Effects of recombinant human bone morphogenetic protein-2 on midsagittal sutural bone formation during expansion

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Effects of recombinant human bone morphogenetic protein-2 on midsagittal sutural bone formation during expansion Sean Shih-Yao Liu,a Lynne A. Opperman,b and Peter H. Buschangc Indianapolis, Ind, and Dallas, Tex Introduction: The goal of this study was to evaluate whether human recombinant bone morphogenetic protein-2 (rhBMP-2) enhances sutural bone formation or causes premature sutural fusion. Methods: Thirty 6-week-old rabbits underwent midsagittal sutural expansion. The animals were randomly assigned to receive 0 (control), 0.1 mg per milliliter, or 0.4 mg per milliliter of rhBMP-2, delivered by an absorbable collagen sponge placed over the suture. A 100-g constant force was delivered for 33 days by using a nickel-titanium spring to expand the suture between 2 miniscrew implants anchored in the frontal bone. At days 10, 20, and 30, sutural separation was evaluated and modeled over time as polynomials by using multilevel statistical procedures. Bone formation and sutural gaps were analyzed histomorphometrically between days 10 and 20 and days 20 and 30. Results: The control group showed significantly greater overall sutural bone formation than did the 2 rhBMP-2 groups. Over time, bone formation decreased significantly in all groups. Between days 10 and 20, the 0.4 mg per milliliter group produced significantly more (58%) bone than did the 0.1 mg per milliliter group; there were no significant differences in bone formation between the 2 experimental groups between days 20 and 30. Both 0.1 and 0.4 mg per milliliter of rhBMP-2 in the absorbable collagen sponge caused premature fusion by forming a bony bridge connecting the ectocranial aspect of the sutural margins. Premature fusion significantly reduced sutural separation between 10 and 30 days (to 56% and 62% of control values for the 0.1 and 0.4 mg per milliliter groups, respectively). There were no significant differences in sutural separation between the 0.1 and 0.4 mg per milliliter groups. Conclusions: Compared with the 0.1 mg per milliliter group, 0.4 mg per milliliter of rhBMP-2 accelerated sutural bone formation between days 10 and 20. After 10 to 20 days, rhBMP-2 in the absorbable collagen sponge caused premature sutural fusion, despite the constant expansion forces. (Am J Orthod Dentofacial Orthop 2009;136:768.e1-768.e8)

S

utural expansion is routinely used to widen and protract the maxilla.1,2 Depending on the magnitude of force applied, sutural separation can be controlled.3 The amount of bone that forms at the sutures depends, at least partially, on the amount of sutural separation, which makes it possible to partially control sutural growth.3 However, new bone usually generates at

a Assistant professor, Department of Orthodontics and Oral Facial Genetics, School of Dentistry, Indiana University, Indianapolis. b Professor, Biomedical Sciences Department, Baylor College of Dentistry, Texas A&M University Health Science Center, Dallas, Tex. c Professor, Department of Orthodontics, Baylor College of Dentistry, Texas A&M University Health Science Center, Dallas, Tex. Supported by the Robert E. Gaylord Endowed Chair in Orthodontics. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. 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, December 2008; revised and accepted, March 2009. 0889-5406/$36.00 Copyright Ó 2009 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.03.035

a slower rate than sutural separation, with relapse potential of the segments depending on the size of the sutural gap.4 On this basis, 3 to 6 months of retention have been recommended to allow bone growth to fill the sutural gap after maxillary expansion5; without retention, relapse is immediate and can be as high as 45%.6 To expedite treatment while maximizing stability, an alternative approach to accelerate sutural bone formation is required. Human recombinant bone morphogenetic protein-2 (rhBMP-2) might provide a means to accelerate sutural bone formation. RhBMP-2 stimulates osteoblast differentiation and bone formation.7,8 Randomized controlled trials have shown that rhBMP-2 can be used as an alternative to bone grafts for enhancing bone formation in the human spine9 and the maxillary sinus.10 It also accelerates fracture-healing and wound-healing rates in patients with open tibial fractures.11 Compared with autografts, rhBMP-2 has been shown to increase the success rates and shorten the time required for anterior lumbar fusion.12 In rabbits, rhBMP-2 has been shown to accelerate healing after ulnar osteotomy by 33%.13 Experimental studies have also shown that rhBMP-2 768.e1

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Fig 1. Surgical procedures: A, MSI placement with a manual driver; B, transverse periosteal incision; C, periosteum elevation; D, rhBMP-2 placement; E, sutured placement site and the expansion device at day 10; F, a NiTi coil spring applying 100 g of force between 2 MSIs with a sliding tube.

induces bone formation to fill the cranial bone defects14,15 and enhances callus formation during mandibular distraction osteogenesis.16 The effects of rhBMP-2 on sutural growth are still unknown. It is also unknown whether rhBMP-2 causes premature sutural fusion. Injection of noggin, the antagonist of BMP-2, has been shown to maintain the patency of the mouse posterior frontal suture, which normally fuses within 3 to 4 weeks after birth.17 Premature fusion of sutures with BMP-2 might be expected because sutures are histologically similar to the periodontal ligament (both consist of collagen fibers connecting hard-tissue structures). When rhBMP-2 was applied to regenerate the periodontium, partial ankylosis was often observed between the root and adjacent bone.18-20 The aim of this study was to understand how rhBMP-2 affects sutures that are actively being expanded. We hypothesized that rhBMP-2 enhances sutural bone formation when a constant force is applied.

MATERIAL AND METHODS

The sample included 30 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. All animals underwent midsagittal sutural expansion with a 100-g constant force. They all had absorbable collagen sponges (ACS) surgically placed over the suture. The animals were randomly assigned to 3 groups, including 2 experimental groups treated with 0.1 and 0.4 mg per millileter of rhBMP-2, respectively, and a control group with only buffer solution in the ACS.

All animals were anesthetized with ketamine at 75 mg per kilogram intramuscularly and acepromazine at 5 mg per kilogram intramuscularly. Surgical procedures were performed under sterile conditions. Two small (approximately 2 mm) periosteal incisions were made 4 to 5 mm on either side of the midsagittal suture, midway between the anterior and posterior limits of the orbital rims. Two custom-made miniscrew implants (MSIs) (Dentos, Daegu, Korea), 3.0 mm long and 1.7 mm in diameter, were placed by using a manual driver in the frontal bones through the incisions (Fig 1, A). Four 1.5-mm long and 0.8-mm diameter 99.95% tantalum bone markers were tapped into the frontal bone 2 to 3 mm anterior and 2 to 3 mm posterior to the MSIs by using a custom-made stainless steel appliance. The bone markers were used to radiographically quantify sutural separation. A transverse periosteal incision was made approximately 4 mm anterior to the MSIs (Fig 1, B). The periosteum was detached above the midsagittal suture by using a periosteal elevator, and a tunnel was created to house the ACS (Fig 1, C). The ACS was 6 3 8 3 2 mm, soaked with 0, 0.1, or 0.4 mg per milliliter of rhBMP-2 (Medtronic, Minneapolis, Minn) for 15 minutes, and placed in the tunnel (Fig 1, D). The periosteum and skin were sutured layer by layer (Fig 1, E). A 20-mm long, 0.020-in diameter, stainless steel interabutment guide wire was engaged into the holes in the MSI heads (Fig 1, F). A 15-mm long Sentalloy nickel-titanium (NiTi) open-coil spring (GAC International, Bohemia, NY), delivering a 100-g constant force, was 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 by the NiTi open-coil springs were maintained because the spring remained compressed at lengths from 8 to 12

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Fig 2. Schematic view of ventrodorsal radiographs used to evaluate MSI width, anterior bone marker width, and posterior bone marker width. Gray circles, MSIs; red circles, tantalum markers.

mm, which were checked every 10 days.21 When necessary, sliding tubes were added to the guide wire to maintain the springs’ compressed lengths. All animals were given penicillin (60000 IU per pound intramuscularly) immediately after surgery to prevent infection and buprenorphine (0.02 mg per kilogram subcutaneously when necessary) to minimize discomfort. Records, including animal weights, ventrodorsal cephalometric digital radiographs, and MSI widths (measured with calipers) were obtained under anesthesia at 10, 20, and 30 days. By using a customized headholder, digital ventrodorsal radiographs were taken at 65 kVp and 10 mA for 12 seconds at fixed distances. To localize the bone-forming regions of the midsagittal suture, oxytetracycline (13.6 mg per pound intramuscularly; Merial, Duluth, Ga) and calcein (10 mg per kilogram intramuscularly; Sigma, St Louis, Mo) fluorescent labels were administered to all animals. Calcein was given at days 10 and 30; oxytetracycline was given at day 20. All animals were killed at day 33. Blinded biometric assessments were based on body weights, caliper widths, and radiographic widths. Caliper width measurements between each MSI pair (MSIc) were taken at the outer-most margins immediately above the guide wire. The widths of the anterior (AB) and posterior bone markers (PB) and outermost margins of the MSIs (MSIr) were measured on the radiographs with Visix software (Fig 2; Air Techniques, Melville, NY). After 2 weeks, 80 radiographs were remeasured for evaluating intraexaminer measure error (AB, 0.098 mm; MSIr, 0.118 P mm; PB, 0.089 mm) with Dahlberg’s formula, O( d2/2n).22 The amount of sutural separation was calculated based on the averaged AB and PB changes over time. After 33 days of sutural expansion, the rabbits were killed. A standardized region, including the midsagittal suture and adjacent bone, was dissected and fixed with

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70% ethanol for 2 weeks without decalcification. After dehydration with an ascending series of ethanol (70%-100%), each specimen was embedded in methylmethacrylate and sectioned (approximately 60 mm) coronally by using a diamond saw (3 sequential sections per animal, n 5 90), followed by grinding and polishing. Images were captured by an 80i epifluorescence microscope (excitation wave lengths of 390 nm for oxytetracycline and 485 nm for calcein) (Nikon, Melville, NY) with a Coolsnap K4 camera (Photometrics, Tucson, Ariz) and MetaMorph softward (version 6.3, Molecular Devices, Sunnyvale, Calif). Using Biquant Osteo II software (Bioquant Image Analysis Corporation, Nashville, Tenn), a blinded examiner traced the bone labels. Bone formation (interbone label widths) of the entire suture, from the ectocranial surface to the endocranial surface, was automatically measured every 10 mm between the leading edges of label pairs (Fig 3). The first pair of labels quantified bone formation between days 10 (calcein) and 20 (oxytetracycline); the second pair quantified bone formation between days 20 (oxytetracycline) and 30 (calcein).23 Sutural gaps were measured every 10 mm between the 2 trailing edges of bone labels at day 30. Measurements of bone formation and sutural gaps were made by using the 3 serial sections for each animal; these were averaged for the analyses.

Statistical analysis

All statistical procedures were performed by using Multilevel Win software (version 2.0, University of Bristol, Bristol, United Kingdom) and a 95% confidence interval (P\0.05). The curves describing the changes of the repeated caliper, radiographic widths, and weight measurements were modeled over time as polynomials. The fixed part of the models described changes as a function of time and statistically compared the groups. The random part of the models had animals at the higher level and their repeated measures at the lower level, nested in the higher level. Iterative generalized least squares were used to estimate the polynomials. To statistically evaluate differences at the end of the experiment, the constant of each polynomial was fixed at day 30. Statistical analyses of the histomorphometric measurements were performed with SPSS software (version 15.0, SPSS, Chicago, Ill) and Grism (version 5.01, Graphpad, La Jolla, Calif). Kruskal-Wallis analyses were used to compare the groups, followed by Dunn post-hoc tests for the paired comparisons. Bone formation between days 10 and 20 and days 20 and30 was compared by using the Wilcoxon signed rank test.

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Fig 3. Histomorphometric measures: A, interbone label width between oxytetracycline and calcein injected at day 20 and 30; B, sutural gap width at day 30.

0 mg/mL

0.1 mg/mL

0.4 mg/mL

5

mm

4 3 2 1 0 0

10

20

30

Days Fig 4. Sutural separation over time in the 0 (control), 0.1, and 0.4 mg per milliliter of rhBMP-2 groups.

RESULTS

The animals’ weights increased by 1093 to 1218 g (78%-94%) during the study. There were no significant weight differences between the 3 groups, no infections around the MSIs, and no obvious signs of discomfort during the study. One MSI was mobile at day 10; a new MSI was placed lateral to the mobile implant site and served as anchorage until the end of the experiment. The MSIc and MSIr measurements of this animal before day 10 were treated as missing. The overall MSI success rate was 98% (59 of 60). The biometric measurements showed significant increases in sutural separation over time. The control group had a curvilinear—decelerating—pattern of sutural separation (Fig 4). The patterns of sutural separation for the 0.1

and 0.4 mg per milliliter groups were similar to that of the control group before day 10. However, the rates of sutural separation in the 2 experimental groups rapidly declined between days 10 and 20. After 30 days, the control group had undergone 4.6 mm of sutural separation, compared with 2.6 mm (56% of the controls) in 0.1 mg per milliliter group and 2.9 mm (62% of the controls) in 0.4 mg per milliliter group. Multilevel comparisons showed that the controls’ pattern of sutural separation was statistically different from those of the experimental groups; the amount of separation was also statistically greater in the control group than in the 2 experimental groups. There was no difference in the patterns of sutural separation between the 0.1 and 0.4 mg per milliliter groups. The fluorescent bone labels showed distinctly wider sutural gaps in the control group than in the experimental groups (Fig 5). For all but 1 animal in the 0.4 mg per milliliter group, the superior (ectocranial) 10% to 20% of the sutural margins had fused prematurely. The more inferior aspect of the sutures remained patent, but the sutural gaps appeared narrow. Most of the fused bony bridges showed the bone label calcein, which was injected on day 20. The widths between bone labels in the controls were wider than the corresponding widths in both rhBMP-2 treated groups, indicating greater amounts of bone formation between days 10 and 20 and days 20 and 30. All groups showed significantly greater bone formation between days 10 and 20 than between days 20 and 30 (Table, Fig 6). Between 10 and 20 days, bone formation was approximately 2 times greater in the control group than the 0.1 mg per milliliter group, followed by the 0.4 mg per milliliter group (87% greater). The 0.4 mg per

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DISCUSSION

Fig 5. Fluorescent bone labels after 33 days of midsagittal sutural expansion: A, the 0 mg per milliliter (control) group; B, the 0.1 mg per milliliter group; C, the 0.4 mg per milliliter group. Calcein (green) was injected on day 10, oxytetracycline (red) was injected on day 20, and calcein (green) was injected on day 30.

milliliter group produced significantly (P \0.001) more bone than the 0.1 mg per milliliter group between days 10 and 20. The control group also produced significantly more bone between days 20 and 30 than either rhBMP-2 group (.5 times the bone formation observed in the 0.1 mg per milliliter group and approximately 5 times greater than the 0.4 mg per milliliter group). The experimental groups showed no significant difference in bone formation between days 20 and 30. At the end of the study, sutural gaps in the control group were significantly greater than in the 0.1 and 0.4 mg per milliliter groups by 3.5 and 3 times, respectively. There was no significant difference in sutural gap width between the 2 rhBMP-2 groups (Table, Fig 6).

Sutural separation without rhBMP-2 application had a decelerating pattern. The rates of separation were greatest at the beginning of the experiment and then gradually decreased. Our previous study showed a similar decelerating pattern in sutural separation over 42 days of expansion with a constant 100-g expansion force.3 Decreasing rates indicate that resistance might prevent the suture from separating. The resistance might be due to contact with the frontal bone or other surrounding articulated bones.4,24 The resistance could also have been due to connective tissue in the suture. It has been previously shown that connective tissues limit sutural separation, even after the surrounding structures have been surgically removed.25 Alternatively, the sutures might be less responsive to tension as the animal age. Only limited amounts of sutural separation were produced with rhBMP-2 applied with an ACS. The rates of sutural separation in both rhBMP-2 groups rapidly declined after day 10, and sutural separation stopped by day 20. The sudden changes in rates of separation indicated the resistance induced by rhBMP-2. The histologic sections showed that premature sutural fusion was caused by bony bridges connecting the superior aspect of the sutural margins in both rhBMP-2 groups. Based on the patterns of sutural separation observed, it was postulated that there was bony bridging as early as day 10. Since the ACS was placed between the cranial bone and the periosteum, these results indicate that rhBMP-2 induced bone growth in the ACS and, perhaps, beyond the physical limits of the sponge. It was previously shown that the ACS provides a scaffold for bone formation.16,26 However, bone formation in our study appeared to be evident in the suture. This supports the experimental work by Bouxsein et al,13 who showed that rhBMP-2 increased bone formation in osteotomy sites adjacent to ACSs wrapped around the rabbits’ ulnas. Clinically, it has also been shown that rhBMP-2 and ACS wrapped around tibial fractures accelerates healing and enhances success rates.11 Together, the findings imply that the effects of rhBMP-2 are not restricted to the ACS, but the limits of its effects are unknown. Whereas all fluorescent labels were shown in the bony bridges, calcein (at day 20) was the dominant label. This indicates that most bone formation occurred between 10 and 20 days; this was further supported by the patterns of sutural separation observed. Similar timing of rapid onset of ectopic bone formation with rhBMP-2 was previously demonstrated.21 When rhBMP-2 and collagen membrane were applied between the dental root surface and gingival tissue, new

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Table. Medians and interquartile ranges (IR) of bone formation (BF) between days 10, 20, and 30, and sutural gaps at day 30 for the experimental and control groups, with comparisons between and within groups RhBMP-2 Mg/mL 0.0 0.1 0.4 All groups Post-hoc

0.0 vs 0.1 0.0 vs 0.4 0.1 vs 0.4

BF days 10-20 (mm)

BF days 20-30 (mm)

Median

IR

Median

IR

BF days 10-20 vs days 20-30 P value

728.3 246.9 388.9 \0.001* \0.001* \0.001* 0.001*

469.5 147.2 271.1

381.3 58.5 63.3 \0.001* \0.001* \0.001* NS

174.0 24.8 81.3

\0.001* \0.001* \0.001*

Sutural gap (mm) Median

IR

552.1 122.8 138.4 \0.001* \0.001* \0.001* NS

387.9 76.7 123.7

*P \0.05; NS, not significant. BF Days 10-20

BF Days 20-30

Sutural gap

1800 1600 1400

µm

1200 1000 800 600 400 200 0 0

0.1

0.4

RhBMP-2 (mg/mL)

Fig 6. Bone formation (BF) between days 10 and 20 and days 20 and 30, and sutural gaps at day 30 for the 0 (control), 0.1, and 0.4 mg per milliliter rhBMP-2 groups.

bone formation was observed within 10 days. After a rhBMP-2 and porous polylactic acid composite scaffold was implanted in muscle tissue, new osteoid with bone trabaculae was also found within 2 weeks.27 Interestingly, rhBMP-2 has been shown to accelerate bone healing of human tibial fractures up to 6 months after ACS placement, suggesting that its effects in this study were maintained throughout the experiment.11 Premature fusion with rhBMP-2 limited sutural bone formation. Compared with the controls, overall sutural bone formation was reduced by 72% and 59% in the 0.1 and 0.4 mg per milliliter groups, respectively. It was suggested that sutural bone formation is triggered by stretching of the connected collagen fibers.4,28 It was recently shown that the rates of bone formation are directly related to the rates of sutural separation.3 The bone bridges caused sutural separation rates to dramatically decrease between days 10 and 30 in the rhBMP-2 groups. This reduction resulted in significantly less

bone formation in the patent portion of the suture, presumably due to less stretching of the sutural fibers. Less stretching could explain the reduction in the areas for bone formation observed. Between days 10 and 20, the 0.4 mg per milliliter group produced significantly greater (58%) sutural bone than did the 0.1 mg per milliliter group. Although the sutures were still patent, greater amounts of bone were produced by 0.4 mg per milliliter of rhBMP-2 than by 0.1 mg per milliliter of rhBMP-2, even though the amounts of sutural separation were similar. It was previously shown that rhBMP-2 with an ACS caused a dosedependent response in bone formation.11 Patients with open tibial fractures have shown consistently greater healing rates over 52 weeks with 1.5 mg per milliliter of rhBMP-2 with an ACS than with 0.75 mg per milliliter. Also, when rhBMP-2 with an ACS was implanted for maxillary sinus-floor augmentation, 1.5 mg per milliliter of rhBMP-2 with an ACS produced higher bone density

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than did 0.75 mg per milliliter after 4 months.10 After 20 days, we found no significant differences in bone formation between the 0.1 and 0.4 mg per milliliter groups, presumably because of the bony bridges that had fused above the sutures; these limited expansion. Premature fusion prevented sutural separation, but bone growth continued, resulting in narrower sutural gaps. At the end of the experiment, the sutural gaps of the control group were more than 3 times wider than the gaps of the 2 rhBMP-2 groups. In the control group, the rate of bone formation was less than the rate of sutural separation, resulting in a larger sutural gap and greater relapse potential.6 If the effects of rhBMP-2 could be better controlled, perhaps by titrating its concentration or by increasing the expansion forces, it might ensure that bone formation more closely approximates sutural separation. Such control has important clinical implications; it suggests that it might be possible to make sutural expansion more efficient by reducing the retention phase of treatment. Short MSIs have great potential for sutural modification. All but 1 of the 3-mm MSIs withstood the 100-g force over 33 days, resulting in a 98% success rate (59 of 60). Using the same MSIs, we showed in our previous studies 86% (24 of 28)29 and 88% (65 of 74)3 success rates. The overall success rate of all 3 studies was 97.5% (158 of 162). Similar or lower success rates with 6-mm long MSIs in dogs30,31 and humans (71%)32 have been previously reported. Because traditional MSIs can damage dental roots during placement, shorter MSIs might some day be used for sutural expansion or compression to prevent damage.33,34 Because of the amount of force applied, shorter MSIs also have great potential for tooth movements. CONCLUSIONS

1. 2.

RhBMP-2 enhances sutural bone formation during sutural separation. Both 0.4 and 0.1 mg per milliliter of rhBMP-2 with an ACS caused premature fusion at the ectocranial sutural margins.

We thank GAC International for providing the NiTi coil springs, Medtronic for the rhBMP-2, Dentos for the miniscrew implants, and E. Gerald Hill for invaluable assistance with animal care and the surgical procedures. REFERENCES 1. Keim RG, Gottlieb EL, Nelson AH, Vogels DS 3rd. 2002 JCO study of orthodontic diagnosis and treatment procedures. Part 1. Results and trends. J Clin Orthod 2002;36:553-68.

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2. Gallagher RW, Miranda F, Buschang PH. Maxillary protraction: treatment and posttreatment effects. Am J Orthod Dentofacial Orthop 1998;113:612-9. 3. Liu SS, Opperman LA, Kyung HM, Buschang PH. Is there an optimal force level for sutural expansion? Am J Orthod Dentofacial Orthop 2009 (in press). 4. Storey E. Tissue response to the movement of bones. Am J Orthod 1973;64:229-47. 5. Ekstrom C, Henrikson CO, Jensen R. Mineralization in the midpalatal suture after orthodontic expansion. Am J Orthod 1977;71:449-55. 6. 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. 7. Kawasaki K, Aihara M, Honmo J, Sakurai S, Fujimaki Y, Sakamoto K, et al. Effects of recombinant human bone morphogenetic protein-2 on differentiation of cells isolated from human bone, muscle, and skin. Bone 1998;23:223-31. 8. Kobayashi M, Takiguchi T, Suzuki R, Yamaguchi A, Deguchi K, Shionome M, et al. Recombinant human bone morphogenetic protein-2 stimulates osteoblastic differentiation in cells isolated from human periodontal ligament. J Dent Res 1999;78:1624-33. 9. Burkus JK, Sandhu HS, Gornet MF, Longley MC. Use of rhBMP-2 in combination with structural cortical allografts: clinical and radiographic outcomes in anterior lumbar spinal surgery. J Bone Joint Surg Am 2005;87:1205-12. 10. Boyne PJ, Lilly LC, Marx RE, Moy PK, Nevins M, Spagnoli DB, et al. De novo bone induction by recombinant human bone morphogenetic protein-2 (rhBMP-2) in maxillary sinus floor augmentation. J Oral Maxillofac Surg 2005;63:1693-707. 11. Govender S, Csimma C, Genant HK, Valentin-Opran A, Amit Y, Arbel R, et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002;84-A:2123-34. 12. Burkus JK, Dorchak JD, Sanders DL. Radiographic assessment of interbody fusion using recombinant human bone morphogenetic protein type 2. Spine 2003;28:372-7. 13. Bouxsein ML, Turek TJ, Blake CA, D’Augusta D, Li X, Stevens M, et al. Recombinant human bone morphogenetic protein-2 accelerates healing in a rabbit ulnar osteotomy model. J Bone Joint Surg Am 2001;83-A:1219-30. 14. Por YC, Barcelo CR, Salyer KE, Genecov DG, Troxel K, Gendler E, et al. Bone generation in the reconstruction of a critical size calvarial defect in an experimental model. J Craniofac Surg 2008;19:383-92. 15. Elsalanty ME, Por YC, Genecov DG, Salyer KE, Wang Q, Barcelo CR, et al. Recombinant human BMP-2 enhances the effects of materials used for reconstruction of large cranial defects. J Oral Maxillofac Surg 2008;66:277-85. 16. Nunotani Y, Abe M, Shirai H, Otsuka H. Efficacy of rhBMP-2 during distraction osteogenesis. J Orthop Sci 2005;10:529-33. 17. Warren SM, Brunet LJ, Harland RM, Economides AN, Longaker MT. The BMP antagonist noggin regulates cranial suture fusion. Nature 2003;422:625-9. 18. Takahashi D, Odajima T, Morita M, Kawanami M, Kato H. Formation and resolution of ankylosis under application of recombinant human bone morphogenetic protein-2 (rhBMP-2) to class III furcation defects in cats. J Periodontal Res 2005;40:299-305. 19. Sigurdsson TJ, Lee MB, Kubota K, Turek TJ, Wozney JM, Wikesjo UM. Periodontal repair in dogs: recombinant human bone morphogenetic protein-2 significantly enhances periodontal regeneration. J Periodontol 1995;66:131-8.

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20. King GN, King N, Cruchley AT, Wozney JM, Hughes FJ. Recombinant human bone morphogenetic protein-2 promotes wound healing in rat periodontal fenestration defects. J Dent Res 1997;76:1460-70. 21. von Fraunhofer JA, Bonds PW, Johnson BE. Force generation by orthodontic coil springs. Angle Orthod 1993;63:145-8. 22. Dahlberg G. Statistical methods for medical and biological students. New York: Interscience Publications; 1940. 23. 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. 24. Isaacson RJ, Wood JL, Ingram AH. Forces produced by rapid maxillary expansion. I. Design of the force measuring system. Angle Orthod 1964;34:256-70. 25. 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. 26. Hong L, Mao JJ. Tissue-engineered rabbit cranial suture from autologous fibroblasts and BMP2. J Dent Res 2004;83:751-6. 27. Chang PC, Liu BY, Liu CM, Chou HH, Ho MH, Liu HC, et al. Bone tissue engineering with novel rhBMP2-PLLA composite scaffolds. J Biomed Mater Res A 2007;81:771-80. 28. Ten Cate AR, Freeman E, Dickinson JB. Sutural development: structure and its response to rapid expansion. Am J Orthod 1977;71:622-36.

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29. Liu SS, Kyung HM, Buschang PH. Continuous forces are more effective than intermittent forces in expanding sutures. Eur J Orthod 2009 (in press). 30. 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. 31. 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. 32. 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. 33. Hembree M, Buschang PH, Carrillo R, Spears R, Rossouw PE. Effects of intentional damage of the roots and surrounding structures with miniscrew implants. Am J Orthod Dentofacial Orthop 2009;135:280. e1-9. 34. Brisceno CE, Rossouw PE, Carrillo R, Spears R, Buschang PH. Healing of the roots and surrounding structures after intentional damage with miniscrew implants. Am J Orthod Dentofacial Orthop 2009;135:292-301.