Expression of osteoclastogenic and anti-osteoclastogenic cytokines differs in mouse gingiva injected with lipopolysaccharide, peptidoglycan, or both.

OBJECTIVE: Bacterial substances in subgingival biofilm evoke alveolar bone resorption. We previously reported that gingival injection of bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN) induced alveolar bone resorption in mice. However, the mechanism by which LPS and PGN induce osteoclast formation has not been investigated. The aim of this study is to clarify the role of osteoclastogenic and anti-osteoclastogenic cytokines in the alveolar bone resorption induced by LPS and PGN. MATERIALS: LPS from Escherichia coli, PGN from Staphylococcus aureus, or both were injected into the gingiva of mice every 48 h for a total of 13 times. Alveolar bone resorption was assessed histochemically by tartrate-resistant acid phosphatase staining. Expression of the receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor (TNF)-α, interleukin (IL)-17, and IL-10 were analyzed by immunostaining. To analyze the role of these cytokines, RANKL-pretreated mouse bone marrow macrophages were stimulated with LPS, PGN, or LPS + PGN with or without anti-TNF-α antibody, IL-17, or IL-10. RESULTS: Alveolar bone resorption was induced by both LPS and PGN and exacerbated by LPS + PGN. LPS induced higher RANKL expression than PGN. Expression of TNF-α and IL-10 was correlated with bone resorption. PGN injections induced the strongest expression of IL-17, followed by LPS + PGN and LPS. In an in vitro osteoclastogenesis assay, anti-TNF-α antibody and IL-10 inhibited osteoclast formation, but IL-17 promoted it. CONCLUSION: LPS, PGN, or LPS + PGN injections induce distinctive expression of TNF-α, IL-10, and IL-17, suggesting that the composition of these bacterial ligands in dental plaque is critical for alveolar bone resorption.

The efficacy of the novel zinc-containing desensitizer CAREDYNE Shield on dentin hypersensitivity: a study protocol for a pilot randomized controlled trial.

BACKGROUND: Dentin hypersensitivity (DH) is a condition characterized by short and sharp episodes of pain which will arise in response to tactile, chemical, thermal, evaporative or osmotic stimuli. The painful symptoms cause discomfort in patients and reduce their quality of life. Recently, the novel zinc-containing desensitizer CAREDYNE Shield has been developed as a new type of desensitizer that acts by inducing chemical occlusion of dentinal tubules, and releasing zinc ion for root caries prevention. However, the clinical effectiveness of CAREDYNE Shield on DH remains unclear. Therefore, the aim of this study is to evaluate the effectiveness of CAREDYNE Shield on DH by comparing with that of another desensitizer, Nanoseal, commonly used in Japan. METHODS/DESIGN: This study protocol is a two-arm, parallel, pilot randomized controlled trial. Forty DH patients will be randomly allocated to two groups. Participants in the intervention group will be treated with CAREDYNE Shield, while those in the control group will be treated with Nanoseal. The primary outcome is the reduction of pain intensity in response to air stimuli measured with a 5-point verbal response scale from baseline to 4 weeks after the intervention, and Fisher's exact test will be used for analyses. DISCUSSION: CAREDYNE Shield can be casually applied to subgingival areas and proximal surfaces because it reacts with only tooth substance. Furthermore, zinc has been reported to reduce the demineralization of enamel and dentin and inhibit biofilm formation, plaque growth and dentin-collagen degradation. Therefore, CAREDYNE Shield may be expected to be a useful novel desensitizer that acts not only as a desensitizer but also as a root caries inhibitor. TRIAL REGISTRATION: UMIN Clinical Trials Registry (UMIN-CTR), ID: UMIN000038072. Registered on 21 September 2019. TRIAL STATUS: This study (protocol version number: version 1.4.0; approved on 22 October 2019) is ongoing. The recruitment of participants began in December 2019 and will be continued until November 2020 (Hanke, Am Dent Assoc 27:1379-1393, 1940).

Cell migration capability of vascular endothelial growth factor into the root apex of a root canal model in vivo.

Once a tooth develops deep caries and the dental pulp tissue is irreversibly infected, the infected dental pulp tissue should be removed, and filling material should be placed in the root canal. Endodontically treated teeth are prone to root fracture or periapical periodontitis; however, dental pulp tissue has the potential to prevent root fracture or periapical periodontitis. Therefore, dental pulp regeneration after pulpectomy may help prolong tooth life. In this study, a new method of dental pulp regeneration was developed. Vascular endothelial growth factor-adsorbed collagen gel was injected into the root canal of a prepared root canal model, placed into the dorsum of a rat, and cultured for 3 weeks. After retrieving the implant, histological analysis was performed. It was found that rat somatic cells were recruited into the root apex of the transplanted root canal model. These findings suggest a new potential technique for engineering dental pulp tissue.

Early gene expression analyzed by cDNA microarray and real-time PCR in osteoblasts cultured with chitosan monomer.

Chitosan has a variety of biological activities. Although it has been reported that chitosan promotes osteogenesis in bone lesions, little is known about how it modulates the hard tissue forming cells at the gene level. This study focused on gene expressions in osteoblasts cultured with a super-low concentration of chitosan monomer. cDNA probes were synthesized from isolated RNA and labeled with fluorescent dye. They were hybridized with Human 3.8 II cDNA microarray, and the fluorescent signal was analyzed. cDNA microarray analysis revealed that 10 genes concerning to various signaling-related molecules were expressed at > or =2.0-fold higher signal ratio levels in the experimental group when compared with the control group after 3 days. Real-time PCR analysis showed that chitosan monomer induced an increase in the expression of four signal transduction genes, mitogen-activated protein kinase (MAPK)K3, MAPKKK11, Rac1 and Shc1, together with the alkaline phosphatase gene. These results suggest that a super-low concentration of chitosan monomer could modulate the activity of osteoblastic cells through mRNA levels and that chitosan monomer directly affects signal transduction inside cells.

Chitosan monomer promotes tissue regeneration on dental pulp wounds.

The present study was undertaken to evaluate the applicability of chitosan monomer (D-glucosamine hydrochloride) as a pulp capping medicament. Both in vitro and in vivo experiments were carried out to study the cell metabolism and wound healing mechanisms following the application of chitomonosaccharide. After 3 days of osteoblast culture, alkaline phosphatase (ALP) activity significantly increased in the chitosan group. Reverse transcription polymerase chain reaction analysis revealed that chitosan induced an increase in the expression of ALP mRNA after 3 days and bone morphogenetic protein-2 mRNA after 7 days of osteoblast incubation. Inflammatory cytokine, interleukin (IL)-8, synthesis in fibroblasts was strongly suppressed in the medium supplemented with chitosan monomer. Histopathological effects were evaluated in rat experiments. After 1 day, inflammatory cell infiltrations were observed to be weak when compared with the application of chitosan polymer. After 3 days, a remarkable proliferation of fibroblasts was seen near the applied chitosan monomer. The inflammatory cell infiltration had almost completely disappeared. After 5 days, the fibroblastic proliferation progressed, and some odontoblastic cells appeared at the periphery of the proliferated fibroblasts. These findings indicate that the present study is the first report that chitosan monomer acts as a biocompatibility stable medicament even at the initial stage of wound healing in comparison with the application of chitosan polymer.

Early gene expression analyzed by cDNA microarray and RT-PCR in osteoblasts cultured with water-soluble and low molecular chitooligosaccharide.

Chitosan has a variety of biological activities. However, little is known about how chitosan modulates the hard tissue forming cells. When we cultured an osteoblastic cell line in alpha-MEM supplemented with 10% FBS and 0.005% chitooligosaccharide for 3 days, alkaline phosphatase (ALP) activity was significantly high compared with the control culture group (p<0.05). This study was focused on gene expression in osteoblasts cultured with water-soluble chitooligosaccharide. cDNA probes were synthesized from isolated RNA and labeled with fluorescent dye. They were hybridized with Human 1.0((R)) cDNA microarray, and fluorescent signal was analyzed. cDNA microarray analysis revealed that 16 genes were expressed at >/=1.5-fold higher signal ratio levels in the experimental group compared with the control group after 3 days. RT-PCR analysis showed that chitosan oligomer induced an increase in the expression of two genes, CD56 antigen and tissue-type plasminogen activator. Furthermore, the expression of mRNAs for BMP-2 was almost identical in the experimental and control groups after 3 days of culture, but slightly increased after 7 days of culture with chitosan oligomer. These results suggest that a super-low concentration of chitooligosaccharide could modulate the activity of osteoblastic cells through mRNA levels and that the genes concerning cell proliferation and differentiation can be controlled by water-soluble chitosan.

Potency of fish collagen as a scaffold for regenerative medicine.

Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD) motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine.

D-glucosamine promotes transfection efficiency during electroporation.

D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes) in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP) in the cultured cells (osteoblasts; NOS-1 cells). The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.

Fabrication and characteristics of chitosan sponge as a tissue engineering scaffold.

Cells, growth factors, and scaffolds are the three main factors required to create a tissue-engineered construct. After the appearance of bovine spongiform encephalopathy (BSE), considerable attention has therefore been focused on nonbovine materials. In this study, we examined the properties of a chitosan porous scaffold. A porous chitosan sponge was prepared by the controlled freezing and lyophilization of different concentrations of chitosan solutions. The materials were examined by scanning electron microscopy, and the porosity, tensile strength, and basic fibroblast growth factor (bFGF) release profiles from chitosan sponge were examined in vitro. The morphology of the chitosan scaffolds presented a typical microporous structure, with the pore size ranging from 50 to 200 µm. The porosity of chitosan scaffolds with different concentrations was approximately 75-85%. A decreasing tendency for porosity was observed as the concentration of the chitosan increased. The relationship between the tensile properties and chitosan concentration indicated that the ultimate tensile strength for the sponge increased with a higher concentration. The in vitro bFGF release study showed that the higher the concentration of chitosan solution became, the longer the releasing time of the bFGF from the chitosan sponge was.

Biological safety of fish (tilapia) collagen.

Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine.

Early gene and protein expression associated with osteoblast differentiation in response to fish collagen peptides powder.

This study was designed to investigate the biological effects of fish collagen peptide (FCP) on human osteoblasts. Human osteoblasts were treated with 0.1% FCP, which was the optimal concentration confirmed by the increase in alkaline phosphatase activity. After one, three, five and seven days of culture, the number of FCP-treated cells increased significantly compared with untreated cells. In a real-time PCR analysis, the expression of osteocalcin, osteopontin, BMP-2 and integrin ß3 mRNAs in FCP-treated cells showed increases compared with untreated cells after three days of culture. After seven days of culture, the expression levels of osteopontin and integrin ß3 were still higher in the FCP-treated cells than in untreated cells. The production of osteocalcin, osteopontin and integrin ß3 proteins in FCP-treated cells also showed increases after seven days of culture. Furthermore, FCP accelerated matrix mineralization in the cultures. The present study indicates the potential utility of FCP as a biomaterial.

Early gene expression analyzed by a genome microarray and real-time PCR in osteoblasts cultured with a 4-META/MMA-TBB adhesive resin sealer.

OBJECTIVES: Adhesive resin sealer systems have been applied in endodontics to seal the root canal system. This study was designed to confirm the mechanism of intracellular molecular events in an in vitro cell culture system with a 4-methacryloxyethyl trimellitate anhydride/methylmethacrylate-tri-n-butyl borane (4-META/MMA-TBB) adhesive resin sealer. STUDY DESIGN: The gene expression patterns relating to cell growth and differentiation were examined using a human genome expression microarray and real-time polymerase chain reaction analyses in hard tissue-forming osteoblasts cultured with and without a 4-META/MMA-TBB resin sealer. RESULTS: There was no significant difference in the cell number between the control and adhesive sealer groups. An increased expression of integrin beta, transforming growth factor beta-related protein, craniofacial development protein 1, and PI3K genes was demonstrated. The integrin beta and PI3K genes showed extremely high ratios. CONCLUSIONS: The signal transduction pathway, at least through the PI3K/Akt cascade for cell proliferation and differentiation, can be controlled by some components of this type of adhesive resin sealer.