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Breaking Biological Barriers with a Toothbrush
K. Amano1, ,
K. Miyake2,,
J.L. Borke3 and
P.L. McNeil1,2,*
1 Department of Cellular Biology and Anatomy,
2 Institute of Molecular Medicine and Genetics, and
3 Department of Oral Biology and Maxillofacial Pathology, Medical College of Georgia, Augusta, GA 30912-2000, USA
Correspondence: * corresponding author, pmcneil{at}mail.mcg.edu
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ABSTRACT
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Toothbrushing exposes epithelia and other tissues of the oral cavity to mechanical stress. Here, we investigated whether brushing induces cell wounding—plasma membrane disruption—in epithelial and other cell types in the oral cavity. Brushing of the gingivae and tongues of rats resulted in a striking increase in the number of cells positive for a marker of disruption injury. These cells included those in all strata of the gingival epithelium, and in the skeletal muscle of the tongue. Additionally, we found that brushing resulted in an increase in c-fos expression by junctional epithelial and skeletal muscle cells. Epithelial barrier function, however, was not overtly affected by brushing, despite the observed individual injuries to cells. We concluded that brushing disrupts cell plasma membrane barriers in the oral cavity and activates gene expression events that may lead to local adaptive changes in tissue architecture beneficial to gingival health.
Key Words: cell injury • plasma membrane disruption • c-fos • oral epithelium
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INTRODUCTION
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Plasma membrane disruption, or cell wounding, is a common and normal event in many mechanically active mammalian tissues. It has been documented, for example, under physiological conditions in epithelial cells lining the stomach and intestine (McNeil and Ito, 1989), in endothelial cells lining the aorta (Yu and McNeil, 1992), and, most strikingly, in cardiac muscle (Clarke et al., 1995), orthodontic tooth movement (Orellana et al., 2002), and skeletal muscle undergoing high force contractions (McNeil and Khakee, 1992).
A plasma membrane disruption is a potentially lethal cell injury, and therefore adaptations exist at the cellular level (and likely at higher and lower levels), for both preventing and repairing them (McNeil and Steinhardt, 2003). Failure of either mechanism results in disease in skeletal muscle, and perhaps in other tissues as well. Moreover, plasma membrane disruptions may act as potent mechanotransducers (Grembowicz et al., 1999; Togo et al., 2003). A disruption provides a direct route into a cell for known intracellular signals, such as Ca2+, and, conversely, for exit of potent extracellular signaling molecules, such as polypeptide growth factors, capable of stimulating neighboring cells.
The oral cavity is a mechanically active environment subject to shear, compression, and tensile forces both during mastication and, in humans, during brushing of the teeth, gingiva, and tongue. Does brushing of these oral cavity soft tissues induce cell wounding and, if so, can a local biologic response be detected? We here address these questions using established techniques for identifying wounded cells in vivo, and for assessing local c-fos expression levels.
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MATERIALS & METHODS
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Animals
Ten-week-old male rats (Harlan Sprague Dawley, Indianapolis, IN, USA), weighing from 350 to 400 g each, were used in all experiments, which were conducted under an approved protocol (Committee for Care and Use of Laboratory Animals at the Medical College of Georgia).
Brushing
For the detection of cell-wounding events, deeply anesthetized rats (Nembutal, i.p. injection, 1.0 mg/kg body weight) were injected intravenously with 1.0 mL of phosphate-buffered saline (PBS) containing fluorescein-labeled, lysine-fixable, 10,000 average-molecular-weight dextran (fluorescein-labeled dextran) at 10 mg/mL. Fifteen min after injection, an interval previously found to be sufficient for effective fluorescein-labeled dextran delivery via the circulation to peripheral tissues, the gingiva and tongue were each brushed for 2 min by means of an electric toothbrush (Braun Oral-B Plak Control, Battery Toothbrush, South Boston, MA, USA) fitted with medium hardness bristles, most of which were trimmed off to produce a working surface appropriate to the rat anatomy (an ~ 3 x 5 mm oval array), and operating at 9600 oscillations/min and 5 gm of applied force (0.25 gm/cm2).
For the detection of epithelial barrier disruptions, fluorescein-labeled dextran (20 mg/mL in PBS) was repeatedly pipetted directly onto the gingivae and tongues of deeply anesthetized rats. The tongues and gingivae were then brushed as described above.
Tissue Processing for Microscopy
Rats were perfused with 250 mL of warm PBS, followed by an equal volume of freshly prepared 4% paraformaldehyde, as previously described (McNeil and Khakee, 1992). One hr later, the tongue and both the maxillary and mandibular jaws were excised and immersed for an additional hour in fixative. Fixed tissue samples were then washed 3 times in PBS, infiltrated with sucrose over a 24- to 48-hour period, mounted on stubs in a freezing medium (Tissuetech OTC, Miles Laboratories, Naperville, IL, USA), snap-frozen in liquid isopentane/nitrogen, and sectioned on a freezing microtome (Zeiss HM 500 Cryostat, Carl Zeiss, Oberkochen, Germany). Sections (from 10 to 50 µm thick) were picked up onto glass slides (Superfrost® Plus, Fisher, Pittsburgh, PA, USA) and mounted in an anti-fading agent (ProLong Antifade, Invitrogen, Carlsbad, CA, USA) or further processed for antibody staining. Sections for immunostaining were permeablized by a 10-minute incubation in 0.2% Triton X-100 (in PBS), washed several times in PBS, and then incubated for 2 hrs at 37°C in PBS containing 2% BSA and a rabbit polyclonal antibody raised against c-fos (Calbiochem, EMD Biosciences, Inc., San Diego, CA, USA) diluted 1:50 and/or a peroxidase antibody raised against rat albumin (MP Biomedicals, Solon, OH, USA) diluted 1:100. After being washed several times in PBS, slides were then incubated for 1 hr at 37°C in a secondary antibody (anti-rabbit IgG-Cy3 conjugate or anti-peroxidase-Cy2 conjugate, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), then were washed and mounted in an anti-fade reagent (Invitrogen, Carlsbad, CA, USA).
Microscopy and Image Analysis
Sectioned tissue was imaged by laser scanning confocal microscopy (Zeiss LSM 510 Meta, performed at the Cell Imaging Core Facility of the Medical College of Georgia), and images were recorded onto an optical disc drive for further analysis. We quantitatively assessed levels of fluorescein-labeled dextran staining in the gingival epithelium (Zeiss ’Physiology’ image analysis software) in these images by drawing a single analysis window around all epithelium present in randomly selected images of experimental (brushed) or control (not brushed) samples, and then measuring the average fluorescence intensity within each window domain. Three animals in each treatment group and 5 sections from each animal, through the relevant epithelium, were analyzed as described above. Analysis of skeletal muscle cell wounding was performed as described previously (McNeil and Khakee, 1992). Briefly, a circular measurement window was placed in the center of fibers in randomly selected micrographs of experimental and control (undisturbed) groups, and the average intensity was measured and recorded. Three animals in each group were analyzed, and, for each animal, 10 sections through tongue skeletal muscle were analyzed. Analysis of c-fos staining was carried out as previously described (Grembowicz et al., 1999). Briefly, the number of fluorescence-positive nuclei residing in analysis windows (200 µm x 50 µm rectangle), drawn at random over epithelia or skeletal muscle, was counted.
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RESULTS
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The effect of brushing on cell plasma membrane integrity was examined in rats injected intravenously with fluorescein-labeled dextran. Fluorescein-labeled dextran is normally a membrane-impermeable molecule that enters and stains cell cytosol only when a repairable break in plasma membrane integrity occurs. The epithelium of the undisturbed rat maxillary gingiva typically contained few, if any, fluorescein-labeled, dextran-positive, wounded cells (Fig. 1A ). In contrast, the epithelium of mandibular gingiva of the same animal (Fig. 1B), which was brushed 15 min before tissue preparation for microscopy, typically contained numerous fluorescein-labeled, dextran-positive, wounded cells. Labeling of epithelial cells with fluorescein-labeled dextran was still present 3 hrs after brushing, indicating that the tracer had entered through a disruption that was repaired by the wounded cell, an event that irreversibly traps the tracer in wounded cell cytoplasm (Fig. 1C). Quantitative analysis confirmed that brushing induced a statistically significant increase in epithelial labeling with fluorescein-labeled dextran (Fig. 1G).
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Figure 1. Cell wounding in the gingiva and tongue induced by brushing. (A) Confocal fluorescence image of epithelium and underlying connective tissue from the undisturbed (not brushed) maxillary gingiva. Epithelial cells labeled with the cell wound marker, fluorescein-labeled dextran, are not observed. (B) Confocal fluorescence image of epithelium and underlying connective tissue from the brushed, mandibular gingiva (taken 15 min after brushing). Epithelial cells labeled with fluorescein-labeled dextran are observed in all strata. (C) Confocal fluorescence image of epithelium and underlying connective tissue from the brushed mandibular gingiva (taken 3 hrs after brushing). Epithelial cells labeled with fluorescein-labeled dextran are observed in all strata. (D) Skeletal muscle of a tongue, which was not brushed. Fluorescein-labeled dextran is observed in the connective tissues surrounding the individual myocytes. The myocytes themselves are not labeled intracellularly with the fluorescein-labeled dextran. (E) Skeletal muscle of a tongue 15 min after brushing. Many of the myocytes display strong cytoplasm labeling with fluorescein-labeled dextran. (F) Skeletal muscle of a tongue 3 hrs after brushing. Again, many of the myocytes are strongly labeled with fluorescein-labeled dextran. (G) Quantitative analysis of fluorescein-labeled dextran labeling of epithelial cells. The average fluorescence intensity resulting from fluorescein-labeled dextran staining of cells was measured over randomly assigned domains of the epithelium of the undisturbed (not brushed) maxillary gingiva and brushed mandibular gingiva at two intervals after brushing, 15 min and 3 hrs. Significantly more fluorescence was measured at both time points in the brushed mandibular gingiva. Error bar = SEM; asterisk indicates a p value of < 0.001 (n = 3 animals, 5 sections each, 18–20 images each section), when paired with the undisturbed (not brushed) control, Mann-Whitney test. (H) Quantitative analysis of fluorescein-labeled dextran labeling of skeletal muscle cells. The average fluorescence intensity of individual myocytes was measured in undisturbed and brushed (15 min, 3 hrs) tongues. Error bar = SEM. Asterisk indicates a p value of < 0.001 (n = 3 animals, 5 sections each, 18–20 images each section) when paired with the undisturbed (not brushed) control, Mann-Whitney test. Magnification bar = 25 µm.
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In the brushed tongue, few labeled epithelial cells were observed (data not shown). However, compared with undisturbed controls that were not brushed (Fig. 1D ), brushing induced a striking increase in the labeling of skeletal muscle cells, in samples taken both 15 min (Fig. 1E) and 3 hrs (Fig. 1F) after this mechanical challenge. Quantitative analysis confirmed that brushing resulted in a statistically significant increase in myocyte labeling with fluorescein-labeled dextran (Fig. 1H).
Cell wounding, as well as other types of cell injury, has been associated with activation of immediate early genes, such as c-fos (Grembowicz et al., 1999). We therefore asked whether expression of this gene, normally not detectable in gingival epithelium (Fisher et al., 1991), is activated by brushing. Strong activation of c-fos expression was induced by brushing: Compared with undisturbed controls that were not brushed (Figs. 2A–2D), the epithelium of brushed tongue and gingiva (Figs. 2E, 2F), and the skeletal muscle of the tongue (Figs. 2G, 2H) showed a high density of cell nuclei positively stained with an antibody against c-fos. Quantitative analysis demonstrates the statistical significance of the increase in c-fos expression induced by brushing of the gingiva and the skeletal muscle of the tongue (Fig. 2I).
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Figure 2. Induction of c-fos expression by brushing. Paired differential interference contrast (DIC) and fluorescence (c-fos) confocal images are shown. (A,B) In the undisturbed maxillary gingiva epithelium, very few nuclei labeled with an antibody against c-fos are observed in either the epithelium or connective tissue. (C,D) In the undisturbed tongue, very few c-fos-positive nuclei are observed in the cells of the epithelium, connective tissue, or skeletal muscle. (E,F) In the brushed mandibular gingiva, many c-fos-positive nuclei are observed in both the epithelium and connective tissue. (G,H) In the brushed tongue, many c-fos-positive nuclei are observed in cells of the epithelium, connective tissue, and skeletal muscle. (I) Quantitative analysis of c-fos expression as a function of brushing. The number of c-fos-positive cells counted in standardized measurement areas (200 µm X 50 µm) over the gingival epithelium (Gingiva) and over tongue skeletal muscle (Tongue) are indicated for undisturbed tissue (Undisturbed) and brushed tissue (Brushed). Error bar = SEM; asterisk denotes a p value of < 0.001 (n = 3 animals, 5 sections each, 6 images each section) when paired with the undisturbed (not brushed) control, Mann-Whitney test. Magnification bar = 20 µm.
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Serum albumin, a protein present at high concentrations in most extracellular spaces of the mammalian body, is an alternative to injected fluorescein-labeled dextran as a cell-wound marker (Clarke, 1999). To establish that the wounding and c-fos expression events reported above were not artifacts induced by a foreign molecule (fluorescein-labeled dextran), we also used albumin as a wound marker to evaluate wounding incidence, and re-evaluated, in this context, c-fos expression as a function of brushing. Omission of fluorescein-labeled dextran did not perceptibly change either the pattern of wounding or the c-fos expression pattern. Brushing greatly enhanced the number of cells labeled fluorescently with an anti-albumin antibody in the gingiva and the tongue (Figs. 3A–3D). Moreover, brushing in the absence of fluorescein-labeled dextran was observed to increase c-fos expression (Figs. 3E–3H).
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Figure 3. Serum albumin as an alternative probe for wounding. (A) Undisturbed maxillary gingiva was immunostained with an antibody directed against mouse serum albumin. As was the case when fluorescein-labeled dextran was used as a cell wound probe, very little intracellular labeling of epithelial cells with this antibody is observed. (B) Brushed mandibular gingiva immunostained for serum albumin. In contrast to undisturbed gingiva, brushed gingiva displayed heavy labeling of surface epithelial cells. (C) Undisturbed tongue skeletal muscle was immunostained for serum albumin. Positively stained skeletal muscle fibers were rarely observed. (D) Brushed tongue was immunostained for serum albumin. Positively stained myofibers were abundant. (E) The same section of undisturbed gingiva was immunostained with an antibody against c-fos. As was the case in fluorescein-labeled dextran-treated tissues, very few positively stained nuclei were observed. (F) Corresponding immunostaining for c-fos. Numerous c-fos-positive nuclei were observed in brushed epithelium. (G) Corresponding immunostaining for c-fos. Myofiber nuclei, positively stained for c-fos, were not observed. (H) Corresponding immunostaining for c-fos. Positively stained myofiber nuclei were abundant. Magnification bar = 20 µm.
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Finally, given the level of epithelial cell plasma membrane injury detected above, we asked if the epithelial diffusion boundary—composed of multiple epithelial membranes and extracellular lipid—was also broken by brushing. For these experiments, the oral tissues to be examined were first rinsed with fluorescein-labeled dextran in physiological saline, and then were brushed in the presence of this marker, or left undisturbed. We found, in line with the work of other investigators (Squier and Hall, 1984, 1985), that, in the undisturbed gingiva and tongue, a macromolecular tracer, fluorescein-labeled dextran in this case, did not penetrate beneath the outermost squamous strata of the epithelium (Figs. 4A–4D). We here report that brushing did not alter this result (Figs. 4E–4H). Thus, brushing, under the conditions we used in the rat, did not appear to disrupt epithelial barrier functioning.
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Figure 4. Brushing induced penetration from the oral cavity of fluorescein-labeled dextran into underlying connective tissue. Paired DIC (DIC) and fluorescein dextran-derived fluorescence (FDx) images are shown. (A,B) The maxillary gingival epithelium was bathed for 1 min in fluorescein-labeled dextran but was not otherwise perturbed. No penetration of the fluorescein-labeled dextran beneath the outermost strata of the epithelium is observed. (C,D) The tongue was bathed in fluorescein-labeled dextran, but was not otherwise disturbed. As in the case of undisturbed gingival epithelium, no penetration of fluorescein-labeled dextran was observed beneath the most superficial strata of the epithelium. (E,F) The mandibular gingiva was bathed for 1 min in fluorescein-labeled dextran and then brushed for another min. Penetration of fluorescein-labeled dextran between epithelial cells and into the underlying connective tissue is not observed. (G,H) The tongue was bathed in fluorescein-labeled dextran and then brushed. Enhanced penetration of the fluorescein-labeled dextran probe was not induced by brushing of this epithelium. Magnification bar = 50 µm.
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DISCUSSION
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What might be the biological consequences in the oral cavity of these brushing-induced disruptions in a major biological barrier, the cell plasma membrane? Damage induced by mechanical stress in a tissue often results in an adaptive response, which serves to prepare the tissue for future mechanical onslaughts of a similar nature. This is most clearly the case for skeletal and cardiac muscle, both of which undergo hypertrophy in response to repetitive or continuous impositions of increased mechanical load. Increases in mechanical load in cardiac and skeletal muscle lead to increases in plasma membrane disruption frequency in these tissues, and, concomitantly, increased release of the polypeptide growth factor, fibroblast growth factor 2 (FGF2) (Clarke et al., 1993). In vitro studies have clearly demonstrated that FGF2, which lacks a classic signal sequence for export via an exocytotic pathway, is released from cells that have suffered sublethal, plasma membrane disruption injuries (McNeil et al., 1989; Muthukrishnan et al., 1991), such as are reported here to occur in the oral epithelium. In FGF2 null mice, there is a failure of the cardiac hypertrophy (Pellieux et al., 2001) normally produced by mechanical overload associated with membrane disruptions (Fischer et al., 1997). Though it is unclear whether the epithelial cells of the gingiva produce FGF2—it has been detected by immunostaining only in mast cells, macrophages, and endothelial cells of this tissue (Murata et al., 1997)—this general, disruption-mediated mechanism of growth factor release in the oral cavity clearly merits further investigation.
However, the c-fos expression response to brushing might be a direct consequence of a plasma membrane disruption event, not mediated, that is, by the release of factors into the extracellular environment. It is important to note that the cell injuries detected here were sublethal: Dextran-trapping in cytosol, used to identify wounded cells, is possible only if a cell suffers and repairs a membrane disruption. Previously, we have shown, in vitro, that not only does c-fos activation correlate spatially and quantitatively on a cell-by-cell basis with the occurrence of a disruption event, but also that this response is eliminated in a culture of wounded cells, if those suffering membrane disruptions are precluded from responding (Grembowicz et al., 1999): The wounded cell itself, not its undisturbed neighbor in the same culture, was the responder. This suggests that a diffusible factor, released from wounded cells, such as FGF2, is not required for membrane-disruption-induced c-fos expression. Instead, entry of a signal directly into a sublethally wounded cell, such as Ca2+, might be important.
Friction has long been known to stimulate oral epithelium. For example, chronic treatment of the hamster cheek pouch with a rotating cotton mop resulted in an increase in stratum corneum thickness, the number of cells in the stratum Malphighii, and an increase in mitotic figures (Mackenzie and Miles, 1973). Moreover, numerous more recent studies have shown that brushing induces epithelial cell proliferation, and induces fibroblasts and endothelial cells to proliferate and collagen to be synthesized in gingival connective tissue (Horiuchi et al., 2002; Tomofuji et al., 2002, 2003; Sakamoto et al., 2003; Yamamoto et al., 2004; Kusano et al., 2006). We propose that one mechanism by which brushing contributes to oral health is, paradoxically, via cell injury, namely, plasma membrane disruption. The firm, well-vascularized tissue thought to represent healthy gingiva (Muller and Kononen, 2005) may, in other words, be the result of an adaptive response to a repeated, and initially damaging, mechanical stimulus. Clearly, identifying the signals that could promote this adaptive response must be an important goal of future research. However, we have already alluded to two possibilities. Growth factors such as FGF2 can promote both collagen production and angiogenesis. FGF2 is indeed capable of stimulating human oral cavity cells to divide and differentiate (Takayama et al., 2002). Ca2+ entry might induce c-fos and other gene expression events: Expression of this gene is a common response to numerous kinds of cell injury. C-fos expression might be directly involved in tissue adaptation: It has been linked, for example, not only to cell proliferation but also, in stratified epithelia, to keratinization (Fisher et al., 1991). In conclusion, we suggest that, in addition to its well-known ability to remove bacteria and their harmful products from teeth, brushing may, by causing plasma membrane disruptions, lead to local cell-adaptive responses ultimately of benefit to gingival health.
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ACKNOWLEDGMENTS
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The authors thank M. Baker for technical assistance. P.L.M. was supported by a grant from NASA.
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FOOTNOTES
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 authors contributing equally to the work 
 present address, The First Department of Anatomy, Kyorin University of Medicine, Tokyo, Japan
Received for publication August 15, 2006.
Revision received February 8, 2007.
Accepted for publication April 17, 2007.
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Journal of Dental Research, Vol. 86, No. 8,
769-774 (2007)
DOI: 10.1177/154405910708600816
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ABSTRACT
INTRODUCTION