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1

Electronic Resource

Osteogenic inhibition by rat periodontal ligament cells: modulation of bone morphogenic protein-7 activity in vivo (1998)

Rajshankar, Dhaarmini ; McCulloch, Christopher A. G. ; Tenenbaum, Howard C. ; [et al.]

Springer

Cell & tissue research 294 (1998), S. 475-483

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ISSN: 1432-0878

Keywords: Key words Periodontal ligament ; α-Smooth muscle actin ; Osteopontin ; Bone sialoprotein ; Bone morphogenic protein ; Rat (Wistar)

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by 3H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of α-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P〈0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P〉0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P〈0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for α-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s004410051199

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2

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Relationship of cellular proliferation to expression of osteopontin and bone sialoprotein in regenerating rat periodontium (1996)

Lekic, Predrag ; Sodek, Jaro ; McCulloch, Christopher A. G.

Springer

Cell & tissue research 285 (1996), S. 491-500

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ISSN: 1432-0878

Keywords: Key words: Periodontium ; Osteopontin ; Bone sialoprotein ; Cell proliferation ; Rat (Wistar)

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract. Cellular repopulation was studied in a model in which adjacent mineralising and soft connective tissue matrices are regenerated. Window wounds were created through alveolar bone, with either preservation or removal of periodontal ligament, in 30 male Wistar rats. Three animals per time period were killed on days 1, 3, 7, 14, and 28 after surgery for each wound type. Cellular proliferation in alveolar bone and periodontal ligament was assessed by 3H-thymidine labelling 1 h before death, followed by radioautographic analysis. Cellular differentiation was determined by the temporal expression of the bone-related markers osteopontin and bone sialoprotein, using immunohistochemical methods. In regenerating periodontium, osteopontin was expressed earlier than bone sialoprotein, which was restricted to alveolar bone. After wounding, transient expression of osteopontin was detected in the periodontal ligament at days 1 and 3. In general, wounding induced fivefold higher proliferation and clonal growth of periodontal ligament cells compared to the unwounded (control) side. Combined immunostaining and radioautography demonstrated colocalisation of osteopontin in sites with high numbers of labelled cells in both nascent periodontal ligament and regenerating alveolar bone at days 3 and 7. In contrast, bone sialoprotein, which appeared in regenerating alveolar bone on days 14–28 after wounding, was expressed much later than the peak of cellular proliferation. We conclude that (1) the intact periodontal ligament influences cell proliferation and osteopontin expression; (2) osteopontin is an early marker of periodontal tissue regeneration that is temporally and spatially associated with intensive cell proliferation and migration in osteogenic and periodontal ligament cell populations; and (3) bone sialoprotein is expressed after proliferation at sites of mineralising bone formation.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s004410050665

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3

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Deficiencies in collagen phagocytosis by human fibroblasts in vitro: A mechanism for fibrosis? (1993)

McCulloch, Christopher A. G. ; Knowles, Gisele C.

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 155 (1993), S. 461-471

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ISSN: 0021-9541

Keywords: Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: 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. © 1993 Wiley-Liss, Inc.

Additional Material: 5 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcp.1041550305

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Slow oscillations of free intracellular calcium ion concentration in human fibroblasts responding to mechanical stretch (1994)

Arora, Pamela D. ; Bibby, Kathryn J. ; McCulloch, Christopher A. G.

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 161 (1994), S. 187-200

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ISSN: 0021-9541

Keywords: Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: Calcium transients in single, human gingival fibroblasts were studied after mechanical stretching of flexible culture substrates. A model system was developed to reproducibly stretch and rapidly (〈 1 sec) refocus cells in the same focal plane so that changes in the concentration of free intracellular calcium ions ([Ca2+]i) were monitored without delay. Attached cells were grown on flexible bottom Petriperm dishes, loaded with fura-2/AM, and stretched by 1% or 2.8% of substrate area. The stretch caused no significant cell detachment or membrane lesions. A 1% stretch induced no calcium response, but a 2.8% stretch stimulated an initial calcium transient and the subsequent generation of [Ca2+]i oscillations of up to 2,000 sec. At 1% stretch, there was no calcium response. Cell shape and plating time were important determinants in the calcium response to mechanical stimulation: the responder cells were small and round without long processes. Major calcium transients were inhibited completely by 5 mM EGTA or by 10 μM gadolinium ions, by 50 μM nifedipine, or 250 μM verapamil, suggesting an influx of calcium through stretch-activated (SA) channels and L-type calcium channels. Depolarization by high KCl (144 mM) in the extracellular medium enhanced the amplitude of calcium transients by 54%. Calcium oscillations were not inhibited by preincubation with thapsigargin, caffeine, cholera toxin, staurosporine or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), indicating that IP3 sensitive pools, IP3 insensitive pools, G5α subunits, and protein kinase C, respectively, were not involved in the generation of calcium oscillations. Pretreatment with genistein, a specific tyrosine kinase inhibitor or cytochalasin D, an inhibitor of actin polymerization, or pertussis toxin, an inhibitor of Giα and Goα subunits, completely abolished calcium transients and oscillations. These results indicate that Ca2+ flux due to mechanical stretching is likely mediated through SA ion channe s and is dependent on tyrosine kinases, pertussis toxin-sensitive subunits of G-proteins, and actin filaments. © 1994 Wiley-Liss, Inc.

Additional Material: 16 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcp.1041610202

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5

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Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro (1991)

Ogiso, Bunnai ; Hughes, Francis J. ; Melcher, Antony H. ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 146 (1991), S. 442-450

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ISSN: 0021-9541

Keywords: Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: 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 105M indomethacin. Dose-dependent inhibition of bone nodule formation was also observed in cultures incubated with prostaglandins E2 (at 10-6M) or F2α(at 10-7). The results indicate that fibroblasts may inhibit osteoblast differentiation and function in part by release of soluble factors including prostaglandins.

Additional Material: 8 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcp.1041460315

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Dependence of collagen remodelling on α-smooth muscle actin expression by fibroblasts (1994)

Arora, Pamela D. ; McCulloch, Christopher A. G.

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 159 (1994), S. 161-175

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ISSN: 0021-9541

Keywords: Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: To study the relation between expression of the putative myofibroblast marker α-smooth muscle actin and the remodelling of extracellular matrix, immunocytochemical, gel electrophoresis, and collagen gel contraction studies were performed on two human fibroblast subtypes. Double immunolabelling for total actins and α-smooth muscle (sm) actin as well as affinity labelling of filamentous and monomeric actins in gingival fibroblasts demonstrated that α-sm was colocalized in stress fibres and in regions with high levels of monomeric actin throughout the cytoplasm. α-sm comprised up to 14% of total cellular actin as assessed by 2D gel electrophoresis. Thirteen different gingival and seven different periodontal ligament fibroblast lines constitutively expressed on α-sm actin. These cells exhibited up to 60% inter-line variations of fluorescence due to α-sm actin and up to 70% and 45% inter-line variation in the rate of collagen gel contraction. Quantitative, single cell fluorimetry of α-sm actin immunoreactivity demonstrated a linear relation between gel contraction and α-sm actin (correlation coefficients of 0.71 for gingival and 0.61 for periodontal ligament cells), but there was no detectable relationship between total actin content and gel contraction. In contrast, flow cytometry demonstrated that 99% of the total gated cells from cell lines exhibiting rapid gel contraction showed α-sm actin staining above background fluorescence as compared to only 35% of cells with slow rates of gel contraction. Contracting collagen gels stained with FITC-phalloidin showed cells with well-developed stress fibres that were progressively more compact and elongated during the time of maximal gel contraction. To examine the dependence of gel contraction on assembly of monomeric actin into actin filaments, cells were electroporated in the presence of phalloidin or cytochalasin D. Collagen gels exhibited up to 100% inhibition of gel contraction that was dose-dependent. Gel contraction was inhibited 93% by electroinjection of cells with α-sm actin antibody prior to incubation, but the antibody did not inhibit actin assembly after attachment and spreading on substrates. These data indicate that gel contraction is dependent on α-sm actin expression and that α-sm actin is a functional marker for a fibroblast subtype that rapidly remodels the extracellular matrix. © 1994 wiley-Liss, Inc.

Additional Material: 9 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcp.1041590120

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