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Effect of therapeutic ultrasound on endochondral ossification
Ultrasound in Med. & Biol., Vol. 21, No. I, pp. 121-127,
1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0301.5629195 $9.50 + .OO
Pergamon 0301-5629(94)00092-l
@Original Contribution
EFFECT
ANNEKE
OF THERAPEUTIC ULTRASOUND ENDOCHONDRAL OSSIFICATION
WILTINK,+* PETER J. NIJWEIDE,~ WIM
ON
A. OOSTERBAAN,”
ROB T. HEKKENBERG” and PAUL J. M. HELDERS~ Departments of ‘Cell Biology and Histology and tPhysiology and Physiological Physics, Leiden University, Leiden, The Netherlands; *TNO, Leiden, The Netherlands; and $Department of Pediatrics, Utrecht University, The Netherlands (Received
9 February
1994; in final
formIO March 1994)
Abstract-The effect of therapeutic doses of ultrasound was tested on endochondral ossification of in vitro developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice. Bone growth, calcification and resorption following exposure to several doses of pulse-wave (PW) or continuous-wave (CW) ultrasound were examined. PW was applied at intensities between 0.1 W cm-’ and 0.77 W cm-’ (I,,,) and CW intensities were 0.1 W cm-’ or 0.5 W cm-’ (I.,,). After 1 week of culture, the metatarsal long bone rudiments were fixed and paraffin sections were prepared for histological evaluation and for measurement of the relative contribution of the various cartilage zones to the total bone length. In contrast to treatment with CW ultrasound, treatment of lCday-old metatarsal long bone rudiments with PW ultrasound resulted after 4 days of culture in significantly increased longitudinal growth. Histology revealed a significant increased length of the proliferative zone, whereas the length of the hypertrophic cartilage zone was unaltered. This might indicate that proliferation of the cartilage cells is stimulated without influence on cell differentiation. Key Words: Therapeutic ultrasound. cation. Mechanical effects.
Endochondral
ossification,
Metatarsae,
Cartilage,
Resorption,
Calcifi-
(Carstensen 1986; Chapman et al. 1979; Dinno et al. 1989a; Dinno et al. 1989b; Mortimer and Dyson 1988). Such permeability changes may reflect activation of stretch activated ion channels in the cell membrane due to membrane perturbation (for review see Sachs 1988). This could then result in a change in Ca*+ ion permeability causing Ca2+ influx into the cell and subsequent increase in the intracellular calcium concentration, as was described for fibroblasts upon ultrasound treatment (Mortimer and Dyson 1988). Calcium is one of the most important intracellular messengers for extracellular signals known so far (for review see Berridge 1993). At higher therapeutic intensities, or if standing waves can develop resulting from interference of ultrasound waves due to reflection on, e.g., mineralized matrix of bone tissue, there is a potential hazard of collapse cavitation. This situation, which should be avoided, is damaging for the surrounding tissue due to formation of free radicals, extremely high local temperatures and high pressure waves (NCRP 1983; Carstensen 1986; Holland and Apfel 1990). Especially in tissues with different acoustic impedances, such as in bone tissue, where a mineralized matrix is surrounded
INTRODUCTION Therapeutic ultrasound may cause various physiological effects, generally divided into thermal effects at higher doses and nonthermal, mechanical effects resulting from stable cavitation at lower doses (NCRP 1983; Dyson 1987; Kitchen and Partridge 1990; Nyborg and Ziskin 1985; Holmes and Rudland 1991; Maxwell 1992). Whether thermal effects will occur depends on several factors such as duration of the treatment, intensity of the ultrasound beam, frequency-dependent absorption and thermal diffusivity (for review see NCRP 1983; Dyson 1987; Drewniak et al. 1989). In addition, for therapeutic thermal effects to arise, tissue temperature should remain between 40°C and 45°C for more than 5 min (Nyborg and Ziskin 1985; Dyson 1987). Therapeutic mechanical effects are most likely due to stable cavitation in combination with acoustic streaming of extracellular fluid, resulting in changes of the cell membrane permeability of adjacent cells Address correspondence to: A. Wiltink, Department of Physiology and Physiological Physics, Leiden University, P.O. Box 9604, 2300 RC Leiden, The Netherlands. 121
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culture medium, consisting of alpha minimal essential medium (MEM), 10% chicken embryonic extract, 10% cock serum, 20% cock plasma, 85 yglmL gentamycin. In some experiments, 1 mM or 0.1 mM beta-glycerophosphate was added to stimulate calcification. MT were cultured in a humidified atmosphere of 5% COz. in air at 37°C.
Fig. 1. Phase contrast photograph of 16-day-old fetal mouse metatarsal long bone rudiments (MT) in primary culture. MT (II-IV) were dissected along with metatarsus V to facilitate orientation of the triplets. The MT were cultured for 8 days
on semisolid culture medium, consisting of alpha-MEM, 10% chicken embryonic extract, 10% cock serum, 20% cock plasma and 85 bg/ml gentamycin. Note the area of calcified tissue (black area) in the center of each bone rudiment. Magnification
30X
by periosteum, reflection of the ultrasound beam and subsequent interference can result in peaks in ultrasound intensity (Wells 1977). In the case of bone this may cause damage to the periosteal cells. On the other hand, bone repair can be stimulated by ultrasound treatment via therapeutic effects on cells involved in fracture healing (Dyson and Brookes 1983; Tsai et al. 1992). One of the contraindications to ultrasound therapy of children or young adults is treatment of areas in the vicinity of developing tissue such as the epiphyseal discs, since it is generally assumed that insonation of these discs may influence normal bone development (NCRP 1983). Developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice can be used as an in vitro model system for endochondral ossification (Gaillard et al. 1977; Nijweide et al. 1978) as occurs in the epiphyseal discs of children and young adults. To assess the possible influence of therapeutic ultrasound on bone development or cartilage cell proliferation and differentiation, we tested therapeutic doses of ultrasound in this model. MATERIALS
AND
METHODS
Tissue culture Paired metatarsal long bone rudiments (II-IV) were dissected from 16- and 17-day-old fetal mice along with metatarsus V to facilitate orientation of the triplets (MT). The tissue between the metatarsae was not disrupted to avoid damage to the developing bone rudiments (Fig. 1). The MT were cultured on semisolid
Ultrasound treatment After 24 h of culture, MT were treated for 5 min or 1 min with l-MHz ultrasound (day 0). Pulse-wave ultrasound (PW; pulse frequency 100 Hz, pulse length 2 ms) was applied at intensities of 0.1 W cm-‘, 0.33 W cm-‘, 0.49 W cmp2 or 0.77 W cmp2 (Is+, spatial average temporal peak intensity), continuous-wave (CW) ultrasound had intensities of 0.1 W cm-’ or 0.5 W cmm2 (I,,,,, spatial average temporal average intensity). The experimental setup consisted of an ultrasound therapy unit (Sonopuls 464, Delft Instruments), a measurement tank filled with a freshly degassed standard extracellular solution (S-ECS) and Petriperm culture dishes (Dijkstra Vereenigde BV) as the exposure chambers containing the MT (Figs. 2 and 3). The SECS solution contained (in mM): 135 NaCl, 5.4 KCl, 0.45 KH,PO,, 0.42 Na,HPO,, 4.2 NaHCO,, 0.8 MgS04.7H20, 5.5 o-glucose, 1.25 CaC12 and 10.9 HEPES. The walls and bottom of the measurement tank were lined with rubber (Wallgone Model 431,
5cm n
3 Fig. 2. The experimental setup, consisting of the ultrasound therapy unit (1) the transducer (ERA 5 cm*) fixed to a metal rod (2), metal support (3) measurement tank filled with freshly degassed standard extracellular solution (S-ECS) and lined with ultrasound absorbing Wallgone rubber (4), Wallgone rubber mat (see Materials and Methods) (5) rubber ring (6), location of the exposure chambers with the treated MT (7) and paired control MT (8).
Effect of therapeutic
US 0 A. WILTINK et al.
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sured effective radiation area (ERA) by means of a radiation balance and a beamplot setup with a hydrophone (Hekkenberg and Oosterbaan 1986). Bone development
Fig. 3. The Petriperm culture dish (bottom up) used during the experiments, with dialysis tube (1) and fixation device (2). The dialysis tube is fixed to the culture dish, without allowing air bubbles to be included. Rotation of the fixation
device results in stretching the band around the culture dish, thereby firmly attaching the dialysis tube to the dish. During experiments MT were put in the center of the dish (arrow).
Consumer to reduce
Usage
Laboratories
Inc.)
specially
devised
reflection and to absorb the ultrasound energy. The Petriperm culture dishes had a thin bottom layer. MT were put on the backside of the bottom layer in 500-PL S-ECS, 10% cock serum and 85 yg/mL gentamycin. Dialysis tube was used to close the exposure chamber (Fig. 3). Special care was taken to exclude air bubbles from the inside of the exposure chamber. A near fully transmission of ultrasound was assumed and confirmed by visual observation using a Schlieren optical system. No reflection on the surfaces was observed. MT that were treated were put at a distance of 10 cm from the face of the ultrasound transducer (Fig. 2). This distance was chosen since it is equal to the near field length, so the treated MT were located at the start of the homogeneous far field of the ultrasound beam. After transmission through the exposure chamber, the ultrasound beam hit a specially designed absorbing device consisting of Wallgone rubber with a defined wedged surface to avoid reflection from the bottom of the measurement tank. Therefore, the treated MT were exposed to a homogeneous ultrasound beam with free field conditions. Before the start of the experiments, a conical rod positioned at the place of the transducer was used to point the centre of the ultrasonic beam on the center of the exposure chamber where the MT were located. The paired control MT were put in an identical exposure chamber in the same measurement tank behind a rubber wall (Wallgone). The ultrasound physical therapy unit was calibrated to apply a defined ultrasound intensity. This intensity calibration was performed by calculating the ratio of the measured ultrasonic power and the mea-
During subsequent culture, the length of the MT and the length of the calcification zone were measured daily, using a measuring ocular, and resorption was evaluated by appearance of translucent spots in the mineralized zone. The third MT (MT,,,) was used for all measurements. Phase contrast pictures were taken at day 7 and day 8 of culture. The 16-day-old and 17day-old MT were fixed and decalcified after 8 and 7 days of culture, respectively. After overnight fixation in a 4% buffered formalin solution at 4”C, decalcification was performed in a mixture of 5% (v/v) formic acid and 5% (v/v) formalin for 3 to 6 h at 4°C. The presence of osteoclasts in the 17-day-old MT was examined using the staining method for tar&ate-resistant acid phosphatase (TRAcP) activity described by Scheven et al. (1986). Naphtol-ASBI-phosphate (Serva) was used as substrate, pararosanilin (Gurr) as coupler (Barka and Anderson 1962) and 10 mM L(+)tartaric acid (Sigma) as inhibitor. The 16- and 17-dayold MT were embedded in paraffin and serial sections of 5-pm thickness were counterstained with Ehrlich’s haematoxylin (Romeis 1968). The sections were exam-
60
40
20
0 o
1
2
3
Time
4
5
6
7
(days)
Fig. 4. Effect of ultrasound treatment on longitudinal growth of 16-day-old metatarsal long bone rudiments (MT), quantified as percentage increase in length (%increase) of MT,,,. MT (m) were treated with 0.77 W cm-* pulse-wave ultrasound (PW; Z\.tp) for 5 min at day 0. Paired control MT (A) were sham treated (see Materials and Methods section). Error bars represent SEM. Following day 4, %increase of the treated MT was significantly different from the paired control group (paired t test, *p < 0.05, **p < 0.01).
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Table 1. Effect of pulse-wave (PW) ultrasound treatment on longitudinal growth of 16-day-old metatarsal rudiments (MT), quantified as percentage increase in length (%increase) of MT,,,.
long bone
Intensity, W cm2 (I,,,,)
n
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
0. I
3
6.2 (0.X) 6.2 (2.2) 7.3 (4.4) 1.9 (4.5) 6.0 (4.0) 5.9 (3.5) 5.5 (3.9)
12.3 (2.2) I I.8 (2.2) 16.X (2.X) 15.0 (7.0) 13.4 (6.2) 14.3 (6.4) 15.8 (6.X)
23.6 (5.1) 21.1 (3.9) 26.0 (3.6) 24.X (9.X) 23.0 (10.6) 24.X (5.0) 24.4 (6.1)
32.1 (2.5) 25.4 (6.3) 36.7 (5.2) 35.1 (9.6) 33.1 (10.X) 35.x (5.6) 36.6 (9.5) *
3x.3 (5.5) 29.X (5.0) 46.7 (5.1) 42.0 (12.3) 41.5 (11.3) 42.X (7.4) 43.7 (X.3) **
44.5 (4.9) 36.2 (2.9) 5 I .6 (6.6) 49.6 (I 1.5) 49.6 (12.3) 4x.7 (7.7) 51.3 (10.7) **
50. I (6.6) 44.x (5.6) 56.6 (4.4) 54.9 (13.6) 54.0 (I 1.9) 52.X (X.1) 56.5 (I 1.2) **
4.6 (2.3)
12.7 (5.2)
21.3 (X.X)
30.3 (7.0)
36.0 (9.0)
40.9 (X.2)
46.2 (X.9)
Control
0. I
0.33 Control
6 0.33
0.49 Control
6 7
0.49
0.71
Control
3
7 5
0.77
5
MT were treated at day 0 for 5 min with various intensities PW ultrasound (I,,,,). Paired control MT were sham-treated (see Materials and Methods section). Results are presented as mean + SD (SD in parentheses) for a given number of experiments (N). Starting from day 4, %increase of MT treated with 0.77 W cm-’ is significantly higher than %increase of the paired control MT (paired t test. * p < 0.05; ** p < 0.01).
ined for general morphology and for the presence of resorption. In the most central of the axial sections the length of the different cartilage zones was measured (Gaillard et al. 1977; Nijweide et al. 1978). Statistical analysis was performed using the Student paired t test. Longitudinal growth of the MT was quantified as the percentage increase in length (%increase) relative to the length of the MT at day 0. Results are presented as mean ? SD or, as indicated, mean 5 SEM for a given number of experiments (n).
? 0.03 (n = 5) in the paired control MT. No difference was found in the length of the hypertrophic cartilage zone between the treated MT and control MT (n = 5)
+*
I
RESULTS Treatment of the 16-day-old MT for 5 min at day 0 with pulse-wave (PW) ultrasound resulted in accelerated MT growth, quantified as the percentage increase in length (%increase) of the MT (Table 1). The difference between the treated MT and the paired control group was significant in the group of MT that was treated with a dose of 0.77 W cm-’ (p < 0.01, paired t test; Table 1, Fig. 4). This accelerated longitudinal growth upon PW ultrasound treatment is again shown in a dose-response curve of the difference in % increase between the treated MT and the paired control group at day 7 (Fig. 5). Histological examination of the 16-day-old MT treated with 0.77 W cm-* PW ultrasound revealed that the length of the proliferative cartilage zone was increased significantly (p < 0.02, paired t test) in comparison to the paired control MT. The fraction of the proliferative cartilage zone at day 7 as compared to the total length of the MT at day 7 was 0.56 k 0.03 (n = 5) in the treated MT group and 0.47
PW intensity
(W
cmm2)
Fig. 5. Dose-response curve of effect of pulse-wave (PW; Z5atp)ultrasound treatment on longitudinal growth of 16-dayold metatarsal long bone rudiments (MT). The difference in percentage increase in length (A %increase) between the treated MT and the respective paired control MT at day 7 is plotted against the PW ultrasound intensity. Error bars represent SEM. The %increase of MT treated with 0.77 W cm-’ was significantly different from the paired control group (paired t test, **p < 0.01).
Effect of therapeutic
US 0 A. WILTINK et al.
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Table 2. Effect of continuous-wave (CW) ultrasound treatment on longitudinal growth of 16-day-old metatarsal rudiments (MT), quantified as percentage increase in length (%increase) of MT,,,. Intensity, W cm-’ (L) 0.1, 5 min Control,
0.
I, 5 min
0.5,
I
7.1 (3.7) 7.4 (3.6) 5.2 (4.6) 7.8 (5.1) 8.5 (1.5) 7.7 (4.0)
6
0.5, 5 min
6
I min
Control,
Day
7
0.5, 5 min Control,
n
5
0.5, 1 min
5
I
long bone
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
14.9 (4.8) 15.9 (4.1) 9.6 (4.4) 16.2 (7.0) 16.7 (3.6) 17.8 (3.0)
25.3 (4.3) 25.8 (6.0) 18.4 (6.8) 26.5 (6.0) 21.4 (2.5) 27.5 (5.4)
33.1 (5.3) 36.4 (6.6) 28.0 (9.5) 36.8 (7.2) 31.1 (8.9) 32.1 (5.5)
43.6 (9.3) 45.2 (11.2) 34.6 (10.0) 41.6 (7.8) 38.9 (5.7) 43.3 (3.4)
48.8 (10.3) 50.6 (7.4) 40.6 (10.2) 50.0 (5.9) 43.6 (7.3) 45.7 (6.9)
53.7 (10.5) 57.4 (8.9) 45.7 (12.1) 57.5 (6.3) 48.9 (12.2) 53.3 (4.5)
MT were treated at day 0 for 5 min or 1 min with various intensities CW ultrasound (I,,,,). Paired control MT were sham-treated (see Materials and Methods section). Results are presented as mean 2 SD (SD in parentheses) for a given number of experiments (n).
indicating that the accelerated MT growth was mainly due to increase in growth of the proliferative cartilage zone. Doses of 0.1 or 0.5 W cm-’ continuous-wave (CW) ultrasound, applied for 1 or 5 min, had no significant effect on the longitudinal growth of the 16day-old MT during culture, although the treated samples tended to show decreased growth (Table 2). Treatment of 17-day-old MT with PW ultrasound for 5 min at day 0 resulted in significantly higher %increase only at day 3 of the MT treated with 0.5 W cm-* (paired t test, p < 0.05; Table 3). No difference was found between treated MT and paired control MT in resorption and in length of the calcification zone (data not shown). We did not find an overt difference in estimated number of osteoclasts between treated MT and paired control MT (data not shown).
DISCUSSION Ultrasound treatment in physical therapy is used extensively as a therapeutic tool, although few clinical trials have been performed to assess its effectiveness (for review see Holmes and Rudland 1991; Bouter et al. 1992). In addition, it is unclear at the tissue level what kind of mechanisms result in physiological effects. One of the contraindications to treatment is insonation of the epiphyseal discs of growing long bones. To gain more insight into the concept that ultrasound treatment might influence normal endochondral ossification of epiphyseal discs, we used an in vitro model system of fetal mouse metatarsal long bone rudiments (MT). Treatment of 16-day-old MT with PW ultrasound with a dose of 0.77 W cme2 (Is,,,,; 2.54 W cmp2 I,,,,)
Table 3. Effect of pulse-wave (PW) ultrasound treatment on longitudinal growth of 17-day-old metatarsal rudiments (MT), quantified as percentage increase in length (%increase) of MT,,,. Intensity W cm-’ (I,,,,)
n
0.1
5
Control,
0.1
0.5
Control,
4
0.5
0.77 Control,
5
4 3
0.77
3
1
long bone
Day 2
Day 3
Day 4
Day 5
Day 6
5.6 (3.6) 6.4 (3.1) 4.8 (1.4)
12.6 (5.1) 13.6 (4.6) 11.1 (5.8)
18.9 (6.6) 22.4 (5.3) 20.1 (5.0) *
26.1 (5.2) 28.6 (4.4) 25.6 (3.9)
32.0 (10.5) 33.3 (5.4) 29.1 (2.1)
36.0 (10.7) 36.4 (4.8) 34.2 (6.5)
4.8 (4.2) 7.6 (4.4) 3.3 (1.6)
5.9 (4.4) 15.0 (5.9) 15.7 (9.3)
12.7 (7.4) 26.9 (12.9) 20.2 (3.2)
20.2 (5.8) 28.9 (9.7) 28.8 (3.0)
28.1 (3.1) 36.8 (11.4) 35.9 (3.7)
31.1 (7.7) 39.9 (8.8) 40.3 (5.2)
Day
MT were treated at day 0 for 5 min with various intensities PW ultrasound (I,,,,). Paired control MT were sham-treated (see Materials and Methods section). Results are presented as mean ir SD (SD in parentheses) for a given number of experiments (n). The Rincrease of MT treated with 0.5 W cm-’ at day 3 (* p < 0.05) is significantly higher than the %increase of the paired control MT.
126
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resulted in a significantly accelerated percentage increase in length (%increase) of the treated MT. Histological examination revealed that the length of the proliferative cartilage zone was increased in the treated MT. As measured with a beamplot setup and a calibrated hydrophone, a dose of 0.77 W cm-’ causes an acoustic pressure amplitude of 276 kPa in the spatial peak in degassed water (data not shown). This value is sufficient for stable cavitation (Daniels et al. 1987; ter Haar and Daniels 1981) and possibly also for collapse cavitation (Dyson 1986; Nyborg and Ziskin 1985). The fact that cartilage proliferation seemed to be stimulated points to stable cavitation rather than to the deleterious effects of collapse cavitation. In addition to mechanical effects ultrasound can exert its action on tissue via thermal effects (Dyson 1987; Nyborg and Ziskin 1985; Drewniak et al. 1989; Kitchen and Partridge 1990; Holmes and Rudland 1991). PW ultrasound is thought to cause mainly mechanical effects in the treated tissue. In our experiments, local heating of the bone rudiments is unlikely, due to the relatively low intensities used (NCRP Report No. 113 1992). In contrast to the 16-day-old MT treated with PW ultrasound, the 17-day-old MT showed a transient stimulation of the %increase in length at day 3 (0.5 W cm-‘) only. This contrast between the two groups might result from the difference in developmental state of the MT. Although not significantly, the percentage increase in length of IBday-old MT treated with CW ultrasound tended to be decreased as compared to the control MT. PW ultrasound treatment of 16-day-old MT resulted in stimulation of the proliferative cartilage zone without affecting the hypertrophic cartilage zone. This suggests that the proliferation of cartilage cells is stimulated without influence on their differentiation. This corroborates with the stimulation of cartilage proliferation during fracture healing, as was described by Dyson and Brookes (1983). Ultrasound treatment was shown to stimulate calcium uptake in fibroblasts (Mortimer and Dyson 1988). In addition, in preosseous chondrocytes a correlation was found between elevation of the cytosolic calcium concentration and increase in cell proliferation of resting chondrocytes, whereas in hypertrophic cells no such correlation was found (D’Andrea et al. 1990). It might be, therefore, that ultrasonic treatment results in increased permeability of the cell membrane for calcium ions, resulting in elevated levels of the intracellular calcium concentration and influence on cartilage cell proliferation. The experimental setup described in this study fulfils the requirements for a relatively simple and accurate in vitro study of the effects of ultrasound treatment on bone development. Our first, explorative, studies indicate that ultrasound treatment may influence
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cartilage proliferation during normal bone development of long bones. Although caution is needed in extrapolating data from in vitro studies to clinical use of ultrasound, the data in the present study confirm that care should be taken in the application of ultrasonic therapy in children and young adults. Ackno~~led~emerrrs-The authors thank Mr. A. van der Plas, Mrs. T. Vrijheid-Lammers and Dr. B. Van Duijn for advice. This work was partly supported by The Netherlands Organization for Scientitic Research (NWO) through a grant from the Dutch Foundation for Biophysics to A. W.
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ultrasonics.
London:
Academic
Press;
1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0301.5629195 $9.50 + .OO
Pergamon 0301-5629(94)00092-l
@Original Contribution
EFFECT
ANNEKE
OF THERAPEUTIC ULTRASOUND ENDOCHONDRAL OSSIFICATION
WILTINK,+* PETER J. NIJWEIDE,~ WIM
ON
A. OOSTERBAAN,”
ROB T. HEKKENBERG” and PAUL J. M. HELDERS~ Departments of ‘Cell Biology and Histology and tPhysiology and Physiological Physics, Leiden University, Leiden, The Netherlands; *TNO, Leiden, The Netherlands; and $Department of Pediatrics, Utrecht University, The Netherlands (Received
9 February
1994; in final
formIO March 1994)
Abstract-The effect of therapeutic doses of ultrasound was tested on endochondral ossification of in vitro developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice. Bone growth, calcification and resorption following exposure to several doses of pulse-wave (PW) or continuous-wave (CW) ultrasound were examined. PW was applied at intensities between 0.1 W cm-’ and 0.77 W cm-’ (I,,,) and CW intensities were 0.1 W cm-’ or 0.5 W cm-’ (I.,,). After 1 week of culture, the metatarsal long bone rudiments were fixed and paraffin sections were prepared for histological evaluation and for measurement of the relative contribution of the various cartilage zones to the total bone length. In contrast to treatment with CW ultrasound, treatment of lCday-old metatarsal long bone rudiments with PW ultrasound resulted after 4 days of culture in significantly increased longitudinal growth. Histology revealed a significant increased length of the proliferative zone, whereas the length of the hypertrophic cartilage zone was unaltered. This might indicate that proliferation of the cartilage cells is stimulated without influence on cell differentiation. Key Words: Therapeutic ultrasound. cation. Mechanical effects.
Endochondral
ossification,
Metatarsae,
Cartilage,
Resorption,
Calcifi-
(Carstensen 1986; Chapman et al. 1979; Dinno et al. 1989a; Dinno et al. 1989b; Mortimer and Dyson 1988). Such permeability changes may reflect activation of stretch activated ion channels in the cell membrane due to membrane perturbation (for review see Sachs 1988). This could then result in a change in Ca*+ ion permeability causing Ca2+ influx into the cell and subsequent increase in the intracellular calcium concentration, as was described for fibroblasts upon ultrasound treatment (Mortimer and Dyson 1988). Calcium is one of the most important intracellular messengers for extracellular signals known so far (for review see Berridge 1993). At higher therapeutic intensities, or if standing waves can develop resulting from interference of ultrasound waves due to reflection on, e.g., mineralized matrix of bone tissue, there is a potential hazard of collapse cavitation. This situation, which should be avoided, is damaging for the surrounding tissue due to formation of free radicals, extremely high local temperatures and high pressure waves (NCRP 1983; Carstensen 1986; Holland and Apfel 1990). Especially in tissues with different acoustic impedances, such as in bone tissue, where a mineralized matrix is surrounded
INTRODUCTION Therapeutic ultrasound may cause various physiological effects, generally divided into thermal effects at higher doses and nonthermal, mechanical effects resulting from stable cavitation at lower doses (NCRP 1983; Dyson 1987; Kitchen and Partridge 1990; Nyborg and Ziskin 1985; Holmes and Rudland 1991; Maxwell 1992). Whether thermal effects will occur depends on several factors such as duration of the treatment, intensity of the ultrasound beam, frequency-dependent absorption and thermal diffusivity (for review see NCRP 1983; Dyson 1987; Drewniak et al. 1989). In addition, for therapeutic thermal effects to arise, tissue temperature should remain between 40°C and 45°C for more than 5 min (Nyborg and Ziskin 1985; Dyson 1987). Therapeutic mechanical effects are most likely due to stable cavitation in combination with acoustic streaming of extracellular fluid, resulting in changes of the cell membrane permeability of adjacent cells Address correspondence to: A. Wiltink, Department of Physiology and Physiological Physics, Leiden University, P.O. Box 9604, 2300 RC Leiden, The Netherlands. 121
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culture medium, consisting of alpha minimal essential medium (MEM), 10% chicken embryonic extract, 10% cock serum, 20% cock plasma, 85 yglmL gentamycin. In some experiments, 1 mM or 0.1 mM beta-glycerophosphate was added to stimulate calcification. MT were cultured in a humidified atmosphere of 5% COz. in air at 37°C.
Fig. 1. Phase contrast photograph of 16-day-old fetal mouse metatarsal long bone rudiments (MT) in primary culture. MT (II-IV) were dissected along with metatarsus V to facilitate orientation of the triplets. The MT were cultured for 8 days
on semisolid culture medium, consisting of alpha-MEM, 10% chicken embryonic extract, 10% cock serum, 20% cock plasma and 85 bg/ml gentamycin. Note the area of calcified tissue (black area) in the center of each bone rudiment. Magnification
30X
by periosteum, reflection of the ultrasound beam and subsequent interference can result in peaks in ultrasound intensity (Wells 1977). In the case of bone this may cause damage to the periosteal cells. On the other hand, bone repair can be stimulated by ultrasound treatment via therapeutic effects on cells involved in fracture healing (Dyson and Brookes 1983; Tsai et al. 1992). One of the contraindications to ultrasound therapy of children or young adults is treatment of areas in the vicinity of developing tissue such as the epiphyseal discs, since it is generally assumed that insonation of these discs may influence normal bone development (NCRP 1983). Developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice can be used as an in vitro model system for endochondral ossification (Gaillard et al. 1977; Nijweide et al. 1978) as occurs in the epiphyseal discs of children and young adults. To assess the possible influence of therapeutic ultrasound on bone development or cartilage cell proliferation and differentiation, we tested therapeutic doses of ultrasound in this model. MATERIALS
AND
METHODS
Tissue culture Paired metatarsal long bone rudiments (II-IV) were dissected from 16- and 17-day-old fetal mice along with metatarsus V to facilitate orientation of the triplets (MT). The tissue between the metatarsae was not disrupted to avoid damage to the developing bone rudiments (Fig. 1). The MT were cultured on semisolid
Ultrasound treatment After 24 h of culture, MT were treated for 5 min or 1 min with l-MHz ultrasound (day 0). Pulse-wave ultrasound (PW; pulse frequency 100 Hz, pulse length 2 ms) was applied at intensities of 0.1 W cm-‘, 0.33 W cm-‘, 0.49 W cmp2 or 0.77 W cmp2 (Is+, spatial average temporal peak intensity), continuous-wave (CW) ultrasound had intensities of 0.1 W cm-’ or 0.5 W cmm2 (I,,,,, spatial average temporal average intensity). The experimental setup consisted of an ultrasound therapy unit (Sonopuls 464, Delft Instruments), a measurement tank filled with a freshly degassed standard extracellular solution (S-ECS) and Petriperm culture dishes (Dijkstra Vereenigde BV) as the exposure chambers containing the MT (Figs. 2 and 3). The SECS solution contained (in mM): 135 NaCl, 5.4 KCl, 0.45 KH,PO,, 0.42 Na,HPO,, 4.2 NaHCO,, 0.8 MgS04.7H20, 5.5 o-glucose, 1.25 CaC12 and 10.9 HEPES. The walls and bottom of the measurement tank were lined with rubber (Wallgone Model 431,
5cm n
3 Fig. 2. The experimental setup, consisting of the ultrasound therapy unit (1) the transducer (ERA 5 cm*) fixed to a metal rod (2), metal support (3) measurement tank filled with freshly degassed standard extracellular solution (S-ECS) and lined with ultrasound absorbing Wallgone rubber (4), Wallgone rubber mat (see Materials and Methods) (5) rubber ring (6), location of the exposure chambers with the treated MT (7) and paired control MT (8).
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sured effective radiation area (ERA) by means of a radiation balance and a beamplot setup with a hydrophone (Hekkenberg and Oosterbaan 1986). Bone development
Fig. 3. The Petriperm culture dish (bottom up) used during the experiments, with dialysis tube (1) and fixation device (2). The dialysis tube is fixed to the culture dish, without allowing air bubbles to be included. Rotation of the fixation
device results in stretching the band around the culture dish, thereby firmly attaching the dialysis tube to the dish. During experiments MT were put in the center of the dish (arrow).
Consumer to reduce
Usage
Laboratories
Inc.)
specially
devised
reflection and to absorb the ultrasound energy. The Petriperm culture dishes had a thin bottom layer. MT were put on the backside of the bottom layer in 500-PL S-ECS, 10% cock serum and 85 yg/mL gentamycin. Dialysis tube was used to close the exposure chamber (Fig. 3). Special care was taken to exclude air bubbles from the inside of the exposure chamber. A near fully transmission of ultrasound was assumed and confirmed by visual observation using a Schlieren optical system. No reflection on the surfaces was observed. MT that were treated were put at a distance of 10 cm from the face of the ultrasound transducer (Fig. 2). This distance was chosen since it is equal to the near field length, so the treated MT were located at the start of the homogeneous far field of the ultrasound beam. After transmission through the exposure chamber, the ultrasound beam hit a specially designed absorbing device consisting of Wallgone rubber with a defined wedged surface to avoid reflection from the bottom of the measurement tank. Therefore, the treated MT were exposed to a homogeneous ultrasound beam with free field conditions. Before the start of the experiments, a conical rod positioned at the place of the transducer was used to point the centre of the ultrasonic beam on the center of the exposure chamber where the MT were located. The paired control MT were put in an identical exposure chamber in the same measurement tank behind a rubber wall (Wallgone). The ultrasound physical therapy unit was calibrated to apply a defined ultrasound intensity. This intensity calibration was performed by calculating the ratio of the measured ultrasonic power and the mea-
During subsequent culture, the length of the MT and the length of the calcification zone were measured daily, using a measuring ocular, and resorption was evaluated by appearance of translucent spots in the mineralized zone. The third MT (MT,,,) was used for all measurements. Phase contrast pictures were taken at day 7 and day 8 of culture. The 16-day-old and 17day-old MT were fixed and decalcified after 8 and 7 days of culture, respectively. After overnight fixation in a 4% buffered formalin solution at 4”C, decalcification was performed in a mixture of 5% (v/v) formic acid and 5% (v/v) formalin for 3 to 6 h at 4°C. The presence of osteoclasts in the 17-day-old MT was examined using the staining method for tar&ate-resistant acid phosphatase (TRAcP) activity described by Scheven et al. (1986). Naphtol-ASBI-phosphate (Serva) was used as substrate, pararosanilin (Gurr) as coupler (Barka and Anderson 1962) and 10 mM L(+)tartaric acid (Sigma) as inhibitor. The 16- and 17-dayold MT were embedded in paraffin and serial sections of 5-pm thickness were counterstained with Ehrlich’s haematoxylin (Romeis 1968). The sections were exam-
60
40
20
0 o
1
2
3
Time
4
5
6
7
(days)
Fig. 4. Effect of ultrasound treatment on longitudinal growth of 16-day-old metatarsal long bone rudiments (MT), quantified as percentage increase in length (%increase) of MT,,,. MT (m) were treated with 0.77 W cm-* pulse-wave ultrasound (PW; Z\.tp) for 5 min at day 0. Paired control MT (A) were sham treated (see Materials and Methods section). Error bars represent SEM. Following day 4, %increase of the treated MT was significantly different from the paired control group (paired t test, *p < 0.05, **p < 0.01).
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Table 1. Effect of pulse-wave (PW) ultrasound treatment on longitudinal growth of 16-day-old metatarsal rudiments (MT), quantified as percentage increase in length (%increase) of MT,,,.
long bone
Intensity, W cm2 (I,,,,)
n
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
0. I
3
6.2 (0.X) 6.2 (2.2) 7.3 (4.4) 1.9 (4.5) 6.0 (4.0) 5.9 (3.5) 5.5 (3.9)
12.3 (2.2) I I.8 (2.2) 16.X (2.X) 15.0 (7.0) 13.4 (6.2) 14.3 (6.4) 15.8 (6.X)
23.6 (5.1) 21.1 (3.9) 26.0 (3.6) 24.X (9.X) 23.0 (10.6) 24.X (5.0) 24.4 (6.1)
32.1 (2.5) 25.4 (6.3) 36.7 (5.2) 35.1 (9.6) 33.1 (10.X) 35.x (5.6) 36.6 (9.5) *
3x.3 (5.5) 29.X (5.0) 46.7 (5.1) 42.0 (12.3) 41.5 (11.3) 42.X (7.4) 43.7 (X.3) **
44.5 (4.9) 36.2 (2.9) 5 I .6 (6.6) 49.6 (I 1.5) 49.6 (12.3) 4x.7 (7.7) 51.3 (10.7) **
50. I (6.6) 44.x (5.6) 56.6 (4.4) 54.9 (13.6) 54.0 (I 1.9) 52.X (X.1) 56.5 (I 1.2) **
4.6 (2.3)
12.7 (5.2)
21.3 (X.X)
30.3 (7.0)
36.0 (9.0)
40.9 (X.2)
46.2 (X.9)
Control
0. I
0.33 Control
6 0.33
0.49 Control
6 7
0.49
0.71
Control
3
7 5
0.77
5
MT were treated at day 0 for 5 min with various intensities PW ultrasound (I,,,,). Paired control MT were sham-treated (see Materials and Methods section). Results are presented as mean + SD (SD in parentheses) for a given number of experiments (N). Starting from day 4, %increase of MT treated with 0.77 W cm-’ is significantly higher than %increase of the paired control MT (paired t test. * p < 0.05; ** p < 0.01).
ined for general morphology and for the presence of resorption. In the most central of the axial sections the length of the different cartilage zones was measured (Gaillard et al. 1977; Nijweide et al. 1978). Statistical analysis was performed using the Student paired t test. Longitudinal growth of the MT was quantified as the percentage increase in length (%increase) relative to the length of the MT at day 0. Results are presented as mean ? SD or, as indicated, mean 5 SEM for a given number of experiments (n).
? 0.03 (n = 5) in the paired control MT. No difference was found in the length of the hypertrophic cartilage zone between the treated MT and control MT (n = 5)
+*
I
RESULTS Treatment of the 16-day-old MT for 5 min at day 0 with pulse-wave (PW) ultrasound resulted in accelerated MT growth, quantified as the percentage increase in length (%increase) of the MT (Table 1). The difference between the treated MT and the paired control group was significant in the group of MT that was treated with a dose of 0.77 W cm-’ (p < 0.01, paired t test; Table 1, Fig. 4). This accelerated longitudinal growth upon PW ultrasound treatment is again shown in a dose-response curve of the difference in % increase between the treated MT and the paired control group at day 7 (Fig. 5). Histological examination of the 16-day-old MT treated with 0.77 W cm-* PW ultrasound revealed that the length of the proliferative cartilage zone was increased significantly (p < 0.02, paired t test) in comparison to the paired control MT. The fraction of the proliferative cartilage zone at day 7 as compared to the total length of the MT at day 7 was 0.56 k 0.03 (n = 5) in the treated MT group and 0.47
PW intensity
(W
cmm2)
Fig. 5. Dose-response curve of effect of pulse-wave (PW; Z5atp)ultrasound treatment on longitudinal growth of 16-dayold metatarsal long bone rudiments (MT). The difference in percentage increase in length (A %increase) between the treated MT and the respective paired control MT at day 7 is plotted against the PW ultrasound intensity. Error bars represent SEM. The %increase of MT treated with 0.77 W cm-’ was significantly different from the paired control group (paired t test, **p < 0.01).
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Table 2. Effect of continuous-wave (CW) ultrasound treatment on longitudinal growth of 16-day-old metatarsal rudiments (MT), quantified as percentage increase in length (%increase) of MT,,,. Intensity, W cm-’ (L) 0.1, 5 min Control,
0.
I, 5 min
0.5,
I
7.1 (3.7) 7.4 (3.6) 5.2 (4.6) 7.8 (5.1) 8.5 (1.5) 7.7 (4.0)
6
0.5, 5 min
6
I min
Control,
Day
7
0.5, 5 min Control,
n
5
0.5, 1 min
5
I
long bone
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
14.9 (4.8) 15.9 (4.1) 9.6 (4.4) 16.2 (7.0) 16.7 (3.6) 17.8 (3.0)
25.3 (4.3) 25.8 (6.0) 18.4 (6.8) 26.5 (6.0) 21.4 (2.5) 27.5 (5.4)
33.1 (5.3) 36.4 (6.6) 28.0 (9.5) 36.8 (7.2) 31.1 (8.9) 32.1 (5.5)
43.6 (9.3) 45.2 (11.2) 34.6 (10.0) 41.6 (7.8) 38.9 (5.7) 43.3 (3.4)
48.8 (10.3) 50.6 (7.4) 40.6 (10.2) 50.0 (5.9) 43.6 (7.3) 45.7 (6.9)
53.7 (10.5) 57.4 (8.9) 45.7 (12.1) 57.5 (6.3) 48.9 (12.2) 53.3 (4.5)
MT were treated at day 0 for 5 min or 1 min with various intensities CW ultrasound (I,,,,). Paired control MT were sham-treated (see Materials and Methods section). Results are presented as mean 2 SD (SD in parentheses) for a given number of experiments (n).
indicating that the accelerated MT growth was mainly due to increase in growth of the proliferative cartilage zone. Doses of 0.1 or 0.5 W cm-’ continuous-wave (CW) ultrasound, applied for 1 or 5 min, had no significant effect on the longitudinal growth of the 16day-old MT during culture, although the treated samples tended to show decreased growth (Table 2). Treatment of 17-day-old MT with PW ultrasound for 5 min at day 0 resulted in significantly higher %increase only at day 3 of the MT treated with 0.5 W cm-* (paired t test, p < 0.05; Table 3). No difference was found between treated MT and paired control MT in resorption and in length of the calcification zone (data not shown). We did not find an overt difference in estimated number of osteoclasts between treated MT and paired control MT (data not shown).
DISCUSSION Ultrasound treatment in physical therapy is used extensively as a therapeutic tool, although few clinical trials have been performed to assess its effectiveness (for review see Holmes and Rudland 1991; Bouter et al. 1992). In addition, it is unclear at the tissue level what kind of mechanisms result in physiological effects. One of the contraindications to treatment is insonation of the epiphyseal discs of growing long bones. To gain more insight into the concept that ultrasound treatment might influence normal endochondral ossification of epiphyseal discs, we used an in vitro model system of fetal mouse metatarsal long bone rudiments (MT). Treatment of 16-day-old MT with PW ultrasound with a dose of 0.77 W cme2 (Is,,,,; 2.54 W cmp2 I,,,,)
Table 3. Effect of pulse-wave (PW) ultrasound treatment on longitudinal growth of 17-day-old metatarsal rudiments (MT), quantified as percentage increase in length (%increase) of MT,,,. Intensity W cm-’ (I,,,,)
n
0.1
5
Control,
0.1
0.5
Control,
4
0.5
0.77 Control,
5
4 3
0.77
3
1
long bone
Day 2
Day 3
Day 4
Day 5
Day 6
5.6 (3.6) 6.4 (3.1) 4.8 (1.4)
12.6 (5.1) 13.6 (4.6) 11.1 (5.8)
18.9 (6.6) 22.4 (5.3) 20.1 (5.0) *
26.1 (5.2) 28.6 (4.4) 25.6 (3.9)
32.0 (10.5) 33.3 (5.4) 29.1 (2.1)
36.0 (10.7) 36.4 (4.8) 34.2 (6.5)
4.8 (4.2) 7.6 (4.4) 3.3 (1.6)
5.9 (4.4) 15.0 (5.9) 15.7 (9.3)
12.7 (7.4) 26.9 (12.9) 20.2 (3.2)
20.2 (5.8) 28.9 (9.7) 28.8 (3.0)
28.1 (3.1) 36.8 (11.4) 35.9 (3.7)
31.1 (7.7) 39.9 (8.8) 40.3 (5.2)
Day
MT were treated at day 0 for 5 min with various intensities PW ultrasound (I,,,,). Paired control MT were sham-treated (see Materials and Methods section). Results are presented as mean ir SD (SD in parentheses) for a given number of experiments (n). The Rincrease of MT treated with 0.5 W cm-’ at day 3 (* p < 0.05) is significantly higher than the %increase of the paired control MT.
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resulted in a significantly accelerated percentage increase in length (%increase) of the treated MT. Histological examination revealed that the length of the proliferative cartilage zone was increased in the treated MT. As measured with a beamplot setup and a calibrated hydrophone, a dose of 0.77 W cm-’ causes an acoustic pressure amplitude of 276 kPa in the spatial peak in degassed water (data not shown). This value is sufficient for stable cavitation (Daniels et al. 1987; ter Haar and Daniels 1981) and possibly also for collapse cavitation (Dyson 1986; Nyborg and Ziskin 1985). The fact that cartilage proliferation seemed to be stimulated points to stable cavitation rather than to the deleterious effects of collapse cavitation. In addition to mechanical effects ultrasound can exert its action on tissue via thermal effects (Dyson 1987; Nyborg and Ziskin 1985; Drewniak et al. 1989; Kitchen and Partridge 1990; Holmes and Rudland 1991). PW ultrasound is thought to cause mainly mechanical effects in the treated tissue. In our experiments, local heating of the bone rudiments is unlikely, due to the relatively low intensities used (NCRP Report No. 113 1992). In contrast to the 16-day-old MT treated with PW ultrasound, the 17-day-old MT showed a transient stimulation of the %increase in length at day 3 (0.5 W cm-‘) only. This contrast between the two groups might result from the difference in developmental state of the MT. Although not significantly, the percentage increase in length of IBday-old MT treated with CW ultrasound tended to be decreased as compared to the control MT. PW ultrasound treatment of 16-day-old MT resulted in stimulation of the proliferative cartilage zone without affecting the hypertrophic cartilage zone. This suggests that the proliferation of cartilage cells is stimulated without influence on their differentiation. This corroborates with the stimulation of cartilage proliferation during fracture healing, as was described by Dyson and Brookes (1983). Ultrasound treatment was shown to stimulate calcium uptake in fibroblasts (Mortimer and Dyson 1988). In addition, in preosseous chondrocytes a correlation was found between elevation of the cytosolic calcium concentration and increase in cell proliferation of resting chondrocytes, whereas in hypertrophic cells no such correlation was found (D’Andrea et al. 1990). It might be, therefore, that ultrasonic treatment results in increased permeability of the cell membrane for calcium ions, resulting in elevated levels of the intracellular calcium concentration and influence on cartilage cell proliferation. The experimental setup described in this study fulfils the requirements for a relatively simple and accurate in vitro study of the effects of ultrasound treatment on bone development. Our first, explorative, studies indicate that ultrasound treatment may influence
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cartilage proliferation during normal bone development of long bones. Although caution is needed in extrapolating data from in vitro studies to clinical use of ultrasound, the data in the present study confirm that care should be taken in the application of ultrasonic therapy in children and young adults. Ackno~~led~emerrrs-The authors thank Mr. A. van der Plas, Mrs. T. Vrijheid-Lammers and Dr. B. Van Duijn for advice. This work was partly supported by The Netherlands Organization for Scientitic Research (NWO) through a grant from the Dutch Foundation for Biophysics to A. W.
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