2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside Isolated from Polygoni Multiflori Ameliorates the Development of Periodontitis.

Periodontitis, a chronic infection by periodontopathic bacteria, induces uncontrolled inflammation, which leads to periodontal tissue destruction. 2,3,5,4'-Tetrahydroxystilbene-2-O-beta-glucoside (THSG), a polyphenol extracted from Polygoni Multiflori, reportedly has anti-inflammatory properties. In this study, we investigated the mechanisms of THSG on the Porphyromonas gingivalis-induced inflammatory responses in human gingival fibroblasts and animal modeling of ligature-induced periodontitis. Human gingival fibroblast cells were treated with lipopolysaccharide (LPS) extracted from P. gingivalis in the presence of resveratrol or THSG to analyze the expression of TNF-α, IL-1ß, and IL-6 genes. Increased AMP-activated protein kinase (AMPK) activation and SirT1 expression were induced by THSG. Treatment of THSG decreased the expression of LPS-induced inflammatory cytokines, enhanced AMPK activation, and increased the expression of SirT1. In addition, it suppressed the activation of NF-κB when cells were stimulated with P. gingivalis LPS. The anti-inflammatory effect of THSG and P. Multiflori crude extracts was reproduced in ligature-induced periodontitis animal modeling. In conclusion, THSG inhibited the inflammatory responses of P. gingivalis-stimulated human gingival fibroblasts and ameliorated ligature-induced periodontitis in animal model.

NDAT suppresses pro-inflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells.

Nano-diamino-tetrac (NDAT), a tetraiodothyroxine deaminated nano-particulated analog, has shown to inhibit expression of pro-inflammatory genes. NDAT inhibits expression of programmed death-ligand 1 (PD-L1). On the other hand, in addition to inhibiting inflammatory effect, the stilbene, resveratrol induces expression of cyclooxygenase-2 (COX-2) and its accumulation. Sequentially, inducible COX-2 complexes with p53 and induces p53-dependent anti-proliferation. In current study, we investigated mechanisms involved in combined treatment of NDAT and resveratrol on anti-proliferation in human oral cancer cells. Both resveratrol and NDAT inhibited expression of pro-inflammatory IL-1ß and TNF-α. They also inhibited expression of CCND1 and PD-L1. Both resveratrol and NDAT induced BAD expression but only resveratrol induced COX-2 expression in both OEC-M1 and SCC-25 cells. Combined treatment attenuated gene expression significantly compared with resveratrol treatment in both cancer cell lines. Resveratrol reduced nuclear PD-L1 accumulation which was enhanced by a STAT3 inhibitor, S31-201 or NDAT suggesting that NDAT may inactivate STAT3 to inhibit PD-L1 accumulation. In the presence of T4, NDAT further enhanced resveratrol-induced anti-proliferation in both cancer cell lines. These findings provide a novel understanding of the inhibition of NDAT in thyroxine-induced pro-inflammatory effect on resveratrol-induced anticancer properties.

Adenovirus-mediated gene therapy for human head and neck squamous cell cancer in a nude mouse model.

Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir administration was used to treat human head and neck cancer in nude mice. Tumors were generated by transcutaneous needle injection of 6 x 10(6) human squamous carcinoma cells into the floor of the mouth. After 14 days, 10(10) particles of a replication-defective recombinant adenovirus containing the herpes simplex virus thymidine kinase gene (ADV/RSV-tk) were injected directly into the tumors. The mice subsequently received ganciclovir injections for six consecutive days and were sacrificed at 21 days post tumor cell implantation. Clinical response to the treatment was assessed by computer-imaged morphometric analysis of cross sectional area of nonnecrotic tumor and mitotic activity, which were used for the calculation of a tumor index. The median tumor index value of the treatment group was 280- to 2400-fold smaller than controls which did not receive the therapeutic gene (P < 0.001-0.016), and three-quarters of the treatment group had tumor index values that were indicative of near total tumor regression. Survival studies show that 50% of the ADV/RSV-tk-treated mice are free of tumor at 160 days post adenovirus injection, while all controls died or required sacrifice within 43 days. These results demonstrate that clinically effective in vivo treatment of human squamous cell cancer can be achieved using adenovirus-mediated gene therapy.

Combination gene therapy for oral cancer in a murine model.

Combination therapy involving adenovirus-mediated transfer of the genes for herpes thymidine kinase (tk) and murine interleukin 2 (mIL-2) was used to treat head and neck cancer in C3H/HeJ mice. Tumors were generated by transcutaneous injection of 5 X 10(5) murine squamous carcinoma cells into the floor of the mouth of these syngeneic mice. After 1 week, recombinant adenoviral vectors containing both therapeutic and control genes in various combinations were injected directly into the established tumors, and subsequently all mice were administered ganciclovir twice daily (25 mg/kg) for 6 days. Animals receiving either tk alone or tk + mIL-2 demonstrated significant tumor regression compared to mIL-2 alone or control vector-treated mice (P < 0.008). Mice receiving both tk + mIL-2, however, also demonstrated a significantly greater regression of tumors compared to those treated with tk alone (P<0.008), indicating a synergistic effect of the combination gene therapy. This synergism was confirmed in survival studies because tk + mIL-2 treated mice showed increased survivals (P=0.0002). Clinical and microscopic exam of regional surrounding tissues and distant organs showed no evidence of cytotoxicity for representative animals in each experimental group. These results suggest that combination tk and mIL-2 gene therapy may provide a powerful new modality for the treatment of head and neck cancer.

The role of interleukin-2 in combination adenovirus gene therapy for head and neck cancer.

Interleukin-2 (IL-2) gene therapy alone and in combination with the herpes thymidine kinase gene (tk) was used to evaluate immunological responses and antitumor effects in head and neck cancer. Established floor of mouth squamous cell carcinomas in C3H/HeJ mice were directly injected with recombinant adenoviral vectors carrying both therapeutic and control genes. One week after adenoviral gene transfer, only the animals treated with combination IL-2+tk or tk alone demonstrated significant tumor regression. Residual tumors were harvested for microscopic evaluation and immunohistochemistry staining, which revealed a predominance of CD8+ lymphocytes in the tumor beds of the animals treated with IL-2. To evaluate the systemic immune effects of IL-2, animals treated with single or combination gene therapy received a second site challenge with parental tumor cells or a heterologous but syngeneic sarcoma cell line. Mice treated with combination IL-2 and tk demonstrated a protective systemic immunity specific to the parental tumor cell line, whereas no systemic immune response was evident in mice receiving IL-2 alone. In a separate experiment, a range of concentrations of the adenovirus IL-2 vector were used to treat established tumors. Even with the maximal single-dose adenovirus concentration, IL-2 alone was ineffective as a single therapy. These results support the use of adenovirus-mediated gene transfer of IL-2 as an effective immunotherapy when used adjuvantly with the tk "suicide gene".

Development of biodegradable Zn-1X binary alloys with nutrient alloying elements Mg, Ca and Sr.

Biodegradable metals have attracted considerable attentions in recent years. Besides the early launched biodegradable Mg and Fe metals, Zn, an essential element with osteogenic potential of human body, is regarded and studied as a new kind of potential biodegradable metal quite recently. Unfortunately, pure Zn is soft, brittle and has low mechanical strength in the practice, which needs further improvement in order to meet the clinical requirements. On the other hand, the widely used industrial Zn-based alloys usually contain biotoxic elements (for instance, ZA series contain toxic Al elements up to 40 wt.%), which subsequently bring up biosafety concerns. In the present work, novel Zn-1X binary alloys, with the addition of nutrition elements Mg, Ca and Sr were designed (cast, rolled and extruded Zn-1Mg, Zn-1Ca and Zn-1Sr). Their microstructure and mechanical property, degradation and in vitro and in vivo biocompatibility were studied systematically. The results demonstrated that the Zn-1X (Mg, Ca and Sr) alloys have profoundly modified the mechanical properties and biocompatibility of pure Zn. Zn-1X (Mg, Ca and Sr) alloys showed great potential for use in a new generation of biodegradable implants, opening up a new avenue in the area of biodegradable metals.

Electron-capture gas chromatographic determination of cyanide, iodide, nitrite, sulfide, and thiocyanate anions by phase-transfer-catalyzed derivatization with pentafluorobenzyl bromide.

A sensitive gas chromatographic method has been established for the simultaneous determination of biologically active inorganic anions, including cyanide, iodide, nitrite, sulfide, and thiocyanate anions as their volatile organic derivatives. The method is based on the formation of ion pairs from the anions and a complex cryptand and on the resulting neutral ion-pair partition to an organic phase for derivatization with pentafluorobenzyl bromide. Several parameters affecting the partition and derivatization of the anions were investigated. Individual and simultaneous determination of the anions can be achieved at sub-nmol levels with an electron-capture detector. Partial application of the method for the analysis of cyanide, nitrite, and thiocyanate in real samples proved satisfactory.

Determination of thiocyanate anion by high-performance liquid chromatography with fluorimetric detection.

A simple and sensitive high-performance liquid chromatographic (HPLC) method was established for the trace determination of thiocyanate anion as a fluorogenic derivative. The method is based on the chemical derivatization of thiocyanate anion with 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one. The resulting derivative was separated by a Nova-Pak C18 reversed-phase column. Optimization conditions for the derivatization of thiocyanate anion were investigated by HPLC with fluorimetric detection. The linear range for the quantitation of thiocyanate anion was 1-0.05 nmol in 0.1 mL of sample; the detection limit (with a signal-to-noise ratio of 5) of a 20-microL injected aliquot was approximately 3.3 +/- 1.2 fmol. Application of the method to the analysis of thiocyanate anion in saliva and plasma proved to be feasible.

Mandibular osteomyelitis caused by Blastoschizomyces capitatus in a child with acute myelogenous leukemia.

A 6-year-old girl with acute myelogenous leukemia (AML) developed fungal mandibular osteomyelitis during chemotherapy. Blastoschizomyces capitatus was recognized histologically by its yeast-like morphology and formation of annelloconidia, and was confirmed by culture. The fungal osteomyelitis of the mandible was treated successfully with prolonged antifungal medication, extensive surgical debridement and an oral care program, without interrupting leukemia chemotherapy. B. capitatus osteomyelitis of the mandible may occur during chemotherapy in AML patients with poor dental condition. Successful treatment can be achieved by careful management without interruption of antineoplastic chemotherapy.

Combined cleft and craniofacial team--multidisciplinary approach to cleft management.

A combined cleft palate and craniofacial was organized at Chang Gung Memorial Hospital, Taipei, in 1980. The team includes four plastic surgeons, four orthodontists, one prosthodontist, one otolaryngologist, one pediatrician, two speech pathologists, one social worker, one psychologist, two neurosurgeons, one ophthalmologist and a medical artist. Weekly combined clinics and meetings are held regularly to evaluate cleft lip/palate and all craniofacial cases. The team members set up treatment plans for each cleft palate and craniofacial patient, and follow up the patient's facial growth with critical evaluation of the result, complication, relapse and patient's or parent's satisfaction after surgery. Parents' support groups, summer speech camps and conferences between patients, parents and other public resources are held regularly. Longitudinal studies are strictly carried out on specific groups of cleft patients and major craniofacial patients.

An experimental study of osteogenesis in explants of rat dental pulp, periodontal ligament, bone marrow and muscle cells in vitro.

Cells of dental pulp, periodontal ligament, bone marrow and muscle derived from the same rat were cultured in vitro. Outgrowth of cells from these four explants were examined using phase-contrast microscope, light microscope and scanning electron microscope. Furthermore, cell lengths were measured randomly from SEM photographs and growth curves were plotted by calculating cell numbers. These cells had different morphology in both shape and length. The bone marrow cells showed the most rapid proliferation rate followed by muscle, periodontal ligament and most slowly in dental pulp cells.

Induction of cartilage and bone formation by cells from explants of various oral tissues in vitro.

Cells with both osteogenic and chondrogenic capabilities from various connective tissues, dental pulp, periodontal ligament, bone marrow, muscle and subcutaneous connective tissues of the rat were investigated by using either mineralized or demineralized bone and dentin matrix in vitro. After 28 days of the cell culture, explant cells were examined histologically. Bone and cartilage formations were assessed by counting their numbers in serial sections. Induction of cartilage was occurred only in muscle. New bone formation was occurred most frequently in dental pulp, followed in order by bone marrow and periodontal ligament. These results suggest that osteogenic cells, dental pulp, bone marrow stromal cells, and periodontal ligament can not response to inductive stimuli such as the bone morphogenetic protein, BMP.

Osteogenic activity of cells from dental pulp, periodontal ligament, bone marrow and muscle in vitro: an ultrastructural study and alkaline-phosphatase activity.

Dental pulp, periodontal ligament, bone marrow and muscle tissue from the same rat were cultured in vitro in order to investigate their osteogenic activity by transmission electron microscope. Immunohistochemical methods with various antibodies were utilized and alkaline-phosphatase (ALPase) activities of these cells were also measured biochemically. Dental pulp cells were stellate in shape, showed an intense ALPase reaction, and had lipid-like droplets. Periodontal ligament cells were composed of spindle fibroblasts and epithelial cells. The former revealed a positive reaction for ALPase and possessed microfilaments. Bone marrow cells were spindle shaped, resembling fibroblasts, but some of them were similar to osteoblasts. Muscle cells were long, slender in shape, and showed no positive reaction for ALPase. The cells from pulp tissue showed the highest activity of ALPase, followed by periodontal ligament and bone marrow; there was no activity in muscle tissue. All the cells except the epithelial-like cells of the periodontal ligament and muscle cells were positive in reaction with ALPase which is a marker for osteogenic cells, and vimentin which is a marker of fibroblastic characteristics. Osteogenic activity and cellular differentiation of these cells were discussed.

Ameloblastoma and its relationship to ameloblastic fibroma: their histogenesis based on an unusual case and review of the literature.

The present paper describes the relationship between ameloblastoma and ameloblastic fibroma deduced from a case diagnosed as "ameloblastoma combined with ameloblastic fibroma" arising in the mandible of a 5-year-old boy. Histologically, the tumor consisted of ameloblastoma in the central area and ameloblastic fibroma in the peripheral area; it clinically fits the characteristics of ameloblastic fibroma based on predominant age, manner of growth, and encapsulation. We reviewed the literature and discussed the relationship between ameloblastoma a ameloblastic fibroma in terms of tumorigenesis. It is assumed that ameloblastic fibroma can also be transformed into ameloblastoma, if the succeeding hard tissues are not formed, and the collagenous connective tissue substituting for the stromal mesenchymal tissue is formed by the inductive effect of the epithelial strands or other unknown factors. Several possibilities relative to the pathogenesis of ameloblastoma have been proposed by oral pathologists; however, to our knowledge, "ameloblastic fibroma can be transformed into ameloblastoma" has not hitherto been reported. The case we experienced here may be thought as an intermediate tumor pattern between ameloblastic fibroma and ameloblastoma.

PLGA/TCP composite scaffold incorporating bioactive phytomolecule icaritin for enhancement of bone defect repair in rabbits.

Bone defect repair is challenging in orthopaedic clinics. For treatment of large bone defects, bone grafting remains the method of choice for the majority of surgeons, as it fills spaces and provides support to enhance biological bone repair. As therapeutic agents are desirable for enhancing bone healing, this study was designed to develop such a bioactive composite scaffold (PLGA/TCP/ICT) made of polylactide-co-glycolide (PLGA) and tricalcium phosphate (TCP) as a basic carrier, incorporating a phytomolecule icaritin (ICT), i.e., a novel osteogenic exogenous growth factor. PLGA/TCP/ICT scaffolds were fabricated as PLGA/TCP (control group) and PLGA/TCP in tandem with low/mid/high-dose ICT (LICT/MICT/HICT groups, respectively). To evaluate the in vivo osteogenic and angiogenic potentials of these bioactive scaffolds with slow release of osteogenic ICT, the authors established a 12 mm ulnar bone defect model in rabbits. X-ray and high-resolution peripheral quantitative computed tomography results at weeks 2, 4 and 8 post-surgery showed more newly formed bone within bone defects implanted with PLGA/TCP/ICT scaffolds, especially PLGA/TCP/MICT scaffold. Histological results at weeks 4 and 8 also demonstrated more newly mineralized bone in PLGA/TCP/ICT groups, especially in the PLGA/TCP/MICT group, with correspondingly more new vessel ingrowth. These findings may form a good foundation for potential clinical validation of this innovative bioactive scaffold incorporated with the proper amount of osteopromotive phytomolecule ICT as a ready product for clinical applications.

Comparative study of osteogenic potential of a composite scaffold incorporating either endogenous bone morphogenetic protein-2 or exogenous phytomolecule icaritin: an in vitro efficacy study.

A local delivery system with sustained and efficient release of therapeutic agents from an appropriate carrier is desirable for orthopedic applications. Novel composite scaffolds made of poly (lactic-co-glycolic acid) with tricalcium phosphate (PLGA/TCP) were fabricated by an advanced low-temperature rapid prototyping technique, which incorporated either endogenous bone morphogenetic protein-2 (BMP-2) (PLGA/TCP/BMP-2) or phytomolecule icaritin (ICT) (PLGA/TCP/ICT) at low, middle and high doses. PLGA/TCP served as control. In vitro degradation, osteogenesis and release tests showed statistical differences among PLGA/TCP/ICT, PLGA/TCP and PLGA/TCP/BMP-2 groups, where PLGA/TCP/ICT had the desired slow release of bioactive icaritin in a dose-dependent manner, whereas there was almost no BMP-2 release from the PLGA/TCP/BMP-2 scaffolds. PLGA/TCP/ICT significantly increased more ALP activity, upregulated mRNA expression of osteogenic genes and enhanced calcium deposition and mineralization in rabbit bone marrow stem cells cultured on scaffolds compared with the other two groups. These results indicate the desired degradation rate, osteogenic capability and release property in PLGA/TCP/ICT composite scaffold, as icaritin preserved its bioactivity and structure after incorporation, while PLGA/TCP/BMP-2 did not show an initially expected osteogenic potential, owing to loss of the original bioactivity of BMP-2 during its incorporation and fabrication procedure. The results suggest that PLGA/TCP composite scaffolds incorporating osteogenic ICT might be a promising approach for bone tissue bioengineering and regeneration.

Analysis of DNA fragments by microchip electrophoresis fabricated on poly(methyl methacrylate) substrates using a wire-imprinting method.

Microfluidic devices were fabricated on poly(methyl methacrylate) (PMMA) substrate using two small-diameter (79 microm) wires to create a cross impression in plastics softened by low-temperature heating. The resulting channels had a rounded shape and 75 microm in depth. The variability of the channel dimensions was found to be less than 6% from different locations of the same channel and less than 10% between chips. Moreover, the fabricated PMMA chip appeared to sustain an electric field strength up to 300 V/cm without significant Joule heating. The function of resulting devices for electrophoretic injection and separation of a DNA size marker, HaeIII digest of (phiX174, was also characterized. Results indicated that all of the 11 DNA fragments of the size marker could be identified in less than 3 min with relative standard deviations less than 0.4% and 8% for migration time and peak area, respectively. Moreover, with the use of near infrared (IR) dye, fluorescence signals of the higher molecular weight fragments (> 603 bp in length) could be detected at total DNA concentrations as low as 0.1 microg/mL (S/N = 4.2). In conclusion, the performance of wire-imprinted devices on PMMA substrate were comparable to those fabricated by other professional means.

Polymer solution-filled column for the analysis of antisense phosphorothioates by capillary electrophoresis.

A capillary electrophoresis (CE) column filled with 13% poly(ethylene glycol) (PEG) solution is demonstrated to resolve different lengths of antisense phosphorothioates in 100 mM Tris-borate (pH 9.0) buffer containing 30% formamide at 50 degrees C. Two sets of mixtures composed of 15-20 mers of either antisense phosphorothioate or phosphodiester oligonucleotides were synthesized based on a sequence of the antisense orientation directed against DNA-methyltransferase (denoted as MT-AS) and were used as model compounds. It was found that column coating reduced electroosmotic flow, as well as wall adsorption, and led to the separation of both phosphorothioate and phosphodiester molecules. Substantial peak broadening, however, specifically occurred to the phosphorothioates and was reduced statisfactorily by the addition of formamide into the buffer solution, raising the temperature, and raising the pH value. Under experimental conditions, a linear relationship between the migration time and the base number was observed, indicating that no peak compression artifacts existed. Without tedious pretreatment, antisense phosphorothioates were spiked into human serum, followed by water dilution, and then directly injected into the column. Separation of different lengths of phosphorothioates was observed using pressure injection, which did not suffer from injection bias.

Treatment of platelet alloimmunization with intravenous immunoglobulin in a child with aplastic anemia.

A 9-year-old girl with severe refractory aplastic anemia had become refractory to platelet transfusion due to platelet alloimmunization. As a result, a huge right parieto-temporo-occipital cerebral hematoma and a subarachnoid hemorrhage subsequently occurred. Intravenous immunoglobulin (IVIG) (Green Cross, Taiwan) at a dose of 0.4 g/kg/day for 5 days, followed by another 3 doses during the following 5 days, and combined with large-dose platelet transfusions effectively controlled the bleeding by elevating the platelet count to above 100 x 10(3)/mm3. The patient soon recovered with only a mild sequela. Two months later, because of multiple caries and a periodontal abscess, dental extraction needed to be performed. IVIG (0.4 g/kg/day x 6 days) enabled surgery to proceed by elevating the platelet count to above 55 x 10(3)/mm3, and no bleeding complications occurred during or after the procedure. The potential benefit of high-dose IVIG in modulating platelet alloimmunization is a result of increasing the survival of transfused platelets. Thus, this therapy is recommended when patients with platelet alloimmunization have critical bleeding episodes or undergo surgical procedures.

Determination of thiocyanate anion as an organic derivative by gas chromatography.

A simple and sensitive gas chromatographic method has been established for the determination of biologically active thiocyanate anion as pentafluorobenzyl thiocyanate. The method is based on the partition of an ion-pair from the thiocyanate anion and a complex crypstand to a benzene layer for derivatization with pentafluorobenzyl bromide. The resulting derivative was analyzed by gas chromatography with electron capture detection. The quantitation range for thiocyanate anion was over 0.086 and 3.45 nmol. The detection limit of thiocyanate in 0.2 ml sample is about 34 pmol. The effects of several parameters such as base or acid, reaction time and the amount of pentafluorobenzyl bromide were evaluated for the optimization of partition and derivatization of thiocyanate anion. Application of the method to the analysis of thiocyanate in waste water and human saliva proved to be feasible.