| Objectives: Suitable occlusal vertical dimension(OVD) is key for fully playing the chewing efficiency, reducing masticatory muscle damage and obtaining balance and coordinate of facial proportions. On the other hand,normal muscle structure and function is anatomical basis of maintaining normal occlusal vertical dimension.Increasing occlusal vertical dimension(i OVD) cases are often involved in the process of orthodontic treatment. For most of patients, the treatment is successful.Though a part of clinical patients with deep overbite are relapse. Some scholars believe that establishing the appropriate muscle balance is very important for the stability of orthodontics.Retention period depends on the organizational structure and functional reconstruction. Therefore, less adaptive change on the structure and function of the muscle could be one of the important reasons of relapse.As the main jaw-elevators in masticatory muscles, the function of masseter is closely related to the occlusal vertical dimension. Increasing the occlusal vertical dimension is bound to cause muscle fiber stretch, which causes mechanical load rising. Skeletal muscle of mammals show a high plasticity, which can change its anatomical, physiological function,histochemical and biochemical characteristics according to muscle loading.The change of MHC isoforms makes skeletal muscles optimize the demand for energy. Howerver, there are few reports on whether masseter will change similarly as other skeletal muscle stretching. Meanwhile the effects of increasing the vertical dimension on adult animals have not been reported.In present study, we researched the influence of i OVD on masseter and myogenic regulatory factors through establishment the animal model of increasing occlusal vertical dimension. Also the underlying molecular signal mechanism was investigated. This research will provide theoretical basis forlong-term stability of clinical i OVD patients of different age.Methods:Part I Establishment animal model of increasing occlusal vertical dimen-sion1 The experimental animals and groupingA total of forty eight male Wistar rats were divided into two groups according to the age: teenager group(n=24, 1.5 months) and young adult group(n=24, 8 months). Both of teenager and young adult rats were randomized divided into two subgroups: control group(n=12) and experimental group(n=12). So there were four groups according to the age and experimental intervention, namely teenager control group, teenager experimental group, young adult control group and young adult experimental group. The experimental groups were increased the vertical dimension by placing the composite resinon all maxillary molars. The control groups were performed as operation control.2 Increased the vertical dimensionRats in the experiment group were anesthetized with 10% chloral hydrate by intraperitoneal injection(0.3ml/100 g of the body weight). Both left and right upper molars were etched using 35% phosphate gel for 20 s. After rinsing completely, the adhesive was coated on the surface of the molars and light-cured for 10 s. The composite resin was applied on molars making the first molar 1.5mm thick and the third molar 1mm thick then light-cured for 20 s. The occlusal interference points were removed by grinding machine.3 Manipulations of the control groupsThe control groups were performed all the procedures above expect placing the composite resin.4 Evaluation of increasing OVD(i OVD)4.1 Observation of the general conditionSplint maintenance, behavioral observation and body weights were recorded twice in fixed time during the whole experiment period.4.2 Occlusal changesThe occlusal changes also were observed and recorded by photograghs before OVD(T0), 0 day(T1), 7 day(T7) and 14 day(T14) after OVD.4.3 Cephalometric observationTo evaluate the degree and stability of OVD amount during the experimental period, radiographs was obtained from two groups on T0, T1, T7 and T14 after OVD using X-ray machine. The cephalometric landmarks were determined by FUJI PRIMA Console software referencing the method described by Yagi. Each radiograph was measured twice repeatedly by different observers. The difference of vertical dimension(VT) between experiment group and control group were counted. Also Δ VT values of teenager and young adult rat on different time points were measured.4.4 HE stainAfter bite-rising for 7 days and 14 days, six rats were randomly chosen from the four subgroups and sacrificed. The mid-portion of the deep masseter was excised perpendicularly to the direction of the muscle fibers. The size of the tissue block is 8.0mm×5.0mm×3.0mm. The muscles were rapidly frozen in isopentane cooled by liquid nitrogen and stored at-80℃. HE staining was used to observe i OVD impact on masseter.Part II Influence of i OVD on histology and morphology of rat deep masseter at different age1 The experimental animals and grouping in accordance with the first part2 Collection of the specimenAfter bite-rising for 7 days and 14 days, six rats were randomly chosen from the four subgroups and sacrificed. Collection method of specimens used for enzyme histochemistry was same as HE stain in part I. Specimens used for electron microscopy is 1.0mm × 1.0mm × 3mm and fixed in 4%glutaraldehyde for 2 hours before inspection.3 Enzyme histochemistrySerial sections were stained for myofibrillar ATPase reactivity(alkalinepreincubation, p H=10.6; acid preincubation, p H=4.5) and nicotinamide adenine dinucleotide tetrazolium(NADH-TR). Based on these reactions, the myofibers were classified asⅡa, Ⅱx/d and Ⅱb. The MHC composition ratio of masseter muscle was analyzed using Image-Pro Plus software.4 Transmission electron microscopy observationThe filament, sarcomere and mitochondria were observed under transmission electron microscopy and recorded.Part III Influence of i OVD on MHC m RNA relaive expression level of rat deep masseter at different age and mechanism discussion1 The experimental animals and grouping in accordance with the first part2 Collection of the specimenAfter bite-rising for 7 days and 14 days, six rats were randomly chosen from the four subgroups and sacrificed. The mid-portion of the deep masseter was excised perpendicularly to the direction of the muscle fibers. After flushing blood by cooled physiological saline, muscles were rapidly frozen in liquid nitrogen and stored at-80℃.3 Detection of MHC m RNA levels and myogenin:Myo D m RNA ratio using relative quantitative real-time PCR technologyThe main steps including: tissue homogenation, total RNA extraction by Trizol method, RNA integrity detection, primer design, reverse transcription,PCR amplification. ??CT method was used to test the expression of MHC m RNA levels and MRFs m RNA levels. The relationships between MHC m RNA levels and Myo D:myogenin m RNA ratio were analysed by correlation analysis.Results:Part I Establishment animal model of increasing occlusal vertical dimension1 Observation of the general condition1.1 Observation of splint maintenanceThe splint had good retention in different groups during the experiment.1.2 Behavioral observationThe adverse reaction of wearing biteplate improved gradually after a week. The behavior reaction of experimental group was mainly identical with that of the control group.1.3 Body weight changesThere was no significant statistical difference in body weight between control group and experimental group(P>0.05).2 Occlusal changesThe upper incisor of rat at different age covered on the lower incisor about 2 mm when biting passively from the extroral view. The occlusal state of group TE recovered to normal state quicker than that of in young adult group.The oral health status were fine during the experiment period of different groups. There was no obvious uneven abrasion of the splint.3 Cephalometric observationDuring the observation period, VT values were statistically significant the control and experimental group of different age(P<0.05). While in the same observation period, there was no significant difference of ΔVT in different age(P>0.05). Also, there was no significant difference between ΔVTTG and ΔVTYAG on T1, T7 or T14(P>0.05).4 HE stainThere was no obvious difference of morphology of muscle fibers and endomysium in control and i OVD group at different age.Part II Influence of i OVD on histology and morphology of rat deep masseter at different age1 Enzyme histochemistryATPase staining proved deep masseter muscle fiber type containing only the MHC Ⅱ fibers. The myofibers were further divided into three subtypes by NADH-TR: MHCⅡa, MHCⅡx/d and MHCⅡb.Deep masseter of teenager group mainly consisted of MHCⅡb type fiber,followed by MHCⅡa and MHCⅡx. With the observation time, MHCⅡatype fiber decreased and MHCⅡb increased(P<0.05). When i OVD 7 days,the proportion of MHCⅡa fiber was 25.24±2.70% and 45.50±1.89% in the group TC and the experimental group respectively. Howerver, the proportion of MHCⅡb and MHCⅡx decreased. On the 14-day of i OVD, the proportion of MHCⅡa fiber declined and MHCⅡb fiber proportion increased.Deep masseter of group YAC mainly consisted of MHCⅡb type fiber,followed by MHCⅡx and MHCⅡa. With the observation time, the change between MHC Ⅱfibers of group YAC was not obvious. When i OVD 7 days,there was no significant change in MHCⅡa and MHCⅡb. On the 14-day of i OVD, the proportion of MHCⅡa fiber increased and MHCⅡb fiber proportion declined.2 The transmission electron microscopy observationIncreasing occlusal vertical dimension had impact on masseter fiber arrangement and mitochondria structure. The phenomenon of muscle fiber reconstruction and compensatory increasing number and volume of mitochondria appeared earlier in group TE. There are less cellular traumatic reaction than that in group YAE.Part III Influence of i OVD on MHC m RNA relaive expression level of rat deep masseter at different age and mechanism discussion1 Analysis of influence of i OVD on MHC m RNA relaive expression level of rat deep masseter at different age1.1 Influence of age on the changes of MHC m RNA relaive expression levelThe relative level of MHCⅡa m RNA in group TC was more than that in group YAC, however, the relative level of MHCⅡb m RNA and MHCⅡx m RNA was less than that in group YAC.1.2 Influence of i OVD on the changes of m RNA relaive expression levelIncreasing the vertical dimension made the level of MHCⅡa m RNA increased and made the level of MHCⅡb m RNA and MHCⅡx m RNA decreased gradually, both in the teenager and young adult rats. These changes occured later in young adult rats.The relative level of MHCⅡa m RNA in group TE was more than that ingroup YAE. The relative level of MHCⅡb and m RNA was less than that in group YAE.1.3 Influence of time on the changes of m RNA relaive expression levelThe relative level of MHCⅡa m RNA tended to decrease gradually in the group TC, and the MHCⅡb m RNA tended to increase with time However,there was no obvious change in the relative level of MHCⅡa Ⅱx and Ⅱb m RNA in the group YAC.The relative level of MHCⅡa m RNA decreased in the group TE. The level of MHCⅡa m RNA increased on 14-day in the group YAE. The change of MHCⅡb m RNA level was opposite to MHCⅡa m RNA in the two groups.2 Analysis of influence of i OVD on Myo D and myogenin m RNA relaive expression level of rat deep masseter at different age2.1 Influence of age on the changes of Myo D and myogenin m RNA relaive expression levelThe relative level of Myo D and myogenin m RNA in teenager rats was more than that of in young adult rats.2.2 Influence of i OVD on the changes of Myo D and myogenin m RNA relaive expression levelIncreasing the vertical dimension could increase the Myo D and myogenin m RNA levels in both teenager and young adult group of rat masseter.2.3 Influence of time on the changes of Myo D and myogenin m RNA relaive expression levelMyo D and myogenin m RNA level decreased gradually in both of teenager and young adult control group with research time except the level of myogenin in group YAC.3 Analysis of influence of i OVD on myogenin:Myo D m RNA ratio of rat deep masseter at different age3.1 Influence of age on the changes of myogenin:Myo D m RNA ratioThe myogenin:Myo D m RNA ratio in teenager rats was more than that of in young adult rats.3.2 Influence of i OVD on the changes of myogenin:Myo D m RNA ratioIncreasing the vertical dimension could increase myogenin:Myo D m RNA ratio in both group TE and group YAE, These changes occured later in group YAE.3.3 Influence of time on the changes of myogenin:Myo D m RNA ratioThe myogenin:Myo D m RNA ratio decreased gradually in group TC and the ratio increased in group YAE with time.4 The correlation analysis between MHC m RNA levels and Myo D:myogenin m RNA ratioThere was positive correlation between the myogenin:Myo D m RNA ratio and MHCⅡa m RNA(r=0.954). However, there was negative correlation between the proportions of myogenin:Myo D m RNA ratio and MHCⅡb m RNA(r=-0.755)Conclusions:1 Establishment animal model of increasing occlusal vertical dim--ension by placing the composite resinon on all maxillary molars occlusal surface is stable and operated simply.2 The deep masseter of teenager and young adult rat in experimental group could change its structure to slower phenotype in the level of histology,m RNA when increasing the vertical dimension.3 The deep masseter of teenager rat in experimental group changes its structure to slower phenotype earlier and more than that of young adult rat in experimental group. It reminds that the OVD in teenager probable be more stable than young adult treating the patients with deep overbite in clinical treatment. Also i OVD treatment in young adult requires enough treatment time and maintains for a long-time after treatment.4 The Myo D and myogenin which belong to myogenic regulatory factors plays an important role in fiber reconstruction. |
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