Yoshinobu Shibasaki

Clinical application of prostaglandin E1 (PGE1) upon orthodontic tooth movement

Chemically produced prostaglandin E1 (PGE1) was administered in clinical cases of orthodontic tooth movement. In the first phase, lingual arch springs were applied on both sides of the maxilla to upper first premolars which were scheduled for extraction. One side received submucosal injections of PGE1 and the other received vehicle injections. The rate of tooth movement in the buccal direction approximately doubled on the side of several PGE1 injections as compared to the control side. In the second phase, the PGE1 injections were applied in canine-retraction cases for up to 3 weeks in first-premolar-extraction cases. The rate of distal canine movement was almost double on the side receiving PGE1 injections as compared to the vehicle-injected side. In the third phase, the PGE1 injections were applied on routine canine retraction in first-premolar-extraction cases. The rate of distal canine movement was almost 1.6-fold on the side of PGE1 injections as compared to the vehicle-injected side. Throughout this study, no side effects were observed macroscopically in the gingiva and roentgenographically in the alveolar bone, except for a slight pain reaction consistent with orthodontic tooth movement.

Activation of cytosolic phospholipase A2 by platelet-derived growth factor is essential for cyclooxygenase-2-dependent prostaglandin E2 synthesis in mouse osteoblasts cultured with interleukin-1.

The synthesis of prostaglandins (PGs) is regulated by the arachidonic acid release by phospholipase A2 (PLA2) and its conversion to PGs by cyclooxygenase (COX). In the present study, we examined the regulation of PG synthesis by interleukin (IL)-1α in primary mouse osteoblastic cells isolated from mouse calvaria. Although IL-1α greatly enhanced cox-2 mRNA expression and its protein levels, PGE2 was not produced until 24 h. When arachidonic acid was added to osteoblastic cells precultured with IL-1α for 24 h, PGE2 was produced within 10 min. Of several growth factors tested, platelet-derived growth factor (PDGF) specifically initiated the rapid synthesis of PGE2, which was markedly suppressed by a selective inhibitor of cox-2 (NS-398). In mouse osteoblastic cells, cytosolic PLA2 (cPLA2) mRNA and its protein were constitutively expressed and increased approximately 2-fold by IL-1α, but secretory PLA2 mRNA was not detected. PDGF rapidly stimulated PLA2 activity, which was blocked completely by a cPLA2 inhibitor (arachidonyltrifluoromethyl ketone). The PDGF-induced cPLA2 activation was accompanied by phosphorylation of its protein. These results indicate that cox-2 induction by IL-1α is not sufficient, but cPLA2 activation by PDGF is crucial for IL-1α-induced PGE2 synthesis in mouse osteoblasts.

Osteoclast induction in periodontal tissue during experimental movement of incisors in osteoprotegerin-deficient mice

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor (TNF) receptor superfamily that negatively regulates osteoclastogenesis. The receptor activator of the NFKB ligand (RANKL) is one of the key regulatory molecules in osteoclast formation and binds to OPG. In this study, it was suggested that OPG and RANKL are involved in alveolar bone remodeling during orthodontic tooth movement. We examined RANKL localization and osteoclast induction in periodontal tissues during experimental movement of incisors in OPG‐deficient mice. To produce orthodontic force, an elastic band was inserted between the upper right and left incisors for 2 or 5 days, and the dissected maxillae were examined for cytochemical and immunocytochemical localization of tartrate‐resistant acid phosphatase (TRAP), vacuolar‐type H+‐ATPase, and RANKL. Compared to wild‐type OPG (+/+) littermates, TRAP‐positive multinucleated cells were markedly induced in the periodontal ligament (PDL) on the compressed side and in the adjacent alveolar bone of OPG‐deficient mice. These multinucleated cells exhibited intense vacuolar‐type H+‐ATPase along the ruffled border membranes. Because of accelerated osteoclastic resorption in OPG‐deficient mice, alveolar bone was severely destroyed and partially perforated at 2 and 5 days after force application. In both wild‐type and OPG‐deficient mice, RANKL expression became stronger at 2 and 5 days after force application than before force application. There was no apparent difference in intensity of RANKL expression between OPG (+/+) littermates and OPG‐deficient mice. In both wild‐type and OPG‐deficient mice, expression of RANKL protein was detected in osteoblasts, fibroblasts, and osteoclasts mostly located in resorption lacunae. These results suggest that during orthodontic tooth movement, RANKL and OPG in the periodontal tissues are important determinants regulating balanced alveolar bone resorption. Anat Rec 266:218–225, 2002.

Regulation of osteoclast differentiation and function by receptor activator of NFkB ligand and osteoprotegerin

The differentiation and functions of osteoclasts (OCs) are regulated by osteoblast‐derived factors. Receptor activator of NFkB ligand (RANKL) is one of the key regulatory molecules in OC formation. Osteoprotegerin (OPG) is a novel secreted member of the TNF receptor superfamily that negatively regulates osteoclastogenesis and binds to RANKL. We examined the biological actions of macrophage‐colony‐stimulating factor (M‐CSF), RANKL, and OPG on the differentiation of OCs isolated from cocultures of mouse osteoblastic cells and bone marrow cells. Preosteoclasts (pOCs) and OCs were characterized by their ultrastructure and the expression of OC markers such as tartrate‐resistant acid phosphatase (TRAP) and vacuolar‐type H+‐ATPase. pOCs formed without any additives expressed TRAP, but showed little resorptive activity on cocultured dentine slices. TRAP‐positive pOCs treated with M‐CSF began to fuse with each other, but lacked a ruffled border (RB) and showed almost no resorptive activity. pOCs treated with RANKL became TRAP‐positive multinucleated cells, which expressed intense vacuolar‐type H+‐ATPase along the RB membranes and exhibited prominent resorptive activity. Such effects of RANKL on pOCs were completely inhibited by the addition of OPG. OPG inhibited RB formation in mature OCs and reduced their resorptive activity, and also induced apoptosis of some OCs. These results suggest that 1) RANKL induces differentiation of functional OCs from pOCs, 2) M‐CSF induces macrophage‐like multinucleated cells, but not OCs, 3) OPG inhibits RB formation and resorptive activity in mature OCs, 4) OPG also induces apoptosis of OCs, and 5) RANKL and OPG are, therefore, important regulators of not only the terminal differentiation of OCs but also their resorptive function. Anat Rec 268:137–146, 2002.

Immunolocalization of vacuolar‐type H+‐ATPase, cathepsin K, matrix metalloproteinase‐9, and receptor activator of NFkB ligand in odontoclasts during physiological root resorption of human deciduous teeth

To investigate the cellular mechanisms of physiological root resorption in human deciduous teeth, the authors examined the immunocytochemical localization of vacuolar‐type H+‐ATPase, a lysosomal cysteine proteinase, cathepsin K, matrix metalloproteinase‐9 (MMP‐9), and receptor activator of NFKB ligand (RANKL) in odontoclasts. H+‐ATPase, cathepsin K, and MMP‐9 are the most important enzymes for decalcification of apatite crystals and degradation of type‐I collagen. In addition, RANKL is one of the key regulatory molecules in osteoclast formation and functions. Odontoclasts developed extensive ruffled borders and clear zones apposed to the resorbing root dentine surfaces. On immunoelectron microscopy, the expression of vacuolar‐type H+‐ATPase was detected along the limiting membranes of pale vacuoles and the ruffled border membranes of odontoclasts. Cathepsin K in odontoclasts was localized within pale vacuoles, lysosomes, the extracellular canals of ruffled borders, and the underlying resorbing dentine surfaces. MMP‐9 localization in odontoclasts was similar to those of cathepsin K. RANKL was detected in both mononuclear stromal cells and odontoclasts located on resorbing dentine surfaces. These results suggest that (1) odontoclasts are directly involved in decalcification of apatite crystals by active extrusion of proton ions mediated by H+‐ATPase and (2) extracellular degradation of dentine type‐I collagen by both cathepsin K and MMP‐9, and (3) odontoclast differentiation and activity are regulated, at least in part, by RANKL, possibly produced by mononuclear stromal cells and odontoclasts themselves in the resorbing tissues. Thus, the cellular mechanisms of physiological root resorption appear to be quite similar to those of osteoclastic bone resorption. Anat Rec 264:305–311, 2001.

Distributional Changes of Osteoclasts and Pre-osteoclastic Cells in Periodontal Tissues during Experimental Tooth Movement as Revealed by Quantitative Immunohistochemistry of H+-ATPase:

To investigate the mechanism of alveolar bone remodeling in response to orthodontic force application, we examined the distribution of osteoclasts and pre-osteoclastic cells using quantitative immunohistochemistry of vacuolar-type H+-ATPase. For orthodontic force to be produced by the Waldo method, an orthodontic elastic band was inserted between the upper first and second molars of rats. The observed areas of periodontal tissues around second molars were the distal surfaces of mesial roots, as the pressure side, and the mesial surfaces of distal roots, as the tension side. Specific expression of vacuolar-type H+-ATPase at the ultrastructural level was detected in mononuclear and multinucleated pre-osteoclastic cells, as well as osteoclasts with ruffled borders on bone surfaces. At 6 hrs after orthodontic force application, many osteoclasts and pre-osteoclastic cells with H+-ATPase expression were first observed in vascular canals of the alveolar bone crest near the pressure side of the periodontal ligament, but the number of osteoclasts was not increased in the periodontal ligament. On day 1 after tooth movement, osteoclasts were increased in number in the periodontal ligament and in adjacent alveolar bones on the pressure side, but were seldom observed in corresponding areas on the tension side. The number of osteoclasts increased until day 7, but had decreased by day 14. These results suggest that, in bone remodeling during experimental tooth movement, (1) osteoclasts and pre-osteoclastic cells can be identified by H+-ATPase immunohistochemistry, (2) osteoclasts and pre-osteoclastic cells are rapidly induced after force application, (3) osteoclast induction first occurs in vascular canals of the alveolar bone crest on the pressure side, and then, (4) the number of osteoclasts increases in the periodontal ligament on the pressure side.

Bisphosphonate administration alters subcellular localization of vacuolar-type H(+)-ATPase and cathepsin K in osteoclasts during experimental movement of rat molars.

This study was designed to clarify the effects of bisphosphonate (BP) administration on structure and functions of osteoclasts in alveolar bone resorption during experimental movement of rat molars. To produce orthodontic force, elastic band was inserted between the upper first and second molars for 4 days, and dissected maxillae were then examined by means of light and electron microscopic immunocytochemistry for vacuolar‐type H+‐ATPase and lysosomal cystein proteinase, cathepsin K in osteoclasts. Vacuolar‐type H+‐ATPase and cathepsin K in osteoclasts are the most important enzymes for demineralization of apatite crystals and degradation of bone type‐I collagen, respectively. At 1 day before elastic band insertion, BP was administered intraperitoneally. Control rats received the same volume of physiologic saline. In BP‐administered rats, most osteoclasts exhibited either irregularly‐formed ruffled borders and clear zones or only clear zones of various degrees of extension. Subcellular localization and expression of both vacuolar‐type H+‐ATPase and cathepsin K was significantly decreased in such osteoclasts with impaired ruffled borders and/or only clear zones by BP administration. In particular, cathepsin K secretion by osteoclasts towards resorption lacunae was markedly inhibited by BP administration. Our results indicate for the first time that BP administration significantly impair the osteoclast structure and reduces expression of both vacuolar‐type H+‐ATPase and cathepsin K in osteoclasts during tooth movement. Anat Rec 260:72–80, 2000.

Changes in Cortical Bone Mineralization in the Developing Mandible: A Three‐Dimensional Quantitative Computed Tomography Study

Quantitative computed tomography (QCT) was completed in 34 subjects between the ages of 9 and 33 years with symmetrical mandibles in order to investigate the three‐dimensional cortical bone mineral density (BMD) distribution in the mandible. The number and distribution of the pixels were determined at three levels: (1) representing the entire mandibular bone; (2) the cortical bone at 60% above the baseline defined as the segmentation level (around 1050 mg/cm3) and representative of only cortical bone; and (3) the highest mineralized cortical bone (>1250 mg/cm3). The geometrical distribution of the highest mineralized areas was evaluated by three‐dimensional reconstruction of the images. The total number of pixels for the entire mandible increased significantly at each time point represented at four increasing ages groups (9–11 years of age, 12–14 years of age, 15–17 years of age, and >18 years of age). The male and female subjects had a similar total number of pixels for the entire mandible before the age of 11, but the male subjects showed a significantly larger total number of mandibular pixels after that age. Comparison of the number of pixels for pure cortical bone (60% segmentation level) and the highest mineralized cortical bone indicated a significant increase with maturation with the greatest change occurring between the 13‐year and 16‐year age groups. However, the ratio of cortical bone/total bone increased at a more rapid rate in the male subjects and reached a plateau by the 16‐year age group, showing distinct differences in mineralization of the mandible between the sexes.

Behavior of Mast Cells in Periodontal Ligament Associated with Experimental Tooth Movement in Rats

Orthodontic mechanical stress reduced the frequency of the appearance of mast cells stainable with toluidine blue in the periodontal ligament on both the pressure and tension sides of rat molar teeth. This effect was seen immediately after insertion of elastic bands between the first and second molars, and the greatest reduction in the number of mast cells stainable with toluidine blue was observed 15 min after insertion.

A stable analogue of throm☐ane A2, 9,11-epithio-11,12-methanothrom☐ane A2, stimulates bone resorption in vitro and osteoclast-like cell formation in mouse marrow culture

Thromboxane A2 (TXA2) is a powerful promoter of platelet aggregation and smooth muscle contraction. However, this compound is highly unstable and is rapidly hydrated to a more stable metabolite, thromboxane B2 (TXB2). TXA2 has been considered to be involved in bone resorption, in particular bone loss caused by inflammatory diseases and by orthodontic treatment. However precise mechanisms of bone resorption caused by TXA2 have not yet been proved because of its highly unstable nature. Recently, a chemically stable analogue of TXA2, 9,11-epithio-11,12-methanothromboxane A2 (STA2), was successfully synthesized. Using this synthetic compound, we examined its in vitro bone resorbing activity and induction of osteoclast-like cells in a mouse marrow culture system in comparison with related compounds with bone resorbing activity. Like prostaglandin E2 (PGE2), a well-known bone resorbing agent, STA2 time- and dose-dependently stimulated the release of 45Ca from prelabelled mouse calvariae. Both STA2 and PGE2 induced the accumulation of cAMP in mouse calvariae. The TXA2 antagonist, ONO-3708, inhibited STA2-induced release of 45Ca. TXB2 induced neither bone resorption nor cAMP accumulation. When mouse marrow cells were cultured with STA2 for 8 days, osteoclast-like multinucleated cells appeared in parallel with the increase of the amount of STA2 added. Again TXB2 showed no effect on osteoclast-like cell formation. These results indicate a role for TXA2 in some form of bone resorption.