| Background and ObjectivesRemodeling changes in periodontal tissues are thought to be essential in tooth movement induced by orthodontic force application in the treatment of malocclusion deformity. When exposed to mechanical loading, periodontal tissues show extensive macroscopic and microscopic changes. Various molecules are activated, which ultimately stimulate localized bone resorption at the compression side and bone deposition at the tension side.High mobility group box protein 1 (HMGB1), a non-chromosomal nuclear protein, has gained much scientific attention because of its dual function as a gene transcription regulator and proinflammatory cytokine. It can be actively secreted by activated immune cells or passively released by injured/dead cells. In case of these conditions, HMGB1 can be translocated from the cell nucleus to cytoplasm and further to extracellular space. In recent papers, HMGB1 has been found basally expressed in periodontal ligament (PDL) cells, and released by PDL cells extracellularly and thus unregulated the expression of various osteoclastogenic cytokines to modulate alveolar bone resorption by binding to its receptor for advanced glycation end products (RAGE). Tooth movement was performed according to Waldo’s method in our experiment. The first part of this study aimed to investigate the expression of HMGB1 in periodontal tissue during orthodontic tooth movement in mice.Periostin, a disulfide linked 90 k Da multifunctional extracellular matrix protein, is primarily expressed in the periosteum and PDL, which plays a crucial role in the PDL remodeling induced by orthodontic force. Periostin-null mice showed a dramatic of loss of periodontal tissue support and a compromised attachment at 3 month old. It is not only essential for the integrity and function of the PDL during orthodontic tooth movement, but also is essential for the progress of periodontal disease and remolding of the PDL. The aim of the second part of the experiment was to investigate the effect of periostin gene knockout on HMGB1 expression in periodontal ligament of mice during orthodontic tooth movement. To understand the relationship of periostin and HMGB1 would provide a theoretical basis of the mechanism of orthodontic tooth movement.Materials and Methods1. Expression of HMGB1 in periodontal ligament during orthodontic tooth movement in miceWe used 8-week-old C57BL6 male mice for this experiment (Every groups contains six animals). Tooth movement was performed according to Waldo’s method, in which 0.5-mm-thick elastic bands were inserted between the right first and second upper molars under anesthesia to induce tooth movement. The untreated sides were used as controls. At 3 days after orthodontic force application, all animals were subjected to transcardial perfusion with 4% paraformaldehyde. Following the fixative, the maxilla of each animal was dissected, divided in two halves carefully. After that the samples underwent dehydration and were embedded in paraffin.5 u m thickness serial sections were prepared for following histological analysis. Hematoxylin and eosin (H&E) staining was performed to investigate the morphology of the periodontal ligament in each group. Immunohistochemical staining was performed to research the expression of HMGB1 in periodontal ligament of each group.6 tissue sections were selected from each sample and 3 randomly selected non-overlapping microscopic fields were selected in each section. The width of periodontal ligament and immunostaining intensity were analyzed using Image-Pro Plus 6.2 software. All statistical analyses were performed using SPSS software and all values are presented as mean ± standard deviation.2. Effect of periostin gene knockout on HMGB1 expression in periodontal ligament of mice during orthodontic tooth movementWe used 8-week-old male mice homozygous for the disrupted periostin gene (Pn-/-) and their wild-type (WT) littermates. Every group contains six animals. The method of the experiment and result analysis are identical to part one.Results1. Expression of HMGB1 in periodontal ligament during orthodontic tooth movement in miceIn the untreated group, the width of the mesial and distal PDL surrounding the root showed no apparent difference. The fiber arranged in rule, the fibroblasts and the capillary distributed in PDL. Following orthodontic tooth movement, the width of PDL at the tension side was increased as stretched by the mechanical force compared to control group. The fiber was stretched longer, and the capillary was expanded with irregular shape. However, the PDL at compression side was squeezed and extensive cell-free hyaline zones were visible. Meanwhile, the mechanical force cusses occlusion of blood vessels and cell apoptosis at this side.Immunohistochemistry studies revealed that PDL at both side showed similar basal HMGB1 immunoreactivity in the untreated group. Following orthodontic tooth movement, HMGB1 expression in the tension side was increased significantly compared with the untreated group. However, the immunolabeling for HMGB1 at the compression side was almost invisible.2. Effect of periostin gene knockout on HMGB1 expression in periodontal ligament of mice during orthodontic tooth movementIn Pn-/- and WT untreated groups, the width of the mesial and distal PDL surrounding the root showed no apparent difference. Following orthodontic tooth movement, both groups showed a wider tensed PDL at tension side, and WT mice showed a wider tensed PDL than Pn-/- mice. However, the width of compressed periodontal ligament in the Pn-/- group was wider than that of WT group.Immunohistochemistry studies revealed that PDL at both sides in Pn-/- mice exhibited a higher basal expression of HMGB 1 compared with WT mice. After 3 days of orthodontic tooth movement, the numbers of HMGB 1-positive cells in PDL of tension side from WT and Pn-/- mice were both increased compared with their respective unloaded counterparts, but there was no significant difference between the two groups. In compression side, the PDL was almost replaced by cell-free hyaline zones filled with disorganized collagen fibers in both Pn-/- and WT groups, thus no visible HMGB 1-positive cells were observed in these areas.Conclusions1. HMGB1 was mainly located at the cell nucleus of fibroblasts. Following orthodontic force application, the divergent expression of HMGB 1 expression was observed in tension and compression side of periodontal ligament:increased at the tension side and decreased at the compression side.2. Compared with WT mice, PDL in Pn-/- mice exhibited a higher basal expression of HMGB1. Following orthodontic tooth movement, HMGB1 expression in PDL of tension side was increased significantly compared with the control group. However, there was no significant difference between the Pn-/- and WT groups. In the compression side, the PDL was almost replaced by cell-free hyaline zones, thus the HMGB1 expression decreased. Our findings suggest an inhibitory effect of periostin on HMGB1 production by PDL, although mechanical loading is the predominant stimulant of HMGB1 expression relative to periostin deficiency. |
