Ultrasoundin Med. & BioL Vol. 16, No. 8, pp. 801-807, 1990 Printed in the U.S.A.

0301-5629/90 $3.00+ .00 © 1990PergamonPressplc

OOriginal Contribution THE

BIOMECHANICAL EFFECTS OF LOW-INTENSITY ULTRASOUND ON HEALING TENDONS

CHUKUKA S. ENWEMEKA, t$ OSCAR RODRIGUEZ $ and SONIA MENDOSA* *Division of Physical Therapy, Department of Orthopaedics and Rehabilitation, University of Miami School of Medicine and Department of Veterans Affairs Medical Center, Miami, FL; *Department of Veterans Affairs, Audie L. Murphy Hospital, San Antonio, TX; *University of Texas at San Antonio, San Antonio, TX (Received 24 January 1990; in final f o r m 10 M a y 1990)

Abstract--The effect of low-intensity ultrasound on the healing strength of tendons was studied in experimentally tenotomized, repaired and immobilized right tendo calcaneus (Achilles tendon) of 24 rabbits. Ten tendons were sonicated in continuous waves at a space-averaged intensity of 0.5 W cm -2 for 5 min every day. The remaining 14 tendons were mock-sonicated as controls. After nine consecutive treatments, the tendons were excised under anesthesia and compared for differences in tensile strength, tensile stress and energy absorption capacity. Sonication at 0.5 W cm -2 induced a significant increase in the tensile strength (p < .02), tensile stress (p < .005) and energy absorption capacity (p < .001) of the tendons. These findings suggest that high-intensity sonication may not be necessary to augment the healing strength of tendons and that sonication at similarly low intensities may enhance the healing process of surgically repaired human tendo calcaneus.

Key Words: Tendons, Wound healing, Therapeutic ultrasound, Tensile strength.

INTRODUCTION

Lindholm 1959; Ma and Griffith 1977), and thrombophlebitis (Christiansen 1954; Ma and Griffith 1977), attempts have been made to modify current treatment procedures. More than a decade ago, a change in surgical technique and carbon and polyester fiber implantation were simultaneously proposed as means of overcoming these problems (Jenkins et al. 1977; Ma and Griffith 1977). Subsequently, two new surgical techniques, percutaneous approximation of tendon ends (Ma and Griflith 1977) and external fixation (Nada 1985), were developed and found useful in preventing skin necrosis, tendocutaneous adhesion and rerupture, but other complications of prolonged immobilization persist as more than 6 weeks of immobilization remain necessary. Although carbon and polyester implants were said to enhance healing, they still require 8 or more weeks of cast immobilization (Jenkins and McKibbin 1980; Alexander et al. 1981; Howard et al. 1984; Amis et al. 1985). These findings suggest that new ways must be found to overcome the post-immobilization complications of tendon surgery. If healing can be quickened, then the duration of cast immobilization may be reduced to minimize all the complications that retard rehabilitation and recovery. Accumulating evidence suggests that thera-

Traumatic rupture of the tendo calcaneus (Achilles tendon) is a debilitating injury requiring at least 6-8 weeks of cast immobilization after surgical or nonsurgical intervention (Lawrence et al. 1955; Gillespie and George 1969; Ralston and Schmidt 1971; Lea and Smith 1972; Gilles and Chalmers 1977; Sjostrom et al. 1978; Quigley and Scheller 1980; Nistor 1981; Shields 1982; Sjostrom and Nystrom 1983). Because such prolonged periods of immobilization precipitate muscle atrophy (Christiansen 1954; Arner and Lindholm 1959; Booth 1982, 1987; Carden et al. 1987), osteoarthritis (Hall 1964, 1969; Thaxter et al. 1965; Langenskiold et al. 1979), atrophy and ulceration of joint cartilage (Thaxter et al. 1965; Enneking and Horowitz 1972; Woo et al. 1975), skin necrosis (Christiansen 1954; Ma and Griffith 1977), infection (Christiansen 1954; Arner and Lindholm 1959), tendocutaneous adhesion (Christiansen 1954; Arner and

Address correspondence to: Dr. Chukuka S. Enwemeka, Division of Physical Therapy, Department of Orthopaedics and Rehabilitation, University of Miami School of Medicine, 5915 Ponce de Leon Blvd., 5th Floor, Coral Gables, FL 33146. Supported in part by grants from the VA-RR&D and the Foundation for Physical Therapy. 801

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peutic ultrasound facilitates fibroplasia and protein synthesis (Drastichova et al. 1973; Harvey et al. 1975; Popspisilova 1976; Popspisilova and Rottova 1977; Morcos and Aswad 1978; Edmond and Ross 1988). Cultured human fibroblasts exposed to 0.5-2.0 W cm 2 continuous-wave (CW) ultrasound of 3 MHz, were shown by 3H-proline analysis to synthesize more collagen than controls (Harvey et al. 1975). Similarly, C1300 neuroblastoma cells (N2A) sonicated with 1 MHz "burst-mode" ultrasound at 3.4 W cm -2 for 5 min synthesized more protein as judged by 3H-leucine uptake (Edmond and Ross 1988). Experimental rat skin granulomas have also been shown to synthesize more collagen, when exposed to 10 doses of ultrasound at 1 W cm -2 for 5 min (Popspisilova and Rottova 1977). Others have also shown that exposure of skin incisions to two 5-min doses of 1.5 W cm -2 promotes collagen synthesis in guinea pigs (Drastichova et al. 1973). Thus, in both in vitro and in vivo experimental models, various intensities of therapeutic ultrasound have been shown to augment protein synthesis. In addition, however, recent studies suggest that the beneficial effects of ultrasound may be enhanced by sonicating at lower intensities rather than at higher intensities (Anderson and Barrett 1981; Saad and Williams 1982, 1986; Mortimer and Dyson 1988; Kerr et al. 1989). For example, exposure of cultured fibroblasts to sonication intensities of 0.5-1.0 W cm -2 for 5 min has been shown to augment intracellular calcium, a well-known mediator of numerous cellular processes including protein synthesis (Mortimer and Dyson 1988). This same effect is not produced when sonication intensity is raised to 1.5 W cm -~ (Mortimer and Dyson 1988). Similarly, exposure of pig ears to 750 kHz ultrasound of 1.5 W cm -2 space-averaged intensity for 15 min, produces hyperthermia and venous "damage" (Kerr et al. 1989). This deleterious effect is not observed when sonication intensity and treatment duration are reduced to 1.2 W cm -2 and 10 min respectively (Kerr et al. 1989). In the same vein, therapeutic intensities of CW 1.65 MHz ultrasound applied to the umbilical area of rats produce a reversible dose-dependent decrease in the rate of removal of 99 TM technitium radio-labelled sulphur colloid (Saad and Williams 1982). The magnitude of this effect has been found to be directly proportional to the product of sonication intensity and treatment duration (Saad and Williams 1982). Follow-up studies (Saad and Williams 1986) have demonstrated a direct relationship between the total amount of sound energy applied and the rate of removal of the labelled colloid, thus suggesting that the immunosuppressive effect of therapeutic ultrasound

Volume 16, Number 8, 1990

(Anderson and Barrett 1981; Saad and Williams 1982, 1986; Kerr et al. 1989) may be minimized either by reducing the intensity of sonication or by decreasing the duration of treatment. Immune cells, such as monocytes and macrophages, cooperate with fibroblasts in collagen biosynthesis and degradation (Laub et al. 1982; Laub and Vaes 1982). A macrophage-dependent factor is also known to stimulate the proliferation of fibroblasts in vitro (Leibovich and Ross 1976). Therefore, high-intensity ultrasound may hinder fibroplasia and collagen synthesis and hence impair the healing process of tendons. Indeed, Turner et al. (1989) have shown that 3 MHz pulsed ultrasound of 1.0 W cm -2 applied three times per week for a prolonged period of 5 weeks does not alter the mechanical strength of surgically repaired cockerel tendons after 6 weeks of healing. Because of these recent findings, we expanded our studies of ultrasound to include the effects of a lower intensity of sonication on the healing process of tendons. Specifically, our aims were to determine the effects of low-dose 0.5 W cm -2 ultrasound on the tensile strength and energy absorption capacity of healing rabbit tendo calcaneus (Achilles tendon). METHODS

Subjects Twenty-four rabbits, weighing 2.58 _+ 0.64 kg were used for this study. The animals were fed highfiber rabbit chow and water ad libitum, and housed one per standard 30.5 X 71 × 51 cm rabbit cage in an environment maintained at 21-23°C. Surgical procedure Each animal was weighed, then anaesthetized with a mixture of 180 mg xylazine, 30 mg acepromazinc and 900 mg ketamine, by intravenous injection of 1.0 mL per 1.5 Kg body weight. The skin over and around the right tendo calcaneus was prepped; thereafter, the tendon was approached by a medial skin incision, freed from surrounding tissue, and cut sharply and transversely midway between its calcaneal insertion and the musculotendinous junction. The severed ends of the tendon were immediately sutured with three loops of 3.0 silk. Thereafter, the skin incision was closed and the limb immobilized in a light-weight fiberglass cast with the ankle fully plantarflexed and the knee flexed to 90 °. Limb immobilization was performed such that a window, approximately 20 cm z, was left at the site of tenotomy to permit sonication of the tendon without cast removal. All surgical procedures were performed under aseptic conditions.

The biomechanical effects of low-intensity ultrasound • C. S. ENWEMEKAet al.

Ultrasound treatment After surgery, the animals were r a n d o m l y assigned to either a treatment group or a control group. To permit sonication under water, each rabbit was positioned on a platform with the experimental hindlimb alone immersed in a bowl of 12-16 L ofdegased and deionized water. The water t e m p e r a t u r e was maintained at 38°C + 1.5°C. A brand new factorycalibrated clinical ultrasound unit, Sonicator 705 a was used for each treatment. The machine emits a collimated s o u n d b e a m at a frequency o f 1 M H z + 5% via a 5 cm 2 applicator, and has a peak acoustic intensity to average acoustic intensity ratio (i.e., beam nonuniformity ratio) of 6.0. The applicator was moved continuously within the area o f the 20 cm 2 window, but kept 1-2 cm away from the exposed tissue surface during each treatment. Beginning from the first post-operative day, 10 tendons were sonicated daily at a space-averaged intensity of 0.5 W cm 2 for 5 min. A total o f nine consecutive treatments were given before the tendons were excised for testing on the 10th post-operative day. The remaining 14 tendons served as controls; thus, they were handled in the same m a n n e r as the experimental tendons but without exposure to ultrasound, i.e., they were mock-sonicated.

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with a standard 10 N weight as described in the Instron manual. To prevent tendon slippage, the Instron clamps were modified by applying a piece of 240 grit (40 m m ) sand paper to each surface of the clamp in contact with the tendon. An air pressure of 400,000 pascals was then used to close the clamps, before each tendon was pulled to rupture at a crosshead speed o f 8.33 m m S -J. S i m u l t a n e o u s l y , the load-deformation curve was recorded at a chart speed of 16.67 m m S -l. The tensile strength and other biomechanical characteristics of the tendons were read from the load-deformation curves obtained. The energy absorption capacity o f each tendon was derived f r o m the c u r v e s by e l e c t r o n i c m e a s u r e m e n t , by drawing a vertical line from the failure point on each curve to the x-axis. Then, a Jandel Scientific digitizer c interfaced to an IBM P C - X T c o m p u t e r d that had Sigma-Scan, e a software designed for area and morphometric measurements, was calibrated as described in the Sigma-Scan manual and used to measure the area.

Data analysis" The data were analyzed by independent samples t-test and reported below as mean + standard error. RESULTS

Tendon excision On the 10th post-operative day, the immobilization splint of each rabbit was removed, and the animal weighed a n d anaesthetized as previously described. Thereafter, the skin incisions were reopened. After removing the tendon sutures, the tendo calcaneus was freed from the surrounding tissue and excised. The intact nontenotomized left tendons were also removed before each animal was sacrificed with an overdose ofpentobarbital sodium (nembutal). The excised tendons were quickly frozen in 0.09% NaCI solution at - 7 0 ° C . Evidence indicates that this manner of preservation does not affect biomechanical test results (Mathews and Ellis 1968; W o o et al. 1986). B i o m e c h a n i c a l tests Each tendon was first thawed to r o o m temperature and their cross-sectional areas determined by linear and volumetric measurements (Ellis 1969) before b i o m e c h a n i c a l analysis. A P n e u m a t i c Action Grip was then used to attach each tendon to the cross heads o f an I n s t r o n b Materials Testing System (MTS). On each test date, the MTS was calibrated

a Metier Electronics Inc., Anaheim, CA. b Instron Inc., Houston, TX.

There was no significant weight difference between the two groups of rabbits, either before tenot o m y or prior to sacrifice (p > 0.10). Irrespective of treatment, the tenotomized right tendons were consistently larger in cross-sectional area than the intact nontenotomized left tendons (p < .001) (Fig. 1). When the mean tensile strength and energy absorption capacity of the tendons were compared, the effect o f sonication at 0.5 W cm 2 was unequivocal. Exposure to ultrasound induced a statistically significant increase in tensile strength (p < .02), tensile stress (p < .005) and energy absorption capacity (p < .001) of the tendons (Figs. 2-4). Similar differences were not observed between the corresponding intact nontenotomized left tendons (p > 0.10). Strain data were inconsistent and did not differ significantly between groups. DISCUSSION These findings warrant the conclusion that sonication at 0.5 W cm -2 for 5 min daily for 9 days aug-

cJandel Scientific, Corte Madera, CA. a International Business Machines Corp., Boca Raton, FL. eJandel Scientific, Corte Madera, CA.

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Ultrasound in Medicine and Biology

Volume 16, Number 8, 1990

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Fig. 1. The relative cross-sectional areas of the intact left tendons and the right sonicated tendons. Note the significant increase in the sizes of right tenotomized tendons over the sizes of the left intact tendons (p < .001). ments the healing strength of regenerating rabbit t e n d o c a l c a n e u s . A l t h o u g h a s i m i l a r effect was obs e r v e d in a p r e v i o u s s t u d y in w h i c h r e g e n e r a t i n g rabbit t e n d o c a l c a n e u s were s o n i c a t e d at 1.0 W c m -2 ( E n w e m e k a 1989), o u r p r e s e n t results suggest t h a t s o n i c a t i o n i n t e n s i t i e s as high as 1.0 W c m 2 m a y n o t be n e c e s s a r y to p r o m o t e t e n d o n healing. I n d e e d , the s u p e r i o r i t y o f t h e l o w e r i n t e n s i t y m a y be seen in the d a t a o f t h e t w o studies. W h e r e a s t h e tensile strength,

tensile stress a n d e n e r g y a b s o r p t i o n c a p a c i t y o f the t e n d o n s s o n i c a t e d at 1.0 W c m 2 were 45.63 + 6.51 N, 114.01 + 11.67 N c m -2 a n d 125.28 +_ 10.49 m J , r e s p e c t i v e l y , t h e c o r r e s p o n d i n g v a l u e s for t e n d o n s s o n i c a t e d at 0.5 W c m -2 were 49.32 + 9.32 N, 150.69 + 18.68 N c m -2 a n d 189.77 + 46.16 mJ. T h e larger m e a n cross-sectional a r e a o f t h e right t e n o t o m i z e d t e n d o n s p e r h a p s relates to the fact t h a t r e g e n e r a t i n g t e n d o n s h a v e larger a n d highly m e t a -

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The biomechanical effects of low-intensity ultrasound • C. S. ENWEMEKAet

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Fig. 3. Tensile stress developed by the two groups of tendons. The means tensile stress of control and sonicated tendons were 101.83 __+ 12.36 and 150.69 + 18.68 N cm 2, respectively. The mean tensile stress of sonicated tendons was significantly higher than control values (p < .005).

bolic fibroblasts and numerous collagen fibrils that are scattered, disoriented and not as closely packed as those o f intact n o n t e n o t o m i z e d tendons (Salamon and H a m o r i 1966; C o n w a y 1967; R o k k a n o n and Vainio 1971; Miller and Parry 1973; Postacchini et al. 1978; Postacchini and De Martino 1980). Because it is well k n o w n that the strength o f regenerating tendons bears a direct relationship with the duration of healing, and the quantity and cross-sectional areas o f

its collagen fibrils (Elliot 1965; Vogel 1974; Parry et al. 1978; Williams et al. 1985), the higher tensile strength and energy absorption capacity induced by s o n i c a t i o n indicate that t h e r a p e u t i c u l t r a s o u n d quickens the healing process of tenotomized rabbit tendo calcaneus. Tensile stress represents the force per unit area developed by a tissue as it is pulled to rupture (Frankel and Nordin 1980). Thus, the higher tensile stress developed by the sonicated tendons sug-

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