THERMAL EFFECTS of FOCUSED ULTRASOUND ENERGY ON.Pdf

Ultrasound in Med. & Biol., Vol. 27, No. 10, pp. 1427–1433, 2001 Copyright © 2001 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/01/$–see front matter PII: S0301-5629(01)00454-9

● Technical Note

THERMAL EFFECTS OF FOCUSED ULTRASOUND ENERGY ON BONE TISSUE

† ‡ NADINE BARRIE SMITH*, JOSHUA M. TEMKIN, FREDERIC SHAPIRO and KULLERVO HYNYNEN* *Brigham and Women’s Hospital, Harvard Medical School, Department of Radiology, Division of MRI, Boston, MA, USA; †Tufts University, Department of Biology, Medford, MA, USA; and ‡Children’s Hospital, Harvard Medical School, Orthopaedic Research Laboratories, Department of Orthopaedic Surgery, Boston, MA, USA

(Received 5 April 2001; in ﬁnal form 31 July 2001) Abstract—The effects of focused ultrasound (US) at therapeutic acoustic power levels were studied in vivo on the bone-muscle interface in rabbit thighs. The purpose of this study was to provide direction in establishing safety guidelines for treating tissue masses using focused US on or near bone. A positioning device was used to manipulate a focused US transducer (1.5 MHz) in a magnetic resonance imaging (MRI) scanner. This system was used to sonicate the femurs of 10 rabbits at acoustic power levels of 26, 39, 52 and 65 W for 10 s. The rabbits were euthanized either4hor28days after the sonications and the bone samples were harvested for histology examinations. In the femurs studied, acoustic power levels from 39 to 65 W resulted in soft tissue damage characterized grossly by coagulated tissue and bone damage depicted by yellow discoloration. Histologic examination of lesions from sonications from 39 to 65 W demonstrated that osteocyte damage and necrosis, characterized by pyknotic cells and empty lacunae, occurred within the ablation area extending through the bone. The follow-up MR images demonstrated an increase in the amount of damage in the femurs at 28 days posttreatment in comparison to images taken immediately after treatment. Focused US directed at the femur caused immediate signiﬁcant thermal damage to bone in the form of osteocyte necrosis extending through the (approximately) 1 cm bone in this study. The results suggest that, when focused US energy is directed at or near bone-muscle interfaces, precautions should be taken to avoid thermal damage to the bone that can compromise its strength for extended periods. (E-mail: [email protected]) © 2001 World Federation for Ultrasound in Medicine & Biology. Key Words: HIFU, Focused ultrasound, Bone, Muscle, Femur, Necrosis, Coagulation, Thermal.

INTRODUCTION care must be taken during treatment due to the proximity of the prostate to the bony pelvis. Studies have shown Many studies have experimented with focused ultra- that high temperatures are reached at the soft tissue/bone sound (US) as a noninvasive method for treating soft interface during exposure to US (Lehmann et al. 1967, tissue masses and for hyperthermic heating of tissue volumes (ter Haar et al. 1980; Ebbini and Cain 1991; 1978; Hynynen and DeYoung 1988). This temperature Goss et al. 1996; McGough et al. 1996; Fujii et al. 1999; elevation has caused pain during long US hyperthermia Hurwitz et al. 2001). In many cases, the mass of tissue treatments, limiting the amount of power and, thus, tem- that is treated is on or near bone (Lin et al. 2000; Lu et peratures in the tumor and in the bone. During US al. 2000). For example, a high-intensity focused US surgery, the patients are frequently sedated or anesthe- device for the treatment of benign prostatic hyperplasia tized; this allows tumors next to the bone to be treated, (BPH) has been used in clinical studies (Sanghvi et al. but may expose the bone surface to high acoustic powers 1999). This clinical device has been successfully used for and temperatures. Therefore, the effects of the high- thermal ablation of diseased prostate tissue, although temperature elevations on the bone should be studied before such treatments are performed. It is generally assumed that thermal damage to the bone would be Address correspondence to: Nadine Smith, Ph.D., Assistant Pro- superﬁcial due to the sharp temperature gradient (Leh- fessor of Bioengineering, The Pennsylvania State University, 219 Hal- lowell Building, University Park, PA 16802 USA. E-mail: mann et al. 1967, 1978; Hynynen and DeYoung 1988). [email protected] However, there are no studies demonstrating the extent

1427 1428 Ultrasound in Medicine and Biology Volume 27, Number 10, 2001 of bone damage caused by a focused US surgery device. Given the increasing interest in using US for noninvasive treatment, the purpose of this study was to investigate thermal damage caused by focused US energy on bone tissue.

MATERIALS AND METHODS Equipment The US field was generated by a single focused PZT, air-backed US transducer with diameter, radius of curvature and resonant frequency of 100 mm, 80 mm (F number ϭ 0.8) and 1.5 MHz, respectively. The radiofre- quency (RF) signal feeding the transducers was generated by a frequency generator (Wavetek Inc., San Diego, CA, Model 271) and amplified by an RF amplifier (ENI Inc., Rochester, NY, Model 2100L). The electrical impedance of the transducer was matched to the output impedance of the amplifier by an external LC (L, induc- tor ϭ 3.4 ␮H; C, capacitor ϭ 3588 pF) matching net- work. The forward and reflected electrical powers were measured using a digital power meter (Bird, Model 4421) and a dual directional coupler (Werlatone, Model Fig. 1. Schematic diagram displaying the MR-guided focused C1373). The transducer was aimed so that the focus US surgery system used for the in vivo animal experiments. could be set above the water surface within the rabbit thigh. Movement of the transducer in the x, y or z direction was performed by the use of a magnetic resonance (MR)-compatible (GE Medical Systems, Milwau- respectively. Focal lesions that approximate the half- kee, WI) mechanical positioning system (Hynynen et al. intensity contour in tissue were anticipated because 1996). A plastic membrane covered the Plexiglas box blood flow minimally affects lesion size during short that was embedded in the standard magnetic resonance sonications (Fry 1993). imaging (MRI) patient bed (Fig 1). The entire setup was placed in the GE Medical Systems 1.5 Tesla Signa scan- Animal experiments ner. A 12.7-cm diameter surface coil was used in the A total of 10 New Zealand rabbits (2 kg, 3 months experiments to improve the signal-to-noise ratio (SNR). old) were studied. Of these, 5 rabbits were used in the acute (nonsurvival) thermal effects study and 5 were used in the thermal effects survival study. Each animal Ultrasound calibration was anesthetized with a combination of ketamine hydro- The acoustical efficiency of the transducer was chloride (Ketaset, 40 mg/kg IM, Fort Dodge Laborato- measured using a radiation force technique (Hynynen ries, Fort Dodge, IA) and xylazine hydrochloride (10 1993). The transducer was placed in a tank filled with mg/kg IM, Fermenta Animal Health Co., Kansas City, degassed water and the US field aimed toward an acous- MO). The rabbits were provided housing, food and vet- tical absorber. Acoustic efficiency was determined to be erinary care according to procedures approved by the 65%. At the focus, the intensity was determined from the Harvard Medical Area Standing Committee on Animals. total acoustic power output when its distribution at the The thighs were shaved using an electric shaver to re- focus was known from the beam plots (Herman and move the hair and a depilatory agent was applied to the Harris 1982). Assuming a 15-mm depth in muscle with Ϫ exposed skin to eliminate any remaining hair. Through- an amplitude attenuation coefficient of 4.1 Np m 1 Ϫ out the US therapy, the rabbit’s body temperature was MHz 1 (Moros et al. 1993), the peak intensity (W/cm2) constantly monitored with a copper-constantan thermo- in the muscle can be estimated by multiplying the acous- Ϫ couple placed in the rectum. tic power (W) by 70 cm 2. This is based on the obser- vation that the intensity is directly proportional to the Experiment I acoustic power applied with sharply focused transducers at the power levels used (Hynynen 1991a). The half- Acute study. The five rabbits were placed on their intensity beam diameter and length were 1.0 and 4.8 mm, sides on the top of the transducer positioning system and Focused ultrasound on bone ● N. B. SMITH et al. 1429 sonicated according to the study protocol. Eight femurs in five rabbits were sonicated. Each femur was sonicated at four different locations spaced by 10 mm. The acoustic powers used were 26, 39, 52 and 65 W. Location and order of sonication was varied from animal to animal. The femur was located using T1 weighted images and the US was aimed at the bone-muscle interface using these images. After the sonications, the tissue damage was imaged. At the end of the experiment, animals were euthanized with an injection of Fatal-Plus (pentobarbitone sodium, 1 mL per 4.5 kg IV, Vortech Pharmaceuticals, Dearborn, MI). Following dissection, lesions on the femur and the surrounding tissue were recorded. Damaged bone and muscle lesion areas were characterized by discoloration compared with the healthy surrounding tissue. Histologic examination was performed on two of the femurs. The entire femurs were removed and fixed in 10% neutral buffered formalin for a minimum of 2 weeks. After fixation, femurs were decalcified in 25% formic acid for 4 weeks, trimmed to segments containing the treated bone plus surrounding bone, embedded in paraffin, sectioned at 5-␮m thickness and stained with hematoxylin and eosin (H & E) or 1% toluidine blue. Multiple long axis sections were cut for light micro- Fig. 2. (a) A T1-weighted axial view of the pretreatment rabbit scopic assessment to evaluate cellular damage (Shapiro shows the location of the transducer. The arrow within the 1988, 1992). rabbit shows the location of the femur-muscle interface to be sonicated. (b) During a 52-W acoustic power sonication, temperature change from the transducer can be seen from an MR Experiment II phase-difference image showing the focused US beam as a dark area indicated by the arrow. (c) Immediately posttreatment, Survival study. Five femurs in five rabbits were lesions (arrow) to the tissue and bone can be seen in the sonicated for 10 s at 39, 52 and 65 W and the animals T1-weighted contrast enhanced images. (d) At 28 days post- survived. Follow-up imaging using T1- and T2-weighted treatment, damage to the bone can still be seen in the T1- images (axial, sagittal and coronal planes) with contrast weighted contrast-enhanced images. was performed immediately after the treatment and at 14 and 28 days following treatment to assess the extent of soft tissue damage present. Following the final MR im- al. 1996). The phase shift was calculated using a fast aging at 28 days posttreatment, the rabbits were eutha- spoiled gradient echo sequence (FSPGR) (Ishihara et al. nized. Histologic examination of the femur was per- 1995; Chung et al. 1996). After sonications, T1- and formed as described above. T2-weighted images, with and without contrast, were used to determine the extent of the treatment. A bolus of MR imaging contrast (Gd-DTPA, 0.2 mL/kg IV, Magnesvist, Berlex For localizing the femur-muscle interface and treat- Imaging, Wayne, NJ) was injected in the ear veins of the ment planning, a T1-weighted fast spin-echo imaging rabbits and images were collected until the signal had sequence was used: echo time (TE) ϭ 16 ms; repetition peaked on MR images. time (TR) ϭ 500 ms; echo train length ϭ 4; FOV ϭ 16 cm; bandwidth ϭ 16 kHz; slice thickness ϭ 3 mm; RESULTS matrix size ϭ 256 ϫ 128; number of excitations (NEX) ϭ 1. Phase-difference imaging was used to lo- A typical pretreatment, axial T1-weighted scan of a calize the US focus and determine the proton resonant rabbit and transducer shows the location of the transducer frequency (PRF) shifts. A reference (baseline) scan and with respect to the femur-muscle interface (Fig. 2a). The the measurement scan were obtained and phase subtrac- depth of the heating from the transducer can be seen from tion was carried out to compute the PRF shift (Chung et a phase difference image (Fig. 2b) showing the focused US 1430 Ultrasound in Medicine and Biology Volume 27, Number 10, 2001

tissue surrounding the femur, at 4 h and 28 days posttreatment increased in size with increasing acoustic power. Within the discoloration, browning of the muscle tissue was observed for many of the thighs at 52 and 65 W. On the femurs, circular lesions were characterized by a yellow discoloration on the bone and were observed from 39 to 65 W. Additionally, excised femurs at 28 days posttreatment exhibited small areas of new bone growth at the sonication sites, characterized by increased struc- tural calcification. Two femurs from the acute study (4 h) were decalcified and processed for light microscopic evaluation. Soni- cation areas on these femurs exhibited yellow discoloration grossly compared to nonsonicated areas. Evaluation of the histologic slides showed osteocyte damage at 39, 52 and 65 W. The bone was assessed from three regions: (a) area beyond the ablation periphery; (b) periphery of the ablation area; and (c) ablation area. The area beyond the sonication site (a) was characterized by normal healthy bone. A toluidine blue-stained slide of an area outside the ablation area shows normal osteocytes and no empty lacunae. A high- power photomicrograph shows a normal osteocyte with a dark central nucleus and lighter staining cytoplasm in each lacuna. In Fig. 4a, arrow 1 shows a blood vessel and arrow 2 points to a normal osteocyte. The area at the periphery of the ablation site (b) is characterized by a mixture of empty Fig. 3. T1-weighted images with contrast of a femur (a) imme- lacunae and normal cells (Fig. 4a). The line of demarcation, diately after sonication; and at (b) 28 days posttreatment, show- however, is quite well defined. Figure 5b shows an area that ing an increase of tissue damage. T2-weighted images were also used to determine the extent of the treatment (c) immedi- is filled with empty lacunae, representing necrotic bone, and ately after treatment and at (d) 28 days posttreatment. normal-appearing osteocytes peripheral to this. Arrow 1 in Fig. 4b shows a normal osteocyte with nucleus present and arrow 2 indicates an empty lacuna. The ablation site (c) is beam (dark area indicated by an arrow) in rabbit muscle at characterized by completely empty lacunae (arrow), evi- the femur during a 52-W acoustic power sonication. Dam- dence of osteocyte necrosis (Fig. 4c). age (arrow) to the tissue and bone can be seen immediately At 28 days postsonication, the femurs of two of the posttreatment in the T1-weighted contrast enhanced images survival rabbits were used for light microscopic evalua- (Fig. 2c). Using the same rabbit and examining the same tion. There is bone cell damage for the 39-, 52- and 65-W focal area, damage to the bone can still be seen at 28 days samples for both femurs. Histologic sections show areas (survival) posttreatment (Fig. 2d). of healthy bone containing normal osteocytes (Fig. 5a) The femurs showed visible lesions and tissue dam- and adjacent damaged bone (Fig. 5b) with empty lacunae age for sonications at 39, 52 and 65 W in both the T1- similar to the bones from the acute study. As with the and T2-weighted images immediately after (acute) and at acute study, bone damage is characterized by empty 28 days posttreatment. Figure 3 displays the immediate lacunae, evidence of osteocyte necrosis (Fig. 5b, c, d). and long-term effects of the thermal energy of focused These bones show a high level of cell death at sonication US on the surrounding soft tissue as revealed by the (a) site with a sharp delineation between the area of live and acute, T1-weighted; (b) survival, T1-weighted; (c) acute, dead bone (Fig. 5d). The areas of cell death were often T2-weighted; and (d) survival, T2-weighted images. continuous across the bone. Periosteal metaplasia was These images exhibited delineated lesions with signifi- also evident overlying necrotic bone with both bone and cant soft tissue damage and edema both on the bone and cartilage formation occurring (Fig 5c). in the surrounding tissue for all 39 to 65 W. Visual examination at 4 h and 28 days posttreatment DISCUSSION showed extensive soft tissue damage surrounding lesions at acoustic powers from 39 to 65 W. Soft tissue damage, At 23 W acoustic power, no significant bone dam- characterized by the white discoloration of the muscle age was observed. This indicates a maximum safety level Focused ultrasound on bone ● N. B. SMITH et al. 1431

Fig. 4. (a) Histologic sections of femoral cortex outside the ablation area (acute study) 4 h posttreatment show normal blood vessel (arrow 1) and osteocytes (arrow 2) with no empty lacunae. (b) The area at the periphery of an ablation site shows normal osteocytes (arrow 1) with nuclei present and arrow 2 indicates empty lacunae of necrotic bone. (c) Empty lacunae and missing osteocytes are evident within an ablation site. The arrow indicates an empty lacuna where osteocyte necrosis and lysis has occurred.

that would be acceptable during surgery at the bone bone with empty lacunae. Areas of empty lacunae, evi- surface. For both the acute and survival experiments of dence of osteocyte necrosis, surrounded by healthy cells, focused US near bone, the results indicate muscle and were observed. Demarcation between dead and living bone damage at sonication sites at power levels used in tissue was usually quite clear (Fig. 5d, arrows). Quanti- focused US surgery. Protein coagulation and consequent tative studies with serial sections would be needed to soft tissue damage in the form of necrosis resulted from correlate the extent of the cell death with sonication the temperature elevation. Based on previous research, intensity. the measured cavitation pressure amplitude in dog thigh The T1- and T2-weighted images indicated an in- muscle was found to depend linearly on frequency with crease in the amount of edema in the surrounding tissue a slope of 5.3 MPa MHzϪ1 (Hynynen 1991b). From at 14 and 28 days posttreatment. Comparison of the 14- these experiments for tissue alone at 1.5 MHz, the and 28-day images shows less edema and vascular dam- threshold would be 8 MPa. The highest acoustic power age at 28 days, which indicates some healing. Addition- used (65 W) approached the cavitation threshold as de- ally, penetration of the thermal damage was continuous termined from hydrophone measurements, although most across the entire lateral cortex. Although the length of of the treatments were probably in the thermal range this study was only to 28 days, a longer study might Focused US was directed at the femur-muscle in- show more healing of the damaged tissue and bone at the terface. The histology performed here clearly delineates sonication site. areas of healthy bone with intact osteocytes and necrotic Histology on the 28-day bones continued to show 1432 Ultrasound in Medicine and Biology Volume 27, Number 10, 2001

Fig. 5. Histologic evaluation of the femurs of two survival rabbits showed cellular damage due to the US at 28 days posttreatment. These femurs showed areas of (a) healthy bone (normal osteocytes) with no empty lacunae and (b) adjacent damaged bone (empty lacunae, evidence of osteocyte necrosis). (c) In adjacent damaged bone, periosteal metaplasia was also evident overlying necrotic bone with both bone (top left) and cartilage (top right) formation occurring. (d) At the sonication site, the femoral cortical bone showed a sharp delineation (arrows) between the area of live (left) and dead (right) bone.

distinct bone necrosis, which seemingly indicates a spe- an increase in damage 2 weeks after treatment. The cific dose/distance effect (Fig. 5b). The area of dead bone damage most likely occurs immediately, but tissue was sometimes continuous across the entire cortical changes evolve over a period of time. This is consistent thickness. The periosteum was stimulated to new bone with laser findings that report that osteocyte damage is and cartilage formation (Fig. 5c). With time, bone heal- more pronounced during the first few weeks of recovery ing should occur from the periosteum and from vascular (Stein et al. 1990). ingrowth from the adjacent living cortical bone. There is evidence to suggest that focused US di- In comparison to the various types of clinical lasers rected at the skeletal system can cause immediate dam- used in thermal ablation of bone, focused US energy can age. This thermal damage could explain earlier clinical generate the same types of bone lesions based on the reports of periosteal pain during treatments (Marmor et resulting gross appearance and histologic changes (Zhao- al. 1979) or the pain can be attributed to nerve-ending Zhang et al. 1992; Rayan et al. 1992; Stein et al. 1990; exposure. Although the eventual complete healing of the Lustmann et al. 1992). Results here showed significant femur has not been determined, laser studies have shown immediate damage to the osteocytes within the ablation a delayed healing response following ablation but, ulti- area. Osteocyte necrosis was evident and characterized mately, the bone does repair itself (Lustmann et al. 1992; by empty lacunae. Although long-term histologic data Rayan et al. 1992; Buchelt et al. 1994). It is, therefore, were not available for this paper, MR imaging suggests believed that the potential thermal damage to the tissue Focused ultrasound on bone ● N. B. 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