Yoshio Shimabukuro

Periodontal Regeneration by FGF-2 (bFGF) in Primate Models

We recently demonstrated that a topical application of basic fibroblast growth factor (FGF-2 ; bFGF) to alveolar bone defects in beagle dogs enhanced periodontal regeneration. The purpose of this study was further characterization of the biological effects of FGF-2 in non-human primates. Thirty-two inflamed furcation class II defects were surgically created in 4 male primates. The gelatinous carrier alone or the carrier containing 0.1 or 0.4% human recombinant FGF-2 was topically applied to the defects and compared with no treatment. Eight weeks after application, the periodontal regeneration in those defects was analyzed. In all FGF-2-treated sites, significant periodontal regeneration was dose-dependently observed in greater amounts than in the carrier-treated or non-treated sites. No instances of epithelial down-growth, ankylosis, or root resorption were observed in the FGF-2-treated sites. These results indicate that a topical application of FGF-2 can enhance considerable periodontal regeneration in surgically created furcation class II defects of non-human primates.

Fibroblast growth factor-2 regulates the synthesis of hyaluronan by human periodontal ligament cells.

Basic fibroblast growth factor (FGF‐2) can enhance biological potentials of periodontal ligament cells and its topical application induces considerable periodontal tissue regeneration in vivo. In this study, we examined the effect of FGF‐2 on the production of hyaluronan (HA), an extracellular matrix playing important roles in homeostasis and inflammatory/wound healing responses, by human periodontal ligament (HPDL) cells. An inhibition binding‐protein assay revealed that FGF‐2 significantly increased HA production by HPDL cells in a dose dependent manner. Analysis by HPLC revealed that in conditioned medium of FGF‐2‐treated HPDL cells HA had a higher molecular mass, compared to that of untreated HPDL cells. RT‐PCR analysis revealed the enhancement of mRNA expression of hyaluronan synthase (HAS) 1 and HAS 2, both of which contribute to the production of HA with a high molecular mass, but not HAS 3 in the FGF‐2‐treated HPDL cells. In contrast, three isoforms of hyaluronidase (HYAL) transcript were unchanged in the FGF‐2‐treated HPDL cells. These results provide new evidence for the possible involvement of FGF‐2 in the regulation of HA production and its appreciable roles in not only homeostasis but also regeneration of periodontal tissues.

Quantitative Analysis of Immunocompetent Cells in Human Periapical Lesions: Correlations with Clinical Findings of the Involved Teeth

Human periapical lesions develop as a result of a pathological immune response to continuous stimuli from infected root canals. This study identified the immunocompetent cells in such lesions immunohistochemically and quantified them to examine their interrelationships and correlations with clinical findings. The number of IgG-containing cells in CD4+ cell (Th/i)-rich lesions (> or = 55 CD4+ cells/2 x 10(4) microns 2) was significantly larger than in CD4+ cell-poor lesions (< 55 CD4+ cells/2 x 10(4) microns 2). This indicated that the CD4+ cells and the IgG-containing cells acted together against antigenic stimuli. The proportion of T cells in the mononuclear infiltrates varied with the size of the periapical lesions. The proportion of CD11+ cells (monocytes/macrophages) was significantly larger in lesions which showed a positive reaction to percussion or were tender on palpation than in the lesions without these symptoms. These observations suggest that T cells may play an important role in the development of periapical lesions and that CD11+ cells may be involved in the development of symptoms.

Fibroblast growth factor-2 regulates the cell function of human dental pulp cells.

INTRODUCTION Homeostasis and tissue repair of dentin-pulp complex are attributed to dental pulp tissue and several growth factors. Dental pulp cells play a pivotal role in homeostasis of dentin-pulp complex and tissue responses after tooth injury. Among these cytokines, fibroblast growth factor (FGF)-2 has multifunctional biologic activity and is known as a signaling molecule that induces tissue regeneration. In this study, we examined the effects of FGF-2 on growth, migration, and differentiation of human dental pulp cells (HDPC). METHODS HDPC were isolated from healthy dental pulp. Cellular response was investigated by [(3)H]-thymidine incorporation into DNA. Cytodifferentiation was examined by alkaline phosphatase (ALPase) assay and cytochemical staining of calcium by using alizarin red. Migratory activity was determined by counting the cells migrating into cleared area that had introduced with silicon block. RESULTS FGF-2 activated HDPC growth and migration but suppressed ALPase activity and calcified nodule formation. Interestingly, HDPC, which had been pretreated with FGF-2, showed increased ALPase activity and calcified nodule formation when subsequently cultured without FGF-2. These results suggest that FGF-2 potentiates cell growth and accumulation of HDPC that notably did not disturb cytodifferentiation of the cells later. Thus, FGF-2 is a favorable candidate for pulp capping agent. CONCLUSIONS These results provide new evidence for the possible involvement of FGF-2 not only in homeostasis but also in regeneration of dentin-pulp complex.

Fibroblast growth factor-2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction.

Fibroblast growth factor‐2 (FGF‐2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF‐2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF‐2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3‐kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF‐2‐stimulated migration of MPDL22 cells. Moreover, in response to FGF‐2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)‐1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF‐2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti‐CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF‐2. Furthermore, an anti‐CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF‐2‐induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF‐2‐mediated cell motility of PDL cells, suggesting that FGF‐2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production. J. Cell. Physiol. 226: 809–821, 2011.

Direct Interaction between Gingival Fibroblasts and Lymphoid Cells Induces Inflammatory Cytokine mRNA Expression in Gingival Fibroblasts

In inflamed periodontal lesions, dense infiltration of lymphocytes is usually observed in the extravascular periodontal connective tissue, adjacent to gingival fibroblasts. Our previous study revealed that activated lymphocytes can adhesively interact with gingival fibroblasts in vitro. In the present study, we investigated whether gingival fibroblasts are activated through direct interaction with lymphoid cells by monitoring the expression of inflammatory cytokine mRNA in human gingival fibroblasts (HGF). Co-culture with various human lymphoid cells in vitro resulted in a marked increase in the expression of IL-la, IL-1β, and IL-6 mRNA by the HGF. In addition, expression of the mRNA of the IL-1β-converting enzyme (ICE), which is essential to produce the mature form of IL-1β, was constitutively observed in the HGF, suggesting that mature IL-lp is produced by these cells. When HGF were cultured with the culture supernatant of the lymphoid cells, the increase in the inflammatory cytokine mRNA expression was not observed. Similarly, when HGF and lymphoid cells were cultured in the same well but separated by a membrane which prevented direct contact between the cells, no increase in inflammatory cytokine mRNA expression was observed. These results strongly indicate that direct interaction between these heterotypic cell types transduces activation signals into HGF that induce an increase in inflammatory cytokine mRNA expression. Furthermore, IL-1β mRNA expression in the HGF was synergistically increased when HGF directly interacted with lymphoid cells in the presence of exogeneous IL-1β. The present study demonstrates that direct interaction between HGF and lymphoid cells stimulates HGF to increase inflammatory cytokine mRNA expression, and raises the possibility that heterotypic cell-cell interaction may facilitate local inflammatory reactions.

Fibroblast growth factor-2 regulates expression of osteopontin in periodontal ligament cells

Osteopontin is a protein found in the bone‐related matrix and plays multiple regulatory roles in mineralizing and non‐mineralizing tissue. In osteogenic cell‐lines, the expression of osteopontin increases with the progression of differentiation, but both the expression and function of osteopontin vary with the cell type and its activation state. In this study, we examined the expression of osteopontin by clones established from mouse periodontal ligament, in response to inorganic phosphate and fibroblast growth factor (FGF)‐2, which can induce periodontal tissue regeneration. The involvement of inorganic phosphate in the expression of osteopontin during the course of cell differentiation of a clone MPDL22 was confirmed by addition of foscarnet, an inorganic phosphate transport inhibitor. Although FGF‐2 decreased the mRNA expression of almost every bone‐related protein in MPDL22, FGF‐2 upregulated the expression of osteopontin in MPDL22 at both mRNA and protein levels. Interestingly, FGF‐2 enhanced the concentration of osteopontin in the culture supernatant of MPDL22, whereas inorganic phosphate did not. The FGF‐2‐induced osteopontin in the culture supernatant seems to be involved in cell survival activity. An immunohistochemical study showed that the FGF‐2‐induced osteopontin was mainly present in perinuclear matrices while the inorganic phosphate‐induced osteopontin was associated with extracellular matrices in addition to perinuclear matrices. The present results indicated that FGF‐2 induces unique expression of osteopontin, which may play a role different from the other bone‐related proteins during the process of periodontal tissue regeneration by FGF‐2. J. Cell. Physiol. 216: 640–650, 2008,

Nicotine Inhibits Mineralization of Human Dental Pulp Cells

Nicotine is a major component of tobacco smoke, and signals via nicotinic acetylcholine receptors (nAChR). However, little is known about the effects of nicotine on human dental pulp cells (HDPCs). In this study, we assessed the effects of nicotine on mineralization in HDPCs. We confirmed messenger RNA expression of nAChR subunits and examined the effects of nicotine on expression of extracellular matrices (ECMs), alkaline phosphatase (ALP) activity, and mineralized nodule formation by HDPCs. Gene expression of nAChR subunits alpha1, alpha2, alpha 4, alpha 5, alpha 6, alpha 7, beta1, beta2, and beta 4 was detected in HDPCs. Interestingly, the messenger RNA expression of dentin matrix acidic phosphoprotein-1, bone sialoprotein, and ALP activity were significantly reduced in nicotine-treated HDPC. In addition, mineralized nodule formation, which was examined by alizarin red staining, was also inhibited in HDPCs by the same treatment. These results indicate that nicotine suppresses the cytodifferentiation and mineralization of HDPCs, possibly via nAChR.

Activation of Adenosine-receptor-enhanced iNOS mRNA Expression by Gingival Epithelial Cells

A series of reports has revealed that adenosine has a plethora of biological actions toward a large variety of cells. In this study, we investigated the influence of adenosine receptor activation on iNOS mRNA expression in human gingival epithelial cells (HGEC) and SV-40-transformed HGEC. HGEC expressed adenosine receptor subtypes A1, A2a, and A2b, but not A3 mRNA. Ligation of adenosine receptors by a receptor agonist, 2-chloroadenosine (2CADO), enhanced iNOS mRNA expression by both HGEC and transformed HGEC. In addition, the adenosine receptor agonist enhanced the production of NO2 -/NO3 -, NO-derived stable end-products. An enhanced expression of iNOS mRNA and NO2 -/NO3 - was also observed when SV40-transformed HGEC were stimulated with CPA or CGS21680, A1- or A2a-selective adenosine receptor agonists, respectively. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses by HGEC in periodontal tissues.

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells ☆

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.