Mechanical strain inhibits repair of airway epithelium in vitro

Ushma Savla1 and Christopher M. Waters1,2

Departments of 1 Biomedical Engineering and 2 Anesthesiology, Northwestern University, Chicago, Illinois 60611

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The repair of airway epithelium after injury is crucial in restoring epithelial barrier integrity. Although the airway epithelium is stretched and compressed due to changes in both circumferential and longitudinal dimensions during respiration and may be overdistended during mechanical ventilation, the effect of cyclic strain on the repair of epithelial wounds is unknown. Human and cat airway epithelial cells were cultured on flexible membranes, wounded by scraping with a metal spatula, and subjected to cyclic strain using the Flexercell Strain Unit. Because the radial strain profile in the wells was nonuniform, we compared closure in regions of elongation and compression within the same well. Both cyclic elongation and cyclic compression significantly slowed repair, with compression having the greatest effect. This attenuation was dependent upon the time of relaxation (TR) during the cycle. When wells were stretched at 10 cycles/min (6 s/cycle) with TR = 5 s, wounds closed similarly to wounds in static wells, whereas in wells with TR = 1 s, significant inhibition was observed. As the TR during cycles increased (higher TR), wounds closed faster. We measured the effect of strain at various TRs on cell area and centroid-centroid distance (CD) as a measure of spreading and migration. While cell area and CD in static wells significantly increased over time, the area and CD of cells in the elongated regions did not change. Cells in compressed regions were significantly smaller, with significantly lower CD. Cell area and CD became progressively larger with increasing TR. These results suggest that mechanical strain inhibits epithelial repair.

wound healing; lung injury; mechanical ventilation; cell spreading; cell migration

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

AIRWAY EPITHELIAL DAMAGE occurs with mechanical trauma from ventilators (5, 16), in inflammatory diseases such as asthma (13), and in response to a multitude of different insults such as cigarette smoke (2) and bacterial or viral infections (20, 23). The rapid regeneration of a continuous epithelium is critical in maintaining barrier function (5) and in limiting airway hyperreactivity (21). The complex process of reepithelialization involves several steps, including spreading of cells at the wound edge into the denuded surface, migration of cells to the wound site, and, eventually, proliferation of the cells surrounding the wound (34, 35). Zahm et al. (34, 35) showed rapid closure of small wounds in airway epithelial cells (AECs) due to progressive elongation of cells adjacent to the wound and rapid migration rates of cells at the wound edge. Rapid closure of wounds in type II alveolar epithelium has also been attributed to motility and cell spreading (8, 11). In vivo, AEC wound closure was dependent on proliferation after 15-30 h (6). Spreading and migration have been shown to be stimulated by transforming growth factor-alpha (11) and fibronectin (8) in vitro and by local blood flow in vivo (6).

In vivo, the airways undergo cyclic deformation at a frequency dependent on the respiration rate; however, little is known about how such mechanical strain affects cellular motility, spreading, and proliferation. We can define two types of mechanical strain that result from changes in airway geometry: 1) a circumferential wall strain due to changes in airway diameter and 2) a longitudinal wall strain due to airway lengthening during lung expansion. Because strain can be defined by changes relative to a resting position, it can be either positive (elongation) or negative (compression) as may occur during bronchoconstriction. On the basis of reported changes in canine airway cross-sectional area measured using high-resolution computed tomography (4), we estimate that circumferential strain can vary from -42% with breath holding to +32% with deep inspiration.

Recent investigations have shown that mechanical strain affects several processes involved in tissue repair. Stretch influences compensatory lung growth after partial pneumonectomy (22) and increases the proliferation rates of human lung fibroblasts (3) and fetal rat lung cells (14). Cyclic strain has also been shown to increase collagen production by smooth muscle cells (31), and collagen and other extracellular matrix components are important in wound closure (8, 25). However, the role of physical forces in airway wound healing has not been examined. We hypothesized that each component of the repair process (spreading, migration, and proliferation) may be influenced by cyclic mechanical strain. We used an in vitro model to study the repair mechanisms of wounded monolayers of human and cat AECs cultured on elastic membranes and subjected to cyclic strain using the Flexercell Strain Unit (Flexercell, McKeesport, PA). Due to the radially dependent strain profile in Flexercell plates, we were able to compare the effects of both cyclic elongation and compression. We show that both cyclic elongation and compression significantly attenuate wound closure in a frequency-dependent manner.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Phosphate-buffered saline (PBS), minimum essential medium (MEM), Ham's F-12 medium, fetal bovine serum (FBS), gentamicin, trypsin-EDTA, and nonessential amino acids (NEAA) were obtained from GIBCO (Grand Island, NY). Collagenase was obtained from Worthington Biochemical (Freehold, NJ). Bronchial epithelial growth medium (BEGM) was acquired from Clonetics Human Cell Systems (San Diego, CA). All other chemicals were purchased from Sigma (St. Louis, MO). The Flexercell Strain Unit and Flex-I type I collagen-coated six-well culture plates were purchased from Flexercell International.

Calu-3 culture. Calu-3 cells (American Type Culture Collection, Rockville, MD), derived from a human lung adenocarcinoma, have been previously described to resemble tracheal epithelial cells and retain constant properties over repeated passages (27). Cells were maintained in T-150 culture flasks in Calu-3 media (MEM, 1 mM sodium pyruvate, 1 mM NEAA, 0.1% gentamicin, and 10% FBS) before experiments. Cells between passages 18 and 26 were used in experiments 4-5 days after plating into six-well Flex-I plates at 3-3.5 × 105 cells/well (~350,000 cells at confluence). The medium was changed every 2 days.

Cat tracheal epithelial cell culture. Cat tracheal epithelial (CTE) cells were isolated from healthy cats (courtesy of Dr. Robert TenEick, Department of Molecular Pharmacology, Northwestern University) euthanized with an intravenous injection of pentobarbital sodium (Abbott Laboratories, North Chicago, IL). The trachea was dissected from the surrounding tissue, cut from the larynx to the first bifurcation of the bronchi, placed on a gauze-covered sheet of styrofoam, cut longitudinally, and pinned open. Superficial parallel incisions were made longitudinally with a razor blade jig. Strips of epithelium pulled away from the underlying tissue were rinsed two to three times in sterile PBS containing antibiotics and then placed in a solution of 0.02% (wt/vol) type II collagenase, 5 mM dithiothreitol, and 200 U/ml DNase in Ca2+/Mg2+-free PBS containing antibiotics at 37°C. The mixture was placed in a heated shaker or agitated by inversion every 10-15 min. After 60 and 120 min, the cell suspension was removed and washed two times in CTE cell media (Ham's F-12 medium with 10 µg/ml insulin, 7.5 µg/ml endothelial cell growth supplement, 0.5 µg/ml transferrin, 0.4 µg/ml hydrocortisone, 2 µg/ml triidothyronine, 1% antibiotic-antimycotic solution, and 2% sodium bicarbonate). Cells were seeded at 2.5-3 × 105 cells/well and cultured in CTE cell media with 10% FBS (200,000 cells at confluence; CTE cells are larger than other cell types). The medium was changed every 2 days, and the cells were used in experiments on day 6 or 7 of culture.

Normal human bronchial epithelial cell culture. Normal human bronchial epithelial (NHBE) cells were obtained from Clonetics (San Diego, CA) and maintained in BEGM medium (0.5 µg/ml hydrocortisone, 0.5 ng/ml human recombinant epidermal growth factor, 0.5 µg/ml epinephrine, 10 µg/ml transferrin, 5 µg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, and bovine pituitary extract). Passage 2-4 NHBE cells were seeded onto Flex-I six-well culture plates at 1.75 × 104 cells/well, as suggested by Clonetics, to obtain confluence (~350,000 cells). The medium was changed every 2 days, and the cells were used in experiments on day 4 or 5 of culture.

1HAEo- cell culture. AECs transformed with the SV40 virus (1HAEo-) were obtained from Dr. D. Gruenert (University of California, San Francisco) and have been characterized by his laboratory (10). Cells were grown in Eagle's modified medium containing 10% FBS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin G. Cells were seeded onto Flex-I six-well culture plates at 2-3 × 105 cells/well and used on day 4 or 5 of culture (~350,000 cells at confluence). These cells can be passaged >200 in culture.

Cyclic strain. The Flexercell Strain Unit has been described in detail (1, 9, 30). The system utilizes vacuum pressure regulated by a solenoid valve to deform a silicone rubber substrate on which the cells have been cultured. When the vacuum is applied under the plate, the Silastic culture plate bottoms are deflected downward to a known elongation, which is translated to the cells. Upon release of the vacuum, the Silastic substrate returns to its original unstretched conformation. The frequency, duration, and magnitude of applied strain can be varied in this system by computer. When cells were stretched at 30 cycles/min (cpm), the membrane was stretched for 1 s and relaxed for 1 s; for 10 cpm, the membranes were stretched for various durations (1-5 s) and relaxed for various durations (1-5 s) within a 6-s cycle. Finite-element analysis of the membrane strain has shown that, upon deformation, the Flex-I plate yields a nonhomogeneous radial strain profile that is maximum at the periphery and minimal at the center (see Fig. 2A), with 1-2% compression seen in the center of the stretched wells (9).

Experimental protocol. Cells were grown to confluence, and wounds of ~500-µm width were produced by scraping the monolayers with a metal spatula across the diameter of the well. In each experiment, duplicate paired plates were either subjected to cyclic strain using the Flexercell apparatus or maintained static as a control. The frequency of strain was varied from 10 to 30 cpm, and the maximal elongation was 20-22% in all experiments (mean strain 9% across the well; see Ref. 8). Before the initiation of strain, the cells were rinsed one time with PBS to remove cellular debris. Two milliliters of complete growth medium were added to the wells; this medium was not removed during the course of the experiment. Images were obtained at the initial time of wounding and at various times up to 48 h postwounding. Due to the nonuniform radial strain profile in the strained wells, images of the wounds were obtained across the entire well.

Imaging the wound. Images of the wounds were collected at specified times with a Nikon Diaphot 300 inverted microscope equipped with a Hamamatsu integrating CCD camera, an Argus-20 real-time digital-image processor, and a Pentium computer with a frame grabber (Fryer, Huntley, IL). Images were analyzed using Metamorph image-analysis software (Universal Imaging, West Chester, PA). After the image was acquired, it was calibrated from pixels to micrometers. To digitally enhance the contrast, the image was processed through two filters (low pass; Prewitt), and then the filtered image was subtracted from the original image. This processing resulted in a dark line around the wound edge that could be easily visualized. Using Metamorph, an outline of the wound was manually drawn, and the mean wound width, w, was tabulated. The mean wound width, w, is given by
<IT>w</IT> = 0.25<FENCE><IT>P</IT> − <RAD><RCD>(<IT>P</IT><SUP>2</SUP> − 16<IT>A</IT>)</RCD></RAD></FENCE> (1)
where P and A are the perimeter and area of the wound in the image, respectively. Data are expressed as a percentage of the original wound width to normalize variability in wounding from well to well, although similar-sized initial wounds were observed from experiment to experiment.

Proliferation studies. For the growth studies, duplicate paired plates of Calu-3 cells were seeded at 2-2.5 × 105 cells/well (subconfluent) and subjected to cyclic stretch (10-30 cpm, 20% maximal elongation). After 0 and 48 h, the monolayers were trypsinized, and cells were counted using a Coulter Counter (Coulter Electronics, Hialeah, FL). For the bromo-2'-deoxyuridine (BrdU) studies, Calu-3 and CTE cells were grown on Flex-I six-well plates and wounded as described. BrdU (10 µM) was added for 60 min either at the initial time of wounding or at the end of the incubation period. The cells were washed with PBS, fixed in 70% ethanol (in glycine buffer, pH 2.0) for 20 min at -20°C, and washed again. The cells were subsequently incubated with anti-BrdU antibodies (1:10 dilution; Boehringer Mannheim, Indianapolis, IN) for 30 min at 37°C. The cells were rinsed and incubated with anti-mouse-Ig-alkaline phosphatase solution for 30 min at 37°C. After the cells were rinsed, the wells were covered with 13 µl of nitro blue tetrazolium solution and 10 µl of X-phosphate solution (50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt in dimethylformamide) in 3 ml of 100 mmol/l of Tris buffer (pH 9.5) for 30 min at room temperature. After the wells were rinsed, they were removed from the plate and mounted onto coverslips. The cells were viewed with the Nikon Diaphot 300 inverted microscope. The range of pixel intensity values was digitally expanded, resulting in a contrast enhanced image, which was analyzed with Metamorph software. The number of objects within an image with pixel intensity values above a certain threshold value and within a size range (10-2,000 µm2) were counted and considered labeled with BrdU. The accuracy of this measurement was verified by comparing the computed labeling index with that found upon visual inspection of the image. To determine if cellular viability was altered by cyclic stretching, the medium from Calu-3 cells was collected after 48 h of stretch and assayed for lactate dehydrogenase (LDH) using a colorimetric assay from Sigma Chemical.

Cell spreading studies. As a measure of cell spreading and motility, the distance between the centroid of one cell and the centroid of its four neighboring cells was measured in the center and periphery of stretched and unstretched wells after 0, 24, and 48 h in several experiments. Also, the average cell size was determined by tracing cells in wounded monolayers at the center and the periphery of several stretched (10-30 cpm, 20% maximal elongation) and unstretched wells. The accuracy of the cell size measurements was verified by measuring cell size in the same field at higher magnifications. For each experimental condition, four sets of cells at the leading edge of the wound were traced in each experiment (n = 16 pairs of cells in each group).

Statistical analysis. Data are presented as means ± SE for the indicated number of experiments (n). Wound widths from the peripheral regions of each well were averaged, and the mean was treated as a single data point. Comparisons between mean values were made using a repeated-measures analysis of variance and Tukey's modified t-test (the Bonferroni criterion). A P < 0.05 was considered significant. All statistical comparisons were performed using the SigmaStat program (Jandel Scientific, San Rafael, CA).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cyclic strain inhibits wound closure. To investigate the effect of cyclic mechanical strain on AEC wound closure, primary cultures of cat and human AECs (CTE and NHBE) and lines of human AECs (Calu-3 and 1HAEo-) were grown to confluence on elastic membranes, and wounds were induced by scraping with a metal spatula. Figure 1 shows the initial wound (~500 µm) in a CTE monolayer (Fig. 1A), the wounds after 20 h in an unstretched well (Fig. 1B), wounds in the periphery of a well stretched at 30 cpm (Fig. 1C), and wounds in the center of a well stretched at 30 cpm (Fig. 1D). Because the strain within each well depends on the radial position (Fig. 2A), measurements of the healing wounds were taken across the well. In the periphery of the wells, elongation occurs (positive strain), whereas deformation in the center of the well results in compression (negative strain). Wound closure was impaired in regions of cyclic stretch (Fig. 1C) and cyclic compression (Fig. 1D). Similar wound images were observed when Calu-3 cells were cyclically strained at 30 cpm (Fig. 1, E-H).


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Fig. 1.   Wound closure in cat tracheal epithelial (CTE) cells (A-D) and Calu-3 monolayers (E-H). In CTE monolayers, the images show a typical initial wound (A), wounds after 20 h in an unstretched well (B), wounds in the periphery of a well stretched at 30 cycles/min (cpm; C), and wounds in the center of a well stretched at 30 cpm (D). In Calu-3 cells, the images show a typical initial wound (E) and wounds after 24 h in an unstretched well (F), the periphery of a well stretched at 30 cpm (G), and the center of a well stretched at 30 cpm (H).


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Fig. 2.   A: finite-element model estimate for radial strain in Flex-I 6-well plates at a vacuum pressure of 15.2 kPa. Center of the well is at 0 mm, and 12.5 mm is the outer edge of the well. Periphery of the well experiences maximal elongation, whereas the center of the well experiences a small degree of compression. Data were provided by Dr. G. W. Link, Flexercell, McKeesport, PA. B: wound widths across healing wounds in Calu-3 monolayers. Filled symbols represent static wells; open symbols represent 30 cpm wells. Circles show the initial wound, squares show wound widths after 24 h, and triangles denote wound widths after 48 h. Data are from single wells from a representative experiment and are expressed as %original wound width.

To determine the dependence of wound closure on elongation and compression, wound widths were measured at several radial positions (Fig. 2B). Although data presented in Fig. 2 were from single wells, similar results were seen in all wells. Wound closure in static wells was relatively uniform across the well over the time course. In the cyclically strained wells, the original wound was consistent in width across the well, but, after 24 and 48 h, wound closure in all regions of the wells was decreased compared with unstretched wells. The inhibition of closure was greatest in the compressed regions (center). To assess cell toxicity in the different conditions, LDH levels were measured in the media after 48 h. In each case, LDH levels were below the detection limit of the assay.

Wound closure was measured in monolayers of CTE (n = 6), Calu-3 (n = 14), NHBE (n = 6), and 1HAEo- (n = 6) cells (Fig. 3) to determine the extent of inhibition due to elongation or compression. There were differences in the rate of wound closure in the different cell types; whereas wounds in CTE (Fig. 3A) and 1HAEo- (Fig. 3D) monolayers were closed by 24 h, Calu-3 (Fig. 3B) and NHBE (Fig. 3C) monolayers had closed to only ~60% of the original width. In each cell type, cyclic strain (30 cpm, 20% maximal elongation) significantly inhibited wound closure. Cyclic compression in the center of the wells inhibited closure to a greater extent than cyclic elongation in every case except 1HAEo- cells. Wound closure was significantly different in cyclically strained cells by 4 h in CTE cells, by 5 h in Calu-3 cells, by 20 h in NHBE cells, and by 10 h in 1HAEo- cells. With the exception of NHBE cells, these differences suggest that inhibition occurs early, before potential changes in proliferation.


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Fig. 3.   Summary of wound healing in CTE (A), Calu-3 (B), normal human bronchial epithelial (NHBE; C), and 1HAEo- (D) cells in either static (filled symbols) or 30 cpm (open symbols) wells. Circles represent measurements taken from the center of the wound, and squares indicate wound measurements from the periphery of the wells. * Significant difference from static wound widths (P < 0.05). Data are expressed as %original wound width, and error bars represent SE; n = 14 for Calu-3, n = 6 for all others.

Wound closure depends on intervals of strain and relaxation. We investigated the dependence of wound closure on the frequency of cyclic strain by measuring wound widths in Calu-3 monolayers stretched at 10 cpm. When the cells were strained at 30 cpm, the duration of strain per cycle was 1 s, and the membrane and cells were "relaxed" for the remaining 1 s of the cycle. However, when the cells were flexed at 10 cpm, the time of stretch per cycle was varied from 1 to 5 s. Because each cycle was 6 s, the corresponding time of relaxation (TR) was varied from 5 to 1 s. We measured the extent of wound closure in Calu-3 cells after 24 h (%original wound width) as a function of the time of stretch per cycle, as shown in Fig. 4. By 24 h, wounds in unstretched wells closed to ~55% of the original width, whereas cells flexed at 30 cpm (TR = 1 s) had closed to only 68% in the stretched region (periphery) and 77% in the compressed region (center). For the same time of stretch per cycle (1 s), cells flexed at 10 cpm (TR = 5 s) closed to the same extent as in static wells. When TR was decreased to 4 s for the same frequency (10 cpm), there was a significant inhibition of wound closure, and the inhibition increased with shorter TR. The inhibition of closure at TR = 1 s in 10 cpm wells was identical to the inhibition measured at 30 cpm with the same TR.


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Fig. 4.   Extent of wound closure in Calu-3 cells after 24 h is dependent on the time of relaxation (TR). Wounds in 10 cpm wells closed to a lesser extent when TR was reduced. Nearly the same inhibition of closure was observed in wounds with TR = 1 s regardless of frequency. Time of stretch = 0 refers to wounds in static wells. Error bars represent SE. periph, Periphery.

Cyclic strain inhibits proliferation. To determine the extent to which cyclic strain inhibits proliferation, Calu-3 cells were plated at a subconfluent density and subjected to cyclic strain. After 48 h of cyclic strain, the cells were counted using a Coulter Counter (Table 1). Cell number was not different initially. The number of cells in wells strained at 10 cpm (TR = 5 s) was not significantly different from the cell count in the unstretched controls after 48 h. However, cyclic strain at 30 cpm caused a significant reduction in cell number. Cell number in confluent monolayers did not increase over 3 days in stretched or unstretched wells, implying contact inhibition, and no significant cell division after confluence is achieved (data not shown). To assess proliferation in wound closure, BrdU incorporation into cellular DNA was determined in cells at the wound edge. The BrdU labeling index in wounded Calu-3 cells was not significantly different (P > 0.05, n = 3) in static (281 ± 24), stretched (239 ± 64), or compressed (277 ± 21) regions after 24 or 48 h. In CTE monolayers, the BrdU labeling index was not significantly different after 8 h, but by 20 h the labeling index was significantly lower in the compressed regions (167 ± 8, P < 0.05) compared with static control (313 ± 15) and stretched regions of wells (247 ± 7).

                              
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Table 1.   Proliferation of Calu-3 cells is inhibited by cyclic strain

Cyclic strain inhibits cell spreading. To distinguish between the contribution of cell spreading and migration in wound closure, the distance between centroids of adjacent cells at the wound edge was measured (the centroid is the geometric center of the cell). Kheradmand et al. (11) suggested that, in the absence of cellular proliferation, a significant increase in internuclear distance would indicate that cell spreading is the primary mechanism of wound closure. By modifying their approach to measure the centroid-centroid distance (CD), we were able to study cellular spreading. Figure 5A shows a comparison of CD for Calu-3 cells stretched at 10 cpm with varying TR (from the same experiments as Fig. 4). There was no significant difference initially among the treatment groups. CD significantly increased over time in static wells (P < 0.05, n = 80), suggesting that cell spreading was the primary mechanism of wound closure. In contrast, CD in elongated regions (periphery) did not change over time regardless of TR but was significantly less than CD in static wells (P < 0.05, n = 80). CD in the compressed regions (center) significantly decreased over time and was dependent on TR (P < 0.05, n = 16). As TR decreased, CD became progressively smaller (Fig. 5A). Not only were cells prevented from spreading as TR decreased, but the cell size actually decreased (Fig. 5B). As the average cell size in static wells significantly increased over time (P < 0.05, n = 120) and the cell size in elongated regions remained constant, cell size in compressed regions decreased as a function of TR. Shape change in stretched and unstretched cells was also measured by measuring the shape factor, with a value from zero to one describing how closely an object resembles a circle (circle = 1). Although static (0.75) and elongated (0.78) cells retained approximately the same shape over time, cells in the compressed regions became more circular (0.85) over time, independent of TR (n = 120).


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Fig. 5.   Time course of changes in centroid-centroid distance (A) and average cell size (B) in Calu-3 cells. Measurements are from static, elongated (periphery), and compressed (center) regions. * Significant difference from static measurement (P < 0.05). Error bars represent SE. For CD, static n = 80, all others n = 16; for cell size, static n = 120, all others n = 20.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Shedding of airway epithelium is commonly observed in the airways of asthmatics (32), and endotracheal intubation and mechanical ventilation can cause severe epithelial damage in the airways (17). Repair of epithelial damage involves several processes, including migration, cell spreading, and proliferation (8, 34, 35). Although airways undergo significant mechanical strain during the respiratory cycle, there has been no previous investigation of the role of cyclic strain in this repair process. Using an in vitro model of epithelial wound repair, we measured wound closure in airway epithelium from human and feline airways. The major findings of this study are that both cyclic elongation and cyclic compression inhibit the repair of epithelial monolayers and that the extent of inhibition depends on the TR between cycles of strain.

The Flexercell apparatus, with the proper vacuum pressure, leads to both cyclic mechanical elongation and compression within the same well. The resulting forces are transmitted within the plane of the Silastic membrane and the attached cells. The strain levels (20% maximum elongation, 2% compression) are comparable to physiological levels. Cyclic strain at 30 cpm significantly decreased the rate of wound closure in CTE, Calu-3, NHBE, and 1HAEo- cells (Fig. 3). Although the rate of closure was different for each cell type, the inhibitory effects of compression and elongation were similar in each case: cyclic compression attenuated wound closure more extensively than cyclic elongation except in 1HAEo- cells. This was observed even though the magnitude of compression (1-2%) was much less than the magnitude of elongation (up to 20%). If we define the resting state of the airways as that which occurs at functional residual capacity, then both elongation and compression occur in vivo. Circumferential and longitudinal expansion of airways would occur during normal respiration and potentially to a greater extent during mechanical ventilation. Compressive forces can result from the downward and upward pull of the diaphragm, from constriction of smooth muscle around airways, and from gravitational forces (18, 33). Circumferential compression occurs during bronchoconstriction, but it is unclear whether longitudinal compression also occurs. Our results raise questions as to whether prolonged states of bronchoconstriction may impair epithelial healing.

Wound closure was also dependent on TR during each cycle. Although wells strained at 10 and 30 cpm were both maximally strained at 20% and were both compressed ~2%, the extent of wound closure in wells strained at 10 cpm was dependent on TR (Fig. 4). There was no effect of cyclic strain on wound closure at 10 cpm when TR was 5 s, but as TR decreased, there was progressively greater inhibition of wound closure. Despite the difference in frequency of stretch (10 or 30 cpm), wounds in wells with TR = 1 s were similarly inhibited in wound closure. This indicates that the duration of relaxation during the respiratory cycle, rather than the time of strain, is the most important parameter in determining wound closure. Furthermore, wound closure in monolayers strained at 10 cpm (1 s stretch, 5 s relax) was not inhibited. These results suggest that, with TR = 5 s, cells progress through the mechanisms of spreading, migration, and proliferation similarly to unstretched cells, but as TR decreases, one or more of these processes is inhibited.

Significant cell division as a mechanism of wound healing is not likely to occur in cells before 15-24 h (6) but may contribute to wound closure after this time. In CTE monolayers, proliferation did not change significantly in unstretched wells until 20 h, as measured by BrdU labeling. There was no change in BrdU labeling index in stretched regions of wells, but after 20 h, compressed regions displayed significantly less proliferation than those in static wells. The BrdU labeling index of Calu-3 cells did not change significantly in static or flexed wells after 24 and 48 h. Also, cyclic strain decreased proliferation of Calu-3 cells after 48 h, as measured by cell counting (Table 1). This is in contrast with previous reports of strain-induced increases in proliferation of human lung fibroblasts (3) and fetal rat lung cells (14). Strain-induced proliferation in human lung fibroblasts was attributed to production of autocrine growth factors (3); however, it is possible that signal transduction molecules such as protein kinase C (PKC) may be involved. Mechanical strain activated PKC in both bovine aortic endothelial cells (26) and mixed fetal rat lung cells (15), which may trigger downstream events that lead to cell proliferation. However, mechanical strain-induced activation of PKC did not mediate strain-induced proliferation in bovine aortic smooth muscle cells (19), and cyclic stretch inhibited PKC activation in bovine articular chondrocytes (7). In preliminary experiments, wound closure in Calu-3 cells was not affected by the PKC inhibitor calphostin C (data not shown), although we have not investigated the effects of calphostin C on proliferation. The role of PKC in cyclic strain-induced regulation of proliferation remains unclear and may be dependent on the type of cell. Very low levels of LDH in the media 48 h after stretching indicate that no significant cell death is occurring, but it is conceivable that apoptosis may contribute to impairment of closure in stretched wells.

In the absence of significant proliferation, cell spreading and migration are the primary mechanisms of wound closure in the initial 15-24 h (11, 34, 35). In CTE and Calu-3 monolayers, wound closure was significantly less in cyclically strained wells as early as 4 h (Fig. 3, A and B) in both elongated and compressed regions. To assess cell spreading as a mechanism of wound closure in Calu-3 cells, the CD was measured between cells at the leading edge of the wound. According to Kheradmand et al. (11), an increase in internuclear distance suggests that cell spreading is the primary mechanism of wound closure. We utilized CD rather than internuclear distance, but both measurements identify changes in the location of cells over time. CD increased over 48 h in unstretched wells (Fig. 5A), suggesting that cell spreading as an important mechanism of wound closure in these experiments. Furthermore, cell area significantly increased at the wound edge (Fig. 5B) in static wells. In cyclically strained wells, neither CD nor cell area changed in elongated regions of the wells. In compressed regions, there was a significant decrease in CD and cell area, and the extent of the decrease was dependent on TR. According to Zahm et al. (34, 35), spreading of cells at the wound edge pulls the cells behind them, causing progressive deformation and spreading of the cells around the wound edge. Cyclic elongation inhibited this process, and cyclic compression caused cells to become even smaller than control cells. These results are in contrast to cell size measurements in cardiac myocytes in which mechanical overload (cardiac hypertrophy) results in increased cell size (29). The dependence on TR is important, suggesting that the mechanism of spreading is dependent on the ability of cells to spread on a static surface for a critical amount of time (>4 s). We could not directly measure cell migration during cyclic strain because the wells cannot be visualized during strain with the Flexercell apparatus. However, the fact that wound closure occurred in the elongated and compressed regions in the absence of cell spreading suggests that migration did occur. Both cell spreading and migration involve formation of new lamellipodia, cytoskeletal rearrangement, and redistribution of integrin-matrix attachments. Each of these processes may be regulated by the continuous movement of the substrate and by the cyclic changes in cell shape due to elongation or compression. Integrins have also been shown to play an important role in the cellular responses to mechanical stress (29). In future studies, it will be important to investigate cell-matrix interactions, formation of actin stress fibers, and integrin expression in cyclically strained cells.

Clinically induced airway injuries resulting from endotracheal intubations or physician error present the full spectrum of injury, ranging from mild edema to complete destruction of the mucosa and cartilaginous structure of the airways (5, 16). Several studies have indicated that airway damage caused by conventional mechanical ventilators (CMV) is less severe than that caused by high-frequency devices (16, 28). High-frequency jet ventilators (HFJV) and high-frequency positive-pressure ventilators (HFPPV) can cause significant inflammation, erosion, and necrosis at the endotracheal tube tip (28), and high-frequency oscillators (HFO) can lead to bronchopneumonia (24) and necrotizing tracheobronchitis (12). Although the respiratory frequency of mechanical ventilation is usually determined by factors such as patient weight, tidal volume, asthmatic status, and desired blood CO2 concentration, our results suggest that another factor that should be considered is the presence of epithelial damage. In the present study, repair of damaged epithelium was found to be dependent on the TR during the cycle. Figure 6 shows the typical pressure waveforms in CMV, HFPPV, HFJV, and HFO compared with the waveform for the Flexercell device. In Fig. 6, the intrabreath relaxation (when the slope of the pressure curve is ~0) can be equated with TR, or a state of no strain. CMV has the largest TR, with high-frequency devices providing little or no TR, which may be related to the severity of airway damage observed with these ventilator strategies in which short relaxation times may impede repair. Note, however, that changes in strain are lower with HFO due to the smaller tidal volume. The breath and intrabreath activity can be modified by altering the inspiratory flow rate and inspiratory and expiratory time, tidal volume, and lung volume (28). If mechanical ventilator strategies can be designed to provide longer intrabreath relaxation times, epithelial repair may proceed more rapidly.


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Fig. 6.   Magnitude of vacuum pressure for Flexercell at 30 cpm (A) and typical pressure waveforms for conventional mechanical ventilation at 0.5 Hz (CMV; B), high-frequency positive-pressure ventilation at 2 Hz (HFPPV; C), high-frequency jet ventilation at 5 Hz (HFJV; D), and high-frequency oscillation at 7.5 Hz (HFO; E).

This is the first report of the influence of cyclic mechanical strain on wound healing. Wound closure was inhibited by both elongation and compression, and the extent of inhibition was dependent on the relaxation time between cycles.

    ACKNOWLEDGEMENTS

We thank Dr. Steve White (University of Chicago) for helpful discussions regarding these experiments.

    FOOTNOTES

This work was supported by a Whitaker Foundation Special Opportunity Award, the American Lung Association of Metropolitan Chicago, and the Cornelius Crane Asthma Center of Northwestern University. U. Savla is supported by a National Science Foundation Graduate Fellowship.

Address for reprint requests: C. M. Waters, Dept. of Anesthesiology, Northwestern Univ. Medical School, Ward Bldg. 12-189 W139, 303 E. Chicago Ave., Chicago, IL 60611.

Received 21 November 1997; accepted in final form 26 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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