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Mechanical properties of the tibia from chickens with idiopathic scoliosis
0021-9290~83 01005909 c
$03.00 0
1983 Pergamon Press Ltd
MECHANICAL PROPERTIES OF THE TIBIA FROM CHICKENS WITH IDIOPATHIC SCOLIOSIS R. S. RIGGINS, D. A. LEWIS and D. R. BENSON Department
of Orthopaedic
Surgery,
Professional
Building.
4301 X Street, Sacramento.
CA 95817
J. R. MCCARREY Department
of Avian Sciences, University
of California,
Davis, CA 95616
and C. E. FRANYI Department
of Community
Health,
University
of California.
Davis, CA 95616, U.S.A
Abstract-The tibia from six-week old chickens that develop idiopathic scoliosis were studied with stress relaxation experiments and torsional strength testing. Most parameters observed did not show any significant differences between tibias obtained from chickens with scoliosis and tibias from the control birds; however, the rate of stress relaxation of the tibia from the birds with scoliosis was minimally increased over the controls. There were no significant differences noted in ultimate torsional strength, maximum angular deformity or modulae of torsional rigidity of the tibias from scoliotic chickens when compared to tibias from control chickens.
INTRODUCTION
Six male birds from an inbred line of chickens that develop a severe idiopathic scoliosis in over 73 ‘I0of the males were selected for the study. The birds were six weeks old. For controls, six male white leghorn chickens from the University of California stock that were also six weeks old and six male white leghorn chickens matched by weight to the scoliotic birds were
selected. The mean values and standard errors in grams for the animals’ weights were 307 k 11 for weight controls, 388 + 12 for the age controls and 307 + 16 for the scoliotic birds. The ages of the scoliotic birds and age controls were six weeks, whereas the weight controls were only four to five weeks old. The animals were sacrificed with chloroform and their tibias removed and frozen in isotonic phosphate buffer pH 7.4. At the time of testing the bones were thawed and the epiphyses and periosteum removed leaving a hollow tube of bone that was somewhat flattened at each end and larger proximally than distaliy. The testing device was a modification of the torsional bone breaking device designed by Burstein and Frankel (1971) and Riggins et at. (1974). (See Fig. 1.) The device is designed to break the bone rapidly in torsion using a heavy pendulum. The device normally develops a torque of 6.3 x lo3 Nm at the lower portion of its fall and transverses a 30” arch in 0.075 s. The torque is measured with a torsional load cell* and the angular displacement by an appropriate sensor+. The data is displayed on an oscilloscope$ and photographed with a Polaroid camera. The modifications required for the stress relaxation studies were to replace the pendulum with a lever that allowed the manual application of the torque and a clamp that fixed the lever to maintain the deformity. By adding a scale and movable block to which the lever
Rtwiwd jh publicurion 26 Auyusr 1982. Please request reprints from Richard Riggins. M.D.. 69 Butler Street, Atlanta, GA 30306, U.S.A. This study has been supported financially by grant credits AM 2538 and AM 15278.
*Lebow Associates, Oak Park, Michigan. tBrush Metripak Model 3303, Brush Instrument Company. Cleveland, Ohio. *Tektronix 3C66 Carrier amplifier, 2B67 Time base, 564 B Storage Oscilloscope, Beaverton, Oregon.
Idiopathic
scoliosis is a lateral curvature of the spine that most commonly affects the adolescent girl. Tissue from humans with scoiiosis is difficult to obtain, but there is an inbred line of chickens developed from white leghorns that have a hereditary idiopathic scoliosis (Riggins et al., 1977). Studies on these animals suggest a collagen defect may be associated with their scoliosis as these chickens exhibit increased bone collagen solubility (Riggins et al., 1977), increased serum hydroxyproline (Lin et al., 1980) and changes in the viscoelastic properties of their tendons (Cutler er al., 1981). This report concerns a study of the mechanical properties of the tibia from chickens with idiopathic scoliosis. The bone from these animals exhibits changes in its viscoelastic properties as manifested by an increased rate of stress relaxation of the tibia. MATERIALS AND METHODS
59
60
R. S. RIGGINS,D. A. LEWIS, D. R. BENSON,J. R. MCCARREYand C. E. FRANTI
could be pulled, a predetermined angular displacement could be applied rapidly (0.5 s) and the position maintained with the clamp. The deformity applied was 3.5 rad/m based on the length of tibia between the grips and ranged from 7 to 11O.The same load cell that was used for testing the bone to fracture was used for the stress relaxation experiments, but a more stable amplifier was required* and the output was plotted on a strip chart recordert. The grips used were of two varieties. For the rapidly applied force to fracture, the ends of the bone were potted in a rapid setting dental plastic+. The pots would precisely fit the grips in the breaking machine. For the stress relaxation studies the dental plastic was not used as it has a slight stress relaxation of its own. Instead, adjustable brass grips were designed to fit the contours of the proximal and distal ends of the tibia. These grips were tightened down on the bone carefully so as not to damage the cortex. The grips were tested using a 0.32-cm 316 stainless steel rod secured in the brass grips and subjected to twice the torque used on the bones. No Lossof torque was observed over a 12-h period. The specimens were continually irrigated throughout the experiment with isotonic phosphate buffer pH 7.4 to prevent drying. The buffer was circulated through a water bath to provide a constant 37°C temperature and the bone was allowed 5 min or more to equilibrate to the temperature before testing. The bones were initially tested for stress relaxation and the bone ends then potted in the dental plastic. The bones were stored in the phosphate buffer at 4°C at least 24 h prior to destructive testing to give the dental plastic adequate time to set and also to allow the bone to recover from the stress relaxation experiment. The data from the stress relaxation experiment were graphed on semilogarithmic paper plotting the fraction of the initial torque remaining against time on the logarithmic scale. The resulting graph was a straight line over the time observed, 6-1200 s. The line was also calculated using the methods of least squares. The following data were obtained from the rapidly applied torque to fracture: ultimate torsional strength, maximal angular displacement per unit length of tibia between the pots, and a modulus of torsional rigidity or shear modulus of elasticity. The geometry of the specimens was also measnred. This included the length of specimen between the pots, the diameter of the bone at mid-shaft, and the cortical thickness. The midshaft was selected because the fracture began at the midshaft and spiraled at about 3545” toward one or the other end. Secondary fractures occasionally occurred but the initial crack began at the midshaft (Fig. 2). We have performed high
*Valedyne CD19-871, P. 0. Box 9025, Northridge, CA 93128. t Linear 17281 Eastman Avenue, Irvine, CA 92714. $Nu Weld Caulk Co., Milforcl, Delaware.
speed photography of fractures of femurs and tibias of chickens with our testing machine to confirm the location of the initial crack. The diameter of the midshaft was measured twice at 90” apart and the two measurements averaged. At first the values were kept separately, but analysis of the two values showed no significant differences in the values so they were pooled. Similarly the cortical thickness was measured with a micrometer. Two measurements were taken and the values averaged. To calculate a moment of inertia for the bone specimen, the formula for a hollow tube was used. This assumption is only partially correct but adequate for comparison among similar size specimens. TO reduce sample variation, the values obtained from the two tibias were pooled to give one value for each parameter for each bird. In the stress relaxation experiments, variation was further reduced by normalizing the curves to the initial torque applied. Actually, we found no significant differences in the analysis of data if the tibias were treated as individual specimens, i.e. twelve specimens from six birds or the birds were treated as specimens and pooling the results from the two bones from each bird thus only giving six specimens. An analysis of variance was used to evaluate the data. For the stress relaxation data, analysis of variance was also used to determine if within each group a single line could represent the data and if the line was straight when plotted on semilogarithmic paper. The mean values and standard errors were also determined for the various parameters studies and representative graphs were reconstructed from these data.
RESULTS
The analysis of variance of the slopes from the stress relaxation experiments replotted on semilogarithmic paper indicated that within each of the three groups a single line could represent the results of that group as there were no significant differences among the individual slopes in any one group. Furthermore, the tests for linearity of the slope which consisted of an analysis of variance of the individual slopes from a common straight line for each group were not significant, (p > 0.50); that is, there were no significant deviations from the straight line. Analysis of variance testing between the three groups: scoliosis, the age control, and weight control indicated no significant differences in the slopes of the controls, but the slope of the scoliotic birds’ data was significantly different (p < 0.01). There were no significant differences among the mean intercepts of any of the three groups. Reconstructed graphs from the mean values can be found in Fig. 3 plotted on normal graph paper, and Fig. 4 plotted on semilogarithmic paper. The initial torque to obtain the deformity of 3.5 rad/m of tibia between the grips was significantly greater for the controls by age than for the controls by weight or the
Fig. 1. Torsional bone testing machine. The pcnduium can be seen supported by a retainer. The lever arm attached to the rotating grip with its set screw is also visible as is the adjustment stop block. For the stress relaxation test, the block is moved to a predetermined degree and the lever pulled rapidly to the block, thus applying torque to the bone held in the grips. For the destructive test to fracture, the lever arm is removed and the grip can rotate freely. The pendulum at the bottom of its arc contacts a dog attached to the grip, thus breaking the bone. Torsional strain gauge and irrigation piping are also seen. Fig. 2. A chick tibia with a typical fracture created by the torsional bone testing machine of similar design to that of Burstein and Frankel (1971).
61
Mechanical
60
properties
I
I
100
200
I
I
300
400
500 TIME,
Fig. 3. Composite
63
of bone
stress
I
1
600
I
800
700
I
900
-
60
-
I
I
1100
1200
seconds
relaxation curves from the mean values obtained experiments plotted on normal graph paper.
80
I
1000
from
the stress
relaxation
FORCE
50
r
0’
I
I
IO
1000
100 TIME,
seconds
Fig. 4. Same data plotted in Fig. 3 replotted on semilogarithmic paper. The difference in the slope between the controls was not significant. The slope from scoliotic birds was significantly different from the controls p < 0.01.
Table
Birds (number) Scoliotic
(61
1. Stress relaxation
Initial torque* (Nm)
Torque
values (mean + standard
at 1200 s* (Nm)
error)
Intercept (fraction of initial torque)
Slope+ Fraction initial torque vs log time
0.126t0.013
0.075 f 0.0082
0.94 f 0.018
0.112 + 0.007
Control by weight (61
0. I25 f 0.0052
0.087 f 0.0036
0.98 * 0.002
0.092 + 0.001
Control by age (6)
0.166 + 0.011
0.113~0.0072
0.93 * 0.008
0.094 * 0.002
*Age control significant greater than scoliotic or wt control p < 0.025. tThe slope for the scohotic birds was significantly greater p -c 0.01 than controls.
R. S.
64
RIGGINS, D.
A.
LEWIS, D. R. BENSON,J. R. MCCARREY and C. E. FRANTI
scoliotic birds. This difference was still present to a significant degree at the end of the experiment, i.e. 1200 s. These data are presented in Table 1.
Values obtained
in the torsional
modulus of elasticity. These data are also plotted in Fig. 5. The lengths of the test specimens, diameter at midshaft, and cortical thickness can be found in Table 3. The length of the tibia1 shafts was significantly greater for the age control birds but the differences in their diameters and cortical thickness were not significant.
testing studies can
be found in Table 2. There were no significant differences in the ultimate torsional strengths, angular displacements per unit length of tibia between the pots, or the calculated modulae of torsional rigidity or shear
Table 2. Torsional testing values (mean f standard error) Ultimate torsional strength (Nm)
Birds (number)
Ultimate angular displacement * @ad/cm)
Calculated modulus+ of torsional rigidity (gNm- *)
Scoliotic (6)
0.494 * 0.033
0.0960 + 0.0078
3.41 f 0.368
Controls by weight (6)
0.401 f 0.029
0.0928 f 0.007
2.66kO.181
Controls by age (6)
0.529 f 0.046
0.0809 + 0.008
2.6OkO.197
*Angular displacement in radians divided by length of tibia between pots. Kalculated from experiment dataassuming tibia1 shaft was a hollow tube (J = 1l/32 (od4 - id4)). There were no significant differences among values reported in this table.
AGE CONTROL
RADIANS
* cm-
Fig. 5. The composite force deformation curves constructed from mean values obtained deformation to fracture. No significant differences in the curves were noted.
Table 3. Geometry of the tibia (mean &standard error)
Birds (number)
Length of specimen (cm) between pots (grips)
Midshaft diameter (cm)
Thickness of cortex (cm)
Scoliotic (6)
4.02; *0.17
0.386 + 0.009
0.0626 f 0.0039
Control by weight (6)
4.30+0.11
0.398 f 0.006
0.0660 f 0.0022
Control by age (6)
4.59’ * 0.09
0.438 f 0.003
0.0615 f 0.0032
*Significantly different p < 0.025.
by rapid
Mechanical
properties of
DISCUSSION
The purpose of this investigation was to determine if differences in the mechanical properties of the bone from chickens with idiopathic scoliosis existed when compared to bone taken from normal control chickens of similar age and weight. The scoliotic chickens have been shown to develop both sexually and physically slower than white leghorn chickens from which they were originally derived (Lin e? al., 1980). Because of this delayed maturation, two sets of control animals were needed, one set of controls matched by weight as animal body weight affects mechanical strength of bone (Saville, 1967; Rucker et ai., 1975) and one set of controls matched by age as age affects the physical properties of collagen (Viidik, 1973). The larger size of the control chickens. the same age as the scoliotic birds, no doubt accounted for the increased length of their tibia1 shaft and the increased torque required to produce the deformation for the stress relaxation experiments. Ideally we would like to select the spine to study, but our facilities do not have the capacity to mechanically test such small specimens, so the long bone of the tibia was selected. Our previous investigations (Riggins et al., 1977; Cutler et al., 1981) suggest the chicken with idiopathic scoliosis has a generalized collagen defect so if the collagen abnormality affects the mechanical properties of bone, it should affect the tibia as well as the spine. We elected to study the mechanical properties in torsion since specimen orientation in the torsional testing device is less critical than in some other modes of testing. Bone is anisotrophic and the chick tibia1 shaft does not precisely conform to any standard geometrical shape, so specimen orientation in some testing devices could be a problem. We found the shear modulus of elasticity or torsional rigidity for bone to be considerably lower than values reported by several investigators (Wainwright et al., 1976). The difference was probably related to the deviation of the geometry of the tibia1 shaft from our assumption that the shaft was a hollow tube. Other possibilities for the low modulus exist as previous investigators used human or bovine bone and tested in bending, compression or tension. To study the viscoelastic properties of bone, the stress relaxation test was selected as it is a fairly standardized test and has been used previously in bone by other investigators (Lakes and Katz, 1979; Lugassy and Korostaff, 1969). Stress relaxation data are easily determined and fairly immune to experimental error, an important consideration when working with biological material where small differences may be hidden by the individual variation. To further reduce individual variation, the stress relaxation data was normalized to the initial deforming force or torque. The time frame selected was 6-1200 s to cover three decades of time. Observations under 6 s were not attempted, as our equipment did not apply the initial force rapidly
bone
65
enough to prevent some degree of overlap between force application and stress relaxation from occurring in the initial part of the curve. To examine viscoelastic properties in a very short time, dynamic tests are more appropriate. The duration of observation was arbitrarily selected to be 1200 s. By this time, the loss of torque per unit time was becoming very small but still continuing and would probably continue for some time further. Lakes and Katz (1979) found stress relaxation continuing in their bone specimens tested in torsion at IO5 s and Lugassy and Korostaff (1969) estimated from their data that the fully relaxed state of bone in compression would require over 69 days. Currey (1965) in long term creep experiments did not find an end point after 55 days of observation. From our results, 1200 s does not seem to be an adequate time as the amount of stress relaxation that occurred in the threegroups was not statistically different when the data were expressed as a fraction of the initial torque. Because of the increased size of the age controls, the initial torque required to produce the standard angular displacement for the stress relaxation experiments was greater in the age controlled animals as was the torque remaining at 1200 s; however, the percentage change was not different from the other two groups. Since the slope of the curve for the scoliotic birds was statistically greater than the two controls, one would anticipate eventually the amount of stress relaxation would become greater for the scoliotic birds if the tests were carried out long enough. Of course, for very long time periods, if bone does indeed reach a fully relaxed state, the values for the three groups could approach each other once again. Apparently, observations over several months will be required to obtain these data about the fully relaxed state of bone if such a state exists. Several investigators have found differences in the behavior of the metabolism of connective tissues in patients with idiopathic scoliosis. Francis et al. (1976) reported increased collagen solubility in the skin of humans with idiopathic scoliosis, and hydroxyproline excretion in children with idiopathic scoliosis is elevated during certain periods of their development (Laitinen et al., 1966; Zorab, 1968; Zorab et al.. 1971). Ground substance might also be faulty, as Pedrini er al. (1972) reported abnormalities in the glycosominoglycans of the intervertebral disc from children with scoliosis. Balaba (1972) also found disturbances in the glycosominoglycan metabolism in the spine and its ligaments in children with scoliosis. In these patients, hydroxyproline excretion was elevated above normal controls. Stress relaxation experiments have been carried out by other investigators on the tendons and spinal ligaments of humans with scoliosis (Walter and Morris, 1973; Nordwall, 1973). These investigators did find slight increases in stress relaxation from children with idiopathic scoliosis, but the differences were not felt to be significant. We had similar difficulties in demonstrating that the differences betwen our control chickens and the chickens with scoliosis were
R. S. RIGGINS. D. A. LEWIS,D. R. BENSON,J. R. MCCARREYand C. E. FRANTI
66
significant. Only when the data were normalized against the initial deforming torque and plotted against time on the logarithmic scale could significant differences be found. These were differences in the rate of stress relaxation. The observations were not carried out over a sufficient period to determine if differences would exist in the fully relaxed state. In the chickens with idiopathic scoliosis changes in the mechanical properties of their tendons can be demonstrated by an increased rate of stress relaxation in their digital flexor and extensor tendons (Cutler et al., 1981) as well as a slight increased rate of stress relaxation in the bone of their tibias as reported here. Furthermore, with increased bone and cartilage collagen solubility (Riggins et al., 1977) and elevated serum hydroxyproline (Lin er al., 1980) a strong case for a collagen defect in these chickens can be made. Collagen solubihty is related to the number and qualitv of the intermolecular cross-links (Bailev et al.. i974; i(ang and Trelstad, 1973; Piez, 1968, Smith et a/.; 1975) and the tensile strength of tendon is also influenced by the cross-links of its collagen (Bailey, 1968: Bailev. et al.. 1974). Therefore. it seems reasonable to suggest that the defect in collagen from chickens that develop idiopathic scoliosis results from a failure of the collagen to develop the proper cross-links. Similarly, animals fed beta aminoproprionitrile, which reduces the normal cross-links of collagen by interfering with lysyl oxidase (Bailey, 1968; Bailey et al., 1974) I
develop scoliosis and exhibit increased solubility of their collagen (Miller et al., 1967; Ponseti and Baird,
1952). At least two other disorders, in which scoliosis is a prominent feature, have shown defects of collagen cross-linking and increased collagen solubility: homocystinuria (Kang and Trelstad, 1973) and osteogenesis imperfecta
(Smith et al., 1975).
How collagen disorders produce scoliosis remains a mystery. Like man, the chicken is bipedal and maintains its spine in a more vertical posture than quadripeds. Currey (1965) studied creep and creep recovery in bone in long term experiments. He postulated that bone loaded as a column, especially the spine, could be deformed by anelastic* strain although he felt the strains in the spine were generally insufficient. The ligaments and muscle-tendon units are responsible for maintaining the spine against gravitational forces. Possibly minor changes in the viscoelastic properties of the spine and its supporting structures could allow a
eventually becomes fixed bY alterations in sDinal growth. Idiopathic sEolio& in chickens has a number of features in common with idiopathic scoliosis in humans. Both humans and chickens with scoliosis exhibit increased collagen solubility (Francis et al., 1976; Riggins er a/., 1977)and have increased plasma or curve
to develop
which
urinary hydroxyproline (Zorab, 1968; Zorab et al., 1971; Lin er al., 1980). Also both humans and chickens have a more severe expression of the disease in the homogametic sex (McCarrey er al.. 1981; Wynn-Davis, 1968), i.e. male chicken and female human. Chickens with idiopathic scoliosis exhibit differences in the rate of stress relaxation of their tendons and bone when compared to weight and age matched controls. Possibly these mechanical changes could be related to the development of scoliosis.
REFERENCES
Bailey, A. J. (1968) Intermediate labile intermolecular crosslinks in collagen fibers. Eiochi. biophys. Acra 160,447453. Bailey, A. J., Robin, S. P. and Balian, G. (1974) The biological significance of the intermolecular cross-links of collagen. h&we, Lond. 251, 105-109.
Balaba, T. Y. (1972)Some biochemicalaspects of scoliosisand their pathogenic significance. Reconsrrucrice Surg. Trauma 13, 19-209.
Burstein, A. H. and Frankel, V. H. (1971) A stan&rd test for laboratorv animal bone. J. Biomechanics 4. 155-158. Currey, J. b. (1965) Anelasticity in bone and echinoderm skeletons. J. exp. Biol. 43, 279-292. Cutler, A. D. (1980) Investigation of mechanical properties in connective tissue of scoliotic chickens. Masters Thesis, School of Engineering, University of California, Davis.
Cutler, A. D., Riggins, R. S., Lin, H. J., Benson, D. R., Ramey, M. R., Herrmann, L. R., Rucker, R. B. and Abbott. U. K. (1981) Stress relaxation in tendons of chickens with scoliosis. J. Biomechanics 14, 439-441. Francis. M. J. 0.. Sanderson. M. C. and Smith. R. (1976) Skin collagen in idiopathic adolescent scoliosis and Marfan’s
syndrome. C/in. Sci.
clinical significance. Acta
med. &and.
179, 275-254.
Lakes, R. S. and Katz, J. L. (1979) Viscoelastic properties of wet cortical bone-l torsional and biaxial studies. J. Biomechanics
12, 657-678.
Lin. H. J., Benson, D. R., Riggins, R. S., Rucker, R. B. and Abbott, U. K. (1980) Plasma free hydroxyproline. growth and sexual maturation of the hereditary scoliotic chicken. Proc. Sot. Exp. Biol. Med. 165, 345-348.
Lugassy. A. A. and Korostaff, E. (1969)Viscoelasticbehavior of bovine femoral cortical bone and sperm whale dentin. Medical Material (edited KorostafF E.) pp. l-12. McCarrey, J. R., Abbott, U. K., Benson. D. R. and Riggins. R. S. (1981) The genetics of scoliosis in chickens. J. Hered. Reseorrh inDentaland
72, 6-10.
Miller, E. K., Martin, G. R., Piez, K. A. and Powers. M. J. (1967) Characterization of chick bone collagen and compositional changes associated with maturation. J. biol. Chem. 242, 5481-5489. Nordwall, A. (1973) Studies in idiopathic scoliosis. Acro orthop. stand. Supp. 150, I-178. Pedrini. V. A., Ponseti, I. V. and Dohrman, S. C. (1973) Gluc&ominoglycans of intervertebral discs in idiopathic scoliosis. J. Lab. clin. Med. 82, 938-950. Piez, K. A. (1968) Cross-linking of collagen and elastin. A. Rev. Biochem.
*Anelastic strain is recoverable creep, a viscoelastic property of bone.
molec. Med. 51, 467474.
Kang, A. H. and Trelstad, R. L. (1973) A collagen defect in homocystinuria. J. c/in. Invest. 52, 2571-2578. Laitinen, 0.. Nikkila, E. A. and Kivirikko, K. 1. (1966) Hydroxyproline in serum and urine: normal value and
37, 547-570.
Ponseti, 1. V. and Baird, W. A. (1952) Scoliosis and dissecting aneurysm of the aorta in rats fed lathyrus odoratus seeds. Am. J. Path. 20, 1059-1077.
Mechanical properties Riggins, R. S., Zeman, F. and Moon, D. (1974) The effects of sodium fluoride on bone breaking strength. C&if. Tissue Res. 14. 283-289. Riggins, R. S., Abbott, U. K., Ashmore, C. R., Rucker, R. B. and McCarrey, .I. R. (1977) Scoliosis in chickens. J. Bone Jr Surg. 59A, 1020-1026. Rucker, R. B., Riggins, R. S., Laughlin, R., Chan, M. M., Chen, M. and Tom, K. (1975) Effects of nutritional copper deficiency on the biomechanical properties of bone and arterial elastin metabolism in the chick. J. Nurr. 105, 1062-1070. Saville, P. D. (1967) Water fluoridation effect on bone fragility and skeletal calcium content in the rat. J. Nurr. 91, 353-357. Smith, R., Francis, M. J. 0. and Bauze, R. J. (1975) Osteogenesis imperfecta. Q. J. Med. 176, 555-573. Viidik. A. (1973) Functional properties of collagenous tissues,
of bone
67
Int. come&e Tissue Res. 6, 127-216. Wainwright, S. A., Biggs, W. D.. Currey. J. D. and Gosline. J. M. (1976) Mechanical Design in Organisms. Edward Arnold. London. Walter, R. L. and Morris, J. M. (1973) In oitro study of normal and scoliotic interspmous ligaments. J. Biomechanics 6, 343-348. Wynn-Davis, R. (1968) Familial (idiopathic) scoliosis. A family survey. J. Bone Jr Surg. SOB. 2430. Zorab, P. A. (1968) Total hydroxyproline excretion m __ scoliosis. In: Proc. Second Symp. on Scoliosis: Causarton PP. 55-57. Action for the Crinpled Children Monoeraoh. . . London, Churchill Livingston: Zorab, P. A., Clark, S., Cotrel, Y. and Harrison, A. (1971) Bone collagen turnover in idiopathic scoliosis. Estimate from total hydroxyproline excretion. Archs Dis. Childn. 46, 828-832.
$03.00 0
1983 Pergamon Press Ltd
MECHANICAL PROPERTIES OF THE TIBIA FROM CHICKENS WITH IDIOPATHIC SCOLIOSIS R. S. RIGGINS, D. A. LEWIS and D. R. BENSON Department
of Orthopaedic
Surgery,
Professional
Building.
4301 X Street, Sacramento.
CA 95817
J. R. MCCARREY Department
of Avian Sciences, University
of California,
Davis, CA 95616
and C. E. FRANYI Department
of Community
Health,
University
of California.
Davis, CA 95616, U.S.A
Abstract-The tibia from six-week old chickens that develop idiopathic scoliosis were studied with stress relaxation experiments and torsional strength testing. Most parameters observed did not show any significant differences between tibias obtained from chickens with scoliosis and tibias from the control birds; however, the rate of stress relaxation of the tibia from the birds with scoliosis was minimally increased over the controls. There were no significant differences noted in ultimate torsional strength, maximum angular deformity or modulae of torsional rigidity of the tibias from scoliotic chickens when compared to tibias from control chickens.
INTRODUCTION
Six male birds from an inbred line of chickens that develop a severe idiopathic scoliosis in over 73 ‘I0of the males were selected for the study. The birds were six weeks old. For controls, six male white leghorn chickens from the University of California stock that were also six weeks old and six male white leghorn chickens matched by weight to the scoliotic birds were
selected. The mean values and standard errors in grams for the animals’ weights were 307 k 11 for weight controls, 388 + 12 for the age controls and 307 + 16 for the scoliotic birds. The ages of the scoliotic birds and age controls were six weeks, whereas the weight controls were only four to five weeks old. The animals were sacrificed with chloroform and their tibias removed and frozen in isotonic phosphate buffer pH 7.4. At the time of testing the bones were thawed and the epiphyses and periosteum removed leaving a hollow tube of bone that was somewhat flattened at each end and larger proximally than distaliy. The testing device was a modification of the torsional bone breaking device designed by Burstein and Frankel (1971) and Riggins et at. (1974). (See Fig. 1.) The device is designed to break the bone rapidly in torsion using a heavy pendulum. The device normally develops a torque of 6.3 x lo3 Nm at the lower portion of its fall and transverses a 30” arch in 0.075 s. The torque is measured with a torsional load cell* and the angular displacement by an appropriate sensor+. The data is displayed on an oscilloscope$ and photographed with a Polaroid camera. The modifications required for the stress relaxation studies were to replace the pendulum with a lever that allowed the manual application of the torque and a clamp that fixed the lever to maintain the deformity. By adding a scale and movable block to which the lever
Rtwiwd jh publicurion 26 Auyusr 1982. Please request reprints from Richard Riggins. M.D.. 69 Butler Street, Atlanta, GA 30306, U.S.A. This study has been supported financially by grant credits AM 2538 and AM 15278.
*Lebow Associates, Oak Park, Michigan. tBrush Metripak Model 3303, Brush Instrument Company. Cleveland, Ohio. *Tektronix 3C66 Carrier amplifier, 2B67 Time base, 564 B Storage Oscilloscope, Beaverton, Oregon.
Idiopathic
scoliosis is a lateral curvature of the spine that most commonly affects the adolescent girl. Tissue from humans with scoiiosis is difficult to obtain, but there is an inbred line of chickens developed from white leghorns that have a hereditary idiopathic scoliosis (Riggins et al., 1977). Studies on these animals suggest a collagen defect may be associated with their scoliosis as these chickens exhibit increased bone collagen solubility (Riggins et al., 1977), increased serum hydroxyproline (Lin et al., 1980) and changes in the viscoelastic properties of their tendons (Cutler er al., 1981). This report concerns a study of the mechanical properties of the tibia from chickens with idiopathic scoliosis. The bone from these animals exhibits changes in its viscoelastic properties as manifested by an increased rate of stress relaxation of the tibia. MATERIALS AND METHODS
59
60
R. S. RIGGINS,D. A. LEWIS, D. R. BENSON,J. R. MCCARREYand C. E. FRANTI
could be pulled, a predetermined angular displacement could be applied rapidly (0.5 s) and the position maintained with the clamp. The deformity applied was 3.5 rad/m based on the length of tibia between the grips and ranged from 7 to 11O.The same load cell that was used for testing the bone to fracture was used for the stress relaxation experiments, but a more stable amplifier was required* and the output was plotted on a strip chart recordert. The grips used were of two varieties. For the rapidly applied force to fracture, the ends of the bone were potted in a rapid setting dental plastic+. The pots would precisely fit the grips in the breaking machine. For the stress relaxation studies the dental plastic was not used as it has a slight stress relaxation of its own. Instead, adjustable brass grips were designed to fit the contours of the proximal and distal ends of the tibia. These grips were tightened down on the bone carefully so as not to damage the cortex. The grips were tested using a 0.32-cm 316 stainless steel rod secured in the brass grips and subjected to twice the torque used on the bones. No Lossof torque was observed over a 12-h period. The specimens were continually irrigated throughout the experiment with isotonic phosphate buffer pH 7.4 to prevent drying. The buffer was circulated through a water bath to provide a constant 37°C temperature and the bone was allowed 5 min or more to equilibrate to the temperature before testing. The bones were initially tested for stress relaxation and the bone ends then potted in the dental plastic. The bones were stored in the phosphate buffer at 4°C at least 24 h prior to destructive testing to give the dental plastic adequate time to set and also to allow the bone to recover from the stress relaxation experiment. The data from the stress relaxation experiment were graphed on semilogarithmic paper plotting the fraction of the initial torque remaining against time on the logarithmic scale. The resulting graph was a straight line over the time observed, 6-1200 s. The line was also calculated using the methods of least squares. The following data were obtained from the rapidly applied torque to fracture: ultimate torsional strength, maximal angular displacement per unit length of tibia between the pots, and a modulus of torsional rigidity or shear modulus of elasticity. The geometry of the specimens was also measnred. This included the length of specimen between the pots, the diameter of the bone at mid-shaft, and the cortical thickness. The midshaft was selected because the fracture began at the midshaft and spiraled at about 3545” toward one or the other end. Secondary fractures occasionally occurred but the initial crack began at the midshaft (Fig. 2). We have performed high
*Valedyne CD19-871, P. 0. Box 9025, Northridge, CA 93128. t Linear 17281 Eastman Avenue, Irvine, CA 92714. $Nu Weld Caulk Co., Milforcl, Delaware.
speed photography of fractures of femurs and tibias of chickens with our testing machine to confirm the location of the initial crack. The diameter of the midshaft was measured twice at 90” apart and the two measurements averaged. At first the values were kept separately, but analysis of the two values showed no significant differences in the values so they were pooled. Similarly the cortical thickness was measured with a micrometer. Two measurements were taken and the values averaged. To calculate a moment of inertia for the bone specimen, the formula for a hollow tube was used. This assumption is only partially correct but adequate for comparison among similar size specimens. TO reduce sample variation, the values obtained from the two tibias were pooled to give one value for each parameter for each bird. In the stress relaxation experiments, variation was further reduced by normalizing the curves to the initial torque applied. Actually, we found no significant differences in the analysis of data if the tibias were treated as individual specimens, i.e. twelve specimens from six birds or the birds were treated as specimens and pooling the results from the two bones from each bird thus only giving six specimens. An analysis of variance was used to evaluate the data. For the stress relaxation data, analysis of variance was also used to determine if within each group a single line could represent the data and if the line was straight when plotted on semilogarithmic paper. The mean values and standard errors were also determined for the various parameters studies and representative graphs were reconstructed from these data.
RESULTS
The analysis of variance of the slopes from the stress relaxation experiments replotted on semilogarithmic paper indicated that within each of the three groups a single line could represent the results of that group as there were no significant differences among the individual slopes in any one group. Furthermore, the tests for linearity of the slope which consisted of an analysis of variance of the individual slopes from a common straight line for each group were not significant, (p > 0.50); that is, there were no significant deviations from the straight line. Analysis of variance testing between the three groups: scoliosis, the age control, and weight control indicated no significant differences in the slopes of the controls, but the slope of the scoliotic birds’ data was significantly different (p < 0.01). There were no significant differences among the mean intercepts of any of the three groups. Reconstructed graphs from the mean values can be found in Fig. 3 plotted on normal graph paper, and Fig. 4 plotted on semilogarithmic paper. The initial torque to obtain the deformity of 3.5 rad/m of tibia between the grips was significantly greater for the controls by age than for the controls by weight or the
Fig. 1. Torsional bone testing machine. The pcnduium can be seen supported by a retainer. The lever arm attached to the rotating grip with its set screw is also visible as is the adjustment stop block. For the stress relaxation test, the block is moved to a predetermined degree and the lever pulled rapidly to the block, thus applying torque to the bone held in the grips. For the destructive test to fracture, the lever arm is removed and the grip can rotate freely. The pendulum at the bottom of its arc contacts a dog attached to the grip, thus breaking the bone. Torsional strain gauge and irrigation piping are also seen. Fig. 2. A chick tibia with a typical fracture created by the torsional bone testing machine of similar design to that of Burstein and Frankel (1971).
61
Mechanical
60
properties
I
I
100
200
I
I
300
400
500 TIME,
Fig. 3. Composite
63
of bone
stress
I
1
600
I
800
700
I
900
-
60
-
I
I
1100
1200
seconds
relaxation curves from the mean values obtained experiments plotted on normal graph paper.
80
I
1000
from
the stress
relaxation
FORCE
50
r
0’
I
I
IO
1000
100 TIME,
seconds
Fig. 4. Same data plotted in Fig. 3 replotted on semilogarithmic paper. The difference in the slope between the controls was not significant. The slope from scoliotic birds was significantly different from the controls p < 0.01.
Table
Birds (number) Scoliotic
(61
1. Stress relaxation
Initial torque* (Nm)
Torque
values (mean + standard
at 1200 s* (Nm)
error)
Intercept (fraction of initial torque)
Slope+ Fraction initial torque vs log time
0.126t0.013
0.075 f 0.0082
0.94 f 0.018
0.112 + 0.007
Control by weight (61
0. I25 f 0.0052
0.087 f 0.0036
0.98 * 0.002
0.092 + 0.001
Control by age (6)
0.166 + 0.011
0.113~0.0072
0.93 * 0.008
0.094 * 0.002
*Age control significant greater than scoliotic or wt control p < 0.025. tThe slope for the scohotic birds was significantly greater p -c 0.01 than controls.
R. S.
64
RIGGINS, D.
A.
LEWIS, D. R. BENSON,J. R. MCCARREY and C. E. FRANTI
scoliotic birds. This difference was still present to a significant degree at the end of the experiment, i.e. 1200 s. These data are presented in Table 1.
Values obtained
in the torsional
modulus of elasticity. These data are also plotted in Fig. 5. The lengths of the test specimens, diameter at midshaft, and cortical thickness can be found in Table 3. The length of the tibia1 shafts was significantly greater for the age control birds but the differences in their diameters and cortical thickness were not significant.
testing studies can
be found in Table 2. There were no significant differences in the ultimate torsional strengths, angular displacements per unit length of tibia between the pots, or the calculated modulae of torsional rigidity or shear
Table 2. Torsional testing values (mean f standard error) Ultimate torsional strength (Nm)
Birds (number)
Ultimate angular displacement * @ad/cm)
Calculated modulus+ of torsional rigidity (gNm- *)
Scoliotic (6)
0.494 * 0.033
0.0960 + 0.0078
3.41 f 0.368
Controls by weight (6)
0.401 f 0.029
0.0928 f 0.007
2.66kO.181
Controls by age (6)
0.529 f 0.046
0.0809 + 0.008
2.6OkO.197
*Angular displacement in radians divided by length of tibia between pots. Kalculated from experiment dataassuming tibia1 shaft was a hollow tube (J = 1l/32 (od4 - id4)). There were no significant differences among values reported in this table.
AGE CONTROL
RADIANS
* cm-
Fig. 5. The composite force deformation curves constructed from mean values obtained deformation to fracture. No significant differences in the curves were noted.
Table 3. Geometry of the tibia (mean &standard error)
Birds (number)
Length of specimen (cm) between pots (grips)
Midshaft diameter (cm)
Thickness of cortex (cm)
Scoliotic (6)
4.02; *0.17
0.386 + 0.009
0.0626 f 0.0039
Control by weight (6)
4.30+0.11
0.398 f 0.006
0.0660 f 0.0022
Control by age (6)
4.59’ * 0.09
0.438 f 0.003
0.0615 f 0.0032
*Significantly different p < 0.025.
by rapid
Mechanical
properties of
DISCUSSION
The purpose of this investigation was to determine if differences in the mechanical properties of the bone from chickens with idiopathic scoliosis existed when compared to bone taken from normal control chickens of similar age and weight. The scoliotic chickens have been shown to develop both sexually and physically slower than white leghorn chickens from which they were originally derived (Lin e? al., 1980). Because of this delayed maturation, two sets of control animals were needed, one set of controls matched by weight as animal body weight affects mechanical strength of bone (Saville, 1967; Rucker et ai., 1975) and one set of controls matched by age as age affects the physical properties of collagen (Viidik, 1973). The larger size of the control chickens. the same age as the scoliotic birds, no doubt accounted for the increased length of their tibia1 shaft and the increased torque required to produce the deformation for the stress relaxation experiments. Ideally we would like to select the spine to study, but our facilities do not have the capacity to mechanically test such small specimens, so the long bone of the tibia was selected. Our previous investigations (Riggins et al., 1977; Cutler et al., 1981) suggest the chicken with idiopathic scoliosis has a generalized collagen defect so if the collagen abnormality affects the mechanical properties of bone, it should affect the tibia as well as the spine. We elected to study the mechanical properties in torsion since specimen orientation in the torsional testing device is less critical than in some other modes of testing. Bone is anisotrophic and the chick tibia1 shaft does not precisely conform to any standard geometrical shape, so specimen orientation in some testing devices could be a problem. We found the shear modulus of elasticity or torsional rigidity for bone to be considerably lower than values reported by several investigators (Wainwright et al., 1976). The difference was probably related to the deviation of the geometry of the tibia1 shaft from our assumption that the shaft was a hollow tube. Other possibilities for the low modulus exist as previous investigators used human or bovine bone and tested in bending, compression or tension. To study the viscoelastic properties of bone, the stress relaxation test was selected as it is a fairly standardized test and has been used previously in bone by other investigators (Lakes and Katz, 1979; Lugassy and Korostaff, 1969). Stress relaxation data are easily determined and fairly immune to experimental error, an important consideration when working with biological material where small differences may be hidden by the individual variation. To further reduce individual variation, the stress relaxation data was normalized to the initial deforming force or torque. The time frame selected was 6-1200 s to cover three decades of time. Observations under 6 s were not attempted, as our equipment did not apply the initial force rapidly
bone
65
enough to prevent some degree of overlap between force application and stress relaxation from occurring in the initial part of the curve. To examine viscoelastic properties in a very short time, dynamic tests are more appropriate. The duration of observation was arbitrarily selected to be 1200 s. By this time, the loss of torque per unit time was becoming very small but still continuing and would probably continue for some time further. Lakes and Katz (1979) found stress relaxation continuing in their bone specimens tested in torsion at IO5 s and Lugassy and Korostaff (1969) estimated from their data that the fully relaxed state of bone in compression would require over 69 days. Currey (1965) in long term creep experiments did not find an end point after 55 days of observation. From our results, 1200 s does not seem to be an adequate time as the amount of stress relaxation that occurred in the threegroups was not statistically different when the data were expressed as a fraction of the initial torque. Because of the increased size of the age controls, the initial torque required to produce the standard angular displacement for the stress relaxation experiments was greater in the age controlled animals as was the torque remaining at 1200 s; however, the percentage change was not different from the other two groups. Since the slope of the curve for the scoliotic birds was statistically greater than the two controls, one would anticipate eventually the amount of stress relaxation would become greater for the scoliotic birds if the tests were carried out long enough. Of course, for very long time periods, if bone does indeed reach a fully relaxed state, the values for the three groups could approach each other once again. Apparently, observations over several months will be required to obtain these data about the fully relaxed state of bone if such a state exists. Several investigators have found differences in the behavior of the metabolism of connective tissues in patients with idiopathic scoliosis. Francis et al. (1976) reported increased collagen solubility in the skin of humans with idiopathic scoliosis, and hydroxyproline excretion in children with idiopathic scoliosis is elevated during certain periods of their development (Laitinen et al., 1966; Zorab, 1968; Zorab et al.. 1971). Ground substance might also be faulty, as Pedrini er al. (1972) reported abnormalities in the glycosominoglycans of the intervertebral disc from children with scoliosis. Balaba (1972) also found disturbances in the glycosominoglycan metabolism in the spine and its ligaments in children with scoliosis. In these patients, hydroxyproline excretion was elevated above normal controls. Stress relaxation experiments have been carried out by other investigators on the tendons and spinal ligaments of humans with scoliosis (Walter and Morris, 1973; Nordwall, 1973). These investigators did find slight increases in stress relaxation from children with idiopathic scoliosis, but the differences were not felt to be significant. We had similar difficulties in demonstrating that the differences betwen our control chickens and the chickens with scoliosis were
R. S. RIGGINS. D. A. LEWIS,D. R. BENSON,J. R. MCCARREYand C. E. FRANTI
66
significant. Only when the data were normalized against the initial deforming torque and plotted against time on the logarithmic scale could significant differences be found. These were differences in the rate of stress relaxation. The observations were not carried out over a sufficient period to determine if differences would exist in the fully relaxed state. In the chickens with idiopathic scoliosis changes in the mechanical properties of their tendons can be demonstrated by an increased rate of stress relaxation in their digital flexor and extensor tendons (Cutler et al., 1981) as well as a slight increased rate of stress relaxation in the bone of their tibias as reported here. Furthermore, with increased bone and cartilage collagen solubility (Riggins et al., 1977) and elevated serum hydroxyproline (Lin er al., 1980) a strong case for a collagen defect in these chickens can be made. Collagen solubihty is related to the number and qualitv of the intermolecular cross-links (Bailev et al.. i974; i(ang and Trelstad, 1973; Piez, 1968, Smith et a/.; 1975) and the tensile strength of tendon is also influenced by the cross-links of its collagen (Bailey, 1968: Bailev. et al.. 1974). Therefore. it seems reasonable to suggest that the defect in collagen from chickens that develop idiopathic scoliosis results from a failure of the collagen to develop the proper cross-links. Similarly, animals fed beta aminoproprionitrile, which reduces the normal cross-links of collagen by interfering with lysyl oxidase (Bailey, 1968; Bailey et al., 1974) I
develop scoliosis and exhibit increased solubility of their collagen (Miller et al., 1967; Ponseti and Baird,
1952). At least two other disorders, in which scoliosis is a prominent feature, have shown defects of collagen cross-linking and increased collagen solubility: homocystinuria (Kang and Trelstad, 1973) and osteogenesis imperfecta
(Smith et al., 1975).
How collagen disorders produce scoliosis remains a mystery. Like man, the chicken is bipedal and maintains its spine in a more vertical posture than quadripeds. Currey (1965) studied creep and creep recovery in bone in long term experiments. He postulated that bone loaded as a column, especially the spine, could be deformed by anelastic* strain although he felt the strains in the spine were generally insufficient. The ligaments and muscle-tendon units are responsible for maintaining the spine against gravitational forces. Possibly minor changes in the viscoelastic properties of the spine and its supporting structures could allow a
eventually becomes fixed bY alterations in sDinal growth. Idiopathic sEolio& in chickens has a number of features in common with idiopathic scoliosis in humans. Both humans and chickens with scoliosis exhibit increased collagen solubility (Francis et al., 1976; Riggins er a/., 1977)and have increased plasma or curve
to develop
which
urinary hydroxyproline (Zorab, 1968; Zorab et al., 1971; Lin er al., 1980). Also both humans and chickens have a more severe expression of the disease in the homogametic sex (McCarrey er al.. 1981; Wynn-Davis, 1968), i.e. male chicken and female human. Chickens with idiopathic scoliosis exhibit differences in the rate of stress relaxation of their tendons and bone when compared to weight and age matched controls. Possibly these mechanical changes could be related to the development of scoliosis.
REFERENCES
Bailey, A. J. (1968) Intermediate labile intermolecular crosslinks in collagen fibers. Eiochi. biophys. Acra 160,447453. Bailey, A. J., Robin, S. P. and Balian, G. (1974) The biological significance of the intermolecular cross-links of collagen. h&we, Lond. 251, 105-109.
Balaba, T. Y. (1972)Some biochemicalaspects of scoliosisand their pathogenic significance. Reconsrrucrice Surg. Trauma 13, 19-209.
Burstein, A. H. and Frankel, V. H. (1971) A stan&rd test for laboratorv animal bone. J. Biomechanics 4. 155-158. Currey, J. b. (1965) Anelasticity in bone and echinoderm skeletons. J. exp. Biol. 43, 279-292. Cutler, A. D. (1980) Investigation of mechanical properties in connective tissue of scoliotic chickens. Masters Thesis, School of Engineering, University of California, Davis.
Cutler, A. D., Riggins, R. S., Lin, H. J., Benson, D. R., Ramey, M. R., Herrmann, L. R., Rucker, R. B. and Abbott. U. K. (1981) Stress relaxation in tendons of chickens with scoliosis. J. Biomechanics 14, 439-441. Francis. M. J. 0.. Sanderson. M. C. and Smith. R. (1976) Skin collagen in idiopathic adolescent scoliosis and Marfan’s
syndrome. C/in. Sci.
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med. &and.
179, 275-254.
Lakes, R. S. and Katz, J. L. (1979) Viscoelastic properties of wet cortical bone-l torsional and biaxial studies. J. Biomechanics
12, 657-678.
Lin. H. J., Benson, D. R., Riggins, R. S., Rucker, R. B. and Abbott, U. K. (1980) Plasma free hydroxyproline. growth and sexual maturation of the hereditary scoliotic chicken. Proc. Sot. Exp. Biol. Med. 165, 345-348.
Lugassy. A. A. and Korostaff, E. (1969)Viscoelasticbehavior of bovine femoral cortical bone and sperm whale dentin. Medical Material (edited KorostafF E.) pp. l-12. McCarrey, J. R., Abbott, U. K., Benson. D. R. and Riggins. R. S. (1981) The genetics of scoliosis in chickens. J. Hered. Reseorrh inDentaland
72, 6-10.
Miller, E. K., Martin, G. R., Piez, K. A. and Powers. M. J. (1967) Characterization of chick bone collagen and compositional changes associated with maturation. J. biol. Chem. 242, 5481-5489. Nordwall, A. (1973) Studies in idiopathic scoliosis. Acro orthop. stand. Supp. 150, I-178. Pedrini. V. A., Ponseti, I. V. and Dohrman, S. C. (1973) Gluc&ominoglycans of intervertebral discs in idiopathic scoliosis. J. Lab. clin. Med. 82, 938-950. Piez, K. A. (1968) Cross-linking of collagen and elastin. A. Rev. Biochem.
*Anelastic strain is recoverable creep, a viscoelastic property of bone.
molec. Med. 51, 467474.
Kang, A. H. and Trelstad, R. L. (1973) A collagen defect in homocystinuria. J. c/in. Invest. 52, 2571-2578. Laitinen, 0.. Nikkila, E. A. and Kivirikko, K. 1. (1966) Hydroxyproline in serum and urine: normal value and
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Ponseti, 1. V. and Baird, W. A. (1952) Scoliosis and dissecting aneurysm of the aorta in rats fed lathyrus odoratus seeds. Am. J. Path. 20, 1059-1077.
Mechanical properties Riggins, R. S., Zeman, F. and Moon, D. (1974) The effects of sodium fluoride on bone breaking strength. C&if. Tissue Res. 14. 283-289. Riggins, R. S., Abbott, U. K., Ashmore, C. R., Rucker, R. B. and McCarrey, .I. R. (1977) Scoliosis in chickens. J. Bone Jr Surg. 59A, 1020-1026. Rucker, R. B., Riggins, R. S., Laughlin, R., Chan, M. M., Chen, M. and Tom, K. (1975) Effects of nutritional copper deficiency on the biomechanical properties of bone and arterial elastin metabolism in the chick. J. Nurr. 105, 1062-1070. Saville, P. D. (1967) Water fluoridation effect on bone fragility and skeletal calcium content in the rat. J. Nurr. 91, 353-357. Smith, R., Francis, M. J. 0. and Bauze, R. J. (1975) Osteogenesis imperfecta. Q. J. Med. 176, 555-573. Viidik. A. (1973) Functional properties of collagenous tissues,
of bone
67
Int. come&e Tissue Res. 6, 127-216. Wainwright, S. A., Biggs, W. D.. Currey. J. D. and Gosline. J. M. (1976) Mechanical Design in Organisms. Edward Arnold. London. Walter, R. L. and Morris, J. M. (1973) In oitro study of normal and scoliotic interspmous ligaments. J. Biomechanics 6, 343-348. Wynn-Davis, R. (1968) Familial (idiopathic) scoliosis. A family survey. J. Bone Jr Surg. SOB. 2430. Zorab, P. A. (1968) Total hydroxyproline excretion m __ scoliosis. In: Proc. Second Symp. on Scoliosis: Causarton PP. 55-57. Action for the Crinpled Children Monoeraoh. . . London, Churchill Livingston: Zorab, P. A., Clark, S., Cotrel, Y. and Harrison, A. (1971) Bone collagen turnover in idiopathic scoliosis. Estimate from total hydroxyproline excretion. Archs Dis. Childn. 46, 828-832.