Biomacromolecules 2000, 1, 180-185
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The Role of Mineral in the Storage of Elastic Energy in Turkey Tendons Frederick H. Silver,*,† David Christiansen,† Patrick B. Snowhill,† Yi Chen,† and William J. Landis‡ Department of Pathology and Laboratory Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854; and Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272 Received December 15, 1999; Revised Manuscript Received March 22, 2000
Mammals elastically store energy in leg and foot tendons during locomotion. In the turkey, much of the force generated by the gastrocnemius muscle is stored as elastic energy during tendon deformation and not within the muscle. During growth, avian tendons mineralize in the portions distal to the muscle and show increased tensile strength and modulus as a result. The purpose of this study was to evaluate the viscoelastic behavior of turkey tendons and self-assembled collagen fiber models to determine the molecular basis for tendon deformation. The stress-strain behavior of tendons and self-assembled collagen fibers was broken into elastic and viscous components. The elastic component was found to be to a first approximation independent of source of the collagen and to depend only on the extent of cross-linking. In the absence of cross-links the elastic component of the stress was found to be negligible for self-assembled type I collagen fibers. In the presence of cross-links the behavior approached that found for mineralized turkey tendons. The elastic constant for turkey tendon was shown to be between 5 and 7.75 GPa while it was about 6.43 GPa for self-assembled collagen fibers aged for 6 months at 22 °C. The viscous component for mineralized turkey tendons was about the same as that of self-assembled collagen fibers aged for 6 months, a result suggesting that addition of mineral does not alter the viscous properties of tendon. It is concluded that elastic energy storage in tendons involves direct stretching of the collagen triple-helix, nonhelical ends, and cross-links between the molecules and is unaffected by mineralization. Furthermore, it is hypothesized that mineralization of turkey tendons is an efficient means of preserving elastic energy storage while providing for increased load-bearing ability required for locomotion of adult birds. Introduction Collagen fibers, proteoglycans, and mineral and elastic fibers are the major load-bearing elements in extracellular matrix. Collagen fibers function in biological structures to maintain tissue shape, transmit and absorb loads, prevent premature mechanical failure, partition cells and tissues into functional units, and act as a scaffold that supports tissue architecture.1 The primary structural element in mammalian extracellular matrix is fibrillar type I collagen. Type I collagen molecules are rods with limited flexibility2 that self-assemble into quarter staggered arrays of fibrils 20 to several hundred nanometers in diameter. Bundles of fibrils in turn pack in collagen fibers, fascicles and higher ordered tissue structures.3 The structural hierarchy and mechanical properties of type I collagen fibers depend on species and anatomic location; however, in general the ultimate tensile strength of these tissues has been reported to be directly related to the fibril4 and fiber5 diameters. These observations suggest that events involved in fibril diameter growth and stabilization such as * To whom correspondence should be addressed. Telephone: 732-2354027. Fax: 732-235-4825. E-mail:
[email protected]. † UMDNJ-Robert Wood Johnson Medical School. ‡ Northeastern Ohio Universities College of Medicine.
cross-linking may play a significant role in the development of the high strengths required to support collagenous tissue function. During mechanical deformation of tendon, collagen molecules, fibrils, and fibers initially undergo geometrical alignment, molecular stretching, molecular and fibrillar slippage, and failure by defibrillation.6-10 The exact molecular mechanism by which deformation occurs is unclear; however, up to a macroscopic deformation of about 2%, molecular stretching predominates.10 The exact magnitude of the strain at which molecular deformation of the collagen triple helix becomes small compared to fibrillar slippage depends on the strain rate and tissue studied; however, it is clear that the mechanical deformation of tendon is composed of elastic and viscous components.11 The elastic component is the portion of the force or stress that remains after the sample relaxes to its equilibrium length while the viscous component is the difference between the total force and the elastic component. Elastic energy storage in tendons in the legs and feet of many animals is an important mechanism that saves substantial quantities of muscular energy during locomotion.12,13 In animals during normal gait, the body is decelerated as the foot lands on the ground, causing kinetic energy to be
10.1021/bm9900139 CCC: $19.00 © 2000 American Chemical Society Published on Web 05/05/2000
Storage of Elastic Energy in Turkey Tendons
stored as strain energy in the muscles and tendons that are stretched by the impact with the ground. Elastic recoil, primarily by the tendons, converts most of the stored energy back to kinetic energy as the foot leaves the ground.12,13 In the pig, the digital flexor tendons are involved in the elastic storage of strain energy and at maturity they have twice the tensile strength and elastic modulus but only about half of the strain energy dissipation of the corresponding extensor tendons.14 This difference in the elastic strain energy is reported to be attributable to a decreased strain at failure of the flexor tendons (about 6.7%) with respect to the extensor tendons (8.2%). Immature flexor and extensor tendons begin to fail at strains of about 17%. Shadwick14 pointed out that the density and type of cross-links within collagen fibers change with age as well as the fibril morphology. The increased tensile strength and decreased strain at failure of the flexor tendon appear to be a consequence of increased cross-linking within the collagen fibril. In the turkey, direct measurement of force and fiber length in the lateral gastrocnemius muscle reveals that the active muscle produces high force but little work while the tendon produces much of the work because of elastic deformation and recovery.15 Unlike the pig flexor tendon the turkey tendon mineralizes during aging. Results of previous studies on mineralizing turkey tendons conducted in our laboratories indicate that the mechanical properties of the gastrocnemius tendon change during mineralization.16 The elastic modulus increases between weeks 12 and 17 post-hatching of the animals, and this is the time period during which mineralization begins.16 While observations concerning the relationship between changes in modulus and the onset and progression of mineralization have been made, no report has been made concerning the changes in viscoelastic properties of the turkey tendon that are associated with mineralization. The purpose of this study is to evaluate the effect of mineralization on the viscoelastic behavior of turkey tendon. Materials and Methods The viscoelastic behavior of two different collagenous substrates were examined in this study. These included gastrocnemius tendon from domestic turkeys and self-assembled collagen fibers made from soluble collagen. Fresh gastrocnemius tendons were obtained from both male and female slate strains of the domestic turkey, Meleagris gallopaVo, as previously described.16 Birds were sacrificed at intervals between 12 and 16 weeks after hatching. Both left and right gastrocnemius tendons from at least two animals of each age were studied. All such tendons were calcified to varying extents which was verified by X-ray microradiography.16 The upper portion of the tendon was isolated above its natural bifurcation and dissected into two portions as previously detailed, yielding the sections used for mechanical testing.16 The samples were kept frozen at -20 °C in 0.1 M phosphate buffered saline (PBS) pH 7.4 until the day before testing. Prior to testing, the tendons were thawed overnight in fresh PBS at 4 °C. Dimensions of each section were measured (both thickness and width) with a micrometer, and cross-sectional areas were determined by assuming an ellipsoidal geometry. Tendons were mounted in the upper and lower pneumatic grips of an Instron Tester model 1122 (Instron Corp., Canton, MA) at a pressure of 60 psi. All samples were immersed in PBS during testing.
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Figure 1. Incremental stress-strain curve. Diagram illustrating incremental stress-vs-strain curve that is obtained by stretching a specimen by a fixed strain increment and then allowing the stress to relax until it equilibrates. At equilibrium an additional strain increment is added and the cycle is repeated until the specimen fails. The elastic fraction Ef is defined as the ratio of Fe divided by Fi.
Self-assembled collagen fibers were obtained by extrusion of soluble rat tail tendon collagen as discussed previously.17 Solutions of soluble collagen were obtained by acid extraction of rat tail tendon fibers in 0.01 M HCl at 4 °C overnight. The resulting collagen was centrifuged, salt precipitated, redisolved in 0.01 M HCl, filtered through 0.65 and 0.45 µm filters, dialyzed against phosphate buffer, and redissolved in 0.01 M HCl as previously described.17 Collagen fibers were formed by extrusion of collagen solutions in 0.01 M HCl through a dual barrel syringe system containing the collagen solution in one barrel and fiber formation buffer (FFB) in the other barrel.17 The two solutions were extruded through 18 gauge poly(ethylene) tubing into a container filled with FFB at 37 °C. FFB was removed after 24 h of incubation and replaced with a fiber incubation buffer (FIB).17 The fibers were allowed to incubate for an additional 24 h. They were then washed with distilled water for 1 h and air-dried under a slight tension for an additional 24 h. Some of the dried fibers were then processed for immediate mechanical testing while others were allowed to age at room temperature for periods of up to 6 months. Dried self-assembled collagen fibers were mounted on vellum “windows” with a 2 cm gauge length using 5 min epoxy as previously described.17 The fibers were rehydrated in PBS at room temperature for a minimum of 30 min prior to mechanical testing. Fiber diameters were measured from mounted fibers observed through a calibrated eyepiece of a Leitz Pol microscope (Leitz, Rockville, NJ). The diameter was measured and averaged from three locations along the fiber. It was assumed that the fiber cross-section was circular. The volume fraction of collagen was determined on selfassembled collagen fibers by measuring the wet and dry diameters and then calculating the area fraction as the ratio of dry surface area to wet surface area. It was assumed that the fiber length remained unchanged during dehydration. The volume fraction of turkey tendons was calculated from measured fibril diameters previously reported.18 Viscoelastic testing was conducted on wet samples using a hydration at room temperature. Collagen fibers in a hydration chamber and whole turkey tendons were clamped into the Instron grips. Prior to testing, the vellum “window” was cut on selfassembled collagen fibers. The specimen were subjected to either 2% (turkey tendon) or 5% (self-assembled collagen fibers) strain increments at a strain rate of 10% per minute. This procedure resulted in an incremental stress-vs-strain curve (Figure 1). After each strain increment the stress was allowed to decay to equilibrium before an additional strain increment was added. The elastic component of the stress was defined as the stress at equilibrium
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Figure 2. Stress-Strain curves for 12 week old turkey tendon. Total (open squares), elastic (filled diamonds), and viscous (filled squares) stress-strain curves for turkey tendons obtained from incremental stress-strain tensile testing. Typical standard deviations are shown for total and viscous stress measurements. Standard deviations for the elastic stress components are similar to those shown for the total stress.
while the viscous component was calculated from the difference between the total stress and the elastic component. Elastic fraction was defined as the elastic stress divided by the total stress. Lines representing the viscous stress-strain curves obtained from the incremental stress-strain curves were converted into fibril lengths based on estimation of axial ratios and shape factors1 in the following manner. Viscous stress-strain equations were approximated by straight lines as discuused above and the equations were divided by the strain rate (0.1/minute) to give an equation that represented the extensional viscosity vs strain in MPa s. The shear viscosity as a function of strain was then approximated from the extensional viscosity by dividing by a value of 3.0 which is equivalent to the relationship between shear modulus and tensile modulus for isotropic materials with Poisson’s ratio equal to 0.5. Shear viscosity as a function of strain was converted into shape factor, V, by dividing by the solvent viscosity (8.23 × 10-4 MPa s) for water and by dividing by the volume fraction of polymer.1 The volume fraction of polymer was estimated by calculating the ratio of the square of the dry fibril diameters to the square of the wet fibril diameters. It was assumed that the length change was negligible during hydration. The axial ratio, Z, was estimated from eq 1 where k is 0.1395 for collagen. It should be noted that eq 1 is
Z ) (V/k)0.552
(1)
typically used to calculate the axial ratios from the viscosity of polymer solutions where the polymer is rod shaped or a prolate ellipsoid (see ref 1). In this case, eq 1 is used here to estimate the viscosity that is associated with the viscous sliding of fibrils in a swollen tissue. The value of Z can be used to interpret the mechanism of strain-dependent viscosity measurements made on tendons.
Results Typical stress-vs-strain curves were measured for turkey tendons and self-assembled collagen fibers from the incremental stress-strain curve (Figure 1). When the total and elastic stress components were plotted vs strain, the plots were found to be approximated by straight lines (Figure 2). Similar plots were found for self-assembled collagen fibers (Figure 3). The slopes and intercepts of the total and elastic
Figure 3. Stress-strain curves for un-cross-linked collagen fibers. Total (open squares), elastic (filled diamonds), and viscous (filled squares) stress-strain curves for self-assembled un-cross-linked collagen fibers obtained from incremental stress-strain measurements. The fibers were tested immediately after manufacture and were not aged at room temperature. Note lower stress levels compared to turkey tendons. Typical standard deviations are shown for total and viscous stress components. Standard deviations for the elastic stress components are similar to those shown for the total stress.
Figure 4. Elastic fraction, Ef, for turkey tendons. Strain dependence of elastic fraction for 12 week old turkey tendons. Note that values of Ef increase with strain up to a value of about 0.78.
stress-strain curves were determined from linear fits of Figures 2 and 3 and are tabulated in Table 1 along with the correlation coefficients. For samples with failure strains of about 0.15 only two strain increments (0.05 and 0.1) were available for incremental measurements and lines were constructed with only two data points. Therefore, some of the lines used to calculate the slope contained only two points and the correlation coefficient is 1.00 as is listed in Table 1. All turkey tendons tested were found to exhibit stresses between 0 and 80 MPa for strains between 0 and 0.12 (Figure 2) compared to stresses up to about 30 MPa for selfassembled collagen fibers. When the viscous component was plotted vs strain, the curve was found to be nonlinear (Figures 2 and 3); straight lines were used to approximate the slopes. The elastic fraction for turkey tendon was also found to be nonlinear and increased with strain up to a value of about 0.78 (Figure 4). Elastic fraction vs strain is shown for selfassembled collagen fibers in Figure 5. Slopes of the total stress-strain curve listed in Table 1 range from 1.75 MPa for self-assembled collagen fibers to about 485 MPa for turkey tendons from 14 week old turkeys
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Storage of Elastic Energy in Turkey Tendons
Table 1. Slopes and Intercepts of Straight Line Approximations for Total and Elastic Stress-Strain Curves Obtained at a Strain Rate of 10%/min total stress slope, MPa (intercept)
sample turkey tendon 12 week female 14 week male 14 week female 16 week male & female collagen 0 months 3 months 6 months b
CCa
elastic stress slope, MPa (intercept)
CCa
Nb
443 (16.23) 485 (4.85) 360 (12.94) 290 (11.64)
0.975 1.00 0.97 0.039
387 (6.96) 333 (0.1227) 287 (6.37) 249 (4.97)
0.985 1.00 0.865 0.971
7 7 7 6
1.75 (0.0242) 186.7 (-4.85) 394 (-11.51)
0.993 1.00 1.00
0.716 (0.41) 143 (-3.0) 321.7 (-8.77)
0.989 1.00 1.00
9 9 10
a Denotes correlation coefficient. Note: correlation coefficients of 1.0 typically mean that only two strain points were available for slope determination. N ) number of samples tested.
Table 3. Estimation of Axial Ratios and Fibril Lengths from Viscous Stress-Strain Curves
sample turkey tendon 12 week female 14 week male & female 16 week male & female collagen 0 months 3 months 6 months
Figure 5. Elastic fraction, Ef, for self-assembled collagen fibers. Strain dependence of elastic fraction for un-cross-linked (open squares) and cross-linked (filled squares), aged for 6 months at 22 °C and 1 atm pressure, self-assembled collagen fibers. Table 2. Slopes and Intercepts of Straight Line Approximations for Viscous Stress-Strain Curves Obtained at a Strain Rate of 10%/min sample turkey tendon 12 weeks 14 weeks 16 weeks collagen 0 months 3 months 6 months
straight line slope, MPa (intercept)
CCa
Nb
54.7 (9.54) 84.3 (6.36) 41.0 (6.6)
0.793 0.816 0.864
7 14 6
1.008 (-0.0111) 25.2 (-0.293) 45.0 (-0.454)
0.991 0.861 0.891
9 9 10
a Denotes correlation coefficient. Note: correlation coefficients of 1.0 typically mean that only two strain points were available for slope determination. b N ) number of samples tested.
while the slopes of the elastic stress-strain curves were between 0.7 and 387 MPa for self-assembled collagen fibers and turkey tendons, respectively. The slopes of the elastic stress-strain curves were between 249 and 387 MPa for all the samples except the self-assembled collagen fibers aged less than 6 months. Slopes of viscous stress-strain curves for turkey tendons ranged from 41.0 to 84.3 MPa while those for self-assembled collagen fibers were between 1.0 and 45.0 MPa (Table 2). Fibril lengths calculated from the slope of the viscous stressstrain curves ranged from 414 to 616 µm for turkey tendons and from 8.22 to 48.3 µm for self-assembled collagen fibers
axial volume ratioa diam, fraction eq const nm 0.5 0.5 0.5
36 783 46 703 31 372
0.173 0.402 0.495
7348 32 236 43 133
264b 264b 264b 22.4 22.4 22.4
fibril length, µm (initial strain 0.1) 486 (2724) 616 (3459) 414 (2323) 8.22 (46.1) 36.1 (202) 48.3 (266)
a Z ) axial ratio ) constant × strain0.552. b Estimated from Landis and Song.18
(Table 3). Elastic fractions for turkey tendons were found to increase with strain rising from about 0.56 to 0.78 as the strain increased from 0.02 to 0.120 (Figure 4). For selfassembled collagen fibers the elastic fraction was between 0.7 and 0.42 for strains between 0.05 and 0.5, respectively, for unaged fibers, and it was between 0.9 and 0.8 for fibers aged 6 months (Figure 5). The volume fraction of polymer increased from 0.173 to 0.495 for self-assembled collagen fibers aged for 0 and 6 months, respectively. Discussion Previously, mineralization was reported to be associated with an increase in the slope of the stress-strain curve (modulus) as well as an increase in the ultimate tensile strength.16 In this study we compare the viscoelastic behavior of mineralizing turkey tendon to that of purified type I collagen fibers in an attempt to further identify the effect of the mineral. Turkey tendons are known to be composed of a complex assembly of somewhat flexible, highly aligned collagen fibrils with different diameters.18 Smaller collagen fibrils are observed to branch from larger fibrils or to aggregate to fibrils of greater size. Mineral is deposited in the turkey tendon between and within the collagen fibrils. The fibril diameters range from about 25 to 500 nm at about 15 weeks post-hatching with about an equal distribution of small and large fibrils.18 The tendon unit found in the gastrocnemius of the turkey cannot be divided without mechanical disruption so it is not possible to determine whether the mechanical
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properties of tendon unit fragments are similar to the properties of the whole tendon. Previous studies have examined the mechanism by which mechanical energy is translated into molecular and fibrillar deformation in tendon. Sasaki and Odajima10 indicate that up to a strain of 2% in rat tail tendons, molecular stretching predominates while beyond 2%, increases in the collagen D-period occur as a result of molecular slippage.8,20 In theory, the slope of the elastic stress-strain curve is related to the elastic deformation of the collagen molecule. Since the slopes of the elastic stress-strain curves for turkey tendons between 12 and 16 weeks of age and self-assembled collagen fibers aged for 6 months are approximately the same, this reflects the fact that stretching of the triple helix occurs throughout most of the stress-strain curve. Mosler et al.21 have attributed changes in the D-period to increases in the axial rise per residue and stretching of the nonhelical ends of collagen molecules where cross-links are present. Stress-strain measurements on turkey tendons suggested that the elastic modulus of unmineralized turkey tendon is about 100 MPa.16 The modulus increases to about 800 MPa after mineralization.16 Since the elastic moduli reported here for the total stress measurements on turkey tendons were between 290 and 485 MPa, it is concluded that all of the specimens were mineralized to some extent. An interesting finding was that the elastic behavior of self-assembled collagen fibers aged at room temperature for 6 months is very similar to the behavior of collagen fibers from intact turkey tendon. This suggests that mineralization of turkey tendon does not influence the mechanism for molecular deformation. Danielsen21 observed that collagen fibrils reconstituted from neutral salt soluble rat skin collagen incubated at 37 °C for up to 104 days showed increased ultimate tensile strength and modulus. He concluded that the strength gain in vitro was consistent with increases in the amount of crosslinking between collagen molecules and with the conversion of reducible to nonreducible cross-links. The conversion of reducible cross-links to nonreducible cross-links is associated with aging22 as well as the appearance of pyridinium residues.23 To a first approximation the value of the slope of the elastic stress-strain curve is the elastic spring constant, k, for the collagen molecule given by the relationship, F ) kx where f is the force applied to the molecule and x is the axial molecular displacement. Mosler et al.20 indicated that a rat tail tendon fiber stretched by 5% showed an increase in the D-period of 1.5% while the axial separation between amino acids (h spacing) in the triple helix increased by only 0.5%. This means that only about 10% of the macroscopic strain causes direct axial stretching of the collagen triple helix. The rest of the macroscopic displacement is dissipated by the sliding of collagen triple helices relative to each other. Therefore, the spring constant for turkey tendon collagen must be corrected by multiplying by the ratio of the macroscopic strain (5%) to increase in h spacing (0.5%) since not all of the macroscopic strain resulted in axial displacement of the collagen.20 In addition, it is necessary to correct for the area fraction of collagen in the cross section
Silver et al.
(estimated to be 0.5) since the cross section is not composed entirely of collagen molecules. Making these corrections the spring constant for the collagen molecule rises to between 5 and 7.5 GPa for 12-16 week turkey tendons. For selfassembled collagen fibers that have been cross-linked for 6 months, the same correction incorporating 0.472 as the area fraction yields an elastic constant of 6.43 GPa. The elastic constant for the collagen molecule lies between 5 and 7.75 GPa for all collagen specimens tested here when the macroscopic strain is replaced by the amount the triple-helix is stretched. This value is close to the value (9 GPa) reported by Harley et al.24 based on light scattering techniques. Therefore, the slope of the elastic stress-strain curve after correction for the microscopic strain appears equivalent to the elastic constant for the collagen molecule attributable to stretching of the triple-helix. The variation in the value of the elastic constant may reflect differences in the contributions of the nonhelical ends of the collagen molecule, triple helix and regions containing cross-links. Several authors have attempted to calculate Young’s modulus, the slope of the total stress-strain curve, for collagen from mechanical measurements. Shadwick14 reported a value of 0.16 GPa for newborn pig digital tendons and 1.66 GPa for mature digital flexor tendons. The mechanical behavior of adult tendons was reported to be independent of strain rate.14 Values for pig hind leg extensors reported by Smith and co-workers25 were 0.98 GPa, and that reported for bovine Achilles tendon was 2.9 GPa.19 It is clear the value for the Young’s modulus depends on the type of tendon studied and the exact method of determination. In comparison, the elastic constant should be a material constant for collagen and depend only on the amino acid composition. However, the elastic constant for collagen will be affected by stretching of nonhelical ends, the cross-links, and the amino regions devoid of proline and hydroxyproline within the triple helix. It is likely that direct stretching of the rigid portion of the triple helix rich in proline and hydroxyproline only occurs after the more flexible regions are maximally displaced. Furthermore, it is likely that the introduction of cross-links within the nonhelical ends and the regions devoid of proline and hydroxyproline further stiffens the molecule and may raise the elastic constant. If this is the case this would significantly alter the behavior of collagen fibers in adult tissues. When one compares the slopes of the viscous stress-strain curves for the mineralized turkey tendons and self-assembled collagen fibers cross-linked for 6 months, it is immediately apparent that the slopes are similar (Table 2). Therefore, the addition of mineral around and within collagen fibrils does not lead to additional viscous dissipation. The elastic fraction measured for turkey tendon approaches a value of 0.78 at high strains, a value that is consistent with the elastic fraction reported for unmineralized human tendon.11 In contrast, cross-linking appears to increase the effective fibril length for self-assembled collagen fibrils (Table 3). In this study, an attempt was made to estimate the effective fibril length from the viscous stress. The measured viscosity of the fibril is a reflection of the interfibrillar friction which is related to the fibril length and surface properties in a
Storage of Elastic Energy in Turkey Tendons
manner similar to the viscosity of a single macromolecule. It is well-known that macromolecular viscosity is related the shape factor (axial ratio) and second virial coefficient (attractions or repulsions between macromolecules).1 Further studies are necessary to confirm that the fibril length from mechanical measurements can be directly correlated with actual fibril length measurements. The fact that mineralization did not increase the value for the slope of the viscous stress-strain curve suggests that mineralization does not lead to viscous dissipation of elastic energy and the observation supports the conclusion that the elastic storage properties of tendon are not impaired by the presence of mineral. The fact that the elastic fraction was independent of age of the bird suggests that the deformation mechanism probably is not changed during mineralization. However, unlike the pig flexor tendon, which appears to become more crosslinked with age, the turkey tendon becomes mineralized as a means to maintain stored elastic strain energy while at the same time increasing the stress that is borne by the tendon. If we approximate the stress-strain curve as a straight line, then the elastic energy stored during extension is approximately equal to 0.5 times the product of the UTS and the failure strain. Making these calculations during mineralization (at 12 and 16 weeks of age, respectively) then the energy stored during elastic deformation does not decrease with increased degree of mineralization using data reported by Landis et al.16 It would appear then that mineralization of the gastrocnemius tendon in turkey is a mechanism by which increased ultimate tensile strength (UTS) can be achieved as the bird develops without sacrificing the amount of elastic energy stored in the tendon during its deformation. This result is in contrast to that found with the pig extensor tendon in which the increased UTS of this tissue is associated with increased cross-linking and decreased strain-to-failure, characteristics causing a 50% reduction in the elastically stored energy.14 Acknowledgment. The authors would like to acknowledge the assistance of Kahlil Revan, Daniel Behin, and Gerald Vasquez (Biomedical Engineering students at Rutgers University) for collecting the biomechanical data and Ms. Karen Hodgens (Children’s Hospital Medical Center) for collecting the turkey tendons and doing the microradiography. The work was supported by NIH Grant AR41452 (W.J.L.). References and Notes (1) Silver, F. H. Biological Materials: Structure, Mechanical Properties and Modeling of Soft Tissues; New York University Press: New York, 1987; pp 91-110. (2) Birk, D. E.; Silver, F. H. Corneal and scleral type I collagens; Analyses of physical properties and molecular flexibility. Int. J. Biol. Macromol. 1983, 5, 209-214. (3) Silver, F. H.; Kato, Y. P.; Ohno, M.; Wasserman, A. J. Analysis of mammalian connective tissue: Relationship between hierarchical structures and mechanical properties. J. Long-Term Eff. Med. Implants 1992, 2, 165-192.
Biomacromolecules, Vol. 1, No. 2, 2000 185 (4) Parry, D. A. D. The molecular and fibrillar structure of collagen and its relationship to mechanical properties of connective tissue. Biophys. Chem. 1988, 29, 195-209. (5) Doillon, C. J.; Dunn, M. G.; Bender, E.; Silver, F. H. Collagen fiber formation in vivo: Development of wound strength and toughness. Coll. Relat. Res. 1985, 5, 481-492. (6) Torp, S.; Baer, E.; Friedman, B. Effects of age and of mechanical deformation on the ultrastructure of tendon. Colston Pap. 1975, 26, 223-250. (7) McBride, D. J.; Trelstad, R. L.; Silver, F. H. Structural and mechanical assessment of developing chick tendon. Int. J. Biol. Macromol. 1988, 10, 194-200. (8) Folkhard, W.; Mosler, E.; Geercken, W.; Knorzer, E.; NemetshekGansler, H.; Nemetschek, Th.; Koch, H. H. L. Quantitative analysis of the molecular sliding mechanism in native tendon collagen-timeresolved dynamic studies using synchrotron radiation. Int. J. Biol. Macromol. 1987, 9, 169-175. (9) Folkhard, W.; Geercken, W.; Knorzer, E.; Mosler, E.; NemetshekGansler, H.; Nemetschek, Th.; Koch, M. H. Structural dynamic of native tendon collagen, J. Mol. Biol. 1987, 193, 405-407. (10) Sasakai, N.; Odajima, S. Elongation mechanism of collagen fibrils and force-strain relationships of tendon at each level of structural hierarchy. J. Biomech. 1996, 9, 1131-1136. (11) Dunn, M. G.; Silver, F. H. Viscoelastic behavior of human connective tissues: Relative contribution of viscous and elastic components. Connect. Tissue Res. 1983, 12, 59-70. (12) Alexander, R. M. Animal Mechanics; 2nd ed.; Blackwell Scientific: Oxford, U.K., 1983. (13) Alexander, R. M. Elastic energy stores in running vertebrates A. Zool. 1984, 24, 85-94. (14) Shadwick, R. E. Elastic energy storage in tendons: Mechanical differences related to function and age. J. Appl. Physiol. 1990, 68, 1033-1040. (15) Roberts, T. J.; Marsh, R. L.; Weyand, P. G.; Taylor, C. R. Muscular force in running turkeys: The economy of minimizing work. Science 1997, 275, 113-115. (16) Landis, W. J.; LiBrizzi, J. J.; Dunn, M. G.; Silver, F. H. A study of the relationship between mineral content and mechanical properties of turkey gastrocnemius tendon. J. Bone Miner. Res. 1995, 10, 859867. (17) Pins, G. D.; Christiansen, D. L.; Patel, R.; Silver, F. H. Self-assembly of collagen fibers: Influence of fibrillar alignment and decorin on mechanical properties. Biophys. J. 1997, 73, 2164-2172. (18) Landis, W. J.; Song, M. J. Early mineral deposition in calcifying tendon characterized by high voltage electron microscopy and threedimensional graphic imaging. J. Struct. Biol. 1991, 107, 116-127. (19) Sasaki, N.; Odajima, S. Stress-strain curve and Young’s modulus of a collagen molecule as determined by the X-ray diffraction technique. J. Biomech. 1996, 29, 655-658. (20) Mosler, E.; Folkhard, W.; Knorzer, E.; Nemetschek-Gansler, H.; Nemetschek, Th.; Koch, M. H. Stress-induced molecular arrangement in tendon collagen. J. Mol. Biol. 1985, 182, 589-596. (21) Danielsen, C. C. Mechanical properties of reconstituted collagen fibrils. Connect. Tissue Res. 1982, 9, 219-225. (22) Fujii, K.; Tanzer, M. L. Age-related changes in the reducible crosslinks of human tendon collagen. FEBS Lett. 1974, 43, 300-302. (23) Eyre, D. R.; Koor, T. J.; Van Ness, K. P. Quantitation of hydroxypyridinium cross-links in collagen by high-performance liquid chromatogtaphy. Anal. Biochem. 1984, 137, 380-388, 1984. (24) Harley, R.; James, D.; Miller, A.; White, J. W. Phonons and the elastic moduli of collagen and muscle. Nature 1979, 267, 285-287. (25) Smith, C. W.; Young, I. S.; Kearney, J. N. Mechanical properties of tendons: Changes with sterilization and preservation J. Biomech. Eng. 1996, 118, 56-61.
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