Pasutha Thunyakitpisal

Cloning and functional analysis of a family of nuclear matrix transcription factors (NP/NMP4) that regulate type I collagen expression in osteoblasts

Collagen expression is coupled to cell structure in connective tissue. We propose that nuclear matrix architectural transcription factors link cell shape with collagen promoter geometry and activity. We previously indicated that nuclear matrix proteins (NP/NMP4) interact with the rat type I collagen α1(I) polypeptide chain (COL1A1) promoter at two poly(dT) sequences (sites A and B) and bend the DNA. Here, our objective was to determine whether NP/NMP4‐COL1A1 binding influences promoter activity and to clone NP/NMP4. Promoter‐reporter constructs containing 3.5 kilobases (kb) of COL1A1 5′ flanking sequence were fused to a reporter gene. Mutation of site A or site B increased promoter activity in rat UMR‐106 osteoblast‐like cells. Several full‐length complementary DNAs (cDNAs) were isolated from an expression library using site B as a probe. These clones expressed proteins with molecular weights and COL1A1 binding activity similar to NP/NMP4. Antibodies to these proteins disrupted native NP/NMP4‐COL1A1 binding activity. Overexpression of specific clones in UMR‐106 cells repressed COL1A1 promoter activity. The isolated cDNAs encode isoforms of Cys2His2 zinc finger proteins that contain an AT‐hook, a motif found in architectural transcription factors. Some of these isoforms recently have been identified as Cas‐interacting zinc finger proteins (CIZ) that localize to fibroblast focal adhesions and enhance metalloproteinase gene expression. We observed NP/NMP4/CIZ expression in osteocytes, osteoblasts, and chondrocytes in rat bone. We conclude that NP/NMP4/CIZ is a novel family of nuclear matrix transcription factors that may be part of a general mechanical pathway that couples cell structure and function during extracellular matrix remodeling.

Acemannan sponges stimulate alveolar bone, cementum and periodontal ligament regeneration in a canine class II furcation defect model

BACKGROUND AND OBJECTIVE Periodontal disease is a common infectious disease, found worldwide, causing the destruction of the periodontium. The periodontium is a complex structure composed of both soft and hard tissues, thus an agent applied to regenerate the periodontium must be able to stimulate periodontal ligament, cementum and alveolar bone regeneration. Recent studies demonstrated that acemannan, a polysaccharide extracted from Aloe vera gel, stimulated both soft and hard tissue healing. This study investigated effect of acemannan as a bioactive molecule and scaffold for periodontal tissue regeneration. MATERIAL AND METHODS Primary human periodontal ligament cells were treated with acemannan in vitro. New DNA synthesis, expression of growth/differentiation factor 5 and runt-related transcription factor 2, expression of vascular endothelial growth factor, bone morphogenetic protein-2 and type I collagen, alkaline phosphatase activity, and mineralized nodule formation were determined using [(3)H]-thymidine incorporation, reverse transcription-polymerase chain reaction, enzyme-linked immunoabsorbent assay, biochemical assay and alizarin red staining, respectively. In our in vivo study, premolar class II furcation defects were made in four mongrel dogs. Acemannan sponges were applied into the defects. Untreated defects were used as a negative control group. The amount of new bone, cementum and periodontal ligament formation were evaluated 30 and 60 d after the operation. RESULTS Acemannan significantly increased periodontal ligament cell proliferation, upregulation of growth/differentiation factor 5, runt-related transcription factor 2, vascular endothelial growth factor, bone morphogenetic protein 2, type I collagen and alkaline phosphatase activity, and mineral deposition as compared with the untreated control group in vitro. Moreover, acemannan significantly accelerated new alveolar bone, cementum and periodontal ligament formation in class II furcation defects. CONCLUSION Our data suggest that acemannan could be a candidate biomolecule for periodontal tissue regeneration.

Deacetylation affects the physical properties and bioactivity of acemannan, an extracted polysaccharide from Aloe vera.

Acemannan, an acetylated polymannose from Aloe vera, induces tissue repair. We investigated the role of acemannans acetyl-groups on its physical and biological properties. Deacetylated acemannan (DeAcAM) was prepared and characterized. The physical properties and microscopic structure of DeAcAM were evaluated using water solubility, contact angle, X-ray diffraction, and scanning-electron microscopy. The activity of DeAcAM on cell proliferation and gene expression were assessed. Acemannan and DeAcAM structures were simulated and the acemannan tetramer diad and its completely deacetylated structure were also determined. Increased acemannan deacetylation reduced its water solubility and hydrophilicity. Complete deacetylation altered acemannans conformation to a partial crystal structure. The bioactivity of acemannan was reduced corresponding to its deacetylation. Acemannan induced cell proliferation, and VEGF and Collagen I expression; however, 100% DeAcAM did not. The simulated structures of the acemannan diad and the completely deacetylated diad were different. We conclude acetyl-groups affect acemannans structure and physical/biological properties.

The Effect of Chemical Surface Treatments on the Flexural Strength of Repaired Acrylic Denture Base Resin

PURPOSE To investigate the effect of the selected chemical surface treatment agents on the flexural strength of heat-polymerized acrylic resin repaired with autopolymerized acrylic resin. MATERIALS AND METHODS Ninety heat-polymerized acrylic resin specimens (Meliodent) were prepared according to ISO1567 and randomly divided into nine groups: positive and negative control groups (groups I and II), and seven experimental groups (groups III to IX). Specimens in groups II to IX were cut in the middle and beveled 45°. Group III was then treated with methyl methacrylate (the liquid part of Unifast TRAD) for 180 seconds. Group IV was treated with Rebase II adhesive according to the manufacturers instructions. Groups V to IX were treated with methyl formate, methyl acetate, and a mixture of methyl formate-methyl acetate at various concentrations (75:25, 50:50, 25:75% v/v, respectively) for 15 seconds. They were then repaired with autopolymerized acrylic resin (Unifast TRAD). A three-point loading test was performed using a universal testing machine. One-way ANOVA and post hoc Tukeys analysis at p < 0.05 were used for statistical comparison. Failure analysis was then recorded for each specimen. The morphological changes in untreated and treated specimens were observed by scanning electron microscopy. RESULTS The flexural strengths of groups III to IX were significantly higher than that of group II (p < 0.05). The flexural strengths of groups IV to IX showed no significant difference among them (p > 0.05). All specimens in groups V to IX showed 100% cohesive failure, while groups II, III, and IV showed cohesive failure of 10%, 60%, and 60%, respectively. From scanning electron micrographs, the application of methyl formate, methyl acetate, and a mixture of methyl formate-methyl acetate solutions on heat-polymerized acrylic resin resulted in a 3D honeycomb appearance, while specimens treated with methyl methacrylate and Rebase II adhesive developed shallow pits and small crest patterns, respectively. CONCLUSION Treating surfaces with methyl formate, methyl acetate, and a mixture of methyl formate-methyl acetate solutions significantly enhanced the flexural strength of heat-polymerized acrylic denture base resin that had been repaired with autopolymerized acrylic resin.

Sodium-phosphorylated chitosan/zinc oxide complexes and evaluation of their cytocompatibility: An approach for periodontal dressing

The aim of this study was to evaluate the possibility of metal complex formation between sodium-phosphorylated chitosan (PCTS) and ZnO. The polymer–metal complex formation was investigated in terms of thermal degradation. The structure deduction of the PCTS/ZnO complex was investigated by means of Fourier transform infrared spectroscopy and X-ray diffraction (XRD). The PCTS/ZnO complexes were formed by the sharing of lone pairs of electrons from the N atoms in the amine groups and O atoms in the phosphate and hydroxyl groups of PCTS to the protonated hydroxyl species on the ZnO surface. Because complex formation occurred at the surface of ZnO particles, it did not change the ZnO crystalline structure. Cytotoxicity, evaluated by a direct contact test with primary human gingival fibroblast cells, revealed that PCTS was biocompatible and reduced the cytotoxicity of ZnO by complexation, making PCTS/ZnO complexes potentially biocompatible. Within the limits of these data, it appears that PCTS could be used as a reaction rate-modifying agent in periodontal dressings.

Assignment of Nmp4 to mouse chromosome 6 band F1 flanked by D6Mit134 and D6Mit255 using radiation hybrid mapping and fluorescence in situ hybridization

The Nmp4 gene (MGD approved nomenclature) encodes a novel family of Cys2His2 zinc finger proteins, some of which contribute to extracellular matrix gene expression in connective tissue (Alvarez et al., 1998; Thunyakitpisal et al., 1999, Nakamoto et al., 2000; Thunyakitpisal et al., 2001). A human brain partial cDNA clone TNRC1 (alias CAGH1) encodes a protein that likely is the human ortholog of Nmp4 and has been mapped to human chromosome 12p12 (Margolis et al., 1997). Here, we report the isolation of a murine genomic BAC clone containing the Nmp4 gene and the localization of mouse Nmp4 to chromosome 6 band F1, using radiation hybrid mapping and fluorescence in situ hybridization analyses.

Cell proliferative effect of polyxyloses extracted from the rhizomes of wild turmeric, Curcuma aromatica.

Hot water-soluble crude polysaccharides were extracted from the rhizomes of wild turmeric, Curcuma aromatica Salisb. (Zingiberaceae), using dry grinding, boiling water extraction, and then ethanol precipitation. The crude polysaccharide extract was then fractionated by DEAE-cellulose ion exchange column chromatography, and subsequently further purified by Superdex G-200 gel filtration column chromatography, giving two relatively abundant polysaccharide fractions, called P11 and P21, and a much less common fraction P22 obtained in insufficient amounts for further analysis. The two main polysaccharide fractions were evaluated for monosaccharide composition by acid hydrolysis and high performance liquid chromatography (HPLC), whilst the molecular weight and functional groups were determined by gel permeable chromatography (GPC) and FT-IR, respectively. Fractions P11 and P21 were found to be polyxyloses with molecular weight-averages of 469,171 and 157,665 Da, respectively. P11 (100 μg/mL) could significantly induce human gingival fibroblast cells proliferation by 30%, while P21 (100 μg/mL) could significantly inhibit gingival fibroblast cells proliferation by 92%. The in vitro human primary gingival fibroblast cell proliferation in cell culture at a concentration of 100 μg/mL.

Acemannan increases NF-κB/DNA binding and IL-6/-8 expression by selectively binding Toll-like receptor-5 in human gingival fibroblasts

Acemannan, an acetylated polymannose from Aloe vera, has immunomodulatory effects. We investigated whether acemannan induces IL-6 and -8 expression and NF-κB/DNA binding in human gingival fibroblasts. IL-6 and -8 expression levels were assessed via RT-PCR and ELISA. The NF-κB p50/p65-DNA binding was determined. The structures of acemannan mono-pentamers and Toll-like receptor 5 (TLR5) were simulated. The binding energies between acemannan and TLR5 were identified. We found that acemannan significantly stimulated IL-6/-8 expression at both the mRNA and protein level and significantly increased p50/DNA binding. Preincubation with an anti-TLR5 neutralizing antibody abolished acemannan-induced IL-6/-8 expression and p50/DNA binding, and co-incubation of acemannan with Bay11-7082, a specific NF- κB inhibitor, abolished IL-6/-8 expression. The computer modeling indicated that monomeric/dimeric single stranded acemannan molecules interacted with the TLR5 flagellin recognition sites with a high binding affinity. We conclude that acemannan induces IL-6/-8 expression, and p50/DNA binding in gingival fibroblasts, at least partly, via a TLR5/NF-κB-dependent signaling pathway. Furthermore, acemannan selectively binds with TLR5 ectodomain flagellin recognition sites.

Improving shear bond strength between feldspathic porcelain and zirconia substructure with lithium disilicate glass-ceramic liner.

This study investigated the shear bond strength (SBS) between veneering porcelain and zirconia substructure using lithium disilicate glass-ceramic as a liner. The mineral phases and microstructures of lithium disilicate glass-ceramic at temperature range of 800-900°C were preliminarily investigated. SBSs of porcelain-veneered zirconia specimens with and without lithium disilicate glassceramic liner fired at the same temperature were determined. Results showed that SBSs of veneering porcelain and zirconia with lithium disilicate glass-ceramic liner was notably increased (p<0.05). Specimens from the group with the highest SBS (59.7 MPa) were subject to thermocycling up to 10,000 cycles and their post-thermocycling SBSs investigated. Though weakened by thermocycling, SBSs were above the clinically acceptable limit (25 MPa) of ISO 9693. Fractographic analysis revealed mixed cohesive and adhesive failures. It was concluded that lithium disilicate glass-ceramic is a potential liner which generated high SBS between veneering porcelain and zirconia.

Stimulation of Dentin Regeneration by Using Acemannan in Teeth with Lipopolysaccharide-induced Pulp Inflammation

Introduction: This study investigated the effects of acemannan, a polysaccharide from Aloe vera, on human deciduous pulp cells in vitro and the response after vital pulp therapy in dog deciduous teeth. Methods: Human primary dental pulpal cells were treated with acemannan in vitro and evaluated for proliferation, alkaline phosphatase activity, type I collagen, bone morphogenetic protein (BMP‐2), BMP‐4, vascular endothelial growth factor, and dentin sialoprotein expression and mineralization. Osteogenesis‐related gene expression was analyzed by complementary DNA microarray. Pulpal inflammation was induced in dog teeth for 14 days. The inflamed pulp was removed, retaining the healthy pulp. The teeth were randomly divided into 3 treatment groups: acemannan, mineral trioxide aggregate, and formocresol. Sixty days later, the teeth were extracted and evaluated histopathologically. Results: Acemannan significantly increased pulp cell proliferation, alkaline phosphatase, type I collagen, BMP‐2, BMP‐4, vascular endothelial growth factor, and dentin sialoprotein expression and mineralization approximately 1.4‐, 1.6‐, 1.6‐, 5.5‐, 2.6‐, 3.8‐, 1.8‐, and 4.8‐fold, respectively, compared with control. In vivo, partial pulpotomy treatment using acemannan generated outcomes similar to mineral trioxide aggregate treatment, resulting in mineralized bridge formation with normal pulp tissue without inflammation or pulp necrosis. In contrast, the formocresol group demonstrated pulp inflammation without mineralized bridge formation. Conclusions: Acemannan is biocompatible with the dental pulp. Furthermore, acemannan stimulated dentin regeneration in teeth with reversible pulpitis.