Kayoko Nozawa-Inoue

Detailed Process of Bone Remodeling After Achievement of Osseointegration in a Rat Implantation Model

Osseointegration is regarded as the most appropriate implant‐bone interface in dental implantation. However, damaged bone with empty osteocytic lacunae driven by implant cavity preparation remains even after the completion of osseointegration. Although previous studies have suggested the occurrence of bone remodeling around implants, information on its detailed process is meager. Our study aimed to examine the fate of bone around titanium implants after the establishment of osseointegration on an animal model using the rat maxilla. Titanium implants were inserted into prepared bone cavities of the rat maxilla. Bone formation and maturation processes were evaluated by double staining for alkaline phosphatase and tartrate‐resistant acid phosphatase, immunohistochemistry for bone matrix proteins, vital staining with calcein, and elemental mapping with an electron probe microanalyzer. Bone with empty osteocytic lacunae or pyknosis remained between the intact preexisting and newly formed woven bones at post 1 month. It gradually decreased to disappear completely by active bone remodeling with a synchronized coordination of alkaline phosphatase‐positive osteoblasts and tartrate‐resistant acid phosphatase‐reactive osteoclasts at post 3 months, thickening to be replaced by compact bone. Dynamic labeling showed two clear lines in the newly formed bone around the implant through this experimental period. Electron probe microanalyzer analysis demonstrated chronologically increased levels of Ca and P in the newly formed bone identical to those in the surrounding bone at post 2.5 months. These findings indicate that continuous bone remodeling after the achievement of osseointegration causes replacement of the damaged bone by compact bone as well as an improvement in bone quality. Anat Rec, 2009.

Immunolocalization of aquaporin-1 in the mechanoreceptive Ruffini endings in the periodontal ligament.

Previous ultrastructural studies have suggested an axon-Schwann cell interaction in the periodontal Ruffini ending, a primary mechanoreceptor. However, no information is available on the transport mechanism between them. The present study examined the immunolocalization of aquaporin-1 (AQP1) and -4 (AQP4), a member of the water-selective channel, in the periodontal Ruffini endings of the rat incisors and trigeminal ganglion. In addition, the expression of mRNA for AQP1 and 4 was detected in the trigeminal ganglion by a RT-PCR technique. A single PCR product of the sizes anticipated for AQP1 and 4 was detectable in a reverse transcripted cDNA sample from the trigeminal ganglion, whose neurons innervate the periodontal Ruffini endings. An AQP1 immunoreaction was recognizable in the axon terminals of the periodontal Ruffini endings as well as their associated terminal Schwann cells, as confirmed with a double staining with AQP1 and either PGP9.5 or S-100 protein. However, no immunoreaction for AQP4 was found in periodontal Ruffini endings. Although the AQP4 immunoreaction was localized in some satellite cells - but never in neurons - of the trigeminal ganglion, 16.1% trigeminal neurons showed the AQP1 immunoreaction. Furthermore, the AQP1 immunoreaction was found in certain satellite cells which surrounded AQP1-positive or -negative neurons. An analysis of a cross-sectional area of these positive neurons demonstrated that approximately 66.9% of the positive neurons were 400-1000 microm2 (671.4+/-172.4 microm2), indicating that they could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that AQP1 controls water transport in the periodontal Ruffini endings.

Zoledronic acid induces S-phase arrest via a DNA damage response in normal human oral keratinocytes

OBJECTIVE This study aimed to clarify the effects of zoledronic acid (ZOL) on human primary oral mucosal keratinocytes growing in a monolayer culture and on a tissue-engineered oral mucosal construct. DESIGN Changes in the viability and proliferation of oral keratinocytes incubated with ZOL were measured. Following treatment with 10 μM ZOL, histological examinations and immunohistochemical analyses for Ki-67, Geminin, and phospho-histone (γ)-H2A.X were performed on tissue-engineered oral mucosa. Cell cycle distribution and the degree of apoptosis were also measured by flow cytometry. Additionally, we measured the expression of cell cycle regulatory proteins as well as phospho-Chk1 and -Chk2. RESULTS ZOL treatment suppressed cell viability and proliferation in a dose-dependent manner. Compared with untreated tissue-engineered oral mucosa, ZOL treatment resulted in a thinner epithelium in which the basal cells appeared less-organised. This is consistent with the observed significant reduction in the Ki-67 labelling index. In contrast, the Geminin labelling index was significantly higher than that in the untreated sample. In spite of the presence of a few apoptotic cells, ZOL induced an arrest in S-phase, which was confirmed by our observed alterations in the expression levels of cyclin A, B1, p27(KIP1), Rb and phospho-Rb. When the proteasome inhibitor MG132 was added to the ZOL-treated cells, we observed a partial restoration of the expression of cyclin A, cyclin B1, and p27(KIP1). Expression of phospho-Chk1 was detected, and a significant increase in the labelling index of γ-H2A.X was also seen. CONCLUSIONS These results indicate that a 10-μM ZOL treatment induces a DNA damage response in oral keratinocytes that activates the ubiquitin-mediated proteolysis of cell cycle regulators, resulting in cell cycle arrest and repressive effects on cell viability, proliferation, and epithelial turnover.

Detection of acid-sensing ion channel 3 (ASIC3) in periodontal Ruffini endings of mouse incisors.

The acid-sensing ion channel 3 (ASIC3), a member of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily, has been reported to participate in acid sensing, mechanosensation, and nociception. However, no information is available regarding the precise localization and function of this molecule in the periodontal ligament, which contains abundant sensory nerves originating from the trigeminal ganglion. The present study examined the expression of ASIC3 in the lingual periodontal ligament of mouse incisors by immunohistochemistry. Furthermore, the expression of ASIC3 in the trigeminal ganglion - which innervates the periodontal ligament - was investigated at protein (immunohistochemistry and quantitative analysis) and mRNA levels (RT-PCR technique and in situ hybridization histochemistry). Immunohistochemistry for ASIC3 was able to demonstrate dendritic profiles of the periodontal Ruffini endings in the mouse incisors. No thin fibers terminating as nociceptive free nerve endings exhibited ASIC3 immunoreactivity. Double immunofluorescent staining revealed ASIC3 immunoreaction in the axoplasm but not in the ordinary Schwann cells - including the associated terminal Schwann cells. Observation of the trigeminal ganglia showed variously sized neurons expressing ASIC3 immunoreaction; the most intense immunopositivity was found in the small and medium-sized neurons, as confirmed by in situ hybridization histochemistry using a specific cRNA probe. Quantitative analysis on trigeminal ganglion neurons showed that 38.0% of ASIC3 neurons could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that ASIC3 functions as a molecule for mechanosensation in the periodontal Ruffini endings.

Immunocytochemical demonstration of laminin in the synovial lining layer of the rat temporomandibular joint.

This immunocytochemical study describes the distribution of laminin in the synovial lining of the rat temporomandibular joint. Laminin immunostaining was present around some synovial lining cells and blood vessels. Ultrastructurally, immunoreactive products for laminin were deposited around cells with a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they were type B synovial lining cells. The localization of laminin immunoreactivity was not uniform around the cell membrane, the most intense immunoreaction being present on the basal aspect membrane as is seen in the basement membrane of epithelia. In contrast, macrophage-like synovial lining type A cells did not show laminin immunoreactivity. This different immunostaining pattern suggests that laminin acts as an adhesion molecule for the type B cells in their epithelial-like arrangement.

Construction and characterization of a tissue-engineered oral mucosa equivalent based on a chitosan-fish scale collagen composite†

This study was designed to (1) assess the in vitro biocompatibility of a chitosan-collagen composite scaffold (C3) constructed by blending commercial chitosan and tilapia scale collagen with oral mucosa keratinocytes, (2) histologically and immunohistochemically characterize an ex vivo-produced oral mucosa equivalent constructed using the C3 (EVPOME-C), and (3) compare EVPOME-C with oral mucosa constructs utilizing AlloDerm® (EVPOME-A), BioMend® Extend™ (EVPOME-B), and native oral mucosa. C3 scaffold had a well-developed fibril network and a sufficiently small porosity to prevent keratinocytes from growing inside the scaffold after cell-seeding. The EVPOME oral mucosa constructs were fabricated in a chemically defined culture system. After culture at an air-liquid interface, EVPOME-C and EVPOME-B had multilayered epithelium with keratinization, while EVPOME-A had a more organized stratified epithelium. Ki-67 and p63 immunolabeled cells in the basal layer of all EVPOMEs suggested a regenerative ability. Compared with native oral mucosa, the keratin 15 and 10/13 expression patterns in all EVPOMEs showed a less-organized differentiation pattern. In contrast to the β1-integrin and laminin distribution in EVPOME-A and native oral mucosa, the subcellular deposition in EVPOME-C and EVPOME-B indicated that complete basement membrane formation failed. These findings demonstrated that C3 has a potential application for epithelial tissue engineering and provides a new potential therapeutic device for oral mucosa regenerative medicine.

Expression of 25 kDa heat shock protein by synovial type B cells of the mouse temporomandibular joint

Earlier studies have demonstrated immunoreactivity for heat shock protein 25 (Hsp25) in type B synovial lining cells of the rat temporomandibular joint, and also the presence of characteristic cytoplasmic processes in these cells, but it is unclear whether or not the type B cells in other animals possess such elaborate cytoplasmic projections and as there is as yet no evidence for the synthesis of this protein by these cells. For these reasons, the expression of Hsp25 was investigated in the synovial membrane of the mouse temporomandibular joint by immunocytochemistry and by in situ hybridization using a specific cRNA probe. Intense immunoreaction for Hsp25 was found in the cytoplasm of certain synovial lining cells that were identified as type B by immunoelectron-microscopy. These Hsp25-positive cells had slender cytoplasmic processes, either projecting towards or covering the synovial surface. Morphological differences between cytoplasmic processes seemed to depend on the location of the type B cell bodies. In situ hybridization showed intense signals for Hsp25 mRNA in the synovial lining cells, suggesting that the type B cells produce, rather than resorb, Hsp25. These findings indicate that Hsp25 is a useful marker for the identification of the synovial type B cells in the temporomandibular joint. It is further hypothesized that Hsp25 in type B cells is involved in maintaining their specific profile and epithelial-like arrangement, and in protecting against mechanical stress.

Convergence of selected inputs from sensory afferents to trigeminal premotor neurons with possible projections to masseter motoneurons in the rabbit.

Peripheral input convergence on trigeminal premotor neurons in the vicinity of trigeminal motor nucleus has been investigated. Thirty neurons were identified by their antidromic responses to microstimulation of the masseteric subnucleus of trigeminal motor nucleus (NVmot-mass). Peripheral receptive fields were found in the buccal mucosae, periodontal ligaments, palate, tongue and vibrissae for 16 neurons located in the intertrigeminal area (NVint), supratrigeminal area (NVs), main sensory trigeminal nucleus (NVsnpr) and subnucleus gamma of the oral nucleus of the spinal trigeminal tract (NVspo-gamma). Eleven neurons in the NVint, NVs and NVspo-gamma responded to passive jaw opening: nine neurons were activated and two were inhibited. None of the neurons responded to both the orofacial mechanical stimulation and passive jaw opening. Forty-six percent of neurons (13 out of 28 tested) received inputs from the inferior alveolar nerve (IAN) and 53% of neurons (8 out of 15 tested) received inputs from the infraorbital nerve (ION). Out of 15 neurons tested for inputs from the IAN and ION, 7 neurons in the NVsnpr and NVspo-gamma received input from both. Sixteen percent of neurons (4 out of 25) received inputs from the masseteric nerve (MassN). None of the neurons with inputs from IAN and/or ION also received inputs from the MassN. We suggest that trigeminal premotor interneurons with projections to the NVmot-mass fall into two broad categories, those with inputs from the IAN and/or ION and those with inputs from the MassN, possibly muscle spindle afferents, and no neuron receiving inputs from both.

Immunocytochemical detection of S-100β in the periodontal Ruffini endings in the rat incisor

Subcellular localization of S-100 protein, a kind of calcium binding proteins, was examined immunohistochemically in the Ruffini ending, a primary mechanoreceptor, in the periodontal ligament of the rat incisor. The periodontal ligament of the rat incisor was found to contain many S-100beta-immunoreactive (-IR) structures but no S-100alpha-IR elements. The S-100beta-IR structures ramified extensively to form Ruffini endings and were frequently associated with round cells, the terminal Schwann cells, which also showed S-100beta-like immunoreactivity. In many periodontal Ruffini endings, S-100beta-IR products were recognized in the cytoplasm of Schwann cells, but not in the axoplasm. However, some axon terminals which had fewer or shorter axonal fingers, were filled with S-100beta-IR products. The present findings indicated the existence of S-100beta, not S-100alpha, in axon terminals of the periodontal mechanoreceptive endings which were identified as type II Ruffini endings.

Changes in jaw reflexes by stimulation of the hypothalamus in anesthetized rabbits

Changes in the masseteric monosynaptic reflex (MMR) and jaw-opening reflex (JOR) responses resulting from conditioning stimulation in the hypothalamus were studied in anesthetized rabbits. Stimulation of the lateral hypothalamus evoked a facilitation of the MMR and an inhibitory or facilitatory effect on the JOR. The facilitatory effect on JOR was stronger than that on the MMR. The facilitatory effective site for the JOR was in the dorsal and lateral directions as compared to the inhibitory field. The results suggest two functionally distinct regions in the lateral hypothalamus that separately project to the jaw-opening muscles.