Keitaro Isokawa

Effect of compressive force on the expression of inflammatory cytokines and their receptors in osteoblastic Saos-2 cells

OBJECTIVE In orthodontic tooth movement, some cytokines released from periodontal ligament fibroblasts and alveolar bone osteoblasts on the pressure side can alter the normal processes of bone remodelling, resulting in physiological bone resorption. We examined the effect of compressive force and interleukin (IL)-1 type I receptor antagonist (IL-1ra) on the expression of inflammatory cytokines that promote osteoclast formation, as well as on their receptors, in osteoblastic Saos-2 cells. DESIGN The cells were cultured in Dulbeccos modified Eagle medium containing 10% fetal bovine serum with or without continuous compressive force (0.5-3.0 g/cm(2)) and/or IL-1ra for up to 24h. The gene expression levels of the cytokines and their receptors were estimated by determining mRNA levels using real-time PCR; the protein levels were determined using ELISA or immunohistochemical staining. RESULTS The expression of IL-1beta, IL-1 receptor, IL-6, IL-6 receptor, IL-8 receptor, IL-11 and tumor necrosis factor-alpha (TNFalpha) increased depending on the strength and duration of the compressive force, whereas the expression of IL-8, IL-11 receptor and TNFalpha receptor did not change with the application of compressive force. The expression of cytokines and their receptors produced by 3.0 g/cm(2) of compressive force decreased with the simultaneous addition of IL-1ra and the decrease was remarkable in IL-8 receptor, IL-11 and TNFalpha. CONCLUSION These results indicate that mechanical stress induces the production of inflammatory cytokines and their receptors in osteoblasts and the phenomenon is enhanced by the autocrine action of IL-1beta, which is increased in amount by mechanical stress.

CD271/p75(NTR) inhibits the differentiation of mesenchymal stem cells into osteogenic, adipogenic, chondrogenic, and myogenic lineages.

We describe a novel role for CD271 in the differentiation of mesenchymal stem cells (MSCs), including deciduous dental pulp stem cells (DDPSCs) and murine multipotent MSCs (C3H10T1/2 cells). The CD271(+) subpopulation of deciduous dental pulp cells (CD271(+)/DDPSCs) and the forced expression of CD271 in C3H10T1/2 (10T271) were analyzed by fluorescence-activated cell sorting. CD271 expression was detected in DDPSCs that expressed both CD44 and CD90, simultaneously, and the clonogenic capacity of the CD271(+)/DDPSCs was higher than that of the CD271(-)/DDPSCs that expressed both CD44 and CD90. Further, the differentiation of CD271(+)/DDPSCs into osteoblasts and adipocytes was inhibited although CD271(-)/DDPSCs were capable of differentiating into osteoblasts and adipocytes. CD271 was overexpressed in C3H10T1/2 cells, which have the potential to differentiate into osteoblasts, adipocytes, chondrocytes, and myocytes. CD271 inhibited the differentiation of C3H10T1/2 cells into any of these lineages. These results indicate a role for CD271 in inhibiting the differentiation of MSCs.

Effect of Compressive Force on the Production of Prostaglandin E2 and its Receptors in Osteoblastic Saos-2 Cells

In orthodontic tooth movement, prostaglandin E2 (PGE2) released from osteoblasts can alter the normal process of bone remodeling. We examined the effect of compressive force (CF) on PGE2 production, PGE receptors (Ep1–4) expression, phosphorylation of protein kinase A (p-PKA), and calcium content in Saos-2 cells. PGE2 production increased as CF strength. Applying CF of 98 or 294 Pa caused the cells to produce approximately 700 and 1400 pg/mL PGE2, respectively. CF of 98 Pa increased Ep2 gene expression, and 98 and 294 Pa CF increased Ep4. Immunohistochemical staining showed strong expression of Ep2 under 98 Pa and Ep4 under 98 and 294 Pa. The p-PKA increased as the strength of CF or PGE2 concentration. The calcium content was increased by the addition of 700 pg/mL PGE2 but was decreased by 1400 pg/mL. Thus, mechanical stress controls bone formation by stimulating PGE2 production and Ep2 and/or Ep4 expression in osteoblasts.

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation

While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.

The P75 neurotrophin receptor regulates proliferation of the human MG63 osteoblast cell line.

The 75 kDa transmembrane protein, p75(NTR), is a marker of mesenchymal stem cells (MSCs). Isolated MSCs are capable of differentiating into osteoblasts, but the molecular function of p75(NTR) in MSCs and osteoblasts is poorly understood. The aim of this study was to examine the function of p75(NTR) in the human MG63 osteoblast cell line compared to the murine MC3T3E-1 pre-osteoblast cell line. MG63 cells and MC3T3-E1 cells expressing exogenous p75(NTR) protein (denoted as p75-MG63 and p75GFP-E1, respectively) were generated to compare osteogenic differentiation and cell proliferation abilities. Overexpression of p75(NTR) induced alkaline phosphatase activity and the mRNA expression of osteoblast-related genes such as osterix and bone sialoprotein in both p75-MG63 and p75GFP-E1. Interestingly, exogenous p75(NTR) stimulated cell proliferation and cell cycle progression in p75GFP-E1, but not in p75-MG63. To elucidate any different effects of p75(NTR) expression on osteogenic differentiation and cell proliferation, we examined the mRNA expression of tropomyosin receptor kinase (trk) genes (trkA, trkB, trkC) and Nogo receptor (NgR), which are binding partners of p75(NTR). Although trkA, trkB, and trkC were detected in both p75-MG63 and p75GFP-E1, only NgR was detected in p75-MG63. We then used the K252a inhibitor of the trks to identify the signaling pathway for osteogenic differentiation and cell proliferation. Inhibition of trks by K252a suppressed p75(NTR)-mediated osteogenic differentiation of p75GFP-E1, whereas deletion of the GDI domain in P75(NTR) from the p75-MG63 produced enhanced cell proliferation compared to p75-MG63. These results suggest that p75(NTR) signaling associated with trk receptors promotes both cell proliferation and osteoblast differentiation, but that p75(NTR)-mediated proliferation may be suppressed by signaling from the p75(NTR)/NgR complex.

Roles of collagen and periostin expression by cranial neural crest cells during soft palate development.

The tissue in the palatal region can be divided into the hard and the soft palates, each having a specialized function such as occlusion, speech, or swallowing. Therefore, an understanding of the mechanism of palatogenesis in relation to the function of each region is important. However, in comparison with the hard palate, there is still a lack of information about the mechanisms of soft palate development. In this study, the authors investigated the contribution of cranial neural crest (CNC) cells to development of both hard and soft palates. They also demonstrated a unique pattern of periostin expression during soft palate development, which was closely related to that of collagen type I (Col I) in palatine aponeurosis. Furthermore, organ culture analysis showed that exogenous transforming growth factor–β (TGF-β) induced the expression of both periostin and Col I. These novel patterns of expression in the extracellular matrix (ECM) induced by CNC cells suggest that these cells may help to determine the character of both the hard and soft palates through ECM induction. TGF-β signaling appears to be one of the mediators of Col I and periostin expression in the formation of functional structures during soft palate development.

Dilated capillaries, disorganized collagen fibers and differential gene expression in periodontal ligaments of hypomorphic fibrillin-1 mice

The periodontal ligaments (PDLs) are soft connective tissue between the cementum covering the tooth root surface and alveolar bone. PDLs are composed of collagen and elastic system fibers, blood vessels, nerves, and various types of cells. Elastic system fibers are generally formed by elastin and microfibrils, but PDLs are mainly composed of the latter. Compared with the well-known function of collagen fibers to support teeth, little is known about the role of elastic system fibers in PDLs. To clarify their role, we examined PDLs of mice underexpressing fibrillin-1 (mgR mice), which is one of the major microfibrillar proteins. The PDLs of homozygous mgR mice showed one-quarter of the elastic system fibers of wild-type (WT) mice. A close association between the elastic system fibers and the capillaries was noted in WT, homozygous and heterozygous mgR mice. Interestingly, capillaries in PDLs of homozygous mice were dilated or enlarged compared with those of WT mice. A comparable level of type I collagen, which is the major collagen in PDLs, was expressed in PDL-cells of mice with three genotypes. However, multi-oriented collagen fiber bundles with a thinner appearance were noted in homozygous mice, whereas well-organized collagen fiber bundles were seen in WT mice. Moreover, there was a marked decrease in periostin expression, which is known to regulate the fibrillogenesis and crosslinking of collagen. These observations suggest that the microfibrillar protein, fibrillin-1, is indispensable for normal tissue architecture and gene expression of PDLs.

Development of the Tarsometatarsal Skeleton by the Lateral Fusion of Three Cylindrical Periosteal Bones in the Chick Embryo (Gallus gallus)

An avian tarsometatarsal (TMT) skeleton spanning from the base of toes to the intertarsal joint is a compound bone developed by elongation and lateral fusion of three cylindrical periosteal bones. Ontogenetic development of the TMT skeleton is likely to recapitulate the changes occurred during evolution but so far has received less attention. In this study, its development has been examined morphologically and histologically in the chick, Gallus gallus. Three metatarsal cartilage rods radiating distally earlier in development became aligned parallel to each other by embryonic day 8 (ED8). Calcification initiated at ED8 in the midshaft of cartilage propagated cylindrically along its surface. Coordinated radial growth by fabricating bony struts and trabeculae resulted in the formation of three independent bone cylinders, which further became closely apposed with each other by ED13 when the periosteum began to fuse in a back‐to‐back orientation. Bone microstructure, especially orientation of intertrabecular channels in which blood vasculature resides, appeared related to the observed rapid longitudinal growth. Differential radial growth was considered to delineate eventual surface configurations of a compound TMT bone, but its morphogenesis preceded the fusion of bone cylinders. Bony trabeculae connecting adjacent cylinders emerged first at ED17 in the dorsal and ventral quarters of intervening tissue at the mid‐diaphyseal level. Posthatch TMT skeleton had a seemingly uniform mid‐diaphysis, although the septa persisted between original marrow cavities. These findings provide morphological and histological bases for further cellular and molecular studies on this developmental process. Anat Rec 293:1527–1535, 2010.

Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration.

Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration.

Osteogenic cell sheets reinforced with photofunctionalized micro-thin titanium.

Cell sheet technology has been used to deliver cells in single-sheet form with an intact extracellular matrix for soft tissue repair and regeneration. Here, we hypothesized that titanium-reinforced cell sheets could be constructed for bone tissue engineering and regeneration. Fifty-µm-thick titanium plates containing apertures were prepared and roughened by acid etching, some of which were photofunctionalized with 12 min of UV light treatment. Cell sheets were prepared by culturing rat calvarial periosteum-derived cells on temperature-responsive culture dishes and attached to titanium plates. Titanium-reinforced osteogenic cell sheet construction was conditional on various technical and material factors: cell sheets needed to be double-sided and sandwich the titanium plate, and the titanium plates needed to be micro thin and contain apertures to allow close apposition of the two cell sheets. Critically, titanium plates needed to be UV-photofunctionalized to ensure adherence and retention of cell sheets. Single-sided cell sheets or double-sided cell sheets on as-made titanium contracted and deformed within 4 days of incubation. Titanium-reinforced cell sheets on photofunctionalized titanium were structurally stable at least up to 14 days, developed the expected osteogenic phenotypes (ALP production and mineralization), and maintained structural integrity without functional degradation. Successful construction of titanium-reinforced osteogenic cell sheets was associated with increased cell attachment, retention, and expression of vinculin, an adhesion protein by photofunctionalization. This study identified the technical and material requirements for constructing titanium-reinforced osteogenic cell sheets. Future in vivo studies are warranted to test these titanium-reinforced cell sheets as stably transplantable, mechanically durable, and shape controllable osteogenic devices.