Hisashi Hattori

Recombinant Procollagen II: Deletion of D Period Segments Identifies Sequences That Are Required for Helix Stabilization and Generates a Temperature-sensitive N-Proteinase Cleavage Site

A cDNA cassette system was used to synthesize recombinant versions of procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were deleted. All the proteins were triple helical and all underwent a helix-to-coil transition between 25 and 42 °C as assayed by circular dichroism. However, the details of the melting curves varied. The procollagen lacking the D1 period unfolded 3 °C lower than a full-length molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length molecule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unfolded 3 °C lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant procollagens with procollagen N-proteinase indicated that in the procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35 °C that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the α1(II) chain.

Enhancement of malignant properties of human osteosarcoma cells with disialyl gangliosides GD2/GD3

The expression and implications of gangliosides in human osteosarcomas have not been systematically analyzed. In this study, we showed that gangliosides GD3 and GD2 are highly expressed in the majority of human osteosarcoma cell lines derived from oral cavity regions. Introduction of GD3 synthase cDNA into a GD3/GD2‐negative (GD3/GD2−) human osteosarcoma subline resulted in the establishment of GD3/GD2+ transfectant cells. They showed increased cell migration and invasion activities in wound healing and Boyden chamber invasion assays, respectively, compared to the control cells. When treated with serum, GD3/GD2+ cells showed stronger tyrosine phosphorylation of p130Cas, focal adhesion kinase, and paxillin than GD3/GD2− cells. In particular, paxillin underwent much stronger phosphorylation, suggesting its role in cell motility. Furthermore, we tried to dissect the roles of GD3 and GD2 in the malignant properties of the transfectant cells by establishing single ganglioside‐expressing cells, that is, either GD3 or GD2. Although GD3/GD2+ cells showed the most malignant properties, GD2+ cells showed almost equivalent levels to GD3/GD2+ cells in invasion and migration activities, and in the intensities of tyrosine phosphorylation of paxillin. Among Src family kinases, Lyn was expressed predominantly, and was involved in the invasion and motility of GD3‐ and/or GD2‐expressing transfectants. Furthermore, it was elucidated by gene silencing that Lyn was located in a different pathway from that of FAK to eventually lead paxillin activation. These results suggested that GD2/GD3 are responsible for the enhancement of the malignant features of osteosarcomas, and might be candidate targets in molecular‐targeted therapy.

Functional Activation of Src Family Kinase Yes Protein Is Essential for the Enhanced Malignant Properties of Human Melanoma Cells Expressing Ganglioside GD3

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3− cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3− cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3− cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3− cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.

Conditioned medium from the stem cells of human dental pulp improves cognitive function in a mouse model of Alzheimer's disease.

Alzheimers disease (AD) is a progressive, neurodegenerative disease characterized by a decline in cognitive abilities and the appearance of β-amyloid plaques in the brain. Although the pathogenic mechanisms associated with AD are not fully understood, activated microglia releasing various neurotoxic factors, including pro-inflammatory cytokines and oxidative stress mediators, appear to play major roles. Here, we investigated the therapeutic benefits of a serum-free conditioned medium (CM) derived from the stem cells of human exfoliated deciduous teeth (SHEDs) in a mouse model of AD. The intranasal administration of SHEDs in these mice resulted in substantially improved cognitive function. SHED-CM contained factors involved in multiple neuroregenerative mechanisms, such as neuroprotection, axonal elongation, neurotransmission, the suppression of inflammation, and microglial regulation. Notably, SHED-CM attenuated the pro-inflammatory responses induced by β-amyloid plaques, and generated an anti-inflammatory/tissue-regenerating environment, which was accompanied by the induction of anti-inflammatory M2-like microglia. Our data suggest that SHED-CM may provide significant therapeutic benefits for AD.

Nerve terminals extend into the temporomandibular joint of adjuvant arthritic rats

The innervation of the temporomandibular joint (TMJ) has attracted particular interest because of the close association with complex mandibular movement. Although the pathological changes of disk innervation may have a crucial role in the development of TMJ pain, the innervation of the TMJ disk by experimentally induced arthritis has rarely been examined in detail. Arthritic rats were induced by injection with 0.1 ml solution of Complete Freunds adjuvant (CFA). We investigated three‐dimensional distribution of nerve fibers in the TMJ disk using immunohistochemistry for protein gene product‐9.5 (PGP‐9.5) and calcitonin gene‐related peptide (CGRP) in naive and arthritic rats. To clarify the possible role of nerve growth factor (NGF) and its receptor on changes in peripheral innervation of the TMJ, the expressions of trkA and p75 receptor in trigeminal ganglia were examined. Although PGP‐9.5 and CGRP immunoreactive (ir) fibers were seen in the peripheral part of the TMJ disk, they were not seen in its central part. The total length and the length density of PGP‐9.5 ir and CGRP ir nerve fibers increased in arthritic rats. The innervation area of fibers proliferating in the rostro‐medial part merged with that of fibers in the rostro‐lateral part in the arthritic rats. In addition, the ratio of trkA‐ and p75‐positive small‐ and medium‐sized cells increased in trigeminal ganglia. It is assumed that increasing innervation of the TMJ disk may be important for the pathophysiology of TMJ pain. NGF and its receptors are likely involved in pathological changes of the TMJ disk.

Conditioned Medium from the Stem Cells of Human Exfoliated Deciduous Teeth Ameliorates Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a major neuroinflammatory demyelinating disease of the CNS. Current MS treatments, including immunomodulators and immunosuppressants, do not result in complete remission. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells derived from dental pulp. Both SHED and SHED-conditioned medium (SHED-CM) exhibit immunomodulatory and regenerative activities and have the potential to treat various diseases. In this study, we investigated the efficacy of SHED-CM in treating experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE mice treated with a single injection of SHED-CM exhibited significantly improved disease scores, reduced demyelination and axonal injury, and reduced inflammatory cell infiltration and proinflammatory cytokine expression in the spinal cord, which was associated with a shift in the microglia/macrophage phenotype from M1 to M2. SHED-CM also inhibited the proliferation of myelin oligodendrocyte glycoprotein–specific CD4+ T cells, as well as their production of proinflammatory cytokines in vitro. Treatment of EAE mice with the secreted ectodomain of sialic acid–binding Ig-like lectin-9, a major component of SHED-CM, recapitulated the effects of SHED-CM treatment. Our data suggest that SHED-CM and secreted ectodomain of sialic acid–binding Ig-like lectin-9 may be novel therapeutic treatments for autoimmune diseases, such as MS.

Effects of self-assembling peptide hydrogel scaffold on bone regeneration with recombinant human bone morphogenetic protein-2.

PURPOSE The objective of this pilot study was to histologically evaluate bone regeneration using a self-assembling peptide hydrogel scaffold with recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rabbit calvaria model. MATERIALS AND METHODS Five adult New Zealand White rabbits were used for the study. Each received four titanium cylinders, which were placed into perforated slits made in the outer cortical bone of the calvaria. The cylinders were filled with the following test materials: (1) unfilled control; (2) rhBMP-2; (3) PuraMatrix (PM), a synthetic self-assembling peptide (RADA16-I) consisting of a 16-amino acid sequence and with a three-dimensional structure; and (4) PM/rhBMP-2. Each cylinder was covered with a titanium lid. After 8 weeks, the animals were sacrificed, and ground sections were obtained for histomorphometric analysis. RESULTS Histomorphometric analysis showed that regenerated tissue in the cylinder with PM/rhBMP-2 was significantly increased compared to the empty control. The mean area values of regenerated tissue in the cylinders were 35.80% ± 10.35% (control), 47.94% ± 5.65% (rhBMP-2), 48.94% ± 11.33% (PM), and 58.06% ± 14.84% (PM/rhBMP-2). The mean area values of newly formed bone in the cylinders were 9.39% ± 4.34% (control), 14.03% ± 2.25% (rhBMP-2), 13.99% ± 2.15% (PM), and 16.61% ± 3.79% (PM/rhBMP-2). Neither rhBMP-2 nor PM alone significantly enhanced bone regeneration compared to the empty control cylinder. CONCLUSIONS PM with rhBMP-2 significantly enhanced bone regeneration on the bone augmentation model in a rabbit. PM promises to be a useful alternative synthetic material as a carrier for rhBMP-2 for bone regeneration.

Multifaceted Therapeutic Benefits of Factors Derived From Dental Pulp Stem Cells for Mouse Liver Fibrosis

Chronic liver injury from various causes often results in liver fibrosis (LF). Although the liver possesses endogenous tissue‐repairing activities, these can be overcome by sustained inflammation and excessive fibrotic scar formation. Advanced LF leads to irreversible cirrhosis and subsequent liver failure and/or hepatic cancer. Here, using the mouse carbon tetrachloride (CCl4)‐induced LF model, we showed that a single intravenous administration of stem cells derived from human exfoliated deciduous teeth (SHEDs) or of SHED‐derived serum‐free conditioned medium (SHED‐CM) resulted in fibrotic scar resolution. SHED‐CM suppressed the gene expression of proinflammatory mediators, such as TNF‐α, IL‐1β, and iNOS, and eliminated activated hepatic stellate cells by inducing their apoptosis, but protected parenchymal hepatocytes from undergoing apoptosis. In addition, SHED‐CM induced tissue‐repairing macrophages that expressed high levels of the profibrinolytic factor, matrix metalloproteinase 13. Furthermore, SHED‐CM suppressed the CCl4‐induced apoptosis of primary cultured hepatocytes. SHED‐CM contained a high level of hepatocyte growth factor (HGF). Notably, HGF‐depleted SHED‐CM (dHGF‐CM) did not suppress the proinflammatory response or resolve fibrotic scarring. Furthermore, SHED‐CM, but not dHGF‐CM, inhibited CCl4‐induced hepatocyte apoptosis. These results suggest that HGF plays a central role in the SHED‐CM‐mediated resolution of LF. Taken together, our findings suggest that SHED‐CM provides multifaceted therapeutic benefits for the treatment of LF.

Stability of a Locking Plate and Self-Drilling Screws as Orthodontic Skeletal Anchorage in the Maxilla: A Retrospective Study

PURPOSE The aim of this retrospective study was to determine the success rate of an orthodontic skeletal anchorage system consisting of a locking plate and 2 self-drilling screws to intrude the upper molars and detect factors that contribute to its stability. PATIENTS AND METHODS The subjects were 32 orthodontic and generally healthy patients who had skeletal anchorage plates placed supraperiosteally and unilaterally or bilaterally. The anchorage plate was considered successful if the plate remained stable throughout the period of intrusion of the upper molar without any movement, persistent pain, or infection and was then retrieved without difficulty. The success rates of the anchorage plate were statistically analyzed on the basis of clinically categorized variables. RESULTS The 32 patients comprised 6 male and 26 female individuals with ages ranging from 11.4 to 35.1 years. The overall success rate of the total 61 plates was 93.4%. No significant differences were observed among the respective success rates analyzed in accordance with gender, age, side of placement, and length of the screws. The thickness of the bony walls that supported the screws was significantly greater in the success group (mean 1.6 +/- SD, 0.2 vs 1.0 +/- 0.1 mm, P < .001). CONCLUSION Bone thickness is a critical factor in supporting the self-drilling screws and locking plate. Skeletal anchorage combining the plate and 2 screws promises a higher success rate with a thicker bone than with the threshold value of thickness that exists within the 1.1 to 1.4 mm range in the maxillary walls.

An ultrastructural study of extracellular fibrillar components of developing mouse mandibular condyle with special reference to type VI collagen.

The localization of type VI collagen was examined from birth to 8 weeks of age. Immunohistochemical staining with anti-type VI collagen antibody was strongly positive in the hypertrophic zone and moderately positive in the fibrous zone and the outer periphery of the proliferative zone, but negative in the inner area of the proliferative zone and mature zone. After ATP treatment, type VI collagen periodic fibrils with about 80-nm intervals were frequently observed but only in the fibrous zone. They occurred mainly in the superficial area of this zone, where striated collagen fibrils were sparse, while a few were noted in the inner area, where bundles of collagen fibrils were abundant. From these distributional differences of both components, a subzonation of the fibrous zone into superficial and inner area is suggested. Moreover, with ATP treatment there were fewer type VI collagen periodic fibrils formed with increasing age (8 weeks). Testicular hyaluronidase digestion before ATP treatment facilitated the formation of periodic fibrils, in all the ages examined, in the intercellular space and around the fibroblastic cells. The interaction of type VI collagen with other components such as collagen fibrils, glycosaminoglycans or proteoglycans may play a part in maintaining the structural integrity of extracellular matrix in the mouse mandibular condyle.