Kojiro Kurisu

Involvement of α5β1 Integrin in Matrix Interactions and Proliferation of Chondrocytes

Integrins are cell surface receptors involved in cellular processes including adhesion, migration, and matrix assembly. In the present study, we analyzed the possible involvement of α5β1 integrin in the regulation of chondrocyte adhesion, spreading, and proliferation. We found that rabbit growth plate chondrocytes were able to attach to substrates coated with type I collagen, type II collagen, or fibronectin within 24 h of culture. During this time period, attachment to fibronectin appeared to be dependent on α5β1 integrin, whereas adhesion to collagens was not. By day 3 of culture, chondrocytes spread onto all the substrates tested. We found that regardless of the nature of the substrate, cell spreading was reversed by treatment with RGD peptide or antibodies against α5β1 or fibronectin, indicating that cell spreading involved α5β1 and fibronectin endogenously produced and deposited by the chondrocytes themselves. Colony formation by chondrocytes in soft agar was inhibited by treatment with RGD peptides or BIIG2, an antibody that interferes with α5β1 integrin–ligand interactions. Furthermore, DNA content was decreased by treatment with anti‐fibronectin antibody in micromass culture of chondrocytes. Immunohistochemical analysis on tissue sections revealed that the α5 subunit was particularly abundant in the proliferative and hypertrophic zones of growth plate. The results of the study indicate that α5β1 integrin plays multiple roles in chondrocyte behavior and function and appears to be involved in the regulation of both chondrocyte–matrix interactions and proliferation.

Hedgehog Proteins Stimulate Chondrogenic Cell Differentiation and Cartilage Formation

Sonic hedgehog (Shh) and Indian hedgehog (Ihh) are important regulators of skeletogenesis, but their roles in this complex multistep process are not fully understood. Recent studies have suggested that the proteins participate in the differentiation of chondrogenic precursor cells into chondrocytes. In the present study, we have tested this possibility more directly. We found that implantation of dermal fibroblasts expressing hedgehog proteins into nude mice induces ectopic cartilage and bone formation. Immunohistological and reverse‐transcription polymerase chain reaction (RT‐PCR) analyses revealed that the ectopic tissues derived largely if not exclusively from host cells. We found also that treatment of clonal prechondrogenic RMD‐1 and ATDC5 cells in culture with Ihh or recombinant amino half of Shh (recombinant N‐terminal portion of Shh [rShh‐N]) induced their differentiation into chondrocytes, as revealed by cytoarchitectural changes, Alcian blue staining and proteoglycan synthesis. Induction of RMD‐1 cell differentiation by Ihh or rShh‐N was synergistically enhanced by cotreatment with bone morphogenetic protein 2 (BMP‐2) but was blocked by cotreatment with fibroblast growth factor 2 (FGF‐2). Our findings indicate that hedgehog proteins have the ability to promote differentiation of chondrogenic precursor cells and that their action in this process can be influenced and modified by synergistic or antagonist cofactors.

Local and Chronic Application of PTH Accelerates Tooth Movement in Rats

We previously reported that whereas systemic continuous infusion of parathyroid hormone (PTH) accelerated orthodontic tooth movement, systemic but intermittent injection of PTH did not increase the rate of tooth movement. Analysis of these data suggested that continuous administration of PTH could be applicable for orthodontic therapy. In the present study, we investigated whether local and chronic application of PTH(1-34) would accelerate orthodontic tooth movement. To increase the residence time of PTH in the injected area, we used methylcellulose (MC) gel (2% w/v) for a slow-release formulation of PTH. MC gel containing PTH (PTH-MC) continuously released biologically active PTH into the acceptor medium for more than 72 hrs in vitro. When male rats received a local injection of PTH-MC into the subpcriosteum in the mesio-palatal region of the maxillary first molar (M1) every other day, M1 movement, which was mesially drawn by an orthodontic coil spring attached to the maxillary incisors, was accelerated in a dose-dependent manner. PTH-MC injection at 1 μg/400 g body weight caused a 1.6-fold increase in the rate of tooth movement. The acceleration of tooth movement by PTH-MC injection was marked on days 6, 9, and 12. Local injection of PTH dissolved in saline without MC did not significantly accelerate tooth movement on day 6 or later. Histological examination revealed active osteoclastic bone resorption and a widened periodontal space on the compression side of the periodontal tissue in the PTH-MC-injected rats. These results suggest that local injection of PTH in a slow-release formulation is applicable to orthodontic therapy.

Effects of Continuous Infusion of PTH on Experimental Tooth Movement in Rats

Development of new methods for accelerating orthodontic tooth movement has been strongly desired for shortening of the treatment period. The rate of orthodontic tooth movement is dependent on the rate of bone resorption occurring in the compressed periodontium in the direction of orthodontic force applied to the tooth. In the present study, we examined the effects of continuous infusion of parathyroid hormone (PTH) on tooth movement. Male rats weighing 350–400 g were treated with subcutaneous infusion of vehicle or hPTH(1–84) at 1–10 μg/100 g of body weight/day. When the upper right first molar (M1) was moved mesially for 72 h by the insertion of an elastic band between the first and second molars, M1 movement was accelerated by PTH infusion at 10 μg. PTH infusion caused a 2‐ to 3‐fold increase in the number of osteoclasts in the compressed periodontium of M1, indicating that such treatment accelerated tooth movement by enhancing bone resorptive activity induced in the compressed periodontium. When M1 was moved mesially by an orthodontic coil spring ligated between upper incisors and M1 for 12 days, PTH(1–84) infusion at 10 μg caused a 2‐fold increase in the rate of M1 movement. PTH(1–34) infusion at 4 μg had an effect comparable to that of PTH(1–84). However, intermittent injection of PTH(1–34) did not accelerate M1 movement. PTH infusion for 13 days did not affect either bone mineral measurements or the serum calcium level. These findings suggest that continuous administration of PTH is applicable to accelerate orthodontic tooth movement.

Regeneration of periodontal primary afferents of the rat incisor following injury of the inferior alveolar nerve with special reference to neuropeptide Y-like immunoreactive primary afferents.

Regeneration of primary afferents and the expression of neuropeptide Y (NPY) in the lingual periodontal ligament of the rat incisor were examined following different types of injury (resection or crush) of the inferior alveolar nerve (IAN) combined with superior cervical ganglionectomy. In normal animals, protein gene product 9.5 (PGP 9.5)-like immunoreactivity (-LI) was localized in the middle areas of the alveolus-related part of lingual periodontal ligament; some of these nerve fibers showed terminal ramification and morphologies resembling those of the periodontal Ruffini endings, and very few thin varicose NPY-like immunoreactive (-IR) nerve fibers were detected around the blood vessels. Three days following crush injury of the IAN, the number of PGP 9.5-IR nerve fibers decreased, then increased to the normal levels around 10-15 days following injury. NPY-IR primary afferents first appeared around 5 days following crush injury, increased in number gradually, reaching a peak around 14 days, and then decreased. No NPY-IR primary afferents were detected 56 days following crush injury of the IAN. The terminal morphology of NPY-IR primary afferents observed around 10-14 days following injury was similar to that of PGP 9.5-IR nerve fibers in the normal animals, but less expanded. The changes in distribution of PGP 9.5-IR and NPY-IR nerve fibers following resection were similar to those observed following crush injury but regeneration was slightly delayed. The present results suggest that injury-evoked NPY is closely associated with the regeneration process of mechanoreceptors in the periodontal ligament following injury of the IAN.

Cell size-specific appearance of neuropeptide Y in the trigeminal ganglion following peripheral axotomy of different branches of the mandibular nerve of the rat

The effect of peripheral axotomy of the mental nerve (MN) and the cutaneous branch of the mylohyoid nerve (MhN) on the appearance of neuropeptide Y-like immunoreactivity (NPY-IR) in cells in the trigeminal ganglion of the rat was examined with combined retrograde-tracing and immunohistochemistry. Retrograde-tracing with True Blue (TB) revealed that the cell-size spectrum of the trigeminal cells sending peripheral processes to the MN (TB MN cells) ranged from 75.9 to 1560.5 microns2 (or from 9.8 to 44.6 microns in diameter); approximately 53% of TB MN cells were 300-600 microns2. TB MhN cells ranged from 47.7 to 1261.5 microns2 (or from 7.8 to 40.1 microns in diameter); 56% of TB MhN cells were < 300 microns2. In the normal trigeminal ganglion, there were no NPY-IR cells. 14 days after MN transection, approximately 35% of TB MN cells displayed NPY-IR. The distribution of the cross-sectional areas of NPY-IR cells after MN transection was very similar to that of TB MN cells. Transection of MhN also induced the appearance of NPY-IR in the trigeminal ganglion but to a lesser extent (approximately 17% of TB MhN cells). The distribution of the cross-sectional areas of NPY-IR cells after MhN transection was similar to that of NPY-IR cells after MN transection. These results indicate that injury-evoked NPY-IR is specific for the medium- and large-sized ganglion cells.

Expression of cytokeratin 14 in ameloblast-lineage cells of the developing tooth of rat, both in vivo and in vitro

In the search for a cell marker useful for studying tooth development, immunohistochemical studies using antibodies against cytokeratin 14 (K14), c-Met/hepatocyte growth factor receptor and amelogenin were carried out in the developing tooth of the newborn rat and in primary cultured cells of the ameloblast lineage, including inner enamel epithelium cells, preameloblasts and ameloblasts, prepared from the mandibular incisors of postnatal 7-day-old rats. The appearance of K14 was cell- and differentiation-stage specific, i.e. there was a weak expression signal within inner enamel epithelial cells that were in the proliferating stage, and there were strong signals within preameloblasts and ameloblasts that were in the post-proliferating and amelogenesis stages, respectively. In the culture system, c-Met appeared in all cells, whereas K14 and amelogenin appeared mainly in clustered cells that were considered to be in the post-proliferating stage. K14 was detected earlier than amelogenin, and it was also confirmed by immunofluorostaining that c-Met, K14 and amelogenin were coexpressed in ameloblasts. These findings indicate that K14 is a good new marker for ameloblast-lineage cells during rat tooth development both in vivo and in vitro.

Expression of the type I collagen gene in rat periodontal ligament during tooth movement as revealed by in situ hybridization

The in situ hybridization technique used digoxigenin-labelled oligodeoxynucleotide. In untreated molars, cells expressing a positive signal for type I collagen mRNA were distributed uniformly in the periodontal ligament space. After experimental tooth movement, the density of cells expressing a positive signal appeared to be much greater in the tension side than the pressure side. In both sides the distribution of the positively hybridizing cells was uniform along the principal fibres of the ligament. This characteristic distribution appeared at 12 h after the initiation of tooth movement, reached a maximum at 1-3 days, and persisted for about 14 days during the treatment. These results indicate that the remodelling of collagen fibres in periodontal ligament occurs in an orderly manner throughout the principal fibres, mainly on the tension side, and that the recovery of gene expression for type I collagen occurs within the first 14 days in response to experimental tooth movement.

Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp‐to‐cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp‐to‐cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh‐expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

Immunohistochemical detection of an enamel protein-related epitope in rat bone at an early stage of osteogenesis.

Monoclonal antibody MI315 was produced against hamster tooth germ homogenate by in vitro immunization. It was found that MI315 reacted with enamel matrix, ameloblasts, and bone matrix at an early stage of osteogenesis. Decalcified tissues of rat femurs and mandibles were examined with MI315 using indirect immunofluorescence. In endochondral ossification of femurs, immunoreactivity was found in bone extracellular matrix (ECM) deposited on the surface of the cartilage core of primary spongiosa, but not in the cartilage core itself. In intramembranous ossification of 0-day-old rat mandibles, intense immunofluorescence was detected in bone ECM and a few young osteocytes, but not in osteoblasts. Immunoreactivity in bone ECM of 2-day-old rats decreased and almost disappeared from bone ECM of 4-day-old rats. Although in nondecalcified sections of 0-day-old rats, negligible immunofluorescence was detected in bone ECM which showed positive staining in decalcified tissues, the immunostaining appeared after decalcification using ethylenediaminetetraacetic acid (EDTA). These results indicate that a substance(s), which had a common epitope with an enamel-derived protein(s), existed in immature bone ECM of both endochondral and intramembranous ossification, and that it might be masked by bone mineral. Monoclonal antibody MI315 is a useful tool to investigate the time- and position-specific changes in osteogenesis and amelogenesis.