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Biomedical subjects

M I Cho

Publications and source records attributed to M I Cho.

At least 37 records · Page 2Linked to original sources
PubMed1994

Differentiation of periodontal ligament fibroblasts into osteoblasts during socket healing after tooth extraction in the rat.

BACKGROUND: The entire socket after tooth extraction is filled with new bone formed by osteoblasts (Obs), but the origin of these Obs remains unknown. Thus, the proliferation and migration of paravascular and endosteal fibroblastic cells and periodontal ligament (PDL) fibroblasts (Fbs) and their differentiation into Obs during socket healing after extraction of the first maxillary molars of the rat were investigated. METHODS: The proliferative activity and migration of these cells in the sockets after tooth extraction were studied using radioautography and immunohistochemistry after injection of 3H-thymidine and 5-bromo-2'-deoxyuridine (BrdU), respectively. Their morphological changes during differentiation was investigated by transmission electron microscopy. RESULTS: One day after tooth extraction, PDL Fbs were the major cell type in the PDL remnant of the socket. Proliferation was low (labeling index (LI) = approximately 2%) until 16 h after tooth extraction but dramatically increased to a maximum level 1 day postextraction (LI = 23%). Between 1 and 2 days, numerous PDL Fbs in the PDL remnant actively migrated into the coagulum and continued to proliferate. On the basis of the high proliferative activity and small number of cellular organelles responsible for procollagen synthesis, these cells appear immature. At 3 days, Fbs contained more cellular organelles and deposited more collagen fibers as they replaced the coagulum with dense connective tissue and the LI declined. At 4 and 5 days, some of the Fbs began to differentiate into Obs, and the proliferation of Fbs dramatically decreased to baseline values. The migration of PDL Fbs and their differentiation into Obs were investigated by labeling with 3H-thymidine or BrdU 1 day after tooth extraction. Heavily labeled Fbs were observed in the PDL remnant at 1 day, in the coagulum at 2 days, and in the dense connective tissue at 3 days. Labeled Obs associated with new bone were seen 4 days after injection. Endosteal and paravascular Fbs also proliferated, but at a lower level and at later time periods than the PDL Fbs. Surprisingly, endosteal and paravascular Fbs contributed only a small population of Fbs to socket healing. CONCLUSIONS: These results indicate that PDL Fbs after tooth extraction actively proliferative, migrate into the coagulum, form dense connective tissue, and differentiate into Obs which form new bone during socket healing.

Animals↗
PubMed1994

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.

Alveolar Bone Loss↗
PubMed1994

Clinical, microbiological, and histological factors which influence the success of regenerative periodontal therapy.

The primary objectives of this double-blind, controlled clinical trial were to assess factor(s) which affect the success of guided tissue regeneration (GTR) procedures in mandibular Class II buccal furcation defects. Thirty subjects, with mandibular Class II furcation defects, were randomly assigned to one of two treatment groups; patients in Group A received oral hygiene instructions with scaling and root planing, while subjects in Group B received similar treatment but without subgingival scaling and root planing at the affected site. After initial oral hygiene instructions and scaling and root planing, GTR surgery was performed using ePTFE barrier membranes. Membranes were retrieved at 6 weeks and subjected to histological examination. Twelve months after regenerative therapy, clinical measurements and re-entry surgical measurements were repeated. Probing reduction (2.61 mm), horizontal probing attachment gain (2.59 mm), and vertical probing attachment gain (0.95 mm) were all significantly better compared to baseline. Likewise, significant improvements in furcation volume (8.0 microliters) and in bone measurements were observed at re-entry. There was no discernible difference between subjects for whom complete anti-infective therapy was deferred to the time of the surgery (Group B) compared to subjects in whom complete anti-infective therapy was performed as part of the hygienic phase of therapy (Group A). Pre-operative pocket depth was directly correlated with the magnitude of attachment gain as well as the amount of new bone formation in the furcation area. Subjects who maintained good oral hygiene and who had minimal gingival inflammation throughout the study demonstrated consistently better regenerative response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Interaction of HIV-1 and human salivary mucins.

Previous studies have suggested that salivary secretions may act as inhibitors of HIV-1 replication in vitro. This inhibitory activity was determined to be associated mainly with secretions obtained from the human submandibular-sublingual glands, and subsequent electron micrographs revealed the association of viral particles with the salivary sediment. Fractionation of human submandibular-sublingual (HSMSL) saliva by size-exclusion chromatography was initiated, and resulting fractions were tested for their ability to modulate the replication of HIV-1 using a plaque assay on HeLa CD4+ cell monolayers. Results indicated that the filtration-sensitive inhibitory activity was primarily associated with the mucin-rich fractions, and the inhibitory activity was found to reduce the number of infectious units by 75%. To determine the identity of the salivary components involved, adsorption experiments involving the interaction of HIV particles with immobilized salivary components were performed. Immunological counter staining revealed an interaction of HIV particles as well as recombinant gp120 with the lower-molecular-weight mucin. Electron microscopic examination of the mucin-rich fractions-HIV incubates revealed the aggregation of virus particles by salivary components. These results suggest that human salivary mucins may have a role in modulating the infectivity of HIV-1.

Adult↗
PubMed1993

Evidence for up-regulation of epidermal growth-factor receptors on rat periodontal ligament fibroblastic cells associated with stabilization of phenotype in vitro.

This study sought to understand the role of epidermal growth factor receptor (EGF-R) in periodontal ligament (PDL) fibroblasts. Rat PDL fibroblastic cells and ROS 17/2.8 cells (highly differentiated osteoblastic osteosarcoma cells) were cultured and treated with transforming growth factor-alpha (TGF-alpha), EGF, dexamethasone (Dex) or a combination of EGF and Dex. Alkaline phosphatase (ALP) activity, an early differentiation marker for mineralized tissue-forming cells, was measured using p-nitrophenylphosphate as a substrate. For Scatchard analysis of [125I]-EGF binding, cells were incubated in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 0-64 ng/ml of [125I]-EGF for 4 h at 4 degrees C. Also, the synthesis of EGF-R protein and the expression of mRNA for EGF-R were measured by immunoprecipitation and Northern blot analysis, respectively. Untreated PDL fibroblastic cells showed a gradual increase in spontaneous ALP activity from 32.4 U/10(6) cells at 2 days to 49.6 U/10(6) cells at 7 days of culture. ALP activity was further increased to 70.8 U/10(6) cells at 7 days after treatment with Dex, whereas EGF treatment reduced it to 19.4 U/10(6) cells. Culture of PDL fibroblastic cells in the presence of a combination of Dex and EGF decreased the Dex-induced ALP activity from 70.8 U to 41.8 U/10(6) cells at 7 days. A similar inhibitory effect on ALP activity was found after treatment with TGF-alpha. In contrast, ROS cells maintained a high ALP activity (1748 U/10(6) cells) throughout culture, unaffected by EGF. Scatchard analysis demonstrated that PDL fibroblastic cells have both high- and low-affinity forms of EGF-R, while ROS cells did not have any detectable EGF-R. Treatment of PDL cells with Dex for 2 days decreased the synthesis of EGF-R protein, the expression of EGF-R mRNA and the number of EGF-R. In contrast, EGF treatment increased the expression of EGF-R mRNA. These data suggest that PDL fibroblastic cells express numerous EGF-R, but the number decreases during their differentiation into mineralized tissue-forming cells under the influence of Dex. Thus, EGF-R may function in the stabilization of phenotype in PDL fibroblastic cells.

Alkaline Phosphatase↗
PubMed1993

Aggregation of human immunodeficiency virus type 1 by human salivary secretions.

Human immunodeficiency virus (HIV-1) is generally transmitted by parenteral contact with infected body secretions. Although extensive epidemiological data and familial studies have failed to provide any conclusive data that saliva may act as a vehicle for transmission of AIDS, both professional and public anxieties remain. The present study, as well as others, suggests that salivary secretions may act as inhibitors of HIV-1 replication in vitro. In our study, the inhibitory activity was determined to be associated mainly with secretions obtained from the human submandibular-sublingual glands. Human submandibular-sublingual (HSMSL) and parotid (HPS) salivas were collected and tested for their ability to modulate the replication of HIV-1, using a plaque assay on HeLa/CD4+ cell monolayers. Initial results examining freshly collected salivary samples from ten individuals confirmed the results previously obtained by Fox et al. (1988, 1989). An average plaque reduction of approximately 66% was obtained with HSMSL, in contrast to 34% reduction obtained with HPS. Titration of the inhibitory activity in HSMSL showed detectable levels at a 1:500 dilution. Comparison of inhibitory activity of dialyzed and lyophilized saliva to fresh saliva indicated little difference between the two samples when filtration occurred after the addition of HIV-1. However, the effect of filtration was significantly diminished in the lyophilized samples. Electron microscopic examination of the saliva-HIV incubates revealed the aggregation/entrapment of virus particles by salivary components. These results suggest that human salivary secretions (with HSMSL > HPS) may have a role in modulating the infectivity of HIV-1.

Adult↗
PubMed1992

In vitro formation of mineralized nodules by periodontal ligament cells from the rat.

The purposes of this study were to determine whether periodontal ligament (PDL) cells are capable of producing mineralized nodules in vitro and to analyze ultrastructural features of the nodules. Rat PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured at confluence in standard medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. To test mineralized nodule formation, cells were further cultured for an additional 3 weeks in the standard medium containing (1) ascorbic acid (50 micrograms/ml) and sodium beta-glycerophosphate (10 mM), (2) ascorbic acid, sodium beta-glycerophosphate, and dexamethasone (5 microM), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Three-dimensional nodules containing mineralized matrices were formed only when the cells were cultured in the presence of ascorbic acid and dexamethasone. They were composed of multilayered fibroblasts (up to 13 layers), and highly organized collagen fibrils with 64 nm cross-banding patterns between the cell layers. The fibroblasts in the nodules exhibited an elongated shape with a high degree of cytoplasmic polarity throughout the nodule, and have the morphological features of PDL fibroblasts as seen in vivo. Mineral deposition with needle-like crystals was initiated on collagen fibrils located in intercellular spaces of the upper cell layers and became increasingly heavier towards the bottom half of the nodules. X-ray microanalysis and electron diffraction analysis confirmed that mineral deposition contained calcium and phosphate in the form of immature hydroxyapatite.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1992

Characterization of an amylase-binding component of Streptococcus gordonii G9B.

The goal of the present study was to begin characterizing the amylase-binding component(s) on the surface of Streptococcus gordonii G9B. Alkali extracts but not phenol-water extracts of this bacterium inhibited 125I-amylase binding to S. gordonii G9B. To identify the bacterial components involved in amylase binding, the alkali extract was subjected to affinity chromatography on amylase-Sepharose. Immunoblotting with a rabbit antiserum against S. gordonii G9B revealed that a 20-kDa streptococcal component was eluted from the amylase-Sepharose with 1% sodium dodecyl sulfate (SDS), 2 M KSCN, or 0.1 M sodium citrate buffer, pH 4.5. Subsequently, the 20-kDa component was prepared from alkali extracts by electroelution from preparative SDS electrophoresis or by gel filtration chromatography. This component was trypsin sensitive, and an antibody raised against it inhibited the binding of 125I-amylase to S. gordonii G9B. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated that both bound amylase and the 20-kDa component were localized to the cell division septum on dividing cells or to polar zones on single cells. In addition, exponentially growing bacteria bound more 125I-amylase than stationary-phase cells did. Collectively, these results suggest that a 20-kDa amylase-binding component is present on the surface of the nascent streptococcal cell wall.

Amylases↗
PubMed1992

Mitogenic, chemotactic, and synthetic responses of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro.

The mitogenic, chemotactic, and synthetic responses of rat periodontal ligament (PDL) fibroblastic cells to epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), recombinant human platelet-derived growth factor (rhPDGF)-AB, rhPDGF-BB, natural (n) PDGF-AB, and insulin-like growth factor-I (IGF-I) were examined in vitro using PDL cells obtained from the coagulum of healing tooth sockets. PDGFs and IGF-I have potent and comparable mitogenic effects on PDL fibroblastic cells. The maximum mitogenic effect of PDGFs was observed at the concentration of 10 ng/ml, whereas that of IGF-I was seen at concentrations higher than 100 ng/ml. In contrast, EGF induced moderate, and TGF-beta inhibitory mitogenic responses. The combination of rhPDGF-AB with either EGF or TGF-beta demonstrated comparable mitogenic potency, equivalent to the level of PDGF alone regardless of the mitogenic effect of other growth factors. The combination of rhPDGF-AB and IGF-I, however, showed a synergistic effect revealing the highest mitogenic effect among all individual growth factors as well as any combinations of the growth factors tested. Similarly, PDL fibroblastic cells demonstrated strong chemotactic responses to both IGF-I and PDGFs. The maximum effect was observed by IGF-I at concentrations higher than 10 ng/ml, followed by rhPDGF-BB at 0.1 ng/ml, rhPDGF-AB and nPDGF at concentrations ranging from 0.1 to 1 ng/ml. TGF-beta revealed no, and EGF slightly increased, chemotactic effects. IGF-I slightly enhanced the synthesis of total protein, whereas other factors had no significant effect. However, both rhPDGF-AB and TGF-beta stimulated collagen synthesis. On the other hand, IGF-I showed no effect on collagen synthesis, while EGF suppressed collagen synthesis. These findings suggest that rhPDGF-BB and IGF-I stimulate proliferation and chemotaxis of PDL fibroblastic cells. In addition, the combination of these growth factors further increases the mitogenic effect. rhPDGF-AB also stimulates collagen synthesis by PDL fibroblastic cells. Thus, rhPDGF-BB and IGF-I may have important roles in promotion of PDL healing, and consequently, may be useful for clinical application in periodontal regenerative procedures.

Animals↗
PubMed1991

Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.

Bacterial Proteins↗
PubMed1991

Occurrence of epidermal growth factor-binding sites during differentiation of cementoblasts and periodontal ligament fibroblasts of the young rat: a light and electron microscopic radioautographic study.

Occurrence of epidermal growth factor (EGF)-binding sites during differentiation of cementoblasts and periodontal ligament (PDL) fibroblasts was investigated using radioautography after I. V. injection of 125I-EGF to 14-day-old rats. During differentiation of cementoblasts, a very low level of EGF-binding sites was present on the mesenchymal cells in dental follicle proper, precementoblasts, and cementoblasts. On the other hand, during differentiation of PDL fibroblasts, numerous EGF-binding sites were observed on the undifferentiated paravascular cells and on the perifollicular mesenchymes representing the major source of PDL fibroblast precursor cells. Also heavy labeling was observed throughout their differentiation to PDL fibroblasts, as well as during full synthetic activity as mature cells. Quantitative analysis of the light microscopic radioautographs revealed that these cells demonstrated approximately 4 grains per 100 microns 2 of cell area. These results suggest that EGF plays an important role in differentiation of PDL fibroblasts, but not in that of cementoblasts. Furthermore, the well-known in vivo effect of EGF in producing precocious eruption of teeth may be a consequence of a more extensive effect of EGF throughout differentiation of PDL fibroblasts as well as during full synthetic activity as mature cells.

Animals↗
PubMed1991

Purification, characterization, and localization of a major membrane protein antigen from Porphyromonas (bacteroides) gingivalis.

Humans and rats infected with P. gingivalis develop a strong immune response to a 75 kDa major membrane component of P. gingivalis and hence knowledge of the nature of this molecule may aid in understanding the host response to P. gingivalis during infection. Purification of the 75 kDa protein was achieved by repeated precipitation from a crude sonicate of P. gingivalis 2561 at pH 5.0. Homogeneity of the purified 75 kDa protein was confirmed by SDS-PAGE and Western immunoblot analysis using monoclonal and polyclonal antibodies. The purified protein revealed an apparent molecular mass of 300 kDa in native form. Although most of the strains of P. gingivalis tested showed a major membrane protein band in the range of 61 to 78 kDa, on Western immunoblot analysis only the strains which have proteins in the range of 75 to 78 kDa were reactive with anti-75 kDa protein polyclonal antibodies. Affinity purified polyclonal antibodies were used to localized 75 kDa protein on the cell surface of P. gingivalis 2561 by immunogold electron microscopy. Immunolabeling of the 75 kDa protein demonstrated specific localization of the protein along the outer cell membrane, but not on the fimbriae. Furthermore, immunogold labeling of the 75 kDa protein on the thin sections showed that the 75 kDa component was present on not only the outer membrane, but also on the cell membrane, and on membrane bound organelles. Localization of this protein suggests that the 75 kDa component is a membrane-associated protein.

Amino Acids↗
PubMed1990

Parameters affecting the inhibition of Candida albicans GDH 2023 and GRI 2773 blastospore viability by purified synthetic salivary histidine-rich polypeptides.

Purified synthetic salivary histidine-rich polypeptides, HRPs 2, 3, 4, 5, and 6, were observed to inhibit Candida albicans blastospore viability at yeast cell concentrations ranging from 10(2) to greater than 10(6) colony forming units per ml. Among the HRPs, HRP-4 was the best inhibitor with significant killing activity noted at a peptide concentration of 0.5 microgram per ml. Antifungal potency under growth conditions was observed to be dependent upon pH. In contrast, killing did not vary throughout the pH range tested under non-growth conditions. Electron microscopy results demonstrated HRP damage at pH 5 which appeared to be initiated at the membrane. At pH 7.4, micrographs revealed clear evidence of intracellular destruction suggesting more extensive damage at neutral as compared to acidic pH. These results suggest that within the changing realm of the oral cavity, the HRPs would be expected to be potent killers of C. albicans.

Candida albicans↗
PubMed1990

Human immunodeficiency virus type 1 Pr55gag and Pr160gag-pol expressed from a simian virus 40 late replacement vector are efficiently processed and assembled into viruslike particles.

Human immunodeficiency virus type 1 (HIV-1) gag and pol genes were expressed by using fragments of the BH10 clone of HIV inserted into a simian virus 40 late replacement vector. An initial construct containing the entire coding regions of gag, pol, and vif produced only minute amounts of the gag precursor, Pr55gag. However, high-level expression was obtained when an additional sequence from the env gene (the rev-responsive element) was inserted 3' of vif in the correct orientation, and rev was provided in trans from a second vector. Western immunoblot analysis of transfected cells showed the presence of large amounts of both Pr55gag and Pr160gag-pol as well as all of the expected cleavage products. Electron microscopy of thin sections of transfected cells showed a multitude of viruslike particles. Both immature particles in the process of budding and particles containing the condensed core characteristic of HIV were observed. Analysis of the released viruslike particles showed the presence of active reverse transcriptase. Sucrose gradient analysis of particles produced from [3H]uridine-labeled cells indicated a peak of radioactivity which cosedimented with a peak of p24, suggesting that the particles contained RNA.

Animals↗
PubMed1989

Radioautographic study of [3H]mannose utilization during cementoblast differentiation, formation of acellular cementum, and development of periodontal ligament principal fibers.

The formation of acellular cementum and the deposition of [3H]mannose-labeled extracellular matrix were studied in 14-day-old Sprague-Dawley rats. The sequential events of cementogenesis and periodontal ligament formation observed by light and electron microscopy were described from the stage of an intact root sheath to postcementogenesis. Ultrastructural examination of cementoblasts and periodontal ligament fibroblasts revealed [3H]mannose labeling of the Golgi apparatus at 10 minutes, collagen secretion granules at 30 minutes, and the extracellular matrix beginning at 30 minutes. The extracellular matrix between cementoblasts and dentin was heavily labeled at 1 and 4 hours. Newly formed principal fibers of the periodontal ligament were also heavily labeled at 4 hours. Fully differentiated cementoblasts exhibited the largest sectional profiles and the highest number of silver grains per unit area of cytoplasm. The morphologic and radioautographic data suggest that during the formation of acellular cementum, the cementoblast phenotype is expressed for a short period of time, after which cementoblasts appear to mix with the fibroblasts of the periodontal ligament.

Animals↗
PubMed1988

Radioautographic demonstration of receptors for epidermal growth factor in various cells of the oral cavity.

Mouse iodinated epidermal growth factor (EGF) was localized by light and electron microscopic radioautography in basal cells of oral epithelium, papillary cells of the enamel organ, periodontal ligament fibroblasts, preodontoblast precursor cells, and preosteoblasts of the alveolar bone of 13-day-old Sprague-Dawley rats. The specificity of binding in these cells was suggested by an observed reduction of about 90% in the labeling when excess unlabeled EGF was injected along with the 125I-EGF. In contrast, fully differentiated cells, such as ameloblasts, odontoblasts, and osteoblasts, were only poorly labeled. Quantitative analysis of the light microscopic radioautographs revealed that the papillary cells had the highest level of labeling (5.5 grains per 100 micron 2 of cell area). The significance of the rather high labeling of the preosteoblasts of the alveolar bone and the fibroblasts of the periodontal ligament is unknown. However, the well-known effect of EGF in producing precocious eruption of teeth may be a consequence of an effect on these two cell types.

Animals↗