Macrophage responsiveness to therapeutic ultrasound

Ultrasound in Med. & BioL Vol. 16, No. 8, pp. 809-816, 1990 Printed in the U.S.A.

0301-5629/90 $3.00 + .00 © 1990 Pergamon Press plc

OOriginal Contribution MACROPHAGE

RESPONSIVENESS

TO THERAPEUTIC

ULTRASOUND

S. R. YOUNG a n d M . DYSON Tissue Repair Research Unit, Department of Anatomy, United Medical and Dental Schools of Guy's and St. Thomas's Hospitals (Guy's Hospital Campus), London, SE 1 9RT, England (Received 5 March 1990; in final form 26 April 1990) Abstract--Macrophages are a source of many important growth factors which can act as wound mediators during tissue repair. The aim of this work was to find out if levels of ultrasound which accelerate repair could stimulate the release of fibroblast mitogenic factors from an established macrophage-like cell line (U937). The U937 cells were exposed in vitro to continuous ultrasound at a space average, temporal average intensity of 0.5 W / e m 2 at either 0.75 M H z or 3.0 M H z , for 5 min. The macrophage-conditioned medium was removed either 30 min or 12 h after exposure, and placed on 3T3 fibroblast cultures. Fibroblast proliferation (defined here as increase in cell number) was assessed over a 5-day period. The results showed that 0.75 M H z ultrasound appeared to be effective in liberating preformed fibroblast affecting substances from the U937 cells, possibly by producing permeability changes, whereas 3.0 M H z ultrasound appeared to stimulate the cell's ability to synthesize and secrete fibroblast mitogenic factors.

Key Words: Acoustics, Ultrasonics, Tissue repair, Macrophages, Growth factors, Wound healing.

macrophages. The PMN is the predominant cell type in the first few days after injury, but by about the fourth or fifth day the macrophage predominates and remains throughout the subsequent proliferative phase of repair, when granulation tissue develops at the wound site. The presence of macrophages in close proximity to the fibroblasts in the wound bed suggests that there may be an interaction between the two cell populations. This was substantiated by an in vivo study showing impaired fibroplasia in monocytopenic animals (Leibovich and Ross 1975). Macrophage-conditioned medium, i.e., culture medium from macrophages maintained in vitro, was found to stimulate fibroblast proliferation and DNA synthesis (Leibovich and Ross 1976). Rutherford et al. (1982) reported the discovery of a fibroblast mitogenic factor derived from h u m a n monocytes stimulated with muramyl dipeptide. Factors mitogenic for fibroblasts were also reported to be released from the murine macrophage-like tumour cell line P388D (Wharton et al. 1982) and the human histiocytic lymphoma cell line U937 when stimulated (Wharton 1983). Korn et al. (1980) found that medium conditioned by human blood monocytes inhibited the growth of dermal fibroblasts in 20% human serum,

INTRODUCTION The initial cellular responses immediately after wounding involve the interaction of platelets with thrombin and collagen, which results in local blood coagulation, accompanied by mast cell degranulation, which results in the liberation of an array of chemical mediators many of which are involved in the inflammatory phase of tissue repair (Yurt 1981). Examples of pharmacologically active agents derived from mast cells include histamine which stimulates exudation, heparin which stimulates angiogenesis, and other numerous chemical mediators which stimulate invasion by leucocytes, cells which in turn release factors which attract fibroblasts and endothelial cells to the injury site and stimulate their proliferation. On completion of the coagulation process there begins a chronological sequence of events characterised by the appearance of various cellular infiltrates in the wound. This sequence was first recognised by Metchnikoff ( 1891) and was quantified by Ross and Benditt (1961). Within a few hours, the edge of the injured area is infiltrated with polymorphonuclear leucoeytes (PMNs) and monocytes, their number increasing over the following few days. On arrival at the wound site, the monocytes develop into activated 809

810

Ultrasound in Medicine and Biology

but stimulated their production o f P G E 2 . Prostaglandin was believed to be the cause of this inhibition since it was abolished when indomethacin, a prostaglandin inhibitor, was added to the fibroblast culture along with the monocyte-conditioned medium. This dialysable growth-suppressive factor was also noted by Leibovich (1975) and Diegelmann et al. (1982). In summary, it can be said that macrophages play a crucial role in wound repair both as scavenger cells involved in the debridement of wounds and as a source o f valuable growth factors stimulating wound repair. The purpose of this investigation was to find out if levels o f ultrasound which have been shown to accelerate tissue repair (Young and Dyson 1990a) could stimulate macrophages to produce fibroblast mitogenic factors using the established cell line U937. These cells have been shown to synthesize and secrete chemical mediators which stimulate the division of cultured fibroblasts (Wharton et al. 1982). The U937 cell line has a fast population doubling time (20-48 h), so vast numbers of similar cells can be acquired rapidly. In addition, large volumes o f conditioned medium can be inexpensively and rapidly obtained (Sundstrom and Nilsson 1976). M A T E R I A L S AND M E T H O D S U937 Cell culture The U937 cells were grown in Rose Park Memorial Institute (RPMI) 1640 (Flow Laboratories). The medium was supplemented with 1% penicillin and streptomycin ( G I B C O Ltd., Uxbridge, Middlesex, England) and 10% heat-inactivated fetal calf serum (GIBCO Ltd.). The cells were subcultured approximately every 2 days, and were grown in 50 m L Nunc culture flasks at 37°C in an incubator with a 5% CO2 atmosphere. Each confluent flask contained approximately l million cells per mL. Trypsinization was not necessary as the cell line does not attach firmly to the substratum.

Cell irradiation All irradiations were carried out in sterile centrifuge tubes (GIBCO Ltd.). The centrifuge tubes were made from polyethylene and the dimensions were 100 × 13 mm. By inserting a PZT5 detector into a centrifuge tube and placing the tube into the ultrasound field, the attenuative properties of the centrifuge tubes were assessed. It was found that the peak pressure amplitude fell by approximately 6% when the detector was encased in the tube. When confluent, the cells were transferred to the centrifuge tubes in 5 m L volumes, with cell densities of 1 million/mL. The dilutions were made up with fresh RPMI.

Volume16, Number 8, 1990 The centrifuge tubes were then placed in the irradiation tank (8 cm from the ultrasound source), maintained at 37°C, and subjected to various exposures o f ultrasound (Fig. l). The ceils were exposed to continuous ultrasound at an intensity of 0.5 W/cm 2 Spatial Average, Temporal Average (SATA). The frequencies used were either 0.75 or 3.0 MHz. Ultrasound calibration was carried out using the method described in detail by Y o u n g and Dyson (1990b). Briefly, the m e t h o d involved measuring the total power output using a tethered float radiometer and dividing this value by the effective radiating area of the transducer (obtained from a field plot across the face of the applicator head), to give an intensity measurement in W/cm 2. All irradiations were carried out in a water bath with the transducer 8 cm from the centre of the centrifuge tube (within the near field). After irradiation, the cells were left for either 30 rain or 12 h in the incubator and then centrifuged for 10 min at 500 rpm; the supernatant was collected and stored in the freezer at - 2 5 ° C . These times were chosen to allow for the possibility of the ultrasound having a two-stage effect, i.e., a rapid effect of causing the release o f preformed products in the cell cytoplasm, e.g., various enzymes and bioactive lipids etc., or a delayed effect, e.g., the activated synthesis and release of factors such as fibroblast mitogenic monokines. Below is a description of the treatment regimes.

Group 1: Control. Sham-irradiated for 5 rain and left in the incubator for 30 min, then centrifuged at 2000 rpm for 4 min, and the supernatant removed and frozen. Group 2: Test. Treated with continuous ultrasound at a frequency of 0.75 M H z and an intensity of 0.5 W/cm 2 SATA for 5 min. Returned to the incubator for 30 min, then centrifuged, and the supernatant removed and frozen. Group 3: 7"est. Treated with continuous ultrasound at a frequency of 3.0 MHz and an intensity of 0.5 W / c m 2 SATA for 5 min. Returned to the incubator for 30 min, then centrifuged, and the supernatant removed and frozen. Group 4: Control. Sham-irradiated for 5 min, left in the incubator for 30 min, and washed in fresh RPMI. Returned to the incubator for 12 h, then centrifuged, and the supernatant removed and frozen. Group 5: Test. Treated with continuous ultrasound at a frequency o f 0.75 M H z and an intensity of 0.5 W / c m 2 SATA for 5 min, left in the incubator for 30 min, and washed with fresh RPMI. Returned to the incubator for 12 h, then centrifuged, and the supernatant removed and frozen.

Macrophage responsiveness to therapeutic ultrasound • S. R. YOUNGand M. DYSON

811

Fig. I. Photograph showing the experimental arrangement for the exposure of the U937 cells to either sham or ultrasound irradiation, a" ultrasound applicator head; c" U937 cell suspension in centrifuge tube; g" ultrasound generator; w: degassed water (37°C).

Group 6: Test. Treated with continuous ultrasound at a frequency o f 3.0 M H z and an intensity of 0.5 W / c m 2 SATA for 5 min, left in the incubator for 30 min, and washed with fresh RPMI. Returned to the incubator for 12 h, then centrifuged, and the supernatant r e m o v e d and frozen. All irradiation procedures were repeated 6 times. Cell viability test At the end o f each treatment, the U937 cells were tested for viability using the trypan blue exclusion test (Tennant 1968), and counted using a Neubauer counting chamber. Six counts were carried out for each group.

Fibroblast culture and proliferation assay The supernatants were assayed for their mitogenic activity on fibroblast p o p u l a t i o n s using the m e t h y l e n e - b l u e assay described by M a r t i n et al. (1978). T h e fibroblasts used were Swiss 3 T 3 k m o u s e kidney (supplied by the Imperial Cancer Research central services, London, England). The fibroblasts were grown in D M E M supplem e n t e d with 10% FCS and 1% penstrep. The incubator was maintained at 37°C and had a 5% C ( 2 a t m o -

sphere. The cultures were confluent 3 days after subculture. When confluent each culture flask contained approximately 3 × 106-4 X 106 cells. The cells were detached from the flask surface using a 0.025% trypsin solution in phosphate buffered saline (PBS). T h e assay involved the plating out o f k n o w n numbers of fibroblasts into each well of a 96-well microplate (Flow Laboratories). The fibroblasts were covered with k n o w n concentrations o f the supernatant from the U937s, and then allowed to grow in the incubator for 3 days. At the end of the test period the fibroblasts were fixed with 100% m e t h a n o l a n d stained with methylene blue (0.1% in 10 m M borate buffer). The a m o u n t of stain which is taken up relates directly to the n u m b e r o f cells present in the wells. Therefore, by reading the absorbance of each well in a spectrophotometer, we obtain data which can be related to cell number, using a standard curve constructed as follows. A range of fibroblast densities (0-20,000 fibroblasts/100 v L culture m e d i u m ) was pipetted into a 96-well microplate. The plate was incubated for 6 h to allow the cells to attach. The methylene blue assay (Martin et al. 1978) was then carried out. F r o m the results obtained, a standard curve relating absorbance (650 nm) to cell n u m b e r was constructed (Fig. 2).

812

U l t r a s o u n d in M e d i c i n e a n d Biology

V o l u m e 16, N u m b e r 8, 1990

.26 .24

.20

E

c .16 o to ¢D itl 07 .12-

0 ¢D .08 -

.04 -

2

4

B

CELL

NUMBER

8

10

12

14

16

(xlO00)

Fig. 2. Standard curve plotting cell number (X 1000) against absorbance at 650 nm. Standard deviations are shown. T o assay the effect of the U937-conditioned med i u m on proliferative activity, the following procedure was carried out. Fibroblasts were pipetted in 100 ~L volumes into a 96-well microplate so that each well contained 1000 cells. The culture m e d i u m in which the cells were suspended was a 50:50 mix of D M E M and the supernatants taken from the various treatment groups of the U937s (as described above). Six plates (one for each irradiation procedure) were set up for each time period, i.e., time 0 (6 h post-plating), 1 'day, 2 days and 3 days. At the end o f each time period, the corresponding 6 plates were prepared for absorbance reading on the s p e c t r o p h o t o m e t e r using the s a m e m e t h o d as used for the standard curve. After the microplates had been read spectrophotometrically, the pooled results from each of the 6 microplates, per time period, were averaged and the absorbance read against the standard curve to give cell n u m b e r per treatment per time period. As stated in the Introduction, the macrophageconditioned m e d i u m has been shown to stimulate the production of fibroblast P G E 2 (Korn et al. 1980). Since PGE2 is an inhibitor of fibroblast proliferation, it is possible that fibroblast proliferation in this experiment m a y be inhibited to some degree by PGE2.

To test this, another microtest plate was set up. Each well was seeded with 1000 fibroblasts in m e d i u m from U937s which had been irradiated with 3.0 M H z ultrasound and left for 12 h. Indomethacin (1 # g / m L concentration) was added to half of the wells to block the synthesis of PGE2. The microplate was prepared for spectrophotometric readings 3 days post-plating. All results were analysed using Student's t-test (n = 6). RESULTS Total cell n u m b e r did not change and no significant change in the viability was observed following the ultrasound irradiations of the U937 cell cultures (Fig. 3). The results o f the fibroblast proliferation assay are shown in Fig. 4. Figure 4a shows the effect of the U937 m e d i u m r e m o v e d 30 m i n after irradiation. Figure 4b shows the effect of the U937 m e d i u m removed 12 h after irradiation. F r o m Fig. 4a it can be seen that m e d i u m from the U937 cells treated with ultrasound at a frequency o f 0.75 M H z stimulated fibroblast proliferation while that from U937 cells treated at a frequency o f 3.0 M H z did not. It can also be seen that there was a

Macrophage responsiveness to therapeutic ultrasound Q S. R. YOUNG and M. DYSON 100-

I

I

x

1

2

3

813

x

75¢0 ._1 I,U

O I¢1 ,-I

50-

m

a~ 25-

4

5

6

GROUP NUMBER

Fig. 3. Bar graph showing the percentage of viable U937 cells remaining in each group after the experimental period. Standard deviations are shown. Group 1: control 30 min post-sham treatment; Group 2" control 12 h post-sham treatment; Group 3:30 min post 0.75 MHz ultrasound treatment; Group 4" 12 h post 0.75 MHz ultrasound treatment; Group 5" 30 min post 3.0 MHz ultrasound treatment; Group 6:12 h post 3.0 MHz ultrasound treatment. t e m p o r a r y fall in cell n u m b e r between day 1 and day 2. F r o m Figure 4b it can be seen that the medium from the U937 cells treated with 0.75 M H z and 3.0 M H z u l t r a s o u n d had a significantly greater (p < 0.001) stimulatory effect on fibroblast proliferation than did the sham-irradiated control group from day 2 onwards. The greatest effect was seen in the 3.0 M H z treated group. There was approximately a 20% increase in fibroblast proliferation (p < 0.001) when the culture was treated with indomethacin (Fig. 5). DISCUSSION 0.75 M H z ultrasound appears to be more effective in stimulating the release o f preformed mitogenic products from the U937 cells than either 3.0 M H z or the sham irradiation. There are two ways in which these products could have been released into the supernatant, either by disruption o f the cell m e m b r a n e or by an increase in either exocytosis or in the permeability o f the membrane. The cell counts and viability tests post-irradiation indicated that the cells were not lethally damaged; therefore, it seems more likely that there was a change in either exocytosis or m e m b r a n e permeability. Because the effect was frequency dependent, and it was the 0.75 M H z and not the 3.0 M H z that caused

the m a x i m u m effect, it is suggested that the mechanism responsible for the release of preformed mitogenic products may have involved cavitation. Measurements of peak negative pressure were made for each frequency. At 0.75 M H z the pressure was 0.178 MPa, and at 3.0 M H z it was 0.171 MPa. These levels are above the threshold pressure at which cavitation can occur (Williams 1987); it is thus possible that cavitation occurred in our treatments. It is unlikely that the effect could be attributed solely to heat as 3.0 M H z would be expected to generate more heat than 0.75 MHz, yet it was 0.75 M H z which caused the greatest release of products which stimulated the proliferation of fibroblasts. The high level of cell viability post-irradiation eliminates transient cavitation as a possible mechanism of action. The most likely mechanism involved in the production of cell permeability changes is stable cavitation, in which gas bubbles a few microns in diameter oscillate in a regular fashion for m a n y cycles. T h e e n h a n c e d m i c r o s t r e a m i n g around them may be responsible, at least in part, for the changes observed here, and also for membrane p e r m e a b i l i t y changes observed by other workers (Williams 1974; Williams et al. 1976; M u m m e r y 1978; Chapman et al. 1979; Mortimer and Dyson 1988), after treatment with therapeutic levels of ultrasound. The reported changes in permeability o f membranes to calcium ions (Mortimer and Dyson 1988)

814

Ultrasound in Medicine and Biology

Volume 16, Number 8, 1990

6

S o o T-

Control

/

5

/

x

4 0.75 MHz

.O

S z o

3 3.0 MHz

2 1 0 0

I 1

I 2

I 3

D a y s Post Plating (a)

11 10

9

/ #

Control

/ S o o

0.75 MHz

i

/

x

/

/ /

3.0 MHz

J~

E z

4

0 3 2

0

I 1

I 2

I 3

D a y s Post Plating (b)

Fig. 4. Graphs plotting the effect of the U937 conditioned medium on fibroblast proliferation. (a) Conditioned medium taken 30 min after irradiations. (b) Conditioned medium taken 12 h after irradiations. All results which are significantly different (p < 0.001 ) from the control group are indicated by an asterisk (*). i n d u c e d by therapeutic ultrasound m a y have dramatic effects on cell behaviour. Calcium ion fluxes act as chemical signals (second messengers) which, in response to m e m b r a n e changes, control the enzymatic activity o f the cell and stimulate, for example,

the increase in synthesis of specific proteins and their secretion (Katz 1966; Douglas 1968; Webster et al. 1978, Fyfe and Chahl 1982). The results illustrated in Fig. 4b indicate that U937 cells irradiated with ultrasound and then left

Macrophage responsiveness to therapeutic ultrasound • S. R. YOUNG and M. DYSON

10

I

O O

rr UJ tIl Z ..I ..I I.U O

A

B

C

GROUP

Fig. 5. Bar graph showing the effect of indomethacin on fibroblast proliferation 3 days post-plating. Standard deviations are shown. Group A: initial cell number; Group B: cells receiving supernatant from U937s 12 h post-irradiation; G r o u p C: cells pretreated with i n d o m e t h a c i n (1 ug/mL) prior to addition of supernatant from U937's 12 h post-irradiation.

for 12 h b e f o r e t h e s u p e r n a t a n t was r e m o v e d released active factors i n t o t h e m e d i u m w h i c h e n h a n c e fibroblast p r o l i f e r a t i o n . T h e degree o f e n h a n c e m e n t was g r e a t e r in this g r o u p o f cells t h a n in t h e g r o u p s w h i c h were left for o n l y 30 m i n after t r e a t m e n t . Also, in t h e g r o u p s left for 12 h after t r e a t m e n t it was t h e 3.0 M H z a n d n o t t h e 0.75 M H z f r e q u e n c y w h i c h h a d t h e greatest s t i m u l a t i n g effect. T h i s is t h e reverse o f t h e effect i l l u s t r a t e d in Fig. 4 a w h e r e 0.75 M H z c a u s e d t h e g r e a t e r effect. T h e r e f o r e , it a p p e a r s t h a t e x p o s u r e to 0.75 M H z u l t r a s o u n d causes a g r e a t e r i m m e d i a t e effect w h e r e a s cells t r e a t e d w i t h 3.0 M H z a p p e a r to n e e d m o r e t i m e t o b e c o m e effective, b u t t h a t after this t i m e has e l a p s e d , t h e i r effectiveness is greater. T h e results o b t a i n e d suggest t h a t 0.75 M H z ultras o u n d c a n s t i m u l a t e t h e l i b e r a t i o n o f m i t o g e n i c subs t a n c e s a l r e a d y in t h e cells, p o s s i b l y b y p r o d u c i n g p e r m e a b i l i t y changes, w h e r e a s 3.0 M H z u l t r a s o u n d a p p e a r s to b e b e t t e r a b l e to s t i m u l a t e t h e cell's a b i l i t y t o s y n t h e s i s e factors w h i c h are r e l e a s e d s o m e t i m e l a t e r b y t h e cell's n o r m a l s e c r e t o r y processes. It is p o s s i b l e t h a t 0.75 M H z u l t r a s o u n d c a n also s t i m u l a t e t h e s y n t h e s i s o f t h e s e a c t i v e factors b u t to a lesser degree t h a n t h e 3.0 M H z .

815

T h e i n c r e a s e in t h e r a t e o f p r o l i f e r a t i o n s e e n w h e n i n d o m e t h a c i n was a d d e d suggests t h a t t h e fib r o b l a s t c u l t u r e s s y n t h e s i z e P G E 2 in r e s p o n s e to t h e U 9 3 7 s u p e r n a t a n t . I n t e r l e u k i n - 1 (IL-1) is o n e o f t h e factors r e s p o n s i b l e for m e d i a t i n g v a r i o u s local a n d s y s t e m i c effects o f i n f l a m m a t i o n . A m o n g these effects is t h e s t i m u l a t i o n o f p r o s t a g l a n d i n p r o d u c t i o n in c o n n e c t i v e tissue cells ( P o s t l e t h w a i t e et al. 1983). I n s u m m a r y , t h e results o b t a i n e d s h o w t h a t t h e U 9 3 7 cells c a n b e s t i m u l a t e d b y t h e r a p e u t i c levels o f ultrasound both to release preformed substances w h i c h are r e s i d e n t in t h e i r c y t o p l a s m , a n d also to start s y n t h e s i s i n g n e w factors. T h e o b s e r v a t i o n t h a t u l t r a s o u n d c a n affect f a c t o r r e l e a s e f r o m m a c r o p h a g e s explains, in part, w h y p h y s i o t h e r a p i s t s find u l t r a s o u n d p a r t i c u l a r l y effective w h e n u s e d d u r i n g t h e i n f l a m m a t o r y p h a s e o f repair, as this is w h e n the m a c r o p h a g e is t h e p r e d o m i n a n t cell type. It is suggested t h a t t h e m a i n m e c h a n i s m i n v o l v e d in p r o d u c ing these effects i n v o l v e s stable c a v i t a t i o n . Acknowledgements--We wish to thank the following: Mr. Peter

Bolton, Ms. Millicent Harrison and Dr. Gay Kingsley for advice on cell culture, Johnson and Johnson for support and advice about the U937 cell line, Dr. John Pond for advice on ultrasound calibration, and Professor Martin Berry in whose department the work was carried out. REFERENCES Chapman, I. V.; Macnally, N. A.; Tucker, S. Ultrasound induced changes in the rates of infux and efflux of potassium ions in rat thymocytes in vitro. Br. J. Radiol. 47:411-415; 1979. Diegelrnann, R. F.; Cohen, I. K.; Kaplan, A. M. Effect of macrophages on fibroblast DNA synthesis and proliferation. Proc. Soc. Exp. Biol. Med. 169:445-451; 1982. Douglas, W. W. Stimulus-secretion coupling: The concept and clues from chromaffin and other cells. Br. J. Pharmacol. 34:453-474; 1968. Fyfe, M. C.; Chahl, L. A. Mast cell degranulation. A possible mechanism of action of therapeutic ultrasound. Ultrasound in Med. & Biol. 8 (Suppl. 1):62; 1982. Katz, B. Nerve, muscle and synapse. New York: McGraw-Hill; 1966:63-85. Korn, J. H.; Halushka, P. V.; Leroy, E. C. Mononuclear cell modulation of connective tissue function. J. Clin. Invest. 65:543-554; 1980. Leibovich, S. J.; Ross, R. The role of the macrophage in wound repair. Am. J. Pathol. 78( 1):71-92; 1975. Leibovich, S. J. Production of macrophage-dependent fibroblaststimulating activity (M-FSA) by murine macrophages. Exp. Cell. Res. 113:47-56; 1975. Leibovich, S. J.; Ross, R. A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vitro. Am. J. Pathol. 84:501-513; 1976. Martin, F.; Martin, M.; Jeannin, J. G.; Lagneau, A. Rat macrophage-mediated cytotoxicity to cancer cells: Effect ofendotoxin and endotoxin inhibitors contained in culture medium. Eur. J. Immunol. 8:607-611; 1978. Metchnikoff, E. Lectures on the comparative pathology of inflammation (Starling, F. A.; Starling, E. H., trans.). New York: Dover; 1891. Mortimer, A. J.; Dyson, M. The effect of therapeutic ultrasound on calcium uptake in fibroblasts. Ultrasound in Med. & Biol. 14(6):499-506; 1988.

816

Ultrasound in Medicine and Biology

Mummery, C. L. The effect of ultrasound on fibroblasts in vitro. Ph.D. thesis, University of London; 1978:265-283. Postlethwaite, A. E.; Lachman, L. B.; Mainardi, C. L.; Kang, A. H. Interleukin I stimulation of collagenase production by cultured fibroblasts. J. Exp. Med. 157:801-806; 1983. Ross, R.; Benditt, E. P. Wound healing and collagen formation 1. Sequential changes in components of guinea pig skin wounds observed in the electron microscope. J. Biophys. Biochem. Cytol. 11:677-700; 1961. Rutherford, B.; Steflin, K.; Sexton, J. Activated human mononuclear phagocytes release a substance(s) that induces replication of quiescent human fibroblasts. J. Reticulo. Soc. 31:281-293; 1982. Sundstrom, C.; Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer. 17:565-577; 1976. Tennant, J. R. Evaluation of the trypan blue technique for the evaluation of cell viability. Transplantation 2:685-694; 1968. Webster, D. F.; Pond, J. B.; Dyson, M.; Harvey, W. The role of cavitation in the in vitro stimulation of protein synthesis in human fibroblasts by ultrasound. Ultrasound in Med. & Biol. 4:343-351; 1978. Wharton, W.; Gillespie, G. W.; Russell, S. W.; Pledger, W. J. Mi-

Volume 16, Number 8, 1990 togenic activity elaborated by macrophage-like cell lines act as competence factor(s) for BALB/c-3T3 cells. J. Cell. Physiol. 110:93-100; 1982. Wharton, W. Human macrophage-like cell line U937-1 elaborates mitogenic activity for fibroblasts. J. Reticuloendothel. Soc. 33:151-156; 1983. Williams, A. R. Release of seratonin from human platelets by acoustic microstreaming. J. Acoust. Soc. Amer. 56:1640-1643; 1974. Williams, A. R.; Sykes, S. M.; O'Brien, W. D. Ultrasonic exposure modifies platelet morphology and function in vitro. Ultrasound in Med. & Biol. 2:311-317; 1976. Williams, A. R. Production and transmission of ultrasound. Physiotherapy 73(3):113-116; 1987. Young, S. R.; Dyson, M. Effect of therapeutic ultrasound on the healing of full-thickness excised lesions. Ultrasonics 28:175180; 1990a. Young, S. R.; Dyson, M. The effect of therapeutic ultrasound on angiogenesis. Ultrasound in Med. & Biol. 16(3):261-269; 1990b. Yurt, R. W. Role of the mast cells in trauma. In: Dineen, P., ed. The surgical wound. Philadelphia: Lea and Febiger; 1981:3762.