Histomorphometric study of bone reactions during orthodontic tooth movement in rats

The biological response to orthodontic tooth movement has generally focused on reactions within the periodontal ligament (PDL), whereas less attention has been paid to the behavior of neighboring bone. The purpose of the study was to describe the influence of orthodontic force on bone surrounding the displaced tooth and the adjacent, untreated teeth. Bone changes in relation to treatment time and different sites were investigated. A mesial tipping of the left maxillary first molar was obtained from 54 adult male Wistar rats. Oxytetracycline was injected subcutaneously 48 h before killing, which took place after 4, 7, or 14 days. The maxilla was fixed in paraformaldehyde and embedded undecalcified in methylmethacrylate. A set of thick horizontal sections was taken from the cervical, intermediate, and apical levels of the roots. The sections were microradiographed and analyzed microscopically under bright-field and fluorescent illumination. Bone fraction and PDL width was measured using a Zeiss Videoplan device equipped with an overlay system. New bone formation was detected by oxytetracycline labels. The analysis showed a consistent, significant decrease of the alveolar bone fraction around both displaced and adjacent teeth at all treatment times. Apposition, indicated by the tetracycline uptake, was found on the periosteal side of the treated hemimaxilla and, after 14 days, also on the surface toward which the tooth was moving and around the adjacent teeth. These results suggest that a time rather than a space relationship exists between bone resorption and formation and that the whole hemimaxilla reacts to the mechanical challenge, resembling the regional acceleratory phenomenon (RAP) observed in other circumstances.     

Coupling of resorption and formation on bone remodeling sequence in orthodontic tooth movement: A histochemical study

Rat's molars were submitted to orthodontic tooth movement. Bone formation areas were detected using lead-labeling technique. Osteoclasts and osteoblasts were detected by enzyme histochemistry using Tartrate resistant Acid phosphatase (TRACPase) and Alkaline phosphatase (ALPase) to determine simultaneously and mark the 2 types of cells on a same section. The sites selected for study were pressure/distal, tension/mesial and transitional areas of second molars. The results showed that: orthodontic force activated bone remodeling sequence throughout the alveolar bone; slight new bone formation was observed on the cement line on the pressure side. ALPase-positive cells were detected on the pressure side neighboring osteoclasts. On the tension side, bone formation was enhanced in the protrusions whereas both resorption and formation were observed in the depressions. In the transitional area, cellular sequence from osteoclastic bone resorption to bone formation was revealed over the cement line. These findings demonstrated that: coupling phenomena occur on the pressure side but with inhibited osteoblast activity. Bone formation on the tension side involves both promotion of bone formation by the traction force; bone remodeling sequence is established on the tension side by the interaction between osteoclastic bone resorption and bone formation that takes place in the depressions; Coupling phenomena occur in the transitional area as well. Our findings on the pressure side led to the consideration that osteoblastic cells in periodontal ligament would be involved in the regulation of osteoclastic bone resorption. Thus, there appears to be an interaction between osteoclastic and osteoblastic cells and an activated bone remodeling sequence involving the coupling phenomena as a mechanical adaptation to orthodontic force.     

Histomorphometric study of alveolar bone turnover in orthodontic tooth movement

Selected histomorphometric parameters were measured in alveolar bone adjacent to rat molars treated with a 40gram tipping force designed to tip the molar mesially and with a sham procedure. Undecalcified parasagittal sections of teeth and surrounding tissues were prepared for static and dynamic histomorphometry at 1, 3, 5, 7, 10, and 14 days. Initial tooth displacement was assessed by measuring differences between groups in the widths of the day-1 periodontal ligaments (PDL) at various vertical locations and correlating these using linear regression analysis. All parameters were measured in the alveolar bone adjacent to four quadrants around the teeth (mesial-distal; occlusalapical). Means and standard errors for each parameter in each group were calculated and compared for time- and group-specific differences using ANOVA and pairwise comparisons with Scheffe's multiple comparison tests. In shamtreated rats, bone resorption predominated on the distal alveolar surface, but significant surface-related differences between mesial and distal surfaces in bone formation were not demonstrated. Time-specific effects in bone resorption were not evident on either surface in the shams. These findings suggested that molar distal drift in the sham rat is facilitated by resorption in the distal alveolar bone. PDL width changes in orthodontically-treated rats were greatest in the mesial occlusal half of the root and decayed linearly toward the apex indicating that the greater initial tooth displacement occurred in the occlusal half of the root surface. Histomorphometric parameters of alveolar bone turnover were also seen to be greater in these locations, suggesting that these processes were sensitive to the increased tooth displacement. In orthodontically-treated rats, the total paradental alveolar bone and both pressure (mesial) and tension (distal) surfaces experienced a wave of resorption that lasted for four days, was preceded by an activation period of two days, and was followed by a wave of formation that persisted until the end of the experiment (14 days). The relative balance between bone formation and resorption waves was location-specific, with formation predominant on the tension side and resorption predominant on the pressure. These findings are consistent with the conclusion that pressure and tension initiate similar bone turnover events, and that bone deposition or removal is controlled by the degree of balance between the relative amounts of bone formation and resorption that occur at these sites.     

Experimental tooth movement and photobiomodulation on bone remodeling in rats

Photobiomodulation (PBM) is a therapy induced by a specific type of laser that affects biologic systems through non-thermal ways. The study of its basic mechanism has gained interest since little is known about the causes of the different effects of this treatment. In the present study, we investigated the action of the PBM application rate changes in the peri-implant tissues in rats subjected to tooth movement in different periods. Wistar rats (±250 g) received an apparatus in the region of the central incisors superiors tightly (70 g) or not, and they were also subjected to one or three PBM sessions. After 7 or 14 days, the rats were subjected to euthanasia and the jaws were dissected and processed for histology. For analysis, serial sections were made that were stained by Picrosirius Red for analysis of collagen fibers, Masson’s trichrome for newly formed bone scan, and Hematoxylin-Eosin for quantification of osteoblasts. PBM applied in one or three sessions increased the population of osteoblasts. Still, the application of three sessions of PBM increased the density of collagen fibers and new bone formation compared to the controls. An increase was observed in the interincisal distance in irradiated groups with three PBM sessions and the application of force for both 7 or 14 days. These findings suggest that PBM can contribute positively to the orthodontic movement. So the laser therapy can be used as an adjunct procedure to be performed concurrently for orthodontic treatment in the dental clinic.     

Effects of low-energy laser irradiation on bone remodeling during experimental tooth movement in rats

BACKGROUND AND OBJECTIVE: Low-energy laser irradiation has many anabolic effects such as the acceleration of bone formation. However, its effects on tooth movement, performed by bone resorption and formation, have not been well characterized. STUDY DESIGN/MATERIALS AND METHODS: A total of 10 g of orthodontic force was applied to rat molars to cause experimental tooth movement. A Ga-Al-As diode laser was used to irradiate the area around the moved tooth, and after 12 days, the amount of tooth movement was measured. Calcein was injected subcutaneously to label the newly formed alveolar bone for quantitative analysis. Immunohistochemical staining of proliferating cell nuclear antigen was performed to evaluate cellular proliferation. TRAPase staining was also performed to facilitate the identification of osteoclasts. RESULTS: In the laser irradiation group, the amount of tooth movement was significantly greater (1. 3-fold) than that of the nonirradiation group in the end of the experiment. The amount of bone formation and rate of cellular proliferation in the tension side and the number of osteoclasts in the pressure side were all significantly increased in the irradiation group when compared with the nonirradiation group (P < 0. 01). CONCLUSION: These findings suggest that low-energy laser irradiation can accelerate tooth movement accompanied with alveolar bone remodeling.     

Remodeling of fracture callus in mice is consistent with mechanical loading and bone remodeling theory

During the remodeling phase of fracture healing in mice, the callus gradually transforms into a double cortex, which thereafter merges into one cortex. In large animals, a double cortex normally does not form. We investigated whether these patterns of remodeling of the fracture callus in mice can be explained by mechanical loading. Morphologies of fractures after 21, 28, and 42 days of healing were determined from an in vivo mid‐diaphyseal femoral osteotomy healing experiment in mice. Bone density distributions from microCT at 21 days were converted into adaptive finite element models. To assess the effect of loading mode on bone remodeling, a well‐established remodeling algorithm was used to examine the effect of axial force or bending moment on bone structure. All simulations predicted that under axial loading, the callus remodeled to form a single cortex. When a bending moment was applied, dual concentric cortices developed in all simulations, corresponding well to the progression of remodeling observed experimentally and resulting in quantitatively comparable callus areas of woven and lamellar bone. Effects of biological differences between species or other reasons cannot be excluded, but this study demonstrates how a difference in loading mode could explain the differences between the remodeling phase in small rodents and larger mammals. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 664–672, 2009     

The relevance of leukotrienes for bone resorption induced by mechanical loading

5-Lipoxygenase (5-LO) metabolites are important pro-inflammatory lipid mediators. However, much still remains to be understood about the role of such mediators in bone remodeling. This study aimed to investigate the effect of 5-LO metabolites, LTB₄ and CysLTs, in a model of mechanical loading-induced bone remodeling. Strain-induced tooth movement and consequently alveolar bone resorption/apposition was achieved by using a coil spring placed on molar and attached to incisors of C57BL6 (wild-type-WT), 5-LO deficient mice (5-LO^−/−) and mice treated with 5-LO inhibitor (zileuton—ZN) or with antagonist of CysLTs receptor (montelukast—MT). The amount of bone resorption and the number of osteoclasts were determined morphometrically. The expression of inflammatory and bone remodeling markers in periodontium was analyzed by qPCR. Osteoclast differentiation and TNF-α production were evaluated in vitro using RAW 264.7 cells treated with LTB₄ or LTD₄. Bone resorption, TRAP⁺ cells and expression of Tnfa, Il10 and Runx2 were significantly diminished in 5-LO^−/−, ZN- and MT-treated mice. The expression of Rank was also reduced in 5-LO^−/− and MT-treated mice. Accordingly, LTB₄ and LTD₄ in association with RANKL promoted osteoclast differentiation and increased TNF-α release in vitro. These data demonstrate that the absence of 5-LO metabolites, LTB₄ and CysLTs reduces osteoclast recruitment and differentiation, consequently diminishing bone resorption induced by mechanical loading. Thus, 5-LO might be a potential target for controlling bone resorption in physiological and pathological conditions.     

Local delivery of osteoprotegerin inhibits mechanically mediated bone modeling in orthodontic tooth movement

INTRODUCTION: The RANKL-OPG axis is a key regulator of osteoclastogenesis and bone turnover activity. Its contribution to bone resorption under altered mechanical states, however, has not been fully elucidated. Here we examined the role of OPG in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement. METHODS: The maxillary first molars of male Sprague-Dawley rats were moved mesially using a calibrated nickel-titanium spring attached to the maxillary incisor teeth. Two different doses (0.5 mg/kg, 5.0 mg/kg) of a recombinant fusion protein (OPG-Fc), were injected twice weekly mesial to the first molars. Tooth movement was measured using stone casts that were scanned and magnified. Changes in bone quantity were measured using micro-computed tomography and histomorphometric analysis was used to quantify osteoclasts and volumetric parameters. Finally, circulating levels of TRAP-5b (a bone resorption marker) was measured using enzyme-linked immunosorbent assay. RESULTS: The 5.0 mg/kg OPG-Fc dose showed a potent reduction in mesial molar movement and osteoclast numbers compared to controls (p<0.01). The molar movement was inhibited by 45.7%, 70.6%, and 78.7% compared to controls at days 7, 14, and 21 respectively, with the high dose of OPG. The 0.5 mg dose also significantly (p<0.05) inhibited molar movement at days 7 (43.8%) and 14 (31.8%). While incisor retraction was also decreased by OPG-Fc, the ratio of incisor to molar tooth movement was markedly better in the high-dose OPG group (5.2:1, p<0.001) compared to the control group (2.3:1) and the low-dose OPG group (2.0:1). CONCLUSIONS: Local delivery of OPG-Fc inhibits osteoclastogenesis and tooth movement at targeted dental sites.     

The endothelin system mediates bone modeling in the late stage of orthodontic tooth movement in rats

The endothelin system is involved in orthodontic tooth movement (OTM). The aim of the study was to examine the role of ET-1, ET(A) and ET(B) in bone modeling during OTM in rats. Male Wistar rats (n=62) were divided into three groups: control animals (n=10; control group) without appliance, and two groups of experimental animals, which were applied a super-elastic closed-coil spring between the first left maxillary molar and the incisors and were treated daily with either TBC3214 (n=10; TBC3214 group) or with saline (n=42; appliance only group). TBC3214 is a highly selective antagonist on ET(A) receptors. The distance between teeth was measured on days 0 and 42. On days 0, 14, 28 and 42 animals of the appliance only group (n=8) were sacrificed and tissue samples were taken. Total RNA and protein contents were isolated. Gene expression levels of ET-1, ET(A) and ET(B) were assessed by means of relative RT-PCR. Protein levels of ET(A) and ET(B) were examined by immunoblotting. Ten animals of each group were sacrificed on day 42 and tissue samples were prepared for histological analysis. Alveolar bone volume, osteoblast and osteoclast volume were determined histomorphometrically. Gene expression levels of ET-1, ET(A) and ET(B) varied throughout the experiment and were significantly up-regulated on day 42 (p<0.001). The immunoreactivity of ET(A) and ET(B) significantly decreased on day 14 (p<0.001) and increased on day 28 (p<0.001). Alveolar bone volume was significantly higher in the TBC3214 group compared to the appliance only group (p<0.001). Osteoclast volume was significantly lower in the TBC3214 group compared to the appliance only group (p<0.05). Gene and protein expression levels of ET-1, ET(A) and ET(B) varied significantly during OTM, suggesting their different roles in the various stages of OTM. TBC3214 significantly increased alveolar bone volume and significantly decreased osteoclast volume, indicating that it decreased bone resorption in stage three of OTM. These data suggest that ET-1 increases osteoclastic bone resorption via ET(A) in the late stage of OTM.     

多西环素对大鼠正畸移动牙根面骨型胶原酶表达的影响

目的:通过建立正畸牙根吸收动物模型,检测牙根面及根周组织中骨型胶原酶(MMP-13)的分布和表达,研究多西环素(DOXY)对根吸收及骨型胶原酶表达的影响。方法:选用SD大鼠36只建立正畸牙移动根吸收的动物模型,随机分为空白对照组、注射DOXY对照组、正常牙加力组、注射DOXY加力组。在加力的0天、7天、10天、14天分别处死各组大鼠。并取相应时段的标本制作组织切片,应用免疫组化技术检测分析各组中骨型胶原酶(MMP-13)在第一磨牙根吸收组织中的定位表达,检测多西环素对根周组织内骨型胶原酶表达及分布的影响。用计算机图像分析技术定量比较上述试验结果。结果:在多西环素的影响下,在第7天和第10天给药受力组的MMP-13的阳性表达低于受力不给药组。给药受力组MMP-13的阳性表达在第10天更低(P0.01)。两实验组MMP-13的阳性表达都在第10天到达峰值后第14天趋于平稳。结论:多西环素可能作为骨型胶原酶的抑制剂对破骨细胞的募集及牙根吸收有抑制作用。     

大鼠上颌第一磨牙正畸移动中牙周膜神经PGP9.5的表达

目的：建立大鼠正畸牙移动模型,观察PGP9.5在牙周膜（PDL）神经中的表达及变化,探讨正畸力转化成神经元的反应的细胞调控机制。方法：成年雄性SD大鼠25只,随机分为5组：加力0、3、7、14、21天组,每组5只。上颌第一磨牙（URM1）施加50g近中向的拉力,按实验期限,分别取不同组大鼠含磨牙及其周围牙槽骨的组织块切片,进行PGP9.5免疫组化染色。镜下观察牙齿压力侧牙周膜神经纤维PGP9.5免疫阳性的变化情况,并进行灰度值分析、统计学方差分析和t检验。结果：压力侧的神经纤维的密度增加。在第7天牙槽骨表面出现类骨质的沉积;第21天根吸收陷窝处可见免疫阳性的牙周神经纤维。第3、7天牙周膜神经纤维PGP9.5的表达最强（P〈0.05）,之后神经的表达开始下降,牙周膜神经密度也逐渐恢复。结论：正畸加力使大鼠牙周膜神经中PGP9.5表达增强,PGP9.5调控神经肽的合成和分泌,可能是神经元细胞调控的重要机制。牙周膜神经在调节骨细胞活性和促进类骨质的形成,以及牙根吸收和修复等过程中可能也发挥重要作用。     

正畸力作用下大鼠炎性牙周组织牙槽骨改建的研究

目的：通过观察正畸力及炎症条件下大鼠牙槽骨高度的变化,研究正畸力和炎症对大鼠牙周组织改建的作用。方法：将60只sD大鼠随机分为加力对照组（P1组）和炎症对照组（P2组）各30只。施加50g近中向正畸力于P1组大鼠的左上第一磨牙;同时建立实验性牙周炎模型于P2组大鼠双侧上颌第一磨牙,2周后去除双侧牙周炎性刺激物,设右上第一磨牙为炎症对照组（P2组）,并施加50g近中向正畸力于左上第一磨牙,为炎症加力组（P3组）。运用组织形态测量法观测各组大鼠上颌第一磨牙加力或去除炎症刺激物后各时点牙槽骨的吸收量。结果：P1组大鼠近中牙槽骨未见明显丧失,P2、P3组可见近远中牙槽骨的丧失,但当去除炎症刺激物后加力,P3组大鼠近中牙槽骨未见明显丧失。结论：只要彻底清除炎性刺激物正畸力不会导致牙槽骨的明显丧失。     

The effect of low-level laser therapy on tooth movement during canine distalization

The aim of the study is to determine the effects of low level laser therapy on tooth movement during canine distalization by evaluating IL-1β, TGF-β1 levels in gingival crevicular fluid. Maxillary first premolars of the 15 Angle Class II division I patients (12–19 years old) were extracted. Right maxillary canines were distalized by standard protocol as control group whereas the left maxillary canines distalized by laser application. A gallium-aluminum-arsenide diode laser with an output power of 20 mW was applied as five doses from the buccal and the palatal side on the day 0, and the 3rd, 7th, 14th, 21th 30th, 33st, 37th, 60th, 63th, and 67th days. Gingival crevicular fluid samples were obtained with filtration paper at the initial, 7th, 14th, and 21th days, and the IL-1ß and TGF-ß1 cytokine levels were analyzed. Orthodontic models and periodontal indices were taken initially and on the days 30th, 60th, and 90th of canine distalization period. Tooth movement was assessed by scanning models (3Shape). The amount of tooth movement in the laser group was 40% more than the control group. First day IL-1ß levels were statistically higher than initial and 21st day levels (P=?0.003, P?=?0.012). The rise in IL-1ß levels caused the negative correlations between 7th day IL-1β and 21st day TGF-β1 levels describes the tissue effects of laser application. Periodontal indices showed no sign of gingival inflammation during canine distalization period. As conclusion, laser does accelerate tooth movement and could shorten the whole treatment duration.     

P物质缓释系统对正畸大鼠牙周破骨细胞数目的影响

目的:探讨P物质纳米缓释微球的生物活性及对正畸牙周改建中破骨细胞数目变化的影响.方法:将P物质-聚乳酸-聚乙醇酸共聚物纳米缓释微球(SP-PLGA NP)注射入正畸大鼠第一磨牙根尖水平龈下,分别在加力后1,3,7,14天后采用HE染色观察牙用组织变化和压力侧破骨细胞数目,并和单纯用p物质以及空白对照组比较.结果:加力后1天,三组间牙周都未发现有破骨细胞;加后3天,破骨细胞数量表现为P物质组＞缓释纳米微球组＞对照组,与对照组有显著性差异(P＜0.05);加力后7天,表现为缓释纳米微球组＞P物质组＞对照组,与对照组有显著性差异(P＜0.05);加力14天后,三组破骨细胞数量都有减少,但缓释纳米微球组数量最多.结论:P物质能促进正畸中牙周破骨细胞形成,而SP-PLGA NP能在较长时间维持这种作用.     

The effect of low-level laser therapy during orthodontic movement: a preliminary study

It has been emphasized that one of the most valuable treatment objectives in dental practice is to afford the patient a pain-free treatment. By the evolution of the laser applications, the dental committee aimed to achieve this goal without analgesic drugs and painful methods. Orthodontic treatment is one of these concerns, that one of the major components of patient to reject this treatment is the pain accompanied during the different treatment phases. Another great concern of the patient is not to get through prolonged periods of treatment. The aim of this study is to evaluate the effect of the low-level (GaAlAs) diode laser (809 nm, 100 mW) on the canine retraction during an orthodontic movement and to assess pain level during this treatment. A group of 15 adult patients with age ranging from 14 to 23 years attended the orthodontic department at Dental School, Damascus University. The treatment plan for these patients included extraction of the upper and lower first premolars because there was not enough space for a complete alignment or presence of biprotrusion. For each patient, this diagnosis was based on a standard orthodontic documentation with photographs, model casts, cephalometric, panorama, and superior premolar periapical radiographies. The orthodontic treatment was initiated 14 days after the premolar extraction with a standard 18 slot edgewise brackets [Rocky Mountain Company (RMO)]. The canine retraction was accomplished by using prefabricated Ricketts springs (RMO), in both upper and lower jaws. The right side of the upper and lower jaw was chosen to be irradiated with the laser, whereas the left side was considered the control without laser irradiation. The laser was applied with 0-, 3-, 7-, and 14-day intervals. The retraction spring was reactivated on day 21 for all sides. The amount of canine retraction was measured at this stage with a digital electronic caliper (Myoto, Japan) and compared each side of the relative jaw (i.e., upper left canine with upper right canine and lower left canine with lower right canine). The pain level was prompted by a patient questionnaire. The velocity of canine movement was significantly greater in the lased group than in the control group. The pain intensity was also at lower level in the lased group than in the control group throughout the retraction period. Our findings suggest that low-level laser therapy can highly accelerate tooth movement during orthodontic treatment and can also effectively reduce pain level.     

局部注射VEGF对大鼠正畸牙齿移动的影响

目的：观察局部注射重组鼠的血管内皮生长因子(rrVEGF)对大鼠正畸牙齿移动的影响，探讨VEGF在正畸牙齿移动中的作用机制。方法：45只雄性SD大鼠，牵引其上颌第一磨牙近中移动，实验中分别将rrVEGF及生理盐水注射入实验组及对照组大鼠右侧上颌第一磨牙腭侧的骨粘膜下，于实验前3天开始注射，每3天一次。分别在加力l、3、7、14、2l天后记录上颌第一磨牙移动距离。然后将各组动物处死，用HE染色观察牙周组织变化情况并采用免疫组织化学方法对组织中表达的VEGF进行分析。结果：实验组大鼠压力侧破骨细胞教和成骨细胞数在实验全过程中均多于对照组，而且，实验组大鼠牙齿移动距离明显大于对照组。结论：进一步证实VEGF作为旁分泌因子，参与了牙齿移动过程中的牙周组织改建，内源性VEGF和注射VEGF都使牙齿移动量显著增加。因此，VEGF在正畸牙齿移动的机制中具有重要意义。