Pluronic polyol effects on human gingival fibroblast attachment and growth.

BACKGROUND: Enhanced speed of human gingival fibroblast (HGF) spreading and attachment, as affected by ionic bonding interactions, may facilitate cell orientation and subsequent collagen synthesis to promote early wound healing. The purpose of this study was to determine the in vitro effects of pluronic polyols, a family of widely used surfactants currently used as drug carriers for antibiotic, anti-inflammatory, and anti-neoplastic agents, on the attachment and growth of human gingival fibroblasts (HGF) to dentin and plastic surfaces using established tissue culture techniques. METHODS: Plastic culture wells containing Eagle's minimal essential media (EMEM) with 10% fetal calf serum and Pluronic F-68 or F-127 in concentrations from 1.2 x 10(-2) to 1.2 x 10(-10) M were incubated with HGF and run in replicates of ten. Attached cells were quantified by measuring the optical density of methylene blue-stained cells. Additional experiments were conducted using human dentin sections as a substrate and Pluronic F-68 or F-127 at a concentration of 1.2 x 10(-8) M. In these experiments, HGF were stained with acridine orange and quantified per unit area of dentin by fluorescence microscopy. RESULTS: Attachment and growth of HGF to both plastic and dentin were significantly increased over serum controls by very low concentrations of Pluronic F-68 and F-127 by 30 minutes, with attachment reaching a plateau at 2 hours. CONCLUSIONS: Pluronic polyols, a family of widely used surfactants, in very low dosages may be beneficial in early postsurgical wound healing by facilitating early attachment and enhancing the growth rate of human gingival fibroblasts.

Astrocytes increase barrier properties and ZO-1 expression in retinal vascular endothelial cells.

PURPOSE: Diabetic retinopathy and other diseases associated with retinal edema are characterized by increased microvascular leakage. Astrocytes have been proposed to maintain endothelial function in the brain, suggesting that glial impairment may underlie the development of retinal edema. The purpose of this study was to test the effects of astrocytes on barrier properties in retinal microvascular endothelial cells. METHODS: Bovine retinal microvascular endothelial cells were exposed to conditioned media from rat brain astrocytes. Transendothelial electrical resistance (TER) was determined on 24-mm Transwell (Cambridge, MA) polycarbonate filters with the End-Ohm device (World Precision Instruments, Sarasota, FL). ZO-1 protein content was quantified by microtiter enzyme-linked immunosorbent assay. RESULTS: Astrocyte-conditioned medium (ACM) significantly increased TER (P < 0.0001) and ZO-1 content (P < 0.01). Both serum-containing and serum-free N1B defined ACM increased ZO-1 expression, but heating abolished the effect. Serum-free ACM decreased cell proliferation by 16%. CONCLUSIONS: Astrocytes release soluble, heat-labile factors that increase barrier properties and tight junction protein content. These results suggest that astrocytes enhance blood-retinal barrier properties, at least in part by increasing tight junction protein expression. Our findings suggest that glial malfunction plays an important role in the pathogenesis of vasogenic retinal edema.

Histamine reduces ZO-1 tight-junction protein expression in cultured retinal microvascular endothelial cells.

We examined ZO-1 protein content in cultured retinal vascular endothelial cells to test the hypothesis that histamine alters tight-junction-protein expression. Histamine (10(-9) -10(-4) M) causes a reversible concentration-dependent reduction of ZO-1 protein content, mediated by both H1 and H2 receptors. Histamine reduces ZO-1 expression within the time associated with increased paracellular permeability. Tight-junction-protein alterations may be a novel explanation for the mechanism by which vasoactive agents increase microvascular permeability.

Protein tyrosine kinase activity in breast cancer and its relation to prognostic indicators.

Tyrosine kinase dependent oncogenes and growth factor receptors are of prognostic importance in breast cancer, but the relation of cytosolic protein tyrosine kinase (PTK) activity to traditional prognostic indicators is poorly defined. We determined cytosolic PTK activity in tumor extracts of 61 women with invasive breast cancer, including 51 primary specimens and 12 nodal or metastatic specimens, 7 women with in situ breast cancer, and 8 control breast specimens. PTK activity (pmol/min/mg) was measured in a dot blot assay using phosphotyrosine antibodies to detect phosphorylated tyrosyl residues in the tissue extracts. Compared with control specimens (mean PTK = 20.5), tyrosine kinase activity was significantly greater in invasive primary cancers (mean PTK = 298.1; p = 0.0008), and nodal/metastatic specimens (mean PTK = 491.5; p = 0.0009). PTK levels of invasive cancers did not correlate with age (p = 0.36), tumor size (p = 0.83), nodal status (p = 0.37), estrogen receptor status (p = 0.66), or progesterone receptor status (p = 0.09). Thus, while tyrosine kinase activity is increased in breast cancer, correlations with traditional prognostic indicators were not found.

Mechanisms regulating skeletal muscle glucose metabolism in sepsis.

Carbohydrate dyshomeostasis is a characteristic feature of sepsis. Sepsis elevates glucose uptake and cellular lactate levels in muscle. The mechanisms responsible for these alterations are unknown. We examined the effects of a chronic, intra-abdominal septic abscess upon glucose uptake, the expression of the insulin receptor, glucose transporter proteins (Glut-1 and Glut-4) and mRNA, and the content of glycolytic intermediates in muscle from the hindlimb. Sepsis caused a 67% increase in glucose uptake compared with control. A differential expression of the Glut-1 and Glut-4 transporter proteins in skeletal muscle of septic rats was observed. Sepsis increased the expression of Glut-1 protein 1.7-fold. The increased Glut-1 protein correlated with a similar increase in the relative abundance of Glut-1 mRNA. In contrast, sepsis did not alter the amount of Glut-4 protein and mRNA or insulin receptor protein. The tissue content of glucose-6-phosphate was increased approximately twofold compared with control. The increase in the glucose-6-phosphate content was not associated with increased glycogen deposition in skeletal muscle of septic animals. Analysis of the glycolytic intermediates showed that only the lactate content of muscles from septic rats was significantly elevated in sepsis. The results are consistent with the hypothesis that sepsis enhances glucose uptake secondary to increased Glut-1 expression. Furthermore, once transported, glucose may be preferentially metabolized to lactate.

Glucose transporter isoform expression in Huntington's disease brain.

Several reports have suggested a characteristic decrease in glucose use in the striatum of patients with Huntington's disease (HD) may contribute to the cellular atrophy of the caudate and putamen. We examined the expression of the two major glucose transporter isoforms of brain, GLUT1 and GLUT3. GLUT1 is found largely in capillary endothelial cells and to a lesser extent in the brain parenchyma, whereas GLUT3 is localized primarily in neurons. Membranes prepared from postmortem samples of HD caudate and cortex and non-HD caudate and cortex were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and probed with antisera to GLUT1 and GLUT3 by western blotting. Compared with controls, GLUT1 and GLUT3 transporter expression in caudate was decreased by three- and fourfold, respectively, in grade 3 of the disease. At earlier stages (grade 1), there was no significant difference in the expression of the two transporter isoforms compared with nondiseased controls. It is surprising that despite a substantial increase in glial fibrillary acidic protein immunoreactivity (an indicator of the extent of gliosis), glucose transporter expression was diminished significantly in HD caudate. The results suggest in the absence of a significant number of neurons, as in grade 3, glial cell GLUT1 and GLUT3 expression is down-regulated, perhaps reflecting the decreased metabolic demand of this brain region in HD.

Evaluation of the effects of diagnostic radiation on titanium dental implant osseointegration in the micropig.

The effect that diagnostic radiation may have on peri-implant supporting alveolar tissues is not well understood. Fifty-four (54) titanium dental implants were inserted into the posterior mandible of nine micropigs. At implant placement surgery, 18 implants were exposed to either 2 (diagnostic) or 10 (excessive) doses of diagnostic radiation; the remaining 36 implants served as controls. Fourteen weeks after implant placement, standardized clinical radiographs were taken, pigs were euthanized, and implants with supporting alveolar tissues were prepared and examined by light and scanning electron microscopy (SEM). Ninety-seven sections were evaluated by SEM for morphometric and morphologic analyses. The mean percent of implant length in contact with bone was 47% for the controls and 53% for the implants receiving radiation. Five implants were lost during the initial healing phase and four implants were clinically mobile at time of euthanasia, giving a success rate of 83% (45/54). Correlative light microscopy of peri-implant supporting tissues revealed no distinct differences between the microvasculature of controls versus implants exposed to radiation. Standardized clinical radiographs revealed crestal saucerization in both control and radiated implants. This study revealed no statistically significant difference for the percent of implant length in contact with alveolar bone for controls or implants exposed to 2 or 10 doses of diagnostic radiation at implant placement time.

An in vitro study of dentinal tubule occlusion by ferric oxalate.

This study examined ferric oxalate's ability to occlude dentinal tubules both in the presence of a smear layer and after its removal. Radicular dentinal chips were prepared with a smear layer created from a high speed carbide bur. The dentinal chips were then grouped as follows: 1) those with the smear layer remaining intact; 2) those sonicated for 7 minutes; 3) those treated with 10% tetracycline HCl; 4) those treated with 0.5M EDTA; 5) those treated with 20% citric acid; or 6) those treated with saturated citric acid. Six percent ferric oxalate was applied for 1 minute to the dentinal chips under blinded conditions. The chips were examined under SEM and the number of small and large crystals formed were counted. The results indicate that a decrease in the number of small crystals occurs following pretreatment of the smear layer by chemical means. An increased variability in size and shape of the crystals is also observed when no chemical pretreatment is used. Thus, relative to the number of crystals that form, no chemical pretreatment of radicular dentin is indicated prior to application of ferric oxalate in the treatment of root sensitivity.

Surgical treatment of induced peri-implantitis in the micro pig: clinical and histological analysis.

The purpose of this pilot study was to determine if lost osseous support adjacent to root form implants could be regenerated using a guided tissue regeneration technique. Three fixtures were placed in each edentulous mandibular bicuspid region of two micro pigs. A total of 6 fixtures were placed in each pig. Due to the presence of a pathologic condition, which was in no way related to the research, the results of one pig were not evaluated. Following osseointegration, peri-implantitis were induced by the use of ligatures and a soft diet. Three modalities of treatment were performed. Utilizing a surgical flap approach, one third of the fixtures (one per quadrant) were covered with expanded polytetrafluoroethylene (ePTFE) membrane and submerged under the soft tissue complex. The second group of fixtures were submerged under the soft tissue complex with no ePTFE membrane. The control fixtures along with their abutments were debrided and remained non-submerged. All fixtures were debrided using an air-abrasive polishing system. The osseous defects around the fixtures were measured from a fixed reference point at the time of surgery and after obtaining block sections.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of chlorhexidine irrigation on tensile wound strength.

Chlorhexidine in an alcohol vehicle with flavoring agents has been used as a mouthrinse to reduce plaque accumulation in periodontal surgery patients. The purpose of this study was to evaluate the effects of a chlorhexidine-containing mouthrinse on the early tensile wound strength of healing surgical wounds in the rat. Standardized transdermal incisions were made on each lateral abdominal wall of 40 Sprague-Dawley rats. Wounds were irrigated with 10 ml of 0.12% chlorhexidine or 10 ml of normal saline prior to closure. Animals were sacrificed at 48 hours and 96 hours, and the wound area was excised by a standardized protocol. Wound strength was measured using constant speed tensiometry to determine the tensile strength of the healing incision. Results revealed a significantly reduced tensile wound strength at 48 hours for the chlorhexidine-treated group (127 +/- 18.5 gm) compared to the saline irrigation group (150 +/- 32.3 gm) (P < 0.001). However, by 96 hours a significantly increased tensile wound strength was demonstrated by the chlorhexidine treated group (202.1 +/- 21.7 gm) compared to the saline irrigation group (183.2 +/- 37.3 gm) (P < 0.05). These data suggest that chlorhexidine-containing mouthrinse irrigation of wounds produced a reduced early tensile wound strength, but ultimately resulted in shorter healing time.

The effect of nicotine on reproduction and attachment of human gingival fibroblasts in vitro.

The ability of fibroblasts to reproduce and attach to teeth is of paramount importance in re-establishing the lost connective tissue attachment after periodontal therapy. This study examined the effect of nicotine, a major component of the particulate phase of tobacco smoke, on human gingival fibroblast (HGF) reproduction and attachment to tissue culture surfaces. Pooled HGF cultures made from explants of gingival biopsies were utilized between passages 5 and 10 and plated in 96-well plates at 1.0 x 10(4) cells per well. Cell numbers were determined using 3-(4,5-dimethylthiazol-2-y)-2,5-diphenyl tetrazolium bromide (MTT), which is a reflection of mitochondrial dehydrogenase activity. The concentrations of nicotine used were 0.025, 0.05, 0.1, 0.2, and 0.4 microM, the average serum concentration for a smoker being approximately 0.1 microM. The effect of continuous nicotine exposure on HGF reproduction was determined by incubating cell cultures and media containing nicotine for up to 48 hours. Residual toxicity was determined by preincubating cells with nicotine for 1 or 6 hours. HGF suspensions and increasing concentrations of nicotine were added together to determine the effect on attachment. Results showed an enhanced effect of nicotine on HGF attachment, with increasing numbers of cells attaching with increasing nicotine concentrations, compared to the control. Low concentrations of nicotine had a stimulatory effect on cell replication, while higher concentrations of nicotine appear to have no significant effect on HGF reproduction. The responses of cells to some concentrations of nicotine may persist after its removal.

Carbonic anhydrase III in obese Zucker rats.

Proteins from 5- to 7-wk-old lean and obese Zucker rats were separated by one-dimensional sodium dodecyl sulfate (SDS) and two-dimensional SDS-isoelectric focusing-polyacrylamide gel electrophoresis. Laser densitometry revealed an obesity-related decrease in the concentration of a 28-kDa cytosolic adipocyte protein, the most abundant protein in adipocytes from lean Zucker rats. Microsequencing revealed the identity of this protein to be carbonic anhydrase III (CA III). The identity and obesity-related decrease was further confirmed using isoform-specific antisera and CA III enzyme activity measurements made by 18O mass spectrometry. Immunoblotting studies also revealed that CA III is present in at least two charge isoforms in adipocytes. Our data indicate that lean Zucker rat adipocytes may represent the richest source of CA III in nature (24% of the cytosolic protein content). An obesity-related decrease in both the concentration and activity of CA III was observed in two lipogenic tissues, liver and white fat, but not in soleus muscle. Adipocyte CA III activity was no longer depressed when hyperinsulinemic obese rats were made insulin deficient by streptozotocin injection. This suggests that the obesity-related decrease in CA III may be related to the hyperinsulinemia as well as to the insulin hyperresponsiveness that adipocytes from obese Zucker rats of this age display.

The effect of titanium implant abutment surface irregularities on plaque accumulation in vivo.

The purpose of this study was 2-fold to: 1) evaluate in vitro the surface texture of titanium implant abutments after exposure to plastic scalers, an air-powder abrasive system, rubber cup polishing with flour of pumice, and untreated control abutments; and 2) compare plaque accumulation in humans on abutments treated with the above methods. In part I, 5.5 mm abutments were instrumented for 30 seconds per 90 degrees segment with the respective methods. The surface character was compared to untreated controls using SEM at 260X magnification. The control abutments revealed prominent milling marks and small pits; plastic scalers slightly smoothed the milling marks and created microscratches; the air-powder abrasive largely obliterated the milling marks and caused some surface pitting; the rubber cup with flour of pumice removed the milling marks and created a smooth swirl pattern. None of the instrumentation appeared to roughen the surface. In the clinical experiment (part II), four abutments, one of each type, were placed in 12 patients for a period of 7 days, during which the patients performed no oral hygiene. At the end of 7 days, the abutments were retrieved and processed for SEM. A digitizer and software program were used to determine the percent of total abutment surface area covered by plaque. The demarcation of supragingival and subgingival plaque was well delineated. The total mean percent surface area of plaque ranged from 52.06% for the air-powder abrasive to 55.29% for the plastic scalers.(ABSTRACT TRUNCATED AT 250 WORDS)

Angioedema as a complication in periodontal surgery: report of a case.

Angioedema is a diffuse swelling of the subcutaneous or submucosal tissues that occurs in both hereditary and non-hereditary forms. It can be a temporarily disfiguring condition, but not usually a serious one unless the airway is compromised. In the majority of cases, no underlying cause can be identified. In this report, a case of "idiopathic" angioedema that occurred while performing a periodontal surgical procedure is presented. This case is interesting because the patient was on long-term use of an angiotensin-converting enzyme [ACE] inhibitor for hypertension, and recent evidence has shown that ACE inhibitors suppress the breakdown of circulating bradykinins. With high plasma levels of bradykinins, a local anesthetic, periodontal surgical procedures, or even emotional stress may trigger an attack of angioedema. Practitioners should be aware of the pharmacologic side effects of ACE inhibitors and be prepared to handle an emergency if a patient's airway becomes compromised.

The effect of maternal diabetes on glycogen metabolism in the embryonic rat.

This study examined the effect of maternal hyperglycemia during pregnancy due to streptozotocin-induced diabetes on the synthesis of glycogen in the brain and liver of embryonic and newborn rats. Maternal hyperglycemia (serum glucose 25.3 +/- 0.9 mM) during gestation had no effect compared to controls (5.7 +/- 0.2 mM) on embryonic and newborn glycogen content in liver. In contrast, embryos experiencing hyperglycemia in utero had a two-fold higher brain glycogen content than controls at term; 1.6 mg/g vs. 0.84 mg/g, respectively. Interestingly there was a significant delay in the mobilization of brain glycogen during the immediate postnatal period in the offspring of diabetic mothers and control animals. These results suggest that uncontrolled maternal diabetes during pregnancy may significantly increase the availability of a potentially important local fuel source for the newborn brain: glycogen.

Rat skeletal muscle, liver and brain have different fetal and adult forms of the glucose transporter.

Rabbit antibodies made against the human erythrocyte glucose transporter were used to determine whether or not embryonic glucose transporters of rat skeletal muscle, liver and brain are identical to the transporters of adult animals. The results indicate that in both skeletal muscle and liver, the transporter switches from a highly antibody-reactive embryonic form to a low antibody-reactive adult form within 2 days of birth. This suggests that there are two different forms of glucose transporter in embryonic and adult skeletal muscle and liver. In contrast, these antibodies have equal reactivity toward the glucose transporters of embryonic and adult brain. In embryonic brain, two forms of the transporter coexist, with different molecular weights (Mr = 45,000 and 40,000), while in the adult brain the Mr = 40,000 form is predominant. The dissociation constant for glucose for the embryonic liver transporter was measured by displacement of bound cytochalasin B. The results indicate that the embryonic liver transporter has a low affinity for glucose and for cytochalasin B, similar to the adult liver transporter, even though the antibody reactivity toward these two transporters is different.

Developmental aspects of the rat brain insulin receptor: loss of sialic acid and fluctuation in number characterize fetal development.

In this study, I have investigated the structure of the rat brain insulin receptor during fetal development. There is a progressive decrease in the apparent molecular size of the brain alpha-subunit during development: 130K on day 16 of gestation, 126K at birth, and 120K in the adult. Glycosylation was investigated as a possible reason for the observed differences in the alpha-subunit molecular size. The results show that the developmental decrease in the brain alpha-subunit apparent molecular size is due to a parallel decrease in sialic acid content. This was further confirmed by measuring the retention of autophosphorylated insulin receptors on wheat germ agglutinin (WGA)-Sepharose. An inverse correlation between developmental age and retention of 32P-labeled insulin receptors on the lectin column was observed. Insulin binding increases 6-fold between 16 and 20 days of gestation [61 +/- 25 (+/- SE) fmol/mg protein and 364 +/- 42 fmol/mg, respectively]. Thereafter, binding in brain membranes decreases to 150 +/- 20 fmol/mg by 2 days after birth, then reaches the adult level of 63 +/- 15 fmol/mg. In addition, the degree of insulin-stimulated autophosphorylation closely parallels the developmental changes in insulin binding. Between 16 and 20 days of fetal life, insulin-stimulated phosphorylation of the beta-subunit increases 6-fold. Thereafter, the extent of phosphorylation decreases rapidly, reaching adult values identical with those in 16-day-old fetal brain. These results suggest that the embryonic brain possesses competent insulin receptors whose expression changes markedly during fetal development. This information should be important in defining the role of insulin in the developing nervous system.

Mitochondrial function after acute alteration of the endogenous insulin-to-glucagon ratio.

Mannoheptulose (2g/kg i.p.) increases serum glucagon and decreases serum insulin via its effect on pancreatic islet cells. These changes in endogenous hormone status had effects on rat liver mitochondria that were comparable to the effects of injecting porcine glucagon (0.5 mg/kg i.p.). Mitochondrial adenine nucleotide content was increased 38 or 39% by mannoheptulose or glucagon respectively, citrulline synthesis by 165 or 193%, pyruvate carboxylation by 113 or 135%, coupled respiration by 34 or 42%, and uncoupled respiration by 40 or 54%. We conclude that the reciprocal changes in endogenous insulin and glucagon brought about by mannoheptulose offer a useful and interesting alternative to glucagon injection for studying the effects of these pancreatic hormones on liver mitochondria.

Regulation of hepatic gluconeogenesis in newborn rabbit: controlling factors in presuckling period.

We have previously shown (Comp. Biochem. Physiol. 77B: 35-39, 1984) that a rapid postnatal increase in hepatic mitochondrial adenine nucleotide content activates pyruvate carboxylation and gluconeogenesis in the newborn rabbit. This study investigated factors limiting flux through the gluconeogenic pathway and examined the physiological stimuli responsible for the activation phenomenon. There is a 2.3-fold increase in total mitochondrial adenine nucleotides, along with a threefold increase in the matrix ATP/ADP ratio, by 2 h after birth, resulting overall in a sixfold increase in the amount of ATP/mg mitochondrial protein. Analysis of gluconeogenic intermediates, measured in freeze-clamped livers between birth and 4 h postnatal, suggests that pyruvate carboxylase controls gluconeogenic flux during this period. Newborn rabbits reared in an hypoxic environment (5% O2) exhibited decreased mitochondrial adenine nucleotide content, decreased rates of pyruvate carboxylation, and depressed blood glucose levels compared with littermates reared in room air or 95% O2. Manipulation of the insulin-to-glucagon ratio in vivo by injecting insulin at birth significantly delayed postnatal increases in the mitochondrial adenine nucleotide content and the rate of pyruvate carboxylation. Conversely, glucagon injection produced a supranormal increase in both mitochondrial adenine nucleotide content and pyruvate carboxylation. In addition, insulin injection prevented, whereas glucagon enhanced, the normal postnatal increase in tissue ATP/ADP. These results suggest that tissue oxygenation and a decreased insulin-to-glucagon ratio promote the rapid influx of adenine nucleotides from the liver cytosol into the mitochondrial matrix, thereby activating pyruvate carboxylation and gluconeogenesis during the presuckling period.

Regional mitochondrial respiratory activity in Huntington's disease brain.

This study investigated mitochondrial respiratory activity in Huntington's disease (HD) brain. Mitochondrial membranes from caudate and cortex of HD and non-HD autopsied brains were assayed for succinate oxidation, cytochrome oxidase activity, and cytochromes b, cc1, and aa3. There was a significant decrease in HD caudate mitochondrial respiration, cytochrome oxidase activity, and cytochrome aa3, whereas cytochromes b and cc1 were normal. These findings are consistent with the hypothesis that mitochondrial dysfunction may contribute to the localized hypometabolism and progressive atrophy of the HD caudate.