Assessing orthostatic hypotension.
Explore the source record for details and available documents.
Aged↗
Biomedical subjects
C A McCulloch
Publications and source records attributed to C A McCulloch.
Deficiencies in collagen phagocytosis by human fibroblasts in vitro: a mechanism for fibrosis?
Degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues. Perturbations of this pathway may provide a mechanism for the development of fibrotic lesions. As collagen phagocytosis may be regulated by either a change of the proportions or the activity of phagocytic cells, we quantified phagocytosis with an in vitro model system. Collagen-coated fluorescent latex beads were incubated with human gingival fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 3 hours contained a mean of 64% phagocytic cells; however, a small subpopulation (10% of phagocytic cells) contained more than threefold higher numbers of beads per cell than the mean. In contrast, cells from fibrotic lesions exhibited a large reduction of the proportions of phagocytic cells (mean = 13.8%) and there were no cells with high numbers of beads. On the basis of 3H-Tdr labeling, cells from fibrotic lesions that had internalized beads failed to proliferate, in contrast to phagocytic cells from normal tissues, which underwent repeated cell divisions. This result was not due to variations of cell cycle phase as there was no preferential internalization of beads during different phases of the cell cycle. The low phagocytic rate of cells from fibrotic lesions was also not due to asymmetric partitioning of phagosomes at mitosis as videocinemicrography of bead-labeled phagosomes in single, pre-mitotic cells demonstrated that > 90% of phagocytic cells equally partitioned beads to daughter cells. To investigate if inhibition of phagocytosis could be replicated in vitro, cells were incubated with the fibrosis-inducing drugs nifedipine or dilantin. These cultures exhibited marked (15-75%), dose-dependent reductions in the proportions of phagocytic cells, but there was no reduction in bead number per cell. Fibrotic lesions appear to contain fibroblasts with marked deficiencies in phagocytosis and the reduced phagocytic activity of these cells may contribute to unbalanced degradation and fibrosis.
Adolescent↗
Induced endocytosis in human fibroblasts by electrical fields.
Electroporation creates transient pores through which exogenous molecules can gain access to the cell cytoplasm. However, the electrical events associated with this phenomenon may perturb membrane-dependent events such as endocytosis. To measure the effect of electroporation on endocytosis, suspensions of human gingival fibroblasts were subjected to 5-ms electrical discharges, allowed to recover for variable periods of time, incubated with fluorescent probes, and then analyzed by flow cytometry. Incubation of electroporated fibroblasts with FITC-conjugated bovine serum albumin (BSA) to label moities on cell membranes nonspecifically demonstrated a time-dependent increase of internalized probe for up to 90 min after electroporation. Pretreatment incubation of cells with cytochalasin D abrogated the increased internalization of FITC-BSA due to electroporation. Compared to controls, fluorescence signals due to internalization of surface glycoproteins with FITC-concanavalin A were 43% higher after electroporation and treatment with endoglycosidase F or H to reduce probe associated with surface membrane. Confocal microscopy confirmed intracellular labeling and reduction of membrane-associated probe by the enzyme. Assessment of nonspecific FITC-Con A labeling of cells by pretreatment with alpha-methyl D-mannoside showed that labeling was largely (92%) specific. Compared to controls, electroporation induced a 60% increase of internalization of lucifer yellow, a fluid-phase endocytosis marker. Dual fluorescence labeling of membrane phosopholipids by FITC and TRITC-DHPE demonstrated an increased acidification after electroporation that was time dependent, indicating that electroporation induced more rapid entry of membrane lipid into endosomal compartments. These data demonstrate that the electrical fields used in electroporation of fibroblasts cause an actin-dependent increase in the internalization of all membrane components examined and an increased rate of probe entry in to acidifying compartments.
Actin Cytoskeleton↗
Bone sialoprotein in developing porcine dental tissues: cellular expression and comparison of tissue localization with osteopontin and osteonectin.
Bone sialoprotein (BSP) is a highly sulphated and glycosylated phosphoprotein that is a major constituent of bone and other mineralized connective tissues. Although BSP can mediate cell attachment through an RGD sequence and binds selectively to hydroxyapatite, its precise function in mineralized tissues is unknown. To provide insights into its possible function, affinity-purified polyclonal antibodies directed against porcine BSP were used to demonstrate the histological distribution of this protein in developing porcine mandibular alveolar bone and the associated tooth tissues from 35- and 50-day fetuses. In addition, a porcine cRNA probe was used to determine the cellular expression of BSP in the same tissues by in situ hybridization. Immunoreactivity to BSP protein was restricted to the cells and matrix of the mineralized tissues of alveolar bone and dentine. In dentine, BSP was localized to the odontoblasts and their processes and to the peritubular dentine. In the alveolar bone, immunoreactivity for BSP was evident in osteoblastic cells and osteocytes and in the bone matrix; the older bone stained more strongly than newly formed bone. In addition, BSP appeared to be concentrated in the reversal lines of the rapidly remodelling bone. The distribution of BSP in these tissues revealed distinct differences when compared to osteopontin and SPARC/osteonectin, which are also prominent non-collagenous proteins of mineralized tissues. Most notable was the localization of osteopontin and especially osteonectin in non-mineralizing tissues. The immunoreactivity of osteoblasts and osteocytes for BSP in bone was consistent with the high levels of BSP mRNA revealed by in situ hybridization. However, much lower levels of hybridization were evident in the odontoblasts of developing mandibular molars. These studies demonstrate that BSP is expressed during the early formation of dentine and alveolar bone and that the protein accumulates in the peritubular dentine and bone matrix.
Alveolar Process↗
Relationship between periodontal probing velocity and gingival inflammation in human subjects.
Little is known about the biophysical characteristics of the dentogingival junction in response to the development or resolution of inflammation. The Toronto Automated Periodontal Probe (TAPP) provides an estimate of the integrity of the dentogingival junction by measuring intrapocket probing velocity. The aim of this study was to measure changes of probing velocity in inflamed human periodontium before and after subgingival debridement. 32 subjects exhibiting gingival inflammation were selected; 29 completed the study. Gingival index (GI), plaque index (PLI), bleeding index (BI) and the rate of gingival crevicular fluid flow (CFF) were measured as concomitant variables. The experimental group (N = 16) received scaling, root planing and oral hygiene instruction at baseline. The control group (N = 13) received no treatment until after 28 days. Subjects were seen at baseline, day 14, 21 and 28 for measurement of probing velocity and concomitant variables on 6 index teeth. At day 28, the control group was treated and then reassessed 28 days later. The experimental group showed a reduction of 51.6% for mean crevicular fluid flow (p < 0.0001), 79.7% for mean plaque index (p < 0.0001), 58.0% for mean gingival index (p < 0.0001), and 72.0% for mean bleeding index (p < 0.01) at day 28, confirming that inflammation was reduced compared with baseline. No significant changes were observed in the control group until after treatment. The velocity of probing and the formation of a plateau in the velocity profile were recorded.(ABSTRACT TRUNCATED AT 250 WORDS)
Dental Plaque Index↗
Disturbances of gingival fibroblast population homeostasis due to experimentally induced inflammation in the cynomolgus monkey (Macaca fascicularis): potential mechanism of disease progression.
We have studied the relationship between perturbations of fibroblast turnover in inflamed gingiva of different severities. To perform detailed spatial analyses of gingival fibroblast progenitor cells, inflammatory cell infiltrates and blood vessels, 3 Cynomolgus monkeys with healthy periodontium and 2 with naturally occurring gingivitis and ligature-induced periodontitis were pulse-labeled with 3H-thymidine. Morphometric analyses of radioautographs from mid-sagittal supra-alveolar gingival connective tissues of incisors were performed in sites subjacent to junctional sulcular and oral epithelium, in the body of the lamina propria and just superior to the alveolar crest. The percentage of fibroblasts incorporating 3H-thymidine label, expressed as the labeling index (LI), was higher subjacent to the sulcular epithelium in periodontitis (1.73 +/- 0.37) than in healthy sites (1.06 +/- 0.22). This was not statistically significant (0.05 < p < 0.1) due to the small number of animals used. The sites subjacent to the sulcular epithelium also exhibited the largest increase in lymphocyte density from health to gingivitis (p < 0.01). In contrast, the LI of fibroblasts subjacent to the oral epithelium was 5-fold higher in healthy (0.82 +/- 0.17) compared to periodontitis sites (0.13 +/- 0.09; p < 0.05). Labeled fibroblasts were found close to blood vessels in all compartments and in all disease states; distance to blood vessels was reduced in inflamed sites (p < 0.10). There were increased numbers of blood vessels per unit area in the lamina propria of gingivitis compared to healthy sites. However, there were no regional differences with respect to blood vessel numbers or area in sites subjacent to junctional epithelium with different disease states. The results indicate that: 1) experimentally-induced inflammation in the gingiva of Cynomolgus monkeys is associated with site-specific perturbations of cell turnover; 2) fibroblast progenitors are preferentially situated adjacent to blood vessels as in the periodontal ligament; 3) the vascular response to inflammation is a generalized increase in blood vessel numbers, but not their size; 4) reactive proliferation of fibroblasts may compensate for cell death in the lamina propria but is not detectable at the site of connective tissue attachment loss subjacent to the junctional epithelium. Failure to maintain the fibroblast progenitor population may be an important component of attachment loss in progressive periodontitis lesions.
Animals↗
Evaluating patient perceptions as short-term outcomes of periodontal treatment: a comparison of surgical and non-surgical therapy.
To quantify patient perceptions of periodontal therapy, we administered a self-report questionnaire employing a visual analog scale (VAS) to 162 patients following surgical and non-surgical treatments. The questionnaire tested for variables of pain, sensitivity, swelling, function, altered appearance, comparison of postoperative discomfort to restorative treatment (crown or filling), discomfort experienced during the procedure, and resultant disability days. Tests of reliability indicated that with the exception of thermal sensitivity, the variables were significantly reproducible (r > 0.45 for all tests, P < 0.002). Analyses of variance indicated that in contrast to non-surgical treatments, surgery produced significantly more postoperative "discomfort" (P < 0.05) for all variables except experience, sensitivity, altered speech, and comparison to crown preparation. When analyzed according to the type of procedure, soft tissue grafts and osseous surgery produced the most postoperative discomfort. Pearson correlation indicated statistically significant (P < .0001) relationships between pain-related measures (postoperative pain, number of postoperative analgesics, days of pain, days of work missed, and sensitivity). Duration of pain was also found to be related to a history of periodontal treatment (P < .02), but not to previous restorative treatment. These data indicate that patient perceptions of periodontal treatment can be reliably measured and may be used to address appropriateness of care issues.
Adolescent↗
Impact of microbiological consultation on clinical decision making: a case-control study of clinical management of recurrent periodontitis.
Data obtained from diagnostic tests may influence the clinician's perception of the patient's state and in some instances may alter subsequent choices of therapeutic interventions. To determine if microbiological consultation influences the clinical management of patients with recurrent periodontitis, an observational, case-control study was conducted to measure the amount and type of periodontal treatment provided by periodontists (n = 13) who had referred patients with recurrent periodontitis for microbiological consultation. The control group consisted of periodontists (n = 10) who had not referred recurrent periodontitis patients for testing. Patients (n = 31; 20 females, 11 males; mean age 49.8 +/- 10.0 years) treated by the case group of periodontists were matched for age and sex to patients (n = 48; 22 females, 26 males; mean age 49.9 +/- 8.5 years) treated by the control group of periodontists. Questionnaires were administered to quantitatively assess the amount and type of treatment before and after receiving the microbiological report. Specific analyses were performed as a function of the time of receipt of the microbiology report. Case-control differences prior to the receipt of the report indicated that the amount of surgery/year was 43% greater for controls (P < 0.04), in spite of control patients exhibiting fewer deep pockets than case patients (P < 0.05). Case-control differences after the receipt of the report indicated that case patients were provided with 45% greater number of appointments/year (P < 0.005), 46% greater scaling/year (P < 0.02), and 79% greater antibiotics/year (P < 0.01) compared with controls. The report influenced 9 (69%) case periodontists to change treatment. Case patients who received a change in treatment (n = 21) exhibited greater number of deep pockets at the time of entry into the study (P < 0.05) as compared with case patients who did not receive a change in treatment. Paired t-tests of differences within groups before and after the report demonstrated that case patients had a significant increase in treatment after the report as shown by 22% greater number of visits/year (P < 0.05) and 54% greater number of antibiotic prescriptions/year (P < 0.01). In contrast, controls demonstrated no significant change in treatment. Further, contrasts of change in treatment before and after the report again indicated that case patients exhibited a significantly higher number of visits/year (P < 0.04) and number of antibiotic prescriptions/year (P < 0.02) compared with control patients.(ABSTRACT TRUNCATED AT 400 WORDS)
Anti-Bacterial Agents↗
Building a professional image: a caring program for gerontological nurses.
Explore the source record for details and available documents.
Empathy↗
Basic considerations in periodontal wound healing to achieve regeneration.
Explore the source record for details and available documents.
Animals↗
Basic considerations in periodontal wound healing to achieve regeneration.
Explore the source record for details and available documents.
Animals↗
Dexamethasone recruitment of self-renewing osteoprogenitor cells in chick bone marrow stromal cell cultures.
Bone marrow stromal cells are a mixed population that contribute to the formation of the hematopoietic microenvironment. The osteogenic lineage includes populations of cells that, in culture, form discrete nodules of mineralized tissue when grown in the presence of ascorbic acid and sodium beta-glycerophosphate. We have used nodule formation to assay for the self-renewal capacity of osteoprogenitor cells in chick bone marrow cultures. To examine the regulatory influence of dexamethasone (Dx), first subcultures were grown continuously or split 1:1 at repeated subculture. Cells in continuous culture exhibited less than two population doublings, while cellular proliferation and alkaline phosphatase area were inhibited by 10(-8) mol/L Dx. Cells in split (redistributed) cultures exhibited up to 14 population doublings and cellular proliferation was also inhibited by Dx. In contrast with continuous cultures, redistributed cultures treated with Dx had increased alkaline phosphatase area and 15-fold larger amounts of mineralized tissue formation than controls. Osteogenesis was sustained for up to four subcultures and the ratio of mineralized tissue area to alkaline phosphotase positive cell area was at most 0.55. These data indicate that the osteogenic lineage of bone marrow stromal cells contains self-renewing progenitors that are recruited by Dx in culture and that at a maximum, only 55% of the alkaline phosphatase-positive cell population contributes to osteogenesis.
Alkaline Phosphatase↗
Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments.
Access to the cell cytoplasm in viable cells may permit direct labeling or manipulation of intracellular molecules and metabolic processes. One method to gain access to the cell cytoplasm is by electroporation, a technique that transiently creates pores in cell membranes by means of applied electrical fields. We used electroporation to introduce large-molecular-mass dextrans and proteins as probes of the cytoplasmic compartment in human gingival fibroblasts. Electrical field strength and pulse decay time were optimized to obtain cellular viability greater than 80%. Analysis by confocal microscopy and by fluorescence spectrophotometry demonstrated that a large proportion of high-molecular-mass probe was membrane-bound after electroporation. Trypsinization did not affect membrane-bound FITC-dextran but eliminated protein probe incorporated into the membrane, thereby permitting measurement of only intracellular, cytoplasmic label. Proteins of up to 66 kDa were incorporated at intracellular concentrations of 10(-15) M. After electroporation under optimal conditions, incorporated anti-vimentin antibodies were capable of binding to vimentin. Cells electroporated in the presence of RNase A exhibited significant reductions of cellular RNA. Electroporation appears to be a useful approach to probe or perturb specific cellular processes by introduction of functional molecular species into the cytoplasm of viable cells.
Cell Membrane↗
Fibroblastic regulation of osteoblast function by prostaglandins.
The effects of osteogenic inhibitory factors secreted by human periodontal ligament fibroblasts were studied in rat bone marrow stromal cell cultures. Serum-free conditioned medium from cultures of fibroblasts strongly depressed formation of mineralized tissue by bone marrow cell cultures. The inhibitory activity was reduced by treatment of fibroblast cultures with indomethacin or by pretreatment of conditioned medium with specific antibodies to prostaglandins (PGs) E2 and F2 alpha. Passage of conditioned medium over octadecyl columns enriched PGs four-fold and significantly increased inhibitory activity. Inhibition of mineralization was replicated by treatment of bone-cell cultures with PGs B2, D2, E2, F2 alpha and I2 at concentrations of 350 ng/ml to 350 pg/ml. All combinations of these agents were inhibitory but PGE2 and PGF2 alpha exhibited the greatest inhibition at low concentrations (350 pg/ml). These experiments indicate that fibroblasts secrete PGs which can inhibit bone formation, and this may be one mechanism whereby fibroblasts can modulate osteogenesis at the interfaces of soft and mineralizing connective tissues.
Animals↗
Osteogenic phase-specific co-regulation of collagen synthesis and mineralization by beta-glycerophosphate in chick periosteal cultures.
Mineralized bone formation in vitro can be induced by the alkaline phosphatase substrate beta-glycerophosphate (GP). GP may not only be essential for mineralization in vitro, but could also modulate other metabolic activities of bone cells, particularly if GP is presented to these cells during different phases of development. To assess GP modulation of bone cell metabolism, biochemical and autoradiographic analyses of chick periosteal cultures treated with GP were performed. About 50% less (p less than 0.05) Type I collagen was produced in periosteal cultures treated with GP. If the fibrous portion of the periostem was microdissected from the osteogenic layer prior to culture, GP inhibition of Type I collagen synthesis was even more marked (60%: p less than 0.05). To define organic phosphate-sensitive phases of osteogenesis, cultures were exposed to GP for various time periods. Mineralization occurred reproducibly when periosteal cultures were treated with GP from the outset of the incubation period (positive control). However, if GP was added after the third day of incubation, phosphate content was the same as in positive control cultures, whereas calcium content was significantly (20%: p less than 0.05) lower. Moreover, if GP was added on day 6, there was virtually no calcium accumulation by day 12, while massive amounts of phosphate had accumulated. Taken together, these findings indicate that organic phosphates may modulate phenotypic expression of osteogenic cells, and that osteogenic cells traverse an organic phosphate-sensitive phase, after which they may be incapable of normal mineralization.
Animals↗
Gingival crevicular fluid gelatinase and its relationship to periodontal disease in human subjects.
Collagenolytic enzymes released by neutrophils are associated with the destruction of periodontium in periodontal diseases. Measurement of these enzymes in gingival crevicular fluid (GCF) could be used to test for periodontal diseases and thereby simplify diagnosis. To test this hypothesis, gelatinase (MMP-9) was analyzed in GCF samples with a simple assay system. GCF was collected by a mouthrinse method from 10 patients with gingivitis (G); 10 well-treated and maintained periodontitis patients (TP) without detectable loss of attachment; and 9 patients with recurrent loss of periodontal attachment (greater than 2 mm) and/or abscess formation (RP). Clinical measurements including tooth mobility (MOB) and gingival attachment level (GAL) were made monthly for a maximum of 10 months. Active and latent forms of gelatinase were measured by a functional assay using gelatin substrate-gel enzymography and the activities were quantified by laser densitometry. Reproducibility analysis demonstrated that the assay (inter-gel, inter-assay, inter-scan) and diurnal variations were small compared to biological variation. The presence of active gelatinase was detected in 97.8% of TP samples, 86.4% of RP samples, but in only 11.4% of G samples. In addition, the mean active gelatinase activity was found to be significantly higher (p less than 0.001) in the RP (71,006 U) than the TP (43,814 U) groups, both of which were higher (p less than 0.001) than the G group (2824 U). During periods of attachment loss, samples from the RP group exhibited a 2-fold increase of mean active gelatinase activity (129,414 U).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Treponema denticola induces actin rearrangement and detachment of human gingival fibroblasts.
Spirochetes are associated with destructive periodontal diseases, and one cultivatable oral species, Treponema denticola, binds to mammalian cells and perturbs metabolism. To evaluate the cytoskeletal responses and attachment functions of human gingival fibroblasts (HGF) exposed to T. denticola, monolayers of HGF were incubated with T. denticola strains ATCC 35405, e, and e' in serum-free medium. HGF retracted pseudopods, rounded up, and ultimately detached from the substratum. Scanning electron microscopy showed spirochetes in close contact with HGF surfaces; occasionally, bacteria were partially submerged between folds in the HGF membrane. Blebbing and numerous microvilli formed on the cell surface as the HGF retracted. By confocal microscopy, spirochetes were detected in contact with the HGF surface but were never found on the ventral surface of fibroblasts between the substratum and cell. Morphological alterations were associated with and preceded by actin assembly, as measured by microscopic fluorimetry: there was a 263% increase in actin fluorescence over controls within 30 min. Detachment of fibroblasts from the substratum was related to incubation time and was dependent on the concentration of T. denticola. Detachment was observed for all strains tested and was also dependent on the viability of T. denticola: UV light, heat, and metronidazole treatment markedly reduced the HGF detachment response. Detachment was also significantly reduced by the protease inhibitor phenylmethylsulfonyl fluoride. HGF viability was not significantly affected by coincubation with spirochetes, as measured by lactate dehydrogenase release. Thus, T. denticola induces rapid cytoskeletal remodelling followed by cell detachment, which might be stimulated by a bacterially associated protease but is not likely directly mediated by proteolytic degradation at the cell-substratum adhesive contact points.
Actins↗
Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.
Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.
Actins↗