springs’ compressed lengths. Records, including animal weights, ventrodorsal cephalometric digital radiographs, and inter-MSI width caliper measurements, were obtained under anesthesia at days 0, 14, 28, and 42 (Fig 3). By using a customized head holder and a metal scale for calibration, standardized ventrodorsal radiographs (no. 4 film; Air Techniques, Melville, NY) were taken at 65 kVp and 10 mA for 12 seconds at fixed distances. To quantify bone formation, oxytetracycline (13.6 mg/lb/im) and calcein (10 mg/kg/im) fluorescent labels were administered to all animals. Oxytetracycline was given on days 18 and 38; calcein was given on day 28. It was previously shown that advancing bone fronts during sutural expansion incorporate fluorescent labels, making it possible to quantify new bone formation. Blinded biometric assessments were based on caliper and radiographic measurements. Caliper width measurements between each MSI pair (MSIc) were taken at the outermost margins immediately above the guide wire (Fig 2, A). The widths between the anterior (AB) and posterior (PB) bone markers and outermost margins of the MSI (MSIr) were measured on the digital radiographs (Fig 2, B) by using Visix software (Air Techniques). After 2 weeks, 80 radiographs were remeasured for establishing the intraexaminer method errors (AB, 0.11 mm; qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi MSIr, 0.10 mm; P PB, ffi 0.10 mm) by using Dahlberg’s formula ( ½ d2 =2n).15 To estimate sutural separation at the implant site, the AB and PB widths were averaged. The MSI lateral displacement during the study was determined by subtracting sutural separation from changes in MSIr width between days 0 and 42. The parallel nature of sutural separation was evaluated based on the differences between the AB and PB widths changes during the study.
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Fig 1. A, Two pilot holes drilled after exposure of the frontal bones and the midsagittal suture; B, 3-mm MSIs under the scanning electron microscope; C, 3-mm long MSI; D, a nickel-titanium open-coil spring telescoped on a guiding wire between the 2 MSIs, with a sliding tube used to maintain the activation level.
After 42 days of expansion, the rabbits were killed with an overdose of beuthanasia (intracardiac injection of 1 cm3 per animal) and perfused with 70% ethanol. A standardized region, including the midsagittal suture and adjacent bone, was dissected and fixed with 70% ethanol for 2 weeks without decalcification. After dehydration with an ascending series of ethanol (70%-100%), the specimens were embedded in methylmethacrylate and sectioned (approximately 60 mm) coronally by using a diamond saw (3 sequential sections per animal), followed by grinding and polishing. Images were captured by an 80i epifluorescence microscope (Nikon, Melville, NY; excitation wave lengths of 390 nm for oxytetracycline and 485 nm for calcein) with a Coolsnap K4 (Photometrics, Tucson, Ariz) camera and MetaMorph software (version 6.3, Molecular
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Devices, Sunnyvale, Calif). Bioquant Osteo II software (Nashville, Tenn) was used to trace the bone labels over the entire suture, from the ectocranial to the endocranial surfaces. All data processing was performed by 1 blinded examiner (S.S.-Y.L.). Bone formation between the leading edges of label pairs was automatically measured every 10 mm along the entire length of the suture (Fig 4). The first pair of labels quantified bone formation between day 18 (oxytetracycline) and day 28 (calcein); the second pair quantified bone formation between day 28 (calcein) and day 38 (oxytetracycline). Sutural gaps after expansion were measured every 10 mm between the 2 trailing edges of the bone labels at day 38 (oxytetracycline). Measurements of bone formation and sutural gaps were based on the averages of the 3 serial sections obtained for each animal.
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calculated. This allowed each animal’s curve to be calculated. The curves were used to provide estimates of the biometric changes between days 18 and 28 and days 28 and 38. Statistical analyses of the histomorphometric measurements were performed by using SPSS software (version 15.0, SPSS, Chicago, Ill). Kruskal-Wallis analyses were performed to compare the groups. The MannWhitney U test was used for paired group comparisons with Bonferroni adjustments. Bone-formation amounts between days 18 and 28 and days 28 and 38 were compared by using the Wilcoxon signed rank test. Spearman correlation analyses were used to evaluate relationships between sutural separation and bone formation. The slopes of the relationship were estimated by using the least squares method. RESULTS
Fig 2. A, Illustration used to evaluate inter-MSIs width (MSIc) with a caliper (note the lateral MSI tipping during expansion); B, schematic of ventrodorsal radiographs used to evaluate inter-MSIs width (MSIr) between the outermost margins of the paired MSIs, the anterior bone marker width (AB) between the centers of the anterior pair bone markers, and the posterior bone marker width (PB) between the centers of the posterior pair bone markers. MSI; bone marker.
Four animals were randomly selected from each force group to evaluate histologic changes. Their sections were stained with Stevenel’s blue and Van Gieson picro-fuchsin and evaluated under a light microscope. Statistical analysis
All statistical procedures were performed by using the Multilevel Win program (version 2.0, Bristol, United Kingdom) with a 95% confidence interval (P 5 0.05). The curves describing the changes of the repeated caliper, radiographic widths, and weight measurements were modeled over time as polynomials, by using the iterative generalized least squares method. The fixed part of the models described the changes and statistically compared the groups. The random part of the models had animals at the higher level and their repeated measures (weeks) at the lower level, nested in the higher level. To evaluate group differences at the end of expansion, the constant of each polynomial was fixed at 42 days. After the order of the models had been statistically verified, the residuals of each animal’s curves were
The animals’ weights increased by 1222 to 1321 g (76%-84%) over the time of treatment. There were no significant weight differences between the 4 groups, no signs of infection around the surgical sites, and no obvious signs of discomfort during the study. A total of 8 MSIs failed during the first 2 weeks, including 2 pairs of control MSIs and 2 pairs of the 50-g group (these animals were reclassified into the control group, but their MSIc and MSIr measurements were treated as missing). One mobile MSI in the 50-g group was identified at week 2; it was immediately replaced with another MSI placed laterally to the original implant site. The overall MSI success rate was 88% (65 of 74). The biometric measurements showed significant increases in sutural separation over time. Multi-level comparisons showed statistically significant differences in the patterns of separations between the 4 force groups for all measures (Table I). All expansion groups had a curvilinear—decelerating—patterns of expansion (Fig 5). The rates of sutural expansion were significantly greater during the first 14 days than during the last 14 days. The 200-g group showed the greatest separation, followed by the 100-g, 50-g, and 0-g groups. The control (0 g) group showed a nearly linear pattern of width increases (Fig 5). MSIc increased only slightly (0.7 mm) between days 0 and 42 in the control group. In contrast, MSIc increased by 5.8, 9.2, and 10.1 mm in the 50-g, 100-g, and 200-g groups, respectively (Fig 5, A). MSIr increased by 5.1, 8.1, and 8.9 mm in the 50-g, 100-g, and 200-g groups, compared with 0.8 mm in the control group (Fig 5, B). AB widths increased by 0.6 mm in the control group and by 3.2, 5.5, and 6.5 mm in the 50-g, 100-g, and 200-g groups (Fig 5, C). PB widths increased by 0.5, 3.1, 4.7, and 5.9 mm in the control, 50-g, 100-g, and
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Fig 3. Experimental timeline for records, including animal weights, caliper measurements, radiographs, and oxytetracycline and calcein bone labels.
Fig 4. Histomorhometric measures of: A, interbone label width between calcein and oxytetracycline administered at day 28 and 38; and B, sutural gap width at day 42.
200-g groups, respectively (Fig 5, D). Sutural separation, based on the average of AB and PB widths, increased by 0.6 mm in the control group, and by 3.2, 5.1, and 6.2 mm, in the 50-g, 100-g, and 200-g groups, respectively. Although the control and the 50-g groups showed minimal differences between AB and PB width increases, AB width increased by 0.74 and 0.53 mm more than PB width in the 100-g and 200-g groups, respectively, but group differences were not statistically significant. MSI lateral displacement between days 0 and 42 was not significant in the control group. The MSIs were displaced laterally in the experimental groups by 1.9, 3.0, and 2.7 mm for the 50-g, 100-g, and 200-g groups, respectively. MSI lateral displacement was statistically less
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in the control group than in the 3 experimental groups. There were no statistically significant differences in lateral displacement between the 3 experimental groups. Stained sections showed some stretching and disruption of the collagen fibers in the 100-g and 200-g groups. There were no obvious signs of sutural damage in the control and 50-g groups. The 3 fluorescent bone labels, administered at days 18 (oxytetracycline), 28 (calcein), and 38 (oxytetracycline), were distinct, narrow, and closely approximated each other in the control group (Fig 6, A). With expansion forces, the bone labels appeared diffused, and interlabel widths became larger. Discontinuities in bone labels were noted in the 3 experimental groups (Fig 6, B-D).
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Table I. Fixed and random parts of the polynomial models for inter-MSI width (mm), based on caliper measures (MSIc), radiographic inter-MSI measures (MSIr), anterior bone marker width (AB), and posterior bone marker width (PB), with the constant fixed at day 42 Fixed part Constant Force (g) 0 50 100 200 MSIr 0 50 100 200 AB 0 50 100 200 PB 0 50 100 200 MSIc
Coefficient 1.06E11 1.55E11 1.82E11 2.01E11 1.05E11 1.48E11 1.77E11 1.88E11 6.94E10 1.03E11 1.26E11 1.43E11 7.41E10 1.01E11 1.17E11 1.29E11
SE 3.39E-1 4.39E-1 4.37E-1 3.97E-1 3.34E-1 4.05E-1 4.30E-1 4.46E-1 2.80E-1 2.59E-1 4.44E-1 4.50E-1 2.28E-1 3.79E-1 3.55E-1 4.11E-1
Linear Coefficient 2.16E-4 3.29E-2 8.86E-3 1.50E-1 1.79E-2 2.25E-2 3.47E-3 6.16E-2 1.40E-2 5.12E-3 1.78E-2 1.20E-1 1.11E-2 4.80E-3 1.15E-2 2.63E-2
SE 9.02E-3 2.08E-2 2.86E-2 6.44E-2 6.13E-3 1.55E-2 2.72E-2 3.84E-2 1.96E-3 1.42E-2 1.79E-2 4.83E-2 1.86E10 1.65E-2 1.41E-2 2.68E-2
Random part Quadratic
Coefficient 4.27E-4 2.50E-3 5.01E-3 6.31E-3
Cubic
SE Coefficient SE 2.10E-4 4.80E-4 6.51E-4 4.61E-3 2.01E-4 6.38E-5
2.35E-3 3.58E-4 4.49E-3 6.20E-4 6.39E-3 8.76E-4 1.67E-3 3.24E-4 2.69E-3 4.08E-4 3.90E-3 3.08E-3 1.63E-3 3.77E-4 2.96E-3 3.21E-4 3.90E-3 6.11E-4
1.13E-4
Between animals
Variance 9.61E-1 1.44E10 1.16E10 9.23E-1 6.96E-1 1.31E10 1.16E10 7.80E-1 7.59E-1 4.66E-1 1.58E10 4.84E-5 1.54E10 4.73E-1 1.11E10 9.97E-1 1.03E10
SE 4.62E-1 7.52E-1 6.17E-1 4.95E-1 3.66E-1 6.39E-1 6.12E-1 4.98E-1 3.46E-1 2.37E-1 7.61E-1 7.58E-1 2.15E-1 5.46E-1 4.87E-1 5.48E-1
Between days Variance 3.83E-2 3.06E-1 5.88E-1 4.97E-1 2.31E-1 1.70E-1 5.31E-1 1.06E10 3.76E-2 1.46E-1 2.30E-1 2.86E-1 3.39E-2 1.97E-1 1.42E-1 5.15E-1
SE 1.31E-2 8.50E-2 1.60E-1 1.35E-1 7.67E-2 4.73E-2 1.45E-1 2.89E-1 9.72E-3 3.96E-2 6.26E-2 7.78E-2 8.76E-3 5.36E-2 3.87E-2 1.40E-1
Width 5 constant 1 (coefficient*day) 1 (coefficient*day2).
Sutural gaps after expansion increased with increasing forces. There were significant differences between the 4 force groups (Table II, Fig 7) in gap widths at the end of the experiment. Bone formation increased with increasing forces up to 100 g; there were no statistically significant differences in bone formation between the 100-g and 200-g groups. The differences in bone formation between the 50-g and the 200-g groups between days 18 and 28 were also not statistically significant. Bone formation decreased significantly over time in each group except for the 200-g group. Spearman correlations showed that individual rates of bone formation were related to rates of sutural separation. The correlations between sutural separation and bone formation were moderately high (r 5 0.84; P \0.001) between days 18 and 28, and moderate (r 5 0.73; P \0.001) between days 28 and 38. Slopes indicated that bone formation increased by 360 mm for every millimeter of sutural separation between days 18 and 28, and by 376 mm for every millimeter of sutural separation between days 28 and 38. There was no significant difference between the 2 slopes, indicating similar relationships over time (Fig 8). DISCUSSION
MSIs provided stable anchorage for suture expansion. Although the thread length was only 3 mm, the overall success rate was high (88%). A lower success rate (67.7%) was previously reported with similar 3-mm
long MSIs in a dog model.16 However, after the authors of that study excluded the MSIs that had sheared off and the MSIs that were placed in 1 dog with unusual chewing activities, their success rates increased to 95% for loaded MSIs and 82% for unloaded MSIs. Owens et al17 and Carrillo et al18 reported 86% and 99% success rates for 6-mm MSIs immediately placed and loaded in the beagle dogs. All the failures in our study occurred during the first 2 weeks; this was consistent with previous reports.16,17,19 This implies that the failures were mainly due to the lack of primary stability, because secondary stability was relatively low during the first few weeks of healing after implant placement.20 Four failures occurred in the control group, and the other 5 occurred in the 50-g group, suggesting that force loads might play a role. Similarly, Garfinkle et al21 reported that loaded MSIs have higher success rate than unloaded ones; they suggested that appropriate force magnitudes bolster initial mechanical retention and stimulate osseous adaptation. All groups, including the controls, showed slightly greater amounts of anterior than posterior expansion. Although higher forces produced greater anterior separation, there were no significant group differences. Clinically, rapid palatal expansion causes a similar fantype of sutural separation because there are more bones articulating in the posterior aspect than in the anterior aspect of the maxilla.22 The rabbit frontal bone articulates anteriorly with the nasal bone. However, it joins posteriorly with the parietal bone, which articulates
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Fig 5. Width changes over time for the 4 force groups: A, inter-MSIs widths measured with calipers (MSIc); B, radiographic inter-MSIs widths measured on the ventrodorsal radiographs (MSIr); C, anterior bone marker widths (AB); D, posterior bone marker widths (BP).
with more skeletal structures than the nasal bone. More posterior articulations seem to provide greater resistance and allow less sutural separation than do anterior articulations. The expansion forces produced significant amounts of lateral MSI movements. Although displacement was not significantly different between the 3 experimental groups, the averages indicated that there might have been a group effect, which the design of the experiment was not powerful enough to detect. It was shown that MSIs can move up to 2 mm in the bone when orthodontic forces are applied.23 Mortensen et al16 demonstrated that pairs of 3-mm long MSIs were displaced significantly more when loaded with 900 g (3.1 mm) than those loaded with 600 g (2.2 mm) of force. This suggests that higher forces produced greater MSI displacement within bone. Moreover, inter-MSI widths measured with calipers tended to be greater than inter-MSI widths measured radiographically. This difference increased with increasing forces and time. Caliper measurements were taken at the outermost MSI margins above the guide wire, whereas the radiographic measurements
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were taken at the intersections between the outermost margins and the wire. On that basis, the differences between the caliper and the radiographic measurements indicate increasing amounts of lateral MSI tipping. Although MSI movements can result from both displacement and tipping, our results could not distinguish between lateral displacement and lateral tipping. Sutural separation followed a decelerating curvilinear pattern over time. The control group showed less than 1 mm of sutural separation over the 42-day experimental period, indicating low growth potential. Inter-MSI and bone marker widths increased rapidly during the first 2 weeks in all experimental groups, with rates gradually decreasing thereafter. The higher force (100 and 200 g) groups showed substantially greater amounts of expansion initially, suggesting that there is only minimal resistance from sutural interdigitation in 6-week-old rabbits. Yen et al8 previously showed that rates of sutural separation gradually decrease over time. Reduced rates of separation might be due to the elastic limit of the connective tissue in the suture4 or the skeletal resistance of the articulated bones,24 including the parietal,
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Fig 6. Representative fluorescent bone labels of the 4 force groups: first label (green) toward bone, oxytetracycline administered on day 18; second label (red), calcein on day 28; third label (green), oxytetracycline on day 38. A, 0-g (control) group; B, 50-g group; C, 100-g group; D, 200-g group. Arrows indicate discontinuities of bone labels.
Table II. Median sutural gap widths and bone formation (BF) between days 18, 28, and 38 for the 4 force groups with
statistical comparisons between and within groups P values of post-hoc paired comparisons
Group Force (g) 0 Sutural gap (mm2) 112.7 51.8 BF d18-28 (mm2) 43.9 BF d28-38 (mm2) BF differences P values 0.001
50 300.7 275.3 166.2 \0.001
100 441.4 370.2 252.4 \0.001
200 740.6 341.8 314.1 NS
Group differences P values \0.001 \0.001 \0.001
0 vs 50 \0.001 \0.001 \0.001
0 vs 100 \0.001 \0.001 \0.001
0 vs 200 50 vs 100 50 vs 200 100 vs 200 \0.001 0.001 \0.001 0.013 \0.001 0.001 NS NS \0.001 0.001 \0.001 NS
NS, Not statistically significant.
zygomatic, and nasal bones. The decreasing rates of expansion were especially evident in the 200-g group, which plateaued after 28 days. The plateau suggests that there might be an upper limit to sutural separation.
Overall, higher forces produced greater sutural separation over time than lower forces. Although sutural separation was significantly different between each of the force groups, the group differences decreased with
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Fig 7. Bone formation between days 18 and 28 (BF days 18-28) and between days 28 and 38 (BF Days 28-38), as well as the sutural gaps between the 2 sutural bone margins for the 4 force groups.
increasing force (ie, the overall separation did not increase in proportion to increasing force magnitudes). This agrees with the results of Parr et al,13 who showed that 3 N of constant force produced less than 3 times the width increase of 1 N of force. This further supports the notion that resistance limits the amount of sutural separation, with higher forces reaching the limit before lower forces. In addition to skeletal resistance, the sutural connective tissues could also play a role.24 Even after eliminating skeletal resistance by removing structures surrounding the suture, separation remained restricted at higher force levels.7 Bone formation rates responded to increased forces but plateaued at the higher force levels. There were no differences in bone formation between the 100-g and the 200-g groups. Parr et al13 expanded the rabbit midnasal suture and showed no differences in bone formation rates between 1 N (about 102 g) and 3 N (about 306 g) of force delivered with coil springs over 35 days. It is possible that both of their groups reached a plateau of bone formation, as in our study. It was also previously shown that the DNA synthesis of fibroblasts and osteoblasts plateaus after a certain magnitude of expansion force.5 This supports the notion that excessive forces inhibit anabolic activities because of rupture of the tissue, bleeding, and so on.5 Excessive forces cause greater amounts of cell death, which could also explain the plateauing observed.12 Bone formation appears to be controlled by sutural separation. The rates of sutural separation were closely related to the rates of bone formation. For every millimeter
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Fig 8. Scatterplots and regression slopes for bone formation as a function of sutural separation between days 18 and 28 and days 28 and 38.
that the sutures were separated, 360 to 370 mm of bone formed. It was suggested that sutural bone formation is triggered by the stretching of sutural fibers connecting the bones.1,3 Our study provides the first direct evidence of a relationship between bone formation and sutural separation. Previous studies have shown differences in the synthesis of osteoblasts and fibroblasts, neither of which provide direct evidence of bone formation over time.5-7 Interestingly, even though the 200-g expansion force produced greater sutural separation than the other 3 force levels, it did not produce more bone formation than did the 100-g force. It has been suggested that heavy forces can stretch connected collagen fibers beyond the elastic limits, causing tissue rupture and limiting sutural growth.4 In the 200-g group, the sutural gap was significantly wider than in the other 3 groups, indicating that the highest forces might contribute more to sutural separation than bone formation. Although higher forces produced greater sutural separation than lower forces, excessive forces cannot be recommended for several reasons. As shown, sutural separation is limited at the highest force magnitudes, because of possible resistance of the articulated skeleton.24 Second, heavy forces can cause tissue damage4 and discomfort or pain in patients.25 Third, higher force levels that do not produce more bone are inefficient; they have greater relapse potential after the forces are removed because of the pullback tendency of the fiber stretching1,4; they also require longer periods of retention. For example, 3 to 6 months of retention has been recommended after rapid palatal expansion to allow the sutural gap to fill with bone26; without retention, relapse can amount to 45% of the sutural separation.27
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CONCLUSIONS
The results support the notion that ideal force levels produce the maximum amount of sutural bone growth. By using varying amounts of constant force anchored by MSIs, the following causal relationships were established. 1.
2. 3.
The amount of sutural separation increases with increasing force levels, but the increases are not proportional. Bone formation increases with increasing force, but the increases are limited at the highest force levels. Bone formation is directly related to the amount of sutural separation produced.
We thank GAC International for the coil springs, Dentos for the miniscrew implants, and E. Gerald Hill for invaluable assistance with animal care and the surgical procedures. REFERENCES 1. Murray JM, Cleall JF. Early tissue response to rapid maxillary expansion in the midpalatal suture of the rhesus monkey. J Dent Res 1971;50:1654-60. 2. Cleall JF, Bayne DI, Posen JM, Subtelny JD. Expansion of the midpalatal suture in the monkey. Angle Orthod 1965;35:23-35. 3. Ten Cate AR, Freeman E, Dickinson JB. Sutural development: structure and its response to rapid expansion. Am J Orthod 1977;71:622-36. 4. Storey E. Tissue response to the movement of bones. Am J Orthod 1973;64:229-47. 5. Morndal O. The importance of force magnitude on the initial response to mechanical stimulation of osteogenic and soft tissue. Eur J Orthod 1987;9:288-94. 6. Steenvoorden GP, van de Velde JP, Prahl-Andersen B. The effect of duration and magnitude of tensile mechanical forces on sutural tissue in vivo. Eur J Orthod 1990;12:330-9. 7. Hickory WB, Nanda R. Effect of tensile force magnitude on release of cranial suture cells into S phase. Am J Orthod Dentofacial Orthop 1987;91:328-34. 8. Yen EH, Yue CS, Suga DM. Effect of force level on synthesis of type III and type I collagen in mouse interparietal suture. J Dent Res 1989;68:1746-51. 9. Hinrichsen GJ, Storey E. The effect of force on bone and bones. Angle Orthod 1968;38:155-65. 10. Southard KA, Forbes DP. The effects of force magnitude on a sutural model: a quantitative approach. Am J Orthod Dentofacial Orthop 1988;93:460-6.
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11. Miyawaki S, Forbes DP. The morphologic and biochemical effects of tensile force application to the interparietal suture of the Sprague-Dawley rat. Am J Orthod Dentofacial Orthop 1987;92: 123-33. 12. Zahrowski JJ, Turley PK. Force magnitude effects upon osteoprogenitor cells during premaxillary expansion in rats. Angle Orthod 1992;62:197-202. 13. Parr JA, Garetto LP, Wohlford ME, Arbuckle GR, Roberts WE. Sutural expansion using rigidly integrated endosseous implants: an experimental study in rabbits. Angle Orthod 1997;67:283-90. 14. von Fraunhofer JA, Bonds PW, Johnson BE. Force generation by orthodontic coil springs. Angle Orthod 1993;63:145-8. 15. Dahlberg G. Statistical methods for medical and biological students. New York: Intersciences Publications; 1940. 16. Mortensen MG, Buschang PH, Oliver DR, Kyung HM, Behrents RG. Stability of immediately loaded 3- and 6-mm miniscrew implants in beagle dogs—a pilot study. Am J Orthod Dentofacial Orthop 2009;136:251-9. 17. Owens SE, Buschang PH, Cope JB, Franco PF, Rossouw PE. Experimental evaluation of tooth movement in the beagle dog with the mini-screw implant for orthodontic anchorage. Am J Orthod Dentofacial Orthop 2007;132:639-46. 18. Carrillo R, Rossouw PE, Franco PF, Opperman LA, Buschang PH. Intrusion of multiradicular teeth and related root resorption with mini-screw implant anchorage: a radiographic evaluation. Am J Orthod Dentofacial Orthop 2007;132:647-55. 19. Melsen B, Costa A. Immediate loading of implants used for orthodontic anchorage. Clin Orthod Res 2000;3:23-8. 20. Raghavendra S, Wood MC, Taylor TD. Early wound healing around endosseous implants: a review of the literature. Int J Oral Maxillofac Implants 2005;20:425-31. 21. Garfinkle JS, Cunningham LL Jr, Beeman CS, Kluemper GT, Hicks EP, Kim MO. Evaluation of orthodontic mini-implant anchorage in premolar extraction therapy in adolescents. Am J Orthod Dentofacial Orthop 2008;133:642-53. 22. Wertz RA. Skeletal and dental changes accompanying rapid midpalatal suture opening. Am J Orthod 1970;58:41-66. 23. Liou EJ, Pai BC, Lin JC. Do miniscrews remain stationary under orthodontic forces? Am J Orthod Dentofacial Orthop 2004;126:42-7. 24. Isaacson JR, Ingram AH. Forces produced by rapid maxillary expansion. II. Forces present during treatment. Angle Orthod 1964; 34:261-9. 25. Needleman HL, Hoang CD, Allred E, Hertzberg J, Berde C. Reports of pain by children undergoing rapid palatal expansion. Pediatr Dent 2000;22:221-6. 26. Ekstrom C, Henrikson CO, Jensen R. Mineralization in the midpalatal suture after orthodontic expansion. Am J Orthod 1977;71: 449-55. 27. Hicks EP. Slow maxillary expansion. A clinical study of the skeletal versus dental response to low-magnitude force. Am J Orthod 1978;73:121-41.
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Is there an optimal force level for sutural expansion? Sean Shih-Yao Liu,a Lynne A. Opperman,b Hee-Moon Kyung,c and Peter H. Buschangd Indianapolis, Ind, Dallas, Tex, and Daegu, Korea
Introduction: The purpose of this study was to establish the causal relationships between expansion force magnitudes, sutural separation, and sutural bone formation. Methods: Thirty-seven 6-week-old rabbits were randomly assigned to 4 force groups (0, 50, 100, or 200 g). Constant forces were delivered for 42 days by nickel-titanium open-coil springs to miniscrew implants (MSIs) placed in the frontal bone on both sides of the midsagittal suture. Inter-MSI and bone marker widths were measured biweekly to quantify sutural separation and MSI movements. Sutural bone formation was quantified based on the incorporation of fluorescent bone labels administered at days 18, 28, and 38. Results: Nine of 74 MSIs failed between days 0 and 14, including 4 in the controls and 5 in the 50-g group. A decelerating curvilinear pattern of sutural separation was evident in the 50-g, 100-g, 200-g groups. Bone markers showed that sutural widths increased by 0.6, 3.2, 5.1, and 6.2 mm in the control, 50-g, 100-g, and 200-g groups, respectively. Except for the 200-g group, significantly greater amounts of bone formation were observed between days 18 and 28 than between days 28 and 38. Sutural bone formation also increased with increasing forces up to 100 g; there was no difference between the 100-g and the 200-g groups. Sutural separation explained 71% and 53% of the variations in bone formation between days 18 and 28 and days 28 and 38, respectively. Conclusions: Within the limits of this study, sutural bone formation is directly related to the amount of sutural separation, which is in turn related to the amount of force applied. The results suggest that there is a level of induced sutural separation that provides the greatest amount of bone formation. (Am J Orthod Dentofacial Orthop 2011;139:446-55)
I
t has been well established that sutural expansion stimulates bone formation. Once expansion forces are applied, tissue trauma occurs with disruption of collagen fibers and blood vessels,1,2 together with cell death of fibroblasts.3 However, most of the collagen fibers and the periosteum remains intact, and the osteoblast layers close to the bone margins survive to form successive lamellae within 3 to 4 days.3 As the suture separates, new bone forms along the collagen fibers aligned perpendicularly across the suture.3 a Assistant Professor, Department of Orthodontics and Oral Facial Genetics, School of Dentistry, Indiana University, Indianapolis. b Professor, Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M Health Science Center, Dallas. c Professor, Department of Orthodontics, Dental School, Kyungpook National University, Daegu, Korea. d Professor, Department of Orthodontics, Baylor College of Dentistry, Texas A&M Health Science Center, Dallas. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Supported financially by Texas A&M Health Science Center, Baylor College of Dentistry’s Department of Orthodontics. Reprint requests to: Sean Shih-Yao Liu, Department of Orthodontics and Oral Facial Genetics, Indiana University School of Dentistry, 1121 W Michigan St, Indianapolis, IN 46202; e-mail, [email protected]. Submitted, January 2009; revised and accepted, March 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.03.056
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Although higher forces have been related to greater sutural separation, the exact nature of the relationship remains unclear. Histologic and biometric studies have shown that short-term sutural separation relates to the magnitude of the expansion forces.4-8 Morndal,5 who expanded rats’ interpremaxillary sutures for 27 hours, confirmed that the amount of sutural separation was linearly related to the magnitude of force. Over a longer time period (28 days), Yen et al8 showed that the springs activated 2 mm with heavier forces produced faster rates of expansion than those activated 2 mm with lighter forces, even though both ultimately produced similar amounts of sutural separation. Importantly, all experimental studies used helical springs. Because the forces of these springs decay as the suture expands, it is difficult to quantify the exact nature of the relationships between forces and sutural separation.9 The relationship between sutural bone formation and the amount of expansion force remains poorly understood. The increases in alkaline phosphatase-stained cells associated with heavy force levels indicate increased bone formation.10,11 It has also been shown that DNA synthesis of fibroblasts and osteoblasts increases with increasing forces, and eventually plateaus at higher force levels.5 DNA synthesis of fibroblasts and osteoblasts has also
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been shown to decrease at higher forces, possibly due to tissue damage.7,12 In the only study that directly evaluated the relationships between bone formation and sutural expansion, Parr et al13 reported no significant difference in bone formation rates between 1-N and 3-N expansion forces. However, their mineral apposition rates suggested a possible difference; because of small sample sizes, the study’s power might have been insufficient to detect the differences. Whether there is an optimal force level to produce the greatest sutural bone formation remains questionable. Our goal was to better understand how sutures respond to varying amounts of force application. Histomorphometric and biometric analyses were performed to test the hypotheses that (1) sutural separation increases as a function of increasing expansion forces, (2) bone formation increases as a function of expansion force magnitude, and (3) bone formation is related to sutural separation. Our clinical objective was to determine whether there is an optimal force that maximizes sutural bone formation. If there is such a force, it provides a potential means of making expansion therapies more effective and efficient. MATERIAL AND METHODS
The sample included 37 six-week-old male New Zealand white rabbits. The housing, care, and experimental protocol were in accordance with the guidelines of the Institutional Animal Care and Use Committee of Baylor College of Dentistry. The rabbits were randomly assigned to 4 groups: 50-g force (n 5 11), 100-g force (n 5 9), 200-g force (n 5 9), or 0-g force (control) (n 5 8). The expansion forces were delivered for 42 days; no forces were used in the control group. All animals were anesthetized with ketamine at 75 mg per kilogram intramuscularly and 0.5% acepromazine at 5 mg per kilogram intramuscularly (Marcaine, Abbott Laboratories, Abbott Park, Ill), with 1:200,000 epinephrine serving as the local anesthetic. All surgical procedures were performed under sterile conditions. After reflecting the skin and the periosteum midway between the anterior and posterior limits of the orbital rims, the midsagittal suture and the frontal bone were exposed (Fig 1, A). Pilot holes were drilled with a size-2 round burr with a low-speed hand piece (\600 rpm) and copious saline-solution irrigation. Two custom-made Dentos (Daegu, Korea) miniscrew implants (MSIs; 3.0 mm long 3 1.7 mm in diameter) were placed approximately 4 mm on either side of the midsagittal suture with a manual driver (Fig 2). Four 99.95% tantalum bone markers (1.5 mm long 3 0.8 mm in diameter) were tapped into the skull 2 to 3 mm anterior and 2 to 3 mm posterior to the MSIs by using a custom-made stainless steel appliance. The bone
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markers were used to radiographically quantify the sutural separation and implant movements. All animals were given penicillin (60000 IU/lb/im) immediately after surgery to prevent infection and buprenorphine (0.02 mg/kg/sc; prn) to minimize discomfort. A 20-mm long and 0.020-in diameter stainless steel interabutment guide wire was engaged in the holes in the MSI heads (Fig 1, D). Fifteen-millimeter long Sentalloy (GAC International, Bohemia, NY) nickel-titanium open-coil springs, delivering 50, 100, or 200 g of force, were telescoped over the wire between the 2 MSIs. Two stop loops were bent to prevent the spring and wire from becoming dislodged. The forces exerted were maintained because the springs remained compressed at lengths ranging from 8 to 12 mm, and the springs’ lengths were checked biweekly.14 When necessary, sliding tubes were added to the guide wire to maintain the springs’ compressed lengths. Records, including animal weights, ventrodorsal cephalometric digital radiographs, and inter-MSI width caliper measurements, were obtained under anesthesia at days 0, 14, 28, and 42 (Fig 3). By using a customized head holder and a metal scale for calibration, standardized ventrodorsal radiographs (no. 4 film; Air Techniques, Melville, NY) were taken at 65 kVp and 10 mA for 12 seconds at fixed distances. To quantify bone formation, oxytetracycline (13.6 mg/lb/im) and calcein (10 mg/kg/im) fluorescent labels were administered to all animals. Oxytetracycline was given on days 18 and 38; calcein was given on day 28. It was previously shown that advancing bone fronts during sutural expansion incorporate fluorescent labels, making it possible to quantify new bone formation. Blinded biometric assessments were based on caliper and radiographic measurements. Caliper width measurements between each MSI pair (MSIc) were taken at the outermost margins immediately above the guide wire (Fig 2, A). The widths between the anterior (AB) and posterior (PB) bone markers and outermost margins of the MSI (MSIr) were measured on the digital radiographs (Fig 2, B) by using Visix software (Air Techniques). After 2 weeks, 80 radiographs were remeasured for establishing the intraexaminer method errors (AB, 0.11 mm; qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi MSIr, 0.10 mm; P PB, ffi 0.10 mm) by using Dahlberg’s formula ( ½ d2 =2n).15 To estimate sutural separation at the implant site, the AB and PB widths were averaged. The MSI lateral displacement during the study was determined by subtracting sutural separation from changes in MSIr width between days 0 and 42. The parallel nature of sutural separation was evaluated based on the differences between the AB and PB widths changes during the study.
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Fig 1. A, Two pilot holes drilled after exposure of the frontal bones and the midsagittal suture; B, 3-mm MSIs under the scanning electron microscope; C, 3-mm long MSI; D, a nickel-titanium open-coil spring telescoped on a guiding wire between the 2 MSIs, with a sliding tube used to maintain the activation level.
After 42 days of expansion, the rabbits were killed with an overdose of beuthanasia (intracardiac injection of 1 cm3 per animal) and perfused with 70% ethanol. A standardized region, including the midsagittal suture and adjacent bone, was dissected and fixed with 70% ethanol for 2 weeks without decalcification. After dehydration with an ascending series of ethanol (70%-100%), the specimens were embedded in methylmethacrylate and sectioned (approximately 60 mm) coronally by using a diamond saw (3 sequential sections per animal), followed by grinding and polishing. Images were captured by an 80i epifluorescence microscope (Nikon, Melville, NY; excitation wave lengths of 390 nm for oxytetracycline and 485 nm for calcein) with a Coolsnap K4 (Photometrics, Tucson, Ariz) camera and MetaMorph software (version 6.3, Molecular
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Devices, Sunnyvale, Calif). Bioquant Osteo II software (Nashville, Tenn) was used to trace the bone labels over the entire suture, from the ectocranial to the endocranial surfaces. All data processing was performed by 1 blinded examiner (S.S.-Y.L.). Bone formation between the leading edges of label pairs was automatically measured every 10 mm along the entire length of the suture (Fig 4). The first pair of labels quantified bone formation between day 18 (oxytetracycline) and day 28 (calcein); the second pair quantified bone formation between day 28 (calcein) and day 38 (oxytetracycline). Sutural gaps after expansion were measured every 10 mm between the 2 trailing edges of the bone labels at day 38 (oxytetracycline). Measurements of bone formation and sutural gaps were based on the averages of the 3 serial sections obtained for each animal.
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B
A
calculated. This allowed each animal’s curve to be calculated. The curves were used to provide estimates of the biometric changes between days 18 and 28 and days 28 and 38. Statistical analyses of the histomorphometric measurements were performed by using SPSS software (version 15.0, SPSS, Chicago, Ill). Kruskal-Wallis analyses were performed to compare the groups. The MannWhitney U test was used for paired group comparisons with Bonferroni adjustments. Bone-formation amounts between days 18 and 28 and days 28 and 38 were compared by using the Wilcoxon signed rank test. Spearman correlation analyses were used to evaluate relationships between sutural separation and bone formation. The slopes of the relationship were estimated by using the least squares method. RESULTS
Fig 2. A, Illustration used to evaluate inter-MSIs width (MSIc) with a caliper (note the lateral MSI tipping during expansion); B, schematic of ventrodorsal radiographs used to evaluate inter-MSIs width (MSIr) between the outermost margins of the paired MSIs, the anterior bone marker width (AB) between the centers of the anterior pair bone markers, and the posterior bone marker width (PB) between the centers of the posterior pair bone markers. MSI; bone marker.
Four animals were randomly selected from each force group to evaluate histologic changes. Their sections were stained with Stevenel’s blue and Van Gieson picro-fuchsin and evaluated under a light microscope. Statistical analysis
All statistical procedures were performed by using the Multilevel Win program (version 2.0, Bristol, United Kingdom) with a 95% confidence interval (P 5 0.05). The curves describing the changes of the repeated caliper, radiographic widths, and weight measurements were modeled over time as polynomials, by using the iterative generalized least squares method. The fixed part of the models described the changes and statistically compared the groups. The random part of the models had animals at the higher level and their repeated measures (weeks) at the lower level, nested in the higher level. To evaluate group differences at the end of expansion, the constant of each polynomial was fixed at 42 days. After the order of the models had been statistically verified, the residuals of each animal’s curves were
The animals’ weights increased by 1222 to 1321 g (76%-84%) over the time of treatment. There were no significant weight differences between the 4 groups, no signs of infection around the surgical sites, and no obvious signs of discomfort during the study. A total of 8 MSIs failed during the first 2 weeks, including 2 pairs of control MSIs and 2 pairs of the 50-g group (these animals were reclassified into the control group, but their MSIc and MSIr measurements were treated as missing). One mobile MSI in the 50-g group was identified at week 2; it was immediately replaced with another MSI placed laterally to the original implant site. The overall MSI success rate was 88% (65 of 74). The biometric measurements showed significant increases in sutural separation over time. Multi-level comparisons showed statistically significant differences in the patterns of separations between the 4 force groups for all measures (Table I). All expansion groups had a curvilinear—decelerating—patterns of expansion (Fig 5). The rates of sutural expansion were significantly greater during the first 14 days than during the last 14 days. The 200-g group showed the greatest separation, followed by the 100-g, 50-g, and 0-g groups. The control (0 g) group showed a nearly linear pattern of width increases (Fig 5). MSIc increased only slightly (0.7 mm) between days 0 and 42 in the control group. In contrast, MSIc increased by 5.8, 9.2, and 10.1 mm in the 50-g, 100-g, and 200-g groups, respectively (Fig 5, A). MSIr increased by 5.1, 8.1, and 8.9 mm in the 50-g, 100-g, and 200-g groups, compared with 0.8 mm in the control group (Fig 5, B). AB widths increased by 0.6 mm in the control group and by 3.2, 5.5, and 6.5 mm in the 50-g, 100-g, and 200-g groups (Fig 5, C). PB widths increased by 0.5, 3.1, 4.7, and 5.9 mm in the control, 50-g, 100-g, and
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Fig 3. Experimental timeline for records, including animal weights, caliper measurements, radiographs, and oxytetracycline and calcein bone labels.
Fig 4. Histomorhometric measures of: A, interbone label width between calcein and oxytetracycline administered at day 28 and 38; and B, sutural gap width at day 42.
200-g groups, respectively (Fig 5, D). Sutural separation, based on the average of AB and PB widths, increased by 0.6 mm in the control group, and by 3.2, 5.1, and 6.2 mm, in the 50-g, 100-g, and 200-g groups, respectively. Although the control and the 50-g groups showed minimal differences between AB and PB width increases, AB width increased by 0.74 and 0.53 mm more than PB width in the 100-g and 200-g groups, respectively, but group differences were not statistically significant. MSI lateral displacement between days 0 and 42 was not significant in the control group. The MSIs were displaced laterally in the experimental groups by 1.9, 3.0, and 2.7 mm for the 50-g, 100-g, and 200-g groups, respectively. MSI lateral displacement was statistically less
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in the control group than in the 3 experimental groups. There were no statistically significant differences in lateral displacement between the 3 experimental groups. Stained sections showed some stretching and disruption of the collagen fibers in the 100-g and 200-g groups. There were no obvious signs of sutural damage in the control and 50-g groups. The 3 fluorescent bone labels, administered at days 18 (oxytetracycline), 28 (calcein), and 38 (oxytetracycline), were distinct, narrow, and closely approximated each other in the control group (Fig 6, A). With expansion forces, the bone labels appeared diffused, and interlabel widths became larger. Discontinuities in bone labels were noted in the 3 experimental groups (Fig 6, B-D).
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Table I. Fixed and random parts of the polynomial models for inter-MSI width (mm), based on caliper measures (MSIc), radiographic inter-MSI measures (MSIr), anterior bone marker width (AB), and posterior bone marker width (PB), with the constant fixed at day 42 Fixed part Constant Force (g) 0 50 100 200 MSIr 0 50 100 200 AB 0 50 100 200 PB 0 50 100 200 MSIc
Coefficient 1.06E11 1.55E11 1.82E11 2.01E11 1.05E11 1.48E11 1.77E11 1.88E11 6.94E10 1.03E11 1.26E11 1.43E11 7.41E10 1.01E11 1.17E11 1.29E11
SE 3.39E-1 4.39E-1 4.37E-1 3.97E-1 3.34E-1 4.05E-1 4.30E-1 4.46E-1 2.80E-1 2.59E-1 4.44E-1 4.50E-1 2.28E-1 3.79E-1 3.55E-1 4.11E-1
Linear Coefficient 2.16E-4 3.29E-2 8.86E-3 1.50E-1 1.79E-2 2.25E-2 3.47E-3 6.16E-2 1.40E-2 5.12E-3 1.78E-2 1.20E-1 1.11E-2 4.80E-3 1.15E-2 2.63E-2
SE 9.02E-3 2.08E-2 2.86E-2 6.44E-2 6.13E-3 1.55E-2 2.72E-2 3.84E-2 1.96E-3 1.42E-2 1.79E-2 4.83E-2 1.86E10 1.65E-2 1.41E-2 2.68E-2
Random part Quadratic
Coefficient 4.27E-4 2.50E-3 5.01E-3 6.31E-3
Cubic
SE Coefficient SE 2.10E-4 4.80E-4 6.51E-4 4.61E-3 2.01E-4 6.38E-5
2.35E-3 3.58E-4 4.49E-3 6.20E-4 6.39E-3 8.76E-4 1.67E-3 3.24E-4 2.69E-3 4.08E-4 3.90E-3 3.08E-3 1.63E-3 3.77E-4 2.96E-3 3.21E-4 3.90E-3 6.11E-4
1.13E-4
Between animals
Variance 9.61E-1 1.44E10 1.16E10 9.23E-1 6.96E-1 1.31E10 1.16E10 7.80E-1 7.59E-1 4.66E-1 1.58E10 4.84E-5 1.54E10 4.73E-1 1.11E10 9.97E-1 1.03E10
SE 4.62E-1 7.52E-1 6.17E-1 4.95E-1 3.66E-1 6.39E-1 6.12E-1 4.98E-1 3.46E-1 2.37E-1 7.61E-1 7.58E-1 2.15E-1 5.46E-1 4.87E-1 5.48E-1
Between days Variance 3.83E-2 3.06E-1 5.88E-1 4.97E-1 2.31E-1 1.70E-1 5.31E-1 1.06E10 3.76E-2 1.46E-1 2.30E-1 2.86E-1 3.39E-2 1.97E-1 1.42E-1 5.15E-1
SE 1.31E-2 8.50E-2 1.60E-1 1.35E-1 7.67E-2 4.73E-2 1.45E-1 2.89E-1 9.72E-3 3.96E-2 6.26E-2 7.78E-2 8.76E-3 5.36E-2 3.87E-2 1.40E-1
Width 5 constant 1 (coefficient*day) 1 (coefficient*day2).
Sutural gaps after expansion increased with increasing forces. There were significant differences between the 4 force groups (Table II, Fig 7) in gap widths at the end of the experiment. Bone formation increased with increasing forces up to 100 g; there were no statistically significant differences in bone formation between the 100-g and 200-g groups. The differences in bone formation between the 50-g and the 200-g groups between days 18 and 28 were also not statistically significant. Bone formation decreased significantly over time in each group except for the 200-g group. Spearman correlations showed that individual rates of bone formation were related to rates of sutural separation. The correlations between sutural separation and bone formation were moderately high (r 5 0.84; P \0.001) between days 18 and 28, and moderate (r 5 0.73; P \0.001) between days 28 and 38. Slopes indicated that bone formation increased by 360 mm for every millimeter of sutural separation between days 18 and 28, and by 376 mm for every millimeter of sutural separation between days 28 and 38. There was no significant difference between the 2 slopes, indicating similar relationships over time (Fig 8). DISCUSSION
MSIs provided stable anchorage for suture expansion. Although the thread length was only 3 mm, the overall success rate was high (88%). A lower success rate (67.7%) was previously reported with similar 3-mm
long MSIs in a dog model.16 However, after the authors of that study excluded the MSIs that had sheared off and the MSIs that were placed in 1 dog with unusual chewing activities, their success rates increased to 95% for loaded MSIs and 82% for unloaded MSIs. Owens et al17 and Carrillo et al18 reported 86% and 99% success rates for 6-mm MSIs immediately placed and loaded in the beagle dogs. All the failures in our study occurred during the first 2 weeks; this was consistent with previous reports.16,17,19 This implies that the failures were mainly due to the lack of primary stability, because secondary stability was relatively low during the first few weeks of healing after implant placement.20 Four failures occurred in the control group, and the other 5 occurred in the 50-g group, suggesting that force loads might play a role. Similarly, Garfinkle et al21 reported that loaded MSIs have higher success rate than unloaded ones; they suggested that appropriate force magnitudes bolster initial mechanical retention and stimulate osseous adaptation. All groups, including the controls, showed slightly greater amounts of anterior than posterior expansion. Although higher forces produced greater anterior separation, there were no significant group differences. Clinically, rapid palatal expansion causes a similar fantype of sutural separation because there are more bones articulating in the posterior aspect than in the anterior aspect of the maxilla.22 The rabbit frontal bone articulates anteriorly with the nasal bone. However, it joins posteriorly with the parietal bone, which articulates
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mm
mm
A
0
7
14
21
28
35
42
Days
Days
Fig 5. Width changes over time for the 4 force groups: A, inter-MSIs widths measured with calipers (MSIc); B, radiographic inter-MSIs widths measured on the ventrodorsal radiographs (MSIr); C, anterior bone marker widths (AB); D, posterior bone marker widths (BP).
with more skeletal structures than the nasal bone. More posterior articulations seem to provide greater resistance and allow less sutural separation than do anterior articulations. The expansion forces produced significant amounts of lateral MSI movements. Although displacement was not significantly different between the 3 experimental groups, the averages indicated that there might have been a group effect, which the design of the experiment was not powerful enough to detect. It was shown that MSIs can move up to 2 mm in the bone when orthodontic forces are applied.23 Mortensen et al16 demonstrated that pairs of 3-mm long MSIs were displaced significantly more when loaded with 900 g (3.1 mm) than those loaded with 600 g (2.2 mm) of force. This suggests that higher forces produced greater MSI displacement within bone. Moreover, inter-MSI widths measured with calipers tended to be greater than inter-MSI widths measured radiographically. This difference increased with increasing forces and time. Caliper measurements were taken at the outermost MSI margins above the guide wire, whereas the radiographic measurements
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were taken at the intersections between the outermost margins and the wire. On that basis, the differences between the caliper and the radiographic measurements indicate increasing amounts of lateral MSI tipping. Although MSI movements can result from both displacement and tipping, our results could not distinguish between lateral displacement and lateral tipping. Sutural separation followed a decelerating curvilinear pattern over time. The control group showed less than 1 mm of sutural separation over the 42-day experimental period, indicating low growth potential. Inter-MSI and bone marker widths increased rapidly during the first 2 weeks in all experimental groups, with rates gradually decreasing thereafter. The higher force (100 and 200 g) groups showed substantially greater amounts of expansion initially, suggesting that there is only minimal resistance from sutural interdigitation in 6-week-old rabbits. Yen et al8 previously showed that rates of sutural separation gradually decrease over time. Reduced rates of separation might be due to the elastic limit of the connective tissue in the suture4 or the skeletal resistance of the articulated bones,24 including the parietal,
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Fig 6. Representative fluorescent bone labels of the 4 force groups: first label (green) toward bone, oxytetracycline administered on day 18; second label (red), calcein on day 28; third label (green), oxytetracycline on day 38. A, 0-g (control) group; B, 50-g group; C, 100-g group; D, 200-g group. Arrows indicate discontinuities of bone labels.
Table II. Median sutural gap widths and bone formation (BF) between days 18, 28, and 38 for the 4 force groups with
statistical comparisons between and within groups P values of post-hoc paired comparisons
Group Force (g) 0 Sutural gap (mm2) 112.7 51.8 BF d18-28 (mm2) 43.9 BF d28-38 (mm2) BF differences P values 0.001
50 300.7 275.3 166.2 \0.001
100 441.4 370.2 252.4 \0.001
200 740.6 341.8 314.1 NS
Group differences P values \0.001 \0.001 \0.001
0 vs 50 \0.001 \0.001 \0.001
0 vs 100 \0.001 \0.001 \0.001
0 vs 200 50 vs 100 50 vs 200 100 vs 200 \0.001 0.001 \0.001 0.013 \0.001 0.001 NS NS \0.001 0.001 \0.001 NS
NS, Not statistically significant.
zygomatic, and nasal bones. The decreasing rates of expansion were especially evident in the 200-g group, which plateaued after 28 days. The plateau suggests that there might be an upper limit to sutural separation.
Overall, higher forces produced greater sutural separation over time than lower forces. Although sutural separation was significantly different between each of the force groups, the group differences decreased with
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BF Days 18-28
BF Days 28-38
800
Sutural gap
700
1600
Days 28-38
600
Bone formation (µm)
1400 1200 µm
Days 18-28
1000 800 600
500 400 300 200 100
400
0
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0
0.2
0.4
0.6
0.8
1
1.2
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1.6
Sutural separartion (mm)
0 0
50
100
200
R=.841; Bone formation18-28=26.013+(359.58*Sutural separation18-28) R=.731; Bone formation28-38=59.232+(375.62*Sutural separation28-38)
Force (g)
Fig 7. Bone formation between days 18 and 28 (BF days 18-28) and between days 28 and 38 (BF Days 28-38), as well as the sutural gaps between the 2 sutural bone margins for the 4 force groups.
increasing force (ie, the overall separation did not increase in proportion to increasing force magnitudes). This agrees with the results of Parr et al,13 who showed that 3 N of constant force produced less than 3 times the width increase of 1 N of force. This further supports the notion that resistance limits the amount of sutural separation, with higher forces reaching the limit before lower forces. In addition to skeletal resistance, the sutural connective tissues could also play a role.24 Even after eliminating skeletal resistance by removing structures surrounding the suture, separation remained restricted at higher force levels.7 Bone formation rates responded to increased forces but plateaued at the higher force levels. There were no differences in bone formation between the 100-g and the 200-g groups. Parr et al13 expanded the rabbit midnasal suture and showed no differences in bone formation rates between 1 N (about 102 g) and 3 N (about 306 g) of force delivered with coil springs over 35 days. It is possible that both of their groups reached a plateau of bone formation, as in our study. It was also previously shown that the DNA synthesis of fibroblasts and osteoblasts plateaus after a certain magnitude of expansion force.5 This supports the notion that excessive forces inhibit anabolic activities because of rupture of the tissue, bleeding, and so on.5 Excessive forces cause greater amounts of cell death, which could also explain the plateauing observed.12 Bone formation appears to be controlled by sutural separation. The rates of sutural separation were closely related to the rates of bone formation. For every millimeter
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Fig 8. Scatterplots and regression slopes for bone formation as a function of sutural separation between days 18 and 28 and days 28 and 38.
that the sutures were separated, 360 to 370 mm of bone formed. It was suggested that sutural bone formation is triggered by the stretching of sutural fibers connecting the bones.1,3 Our study provides the first direct evidence of a relationship between bone formation and sutural separation. Previous studies have shown differences in the synthesis of osteoblasts and fibroblasts, neither of which provide direct evidence of bone formation over time.5-7 Interestingly, even though the 200-g expansion force produced greater sutural separation than the other 3 force levels, it did not produce more bone formation than did the 100-g force. It has been suggested that heavy forces can stretch connected collagen fibers beyond the elastic limits, causing tissue rupture and limiting sutural growth.4 In the 200-g group, the sutural gap was significantly wider than in the other 3 groups, indicating that the highest forces might contribute more to sutural separation than bone formation. Although higher forces produced greater sutural separation than lower forces, excessive forces cannot be recommended for several reasons. As shown, sutural separation is limited at the highest force magnitudes, because of possible resistance of the articulated skeleton.24 Second, heavy forces can cause tissue damage4 and discomfort or pain in patients.25 Third, higher force levels that do not produce more bone are inefficient; they have greater relapse potential after the forces are removed because of the pullback tendency of the fiber stretching1,4; they also require longer periods of retention. For example, 3 to 6 months of retention has been recommended after rapid palatal expansion to allow the sutural gap to fill with bone26; without retention, relapse can amount to 45% of the sutural separation.27
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CONCLUSIONS
The results support the notion that ideal force levels produce the maximum amount of sutural bone growth. By using varying amounts of constant force anchored by MSIs, the following causal relationships were established. 1.
2. 3.
The amount of sutural separation increases with increasing force levels, but the increases are not proportional. Bone formation increases with increasing force, but the increases are limited at the highest force levels. Bone formation is directly related to the amount of sutural separation produced.
We thank GAC International for the coil springs, Dentos for the miniscrew implants, and E. Gerald Hill for invaluable assistance with animal care and the surgical procedures. REFERENCES 1. Murray JM, Cleall JF. Early tissue response to rapid maxillary expansion in the midpalatal suture of the rhesus monkey. J Dent Res 1971;50:1654-60. 2. Cleall JF, Bayne DI, Posen JM, Subtelny JD. Expansion of the midpalatal suture in the monkey. Angle Orthod 1965;35:23-35. 3. Ten Cate AR, Freeman E, Dickinson JB. Sutural development: structure and its response to rapid expansion. Am J Orthod 1977;71:622-36. 4. Storey E. Tissue response to the movement of bones. Am J Orthod 1973;64:229-47. 5. Morndal O. The importance of force magnitude on the initial response to mechanical stimulation of osteogenic and soft tissue. Eur J Orthod 1987;9:288-94. 6. Steenvoorden GP, van de Velde JP, Prahl-Andersen B. The effect of duration and magnitude of tensile mechanical forces on sutural tissue in vivo. Eur J Orthod 1990;12:330-9. 7. Hickory WB, Nanda R. Effect of tensile force magnitude on release of cranial suture cells into S phase. Am J Orthod Dentofacial Orthop 1987;91:328-34. 8. Yen EH, Yue CS, Suga DM. Effect of force level on synthesis of type III and type I collagen in mouse interparietal suture. J Dent Res 1989;68:1746-51. 9. Hinrichsen GJ, Storey E. The effect of force on bone and bones. Angle Orthod 1968;38:155-65. 10. Southard KA, Forbes DP. The effects of force magnitude on a sutural model: a quantitative approach. Am J Orthod Dentofacial Orthop 1988;93:460-6.
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11. Miyawaki S, Forbes DP. The morphologic and biochemical effects of tensile force application to the interparietal suture of the Sprague-Dawley rat. Am J Orthod Dentofacial Orthop 1987;92: 123-33. 12. Zahrowski JJ, Turley PK. Force magnitude effects upon osteoprogenitor cells during premaxillary expansion in rats. Angle Orthod 1992;62:197-202. 13. Parr JA, Garetto LP, Wohlford ME, Arbuckle GR, Roberts WE. Sutural expansion using rigidly integrated endosseous implants: an experimental study in rabbits. Angle Orthod 1997;67:283-90. 14. von Fraunhofer JA, Bonds PW, Johnson BE. Force generation by orthodontic coil springs. Angle Orthod 1993;63:145-8. 15. Dahlberg G. Statistical methods for medical and biological students. New York: Intersciences Publications; 1940. 16. Mortensen MG, Buschang PH, Oliver DR, Kyung HM, Behrents RG. Stability of immediately loaded 3- and 6-mm miniscrew implants in beagle dogs—a pilot study. Am J Orthod Dentofacial Orthop 2009;136:251-9. 17. Owens SE, Buschang PH, Cope JB, Franco PF, Rossouw PE. Experimental evaluation of tooth movement in the beagle dog with the mini-screw implant for orthodontic anchorage. Am J Orthod Dentofacial Orthop 2007;132:639-46. 18. Carrillo R, Rossouw PE, Franco PF, Opperman LA, Buschang PH. Intrusion of multiradicular teeth and related root resorption with mini-screw implant anchorage: a radiographic evaluation. Am J Orthod Dentofacial Orthop 2007;132:647-55. 19. Melsen B, Costa A. Immediate loading of implants used for orthodontic anchorage. Clin Orthod Res 2000;3:23-8. 20. Raghavendra S, Wood MC, Taylor TD. Early wound healing around endosseous implants: a review of the literature. Int J Oral Maxillofac Implants 2005;20:425-31. 21. Garfinkle JS, Cunningham LL Jr, Beeman CS, Kluemper GT, Hicks EP, Kim MO. Evaluation of orthodontic mini-implant anchorage in premolar extraction therapy in adolescents. Am J Orthod Dentofacial Orthop 2008;133:642-53. 22. Wertz RA. Skeletal and dental changes accompanying rapid midpalatal suture opening. Am J Orthod 1970;58:41-66. 23. Liou EJ, Pai BC, Lin JC. Do miniscrews remain stationary under orthodontic forces? Am J Orthod Dentofacial Orthop 2004;126:42-7. 24. Isaacson JR, Ingram AH. Forces produced by rapid maxillary expansion. II. Forces present during treatment. Angle Orthod 1964; 34:261-9. 25. Needleman HL, Hoang CD, Allred E, Hertzberg J, Berde C. Reports of pain by children undergoing rapid palatal expansion. Pediatr Dent 2000;22:221-6. 26. Ekstrom C, Henrikson CO, Jensen R. Mineralization in the midpalatal suture after orthodontic expansion. Am J Orthod 1977;71: 449-55. 27. Hicks EP. Slow maxillary expansion. A clinical study of the skeletal versus dental response to low-magnitude force. Am J Orthod 1978;73:121-41.
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