In vitro effects of therapeutic ultrasound on the nucleus of human fibroblasts.

Key Words: cell culture, Chromosomal aberrations, Spindle, Therapeutic ultrasound, Ultrasound.

The biological and cytological effects of therapeutic ultrasound, in response to sonication at a frequency of 1 MHz and intensities in the range of 0.5 to 3 W/[cm.sup.2], are poorly documented, although ultrasound at this frequency and these intensities is frequently used in combination with other modalities.[1-3]

Studies that focus on the physical effects of therapeutic ultrasound offer little information on the biological implications of sonication.[4,5] Biomechanical studies on surgically repaired rabbit Achilles tendons suggest an increase in tensile strength as a result of ultrasound treatments.[6] In vivo method as are often used to study biological changes.[7-10] An increase in fibroblast profileration[7] and an increase in glycoaminoglycan synthesis[8] after ultrasound treatment have been observed in connective tissues. These cellular effects suggest a supporting role for ultrasound in the treatment of wound healing.[9-11] A report on a possible teratogenic effect of therapeutic ultrasound was inconclusive.[12] An increase in tumor size, however, has been noted using continuous sonication.[13] Dinno et al[14] reviewed the effects of therapeutic ultrasound at the cellular level. They proposed that therapeutic ultrasound results in an increased ion flow across cell membranes and in increased intracellular [Ca.sup.2+] levels. These intracellular changes might result in increased secretion, cell motility, and synthesis of growth factors, aU of which might have beneficial effects on the tissue (wound healing) that is being treated.[14] In the review by Dinno et al,[14] however, no variables such as intensities and mode of application were mentioned.

Another way of evaluating the biological effects of ultrasound is by using cell biology techniques such as cell culture. An in vitro approach might show how ultrasound affects cells and their organelles. Kerr et al,[15] using hela cells, showed disruption of the cell membrane after in vitro ultrasound treatment. A study at the level of the nucleus was performed by Stella et al,[16] who observed an increase in sister-chromatid exchanges in human lymphocytes, but no chromosomal aberrations. Chromosomal aberrations are mutations and have been shown to be related to the formation of neoplasms.[17] Experiments on the possible mutagenic effects of therapeutic ultrasound have been inconclusive.[18,19] A recent study,[20] however, showed a tumor enlargement. No epidemiological evidence has linked cancer to the therapeutic use of ultrasound.[21]

The purpose of this work was to determine whether (1) an in vitro approach can be used to study the effect of therapeutic ultrasound at the cellular level, (2) therapeutic ultrasound induces a change in the number of cells, and (3) therapeutic ultrasound has any effect on the morphology of the chromosomes or the presence of mitotic spindles.

Method and Materials

Ultrasound

A Sonopuls 417 therapeutic ultrasound device(*) was used, which was calibrated by the manufacturer. The ultrasound transducer head (3 cm diameter) was wiped with 700% ethanol to maintain sterility. The sonic head was secured over a 35-mm dish, which was filled with a total volume of 1.5 mL of cell culture medium at 37[degrees]C. The sonic head was positioned perpendicular to the dish and lowered to contact the medium. The distance between the transducer head and the bottom of the dish was approximately 3 to 4 mm. To minimize the heating effect of the ultrasonic energy, a pulsed mode of 2 milliseconds "on" and 8 milliseconds "off" was used. output was set at 1 W/[cm.sup.2] (with the sonication being pulsed 2:8). Dose-response experiments were performed and showed that intensities of more than 1 W/[cm.sup.2] led to a loss of all the cells. For this reason, intensity and pulse mode were held constant and treatment time was the only variable, The cells were treated for 0 (controls), 30, 60, or go seconds; each group consisted of five to six dishes. Cells in the control group underwent the same handling and manipulations as the experimental groups.

Tissue Culture

In this experiment, we used fibroblasts(dagger) from the skin of a genetically normal human male. The cells were cultured in F12 medium containing 15% fetal calf serum, 1% L-glutamine, and 1% penicillin-streptomycin(double dagger) in 50% carbon dioxide and at 95% humidity and 37[degrees]C temperature. Before starting the experiment, the cells were detached from 25-mL plastic culture dishes using a 0.2% trypsin-0.2% ethylenediaminetetraacetic acid (EDTA) solution for 2 to 3 minutes, centrifuged and resuspended. An aliquot was taken to count the cells on a hemacytometer. one milliliter (containing 1 million cells) was resuspended in a 35-mm plastic culture dish. After sonication, the cells and the medium were collected, centrifuged, and resuspended and the cells were counted. The cells were then plated on a 22-mm glass coverslip in 35-mm plastic dishes and cultured for 24 hours. The fibroblasts were stained according to the method described by Wissinger et al.[13] This technique allows a differential staining of chromosomes and mitotic spindles. The cells were fixed in acetic acid: methanol (3:1), containing 4 mmol of magnesium chloride and 1.5 mmol of calcium chloride. After the 14-minute washes in the fixative, the cells were air dried, ribonucleic acid was removed, and the cells were treated for 24 hours in 5% perchloric acid at 4[degrees]C. After the cells were air dried, they were stained for 24 hours in the dark, using a solution consisting of 0.5% safranin 0 and 0.5% brilliant blue R.(sections) After the staining, the cells were washed in distilled water, air dried, and mounted on slides with a synthetic resin mounting medium.

To obtain the mitotic index, mitotic figures of the cells in random fields were scored. A field consisted of a square of 25 [mm.sup.2] obtained through an eyepiece micrometer. The cells were viewed with a X20 objective on a bright-field microscope. Chromosomal aberrations were scored while viewed with a X100 objective and classified according to the method described by Parry et al.[22] A minimum of 400 cells per slide were evaluated. The slide were coded to eliminate any bias from the observer, who counted four to six slides of each treatment. Table 1 describes the classification of the mitotic figures and the chromosomal aberrations as defined by Parry et al.[22] Even though the data were acquired using the extended classification, we reduced the data and represented them in a less elaborate classification (groups I, II, and III in Tab. 1).

[TABULAR DATA OMITTED]

Data Analysis

For all statistical analyses, an alpha level of .05 was used to determine significance. A one-tailed analysis of variance (ANOVA) was used to discern differences in (1) the number of cells recovered as a result of sonication and (2) the increase in mitotic index as a result of the ultrasound treatment. Post hoc tests were performed to detect differences between the treatment groups (0, 30, 60, and 90 seconds). The nonparametric chisquare test was used to examine the relationship between the ultrasound dose and the qualitative classification of mitotic figures.

Results

Cell counts (Fig. 1) revealed that compared with the control group (998[+ or -]53X[10.sup.3] cells/mL), there were 197[+ or -]50X[10.sup.3] cells/mL left after a 30-second pulsed ultrasound treatment. Longer treatments of 60 and 90 seconds resulted in 145[+ or -]34X[10.sup.3] cells/mL and 97[+ or -]25X[10.sup.3] cells/mL, respectively, being present after sonication. The ANOVA and Newman-Keuls post hoc tests indicated that each sonication period added to cell loss.

The mitotic index of the sonicated cells suggested a change in cell proliferation. We analyzed cell proliferation by using the mitotic index, which equals the number of mitotic figures/total cells countedX1,000 (Fig. 2). We saw a fourfold increase in the mitotic index after 30 seconds of sonication and a similar increase after 60 seconds of sonication. Surprisingly, 90 seconds of ultrasound increased the mitotic index threefold. Based on the mitotic index, cell proliferation in the experimental groups was increased compared with that of the control group.

The increase in mitotic index led us to further analysis of the chromosomes and mitotic spindles. Table 2 shows the original data, before they were reduced to the simplified classification (Tab. 1). Classification of the morphology of the chromosomes and the mitotic spindles (Tab. 3) allowed us to observe that the chromosomes of most cells in the control group had a normal morphology. For some reason, however, aberrant chromosomes (group II and group III) were observed in the control group cells. A 30-second in vitro sorication resulted in a nearly eightfold increase in the number of cells with group II aberrations, and cells with a chromosomal morphology classified as group III increased fourfold compared with the cells in the control group. A concomitant decrease in the number of normal mitotic figures was noted. A 60-second ultrasound treatment yielded similar results; aberrations classified as group II (no mitotic spindles) increased ninefold, and aberrations classified as group III (mitotic spindles present) increased threefold. Very few normal mitotic figures were found after the ultrasound treatment (1.39%[+ or -]0.84% with a 30-second treatment).

[TABULAR DATA OMITTED]

Discussion

The nonthermal effects of therapeutic ultrasound at die cellular level are poorly understood. Attention has been paid to the supporting influence of ultrasound in wound healing.[7,9,10] These in vivo experiments seem to indicate a profound effect of ultrasound at the cellular level. An increase in the amount of extracellular matrix components, such as glycoaminoglycans[8] and hydroxyproline (the unique amino acid of collagen),[9] has been reported. The importance of collagen and its interaction with glycoaminoglycans has been well documented and determines the strength and viscoelastic properties of connective tissues.[23]

Besides the stimulatory effect of ultrasound on fibroblasts as demonstrated in studies on wound healing,[7,9,10] therapeutic ultrasound has been reported to stimulate the secretion of mitogenic factors from macrophages.[24] Macrophages are important cells in the immune response to chronic inflammation. The observation that macrophages can be stimulated by ultrasound could support the clinical studies that suggest an optimal use for ultrasound during chronic inflammation rather than during acute inflammation.[25]

The dose-dependent decrease of the number of cells recovered (Fig. 1) demonstrated the destructive effect of nonthermal ultrasound. The decrease in cells recovered could be due to the mechanical, microvibratory effects of ultrasound that would destroy the cells. This lytic effect is nondescriminatory and in chronic inflammation could destroy scar tissue (including collagen and fibroblasts) as well as the macrophages, which clean up the debris. Macrophages, however, migrate to the inflammation site by chemotaxis. The hypothesis that ultrasound might increase chemotaxis for macrophages is appealing but has not been tested.

A lytic effect of ultrasound is thought to be mediated by a disruption of cell membranes.[15] Kerr et al[15] used scanning electron microscopy to demonstrate the appearance of pores in the cell membrane when the cells were sonicated in suspension. Using a similar experimental design, our report describes a dose-dependent cell loss, most likely due to the lytic effect of nonthermal ultrasound on the cultured fibroblasts.

The postsonication cells had a higher mitotic index, which indicates higher cell proliferation (Fig. 2). The mitotic index, however, did not show a dose dependency, suggesting that most of the effect occurred within 30 seconds after sonication (Fig. 1). These findings seem to be similar to those of Young and Dyson,[24] who reported an increase in the number of fibroblasts in vivo. Further analysis, using methods such as determining doubling time and [[H.sup.3]] thymidine incorporation, is needed.

Structural changes of the mitotic apparatus were studied using a detailed classification based on a differential staining of chromosomes and mitotic spindles. The qualitative analysis of the nucleus for the presence of normal chromosomes or mitotic spindles proved to be important in illustrating the extent of the nuclear changes. These data were reduced to a simplified classification of three groups (I, II, and III; see Tab. 1). In our experiments, the data were acquired 24 hours after the cells were treated with ultrasound, demonstrating that the cells were viable. Cell viability of 24 hours, however, does not necessarily imply the capability of cell division. The increased mitotic index (Fig. 2) we noted could be attributed to erroneously counted mitotic figures, which were in reality chromosomal aberrations. Note that the sum of the mitotic figures classified for each group in Table 3 is equal to the mitotic index, shown in Figure 2.

[TABULAR DATA OMITTED]

The increase in spindle/chromosome damage could result from the ultrasound affecting the microtubules/chromatin in the interphase rather than the chromosomes in mitosis. The data show that the biggest increase occurred in the mitotic figures classified as group II (aberrant with no mitotic spindles). If this finding is true, it would suggest a preferential adverse effect on the mitotic spindles or microtubules, more so than on the chromosomes. The interaction of ultrasound with mitotic spindles may be mechanical and unique due to the experimental design, but the results of this study need confirmation.

We documented an increase in chromosomal aberrations in fibroblasts as a result of an extremely short ultrasound treatment. Caution must be exerted in extrapolating results from in vitro experiments to in vivo situations. This is an inherent problem of cell/tissue culture. In our experimental set-up, the cells (in suspension) were directly exposed to the ultrasound. It might be possible that a small dose (30 seconds, 1 W/[cm.sup.2], pulsed 2:8) used in vitro resulted in a magnification of the energy received by the cells possibly through cavitation, acoustic streaming). It may be that the in vitro approach may not mimic the clinical situation and the ultrasound dose received is too strong. The chromosomal aberrations reported here could be so destructive for the cells that even if the cells are viable, they could be incapable of dividing and inducing neoplasms.

Conclusion

This report describes a dose-dependent cell loss resulting in fractionation of cells that could facilitate the resolution of chronic inflammation. Our experiments do not rule out a possible adverse effect of ultrasound at the level of the chromosomes and mitotic spindles. Further research is needed to determine whether the chromosomal aberrations reported here are truly mutagenic or are a result of the experimental design.

(*) Enraf International, 2600 AV Delft, the Netherlands (distributed by Gymna NV, 3740 Bilzen, Belgium). (dagger) Fibroblasts used in this study were donated by the Department of Plastic and Reconstructive Surgery, University of Brussels, Brussels, Belgium. (double dagger) GIBCO-BRL, Life Technologies, Gaithersburg, MD 20884. (sections) Sigma, St Louis, MO 63178.

References

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