| Background and objectionThe long therapeutic course has been an outstanding problem of orthodontic treatment of malocclusion deformity for a long time, which also is one of the critical factors to obtain a successful treatment for many teeth Micromaxillary deformity patients, especially for adult patients. It has become current research difficulty and hotspot to study how to speed up the orthodontic tooth movement and shorten the orthodontic treatment without reversible damage of the periodontal tissue, root and pulp. Now, the research is gradually focusing on the biological response to orthodontic force. It has been found that adhesion teeth without periodontal membrane and dental implants can’t move in orthodontic treatment, which suggests us, only with the force but not periodontal tissue remodeling, teeth can’t move to the ideal location or keep staying in the right location. Therefore,the orthodontic tooth movement depends on the corresponding reaction of periodontal tissue, and orthodontic tooth movement must be on the premise of the remodeling of periodontal ligament and alveolar bone. In the process of orthodontic treatment, mechanical force stimulation passes through the teeth to the periodontal membrane, triggering and activating the biological metabolic activity of its internal various effector cells, thus affects the shaping and rebuilding of the periodontal tissue, eventually making teeth move to the right place. That is to say, the periodontal ligament is the physiological medium of the orthodontic treatment, and it is a variation of the periosteum with obvious bone deposition and bone absorption ability, which is located between cementum and alveolar bone, two kinds of hard tissue, made up of dense connective tissue, mostly arranged in fiber bundles, the two ends are buried in cementum and alveolar bone wall socket, making teeth fixed in alveolar socket. Collagen fiber,arranged in fiber bundles orderly, can buffer and absorption reaction fully, playing a vital role in periodontal tissue repairing and periodontal membrane injury, and after stimulated by mechanical force, it can turn the signal into a biological signal transduction system, causing the change of periodontal tissue metabolism and alveolar bone remodeling,a series of biological responses. This is a very complex process, involving a variety of signal transduction molecules and channel as well as a variety of matrix proteins and cytokines.Periostin (PN) is a kind of new multifunctional extracellular matrix protein, molecular weight of90kD, located in periosteum and periodontal ligament. Periostin can be induced to express by growth factorsβ-1(TGFβ-1), supporting cell adhesion, collagen cross linking and adjusting the fiber to form, which plays an important role in the integrity of the periodontal tissue, tooth development, eruption and tissue repair and regeneration caused by mechanical stress, and is considered to be a critical extracellular matrix proteins in periodontal membrane steady state regulation, it may also plays a key role in periodontal disease and periodontal tissue remodeling during orthodontic tooth movement. The relationship of periostin and TGF-β1is attracting increasing attention from all walks of life. A number of studies have shown that periostin participates in the development of multiple systemic diseases, and there is a close relationship between its expression and TGF-β1,For example, airway epithelial cells secrete periostin,which can activate the TGF-β1and then promote airway fibrosis and reduce airway elastic, causing asthma,the study of heart found that perisotin participates in myocardial collagen synthesis and maturity, knocked out periostin gene of mouse embryonic fibroblasts and stimulated by TGF-β1,the amount of collagen synthesis was only about32%of the wild type,indicating that periostin is the main signal of fibroblasts to synthesize and secret collagen. Norris studied on the lower collagen denaturation temperature of periostin knockout mice cardiac muscle by Differential scanning calorimetry (DSC), which suggests a reduced level of collagen crosslinking. Periostin can adjust the formation of type I collagen fiber, and interacts directly with type I collagen, thus becoming an important function media of fibrous connective tissue biomechanical properties. Rios observed on the periodontal membrane development of periostin knockout mice using X-ray and Micro-ct, there is no difference of the periodontal membrane between PN-null mice and the normal control group during the period from teeth development and eruption to establish normal occlusion, but for the other group that mice with tooth eruption and exposure to masticatory force for3months, there is a significant damage in periodontal tissue, reduced periodontal attachment and alveolar bone resorption, suggesting that periostin plays an indispensable role in maintaining the integrity of the periodontal membrane and functional aspects under the effect of biting force, the change of periostin expression level in the hypofunction group may be considered a form of adaptation to the environment changes.Choi removed the biting force of Wistar rat right upper first molar, and results show that the periodontal membrane fibers became thin and decreased at first, then the fibers and their attached arrangement in alveolar bone became disordered, and finally lost their mesh structure. The expression of periostin mRNA was down-regulated in24hours, which continued in the whole experiment process, confirmed the periodontal membrane fiber system began to degenerate with periostin in the periodontal ligament reducing in the absence of mechanical stress. Wen. explored the function of TGF-(β1and FAK in regulating periostin expression in the periodontal fibroblasts. Periostin mRNA level increased significantly by dealt with TGF-β1, but focal adhesion kinase (FAK) inhibitors have attenuation effect. No periostin mRNA expression was detected in FAK knock out fibroblasts, even after using cyclic stress stimulus. These results suggest that the strong expression of periostin in human periodontal ligament may be involved in the regulation of FAK-dependent pathway. The latest study confirms that the inflammation medium (TNF-a) and bacterial toxin factor (LPS) can significantly reduce the expression of periostin in periodontal ligament fibroblasts, indicating periostin is an important factor in the development of periodontal diseases. It also suggests us what an important premise it is that the oral hygiene care and the control of inflammation during the orthodontic treatment to periodontal patients. Otherwise the periodontal tissue will be damaged, and then orthodontic periodontal tissue and bone remodeling, orthodontic tooth movement is simply impossible.Periostin as an important extracellular matrix protein has aroused widespread attention. It plays an important role in multiple body systems. Now the research of periostin is also increasingly deeper. Periostin as a key factor plays an essential role in maintaining the integrity and function of the periodontal membrane under the action of stress, and the research of periostin is mostly focused on the mechanical stress stimulus of periodontal ligament cells in vitro, in vivo study involving many factors is relatively small. The research about the function of periostin in the process of orthodontic tooth movement and periodontal tissue remodeling through establishing a model of orthodontic tooth movement in mice has yet not been reported. The function and mechanism of periostin in various diseases, especially in periodontal tissue is not clear. This study is the first to establish the mice model of orthodontic tooth movement, using quantitative immunohistochemistry, Micro-ct, RT-QPCR, Western blot and periostin gene knockout mice to study the function of periostin in the periodontal ligament remodeling and the related signaling pathway. Because of too little periodontal ligament in mice, it is difficult to detect its function in protein levels. So our study collects the periodontal ligament of premolars from orthodontic patients, and use RT-QPCR and proteomics technology to further study the related molecular signaling pathways of periostin in orthodontic periodontal tissue remodeling, giving an in-depth discussion about the function and molecular mechanism of periostin in periodontal tissue remodeling during orthodontic tooth movement.The results of this research open up a new way to a further systematic study of the molecular biological mechanism of periodontal tissue remodeling under the action of mechanical force, contributes to a better understanding of the mechanism of orthodontic tooth movement and provides a theoretical basis for orthodontic clinical research of speeding up the movement of orthodontic teeth.Methods:1.Us ing the homemade oral Microscopic fixed traction device establishes an experimental model of orthodontic tooth movementBy using the homemade oral Microscopic fixed traction device and Microscope between mouse incisors and first molars with a Hook and the Upper incisors with a ligature wire with20g force. The coil spring were kept constant and recorded for Id, 3d,5d,7d. Upon completion of experiments, the maxillae were removed. The specimens were fixed in4%paraformaldehyde in0.1M phosphate buffer for24hrs and decalcified in10%ethylene diamine tetraacetic acid (EDTA) at room temperature for5weeks. After being dehydrated in ascending grades of alcohol, cleared in xylene, and paraffin-embedded,3-um serial sections were cut parasagittally on a Microtome. HE staining was observed between morphology and molar periodontal tissue changes.2. Observing the expression of PN Protein in periodontal tissues after orthodontic tooth movementAfter the establishment of an experimental model of orthodontic tooth movement,and adding the force on teeth for1d,3d,5d,7d, then the mice were sacrificed, making immunohistochemistry slices analysis techniques to observe changes in the expression of periodontal ligament PN chemical and quantitative immunohistochemistry images.3. PN gene knock-out mice to identify genotypesClipping the tail of knockout mice0.5cm into the tube labeled good EP, extracted DNA, PCR amplification primers were designed based on amplification products after a1.5%agarose gel electrophoresis to remove placed observed and photographed under UV light. Identification of the gene in mice standards:PN+/+mice genotype PCR product was691bp, PN-/-genotype mice were PCR amplified product of500bp.4. Micro-ct imagingC57BL and PN knockout mice were sacrificed, separating the upper and lower jaw, specimens were selected in each group. Mark the specimens in each group, fixed on a dedicated scanning tube, using Micro-ct imaging system tomography imaging to evaluate the periodontal tissue.5. RT-PCR to observe the PN-related signal pathway regulating factors in the periodontal ligament after orthodontic tooth movementTo an establish experimental model of orthodontic tooth movement, C57mice were divided into two groups, wearing orthodontic device not applying force group and applying force. The experimental mice in group wearing orthodontic device were sacrificed after adding force5d, then put the pulled teeth in lysis buffer containing EP tube, with Real-time PCR detecting the expression of PN-related signaling pathways regulating factors(TGFβ1, FAK)in periodontal ligament,the correlation between PN and the related signaling pathways regulating factors was analyzed..6. RT-PCR to observe the PN-related signal pathway regulating factors in human periodontal ligamentCases of severe congestion was control group, and the experimental group was neat dentition, former arch protrusion patients. Adolescent orthodontic patients in the control group and the experimental group were pulled out premolar. Real-time PCR observed the expression PN, TGF-01, FAK related signaling pathways regulating factors in hunman periodontal ligament, with statistical analysis to detect the correlation between the groups, TGF-β1, FAK expression.7. Westernblot to detect the PN-related signal pathway regulating factors in human periodontal ligamentBased on Westen blot analysis, PN、TGF-β1、FAK、P-Akt AKT related signal pathway regulating factor in human periodontal ligament were detected during orthodontic and not orthodontics teeth. With statistical analysis to detect the correlation between the groups, TGF-β1, FAK.Results1. applying orthdontic force process, the teeth moved in the set direction, stressed group between the second molar and first molar appears gap. Tension side periodontal a gap became wider, periodontal ligament fibers were tuebulent; cellular components increased; active osteoblasts and new bone deposited on alveolar surface; the periodontal ligament on the pressure side became narrow.2. Periostin was expressed at a low level in the mice periodontal tissues, some coloring relatively shallow, more evenly distributed. In Stressed group the expression of periostin increased, periodontal membrane staining became intense. Compared with the control group Od, the expression of periostin was enhanced in each stressed group (P<0.05); Compared with the group1d, the expression of periostin were enhanced in group3d,5d (P<0.05);and group7d showed no statistical significance (P>0.05). Compared with the group3d, group5d,7d showed no statistical significance (P>0.05). Compared with the group5d, group7d was significantly decreased (P<0.01). The expression of periostin was most highest in group7d is the entire tooth movement in mice expressing the strongest period of the PN alterations3.One lane only had one purpose strip, brightness strong bands at the range of691bp, according to criteria determined to WT mice. Lane2also only had one target band, strong brightness strip in the range of about500bp, and therefore is determined PN-/-mice according to criteria.4. Mice MICRO-CT remodeling diagram shows:compared with the control group C57mice, PN-null mice periodontal tissue destructed, significant levels of alveolar bone absorpted, and molar furcation area was completely exposed. Mice MICRO-CT panoramic diagram shows:compared with the control group C57mice, PN-null mice periodontal tissue destructed, apparent absorption of alveolar bone, the alveolar ridge top disappeared, discontinuity of the periodontal ligament damaged5.Compared to the control group C57with no orthodontic force, PN mRNA expression in periodontal ligament of C57mice in the experimental group with orthodontic force increased, the difference was statistically significant (P<0.05); FAK mRNA expression significantly enhanced, the difference was statistically significant (P<0.05); TGF-β1mRNA expression showed significantly enhanced,the difference was statistically significant (P<0.05). Compared with PN-null group with no orthodontic force, In PN-null group with orthodontic force,the FAK mRNA expression in periodontal ligament increased, the difference was statistically significant (P<0.05); TGF-β1mRNA expression significantly enhanced, the difference was statistically significant (P<0.01). Compared with PN-null the control group with no orthodontic force, TGF-β1mRNA expression of periodontal ligament in C57group with orthodontic force significantly decreased, the difference was statistically significant (P<0.01), FAK mRNA expression significantly enhanced, there is statistical difference significance (P<0.01). Compared with PN-null control group with orthodontic force, TGF-β1mRNA expression of periodontal ligament in C57group with orthodontic force decreased, the difference was statistically significant (P<0.05); FAK mRNA expression significantly enhanced, the difference was statistically significant (P<0.01).6.Compared with the control group with no orthodontic force, Periostin mRNA expression in human periodontal ligament orthodontic force in the experimental group was significantly enhanced, difference was statistically significant (P<0.01); FAK mRNA expression significantly enhanced, difference was statistically significant(P<0.01); TGF-β1mRNA expression significantly enhanced, difference was statistically significant(P<0.05).7. Compared with non-orthodontic group, the group of people orthodontic periodontal ligament periostin, TGF-p1, FAK, P-Akt protein expression were significantly increased, difference was statistically significant (P<0.01). Compared with non-orthodontic group, human Orthodontic group Akt, a-tublin protein expression of periodontal ligament were not significantly different, difference was not statistically significant (P>0.05). Conclusion1. The first successful establishment of a mouse model of orthodontic force, during the mice orthodontic tooth movement, PN expression of periodontal tissue enhanced throughout the whole periodontal tissue remodeling process, then determined the PN involved in the process of the mice periodontal ligament remodeling.2. PN is necessary to maintain the integrity of the structure and function of the periodontal ligament, and it is an important molecular link under orthodontic periodontal ligament remodeling process, playing a regulatory role in transduction.3. TGFβ1-PN-FAK signaling pathway involved in the regulation of the remodeling of the periodontal ligament under orthodontic movement, except to TGFβ1-PN-FAK signaling pathway,FAK may be other ways involved in the remodeling of periodontal ligament process.4.TGFβ1-PN-FAK phosphorylation by activating Akt signaling pathway downstream of orthodontic forces regulate the periodontal ligament remodeling. |
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