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C A McCulloch

Publications and source records attributed to C A McCulloch.

At least 127 records · Page 7Linked to original sources
PubMed1992

Direct flow cytometric quantification of alkaline phosphatase activity in rat bone marrow stromal cells.

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.

Alkaline Phosphatase↗
PubMed1992

Serial doxycycline and metronidazole in prevention of recurrent periodontitis in high-risk patients.

The efficacy of metronidazole and doxycycline in preventing recurrent periodontitis was studied in 23 patients. After treatment in the previous 7 months with either bimonthly scaling and 3 weeks of systemic doxycycline (11 subjects) or scaling and placebo (12 subjects), patients were monitored for recurrent periodontitis and were scaled every 2 months. When either a periodontal abscess or greater than 2 mm loss of gingival attachment was observed, metronidazole was administered (250 mg every 8 hours) for 10 days. In the placebo plus metronidazole group, 5 patients (42%) exhibited recurrent periodontitis after the metronidazole regimen compared with only one (9%) in the doxycycline plus metronidazole group (P less than 0.096). Subgingival plaque samples at study and healthy control sites were screened for the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Eikenella corrodens, and Fusobacterium nucleatum by immunofluorescence and for spirochetes using Ryu's stain. Presence/absence analysis of the sum of scores of the 6 individual pathogens demonstrated large reductions (P less than 0.005) in the frequency of detection of pathogens in the former doxycycline compared with the placebo plus metronidazole group at both study and control sites before and one month after metronidazole. By 7 months after metronidazole, there was no detectable difference between groups. These results indicate that prevention of recurrent periodontitis with metronidazole may be enhanced by previous treatment with doxycycline.

Bacteria↗
PubMed1992

Measurement of oral malodor: current methods and future prospects.

Measurement of oral malodor is complicated by a variety of parameters including complexity of gaseous molecular species, sampling difficulties, temporal variations, choice of suitable subject populations, and lack of agreement on reference standards. Since oral malodor is a perceived olfactory stimulus, direct sampling and assessment by human judges may be the most logical measurement approach. However, as with other psychophysical assessments, human malodor measurement by the human nose may vary widely among and between judges, and consequently cannot be confidently reproduced in other laboratories. Such shortcomings have led several investigators to propose quantitative approaches based on measurement of volatile sulfide compounds which are associated with oral malodor. Highly sensitive and discriminatory measurements of volatile sulfides can be made using gas chromatography although for rapid sampling of larger subject populations, portable sulfide monitors may be more appropriate. Future research in this field should consider: 1) improved and simplified instrumentation for more rapid through-put and reliability; 2) development and definition of reference standards for oral malodor assessment; 3) formulation of clinical studies with appropriately sized, well-defined patient populations; and 4) further development of within mouth, site-specific measurements.

Halitosis↗
PubMed1991

Osteogenic progenitor cells in rat bone marrow stromal populations exhibit self-renewal in culture.

Marrow stromal cells are a heterogeneous population, comprising a variety of lineages including osteogenic cells. In the presence of ascorbic acid, sodium beta-glycerophosphate, and dexamethasone, rat bone marrow stromal cells form discrete nodules of mineralized, bonelike tissue. We used nodule formation by rat bone marrow stromal cells to assay for the self-renewal capacity of osteogenic progenitor cell populations. Cultures were subcultured every 5 days up to six times. Osteogenesis was assayed from second to sixth subcultures by counting the number and measuring the areas of mineralized nodules formed in cultures grown with 10(-8) mol/L dexamethasone. Nodule number and area decreased progressively between second and sixth subcultures. Alkaline phosphatase activity associated with individual cells and measured videodensitometrically decreased exponentially between the second and sixth subculture. The number of cells with alkaline phosphatase activity also decreased with progressive subculturing. The proportions of 3H-thymidine-labeled cells after continuous labeling from the beginning of the culture period showed 90% labeling for cells with alkaline phosphatase activity and fibroblastlike cells. Cultures labeled for only the first 3 days exhibited higher labeling of alkaline phosphatase-positive cells than fibroblastlike cells (P less than .05). Cultures that were flash-labeled at the end of the culture period demonstrated low labeling indices for cells with alkaline phosphatase activity and up to 10-fold higher labeling indices for fibroblastlike cells. Separate cultures treated with a cytocidal dose of high specific activity 3H-thymidine did not form nodules. These results indicate that osteogenic progenitor cells or another cell type required for nodules to develop must divide early in culture if nodule formation is to occur, and that osteoprogenitor cells express a limited capacity for self-renewal.

Alkaline Phosphatase↗
PubMed1991

Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro.

The ability of rat skin fibroblasts (RSF) and human periodontal ligament fibroblasts (HPL) to inhibit the formation of mineralised bone nodules in rat bone marrow stromal cell (BMSC) cultures was studied. Co-culture of HPL or RSF with BMSC resulted in a large reduction of bone nodule formation when compared with controls. Conditioned medium from HPL or RSF cultures inhibited bone nodule formation in a dose-dependent manner. HPL-conditioned medium depressed cell proliferation and alkaline phosphatase expression in BMSC cultures. These effects were not due to increased cytotoxicity or nutrient depletion. Inhibitory activity was recovered in a fraction of less than 1 kD following ultrafiltration and was insensitive to freeze-thawing. The inhibitory activity was blocked when HPL cultures were grown in the presence of 10(-5) M indomethacin. Dose-dependent inhibiton of bone nodule formation was also observed in cultures incubated with prostaglandins E2 (at 10(-6) M) or F2 alpha (at 10(-7) M). The results indicate that fibroblasts may inhibit osteoblast differentiation and function in part by release of soluble factors including prostaglandins.

Alkaline Phosphatase↗
PubMed1991

Discrimination of two fibroblast progenitor populations in early explant cultures of hamster gingiva.

Numerous metabolic studies have demonstrated heterogeneity of fibroblast populations in culture, yet little is known about the structure of fibroblast populations in adult tissues in vivo. To determine if populations of both cycling and non-cycling cells are present in gingiva, hamsters were labelled with [3H]-thymidine to label cycling cells in vivo, and explanted biopsies were subsequently incubated with bromodeoxyuridine to label cycling cells in vitro. Cycling cells were identified by combined immunohistochemistry and radioautography. Fibroblasts were recognized by the presence of vimentin and the absence of keratin as determined by immunofluorescence. The largest proportion of cells were double-labelled with [3H]-thymidine and bromodeoxyuridine (43.8%) indicating the presence of actively cycling populations that maintained their proliferative status upon explanation. Cultures also exhibited a second population of cells labelled only with bromodeoxyuridine (38.7%) that did not cycle in vivo, but retained the capacity for proliferation in vitro. However, limiting dilution analysis of single-cell suspensions revealed only a single class of progenitors capable of forming large colonies in vitro. Approximately 1 in 190 plated cells was capable of colony-formation, indicating that, upon explanation, a subset of the cycling cells in vitro exhibits extensive proliferative capacity. There was also a small population of cells unlabeled with either [3H]-thymidine or bromodeoxyuridine (9.4%) that appeared to be terminally differentiated. Different substrates, including glass and thin films of gelatin and collagen, did not significantly alter the fraction of cells labelled with [3H]-thymidine. These data demonstrate the existence of 2 separate progenitor-cell populations with different capacities for proliferation in vivo and in vitro.

Animals↗
PubMed1991

Immunohistochemical localization of bone sialoprotein in foetal porcine bone tissues: comparisons with secreted phosphoprotein 1 (SPP-1, osteopontin) and SPARC (osteonectin).

Bone sialoprotein (BSP) is a prominent component of bone tissues that is expressed by differentiated osteoblastic cells. Affinity-purified antibodies to BSP were prepared and used in combination with biotin-conjugated peroxidase-labeled second antibodies to demonstrate the distribution of this protein in sections of demineralized foetal porcine tibia and calvarial bone. Staining for BSP was observed in the matrix of mineralized bone and also in the mineralized cartilage and associated cells of the epiphysis, but was not observed in the hypertrophic zone nor in any of the soft tissues including the periosteum. In comparison, SPP-1 (osteopontin) and SPARC (osteonectin), which are also major proteins in porcine bone, were observed in the cartilage as well as in the mineralized bone matrix. In addition, SPARC was also present in soft connective tissues. Although SPP-1 distribution was more restricted than SPARC, hypertrophic chondrocytes, periosteal cells and some stromal cells in the bone marrow spaces were stained in addition to osteoblastic cells. The variations in the distribution and cellular expression of BSP, SPARC and SPP-1 in bone and mineralizing cartilage indicate these proteins perform different functions in the formation and remodelling of mineralized connective tissues.

Animals↗
PubMed1991

Role of fibroblast subpopulations in periodontal physiology and pathology.

Fibroblasts are the principal cell type in the soft connective tissues of the periodontium; they perform important functions in development, physiology, and disease. A growing number of reports have indicated site-specific phenotypic variation of fibroblasts. Heterogeneity of metabolic traits has been demonstrated in cells from healthy and diseased tissues. The tissue distribution and relative proportions of fibroblast subpopulations have a significant impact on the regulation of connective tissue function in health and disease.

Cell Differentiation↗
PubMed1991

Collagenase activity in recurrent periodontitis: relationship to disease progression and doxycycline therapy.

Previous reports have suggested that active progression of periodontitis may be correlated with increased collagenolytic activity, and that improved clinical conditions after tetracycline treatment may be explained by inhibition of host collagenase. Eighty-two patients with a recent history of periodontal abscesses and/or loss of gingival attachment level (GAL) despite active periodontal therapy were enrolled in a double-blind, randomized, placebo-controlled trial. Clinical measurements, sampling of gingival crevicular fluid (GCF) and subgingival scaling were performed every 2 months. If any site exhibited greater than 2 mm loss of GAL or a periodontal abscess, patients were administered either 100 mg doxycycline per day for 3 weeks or placebo. During 12 months of monitoring, 55 patients exhibited recurrent active disease and were then randomly assigned to either the doxycycline (n = 30) or placebo (n = 25) groups. Analysis of active collagenase and latent collagenase in GCF samples were determined by functional assays and quantitated after SDS-PAGE and fluorography. Collagenase activities were assayed at sites exhibiting active destruction (study site), at sites with pocket depth comparable to the study site but without active destruction, and at healthy sites. Clinical measurements of GAL and collagenase activity were made at intervals between 1 wk and 7 months after completion of the drug regime. Within 7 months, 15 out of 19 patients on placebo exhibited recurrent disease compared to 13 out of 29 patients on doxycycline. Collagenase activity exhibited large variations among patients and was analyzed as presence or absence of active collagenase with a logistic model.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1991

Evidence for polymorphonuclear leukocyte collagenase and 92-kilodalton gelatinase in gingival crevicular fluid.

Analysis of inflammatory exudate collected from sites of experimental periodontitis in cynomolgus monkeys has revealed the presence of collagenase and a 92-kDa gelatinase that comigrated after electrophoresis with the 92-kDa gelatinase released from polymorphonuclear leukocytes. Since neutralizing antibodies to fibroblast collagenase had no effect on the collagenase activity and bacterial collagenases could not be detected, polymorphonuclear leukocytes appear to be the major source of collagenolytic proteinases in inflammatory fluid from gingiva.

Animals↗
PubMed1991

Quantitative immunohistochemical analysis of keratins and desmoplakins in human gingiva and peri-implant mucosa.

Quantitative immunohistochemistry was used to compare the distributions of keratins and desmoplakins in human gingiva and peri-implant mucosa (three specimens each). In gingiva, keratin 1 (a marker of cornification) and desmoplakins I & II (markers of desmosomes) stained most heavily in granular strata followed by corneal strata; keratin 13, a marker of non-cornifying stratified squamous cells, stained most heavily in suprabasal strata of oral sulcular epithelium. Keratin 19, a marker for junctional epithelium, stained the basal stratum of oral sulcular epithelium most heavily. In peri-implant mucosa, the patterns of staining were similar, except that staining for desmoplakins I & II was generally significantly reduced compared with gingiva, and junctional epithelium co-expressed keratins 13 and 19. Peri-implant junctional epithelial cells attached to titanium implant abutments were removed by trypsin/EDTA digestion, and also exhibited co-expression of keratins 13 and 19. Inflammatory cell infiltration was associated with reduction of keratin 1 staining in gingiva. The data indicate that the epithelia of gingiva and peri-implant mucosa are not composed of identical cell populations.

Analysis of Variance↗
PubMed1991

Reproducibility and sensitivity of oral malodor measurements with a portable sulphide monitor.

Forty-one subjects with bad breath were assessed for oral malodor and periodontal status on three occasions, at intervals of approximately one week. Oral malodor was assessed by measurement of peak and steady-state volatile sulphide levels with a portable sulphide monitor and by organoleptic measurement of whole-mouth, tongue dorsum, and interproximal dental odors by two independent judges. Reproducibility of measurements, assessed by paired t tests and Kappa testing, demonstrated no significant differences between any of the test results from the first and second appointments. Steady-state sulphide levels were the most reproducible of all tests. The ability of the tests to detect an expected reduction of malodor following a 0.2% chlorhexidine mouthrinse regimen was investigated by comparison of test values between the second and third appointments. Following the mouthrinsing treatment, 43% reductions of peak, 47% reductions of steady-state volatile sulphide levels, and 15-58% reductions in all other measurement categories were observed. The majority of the participants (22/41) had no pockets greater than 5 mm and exhibited both moderate gingival inflammation (Mean Gingival Index = 1.17) and moderate plaque accumulation (Mean Plaque Index = 1.84). Plaque Index and presence of pockets greater than 7 mm were weakly related to sulphide measurements. Whereas assessment of steady-state sulphide levels by the sulphide monitor does not constitute a direct measure of oral malodor, its relation to organoleptic measurement, superior reproducibility, objectivity, and sensitivity support the use of the sulphide monitor in clinical studies.

Breath Tests↗
PubMed1991

Quantification of chemotactic response of quiescent and proliferating fibroblasts in Boyden chambers by computer-assisted image analysis.

The chemotactic response of human gingival fibroblasts to platelet-derived growth factor (PDGF) was investigated in 48-well modified Boyden chambers. Results were quantified using computer-assisted image analysis of propidium iodidestained cells and were compared with results obtained using the conventional method of quantification by direct counting. Quantification by image analysis was found to be rapid and accurate, and correlated closely with results obtained by direct counting. Bromodeoxyuridine (BrdU)-labeled proliferating cells were double stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antiserum. The cycling cells showed a markedly reduced chemotactic response to PDGF, whereas cells in S-phase of the cell cycle did not show any response. This method of quantification of results of Boyden chamber assays is simple and reliable, and allows the use of double labels to investigate the chemotactic response of subpopulations of cells within a heterogeneous population of cells.

Cell Cycle↗
PubMed1991

Immunohistochemical localization and quantification of desmoplakins I & II and keratins 1 and 19 in plastic-embedded sections of human gingiva.

We developed immunohistochemical and image analytical techniques to localize and quantify keratins and desmoplakins in sections of plastic-embedded human gingiva. Acetone fixation followed by plastic embedding of gingiva provided excellent morphology and permitted immunohistochemical detection of keratins 1 and 19 and desmoplakins I & II after 2.5-min trypsin digestion. Quantitative image analysis demonstrated that different volume densities of staining of each marker were associated with specific epithelial strata. Keratin 1 stained most heavily in granular strata, followed by corneal and spinous strata; keratin 19 stained most strongly in the basal layer; desmoplakins I & II stained most strongly in the granular and corneal strata. These findings confirm that variations of keratin and desmoplakin expression in these epithelial are associated with regional patterns of epithelial differentiation.

Cell Differentiation↗
PubMed1991

Quantitation of actin polymerization in two human fibroblast sub-types responding to mechanical stretching.

To study early reorganization of the cytoskeleton in response to physical forces, human gingival and periodontal ligament fibroblasts were cultured on flexible plastic substrata and stretched by mechanical deformation of the substratum. F-actin was measured by quantitative spectrofluorimetry of FITC-phalloidin-stained cells. Fluorescence due to FITC-phalloidin was reduced stoichiometrically by co-incubation with unlabeled phalloidin. Increases in stretch-induced fluorescence were blocked by preincubation of cells with cytochalasin D. Compared to baseline values, F-actin in gingival fibroblasts was reduced by 50% at 10 s after stretching but was increased more than 100% at 50 s after stretching. Increases of F-actin were also dependent on the amount of deformation of membrane: 3.3% stretching produced the largest increase and 9.2% stretching produced the least. Stretch-activated actin polymerization was blocked by pre-incubation of cells with 500 ng ml-1 pertussis toxin or 10 mM EGTA. Compared to gingival cells, periodontal ligament fibroblasts exhibited twice the total actin per cell and three times the baseline F-actin per mm2, however, there was no increase in fluorescence after stretching. In both cell types, stretching caused increased ruffling activity and no detectable shape changes at 60 s, but at 180 s the cells shortened and pseudopods retracted. The microfilament system of fibroblasts exhibits rapid dynamic responses to mechanical deformation prior to cell shape changes and appears to be dependent on the cell type.

Actins↗
PubMed1991

Mechanism of collagen phagocytosis by human gingival fibroblasts: importance of collagen structure in cell recognition and internalization.

Phagocytosis of extracellular collagen by fibroblasts appears to be the principal pathway of collagen degradation in the physiological turnover of connective tissues. To study the mechanism of collagen phagocytosis, subconfluent gingival fibroblasts were serum-starved and incubated for up to 16 h with collagen-coated fluorescent latex beads. Internalization of beads was measured either by flow cytometry or by image analysis. Phagocytosis was blocked by inactivation of protein kinase C with staurosporin, and was also decreased significantly (32%) when cells were pre-incubated for 6h with cycloheximide. Phagocytosis of collagen-coated beads appeared to be receptor-mediated, since internalization was inhibited threefold by the cell-attachment blocking peptide (GRGDSP). The process of internalization was influenced by the type of collagen and its molecular structure. Thus, internalization was decreased in the order: type I greater than V greater than III collagen, and internalization of type I collagen was reduced significantly by digestion with either bacterial (45%) or vertebrate (38%) collagenase. However, collagen denaturation, which facilitates binding to fibronectin, did not effect internalization. Although concanavalin A stimulated both phagocytosis (71%) and collagenase synthesis, PMA and IL-1, which also increase collagenase expression, did not affect phagocytosis, indicating that phagocytosis of collagen-coated beads does not require collagenase. Moreover, analysis of tissue inhibitor of metalloproteinase expression revealed no difference between phagocytic and non-phagocytic cells. Collectively, these results demonstrate that collagen phagocytosis is regulated through protein kinase C and is also dependent upon cellular recognition and collagen structure, but not on the expression of collagenase.

Alkaloids↗
PubMed1991

A randomized, placebo-controlled trial of doxycycline: effect on the microflora of recurrent periodontitis lesions in high risk patients.

Twenty-seven patients with a recent history of periodontal abscesses and/or loss of gingival attachment level (GAL) despite active periodontal therapy were enrolled in a double-blind, randomized, placebo-controlled trial. Clinical measurements and subgingival scaling were performed every 2 months. When a site exhibited greater than or equal to 2 mm loss of GAL or a periodontal abscess, patients were administered either doxycycline at a dosage of 200 mg to start and 100 mg per day for 3 weeks, or a placebo. Clinical measurements of GAL and microbial analysis of subgingival plaque at study and control sites were made at the time of active disease and at intervals of 1 week and 7 months after completion of the drug regime. Plaque samples were screened for the presence of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Bacteroides intermedius (Bi), Eikenella corrodens (Ec) and Fusobacterium nucleatum (Fn) by indirect immunofluorescence antibody technique and for spirochetes (Sp) using Ryu's stain. Based on presence or absence analysis of the sum scores of the 6 pathogens, both the placebo (n = 10) and the doxycycline groups (n = 17) exhibited similar scores at the time of detection of active disease (mean placebo = 2.38 +/- 0.32; mean doxycycline = 2.95 +/- 0.27; P = 0.18). One week after treatment, the probability of detection was unchanged in the placebo group (mean placebo = 3.14 +/- 0.47), but was significantly reduced in the doxycycline group (mean doxycycline = 1.77 +/- 0.26; P = 0.0002). Study (active) sites exhibited scores 2 to 3 times higher than control (inactive) sites before doxycycline treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗