Effects of growth hormone and functional ... - Angle Orthodontist

Original Article

Effects of growth hormone and functional appliance on mandibular growth in an adolescent rat model Shuai Wanga; Lu Yea; Mei Lib; Han Zhana; Rui Yec; Yu Lid; Zhihe Zhaoe ABSTRACT Objectives: To investigate the individual and synergistic effects of growth hormone (GH) and functional appliance (FA) on mandibular growth in an adolescent rat model. Materials and Methods: Forty adolescent (6-week-old) female Wistar rats were randomly divided into four groups (10 rats in each group). The control group received a sham treatment (intraabdominal injection of phosphate-buffered saline), the GH group received an intra-abdominal injection of recombinant human growth hormone, the FA group was treated with a mandibular advancement device, and the GHþFA group received both the GH and FA treatments. The amount of mandibular growth in each group was measured quantitatively using cone-bean computed tomography. The growth of condylar cartilage and expression of matrix metalloproteinases–1 and –13 (MMP-1 and MMP-13) and type II and X collagen (Col II and Col X) were assessed using histological staining and immunostaining techniques. Results: After 4 weeks, there was significant mandibular growth in the FA group compared with the control group (P , .05). The GHþFA group had significantly greater mandibular length, thickness of condylar cartilage, and expression of MMP-1, MMP-13, Col II, and Col X in the cartilage than the other groups (P , .05). The GHþFA group and GH group had significantly greater weight than the FA and control groups (P , .05). Conclusions: The FA as well as GHþFA stimulated mandibular growth in adolescent rats. (Angle Orthod. 2018;88:624–631.) KEY WORDS: Growth hormone; Functional appliance; Mandible; Rat

the body of the mandible grows longer by periosteal apposition on its posterior surface, and the ramus grows taller mainly by endochondral replacement at the condyle.2 In addition, mandibular condylar cartilage, an important secondary fibrocartilage, can be stimulated mechanically as well as by loading-induced growth factors.3,4

INTRODUCTION Skeletal Class II malocclusion is one of the most prevalent dentofacial anomalies that affect oral health and facial esthetics. The etiology of a skeletal Class II relationship is most commonly associated with mandibular deficiency or retrusion.1 It has been shown that

a Resident, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China. b Senior Lecturer, Discipline of Orthodontics, Department of Oral Sciences, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand. c Lecturer, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China. d Associate Professor, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China. e Professor and Vice Dean, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China. Corresponding author: Dr Yu Li, Department of Orthodontics, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, 14#, 3rd Section, South Renmin Road, 610041 Chengdu, China (e-mail: [email protected])

Accepted: March 2018. Submitted: December 2017. Published Online: April 30, 2018 Ó 2018 by The EH Angle Education and Research Foundation, Inc. Angle Orthodontist, Vol 88, No 5, 2018

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DOI: 10.2319/120417-829.1

GH AND FA ON MANDIBULAR GROWTH IN ADOLESCENT RAT

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Figure 1. Study flow chart.

Functional appliances (FAs), such as the Forsus, Herbst, and Twin-Block appliances, have been widely used for the treatment of Class II in growing patients by stimulating mandibular growth.5 However, the effect of FAs on mandibular growth is still controversial. It has been reported that there was no significant advantage of two-phase treatment (FA followed by fixed appliances) over one-phase treatment (fixed appliances only) in the long term.6 Growth hormone (GH), one of the most important stimulating factors of human development, has been found to play a significant role in craniofacial growth. For example, it has been found that GH could increase head circumference growth and anterior facial height, influence growth pattern, and regulate mandibular and

condylar growth and remodeling.7–9 Injection of GH has been used clinically for the treatment of short-stature children.10 The effect of GH and FA on mandibular growth is still poorly understood. Most previous studies focused only on one intervention (ie, either GH or FA alone). Some case reports have reported using a combination of GHþFA but reported that GH therapy may cause unpredictable mandibular growth.11–13 It is still unclear whether GH therapy can be combined with FA treatment for children with skeletal Class II malocclusion or whether it is necessary to delay FA treatment until the completion of GH therapy. The aim of this study was to investigate the individual and synergistic effects of GH and FA on Angle Orthodontist, Vol 88, No 5, 2018

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WANG, YE, LI, ZHAN, YE, LI, ZHAO

Figure 2. (A, B) The functional appliance (FA) used to advance rat mandibles in the study. (C, D) Cone-beam computed tomography (CBCT) images showing mandibular advancement by the FA. (E, F) Linear measurements of mandibular growth on the CBCT-generated cephalogram.

mandibular growth in an adolescent rat model. The null hypothesis of the study was that GHþFA treatment has no effect on mandibular growth in rats. MATERIALS AND METHODS Animals and Experimental Protocol The study was approved by the Animal Care and Ethics Committee of the State Key Laboratory of Oral Disease, West China School of Stomatology, Sichuan University, China. All of the experimental procedures followed the experimental protocols guided by the National Institutes of Health, China (Figure 1). Forty 6-week-old female Wistar rats (body weight 120 6 10 g, provided by West China Animal Center, Sichuan University, China) were randomly allocated into four groups. The control group (n ¼ 10) received a sham treatment (an intra-abdominal injection of phosphate-buffered saline [PBS]), the GH group (n ¼ 10) received an intra-abdominal injection of recombinant human growth hormone (rhGH; 0.05 lg/kg/d, ProSpecTany TechnoGene Ltd, Israel), the FA group (n ¼ 10) received functional treatment using a mandibular advancement device14,15 and an intra-abdominal injection of PBS, and the GHþFA group received both an Angle Orthodontist, Vol 88, No 5, 2018

intra-abdominal injection of the rhGH and the FA treatment. The dosage of the rhGH injection was based on the previous literature.16 All rats were fed in separate cages with the same 12-hour light/12-hour dark environment at a constant temperature of 258C and were allowed free access to diet and water. Body weight was recorded every week. The FA device was cemented on the teeth under anesthesia using 10% chloral hydrate (3 mL/kg). The same anesthesia procedure was also performed for the control and GH groups. The mandible was advanced 3.5 mm using the FA that was designed for rat models as previously described14,15 (Figure 2A–D). It consisted of an inclined bite plane that covered the upper incisors and an occlusal plane bonded on the lower incisors. The upper inclined bite plane was made of polymethyl methacrylate heat-cured acrylic resin and bonded using light-cure composite resin (Transbond XT, 3M Unitek, Monrovia, Calif). The lower occlusal plane was made of resilience light-cure cement (Ortho Technology, Lutz, Fla). All rats were fed with the same soft diet during the study. Rats in all groups at the age of 6 weeks old received a daily intra-abdominal injection of GH or PBS


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Figure 3. Body weight gain during the experiment. After 4 weeks of treatment, the GHþFA group and GH group had significantly greater weight than that of the FA group or the control group when the rats were 10 weeks old. **Significant difference of P , .01.

Figure 4. Linear measurements of mandibular growth. The length (Co-A) and height (Co-H) of the mandible and the height of the ramus (Co-B) in the GHþFA group were statistically significantly greater than those in the control group. The heights of the mandible (Co-H) and the ramus (Co-B) in the FA group were statistically significantly greater than those in the control group. *Significant difference of P , .05. **Significant difference of P , .01.

according to the study protocol (Figure 1) for 4 consecutive weeks. Cone-Beam Computed Tomography Imaging and Cephalometric Analysis Three rats in each group were randomly selected and scanned using cone-beam computed tomography (CBCT; MCT1-1 EX-2F, Morita, Japan) at the beginning of the study (age of 6 weeks old) and after treatment (age of 10 weeks old) to evaluate growth of the mandible (Figure 2C,D). The examination was performed under anesthesia using a custom-made head holder to keep the head steady. The linear increment of mandibular growth was measured on the CBCT-generated cephalogram (Figure 2E,F). Briefly, the markers included the top of the condyle (Co), the bottom of the chin (Me), the lingual edge of the lower incisors (A), the midpoint of the mandibular foramen (B), and the anterior and posterior edges of the condyle (C, D). The length (Co-A) and height (Co-H) of the mandible, the length of the ramus (Co-B) and the mandibular body (A-B), and the length of the condyle (C-D) were measured using Mimics software (version 17.0, Materialise, Belgium; Figure 2F). Histology and Immunohistochemistry The rats in each group were euthanized using excessive anesthetic injection after 4 weeks of treatment (at the age of 10 weeks old). The dissected specimens of the mandible and the condyle were fixed in 4% paraformaldehyde for 24 hours and subsequently decalcified in 10% ethylenediamine tetra acetic acid solution until all specimens were softened. Subsequently, conventional dehydration, dipping wax, and paraffin-embedding techniques were performed to obtain condyle sagittal slices with 5-lm thickness. The middle parts of these slices were treated in routine hematoxylin and eosin (HE) staining. Avidin–biotin

complex (ABC) method was used to detect changes in expression of matrix metalloproteinase–1 (MMP-1), matrix metalloproteinase–13 (MMP-13), type II collagen (Col II), and type X collagen (Col X). The primary antibody was anti–MMP-1 (10371-2-AP; rabbit polyclonal, Proteintech Group, Chicago, IL, USA), anti– MMP-13 (ab80576; rabbit monoclonal, Abcam, Cambridge, MA, USA) anti–Col II (sc-28887; rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, Calif), and anti–Col X (sc70869; rabbit polyclonal, Santa Cruz Biotechnology). The HE-stained sections were observed under an inverted microscope system. The mean density (based on the RGB color parameter) and the mean percentage of positive staining were calculated using Image-Pro Plus (version 6.0, Media Cybernetics, Rockville, MD, USA).17 Statistical Analysis Data analysis was performed using SPSS software version 22.0 (Statistical Package for the Social Sciences, SPSS Inc, Chicago, Ill). One-way analysis of variance with post hoc contrasts by Student– Newman–Keuls test was used to detect differences among the groups. A P value of less than .05 was considered to be statistically significant. Data were presented as mean 6 standard deviation (SD). RESULTS Body Weight Gain Body weight gain of the GH group (150 6 8 g) and the GHþFA group (148 6 9 g) was significantly greater than that of the FA group (124 6 8 g) and the control (123 6 7 g) after 4 weeks of treatment (P , .01; Figure 3). Angle Orthodontist, Vol 88, No 5, 2018

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Figure 5. Histological analysis of the central section of the mandibular condyle with hematoxylin and eosin (HE) staining (100-fold magnification). AR indicates articular layer; PR, proliferative layer; CH, chondrogenic layer; HY, hypertrophic layer. The histogram at the bottom of the figure illustrates the statistical comparisons among groups. The thickness of the total layer of the mandible condyle was greatest in the GHþFA group, followed by the FA group, the GH group, and the control. *Significant difference of P , .05. **Significant difference of P , .01.

Mandibular Growth The mandibular parameters were at the same baseline at the beginning of the study. After treatment, the mandibular length (Co-A) and height (Co-H) and the ramus height (Co-B) in the GHþFA group (24.85 6 0.66 mm, 12.51 6 0.62 mm, and 8.71 6 0.30 mm, respectively) were significantly greater than those in the control group after the interventions (22.00 6 1.14 mm, 10.44 6 1.00 mm, and 6.85 6 0.52 mm, respectively; all P , .01). No statistically significant difference was found in mandibular growth between the GHþFA group and the GH or FA groups (P . .05; Figure 4). The Co-H and Co-B measures in the FA group were significantly greater than those in the control group Angle Orthodontist, Vol 88, No 5, 2018

(both P , .05). No significant difference was found between the GH group and the control or FA groups in any of the measurements (all P . .05). There were no significant differences in the length of the mandible (AB) and the length of the condyle (C-D) among the four groups (all P . .05; Figure 4). Condylar Cartilage Thickness The thickness of the total layer of condylar cartilage was greatest in the GHþFA group (140.79 6 6.89 lm), followed by the FA group (128.51 6 4.06 lm) and the GH group (122.07 6 5.51 lm) and then the control (113.27 6 2.35 lm; P , .05; Figure 5). The condylar cartilage thicknesses of the articular layer, the proliferative layer, and the chondroblastic


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Figure 6. Immunohistochemical analysis of the expression of MMP-1, MMP-13, Col II, and Col X in the cartilage (magnification 4003). The expression of MMP-1, MMP-13, Col II, and Col X increased significantly in the treatment groups. *Significant difference of P , .05.

layer in the GHþFA group were also significantly greater (4.11 6 2.02 mm, 7.24 6 3.51 lm, and 9.63 6 1.32 lm, respectively) than those in the control (P , .05 for all; Figure 5). Immunohistochemistry Analysis The immunohistochemical staining of the articular cartilage is shown in Figure 6. The percentage of positive cells of MMP-1 and Col II were highest in the GHþFA group, followed by the GH group and the FA group, and the control group had the lowest percentage (P , .05). The mean density of the MMP-13 was significantly greater in the GHþFA group than in the other groups (P , .05; Figure 6). DISCUSSION This study investigated the effects of GH and FA treatment on mandibular growth in an adolescent rat model by using CBCT imaging, cephalometric analysis, and immunohistochemistry analysis. After 4 weeks of treatment, the GHþFA group had significantly greater mandibular growth, condylar cartilage thickness, and immunohistochemical expression of related proteins than the control. Mandibular advancement treatment with FAs has been commonly used to enhance the growth of the mandible and condyle, which can be influenced by epigenetic factors such as mechanical stimuli and growth factors.18 In addition, other factors such as soft and hard diets may also influence craniofacial morphology.19 Therefore, in this study, all rats were fed with

the same soft diet to minimize the potential impact of diet on mandibular growth. Growth hormone (GH), an important hormone for regulating bone growth and remodeling, has been widely used as one of the routine therapies for shortstatured children, with a positive effect on body growth and no long-term negative effects.11 In addition, GH can increase body weight.20 This is consistent with the finding in this study that the groups that received GH treatment had a greater body weight gain than the groups that did not, indicating a promoting effect of GH on body stature development. The differences in body weight gain were not statistically significant between the GHþFA group and the GH group or between the FA group and the control, indicating that the FA device did not influence the rats’ nutrition during the study. All rats used in this study were female. Male rats have also been used in previous studies on bone development because estrogen may potentially affect bone density.21 However, mandibular advancement therapy is often used before the growth spurt, during which the level of estrogen does not differ significantly between genders. Therefore, female rats have also been used for research on mandibular growth in previous publications,3,4,14 as well as in the current study. Future studies are recommended to include both male and female rats for a subgroup analysis to evaluate the difference between sex in mandibular growth. Various FAs have been used to stimulate mandibular growth in animal research. The bite-jumping mandibular advancement appliance that was used in Angle Orthodontist, Vol 88, No 5, 2018

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this study has been commonly used in previous studies on rats and rabbits because of its convenience of fabrication and employment.14,15 In the rabbit model, local intra-articular injection of growth factor has been reported in some research on mandibular growth.15 However, local administration has limitations, including high technical sensitivity, trauma to experimental animals, and great operative risk in small animals such as rats compared with the intra-abdominal injection that was used in the current study. In this study, mandibular growth (ie, the length [CoA] and height [Co-H] of the mandible) was significantly greater in the GHþFA group in comparison with the control. The FA also enhanced mandibular growth, especially in the vertical direction (ie, heights of the mandible [Co-H] and ramus [Co-B]). This was in agreement with the previous findings in a rabbit model.22 Mandibular condylar cartilage has been found to be generated from differentiated bonelike cells rather than undifferentiated mesenchymal stem cells23 and thus could develop rapidly from former cartilage cells into chondrocytes and hypertrophic chondrocytes. The thickening of the articular, proliferative, and chondroblastic layers of the cartilage in this study demonstrated that the combination of GH and FA might enhance proliferative activity in the condylar cartilage. MMP-1 and MMP-13 are important proteases in cartilage remodeling and mineralization in physiological conditions, which play an important role in the transformation from cartilage into bone, as well as the formation of primary calcification centers and blood vessels during bone reconstruction.24 It has been found that the administration of growth factors along with mandibular repositioning appliances have an almost twofold effect on the induced expression of MMP-1 and MMP-13 in comparison with the administration of mandibular repositioning appliances only.15 This is consistent with the current finding that the GHþFA group had significantly higher levels of MMP-1 and MMP-13 compared with the FA group. Collagen is a major component of the extracellular framework of cartilage. Type II collagen is one of the two major constituents of articular cartilage and provides tissue with strength to withstand tensile forces.25 Type X collagen, considered as a reliable marker of cartilage transforming into bone, has been found to promote endochondral bone formation by regulating mineralization of hypertrophic chondrocyte matrix.26 In this study, the levels of Col II and Col X were significantly higher in the treated groups than in the control, indicating that both collagens might accelerate cartilage transformation into bone in the condyle. The GHþFA treatment enhanced mandibular growth in this study. Future studies, especially well-designed Angle Orthodontist, Vol 88, No 5, 2018


trials with a large sample size, are still needed for a better understanding of the synergistic effect of GH and FA on mandibular growth. CONCLUSION

FA treatment as well as the combination with growth hormone (GHþFA) stimulated mandibular growth in adolescent rats.

ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (11372202).

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