Effects Of Recombinant Human Growth Hormone On Orthodontically Induced Inflammatory Root Resorption And Its Possible Mechanism

Remodeling changes occur in the paradental tissues including periodontal ligament, alveolar bone and gingival during orthodontic tooth movement. When alveolar bone and hyalinised tissue are removed by osteoclastic cells, root cementum and dentine will inevitably be damaged, which eventually leads to root resorption. Two different cell populations play important roles in orthodontically induced inflammatory root resorption (OIIRR). Osteoclasts immigrate to the surface of root and resorb the cementum or/and dentine. Cementoblasts affect the severity of root resorption because they can not only stimulate osteoclasts differentiation and function, but also repair root resorption lacunae by producing cementum. Consequently, any factors changing the conditions of these two cell populations will have a potential to impact OIIRR.Human growth hormone is the most important post-natal growth-promoting hormone, which can influence almost all kinds of tissues and cells. Recent in vitro studies showed that growth hormone (GH) could accelerate osteoclasts formation, differentiation and maturation. Animal and human studies found that GH could stimulate precursor cells to differentiate into mature cementoblasts, and the production of cementum. In consideration of the effects of GH on osteoclasts and cementoblasts both of which are involved in root resorption, we hypothesized that GH may have an influence on OIIRR. Therefore, the aim of this study was to investigate the effects of rhGH on root resorption induced by heavy force and its possible mechanism. This study as a foundation of the future researches provides not only an experimental basis for orthodontists to make treatment plans and evaluate the risk of root resorption for patients who are prescribed rhGH, but also new methods and ideas to prevent and manage root resorption by medications. The study included the following four parts:Part1 Effects of rhGH on orthodontic tooth movement and root resorptionObjective:To explore the effects of rhGH on orthodontic tooth movement and root resorption.Methods:Forty Wistar rats (gender:male; age:7 weeks) were randomly divided into control and experimental groups. A force of 50 g was applied to move the right upper first molars mesially. The experimental and control groups received daily subcutaneous injections of rhGH (2 mg/kg) and equivalent volumes of saline, respectively. The rats were sacrificed on days 1,3,7, and 14. Micro-computed tomography-reconstructed images of the upper right first molars were used to survey root resorption and tooth movement.Results:On days 1 and 3, no resorption lacunae appeared on the compressed side of the distal buccal root in both groups, and there was no significant difference in the distance of tooth movement between the two groups. Indexes of root resorption were lower and tooth movement was faster in the GH-treated groups than in the control groups on days 7 and 14.Conclusion:Short-term GH administration may be a method with which to reduce root resorption and shorten treatment time, especially in patients who are susceptible to root resorption.Part 2 Effects of rhGH on local RANKL, OPG and IGF-I expression during root resorptionObjective:To investigate RANKL, OPG, and IGF-I expression during root resorption induced by heavy force in rhGH-treated rats.Methods:Forty Wistar rats (gender:male; age:7 weeks) were randomly divided into control and experimental groups. A force of 50 g was applied to move the right upper first molars mesially. The experimental and control groups received daily subcutaneous injections of rhGH (2 mg/kg) and equivalent volumes of saline, respectively. The rats were sacrificed on days 1,3,7, and 14. Horizontal sections of the maxillae were prepared for hematoxylin and eosin, tartrate-resistant acid phosphatase, and immunohistochemical staining.Results:More osteoclasts and RANKL-positive PDL cells, and higher RANKL/OPG ratios on day 3, and more IGF-I-and OPG-positive PDL cells, with fewer odontoclasts and lower RANKL/OPG ratios on days 7 and 14 were observed in the GH-treated groups than in the control groups. Conclusion:rhGH can stimulate RANKL, OPG, and IGF-I expression on the compressed sides over different OIIRR stages. The inhibitory effect of rhGH on odontoclasts and root resorption might be mediated by RANKL/OPG and IGF-I.Part 3 Effects of rhGH on cementoblast proliferation and differentiationObjective:To investigate the effects of rhGH on the proliferation and the expression of the mineral associated gene, RANKL and OPG mRNAs in cementoblasts.Methods:Mouse cementoblasts OCCM-30 were cultured. After OCCM-30 cells were treated with different concentrations of rhGH(0,10,50,100 ng/ml) for 24h and 48h, the proliferative activity was measured by MTT assay, and the expression of BSP, OCN, OPN, ALP, RANKL and OPG mRNA were detected by real-time PCR. After incubating OCCM-30 cells in differentiated medium containing different concentrations of rhGH for 7 days, the ALP activity of OCCM-30 cells was detected by ALP assay kit.Results:The number of OCCM-30 cells was significantly increased by 10,50,100 ng/ml rhGH, with a positive correlation between the rate of cell proliferation and the concentration of rhGH or the incubation duration. After OCCM-30 cells were treated with rhGH for 24h, the stimulatory effects were observed in BSP mRNA by 10,50, 100 ng/ml rhGH, in OPN and ALP mRNA by 10ng/ml rhGH, in ALP mRNA by 50ng/ml rhGH, and in OCN and OPG mRNA byl OOng/ml rhGH; and the inhibitory effects were showed in OPN mRNA by 50 and 100ng/ml rhGH. After OCCM-30 cells were treated with rhGH for 48h, the stimulatory effects were observed in OCN, BSP and ALP mRNA by 10ng/ml rhGH, and OPG mRNA by 50 and 100ng/ml rhGH; and the inhibitory effects were showed in ALP mRNA by 50ng/ml rhGH, and in BSP and ALP mRNA by 1 OOng/ml rhGH. RANKL mRNA was not affected by rhGH. The ratio of RANKL/OPG mRNA was decreased by 50 and 100ng/ml rhGH at 24h. The ALP activity was enhanced by 10ng/ml rhGH, while it was decreased by 50 and 100ng/ml rhGH. Conclusion:rhGH can accelerate cementoblast proliferation. rhGH affects cementoblast differentiation in a time- and dose-dependent manner. rhGH may inhibit cementoblast-mediated odontoclastogenesis by reducing the ratio of RANKL/OPG mRNA.Part 4 Effects of rhGH on mitogen-activated protein kinase pathways in cementobiastsObjective:To investigate the effects of rhGH on ERK1/2, JNK/SAPK, and p38MAPK pathways in cementoblasts.Methods:After OCCM-30 cells were treated with 100 ng/ml rhGH for 0,5,10,15, 30,60min, phosphorylation of ERK1/2, JNK/SAPK, p38MAPK were detected by Western blot. Cells were pre-treated with or without ERK inhibitor (U0126) for 1 h, and then treated with or without 100 ng/ml rhGH for 1 Omin; phosphorylation of ERK1/2 was detected by Western blot.Results:Phosphorylation of ERK1/2(p-ERK1/2) in OCCM-30 cells increased after 5min of treatment with rhGH. After reaching the peak at 10min, p-ERK1/2 decreased at 15min, and then returned to the baseline level (Omin) at 30 and 60mins. The level of p-ERK1/2 was not increased by 100ng/ml rhGH in the existence of U0126. There was no obvious change in the phosphorylation of JNK/SAPK and p38MAPK.Conclusion:100ng/ml rhGH can activate ERK1/2 pathway, which is inhibited by ERK1/2 inhibitor.100ng/ml rhGH cannot activate JNK/SAPK and p38MAPK pathways.