Preparation of thermally stable nanocrystalline hydroxyapatite by hydrothermal method.

Thermally stable hydroxyapatite (HAp) was synthesized by hydrothermal method in the presence of malic acid. X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FT-IR), Raman spectroscopy, scanning electron microscopy (SEM), differential thermal analysis (DTA), thermogravimetric analysis (TGA) was done on the synthesized powders. These analyses confirmed the sample to be free from impurities and other phases of calcium phosphates, and were of rhombus morphology along with nanosized particles. IR and Raman analyses indicated the adsorption of malic acid on HAp. Thermal stability of the synthesized HAp was confirmed by DTA and TGA. The synthesized powders were thermally stable upto 1,400 degrees C and showed no phase change. The proposed method might be useful for producing thermally stable HAp which is a necessity for high temperature coating applications.

Establishment of glucagon-producing cells by cell hybridization.

Glucagon-producing cell lines were established by fusing pancreatic islet cells of adult hamster and 6-thioguanine-resistant hamster insulinoma cells. Under phase-contrast microscopy, the morphology of cultured hybrid cells was intermediate between those of the parental cells. The hybrid cells contained A-like granules, though few in number, and were stained with anti-glucagon antibody. The mode of chromosome number decreased to 78 or 79 by 3 mo after hybridization in comparison with the expected chromosome number of the heterokaryon of 104, and showed a minute decrease in 4 of 6 cell lines after 6 mo. The population doubling time ranged from 24 to 38 h, while that of parental insulinoma cells was 22.8 h. There was no correlation between the expression of cellular function and the stability of chromosome number or the length of population doubling time. The capacity of glucagon secretion was between that of the parental cells. The glucagon secreted into the medium, as assayed by the glucagon-specific antibody, was 0.6-2.5 ng/10(6) cells for 2 h, which was about 40% of total glucagon-like immunoreactivity secreted. Secretion of glucagon was not affected by high concentration of glucose, was markedly increased by theophylline, and was suppressed by exogenous insulin. All of the hybrid cells produced tumors on transplantation 6 mo after hybridization. The tumor-bearing hamsters exhibited high levels of plasma glucagon and blood glucose as well as a high level of serum insulin.

Association of polymorphism in the NeuroD/BETA2 gene with type 1 diabetes in the Japanese.

NeuroD/BETA2, a transcription factor of the insulin gene, also plays an important role in the development of pancreatic beta-cells. Recently, the NeuroD/BETA2 gene has been mapped to the long arm of human chromosome 2 (2q32) where the IDDM7 gene has previously been mapped, implying its involvement in diabetes. To identify mutations in the NeuroD/BETA2 gene that may predispose patients to develop diabetes, we studied the gene in 50 Japanese subjects with diabetes (4 with type 1 and 46 with type 2) by the polymerase chain reaction (PCR) followed by single-strand conformation polymorphism and sequencing analyses. Further analysis was performed in 392 Japanese subjects (60 with type 1 and 158 with type 2 diabetes and 174 healthy control subjects) by mismatch PCR restriction fragment length polymorphism. We found a DNA polymorphism of the NeuroD/BETA2 gene. A nucleotide G-to-A transition results in the substitution of alanine to threonine at codon 45 (Ala45Thr). The frequencies of heterozygotes for the Ala45Thr variant were 9.8% in the control subjects, 9.5% in the patients with type 2 diabetes, and 25.0% in the patients with type 1 diabetes, a significant difference (P = 0.006). Because the variant of the NeuroD/BETA2 gene (Ala45Thr) is associated with type 1 but not type 2 diabetes, it may be implicated in the loss of pancreatic beta-cells in type 1 diabetes.

In vitro calcium phosphate growth over surface modified PMMA film.

In vitro nucleation of calcium phosphate phase was studied over functionalized polymethyl methacrylate (PMMA) films using Fourier transform infrared spectroscopy, electron spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. PMMA films were prepared by dissolving commercial grade pellets in chloroform and cast into thin sheets. The films were immersed in a methanol solution of sodium hydroxide before treating with 1.5% solution of adenosine triphosphate (ATP) at a pH of 5.2 for 24 h. ATP treated films were then soaked in saturated lime solution for 4 days to initiate formation of calcium phosphate precursor phase over their surface. The above films immersed in simulated body fluid solution (1.5 x SBF) for more than 5 days led to the nucleation of apatitic calcium phosphate phase all over the film surface. The ATP coupled film not subjected to lime treatment did not show calcium phosphate nucleation behaviour upon immersion in SBF solution. The Ca/P ratio of the calcium phosphate phase increase with increase in soaking time in SBF solution.

Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method.

A porous calcium phosphate coating deposited on chitosan films was studied using scanning electron microscopy, energy-dispersive X-ray analysis, micro-Fourier transform infrared spectroscopy (micro-FTIR) and thin-film X-ray diffractometry (XRD). Chitosan films were first prepared by dissolving chitosan powder in dilute acetic acid and drying in a flat petri dish. The films were phosphorylated using urea and H3PO4 with the P content being 0.1-0.2 wt%. Phosphorylated films soaked in saturated Ca(OH)2 solution for 8 days led to the formation of a calcium phosphate precursor phase over the entire surface. This precursor phase stimulated the growth of a porous coating of calcium-deficient hydroxy apatite when immersed in 1.5 x SBF for more than 20 days. Phosphorylated films not treated with Ca(OH)2 did not show any calcium phosphate growth upon immersion in SBF solution. The precursor phase is thought to be octacalcium phosphate, which nucleates a HAP phase during SBF treatment. Initially, this treatment in SBF results in the formation of a single-layer calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a porous HAP coating. The Ca/P ratio of the HAP coating is a function of SBF immersion time.

Post-infantile giant cell hepatitis in an elderly female patient with systemic lupus erythematosus.

A 69-year-old Japanese female was admitted because of general fatigue. Laboratory data showed elevation of serum total bilirubin, transaminase, gamma-glutamyl transpeptidase, and creatinine levels. An immunological study revealed hypergammaglobulinemia, low titer of complement, and high titers of antinuclear antibody, anti-DNA antibody, and circulating immune complexes. Antibodies to parainfluenza virus 3 were positive. Histology of the liver disclosed numerous giant cell hepatocyte transformations with the lobular architecture being slightly distorted by portal inflammation and fibrosis. These findings led us to make a diagnosis of giant cell hepatitis associated with systemic lupus erythematosus. Prednisolone was effective in improving the anemia and the serum immunoglobulin, immune complex, and antinuclear antibody levels. The addition of cyclosporine to the initial corticosteroid therapy was also beneficial in decreasing the transaminase level and in improving liver histology. The patient died of acute pneumonitis and renal failure on the 166th day after admission. Parainfluenza virus 3 and autoimmune mechanisms were thus considered to be the causes of the giant cell hepatitis.

Growth and adhesion of osteoblast-like cells derived from neonatal rat calvaria on calcium phosphate ceramics.

The effects of biocompatible ceramics on the growth and adhesion of osteoblast-rich rat calvarial cell cultures were investigated. Osteoblast-like cells and mouse fibroblast-like L-929 cells were cultured on composite sinters of hydroxyapatite (HAP) and beta-tricalcium phosphate (TCP) culture carriers, whose Ca/P molar ratios were adjusted to values of 1.50, 1.55, 1.60, 1.64 and 1.67. The growth rates of both cell types were accelerated on the TCP-HAP ceramics as compared to those on polystyrene plastic (LUX) or bioinert zirconia ceramics. The population of osteoblast-like cells reached a density of 2.28 x 10(5) cells/cm2 on 100% HAP (Ca/P ratio 1.67) at 9 d of culture, while the corresponding cell density was 1.66 x 10(5) cells/cm2 on LUX and 1.26 x 10(5) cells/cm2 on zirconia. Adhesion of the osteoblast-like cells on TCP-HAP ceramics was similarly increased as compared with that on LUX or zirconia ceramics. The adhesion of L-929 cells on TCP-HAP ceramics was found to be weaker than that on cultures on LUX or zirconia ceramics. The time-dependent variations in the alkaline phosphatase activity of the osteoblast-like cells showed that the osteoblastic phenotype was potentiated by culturing the cells in calcium-rich media. The surface analyses of the Ca/P ratio and the microstructure by XRD and FTIR suggest that the Ca-rich surface was newly formed by recombination on the surface layer in the culture medium containing fetal bovine serum. These results suggest that the surface of TCP-HAP ceramics, especially that of 100% HAP ceramics, are effective for accelerating growth and differentiation of osteoblast-like cells. This is most probably due to the chemical and physical instability and composition of 100% HAP, which promote the formation of a Ca-rich layer at the cell-material interface and provision of Ca ions to the osteoblast-like cells.

Production of poly-beta-hydroxybutyric acid by microorganisms accumulated from river water using a two-stage perfusion culture system.

The perfusion culture system using a shaken ceramic membrane flask (SCMF) was employed to accumulate microorganisms separated from river water and to produce poly-beta-hydroxybutyric acid (PHB). Using a two-step culture method with a single SCMF, river microorganisms were cultured by separately feeding four representative carbon sources, n-propanol, lactic acid, methanol, and formic acid. After 140 h culture, the cell concentration and PHB content respectively reached 43 g/l and 35% when a propanol medium was fed. Using a two-stage perfusion culture with twin SCMFs, the seed cell mass was increased in the first SCMF and then supplied to the second flask for PHB production. As a consequence, the cellular PHB content rose to 51% in the second SCMF, while the cell concentration gradually increased to 25 g/l after 175 h perfusion culture. These results demonstrated the utility of the two-stage perfusion culture system for developing a cheap means of producing PHB coincident with wastewater treatment.

Initial anchoring and proliferation of fibroblast L-929 cells on unstable surface of calcium phosphate ceramics.

Calcium phosphate ceramics constructed from beta-tricalcium phosphate (TCP) and hydroxyapatite (HAP) have been successfully used as implant materials. However, there is a possibility that these materials are responsible for an unwanted inflammatory response during wound healing. Since TCP is soluble in the body, the instability of this material may contribute to this inflammatory response. Using composite ceramics of TCP and HAP that possessed Ca/P molar ratios of 1.50, 1.55, 1.60, 1.64, and 1.67, the effect of surface instability on fibroblast L-929 cells was investigated. The time-dependent variation of the initial anchoring ratio, cell density, and cell viability were measured. In general, the cells were severely damaged and ruptured on the highly soluble thin surface layer of the TCP-HAP ceramics. The initial anchoring ratio for TCP-HAP ceramics was as high as that for the polystyrene dish (Lux, control). However, viability at 6 h decreased to less than 50% of the initial cell density on ceramics with a Ca/P molar ratio of 1.64 (20% TCP-80% HAP), while 85% of the cells were viable on Lux. The viability on 100% TCP, whose surface is the most highly soluble among the TCP-HAP ceramics used in this study, was reduced to 20%. Morphological observation showed that the anchored cells were ruptured when grown in culture medium on the 100% TCP. Although the high solubility of the thin surface layer on the TCP-HAP ceramics of the carrier was found to be the dominant factor in the decreasing cell viability, the initial viability was enhanced by the stabilization of the surface of the TCP-HAP ceramics by pre-incubating the scaffolds in a culture medium containing 10% fetal bovine serum for 3 d.

[Effectively treated hypertension with minocycline hydrochloride infusion into the cyst in a patient with a multi-septated massive hepatic cyst].

We present a successfully treated case of a multi-septated massive hepatic cyst with repeated injection of minocycline hydrochloride (MINO). A 57-year-old Japanese female complaining of right back pain, hypochondralgia and hypertension had a multi-septated massive hepatic cyst, 25 cm in diameter. Multiple cysts of various sizes were also seen in liver and kidneys. In order to reduce the size of the massive hepatic cyst to relieve the complaints, we performed the reduction therapy of the cyst. After a pig tail catheter was inserted into the cyst, the cystic fluid was aspirated and then a total of 3900 mg of MINO was injected. Red-brownish, serous cystic fluids were obtained. Cytology and bacterial culture were negative, but the LDH (3, 336 IU/l) and CA19-9 (751,500 U/l) concentrations were very high. After the 9 series of the therapy, the cyst was minified on CT and the patient's symptoms were relieved. Furthermore high blood pressure was improved. Thus, the therapy of size-reduction for a massive hepatic cyst is revealed to be very safe and useful.

[Two cases of systemic lupus erythematosus associated with fatty liver and Basedow's disease].

We present two cases of systemic lupus erythematosus (SLE) associated with both Basedow's disease and fatty liver. The first case is a 46-year-old Japanese female who was admitted because of high fever and general fatigue. She had been diagnosed as having Basedow's disease and treated with thiamazole for over 4 years. Since thiamazole-induced lupus was unlikely because of high titer anti-nuclear antibody and anti-DNA antibody and low levels of complements, a diagnosis of SLE was made. The upper abdominal ultrasound study and the specimen obtained by liver biopsy performed before initiating steroid therapy demonstrated marked fatty liver. SLE itself is considered as an etiology of fatty liver in this case. The second case was a 25-year-old Japanese female with SLE. She had been treated with prednisolone for 13 years and was complicated with Basedow's disease 10 years later. Fatty liver was also demonstrated in this patient on ultrasonography, and was thought to be resulted from long-term steroid hormone administration.

Pulsed laser deposition of hydroxyapatite on nanostructured titanium towards drug eluting implants.

Titania nanotubes grown on titanium substrates by electrochemical anodization in glycerol-ammonium fluoride-water system were used to develop efficient drug carrying implants upon coating hydroxyapatite (HA) ceramic. The nanostructured surfaces achieved by anodization were caped with HA crystallites by pulsed laser deposition. The implant substrates were studied for their drug carrying capacity using gentamicin as a model. The nano-tubular surface with HA coating had better drug loading capacity of about 800 µg/cm(2) gentamicin while the bare anodized substrate carried less than 660 µg/cm(2). The HA coating alone stored as low as 68 µg/cm(2) and released the drug within the initial burst period itself. The ceramic coated anodized substrates were found to be more efficient in controlled delivery for longer than 160 h with a drug release of 0.5 µg/cm(2) even towards the end. The substrate with nanostructuring alone delivered the whole drug within 140 h. This study proposes the application of laser deposition of HA over nanostructured titanium, which proves to be promising towards controlled drug eluting bioceramic coated metallic prostheses.

Synthesis and characterization of bioactive hydroxyapatite-calcite nanocomposite for biomedical applications.

In a number of recent reports on the synthesis of hydroxyapatite (HA) by sol-gel method using citric acid as an organic modifier, washing was an essential step to remove the byproducts and citric acid. In the present study we made an attempt to synthesize HA by sol-gel method in the presence of citric acid, wherein we have employed calcination technique instead of the conventional washing process. The products thus obtained were analyzed by X-ray diffraction (XRD), Fourier Transform-Infrared (FT-IR) spectroscopy, thermogravimetry and scanning electron microscopy which confirmed the formation of a nanocomposite of HA and CaCO(3) (calcite) when citric acid was added during synthesis. HA is known to be bioactive and bioresorbable but the rates are too low. On the other hand, CaCO(3) is highly biodegradable. The combination of HA and CaCO(3) compromised the demerits of each others. The dissolved Ca ions from CaCO(3) enhanced the supersaturation of the surrounding fluid which resulted in higher bioactivity of HA.