Long-term orthodontic treatment in mice or rats over 3 or more weeks is scarce due to the challenging experimental setup2,16. We have successfully proceeded the longest studied OTM experiment in mice so far2 and could profoundly investigate the bone and PDL changes after this extended mechanical stimulus, with clear alterations in tooth position, alveolar bone, and PDL morphology. The differences between the bone form in various OTM stages, found in previous works16,27, demonstrate that a thorough understanding of bone remodeling also in the later stages of orthodontic induced periodontal remodeling is highly relevant. This offers an important step for the investigations of interrelation between modifications in alveolar bone microstructure and periodontal apparatus.
The strong variability in tooth translational movement and rotation as well as in their directions, found in this study, points out the simultaneous adaptation of force distribution after each small movement during the 5 weeks of OTM. Although the possible small discrepancies in the orthodontic force direction due to a difficult placing of the NiTi coil in mouse model may also contribute to the OTM discrepancy, these cannot fully explain the large differences in the tooth movement. We also point out the possible other factors, such as biological and habitual differences between the animals, i.e. that may influence the progression of OTM. The fact that the estimated rotational movements were found in both directions despite no external changes on NiTi coil setup, indicate that the center of rotation and the nature of movement adapt to the bone changes over the studied time frame and may thus be inconsistent. For these reasons, we propose that periodontal bone remodeling, especially during long-term orthodontic treatment, cannot be simply distinguished by the compression and tension part without an exact estimation of the tooth movement and rotation. Rather a complex remodeling takes place in a wider area around the tooth roots within the periodontal apparatus. A similar estimation may apply also in the case of shorter studies, as a certain degree of tooth rotation cannot be excluded in earlier stages. It should be noted that certain biological aspects may also contribute to the apparent OTM variance in this study, such as possible geometrical differences between both sides of maxillae.
For the evaluation of bone changes following OTM treatment, several studies applied the division to the tension and pressure side method13,14,15,20,36. Still, the tooth movement complexity during a NiTi-coil-induced OTM in small animal research results in high obstacles for the definition of tension and compression sides. Often, the bone resorption mechanism is prevalent on both sides, leading to higher porosity and lowered BMD and Tr.Th in both regions13,20. Some studies found the opposite remodeling outcome on both sides, leading to lower porosity and thicker or unchanged trabeculae on the tension side, while thinning of trabecular bone, loss of BV/TV and smaller Tr. Sep appeared in the compression region14,15,36. It was usually not examined whether the tooth movement was consistent or whether the constant force led to only translational movement in one direction or whether an additional complete root system rotation took place in the mechanical force application time frame. The OTM mechanism is known to be highly complex and a combination of movements (i.e., translation and rotation) may happen simultaneously and interchangeably16,33. Based on an in vivo study of OTM in male Wistar rats up to 31 days, Zong et al.16 suggested that the tension and pressure side may not be placed on the opposite sides of the tooth root, but rather be adjacent and inter-connected due to the inconsistent OTM rate over the timeline of orthodontic treatment. For that reason, a simple establishing of such areas found in literature and solely based on the the NiTi-coil direction may fall into misleading results regarding various bone remodeling under the opposite mechanical stimuli.
Most of the OTM studies in rodents focus on the alveolar bone in the region of the 1st molar since this is typically the experimental treated molar. Because of the highest expected bone changes in the region between the mesiobuccal and distobuccal root of M1, often a cubic or variably shaped VOI is chosen in this area for the morphometry analysis18,25,35,36,37,38,39,40. In a few studies, the VOI was set up in the region further from the roots14 or directly around the roots21. Still, the exact position, size and form of VOI vary strongly in the literature. Since studies on differences between the bone remodeling process in various regions of alveolar bone and PDL are missing, there is an open question whether only the alveolar socket of M1 is the most affected bone and whether other processes follow in the surrounding regions. The relatively wide VOI might reduce the statistical power because alveolar bone not affected by the movement might be included too. Some studies suggest applying a thin VOI within the region where bone remodeling is expected to take place (of ~ 100 µm thickness)21. It is, however, questionable whether the significant bone changes are restricted only to such relatively small regions close to the stress initiation points or whether wider regions may also be affected, especially after a prolonged orthodontic treatment. Too small VOI may limit the accurate estimation of the parameters, such as Tr.Th and Tr.Sep, if the VOI dimension is comparable to these parameters13. On the other side, Zong et al.16 evaluated the complete alveolar and basal bone around and under M1 and M2. Though, the inclusion of the rather stagnating basal bone into the VOI hinders the detection of smaller changes in the analyzed tissue.
To accurately estimate the change within the alveolar and periodontal bone, we chose to analyze the complete alveolar bone region between the mesiobuccal and distobuccal root (VOI:M1) or between the distobuccal root of M1 and the mesiobuccal root of M2 (VOI:M1-2) and reaching from the furcation up to the tip of the shortest root of M1. In such a case, a maximal VOI is chosen where possible strong changes can be expected while excluding the basal or cortical bone. Also, our VOI dimensions were larger in comparison to the obtained Tr.Th and Tr. Sep. Most importantly, a precise 3D registration ensures an almost identical position of the studied VOI in-between the samples. This allows for a more precise estimation of the morphology parameters as well as for the BMD. To avoid the influence of the cortical bone, the outer boundaries of VOI were set close to the roots center in order to exclude such regions.
The bone resorption as well as bone deposition processes are known to relate to an increased porosity and lower bone volume in the actively regrowing region41. Although no modifications in BV/TV after a short OTM (up to 3 weeks) could be detected in some studies in mice or rats18,42,43, the most of them showed similar reduction of BV/TV and thus an increase of bone porosity in the OTM group13,21,24,25,26,38,44. In our study, both regions of interest showed a significantly lowered BV/TV indicating a still regenerating phase of the alveolar bone. Due to lacking long-term OTM investigations in mice or rats in literature, this study proves in a novel way that previously observed bone remodeling also proceeds after a relatively long force application. Higher and often increased porosity leads to a stronger intratrabecular connectivity. We could see such an increase in both VOI regions while the connectivity increase was larger in VOI:M1. The higher porosity and connectivity of the OTM affected regions indicate a formation of woven bone along the collagenous PDL fibers in the widened PDL-bone attachment region32,41. This initial bone form is known to have an increased porosity comparing to natural alveolar bone and is therefore more susceptible to resorption if inflammation is initiated. In the later stages of bone remodeling, the woven bone will reshape to lamellar bone45.
Alongside these findings, the mineral density seems to be mostly reduced after OTM too13,23,38, which was also confirmed in a study on orthodontic patients22. Interestingly, Wang et al.27 observed a strong reduction of BMD after only 2 weeks of OTM followed by a turnover and its correction within the next 2 weeks of orthodontic treatment. This phenomenon may explain the findings from other studies in mice or rats, where BMD was not significantly different after 3 or more weeks of OTM16,18,43. The possible mineral density regeneration over the period of 5 weeks OTM also corroborates with our findings in both VOIs (i.e., VOI:M1 and VOI:M1-2), where BMD reduction was relatively small or non-significant. The fact that alveolar bone becomes more porous after OTM while mineral density remains relatively stable, indicates the concomitant bone formation and resorption caused by sterile inflammation16. That is an opposite regulation to the known phenomenon of pathological processes during osteoporosis46 or periodontitis47 where both parameters are reduced simultaneously. Thus, the sole BMD parameter is probably not sufficient to study bone changes under mechanical stimulus.
In parallel with bone loss or growth, a thinning of trabeculae has been detected in many studies16,18,20,43. Such reduction of Tr.Th obtained in this study after 5 weeks of OTM in both VOIs was greater than found by Holland et al.48 after 3 weeks of OTM in wild-type mice. Interestingly, this reduction was stronger in the M1 region than in the VOI between M1 and M2. The lowering of Tr.Th is expected, assuming that both bone loss and bone regeneration are happening in VOI:M1, while VOI:M1-2 may prevalently cover a region of bone growth and follows similar trend in other parameters, such as BV/TV, BMD and connectivity. Lower Tr.Th has been found mainly in the compression regions where bone loss process dominates14,36. While Dorchin et al.20 found trabeculae thickening in both tension and compression sides, a constant36, decreased13, or even increased14 value of Tr.Th has been observed on the tension side.
Together with the thinning of trabeculae, a simultaneous increase of Tr. Sep is in agreement with other studies14,18,36 and was found to be significant only in VOI:M1 although both areas presented the same trend. Trabecular separation seems to be a less sensitive parameter of bone remodeling and was found stable even after 3 or 4 weeks of OTM in rats16,43. Bone regrowth on the tension side resulted in a constant Tr.Sep36 or reduction14 leading to structures with smaller pores. Our observation indicates that rather a bone loss mechanism is prevalent in both studied regions.
For a more complete view about the changes in periodontium during orthodontic stimuli, this study emphasizes the importance in the precise evaluation of the periodontal ligament. The PDL assumes a regulation function for the force transfer as a bone-PDL-tooth joint-like system49. An optimal PDL-space biomechanically permits the effective stress redistribution from the tooth to the adjacent tissue. Despite the fact that the exact role of the PDL tissue in bone remodeling under mechanical load hasn’t been fully understood yet, there is a consensus about the high importance of PDL tissue in such processes as well as about its function as stress absorber and redistributor to the adjacent alveolar bone41. Mechanical response and remodeling factors inside the PDL region are expected to be strongly influenced by the heterogeneity of the PDL fiber density and vitality, and thus by the variability of the stress-distribution microregions50. The previous analyses of the PDL system indicate the complexity of this collagenous system and its reactions to external stimuli51. Still, the mechanism of the PDL-space response to variable magnitudes, directions and durations of loading needs to be examined in more detail. Therefore, the understanding of PDL tissue remodeling as a result of force application is an important progress factor for the orthodontic treatment strategy.
Often, bone morphology and PDL shape are studied only qualitatively and only on selected 2D images missing the complete information from the whole bone and PDL volume12,14,21,25,29,30,38,43. Still, for an accurate comparison, the exact position and orientation of the analyzed 2D sections in the control and in OTM treated side is crucial. A volumetric registration of the studied periodontium region shall be an important pre-requisite for such comparative evaluations of PDL thickness. Manually oriented data hinders the validity of the results through the influence of the sectioning angle as well as the position along the molar roots on the estimated PDL thickness. Additionally, the coordinates in which the PDL thickness is measured in 2D are often based on the assumed vector of the orthodontic force applied on the molar tooth. However, our data, confirmed by previous findings31,33, demonstrate the complexity of tooth movement during long term OTM and the simultaneous variability of the force direction during the bone and PDL remodeling process. Therefore, a simple orthodontic force definition without an exact estimation of tooth movement and stress distribution may fall into misleading results.
Moreover, the changes in PDL form found in literature are mostly in agreement with our results of PDL thickening due to orthodontic stimuli. An increase in PDL thickness was also detected by Li et al.43 in rats only after 21 days of OTM. Significant PDL thickening on the compression side of the distobuccal root of M1 was also observed in mice after 12 days of OTM25. A thickened PDL was also found in 1st molar of C57BL/6 wild type mice after occlusal trauma30. A strong widening of PDL in both pressure and tension side after 4 and 2 weeks of OTM, respectively, was shown in rats on histology by Hundson et al.38 and Dorchin et al.20. Nevertheless, only a qualitative evaluation was performed in the mentioned study. Dynamic changes in the PDL form were observed by Laura et al.29. Here, the PDL showed a thickening tendency on the tension side and a thinning on the opposite compression side in the first 3 days of treatment. Though, the original PDL thickness was recovered within the next 2 weeks. Such findings go along with the compressed PDL on the compression side and extended PDL on the tension side observed by Shalish et al.12 after only 4 days of mechanical force application. Both findings indicate the asymmetric changes in PDL thickness caused mainly by tooth movement in the second stage of OTM and assuming no or only negligible effect of rotational forces. Unfortunately, neither work provides verification whether such PDL deformation proceeded also in other root sections.
A full volumetric evaluation of PDL is provided only in a few studies on OTM in mice32,33,34. Here, the PDL thickness was determined by an algorithm that defines the shortest distance from each pixel of the root surface to the surrounding bone surface. For a correct results interpretation, it is important to note that without virtual ‘closing’ of the PDL-bone contact surface, such algorithm also defines the distorted areas and ‘broken pores’ in the alveolar bone as a wider PDL region. Still, the widening of the PDL thickness found in our study, is in agreement with the observations in these studies32,33. A volumetric approach was also applied in the study by Wolf et al.21, where the effect of PDL region extension after mechanical stimuli was confirmed and demonstrated by the significantly lower bone volume inside the 100-µm thick VOI around the molar roots in mice after 11 days of OTM. We note that the additional cementum loss and tooth resorption effect may also contribute to the higher estimated PDL volume after orthodontic treatment2,21.
Considerably a similar trend in the changes of PDL thickness and volume found in our study indicate that the PDL deformation on the side with more compression force is not compensated by its extension in the tension force areas after such long application of orthodontic stimuli. Rather the whole PDL system is deformed and remodeled. Our findings imply that the observed changes within the PDL are not solely due to the dislocation of tooth in the alveolar socket and they do not correlate with the tension and compression side after such long mechanical load. Still, more studies are needed to answer the resulting questions, such as what happens with the PDL and alveolar bone reformation after the orthodontic force is removed; how is the alveolar bone and PDL remodeling influenced by different forces; or how is the length and the process of bone and PDL recovery to its original form at different applied loadings. This break-through study was conceived to show the first evidence about an extended time span-remodeling which was successfully reached through clear significant differences in several morphological parameters even in a relatively small sampling. Future studies with higher sample numbers and analysis of periodontal ligament and microstructures of alveolar bone after removing the appliances may be essential for the better understanding of this phenomena and its application in clinical research.
