Effects Of RhTGF-β₁ On Pyk2 In Osteoclasts During Orthodontic Tooth Movement In Rats

Transforming growth factor beta 1 (TGF-β1) is a multifunctional cytokine that has significant effects on skeletal metabolism. Osteoclasts are multinucleated cells responsible for bone resorption. Osteoclast bone resorption is a complicated multistep process, which starts with matrix recognition, osteoclast attachment, polarization and formation of the sealing zone on the bone, followed by the directional secretion of acids and lysosomal enzymes to the resorbing surface. During the tooth movement, the amount, distribution and functional active status of osteoclasts are associated with the rate of orthodontic tooth movement. Integrinαvβ3 plays a pivotal role in osteoclastic bone resoprtion by mediating both osteoclast attachments on bone matrix and cytoskeleton reorganization. It provides a physical link between the ECM and the cell cytoskeleton, and transduces signals which lead to elevation of cytosolic pH and calcium levels, changes in phospholipid metabolism and ultimately regulate gene expression. Proline-rich tyrosine kinase 2 (Pyk2) is a major tyrosine kinase activated by integrin engagement. In osteoclasts, Pyk2 appears to be the predominant mediator of integrinαvβ3 signaling events that influence osteoclast physiology and pathology. Thus, we conducted this study to investigate the effect of rhTGF-β1 on Pyk2 in osteoclasts during the orthodontic tooth movement using an immunohistochemical and molecular biological approach. Furthermore, we investigated the rhTGF-β1 injection on the rate of tooth movement and bone resportion of osteoclast in order to provide the further evidence for future clinical work.Objective: To investigate the expression of Pyk2 in osteoclasts during orthodontic tooth movement while local injecting rhTGF-β1 in periodontal tissues in Sprague-Dawley(SD) rats. To explore how rhTGF-β1 regulate the signaling transduction pathway of Pyk2 in osteoclasts, and to study the effect of exogenous TGF-β1 on regulating the expression change of Pyk2 in osteoclasts and the mechanism of improving the periodontal tissues remodeling during orthodontic tooth movement.Methods: 80 male SD rats were randomly divided into two groups: The experimental group were injected respectively with rhTGF-β₁ in dosages of 5ng in the buccal submucosal area of the molar every other day, while the Control group received equivalent volumes of PBS. Each group was subdivided equally into five subgroups of eight. An orthodontic appliance, consisting of a 5 mm nickel titanium closed coil spring, was ligated between the maxillary incisors and first molar of each rat to deliver an initial force of 50g. Eight rats in each subgroup were sacrificed at each of five time points (1, 4, 7, 10 and 14 days) after appliance placement. The distance of the tooth movement was measured using the stereomicroscope. Tissue sections around the first maxillary left molar were stained with tartrate-resistant acid phosphatase (TRAP) histochemistry to analyze the changes of the amount and distribution of osteoclasts on the compression side of tooth. The expression of Pyk2 in osteoclasts was detected by immunohistochemical techniques on the same side. Results:â‘ Tooth movement: Molars treated with rhTGF-β₁ moved mesiaily more rapidly than the control group. The distance of tooth movement of experimental group showed a significant increase compared with the control group at day 7(P<0.05) and a highly significant increase compared with the control group at day 10 and 14(P<0.01).â‘¡the amount of osteoclasts: The number of osteoclasts appearing in the periodontal ligament space on the pressure side of the alveolar bone was increased markedly both in two groups. A peak was occurred at day 4 and gradually decreased after that day. There were statistically significant differences in the amount of between experimental and control groups (P <0.01).â‘¢Expression of Pyk2: The time-depended changes in experimental group were similar to those in the control group during orthodontic tooth movement. The expression of Pyk2 in osteoclasts increased and get to the peak at 7 day and gradually decreased after that day. There were significant differences in the magnitude of Pyk2 expression between the two groups (P <0.01).Conclusion: Local injections of exogenous rhTGF-β₁ in the periodontal tissues might accelerate orthodontic tooth movement via enhanced the numbers of osteoclasts and upregulate the expression level of Pyk2 in osteoclasts. At the histologic level, increased numbers of multinucleated osteoclasts were recruited and activated, resulting in greater amounts of alveolar bone resorption on the pressure side of the periodontal ligament. Pyk2 is the downstream signaling molecule of integrinαvβ3 in osteoclasts, which can be upgraded by exogenous TGF-β₁, and has emerged as an important regulator of osteoclast function, involving both its tyrosine kinase activityand its scaffolding properties.