Temperature- and ionic strength-induced conformational changes in the lipid head group region of liposomes as suggested by zeta potential data.

Neutral liposomes composed of DMPC (dimyristoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine) or DSPC (distearoylphosphatidylcholine) are found to exhibit non-zero zeta potentials in an electric field even when they are dispersed in solution at pH 7.4. A model for the orientation of lipid head groups is proposed to explain the observed non-zero zeta potentials. The dependence of the zeta potential on temperature and ionic strength is analyzed via this model to obtain the information on the direction of the lipid head group in the liposome surface region. The direction of the lipid head group is found to be sensitive to the temperature and the ionic strength of the medium. At low ionic strengths, the phosphatidyl groups are located at the outer portion of the head group region. At constant temperature, as the ionic strength increases, the choline group approaches the outer region of the bilayer surface while the phosphatidyl group hides behind the surface. At the phase transition temperature of the lipid, the phosphatidyl group lies in the outer-most region of the surface and the choline group is in the inner-most region.

Pathogenesis of drug-induced gingival overgrowth. A review of studies in the rat model.

Drug-induced gingival overgrowth is a side effect associated principally with 3 types of drugs: anticonvulsant (phenytoin), immunosuppressant (cyclosporine A), and various calcium channel blockers (nifedipine, verapamil, diltiazem). In this review, we describe the features of phenytoin-, cyclosporine A- and nifedipine-induced gingival overgrowth in rats and discuss factors influencing the onset and severity of these disorders. There are several features common to the gingival overgrowth induced by these drugs: 1) gingival overgrowth is more conspicuous in the buccal than in the lingual gingiva and less severe in the maxilla than in the mandible; 2) once the blood concentration of the drug reaches a certain level as a result of increasing the dose, the incidence of gingival overgrowth is 100% and its severity is dependent on the blood level, the most severe overgrowth being induced by cyclosporine A; 3) the duration of drug administration for maximal gingival overgrowth to develop is about 40 days; 4) the gingival overgrowth regresses spontaneously after discontinuing the drug; 5) accumulation of dental plaque is not essential for the onset of overgrowth, but plays a role in its severity; and 6) more severe overgrowth is induced in young than in old rats. Furthermore, male rats are more susceptible than females to nifedipine-induced gingival overgrowth. These results suggest that drug-induced gingival overgrowth in rats is dependent on the oral drug dose, blood drug level, age, and sex and that preexisting gingival inflammation is a factor relevant to its severity. Since these factors have also been suggested to be important determinants for human drug-induced gingival overgrowth, the rat model may prove valuable in the future for elucidating the molecular pathogenesis of the disorder.

Factors influencing nifedipine-induced gingival overgrowth in rats.

Factors such as age, the dose of nifedipine administered in the diet, serum drug level, duration of drug administration, and sex which may influence nifedipine-induced gingival overgrowth were examined in a rat model using 20-, 50-, and 90-days-old male and female rats. Oral administration of nifedipine (50 to 250 mg/kg diet) increased the serum level of the drug in a dose-dependent manner in both males and females. However, a higher serum level was required in females than males to attain the same degree of gingival overgrowth. The minimum dietary concentrations of the drug required to elicit gingival overgrowth in males and females were 150 and 100 mg/kg, respectively, which gave respective minimum serum levels of 800 and 1100 ng/ml. The degree of overgrowth depended on the serum concentration of the drug after it had reached the required minimum in male and female animals. Administration of nifedipine (250 mg/kg diet) for 20 days was enough to induce maximal overgrowth, but this induction occurred only in rats that started to receive the drug when they were 20 days old, not in those that started at 50 and 90 days of age for the same administration period of 55 days, and the overgrowth regressed and the gingiva were normal 40 days after ceasing drug administration. These results suggest that gingival overgrowth occurred in accordance with the drug concentration in the diet, as well as that in the serum, and was more likely to occur in males and younger individuals.

Enantioseparation of beta-blockers labelled with a chiral fluorescent reagent, R (-)-DBD-PyNCS, by reversed-phase liquid chromatography.

A fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfony l)-2,1, 3-benzoxadiazole [R(-)-DBD-PyNCS], has been used for the liquid chromatographic resolution of racemic pairs of beta-blockers. The reagents reacts with beta-blockers at 65 degrees C for 90 min in aqueous acetonitrile containing 0.05% triethylamine to produce the corresponding pair of diastereomers. No racemization occurs during the tagging reaction under these conditions. From results of the time-course study of oxprenolol the reactivities of the enantiomers of beta-blockers with R(-)-DBD-PyNCS are comparable. The optimum excitation and emission wavelengths of the resulting derivatives were ca. 460 and 550 nm, respectively. The derivatives of beta-blockers were efficiently resolved by a reversed-phase column with water-acetonitrile containing 0.1% trifluoroacetic acid as the eluent. The resolution (Rs) values of the diastereomers derived from 10 beta-blockers were in the range of 1.54-4.80. The Rs value for timolol was 0.643. The detection limits (signal-to-noise ratio of 2) were one or two orders of magnitude lower with beta-blockers having the iso-propylamino structure (15-300 fmol) than with those having the tert-butylamino structure (1.25-8.00 pmol). The proposed procedure was applied to the determination of R(+)- and S(-)-propranolol in rat plasma and saliva after oral administration of R(+)-propranolol hydrochloride or S(-)-propranolol hydrochloride.

Aorto-atrial fistula associated with recurrent aortic dissection after ascending aorta replacement.

A 70-year-old woman with a bicuspid aortic valve had undergone ascending aorta replacement for acute DeBakey type I dissection. Computed tomography and aortography 2 months after the operation revealed a thrombosed false lumen in the ascending aorta proximal to the prosthetic graft. However, recurrence of dissection was found at the aortic root proximal to the graft 4 years after the initial operation. Significant aortic stenosis was also noted. Despite intensive medical treatment, she had refractory and progressive heart failure. At the second surgery, an aorto-right atrial fistula, which probably was responsible for the severe heart failure was revealed. Closure of the aorto-right atrial fistula, and aortic root replacement were performed using Piehler's method, with a composite graft. The etiology and management of this rare case are discussed.

In vitro reconstruction of hybrid vascular tissue. Hierarchic and oriented cell layers.

Hybrid vascular tissue was hierarchically reconstructed in vitro. A hybrid medial layer composed of type I collagen gel, in which SMCs derived from a mongrel dog were embedded, was formed on the inner surface of a compliant porous polyurethane graft (internal diameter = 3 mm). Endothelial cells (ECs) from the same animal were seeded and cultured on the hybrid media to build an intimal layer. Subsequently, hierarchically structured grafts constructed in this manner were subjected to pulsatile flow (flow rate: 8.5 ml/min; frequency: 60 rpm; amplitude: 5% of graft outer diameter) of culture medium (Medium 199 supplemented with 20% fetal calf serum). After stress loading for as long as 10 days, tissues were morphologically investigated with a light microscope and a scanning electron microscope. Inner surfaces of the hybrid tissues were covered with EC monolayers that aligned along the direction of the flow (i.e., longitudinally). However, SMCs beneath the intima aligned in the circumferential direction. These cellular orientations resembled those in native muscular arteries. The pulsatile stress loaded hybrid tissue mimicked native muscular arteries with respect to hierarchic structure and cellular orientation. In vitro mechanical stress loading on a hybrid graft might provide a high degree of integrity in terms of tissue structure that promises high tolerance toward hydrodynamic stress and regulation of vasomotor tone upon implantation.

Impregnation of basic fibroblast growth factor on a microporous small caliber graft enhances vascularization.

The incorporation of a cellular adhesive extracellular matrix and a growth factor into a designed concept for a vital, functioning artificial vascular graft may accelerate tissue regeneration, including endothelialization. Microporous polyurethane grafts (inner diameter, 1.5 mm; wall thickness, 100 microns; length, 20 mm; pore size, 100 microns), fabricated using an excimer laser ablation technique, were coated with a mixed solution of photoreactive gelatin, basic fibroblast growth factor (bFGF), and heparin, and were subsequently photocured by ultraviolet irradiation. Control grafts were treated only with photoreactive gelatin. Both bFGF/heparin impregnated grafts (n = 6) and control grafts (n = 9) were implanted in aortas of rats for 4 weeks. All the implanted grafts were patent when harvested. Endothelialization mainly proceeded from anastomotic sites for both groups. The endothelial coverage in the bFGF/heparin impregnated groups was greater than that in the control animals. At the midportion of the grafts, regenerated endothelial and subendothelial layers were seen for the bFGF/heparin groups, but only fibrin layers were seen for the controls. Thus, coimmobilization of bFGF and heparin significantly enhanced neoarterial wall regeneration through perianastomotic as well as transmural tissue ingrowth. The former was more extensive than the latter.

A new microporous polyurethane vascular graft prepared by an excimer laser ablation technique.

Three types of polyurethane (PU) based tubes (internal diameter, 2 mm; wall thickness, 100 microns) with micropores of well controlled size and arrangement were fabricated using an excimer laser (KrF) ablation technique. The pore size (100 microns) and the longitudinal pore-to-pore distance (200 microns) were constant, and the circumferential pore-to-pore intervals were 60 degrees (type 1), 30 degrees (type 2), and 15 degrees (type 3). The surface of the fabricated tube was photochemically modified with photoreactive gelatin. Scanning electron microscopy showed that pore size and arrangement were precisely controlled as designed, and that a gelatinous layer thoroughly covered the luminal surface. The stiffness parameter (beta), inversely related to compliance, was determined from the change in external diameter against intraluminal pressure. An increase in the number of pores around the circumference decreased the beta value. The type 1 tubes implanted preliminarily in rats for 4 weeks showed good patency (80%). The combination of excimer laser-directed microporing and photochemical surface processing techniques enabled the development of a novel compliant small caliber vascular graft, which is expected to show enhanced transmural tissue ingrowth in vivo.

Vascular endothelial growth factor enhances vascularization in microporous small caliber polyurethane grafts.

Neoarterial regeneration in an implanted small caliber vascular prosthesis is complexly controlled by many structural and biologic factors, such as cytokines. The authors designed an artificial graft, which was prepared as follows. Segmented polyurethane tubular film (inner diameter, 1.5 mm; wall thickness, 100 microns; length, 20 mm), in which micropores (pore size, 100 microns) were fabricated by an excimer laser ablation technique, were coated with a mixed solution of photoreactive gelatin and heparin with or without cytokines (vascular endothelial growth factor [VEGF]: 5 or 50 micrograms/ml, basic fibroblast growth factor [bFGF]: 1 microgram/ml). These coated grafts were irradiated by ultraviolet light, and were implanted in aortas of rats for 4 weeks; the VEGF (5 micrograms/ml) group, n = 6; the bFGF group, n = 6; the VEGF (5 micrograms/ml)/bFGF group, n = 11; the VEGF (50 micrograms/ml)/bFGF group, n = 5; and the control group, n = 9. Control grafts were treated without cytokines. Endothelial coverage was greater for the cytokine immobilized groups (approximately equal to 50-60%) than for the control group (approximately equal to 30%). At the midportion of the triple VEGF immobilized group, many capillaries were seen in the neoarterial intima, and in the micropores, although such capillaries were rarely observed in the bFGF and control groups. Thus, impregnation of VEGF in the gelatinous layer of grafts enhanced transanastomotic tissue ingrowth and transmural capillary ingrowth.

Small diameter vascular prostheses with incorporated bioabsorbable matrices. A preliminary study.

The authors have designed small diameter vascular prostheses with incorporated matrices that can be absorbed into a growing anastomotic neointima. First, a gelatin-heparin complex was coated on the inner surface of tubular ultrafine polyester fabrics presealed with heat denatured albumin. Second, to control the bioabsorption rate, the prepared grafts were cross-linked with polyepoxy compounds for 3 days (Group I; n = 14) or 5 days (Group II; n = 4). These grafts, 3 mm in diameter and 4 cm long, replaced the carotid arteries of nine mongrel dogs weighing 9.5-14 kg. Six of eight (75%) grafts in Group I were patent when the animals were killed 4 weeks after surgery. Doppler sound examination revealed that the remaining six were patent for 16 weeks. In Group II, three of four (75%) grafts were patent when the animals were killed 8 weeks after surgery. In both groups, scanning electron microscopic study showed neither platelet aggregation nor fibrin formation on the midportion. However, these two groups significantly differed in histology. In Group I, thin anastomotic neointima advanced over the mostly absorbed gelatin-heparin complex layer. Connective tissue was well formed around the polyester scaffold. In contrast, thick neointima advanced over the gelatin-heparin complex layer that still remained on the luminal surface in Group II. These results suggest that the gelatin-heparin complex, when cross-linked adequately, could simultaneously function as a temporary antithrombogenic surface and as an excellent substructure of an anastomotic neointima.

Localization of sudanophil material at the sites of calcification in dentine, and the compact bone and epiphyseal cartilage plate of tibia in the rat given beryllium carbonate.

The localization of sudanophil material at dentine, the compact bone and epiphyseal cartilage plate of tibia of rat given beryllium carbonate was examined. Sudanophil material was seen at the boundary parts between dentine and widened predentine, and between widened preosseous matrix and calcified bone, but it was not seen at the area corresponding to the zone of provisional calcification. These facts suggest that the localization of sudanophil material in hard tissue of rat with Be rickets was similar to that in vitamin D deficient-induced rickets. This sudanophil material was not disappeared by the enzymes such as papain, pepsin and hyaluronidase as described in vitamin D deficient-induced rickets (Irving, 1960, 1963). Accordingly, it was suggested that the substance was not proteins and mucopolysaccharide.

Urinary mercury monitoring of university staff and students occasionally exposed to mercury vapor.

To monitor the exposure to mercury (Hg) vapor among university staff members and students who occasionally handle elemental Hg in laboratory experiments, urine samples were collected at health examinations conducted by the Health Service Center, University of Tokyo, for six years. Geometric mean of urinary Hg concentrations of 343 samples collected from 234 subjects was 1.61 micrograms Hg/g creatinine (Cr), with the range of 0.30 to 9.31 micrograms Hg/g Cr. Elevated urinary Hg levels, i.e. 3 micrograms Hg/g Cr or higher, were found only among the subjects who worked in several laboratories. This urinary Hg level is judged to correspond to 1-2 micrograms/m3 of air Hg concentration in working areas. The contribution of dental amalgam fillings to urinary Hg excretion, though it exists, was concluded to be small from the result of multiple regression analysis.

Residual 60Co activity in steel samples exposed to the Hiroshima atomic-bomb neutrons.

Residual 60Co radioactivity produced by the neutrons from the Hiroshima atomic bomb has been measured by means of gamma-ray spectrometry for seven steel samples located up to 1,800 m slant range and one control sample obtained at 4.5 km from the hypocenter. After removing the main constituent of iron ions from the steel sample by the solvent extraction method, nickel and cobalt were chemically separated by the ion exchange process. Gamma-ray measurements were performed with a low background well-type germanium detector. Derived specific activities 60Co:Co were compared with previous measurements and with the calculation based on the neutron fluence of current dosimetry system DS86. It has been shown that a systematic discrepancy exists between the measured and calculated activity. The calculated-to-measured ratios for 60Co are consistent with those of 152Eu and 36Cl activity.

Increased success of blind nasotracheal intubation through the use of nasogastric tubes as a guide.

We were able to improve the success rate of blind nasotracheal intubation by using nasogastric tubes as a guide during intubation, first, for passing the endotracheal tube through the nasal cavity, and second, passing it from the pharynx to the larynx. By adding both sedation by modified neuroleptanalgesia (NLA) and topical and transtracheal administration of lidocaine, our technique became safer and smoother. We have completed 36 cases without accident, with an average time for intubation of 8.25 min. The Rüsh spiral tube was thought to be the most suited to this form of intubation because of the 90 degrees cut of its tip, its high-volume cuff, and its flexibility in all directions. These features are useful for hearing breath sounds, raising the tip of the tube by inflation of the cuff, and advancing the tube in a turning motion.

[Resection and reconstruction of full thickness chest wall].

Nine patients underwent full thickness chest wall resection and reconstruction in our department between January 1981 and December 1994. There were chest wall recurrence of breast cancer in 5 cases, primary chest wall chondrosarcoma in 2, primary chest wall malignant fibrous histiocytoma in 1 and metastatic sternal renal cell carcinoma in 1. Seven of 9 cases underwent partial sternal resection. Sizes of chest wall defects were from 10 x 7 cm to 15 x 14 cm. In eight cases of 9, chest wall reconstruction was by double Marlex mesh repairs and various flaps (major pectoral muscle in 3, major pectoral myocutaneous flap in 2, latissimus dorsi myocutaneous flap in 1 and pedicled omentum in 1). The last case underwent repair with rectus abdominis myocutaneous flap without mesh. There was no operative death. Postoperative complications occurred in 4 patients: partial skin necrosis in 2, skin dehiscence in 1 and respiratory failure in 1. Eight cases are alive now from 9 months to 14 years and 8 months after chest wall reconstruction. One patient with metastatic renal cell carcinoma died of recurrence 3 years after operation. Full thickness chest wall resection and reconstruction is a safe operation and may provide a long-term survival.

[Development of a method to prevent degenerative changes of the vein graft for arterial reconstruction].

Autologous vein graft is frequently used for arterial reconstruction in vascular surgery. Its' long-term patency rate, however, is low because of the degenerative changes of the vein wall. Of the basis of our hypothesis that the degenerative changes are mainly caused by an over-distension of vein wall in arterial system, in this study, autologous vein grafts of rabbit (N = 56) were implanted in the carotid arteries. To prevent overdistension, some of the grafts were covered with prosthesis which was made of microporous, compliant biodegradable polyurethane and were compared with those without prosthesis. The integrity of the architecture of the vein wall covered with prosthesis was well preserved at 6 weeks after implantation, where the biodegradation of prosthesis induced an optimal arterialization of the vein grafts. The results indicate that this method may prevent the degenerative changes of vein wall such as fibrosis and intimal hyperplasia, which would cause poor long-term patency of vein graft used for arterial reconstruction.

[Chest wall reconstruction after resection of malignant chest wall tumors].

Full-thickness chest wall resection is performed for complete removal of primary and secondary malignant chest wall tumors. Large defects of the chest wall after resection must be repaired to maintain adequate ventilation, to protect important intrathoracic structures, and to preserve cosmetic integrity. Various materials have been utilized over the years to replace the rigid chest wall. At present, Marlex mesh and a composite of Marlex mesh and methylmethacrylate are frequently used to reconstruct rigid chest wall defects. On the other hand, to replace the soft part of the chest wall and cover the rigid materials, pedicled muscle flaps, myocutaneous flaps, or omentum are used. Major pedicled flaps include the pectoralis major, rectus abdominis and latissimus dorsi muscular, and musculocutaneous flaps. Techniques are now available to repair any chest wall site, and to restore chest continuity in patients whose tumors are curatively resected.

[Late occlusion at the anastomosis site of an extended polytetrafluoroethylene (E-PTFE) graft with small caliber].

Recently expanded polytetrafluoroethylene (E-PTFE) graft is often used in arterial bypass for atherosclerosis obliterans. In our series of 38 patients (48 legs) undergoing femoropopliteal bypass with E-PTFE graft of 6mm-caliber, a cumulative patency rate was 40.8% at 36 months after operation and a mean duration of patency was 26.3 months. To clarify the cause of occlusion, the diseased grafts were removed and histopathologically examined. Macroscopically, the E-PTFE graft was occluded by the intimal hyperplasia, so called pannus ingrowth from the recipient artery into the graft. The pannus formation of the graft was more remarkable in the distal than in the proximal site of anastomosis, suggesting the influence of blood flow on its formation. Microscopically, no endothelial cells were observed inside the E-PTFE graft except for the surface of pannus. Thus, less extension of endothelialization was thought to be a cause of late occlusion in E-PTFE grafts. Most desirable is the autologous saphenous vein for femoropopliteal bypass. However, when it is not available, E-PTFE graft with a larger caliber must be used in combination with postoperative anticoagulant therapy.