Photothermal stress triggered by near-infrared-irradiated carbon nanotubes up-regulates osteogenesis and mineral deposition in tooth-extracted sockets.

PURPOSE: The bone regenerative healing process is often prolonged, with a high risk of infection particularly in elderly and diseased patients. A reduction in healing process time usually requires mechanical stress devices, chemical cues, or laser/thermal therapies. Although these approaches have been used extensively for the reduction of bone healing time, the exact mechanisms involved in thermal stress-induced bone regeneration remain unclear. METHODS: Photothermal stress (PTS) stimulation was carried out using a novel photothermal device, composed of an alginate gel (AG) including carbon nanotubes (CNT-AGs) and their irradiator with near-infrared (NIR) light. We investigated the effects of optimal hyperthermia on osteogenesis, its signalling pathway in vitro and mineral deposition in tooth-extracted sockets in vivo. RESULTS: The PTS (10 min at 42 °C, every day), triggered by NIR-induced CNT, increased the activity of alkaline phosphatase (ALP) in mouse osteoblast MC3T3-E1 cells in a time-dependent manner compared with the non-thermal stress control. PTS significantly induced the expression of osteogenic-related molecules such as ALP, RUNX2 and Osterix in a time-dependent manner with phosphorylated mitogen-activated protein kinases (MAPK). PTS increased the expression of heat shock factor (HSF) 2, but not HSF1, resulting in activation of heat shock protein 27. PTS significantly up-regulated mineral deposition in tooth-extracted sockets in normal and ovariectomised osteoporotic model mice in vivo. CONCLUSIONS: Our novel CNT-based PTS up-regulated osteogenesis via activation of heat shock-related molecules, resulting in promotion of mineral deposition in enhanced tooth-extracted sockets.

Initial clinical trial of a novel hemostat, TDM-621, in the endoscopic treatments of the gastric tumors.

BACKGROUND AND AIM: The feasibility of TDM-621, the synthetic infectious agent-free peptides, was tested in hemostasis of the bleeding after endoscopic treatments of the gastric tumors. METHODS: The patients who underwent endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) were enrolled in the present study. The subject of hemostasis was the oozing after the EMR or ESD. The hemostatic effect, the secondary hemorrhage from one postoperative day to the day before discharge and operability were studied. RESULTS: The hemostatic effects were assessed in 12 patients. It was "remarkably effective" in 11 patients and "effective" in 1 patient. The operability was "very easy" in two patients, "easy" in eight patients and "acceptable" in two patients. No secondary hemorrhage was observed in all of 12 patients. No adverse effect considered to be related to TDM-621 was observed. CONCLUSION: It was shown that hemostasis using TDM-621 was feasible after endoscopic treatments of the gastric tumors without any technical trouble or adverse event.

Risk factors for colonic diverticular hemorrhage: Japanese multicenter study.

BACKGROUND AND AIM: Diverticular hemorrhage is the common cause of lower gastrointestinal bleeding, and its incidence has been increasing in Japan. However, the exact cause of diverticular hemorrhage is not well understood. We investigated the risk factors for diverticular hemorrhage. METHODS: We selected 103 patients with diverticular hemorrhage as cases and patients with colonic diverticulosis without a history of bleeding were selected as control subjects, exactly matched for age and gender. We collected the data from the medical records of each of the patients, such as those related to the comorbidities, medications and findings of colonoscopy, and conducted a matched case-control study to analyze the risk factors for diverticular hemorrhage. RESULTS: Both groups were composed of 75 men and 28 women. The median age of the patients in both groups was 72.0 years (47.0-87.0). The body weight (p = 0.0065), body mass index (p = 0.006), prevalence of hypertension (p = 0.0242), prevalence of ischemic heart disease (p = 0.0015), and frequency of use of low-dose aspirin (p = 0.042) were significantly different between the two groups. The percentage of patients with bilateral diverticula, that is, diverticula on both the right and left hemicolon, was significantly higher in the diverticular hemorrhage group (p = 0.0011). Multiple regression analysis identified only the diverticular location as being significantly associated with the risk of diverticular hemorrhage (p = 0.0021). CONCLUSIONS: Only the diverticular location (bilateral) was found to be an independent risk factor for diverticular hemorrhage.

Clinical characterization of gastric lesions initially diagnosed as low-grade adenomas on forceps biopsy.

AIM: The aim of this study was to elucidate characteristics of gastric lesions that are initially diagnosed as low-grade adenomas and to establish appropriate treatment. METHODS: We retrospectively reviewed 231 lesions initially diagnosed as gastric adenomas. All forceps biopsy samples were histologically diagnosed as category 3 low-grade adenomas according to the revised Vienna Classification. All patients underwent endoscopic resection with endoscopic findings and post-resection diagnoses evaluated subsequently. RESULTS: Sixty-three lesions were initially diagnosed as depressed adenomas, and 168 lesions were diagnosed as protruding adenomas. The depressed lesions were significantly smaller (11.6 ± 5.0 mm) than the protruding lesions (17.0 ± 10.8 mm) (P < 0.001). Diagnoses reclassified to category 4 mucosal high-grade neoplasia (i.e. high-grade adenoma, adenocarcinoma in adenoma and adenocarcinoma) were more frequent among depressed lesions (52.4%) than among protruding lesions (31.0%) (P = 0.004). Multivariate analysis of all 231 lesions showed that lesion size larger than 20 mm (P < 0.001) and depressed appearance (including central depression) (P < 0.001) were significant independent factors suggesting cancer. For the 168 protruding lesions, lesion size larger than 20 mm (P < 0.001) and central depression (P < 0.001) were significant independent factors suggesting cancer. For the 63 depressed lesions, lesion size larger than 15 mm (P = 0.016) and a moth-eaten appearance (P = 0.017) were significant independent factors in the pre-treatment diagnosis of cancer. CONCLUSIONS: Adenocarcinoma lesions were often found in depressed lesions and protruding lesions with central depression. Endoscopic resection for total biopsy is recommended, even if forceps biopsy indicates low-grade adenoma, as pre-treatment biopsy may be inadequate for an accurate histological diagnosis.

Antibody-Conjugated Gel-Coated Single-Walled Carbon Nanotubes as Photothermal Agents.

Photothermal therapy (PTT) using near-infrared (NIR) light is an attractive treatment modality for cancer, in which photothermal agents absorb energy from photons and convert it into thermal energy to lead to cancer cell death. Among the various organic and inorganic materials, single-walled carbon nanotubes (SWCNTs) are promising candidates for NIR photothermal agents due to their strong absorption in this region as well as their high photothermal conversion efficiency. In the development of the SWCNT-based PTT materials, modifications of SWCNTs to offer a stable dispersion for biocompatibility as well as to target the tumor of choice while maintaining their NIR absorption have been required. While modification of SWCNTs through noncovalent methods can be achieved, these modifications can be easily reversed in the body. Contrarily, modifications through covalent attachments, while more desirable, may compromise the NIR absorption characteristics of the SWCNTs. Previously, we reported the development of a synthetic strategy to coat SWCNTs with a cross-linked polymer (i.e., a gel) through a process called CNT Micelle Polymerization and successfully introduced maleimide groups that allowed for postmodification through the ene-thiol reaction without deteriorating the NIR absorption. In this report, we postmodify thiol-containing antibodies (anti-TRP-1, a melanoma specific protein) using maleimide chemistry and find that the SWCNTs conjugated with anti-TRP-1 maintain the characteristic NIR absorption as SWCNTs with dispersion stability. It is estimated that 50 maleimide groups are incorporated in one SWCNT (ca. 280 nm long) and they are modified with 32 TRP-1 fragments. Finally, we successfully use these targeted SWCNTs for the PTT of the melanoma cell line using NIR light (1064 nm; 2 W, 5 min). Our method can be extended to a vast array of specific antibodies as well as other targeting agents.

Drug binding and releasing characteristics of DNA/lipid/PLGA film.

We evaluated the blended compound of DNA/lipid complexes and PLGA (poly(D,L-lactide-co-glycolide)) as a carrier material for drug delivery system (DDS). Transparent, self-standing DNA/lipid/PLGA films were prepared by casting from an organic solvent such as DMSO/chloroform. Daunorubicin hydrochloride (DH) could intercalate and groove bind into DNA in the films, whereby the amount of DH bound to the films was controlled by the latter's immersion period in DH aqueous solution. DH was released from DH films after immersion in PBS solution, whereby release rate was dependent on the chemical structure of lipids. Released DH caused reduction of cell viability during the cell culture of L929 mouse fibroblasts. These results suggested that DNA/lipid/PLGA film was a promising useful material for DDS.

Degradation of methacrylate monomers in human saliva.

This study assessed the effect of the molecular structure of newly synthesized methacrylate monomers on their chemical stability in human saliva, whereby these monomers can be used as dental composite resins. Six model monomethacrylates and two urethane-modified BisGMA monomers were added to human saliva, and their change in concentration after 24, 48, and 72 hours were measured by high-performance liquid chromatography. Degradation of the six model monomethacrylate monomers was found to be influenced by the molecular structure, such as steric hindrance and presence of urethane bond in chemical backbone. Based on the degradation test results of these six monomers, urethane-modified BisGMA derivatives--in which the hydroxyl groups in original BisGMA monomer were substituted with alkyl isocyanate--were synthesized and subjected to degradation test. The urethane-modified BisGMA monomers showed a particular resistance to salivary hydrolysis. Results of this study thus suggested that urethane groups should be considered when designing new monomers for dental composite systems as they demonstrated improved resistance to hydrolysis.

Preparation of carbon nanotube-alginate nanocomposite gel for tissue engineering.

A novel scaffold material based on an alginate hydrogel which contained carbon nanotubes (CNTs) was prepared, and its mechanical property and biocompatibility evaluated. Soluble CNTs were prepared with acid treatment and dispersed in sodium alginate solution as a cross-linker. After which, the mechanical property (elastic deformation), saline sorption, histological reaction, and cell viability of the resultant nanocomposite gel (CNT-Alg gel) were evaluated. The CNT-Alg gel showed faster gelling and higher mechanical strength than the conventional alginate gel. Saline sorption amount of freeze-dried CNT-Alg gel was equal to that of the alginate gel. In terms of histological evaluation and cell viability assay, CNT-Alg gel exhibited a mild inflammatory response and non-cytotoxicity. These results thus suggested that CNT-Alg gel could be useful as a scaffold material in tissue engineering with the sidewalls of CNTs acting as active sites for chemical functionalization.

[The effects of a respiratory education class on psychological status for patients with chronic respiratory disease].

AIM: To educate patients with chronic respiratory disease about respiratory diseases and pulmonary rehabilitation, a respiratory education class was held for patients with chronic respiratory diseases and their families. In this study, we evaluated the psychological effects of the class. METHODS: The respiratory education class was held twice during the 2-year period (2002-2004) while the physician specializing in geriatric and respiratory diseases worked at the hospital. Before, during, and one year after the class, we investigated the mental and physical condition in 24 chronic respiratory patients (aged 72.7 +/- 1.3 years) using the State-Trait Anxiety Inventory (STAI) to measure anxiety, the Self-Rating Depression Scale (SDS) to evaluate depressive state, and the General Self-Efficacy Scale (GSES) to evaluate self-efficacy. RESULTS: Before, during, and one year after the class, the mean STAI scores were 39.2 +/- 2.5, 40.0 +/- 2.5, and 39.4 +/- 2.7, the mean SDS scores were 37.6 +/- 22, 37.0 +/- 2.0, and 38.0 +/- 2.0, and the mean GSES scores were 7.4 +/- 0.7, 8.0 +/- 0.8, and 8.6 +/- 0.8, respectively. No statistically significant difference was observed for scores on the STAI, SDS between the time points. The score during the class significantly (p < 0.05) increased compared to the score before the class, and remained significantly increased one year after the class on the GSES. CONCLUSION: These results suggest that conducting a respiratory education class might be beneficial for patients with chronic respiratory disease.

PBS buffer solutions with different pH values can change porosity of DNA-chitosan complexes.

We examined the effect of phosphate-buffered saline (PBS) solution with different pH values on the formation of porosity in the DNA/chitosan complexes, and evaluated the intercalation behavior of the complexes. Four different PBS solutions with pH = 6.0, 7.0, 7.4, and 7.8 were used for rinsing water-insoluble DNA-chitosan complexes. All complexes showed high porosities ranging from 83 to 95%. Rinsing with PBS at pH 7.0, 7.4, and 7.8 reduced the porosity of the DNA-chitosan complexes. Re-rinsing with PBS at pH 7.4 reduced the porosity of the DNA-chitosan complex rinsed with PBS at pH 6.0. The appearances for porous formation were influenced by the differences in pH of PBS. Daunorubicin hydrochloride intercalated and bound in the grooves of DNA within all of the DNA-chitosan complexes, indicating that DNA in the complexes maintained its double-stranded helical structure. These results suggested that PBS-rinsed DNA-chitosan complex is promising as a scaffold material in tissue engineering.

Estimated molecular structure of a carbon nanotube molecular heater based on binding properties to a target protein.

BACKGROUND: Carbon nanotubes exhibit strong absorbance in the near-infrared (NIR) region and are considered as potent candidates for hyperthermic therapy because they generate significant amounts of heat upon excitation with NIR light. We prepared a single-walled carbon nanotube (SWNT)/IgG complex to use as a "smart molecular heater" for hyperthermic therapy. PURPOSE: The aim of the present study was to assess the binding efficiency of DNA-functionalized SWNT/IgG complexes to a target protein. METHODS: 3 types of complexes with different lengths of spacer arm chain (13.5, 29, and 56 Å) linked to biotinylated IgG were prepared, and we evaluated the effect of the spacer arm length on the specificity, affinity, and capacity of binding to a target protein. RESULTS: Complexes with longer spacer lengths showed increased binding affinity to a target protein. This could be due to a reduction in steric hindrance by increasing the segmental flexibility of the spacer arm. CONCLUSIONS: The results of this study suggested that DNA-functionalized SWNT/IgG complexes could act as a heating nano-device for hyperthermic cancer therapy, and the complexes can bind various types of tumor by modifiying the specific antibody.

Photothermal stress triggered by near infrared-irradiated carbon nanotubes promotes bone deposition in rat calvarial defects.

The bone regenerative healing process is often prolonged, with a high risk of infection particularly in elderly and diseased patients. A reduction in healing process time usually requires mechanical stress devices, chemical cues, or laser/thermal therapies. Although these approaches have been used extensively for the reduction of bone healing time, the exact mechanisms involved in thermal stress-induced bone regeneration remain unclear. In this study, we investigated the effect of optimal hyperthermia on rat calvarial defects in vivo and on osteogenesis in vitro. Photothermal stress stimulation was carried out using a new photothermal device, composed of an alginate gel including in carbon nanotubes and their irradiator with near-infrared light. Photothermal stress (15 min at 42℃, every day), trigged by near-infrared-induced carbon nanotube, promoted bone deposition in critical-sized calvarial defects compared with nonthermal stress controls. We recently reported that our novel DNA/protamine complex scaffold induces bone regeneration in calvarial defects. In this study, photothermal stress upregulated bone deposition in DNA/protamine-engrafted calvarial defects. Furthermore, photothermal stress significantly induced expression of osteogenic related genes in a time-dependent manner, including alkaline phosphatase, osterix, and osteocalcin. This was observed in DNA/protamine cells, which were expanded from regenerated tissue engrafted into the DNA/protamine scaffold, as well as in human MG63 preosteoblasts. In summary, this novel carbon nanotube-based photothermal stress approach upregulated expression of osteogenic-related genes in preosteoblasts, resulting in promotion of mineral deposition for enhanced bone repair.

Estrogenic activity of tissue conditioners in vitro.

OBJECTIVES: In order to assess the estrogenic activities of plasticizers used in tissue conditioners and four commercial tissue conditioners, we carried out in vitro tests. METHODS: Seven plasticizers and two metabolites were diluted to concentrations ranging from 10(-9) to 10(-4)M. Four commercial tissue conditioners were also diluted to concentrations ranging from 2 x 10(-8) to 2 x 10(-3)g/ml. Estrogenic activities were tested by the E-screen test using MCF-7 cells. When estrogen is present, the cells proliferate. Instead of counting the cells or nuclei directly, cell numbers were assessed by measurement of the total protein content using the sulforhodamine B assay. The liquid compositions of the four commercial tissue conditioners were examined by high-performance liquid chromatography. RESULTS: n-Butyl benzyl phthalate, dibutyl phthalate, n-butyl phthalyl n-butyl glycolate, di-2-ethylhexyl phthalate and benzyl salicylate significantly increased proliferation of MCF-7 cells. The remaining two plasticizers, di-2-ethylhexyl adipate and benzyl benzoate, as well as two metabolites of dibutyl phthalate and di-2-ethylhexyl phthalate, i.e. monobutyl phthalate and mono-2-ethylhexyl phthalate, respectively, did not increase proliferation of MCF-7 cells at the concentrations tested. Four commercial tissue conditioners, Coe comfort (CC), Tissue Conditioner (TC), Hydro Cast (HC) and Denture Soft (DS) II, significantly increased proliferation of MCF-7 cells. HPLC data revealed the commercial products contained plasticizers: benzyl benzoate and dibutyl phthalate in CC, dibutyl phthalate in TC, n-butyl benzyl phthalate in HC and n-butyl phthalyl n-butyl glycolate in DS II. SIGNIFICANCE: Except for benzyl benzoate and di-2-ethylhexyl adipate, the plasticizers tested showed estrogenic activity. The four commercial tissue conditioners tested also showed estrogenic activity, and HC showed especially strong estrogenicity.

[The effect of woven glass fiber reinforced composites on the transverse strength of three denture base resins].

PURPOSE: This study evaluated the effect of woven glass fiber reinforced composite (FRC) on transverse strength of denture base materials. METHODS: Three denture base resins (heat-activated resin, autopolymerizing resin and microwave-activated resin) and FRC (Vectris Frame, Ivoclar-Vivadent) were selected for the study. The FRC was light-cured and sandwiched in the middle of denture base resin and then the specimen was polymerized according to manufacturers' instructions in a gypsum mold with a cavity of 65 mm x 10 mm x 2.5 mm. Each specimen was immersed in 37 degrees C distilled water for 50 hours. The control group was without FRC. Ten specimens were fabricated for each group. The transverse strength test was performed on a universal testing machine at a crosshead speed of 5 mm/min. RESULTS: The transverse strengths of all denture base resins reinforced with FRC were significantly higher than that of denture base resin bulk. CONCLUSIONS: Within the limitation of the current study, the FRC has an effect upon reinforcement of denture base resin.

Evaluation of bone formation guided by DNA/protamine complex with FGF-2 in an adult rat calvarial defect model.

DNA/protamine complex paste (D/P) and D/P complex paste with Fibroblast Growth Factor-2 (FGF-2) (D/P-FGF) were prepared to investigate their new bone formation abilities using an ∼40-week-old rat calvarial defect model. It was found that D/P could release FGF-2 proportionally in an in vitro experiment with an enzyme-linked immunosorbent assay. It was also found that aging adversely affected self-bone healing of rats by comparison with the results in a previous study using 10-week-old rats. Microcomputed tomography and histopathological examinations showed that new bone formation abilities of D/P and D/P-FGF were superior to that of the control (sham operation). Control, D/P and D/P-FGF showed newly formed bone areas of 6.7, 58.3, and 67.0%, respectively, 3 months after the operation. Moreover, it was found that FGF-2 could support the osteoanagenesis ability of D/P. It was considered that FGF-2 could play an important role in new bone formation at early stages because it induced the genes such as collagen I, CBFA, OSX, and OPN, which are initiated first in the process of osteogenesis. Therefore, D/P-FGF will be a useful injectable biomaterial with biodegradable properties for the repair of bone defects in the elderly.

Preparation and binding study of a complex made of DNA-treated single-walled carbon nanotubes and antibody for specific delivery of a "molecular heater" platform.

Carbon nanotubes have been explored as heat-delivery vehicles for thermal ablation of tumors. To use single-walled carbon nanotubes (SWNT) as a "molecular heater" for hyperthermia therapy in cancer, stable dispersibility and smart-delivery potential will be needed, as well as lack of toxicity. This paper reports the preparation of a model complex comprising DNA-treated SWNT and anti-human IgG antibody and the specific binding ability of this model complex with the targeted protein, ie, human IgG. Treatment with double-stranded DNA enabled stable dispersibility of a complex composed of SWNT and the antibody under physiological conditions. Quartz crystal microbalance results suggest that there was one immobilized IgG molecule to every 21,700 carbon atoms in the complex containing DNA-treated SWNT and the antibody. The DNA-SWNT antibody complex showed good selectivity for binding to the targeted protein. Binding analysis revealed that treatment with DNA did not interfere with binding affinity or capacity between the immobilized antibody and the targeted protein. The results of this study demonstrate that the DNA-SWNT antibody complex is a useful tool for use as a smart "molecular heater" platform applicable to various types of antibodies targeting a specific antigen.

Dispersion stability and exothermic properties of DNA-functionalized single-walled carbon nanotubes.

Carbon nanotubes act as a photon antenna that serves as an effective "molecular heater" around the near-infrared (NIR) region. This exothermic generation can be used as a possible heating source for hyperthermia therapy. The current study reports the dispersible and exothermic properties with NIR irradiation for single-walled carbon nanotubes (SWNTs) treated with a strong acid (acid-treated SWNTs), and the SWNTs further functionalized with double-stranded DNA (DNA-functionalized SWNTs: DNA-SWNTs). DNA-SWNTs significantly improved the dispersibility of SWNTs when compared with the acid-treated SWNTs. The binding ratio of the acid-treated SWNT and DNA was calculated to be 3.1 (DNA/SWNTs) from the phosphorous content in the DNA-SWNT. This interaction of the SWNTs and DNA would contribute to the stable dispersion of the DNA-SWNTs in a culture medium. With NIR irradiation by a halogen lamp light source, the acid-treated SWNTs and the DNA-SWNTs showed strong heat evolution in vitro (in a culture medium) and in vivo (in the subcutaneous tissue of a mouse) condition without any invasive effect on the non-SWNT area. The results of this study suggested that the functionalization with DNA was an efficient approach to improve the dispersibility of SWNTs in body fluids, and the DNA-SWNT would be a promising source for photo-induced exothermic generation.

Polycationic protamine for water-insoluble complex formation with DNA.

The DNA/protamine complex was prepared by a reaction between DNA and protamine sulfate solutions with stirring, and its cell viability, antibacterial effect and histopathological responses were examined. A water-insoluble white powder, DNA/protamine complex, with a porous structure was obtained. The molar binding ratio of the complex prepared from a solution containing equal amounts of DNA and protamine sulfate by weight was 0.038 and the efficiency of complex formation was 61%. In a cell culture test using MC-3T3-E1 mouse osteoblast cells, the complex showed less cytotoxicity than protamine sulfate alone and cell viabilities were more than 98%. A porous disk could be prepared easily and showed an antibacterial effect against Staphyrococcus aureus, Porphyromonas gingivalis and Prevotella intermedia in an antibacterial sensitivity test and a mild tissue response in vivo test. These results suggested that the DNA/protamine complex could be a useful biodegradable biomaterial with antibacterial effects.

Mold fabrication and biological assessment of porous DNA-chitosan complexes.

A previous study revealed that DNA-chitosan complex prepared from the reaction between native DNA and chitosan in aqueous solution has suitable porosity for cell seeding, is nontoxic, and causes only a mild soft-tissue response. This simple and easy fabrication method for porous DNA-chitosan complex provides for a wide variety of applications as a scaffold material. The present study evaluated whether rinsing with PBS solution can fabricate DNA-chitosan complex in a mold and the histopathological responses of rat soft tissues to fabricated DNA-chitosan complexes. DNA-chitosan complex paste was prepared by mixing distilled water and freeze-dried water-rinsed DNA-chitosan complex powder. A DNA-chitosan complex disk could be fabricated by rinsing with PBS buffer and subsequently freeze-drying the DNA-chitosan complex paste in the mold. Thus, a wide range of applications of DNA-chitosan complex for tissue engineering can be anticipated using the present easy fabrication method. The porosity of the disk was 85%, and many pores were visible in the DNA-chitosan complex (before fabrication) and in the fabricated DNA-chitosan disk. The values of the complex disks gradually reduced in the tissues although 60% of disks remained in the tissues. In conclusion, an easy fabrication method for making porous DNA-chitosan complex disks was developed. It was found that the fabrication method can delay the biodegradation of the DNA-chitosan complex disk without serious tissue responses in vivo. DNA-chitosan complex is promising as a scaffold material, and a wide range of applications of DNA-chitosan complex for tissue engineering are anticipated.