Hironobu Suzuki

PLATELET-RICH PLASMA PROVIDES NUCLEUS FOR MINERALIZATION IN CULTURES OF PARTIALLY DIFFERENTIATED PERIODONTAL LIGAMENT CELLS

SummaryPlatelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-β1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRPs ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.

Capacity of Dental Pulp Differentiation in Mouse Molars as Demonstrated by Allogenic Tooth Transplantation

Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the capability of dental pulp to elaborate bone tissue in addition to dentin by allogenic tooth transplantation using immunohistochemistry and histochemistry. After extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately allografted into the sublingual region in a littermate. In addition, we studied the contribution of donor and host cells to the regenerated pulp tissue using a combination of allogenic tooth transplantation and lacZ transgenic ROSA26 mice. On Days 5-7, tubular dentin formation started next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until Day 14, bone-like tissue formation occurred in the pulp chamber, where intense tartrate-resistant acid phosphatase-positive cells appeared. Furthermore, allogenic transplantation using ROSA26 mice clearly showed that both donor and host cells differentiated into osteoblast-like cells with the assistance of osteoclast-lineage cells, whereas newly differentiated odontoblasts were exclusively derived from donor cells. These results suggest that the odontoblast and osteoblast lineage cells reside in the dental pulp and that both donor and host cells contribute to bone-like tissue formation in the regenerated pulp tissue.

Histological and elemental analyses of impaired bone mineralization in klotho‐deficient mice

The klotho gene‐deficient mouse is known as an animal model for an accelerated gerontic state, mimicking osteoporosis, skin atrophy, ectopic calcification, and gonadal dysplasia. To elucidate the influence of klotho deficiency on bone mineralization, we examined the ultrastructures of osteoblasts and bone matrices in addition to performing the elemental mapping of calcium, phosphorus, and magnesium in the bone. Under anesthesia, 4‐ and 5‐week‐old klotho‐deficient mice (klotho−/–mice) and their wild‐type littermates were perfused with either 4% paraformaldehyde for light microscopic observation or 4% paraformaldehyde and 0.0125% glutaraldehyde for electron microscopic observation. The femurs and tibiae were processed for both observations. Paraffin sections were subject to alkaline phosphatase and tartrate resistant acid phosphatase histochemistry. Semithin and ultrathin sections obtained from epoxy resin‐embedded specimens were used for detecting mineralization – according to von Kossas staining method – and for elemental mapping by electron probe micro‐analyzer, respectively. Alkaline phosphatase‐positive plump osteoblasts adjacent to the growth plate normally developed cell organelles in the klotho−/–metaphyses. This, however, contrasted with the flattened osteoblasts covering the metaphyseal trabeculae and accompanied by small tartrate resistant acid phosphatase‐positive osteoclasts. The wild‐type mice displayed the mineralized matrix at the zone of hypertrophic chondrocyte of the growth plate and well‐mineralized metaphyseal trabeculae parallel to the longitudinal axis of the bone. Alternatively, the klotho−/–mice demonstrated a thick mineralized matrix from the proliferative zone of the growth plate as well as the large non‐mineralized area in the metaphyseal trabeculae. Consistently, electron probe micro‐analysis verified sporadic distributions of higher or lower concentrations of calcium and phosphorus in each trabecule of the klotho−/–mice. The distribution of magnesium, however, was almost uniform. Under transmission electron microscopy, osteoblasts on the metaphyseal trabeculae displayed less‐developed cell organelles in the klotho−/–mice. Thus, the klotho deficiency appears not only to reduce osteoblastic population, but also to disturb bone mineralization.

Pulpal Regeneration Following Allogenic Tooth Transplantation into Mouse Maxilla

Autogenic tooth transplantation is now a common procedure in dentistry for replacing a missing tooth. However, there are many difficulties in clinical application of allogenic tooth transplantation because of immunological rejection. This study aims to clarify pulpal regeneration following allogenic tooth transplantation into the mouse maxilla by immunohistochemistry for 5‐bromo‐2′‐deoxyuridine (BrdU) and nestin, and by the histochemistry for tartrate‐resistant acid phosphatase (TRAP). The upper right first molar (M1) of 2‐week‐old mice was extracted and allografted in the original socket in both the littermate and non‐littermate after the extraction of M1. Tooth transplantation weakened the nestin‐positive reactions in the pulp tissue that had shown immunoreactivity for nestin before operation. On postoperative Days 5–7, tertiary dentin formation commenced next to the preexisting dentin where nestin‐positive odontoblast‐like cells were arranged in all cases of the littermate group until Day 14, except for one case showing immunological rejection in the pulp chamber. In the non‐littermate group, bone‐like tissue formation occurred in the pulp chamber in addition to tertiary dentin formation until Day 14. The rate of tertiary dentin was 38%, and the rate of the mixed form of dentin and bone‐like tissue formation was 23% (the remainder was immunological rejection). Interestingly, the periodontal tissue recovered even in the case of immunological rejection in which the pulp chamber was replaced by sparse connective tissue. These results suggest that the selection of littermate or non‐littermate is decisive for the survival of odontoblast‐lineage cells and that the immunological rejection does not influence the periodontal regeneration. Anat Rec, 2009.

GaAlAs laser irradiation induces active tertiary dentin formation after pulpal apoptosis and cell proliferation in rat molars.

INTRODUCTION This study aimed to clarify pulpal responses to gallium-aluminum-arsenide (GaAlAs) laser irradiation. METHODS Maxillary first molars of 8-week-old rats were irradiated at an output power of 0.5 or 1.5 W for 180 seconds, and the samples were collected at intervals of 0 to 14 days. The demineralized paraffin sections were processed for immunohistochemistry for heat-shock protein (HSP)-25 and nestin in addition to cell proliferation assay using bromodeoxyuridine (BrdU) labeling and apoptosis assay using deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL). RESULTS Intense HSP-25 and nestin immunoreactivities in the odontoblast layer were weakened immediately after 0.5-W irradiation and recovered on day 1, resulting in slight tertiary dentin formation by day 14. On the contrary, 1.5-W irradiation immediately induced the loss of HSP-25 and nestin-immunoreactivities in the odontoblast layer. On day 1, numerous TUNEL-positive cells appeared in a degenerative zone that was surrounded by intense HSP-25 immunoreactivity. BrdU-positive cells occurred within the intensely HSP-25-immunopositive areas during days 2 through 5, whereas TUNEL-positive cells gradually decreased in number by day 5. HSP-25- and nestin-positive odontoblast-like cells were arranged along the pulp-dentin border by day 7, resulting in remarkable tertiary dentin formation on day 14. CONCLUSIONS The output energy determined pulpal healing patterns after GaAlAs laser irradiation; the higher energy induced the apoptosis in the affected dental pulp including odontoblasts followed by active cell proliferation in the intense HSP-25-immunoreactive areas surrounding the degenerative tissue, resulting in abundant tertiary dentin formation. Thus, the optimal GaAlAs laser irradiation elicited intentional tertiary dentin formation in the dental pulp.

Involvement of the klotho protein in dentin formation and mineralization.

Klotho‐deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho‐deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho‐deficient mice–comparing the findings with those from their wild‐type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein‐1 (DMP‐1), terminal deoxynucleotidyl transferase‐mediated deoxyuridinetriphosphate nick end‐labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho‐deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin‐predentinal border at the labial region. Several odontoblast‐like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP‐1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast‐like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast‐like cells and pulpal cells in the klotho‐deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho‐deficient dentin, except for the dentin around abnormal odontoblast‐like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization. Anat Rec, 2007.

Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.