- Email: [email protected]
Ultrasound speed in equine cortical bone: Effects of orientation, density, porosity and temperature
ULTRASOUND SPEED IN EQUINE CORTICAL BONE: EFFECTS OF ORIENTATION, DENSITY, POROSITY AND TEMPERATURE R. N. McCAttTHY.*t
L. B. JEFFCOT?
and R. N. MCCARTNEYS
l Equine Clinical Research Unit. Department of Veterinary Science. University of Melbourne, Veterinary Clinical Centre. Princes Highway. Werribee, Victoria 3030, Australia and $ Cumherland College of Health Sciences, East Street. Lidcombe, New South Wales 2141, Australia
Abstract-Ultrasound speed, as measured by a transmission technique in quine cortical bone, was found to vary markedly with the direction of the ultrasound path through the bone. Using bone samples from the mid-site of the third metacarpus of 20 horses, the ultrasound speed was measured as 4125 ms-’ in the longitudinal direction. 3442 ms-’ m . the circumferential or transverse direction, and 3428 ms- ’ in the radial direction. These results confirm the anisotropic properties of compact bone. Ultrasound speed had a positive linear relationship when compared with hone specific gravity of cortical bone (I ~0.773. n = 35. p
INTRODUCTION Ultrasound speed in cortical bone is dependent on many factors. For cxamplc, the wavelength of ultrasound relative to the dimensions of the bone will effect transmission velocity depending on whether the ultrasound beam is propagated as a bar or a bulk wave (Ashman et ul.. 1984). The direction of the transmission beam through the bone has also been found to influence the wave velocity, which reflects the anisotropic property of cortical bone (Lang, 1970; Van Buskirk et ul.. 1981). Cortical bone has been described as both orthotropic (Van Buskirk et (II.. 1981) and transversely isotropic (Yoon and KatG 1979) in the assessmentof the anisotropic nature of bone and this may reflect a dikrence between primary and secondary bone. The influence of water content and density on the speed of ultrasound in bone has been reported (Lees et al.. 1979) where ultrasound speed was up to 10% faster in dry bone when compared to wet bone and was linearly dependent on the wet and dry bone density. Another factor thought to influence ultrasound speed is the porosity (i.e. the volume fraction of pores) of the bone (Lees et al., 1979). Porosity is also thought to effect the attenuation of ultrasound in bone (Lakes et al., 1984). The influence of porosity on bone stiffness has been demonstrated for cortical bone by mechanical testing (Currey, 1988; SchafRer and Burr, 1988). however a relationship between ultrasound speed and porosity does not appear to have been published. The rdlationship between ultrasound speed
Receivedin jiwl@rm IO May 1990. t Presentaddress: 63 Murray Street, Coolamundra. New South Wales 2590. Australia.
in bone and temperature has been previously described (Bonficld and Tully. IYXZ; Ashman et al.. 1984). The speciesfrom which the cortical bone originates is also important in determining the speed of ultrasound. Species affect the type of bone prcscnt (e.g. proportion of primary and secondary) and also the density of bone present. The age of the individual will also affect the type of bone present; bone from young animals will have a high proportion of primary lamellar bone, while in older animals secondary Haversian bone will predominate. Ultrasound speed measurement in equine cortical bone is usually in young horses which have predominantly primary bone, in humans however this modality may be applied to the adult population whose bone consists of mostly Haversian systems. This paper describes the relationship between orientation, density, porosity and temperature on the transmission speed in cortical bone of the horse. The properties of ultrasound speed in cortical bone have important connotations for assessment of bone strength in racehorses (JeKcott and McCartney, 1985; Jeffcott et al., 1987; McCarthy and Jcffcott. 1988) and are considered to have important possibilities in human medicine (Greenticld et ul., 1981). The measurement of ultrasound speed in bone has been used to successfullydemonstrate changes in bone density in young horses given intcnsivc excrcisc (McCarthy, 1990). MATERIAIS
AND MRTtIODS
Cortical bone specimens were cut from the dorsal corticcs of the third metacarpus from 20 horses (n =40). These metacarpal bones were obtained from
1139
R. N. MCCARTHY ct al.
1140
horses submitted to the Department of Veterinary Paraclinical Sciences for post-mortem. The bones were initially cleaned of soft tissue, wrapped in a clear plastic and then frozen at -9°C. The specimens, oblong in shape (approximately 10 x 10 x 15 mm), were later cut on a mill (Adcock and Shipley, Leeds, U.K.) under constant water irrigation to prevent dehydration and overheating of the bone. The specimens were cut from five sites in the metacarpus [Fig. l(a) and (b)] and placed in plastic vials filled with 0.9% saline and frozen at -9 “C until required for measurement. All the bone specimens were measured for ultrasound transmission speed using a water coupling method (Fig. 2). Two 19 mm, 2.25 MHz transducers (General Electric), of approximately 50 mm focal length, were fixed directly opposing each other on the walls of a perspex water bath. The transducers were set 113 mm apart. The group ultrasound speed of plane waves through water at room temperature was obtained using a pulse module (Panametrics Model 5055PR) to generate a signal at one transducer. The received signal was amplified and displayed together with the trigger signal from the pulse module on a digital storage oscilloscope with signal averaging cap-
Proximal
Site 1
4.0 cm
2
2.0 cm
Site 3
mid-site
Site 4
2.0 cm
Site 5
4.0 cm
Site
Distal
ability (Tektronix Model 468). The cortical bone specimen was then placed between the transducers so that one set of faces were perpendicular to the direct path of the ultrasound wave. The difference, At. between the time of flight for the water alone and for the water with bone inserted was measured on the oscilloscope. The thickness of the specimen was measured using an electronic digital calliper (Max-Cal Model 950-101). The speed of ultrasound in the bone specimen (C,) was calculated from the following equation: C,= looo.d.d_/(d;t-d.Af)(ms-‘)
d=distance between the transducers (mm) d,= thickness of the bone (mm) t = time of flight through water alone (s) At =change of time of flight when the bone is placed between the transducers (s). The group ultrasound speed was measured in three directions, i.e. longitudinal (n = 34). circumferential (n -34) and radial (n=36). at the mid-site, but only in the longitudinal and circumferential directions at the other four sites. Ten of the bone specimens from the mid-site (site 3) were selected to measure the variation of ultrasound speed with alteration in temperature. Ultrasound speed was mcasurcd in the longitudinal direction at seven different temperatures starting at 4” and rising to 42°C and then being repeated at 4°C. The specific gravity of the mid-site specimens was measured by dividing the mass of the bone by its volume which was dctcrmined from the dimensions of the specimen. The bones were then thin-sectioned, with a thickness of 100~. perpendicular to the long axis of the metacarpus using a diamond coated internal-hole saw (Leitz 1600). Microradiographs were then taken of the thin sections. Bone porosity was estimated by viewing the microradiographs under a low power (x63) microscope fitted with a drawing tube and digitising tablet (Mop Videoplan, Kontron Bildanalyze, Munich, F.R.G.). The area of the canals within the field of view was measured on the digitiser as was the entire area of the field of view. The porosity, P, was calculated by: P =
(a)
area of canals in field of view area of field of view
.-. Bone specimen milled from the dorsal cortex
Fig. 1. (a) Diagrammatic representation of the five sites used for obtaining specimensof cortical bone of the third metacarpusof the horse. (b) Location of bone specimens taken from the dorsal corfex of the third metacarpal bone.
(1)
where
x 100%
(2)
and was averaged over five fields of view for each specimen. All results are presented as mean&standard deviation. The diflerence between two group means was tcstcd for significance using a two sample Student’s f test, and the difference between paired data within a group were analysed using a Student’s t test for paired data. Multiple regression analyses were conducted using two explanatory variables. Regression analysis was also used to test equality of slope and intercept of ultrasound velocity in the three major axes vs the specific gravity of the specimen. The significance level was set at P < 0.05.
Ultrasound speed in equine cortical bone
1141
Transmitting Transducer
”
Abnkum, 1 from Pulse
Module
Fig. 2. Diagram of the Lester bath quipment
RESULTS
used lo measure ultrasound speed.
Table 1. Results of ultrasound speed (C,) mea.sunments in three orthogonal directions of bone sections from J sites on the dorsal cortex of Ihe metaphysis of the third metacarpus
The ultrasoubd speed in the longitudinal direction at the mid-site1 was 4125f 143 ms-‘, while in the Ultrasound speed circumferential direction it was 3422 f 138 m s- * and C. in the radial diuection it was 3428 f 140 m s- ’ (Table (m s-l) Site on I). The ratio of .$peedsin the longitudinal compared to Longitudinal Circumferential Radial the circumferetjtial and to the radial direction was dorsal cortex of metacarpus (n = 34) (n f 34) (n = 36) 1.0:0.83:0.83. qe ultrasound speed in the longitudinal and circunbferential directions at the other four 4099 Site 1 3356 sites are shown in Table I. (The table shows results for *I76 *II4 n=36. as som+ of the samples were destroyed by Site 2 4139 3423 *I30 kll7 Instrom comprbsion testing, the results of which were 4125 3422 3428 not included h&e. before all the measurements could Site 3 (mid-&c) f I43 fl38 k140 be made.) The: pooled data for all the metacarpal Sik 4 4106 3423 bones indicatedgood correlations of ultrasound speed il35 *II2 3Y8 I Site 5 3355 between the five diflerent sites. The circumferential &I61 & I45 ultrasound spe+i was found to be significantly lower at the numbers 1 and 5 sitescompared to the other sites. while the longkudinal ultrasound speed was only Table 2. Rcsulu of linear rcgmion analysisof ultrasound lower at the nuknber 5 site. speed(C,) vsspecific gravity,p, for bone specimens from the dorsal cortex of the mid-metacarpus (site 3) The ultrasoubd speed in all three directions at the mid-site (site 3# was found to be significantly correly-Axis Direction of ated to the s&tic gravity of the specimens (Table 2). intcrccpt Slope r ultrasound path n The slope of th# graph of circumferential ultrasound speed against s$ecific gravity (Fig. 3) was found to be I569 34 0.753 II01 Longitudinal 2455 + 235 different from the slope of the radial ultrasound speed 34 0.828 278 1628 Circumferential against specific gravity when tested for parallelism. iI89 f366 The slope of tht regression line of longitudinal ultra36 0.901 -794 2182 Radial sound speed a+inst specific gravity was not difTerent fl80 f 349 from either th# circumferential or radial regression slopes, but the p-axis intercept was. of porosity, the results of the ultrasound speed, specific The ultrasouhd speed in a circumferential direction gravity and porosity of bone from horses less than I2 was found to M inversely related lo the porosity of the months of age were compared to the results from bone (Fig. 4). ‘l/here was a good correlation between the ultrasound1 speed and the porosity (r30.773, n horses older than 12 months that had a similar -34. p=O.ooOt), Specific gravity was also inversely porosity. For a given porosity, the bone from horses lessthan 12 months of age had a lower specific gravity related to porojity (r~0.857, n=34. p=O.OOOl). Muland ultrasound speed (Table 3). tiple regressionhnalysis of ultrasound speed with both The ultrasound speed in the longitudinal direction porosity and qpecific gravity did not significantly in bone was found lo be inversely proporlional to improve the rdlationship. since the variables were temperature (Fig. 5), while the ultrasound speed in highly corretatc/d with each other. It was notedithac Ihe bone from the young horses waler was proportional to temperature. A logarithmic function was found to best fit the relationship of tended to havq a lower ultrasound speed than the ultrasound speed with temperature over the range of older horses.TO determine whether this was a function
1142
R N. MCCAUTHY et al.
4500,
0”
3500.
3000
Fig. 3. Graph of ultrasound speed (C,) for each of the three orthogonal directions vs specific gravity (p) as measured in bone specimens from the dorsal cortex of the midmetacarpus (site 3).
.q
1 0
10
20
30
40
50
Tcmpcrsturc ('C) Fig. 5. Graph of longitudinal ultrasound speed (CJ vs temperature 171. C,=454l-21.3 T”.*”
the repeated ultrasound measurement at 4°C which followed after the bone being measured at 42 “C was consistent with the original measurement. DISCUSSION
0
IO
20
40
30
50
P f%) Fig. 4. Graph of circumferential ultrasound speed (C,) vs bone porosity (P) measured in bone cubes from the dorsal cortex of the mid-metacarpus (site 3). C, = 3614 - 14.3 P.
Table 3. Results of porosity, P. and circumferential ultrasound speed (C,) in bonespecimens from the dorsal cortex of the mid-metacarpus (site 3)
Samples All (n = 34) From horses < I2 months old with a porosity between 7 and I4 % (n=l3) From horses > I2 months old with a porosity between 7 and I4 % (n= 13) *Significantly p=o.O012. tsignificantly p=O.OOOl.
Ultrasound speed
Porosity
C, (m s-l)
(C)
Specific gravity P (g cm-‘)
3422 *I38
12.35 f 7.36
I.938 f 0.069
3405. k58
10.32 f 2.03
1.92st kO.026
3507 *a4
IO.16 f 2.02
I .977 *0.02
lower
than
> 12 month
old
horses,
lower
than
> I2
old
horses,
month
from 14 to 42°C the regression line was essentially linear (r = 0.999, n = 4). The cfTectsof temperature on ultrasound speed in bone over this temperature range were reversible as
4-42 “C. However,
The anisotropic nature of equine cortical bone is similar to that reported for bovine cortical bone (Lees ef al.. 1979) with a corresponding ratio of ultrasound speed in the three different orientations. The ultrasound speed results presented here were somewhat higher than those reported for bovine compact bone by Lees et al. (1979). although the density of the equine cortical bone was generally lower. There was little alteration in the ratio (1.0:0.83:0.83) along the metacarpal shaft. However, there was a trend of reduced anisotropy of the bone near the mctaphyseal ends. This agrees with the results of Pope and Outwater (1974) who found a reduction in the anisotropy of the mechanical properties of cortical bone near the metaphyscs. The reduction of the circumferential speed at the proximal and distal sites may reflect altering bone density and porosity at these sites although this was not investigated here. The reduction of longitudinal speed of the distal shaft (site 5) may reflect lower bone density at this site, as well as an alteration in fibre orientation. The positive linear relationship between ultrasound speed in bone and specific gravity has been reported previously (Lees, 1986) using bone from a wide range of speciesand sites.The relationship described by Lees for ultrasound speed in the longitudinal direction with bone density, although linear, had quite a diflerent slope and intercept compared to that reported here. This was presumably because Lees used bone from many species rather than from just one. The y-axis intercepts that we have presented here for the regression of ultrasound speed against density do not have a physiological meaning and were given to demonstrate the statistical differences between the regression lines for each direction. Transverse isotropy was initially assumed for the distribution of ultrasound speed in these bones and the results of the mid-site ultrasound speed would
Ultrasound speed in quine cortical bone
seem to confirm this (Table 1). However, the regrcssion slopes of radial and circumferential ultrasound speed with specific gravity were differenS the radial ultrasound speed being lower for the bones with low specific gravity compared to the circumferential ultrasound speeds. fhe low specific gravity bones were generally from younger horses that had a primary lamellar structure. while the higher specific gravity bones had a mikture of primary lamcllar and sccondary Haversian structures. The circumferentially orientated lamellae provide many more interfaces to the ultrasound wave in the radial direction which may explain the lower speed measured in this direction. Therefore for primary lamellar bone. orthotropy may be a better description for the distribution of ultrasound speed. Porosity for normal equine cortical bone appears to be a major detetminant of specific gravity as the two parameters were highly correlated. However, there was some tvidcocc that the bone from younger horses may be lessdense for a given porosity. This may have been due to a lawer mineralisation of matrix in these younger horsesas they form new bone. The percentage of ash, or mineral, in metacarpal cortical bone from horses 7-8 months of age is lower than the percentage of ash in adult bone (McCarthy, 1990). The invcrsc relationship between temperature and ultrasound speed in bone confirms the investigations of Bonfield and Tully (1982). and the inverse relationship between stiffness tcstcd mechanically and bone temperature (Sniith and Walmslcy, 1959). The elfcct of temperature appcarcd to be rcvcrsiblc from 4 to 44 “C in our studies. However. BonIicld and Li (1968) found that irreversible damage occurred to the mechanical properties of boqe above SO’XZpossibly resulting from intercollagenous debonding. Ashman er ul. (1984) also reported a simililr alteration of ultrasound speed with temperature. Thley found a change in the axial elastic cocflicicnt with temperature of -0.17 % ‘C-l, which is similar to the decrease in ultrasound speed results presented here ( - 0. I6 % “C - ’ from 14 to 42 “C). The temperature of the testing conditions may also account for some of the differences in ultrasound speed reported in the literature and especially between the ultrasound speed measured in vitro at room temperature (Rabin et 01.. 1983; Jeffcott et al.. 1987) and that measured in viva (McCarthy et ol., 1988). The results pnesented in this paper are important baseline data for the clinical application of ultrasound speed measurements in horses. There appears to be considerable wtential for the assessment of bone quality in horsesi(JeKcottet 01.. 1988) and for interpreting the skeletal tiesponseto the effects of exercise and racing (McCartity and Jeffcott. 1989a. b). REFERENCFS
Ashman,R. B.. Cowin, S. C., Van Buskirk. W. C. and Rice. 1. C. (1984) A cohtinuous wave technique for the measurcment of the c&tic properties of cortical bone. J. Biomechanics 17. 349-361.
1143
Bonficld, W. and Li. C. H. (1968) The temperature dependcna of the deformation of bone. 1. Biumechnnics 1. 323-329. Bonficld, W. and Tully, A. E. (1982) Ultrasonic analysis ol the Young’s Modulus of cortical bone. J. biomrd. Enyny 4. 23-27. Currcy. J. D. (1988) The eficct of porosity and mineral content on the Young’s modulus of elasticity of compact bone. 1. Biomechanics 21, 131-139. Grecnfield. M. A.. Craven. J. D., Huddleston. A.. Kehrer, M. L.. Wishko, D. and Stem, R. (1981) Measurement of the velocity of ultrasound in human cortical bone in oiro. Radiology 138.701-710. JelTcott, L. 8.. Buckingham, S. H. W.. McCarthy. R. N., Cleeland. J. C.. Scotti, E. and McCartney, R. N. (1988) Noninvasive measurement of bone. A review of clinical and research applications in the horse. Equine Vet. J. Suppl, 6. 71-79. JelTcott,L. B., Buckingham.S. H. W. and McCartney. R. N. (1987) Noninvasive measurement of bone quality in horses and changes associated with exercise. In Equine Erurcise Physiology 2 (Edited by Gillespie, J. R. and Robinson, N. E.). pp. 615-630. ICEEP Publications, Davis. CA. JetTcott.L. B. and McCartney. R. N. (1985) Ultrasound as a tool for assessmentof bon; quality.in the horse. Vur. Rec. 116.337-342. Lakes, R.. Yoon. H. S. and Katz, J. L. (1986) Ultrasonic wave propagation and attenuation in wet bone. J. hiomc*d.Enqnq 8, 143-148. Lang. S. B. (1970) Ultrasonic method of measuring elastic cocfflcicnts of bone and results on lrcsh and dried bovine bones. /EEE Trans. biomd Enqng 17. IOI-105. Lees. S. (1986) Sonic properties of miner&cd tissues. In Tissue Chorucfe&nion with Ulrrusounrl (Edited by Grccnlcal. J. F.). pp. 208-226. CRC Press. Baa Raton, FL. Lees. S.. Clcary. P. F.. Hcclry. J. D. and Garcpy. E. L. (1979) Distribution of sonic plcsio-velocity in a compact bone sample. J. Acousr. Sot. Am. 66, 641-646. M&arthy. R. N. (1990) Effects of cxcrcisc on bone dcvrlopment in the horse. Ph.D. thesis, University ol Melbourne. McCarthy, R. N. and JctTcott. L. B. (1988) Monitoring the clTcctsof treadmill cxcrciscon bone by noninvasive means. Equinr Ver. J. Suppl. 6. 88-92. McCarthy, R. N. and Jctfcott, L. B. (1989a) Cortical bone rcsponsc to intense treadmill exercise in young horses I. Noninvasive parameters. C/in. Sporrr Med. (Submitted.) McCarthy, R. N. and JeRcott, L. B. (1989b) Cortical bone response to intense treadmill exercise in young horses 2. Microradiographic and histomorphomctric results. C/in. Sports Med. (Submitted.) McCarthy, R. N.. Jeflcott. L. 8. and McCartney.R. N. (1988) Ultrasonic transmission velocity and single photon absorptiomctric measurement of metacarpal bone strength: an in oiwo study in the horse. Equine Vet. J. Suppl. 6. 80-87. Pope. M. H. and Outwater, J. 0. (1974) Mechanical propcrtics ol bone as a function ol position and orientation. J. Biomechunics 7, 61-66. Rabin, D. S.. Rantanen. N. W.. Seder, J. A., Miller, P. and Hcllhakc. P. (1983) The clinical use of bone strength assessmentin the thoroughbred raahorsc. Proc. Am. Ass. Equine Prucl. 29. 343-351. Schatllcr. M. B. and Burr, D. B. (1988) StitTncssof compact bone: ct&ts of porosity and density. J. Biomrchnics 21. 13-16. Smith, J. W. and Walmslcy. R. (1959) Factors affecting the elasticity of bone. J. A& 93, 503-523. Van Buskirk. W. C.. Cowin. S. C. and Ward. R. W. (19811 Ultrasonic measuicment ol orthotropic e&tic constant; of bovine femoral bone. J. binmech. Engng 103, 67-72. Yoon, H. S. and Katl J. L. (1979) Ultrasonic properties and microtcxture ol human cortical bone. Ulrraronic Tissue Choraclerisorion 2. 189-195.
L. B. JEFFCOT?
and R. N. MCCARTNEYS
l Equine Clinical Research Unit. Department of Veterinary Science. University of Melbourne, Veterinary Clinical Centre. Princes Highway. Werribee, Victoria 3030, Australia and $ Cumherland College of Health Sciences, East Street. Lidcombe, New South Wales 2141, Australia
Abstract-Ultrasound speed, as measured by a transmission technique in quine cortical bone, was found to vary markedly with the direction of the ultrasound path through the bone. Using bone samples from the mid-site of the third metacarpus of 20 horses, the ultrasound speed was measured as 4125 ms-’ in the longitudinal direction. 3442 ms-’ m . the circumferential or transverse direction, and 3428 ms- ’ in the radial direction. These results confirm the anisotropic properties of compact bone. Ultrasound speed had a positive linear relationship when compared with hone specific gravity of cortical bone (I ~0.773. n = 35. p
INTRODUCTION Ultrasound speed in cortical bone is dependent on many factors. For cxamplc, the wavelength of ultrasound relative to the dimensions of the bone will effect transmission velocity depending on whether the ultrasound beam is propagated as a bar or a bulk wave (Ashman et ul.. 1984). The direction of the transmission beam through the bone has also been found to influence the wave velocity, which reflects the anisotropic property of cortical bone (Lang, 1970; Van Buskirk et ul.. 1981). Cortical bone has been described as both orthotropic (Van Buskirk et (II.. 1981) and transversely isotropic (Yoon and KatG 1979) in the assessmentof the anisotropic nature of bone and this may reflect a dikrence between primary and secondary bone. The influence of water content and density on the speed of ultrasound in bone has been reported (Lees et al.. 1979) where ultrasound speed was up to 10% faster in dry bone when compared to wet bone and was linearly dependent on the wet and dry bone density. Another factor thought to influence ultrasound speed is the porosity (i.e. the volume fraction of pores) of the bone (Lees et al., 1979). Porosity is also thought to effect the attenuation of ultrasound in bone (Lakes et al., 1984). The influence of porosity on bone stiffness has been demonstrated for cortical bone by mechanical testing (Currey, 1988; SchafRer and Burr, 1988). however a relationship between ultrasound speed and porosity does not appear to have been published. The rdlationship between ultrasound speed
Receivedin jiwl@rm IO May 1990. t Presentaddress: 63 Murray Street, Coolamundra. New South Wales 2590. Australia.
in bone and temperature has been previously described (Bonficld and Tully. IYXZ; Ashman et al.. 1984). The speciesfrom which the cortical bone originates is also important in determining the speed of ultrasound. Species affect the type of bone prcscnt (e.g. proportion of primary and secondary) and also the density of bone present. The age of the individual will also affect the type of bone present; bone from young animals will have a high proportion of primary lamellar bone, while in older animals secondary Haversian bone will predominate. Ultrasound speed measurement in equine cortical bone is usually in young horses which have predominantly primary bone, in humans however this modality may be applied to the adult population whose bone consists of mostly Haversian systems. This paper describes the relationship between orientation, density, porosity and temperature on the transmission speed in cortical bone of the horse. The properties of ultrasound speed in cortical bone have important connotations for assessment of bone strength in racehorses (JeKcott and McCartney, 1985; Jeffcott et al., 1987; McCarthy and Jcffcott. 1988) and are considered to have important possibilities in human medicine (Greenticld et ul., 1981). The measurement of ultrasound speed in bone has been used to successfullydemonstrate changes in bone density in young horses given intcnsivc excrcisc (McCarthy, 1990). MATERIAIS
AND MRTtIODS
Cortical bone specimens were cut from the dorsal corticcs of the third metacarpus from 20 horses (n =40). These metacarpal bones were obtained from
1139
R. N. MCCARTHY ct al.
1140
horses submitted to the Department of Veterinary Paraclinical Sciences for post-mortem. The bones were initially cleaned of soft tissue, wrapped in a clear plastic and then frozen at -9°C. The specimens, oblong in shape (approximately 10 x 10 x 15 mm), were later cut on a mill (Adcock and Shipley, Leeds, U.K.) under constant water irrigation to prevent dehydration and overheating of the bone. The specimens were cut from five sites in the metacarpus [Fig. l(a) and (b)] and placed in plastic vials filled with 0.9% saline and frozen at -9 “C until required for measurement. All the bone specimens were measured for ultrasound transmission speed using a water coupling method (Fig. 2). Two 19 mm, 2.25 MHz transducers (General Electric), of approximately 50 mm focal length, were fixed directly opposing each other on the walls of a perspex water bath. The transducers were set 113 mm apart. The group ultrasound speed of plane waves through water at room temperature was obtained using a pulse module (Panametrics Model 5055PR) to generate a signal at one transducer. The received signal was amplified and displayed together with the trigger signal from the pulse module on a digital storage oscilloscope with signal averaging cap-
Proximal
Site 1
4.0 cm
2
2.0 cm
Site 3
mid-site
Site 4
2.0 cm
Site 5
4.0 cm
Site
Distal
ability (Tektronix Model 468). The cortical bone specimen was then placed between the transducers so that one set of faces were perpendicular to the direct path of the ultrasound wave. The difference, At. between the time of flight for the water alone and for the water with bone inserted was measured on the oscilloscope. The thickness of the specimen was measured using an electronic digital calliper (Max-Cal Model 950-101). The speed of ultrasound in the bone specimen (C,) was calculated from the following equation: C,= looo.d.d_/(d;t-d.Af)(ms-‘)
d=distance between the transducers (mm) d,= thickness of the bone (mm) t = time of flight through water alone (s) At =change of time of flight when the bone is placed between the transducers (s). The group ultrasound speed was measured in three directions, i.e. longitudinal (n = 34). circumferential (n -34) and radial (n=36). at the mid-site, but only in the longitudinal and circumferential directions at the other four sites. Ten of the bone specimens from the mid-site (site 3) were selected to measure the variation of ultrasound speed with alteration in temperature. Ultrasound speed was mcasurcd in the longitudinal direction at seven different temperatures starting at 4” and rising to 42°C and then being repeated at 4°C. The specific gravity of the mid-site specimens was measured by dividing the mass of the bone by its volume which was dctcrmined from the dimensions of the specimen. The bones were then thin-sectioned, with a thickness of 100~. perpendicular to the long axis of the metacarpus using a diamond coated internal-hole saw (Leitz 1600). Microradiographs were then taken of the thin sections. Bone porosity was estimated by viewing the microradiographs under a low power (x63) microscope fitted with a drawing tube and digitising tablet (Mop Videoplan, Kontron Bildanalyze, Munich, F.R.G.). The area of the canals within the field of view was measured on the digitiser as was the entire area of the field of view. The porosity, P, was calculated by: P =
(a)
area of canals in field of view area of field of view
.-. Bone specimen milled from the dorsal cortex
Fig. 1. (a) Diagrammatic representation of the five sites used for obtaining specimensof cortical bone of the third metacarpusof the horse. (b) Location of bone specimens taken from the dorsal corfex of the third metacarpal bone.
(1)
where
x 100%
(2)
and was averaged over five fields of view for each specimen. All results are presented as mean&standard deviation. The diflerence between two group means was tcstcd for significance using a two sample Student’s f test, and the difference between paired data within a group were analysed using a Student’s t test for paired data. Multiple regression analyses were conducted using two explanatory variables. Regression analysis was also used to test equality of slope and intercept of ultrasound velocity in the three major axes vs the specific gravity of the specimen. The significance level was set at P < 0.05.
Ultrasound speed in equine cortical bone
1141
Transmitting Transducer
”
Abnkum, 1 from Pulse
Module
Fig. 2. Diagram of the Lester bath quipment
RESULTS
used lo measure ultrasound speed.
Table 1. Results of ultrasound speed (C,) mea.sunments in three orthogonal directions of bone sections from J sites on the dorsal cortex of Ihe metaphysis of the third metacarpus
The ultrasoubd speed in the longitudinal direction at the mid-site1 was 4125f 143 ms-‘, while in the Ultrasound speed circumferential direction it was 3422 f 138 m s- * and C. in the radial diuection it was 3428 f 140 m s- ’ (Table (m s-l) Site on I). The ratio of .$peedsin the longitudinal compared to Longitudinal Circumferential Radial the circumferetjtial and to the radial direction was dorsal cortex of metacarpus (n = 34) (n f 34) (n = 36) 1.0:0.83:0.83. qe ultrasound speed in the longitudinal and circunbferential directions at the other four 4099 Site 1 3356 sites are shown in Table I. (The table shows results for *I76 *II4 n=36. as som+ of the samples were destroyed by Site 2 4139 3423 *I30 kll7 Instrom comprbsion testing, the results of which were 4125 3422 3428 not included h&e. before all the measurements could Site 3 (mid-&c) f I43 fl38 k140 be made.) The: pooled data for all the metacarpal Sik 4 4106 3423 bones indicatedgood correlations of ultrasound speed il35 *II2 3Y8 I Site 5 3355 between the five diflerent sites. The circumferential &I61 & I45 ultrasound spe+i was found to be significantly lower at the numbers 1 and 5 sitescompared to the other sites. while the longkudinal ultrasound speed was only Table 2. Rcsulu of linear rcgmion analysisof ultrasound lower at the nuknber 5 site. speed(C,) vsspecific gravity,p, for bone specimens from the dorsal cortex of the mid-metacarpus (site 3) The ultrasoubd speed in all three directions at the mid-site (site 3# was found to be significantly correly-Axis Direction of ated to the s&tic gravity of the specimens (Table 2). intcrccpt Slope r ultrasound path n The slope of th# graph of circumferential ultrasound speed against s$ecific gravity (Fig. 3) was found to be I569 34 0.753 II01 Longitudinal 2455 + 235 different from the slope of the radial ultrasound speed 34 0.828 278 1628 Circumferential against specific gravity when tested for parallelism. iI89 f366 The slope of tht regression line of longitudinal ultra36 0.901 -794 2182 Radial sound speed a+inst specific gravity was not difTerent fl80 f 349 from either th# circumferential or radial regression slopes, but the p-axis intercept was. of porosity, the results of the ultrasound speed, specific The ultrasouhd speed in a circumferential direction gravity and porosity of bone from horses less than I2 was found to M inversely related lo the porosity of the months of age were compared to the results from bone (Fig. 4). ‘l/here was a good correlation between the ultrasound1 speed and the porosity (r30.773, n horses older than 12 months that had a similar -34. p=O.ooOt), Specific gravity was also inversely porosity. For a given porosity, the bone from horses lessthan 12 months of age had a lower specific gravity related to porojity (r~0.857, n=34. p=O.OOOl). Muland ultrasound speed (Table 3). tiple regressionhnalysis of ultrasound speed with both The ultrasound speed in the longitudinal direction porosity and qpecific gravity did not significantly in bone was found lo be inversely proporlional to improve the rdlationship. since the variables were temperature (Fig. 5), while the ultrasound speed in highly corretatc/d with each other. It was notedithac Ihe bone from the young horses waler was proportional to temperature. A logarithmic function was found to best fit the relationship of tended to havq a lower ultrasound speed than the ultrasound speed with temperature over the range of older horses.TO determine whether this was a function
1142
R N. MCCAUTHY et al.
4500,
0”
3500.
3000
Fig. 3. Graph of ultrasound speed (C,) for each of the three orthogonal directions vs specific gravity (p) as measured in bone specimens from the dorsal cortex of the midmetacarpus (site 3).
.q
1 0
10
20
30
40
50
Tcmpcrsturc ('C) Fig. 5. Graph of longitudinal ultrasound speed (CJ vs temperature 171. C,=454l-21.3 T”.*”
the repeated ultrasound measurement at 4°C which followed after the bone being measured at 42 “C was consistent with the original measurement. DISCUSSION
0
IO
20
40
30
50
P f%) Fig. 4. Graph of circumferential ultrasound speed (C,) vs bone porosity (P) measured in bone cubes from the dorsal cortex of the mid-metacarpus (site 3). C, = 3614 - 14.3 P.
Table 3. Results of porosity, P. and circumferential ultrasound speed (C,) in bonespecimens from the dorsal cortex of the mid-metacarpus (site 3)
Samples All (n = 34) From horses < I2 months old with a porosity between 7 and I4 % (n=l3) From horses > I2 months old with a porosity between 7 and I4 % (n= 13) *Significantly p=o.O012. tsignificantly p=O.OOOl.
Ultrasound speed
Porosity
C, (m s-l)
(C)
Specific gravity P (g cm-‘)
3422 *I38
12.35 f 7.36
I.938 f 0.069
3405. k58
10.32 f 2.03
1.92st kO.026
3507 *a4
IO.16 f 2.02
I .977 *0.02
lower
than
> 12 month
old
horses,
lower
than
> I2
old
horses,
month
from 14 to 42°C the regression line was essentially linear (r = 0.999, n = 4). The cfTectsof temperature on ultrasound speed in bone over this temperature range were reversible as
4-42 “C. However,
The anisotropic nature of equine cortical bone is similar to that reported for bovine cortical bone (Lees ef al.. 1979) with a corresponding ratio of ultrasound speed in the three different orientations. The ultrasound speed results presented here were somewhat higher than those reported for bovine compact bone by Lees et al. (1979). although the density of the equine cortical bone was generally lower. There was little alteration in the ratio (1.0:0.83:0.83) along the metacarpal shaft. However, there was a trend of reduced anisotropy of the bone near the mctaphyseal ends. This agrees with the results of Pope and Outwater (1974) who found a reduction in the anisotropy of the mechanical properties of cortical bone near the metaphyscs. The reduction of the circumferential speed at the proximal and distal sites may reflect altering bone density and porosity at these sites although this was not investigated here. The reduction of longitudinal speed of the distal shaft (site 5) may reflect lower bone density at this site, as well as an alteration in fibre orientation. The positive linear relationship between ultrasound speed in bone and specific gravity has been reported previously (Lees, 1986) using bone from a wide range of speciesand sites.The relationship described by Lees for ultrasound speed in the longitudinal direction with bone density, although linear, had quite a diflerent slope and intercept compared to that reported here. This was presumably because Lees used bone from many species rather than from just one. The y-axis intercepts that we have presented here for the regression of ultrasound speed against density do not have a physiological meaning and were given to demonstrate the statistical differences between the regression lines for each direction. Transverse isotropy was initially assumed for the distribution of ultrasound speed in these bones and the results of the mid-site ultrasound speed would
Ultrasound speed in quine cortical bone
seem to confirm this (Table 1). However, the regrcssion slopes of radial and circumferential ultrasound speed with specific gravity were differenS the radial ultrasound speed being lower for the bones with low specific gravity compared to the circumferential ultrasound speeds. fhe low specific gravity bones were generally from younger horses that had a primary lamellar structure. while the higher specific gravity bones had a mikture of primary lamcllar and sccondary Haversian structures. The circumferentially orientated lamellae provide many more interfaces to the ultrasound wave in the radial direction which may explain the lower speed measured in this direction. Therefore for primary lamellar bone. orthotropy may be a better description for the distribution of ultrasound speed. Porosity for normal equine cortical bone appears to be a major detetminant of specific gravity as the two parameters were highly correlated. However, there was some tvidcocc that the bone from younger horses may be lessdense for a given porosity. This may have been due to a lawer mineralisation of matrix in these younger horsesas they form new bone. The percentage of ash, or mineral, in metacarpal cortical bone from horses 7-8 months of age is lower than the percentage of ash in adult bone (McCarthy, 1990). The invcrsc relationship between temperature and ultrasound speed in bone confirms the investigations of Bonfield and Tully (1982). and the inverse relationship between stiffness tcstcd mechanically and bone temperature (Sniith and Walmslcy, 1959). The elfcct of temperature appcarcd to be rcvcrsiblc from 4 to 44 “C in our studies. However. BonIicld and Li (1968) found that irreversible damage occurred to the mechanical properties of boqe above SO’XZpossibly resulting from intercollagenous debonding. Ashman er ul. (1984) also reported a simililr alteration of ultrasound speed with temperature. Thley found a change in the axial elastic cocflicicnt with temperature of -0.17 % ‘C-l, which is similar to the decrease in ultrasound speed results presented here ( - 0. I6 % “C - ’ from 14 to 42 “C). The temperature of the testing conditions may also account for some of the differences in ultrasound speed reported in the literature and especially between the ultrasound speed measured in vitro at room temperature (Rabin et 01.. 1983; Jeffcott et al.. 1987) and that measured in viva (McCarthy et ol., 1988). The results pnesented in this paper are important baseline data for the clinical application of ultrasound speed measurements in horses. There appears to be considerable wtential for the assessment of bone quality in horsesi(JeKcottet 01.. 1988) and for interpreting the skeletal tiesponseto the effects of exercise and racing (McCartity and Jeffcott. 1989a. b). REFERENCFS
Ashman,R. B.. Cowin, S. C., Van Buskirk. W. C. and Rice. 1. C. (1984) A cohtinuous wave technique for the measurcment of the c&tic properties of cortical bone. J. Biomechanics 17. 349-361.
1143
Bonficld, W. and Li. C. H. (1968) The temperature dependcna of the deformation of bone. 1. Biumechnnics 1. 323-329. Bonficld, W. and Tully, A. E. (1982) Ultrasonic analysis ol the Young’s Modulus of cortical bone. J. biomrd. Enyny 4. 23-27. Currcy. J. D. (1988) The eficct of porosity and mineral content on the Young’s modulus of elasticity of compact bone. 1. Biomechanics 21, 131-139. Grecnfield. M. A.. Craven. J. D., Huddleston. A.. Kehrer, M. L.. Wishko, D. and Stem, R. (1981) Measurement of the velocity of ultrasound in human cortical bone in oiro. Radiology 138.701-710. JelTcott, L. 8.. Buckingham, S. H. W.. McCarthy. R. N., Cleeland. J. C.. Scotti, E. and McCartney, R. N. (1988) Noninvasive measurement of bone. A review of clinical and research applications in the horse. Equine Vet. J. Suppl, 6. 71-79. JelTcott,L. B., Buckingham.S. H. W. and McCartney. R. N. (1987) Noninvasive measurement of bone quality in horses and changes associated with exercise. In Equine Erurcise Physiology 2 (Edited by Gillespie, J. R. and Robinson, N. E.). pp. 615-630. ICEEP Publications, Davis. CA. JetTcott.L. B. and McCartney. R. N. (1985) Ultrasound as a tool for assessmentof bon; quality.in the horse. Vur. Rec. 116.337-342. Lakes, R.. Yoon. H. S. and Katz, J. L. (1986) Ultrasonic wave propagation and attenuation in wet bone. J. hiomc*d.Enqnq 8, 143-148. Lang. S. B. (1970) Ultrasonic method of measuring elastic cocfflcicnts of bone and results on lrcsh and dried bovine bones. /EEE Trans. biomd Enqng 17. IOI-105. Lees. S. (1986) Sonic properties of miner&cd tissues. In Tissue Chorucfe&nion with Ulrrusounrl (Edited by Grccnlcal. J. F.). pp. 208-226. CRC Press. Baa Raton, FL. Lees. S.. Clcary. P. F.. Hcclry. J. D. and Garcpy. E. L. (1979) Distribution of sonic plcsio-velocity in a compact bone sample. J. Acousr. Sot. Am. 66, 641-646. M&arthy. R. N. (1990) Effects of cxcrcisc on bone dcvrlopment in the horse. Ph.D. thesis, University ol Melbourne. McCarthy, R. N. and JctTcott. L. B. (1988) Monitoring the clTcctsof treadmill cxcrciscon bone by noninvasive means. Equinr Ver. J. Suppl. 6. 88-92. McCarthy, R. N. and Jctfcott, L. B. (1989a) Cortical bone rcsponsc to intense treadmill exercise in young horses I. Noninvasive parameters. C/in. Sporrr Med. (Submitted.) McCarthy, R. N. and JeRcott, L. B. (1989b) Cortical bone response to intense treadmill exercise in young horses 2. Microradiographic and histomorphomctric results. C/in. Sports Med. (Submitted.) McCarthy, R. N.. Jeflcott. L. 8. and McCartney.R. N. (1988) Ultrasonic transmission velocity and single photon absorptiomctric measurement of metacarpal bone strength: an in oiwo study in the horse. Equine Vet. J. Suppl. 6. 80-87. Pope. M. H. and Outwater, J. 0. (1974) Mechanical propcrtics ol bone as a function ol position and orientation. J. Biomechunics 7, 61-66. Rabin, D. S.. Rantanen. N. W.. Seder, J. A., Miller, P. and Hcllhakc. P. (1983) The clinical use of bone strength assessmentin the thoroughbred raahorsc. Proc. Am. Ass. Equine Prucl. 29. 343-351. Schatllcr. M. B. and Burr, D. B. (1988) StitTncssof compact bone: ct&ts of porosity and density. J. Biomrchnics 21. 13-16. Smith, J. W. and Walmslcy. R. (1959) Factors affecting the elasticity of bone. J. A& 93, 503-523. Van Buskirk. W. C.. Cowin. S. C. and Ward. R. W. (19811 Ultrasonic measuicment ol orthotropic e&tic constant; of bovine femoral bone. J. binmech. Engng 103, 67-72. Yoon, H. S. and Katl J. L. (1979) Ultrasonic properties and microtcxture ol human cortical bone. Ulrraronic Tissue Choraclerisorion 2. 189-195.