J. 3i~c~~~s
Vol. 25, No. 2, pp, 163-173.
1992. (0
Printed in Great Britain
0#21-9290/92 fmo+ .w 199 I Pergamon Press plc
MECHANICAL PROPERTIES OF THE CANINE PATELLAR TENDON: SOME CORRELATIONS WITH AGE AND THE CONTENT OF COLLAGEN ROGERC. HAUT,* RONALD L. LANcAsTERt and CHARLESE. DECAMPS *Department of Biom~h~i~, College of Osteopathic Medicine, A414 East Fee Hall, Michigan State University, East Lansing, MI 48824, U.S.A., ?Johnson & Johnson Orthopaedics, 325 Paramount Drive, Raynham, MA 02767-0350, U.S.A. and $Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, U.S.A. Abstract-Portions of the patellar tendon (PT) are currently used for autogenous and allogeneic reconstruction of a torn or damaged anterior cruciate ligament (ACL). Age-related changes in the mechanical properties of the PT may influence its use in this reco~t~ction procedure. Age-dependent changes in the PT were determined in the dog, which is often used to experimentally study this reconstruction. Tensile failure experiments were performed at 100% s-l on patella-patellar tendon-tibia preparations from dogs aged 0.5-15 yr. The contents of collagen soluble and insoluble in pepsin were also measured at each age. Fifty-nine percent (16/27) of the preparations failed by avulsion at the patella, but neither the failure load nor the mode of failure were a function of age. Failure load and energy were higher for tendon substance failures compared to avulsions of bone from the patella. While a positive, linear correiation was measured between tensile modulus of the PT and age, the slope of regression was not si~ifi~ntly different from zero. The content of total collagen in the PT decreased significantly with age. The content of collagen insoluble in pepsin, however, increased with age and positively correlated with tensile modulus of the tendon. These results are different from those reported for the canine CCL, by others, which degenerates withage. Age-related changes in the mechanical properties of the canine PT are qualitatively similar to earlier, limited data on human patellar tendons. These data from the dog model are discussed in terms of their possible relevance to some questions regarding the outcome of experimental ACL reconstruction using the dog model and its potential relevance to the more aged human patient.
The mechanical
INTRODUCTION
In recent years there has been a significant amount of information documented on the mechanical properties of ligaments and tendons surrounding the knee. Much of this literature has been generated to investigate various methods and consequences of reconstructive surgery to repair a torn or otherwise damaged anterior cruciate ligament (ACL). The use of a portion of an autogenous or allogeneic patellar tendon (PT) is a popular method (Butler, 1989). Frequent rupture of the cranial cruciate ligament (CCL) in the dog has increased its importance as an animal model for human knee ligament research. This research has been conducted to develop surgical techniques, prosthetic implants, and post-operative management methods (Butler et al., 1983; Johnson et al., 1989; Shino et al., 1984; Nikolaou et al., 1986;Vasseur et al., 1987). It is commonly assumed that these data are applicable to the human (Amoczky, 1990). The outcome of experimental reconstructive surgery, however, can depend on graft placement, pretension, and the initial tensile qualities of the graft (Butler, 1989). Normal joint function, and probably the outcome of a reconstruction procedure, may depend on the properties of the graft with respect to the original ACL. Received in jinal form 26 April 1991.
properties
of the canine
CCL de-
pend on age (Vasseur et al., 1985). This structure is analogous to the human ACL. Tensile loading experiments on the canine CCL indicate a significant decrease in material properties (modulus, maximum stress, and failure strain energy density) from 1 to 15 yr of age. Yet animal age is not always controlled in these research projects. Histological studies suggest that degenerative changes in the canine caudal cruciate and collateral ligaments are not as severe; hence, these changes are tissue specific. Vasseur et al. (1985) speculate that CCL degeneration may be more rapid because of a higher physiological state of stress. Guan and Butler (1990) suggest that individual fascicles of the ACL under the greatest tension physiologically have the largest material strength and modulus. Other authors indicate that disease and/or immobilization degrade strength parameters (Woo et al., 1987). With these somewhat contradictory results and the unknown state of stress in the PT compared to the CCL, or ACL, it is difficult to predict age-related changes in the PT based on the above studies. The tensile mechanical properties of soft connective tissues have been correlated with the content, organization and physical properties of collagen (Nordin and Frankel, 1980). The physical properties of collagen are largely dependent on intermolecular and intramol~uiar crosslinkages. During the process of 163
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R. C. HAUT
aging the molecular stability, entropy, and crosslinking of collagen increase (Viidik, 1972; Hamlin and Kohn, 1971; Mitchell and Rigby, 1975). Studies have shown that various mechanical parameters, such as tensile strength, ultimate load and modulus, change with age and correlate with the content of collagen soluble in buffered saline and weak acids (Vogel, 1978). The primary purpose of this study was to document the mechanical properties of the canine patellar tendon at various ages, in part because animal age is not always a tightly controlled parameter in studies of reconstructive surgery. Since most of the previous studies correlating mechanical parameters with contents of soluble collagen were conducted on rats, these biochemical data were also gathered and correlated with various tensile parameters for the canine PT. This information provides new correlations between biochemical and biomechanical parameters on tissues used more often in orthopaedic research. The data also provide important information on trends in the age-related tensile properties of the PT and CCL of the dog model. These relative properties may, in part, dictate the outcome of animal experimentation and its applicability to reconstructive surgery in the human patient. MATERIALS
AND METHODS
Twenty-seven dogs (26.7k8.4 kg) of medium and large breeds were obtained at necropsy. Fourteen animals were male. Medical records were screened to eliminate animals having known histories of musculoskeletal disorders, knee surgery or trauma. The animals were of 18 different ages ranging from 6 to 180 months. After necropsy the hind limbs were disarticulated and frozen at -20°C for an unspecified time (~9 months). Storage at this temperature has been shown not to influence the mechanical properties of ligaments and tendons (Woo et al., 1986). One limb was used per animal for experiments reported here. Prior to tensile tests the limbs were thawed overnight at room temperature (27°C). During isolation of the patella-patellar tendon-tibia complex (PPT), the preparation was kept moistened with a continuous drip of 37°C phosphate-buffered saline (pH 7.2). All soft tissue was removed from the bones, except the patellar tendon. The dimensions of the tendon were measured with vernier calipers by a single investigator (Burks et al., 1990). The initial length was determined from the inferior pole of the patella to the proximal-posterior aspect of the tibia1 attachment. The width and thickness were measured at three locations down the length of the tendon and averaged. A rectangular cross section was assumed. The cross-sectional areas varied by approximately 5% down the lengths. All measurements were made under a slight pretension, sufficient to merely straighten the tendon. For the biomechanical failure experiments, the patella was potted with room temperature curing epoxy in a specially designed box-type fixture using methods
et al.
previously documented (Burks et al., 1990). Care was taken that the epoxy did not contact the tendon. The tibia was potted in a PVC tube which was clamped into a restraint fixture, so that the limb’s flexion angle was approximately 70” (Fig. 1). This configuration oriented the patellar tendon vertically, so it could be stretched in pure tension in a servo-controlled hydraulic testing machine. The specimen was kept moistened with a room temperature, phosphate-buffered saline solution continuously sprayed on the tendon from an overhead drip device during mechanical experiments. Each preparation was cyclically loaded (preconditioned) between 90 and 180 N at 1 Hz for 20 cycles. This range of loading was assumed to be within a physiological range. This was immediately followed by a failure experiment at a nominal strain rate of 100% of the tendon’s length per second. Specimen elongation was monitored as grip-to-grip deformation. This deformation was from the insertion into the patella to the insertion of the PT into the tibia. In earlier studies (Burks et al., 1990) no slippage was observed during experiments filmed with an 8 mm video camera. Resistive loads were recorded continuously. In the cyclic experiments, creep was defined as the change in peak strain (normalized stretch) between the first and fortieth cycle (Fig. 2). A physiological stiffness was determined as the overall average slope of the loading segment in the fortieth cycle. Failure mechanisms were determined from direct post-test examination of the tissue preparation. The elongation and load data were used to determine the failure load and failure energy. Structural stiffness was evaluated by the slope of the response prior to failure in the mid-range, where a linear response was observed between approximately 2 and 8 mm of deformation (Fig. 3). This range was slightly adjusted for each specimen to measure slope in the most representative linear range of response. This corresponded to a range of strains from approximately 5 to 25%. This method of analysis does not take into account nonuniform strains along the length of tendon, shown to exist in the human PT preparation (Stouffer et al., 1985). The data were normalized by dividing load by average cross-sectional area and elongation by initial length to yield stress and strain, respectively. The slope of the stress-strain curve in the linear range was termed the tensile modulus. In cases of mid-substance failure the area under the stress-strain energy curve to the maximum stress was termed the failure strain energy density of the tendon. Immediately after the mechanical tests, the tissues were wrapped in saline moistened gauze for biochemical analyses. Tissue collagen was measured in six separate aliquots (15-29 mg) of pulverized tendon. Tissue was collected away from bone insertions within the middle third of the tendon. Three aliquots were used to measure fractions of collagen soluble in acetic acid and pepsin according to Schnider and Kohn (1981). Following removal of lipids, collagen was extracted overnight three times in 0.5 M acetic acid at
Fig. 1. The tensile loading fixture used to extend the patella-patellar tendon-tibia preparation to structural failure. The limb was flexed so that the patellar tendon was aligned in the servo-hydraulic testing machine.
165
167
Mechanical properties of the canine patellar tendon
TIME
(seconds)
Fig. 2. This typical creep experiment at 1 Hz indicated that the PT continuously extended with each load cycle between 90 and 180 N. The slope of the force4ongation curve in the fortieth cycle was used to determine the physiological stiffness of the PT. No age dependencies were noted in creep compliance or stiffness.
FAILURE LOAD
r
FAILURE ENERGY
\.
T
0
2
4
a
6
DEFORMATION
10
12
(mm)
Fig. 3. A typical plot of the tensile force4ongation response of the canine PT at 100%/s. The failure energy and failure load were computed from these data. Structural stiffness was measured as the slope in the linear region. The data were normalized to compute failure stress, strain and strain energy density.
4°C. Supematants were pooled. The pellet was then digested in 2 ml of pepsin (P7012, Sigma Chemical) at a concentration of 1 mg ml- ’ for 18 h at 4°C. Salt was used to help precipitate the pepsin-soluble fraction. The remaining pellet was the insoluble fraction. Hydroxyproline was measured using the method of Stegemann (1958). Collagen content was quantified as
micrograms hydroxyproline per milligram dry weight of tissue. Regression analyses were used to determine whether parameters varied linearly with animal age. A statistically significant change in any parameter was indicated when the slope of linear regression differed statistically from zero, using the Student’s t-test at a
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R. C. HAUTet al.
level PcO.05. When the analysis of linear regression indicated a slope not statistically different from zero, the experimental data were documented as mean f one standard deviation. A Student’s t-test was used for statistical comparisons of means between any two groups of parameters.
energy for substance failures on an average (26 f 9 J) exceeded that for avulsion types of failure (lo+ 6 J), but the results were not significantly different (P > 0.05). The tensile modulus was computed from the slope of the stress-strain curve, analogous to the computation of structural stiffness from force-elongation responses. While a positive trend was noted in the tensile modulus of the patellar tendon with age, no significance could be attached to this effect (Fig. 5). The tensile modulus of the canine patellar tendon averaged 474_+ 101 MPa between the ages of 6 and 180 months. The tensile failure data were analyzed separately for cases exhibiting substance failures of the tendon. For the eleven experiments in this category maximum stress, strain and failure strain energy density did not vary with animal age, being 122f25.6 MPa, 32.3 + 7.8% and 25.7 + 8.7 J cmw3, respectively. Significant variations were recorded in the contents of collagen in the canine patellar tendon with age of the animal. The concentration of total collagen (measured in three separate aliquots and averaged) decreased significantly with age (Fig. 6). The concentration of collagen soluble in acetic acid was less than 1% for all age groups. The content of collagen insoluble in pepsin showed a significant rise with age (Fig. 7). At six months of age approximately 60% of the collagen was soluble in pepsin, while at 180 months nearly all the collagen was insoluble. Regression analyses were performed to test the dependence of tensile modulus on the total content and the insoluble fraction of collagen. The tensile modulus was positively correlated (P < 0.05) with the content of insoluble collagen in the tendon (Fig. 8). No linear dependence was indicated between total collagen content and tensile modulus. In cases where
RESULTS
Regression analyses indicated that the overall geometry (or size) of the canine patellar tendon in this population of medium and large breeds did not vary with age. The average length, cross-sectional area and volume of the canine patellar tendon was 3.41 kO.65 cm, 0.24kO.07 cm’, 0.82kO.32 cm3, respectively. Low level, preconditioning experiments were conducted to estimate the tensile response under an approximated physiological level of loading. Cyclic creep elongation developed continuously over the entire 20 cycles for all ages. The stiffness of the patellar tendon preparation for the physiological range of loading did not vary with age, being on an average 308 _+66 kN m- I. The corresponding tensile modulus of the tendon within this range of loading was 457 + 98 MPa. Under cyclic loading the tendon elongated 1.4f0.68% for all age groups. In this investigation we also studied the tensile failure properties of the patella-patellar tendon-tibia complex. Fifty-nine percent (16/17) of the preparations failed by an avulsion fracture of bone from the patella (Fig. 4). Neither the failure loads nor the mode of failure was a function of animal age. Failure load, however, was a function of the mode of failure. Avulsion fractures occurred at 208Ok 620 N, while substance failures of the tendon occurred at a significantly higher level of 3250+ 700 N. In a similar fashion,
0
.
0
Avulsion
0
Substance
. .
0 .
0
0
l
.
0
0
Age (months) Fig. 4. Tensile failure loads for the canine PT preparation were not a function of age. Avulsion-type fractures occurred in 50% of the experiments irrespective of age and yielded lower loads on an average than substance failures.
Mechanical properties of the canine patellar tendon
‘
6004
l
l
l
.
.
.
. 500..
l b
l
b
l
400..
0. .
Y :
169
. 300.7
8
. l
zoo-y -0.56x+ 426.45 loo-
00 0
P > 0.05
30
so
60
120
210
165
Age (months)
Fig. 5. The tensile modulus of the canine PT increased slightly with age, but not a statistically significant variation. This contrasts with earlier work of Vasseur et al. (1989, showing that the modulus of the canine ACL decreases significantly with age.
y --0.16x+116.62 P < 0.05
Age (months)
Fig. 6. The total content of collagen in the canine PT decreased significantly with age. The content was measured as micrograms of hydroxyproiine per dry tissue weight. This correlated qualitatively with tensile strength of the patellar tendon. substance failures occurred, the maximum tensile stress did not significantly depend on the total content of collagen or the insoluble fraction. All parameters (geometrical, biomechanical and biochemical) were separately anaIyzed for gender differences. No significant gender dependencies were uncovered in this study. BM 25:2-D
DKSCUSSION The major objective of this research was to document alterations in the tensile mechanical properties of the canine PT due to age. Earlier studies suggest these changes may be tissue-specific and dependent on
R. C. HAUTet of.
170
.
“I
0
CiO
60
90
.
120
150
Iilo
210
Age ~months) Fig. 7. The concentration of insoluble (crosslinked) collagen in the canine PT significantly increased from nearly 40% at 6 months to nearly 100% at 15 yr of age. The collagen concentration was measured as micrograms of hydroxyproline per dry weight. This biochemical parameter correlated with tensile modulus of the canine patellar tendon.
.
. .
I
y * l.69~ + 356.17
I
20
40
60
insoluble
60
Collagen
100
120
140
Content
Fig. 8. The study indicated that a significant, positive linear correlation existed in the canine PT between the content of insoluble (crosslinked) collagen and the tensile modulus of the tendon. The concentration of collagen was measured as micrograms of hydroxyproline per dry weight.
the state of physiology stress in the tissue (Vasseur et al., 1985). The content, organization, and physical properties of collagen have been shown to change with age and correlate with some of the tensile properties of other collagenous tissues (Vogel, 1978). Since the biochemistry of collagen is known to depend on age,
we felt that additions correlations were warranted on tissues more often used in current orthopaedic research. Numerous studies have been and will be conducted dealing with questions of the surgical reconstruction of the ACL with portions of the PT using a canine. model. Relative differences between the ACL
Mechanical properties of the canine patellar tendon
and PT may influence the outcome of these studies. Specimen age could play a significant role. The results of this study on the tensile mechanical properties of the canine PT were quite different from those reported earlier by Vasseur et al. (1985) on the CCL. For a similar population of animals, namely, medium and large breeds from necropsy, the degenerative changes in the CCL were greater than those measured in the PT. Qualitatively the variation in tensile mechanical properties of the canine PT and CCL around a linear regression line were comparable between the two studies. Some variability was expected because the activity level of the animals was not controlled in either study. Immobilization (Woo et al., 1987) and extended cage activity (Noyes et al., 1984) have been shown to significantly influence the strength and modulus of the ACL, MCL and other collagenous structures. The effects of any undetected systemic diseases on the properties of ligaments and tendons are largely unknown (Noyes and Grood, 1976), and may also contribute to variability in the data reported in this study. While the animals were screened, some minor errors in animal age can be expected, since we had to depend on client information on their pets. The covariant type of statistical analysis conducted here helped to handle this by employing numerous ages in the study. In Vasseur et al. (1985) degeneration in mechanical properties of the CCL with age exceeded the variation in test data about the regression line. This was not the case for the canine PT. In the CCL the tensile modulus decreased from approximately 250 MPa at less than 1 yr of age to approximately 200 MPa at 15 yr. The current study indicated that the tensile modulus of the canine PT averaged 474+ 101 MPa for all ages, and even tended to increase with age. The ratio of tensile modulus for the PT to that of the CCL varied from approximately 1.9 at 1 yr to 2.4 at 15 yr, using data from Vasseur et al. (1985) and the current study. The failure mode for the patella-patellar tendon-tibia was also quite different from that documented for the canine CCL. While 41% of the cases failed in the substance for the PT preparation, Vasseur et al. (1985) indicate all substance failures for the CCL. Other studies indicate that for ligaments a higher incidence of avulsion failures is associated with extended periods of immobilization and cage activity (Woo et al., 1987; Noyes and Grood, 1976). Some of the spread in data could, admittedly, be caused by our lack of control in the activity level of these animals. However, it would be very difficult and costly to maintain control of this parameter over a 15 yr span. On the other hand, analysis of only cases in which substance failures occurred (11 specimens aged 0.5-12 yr) also did not suggest age-dependent changes in the maximum stress or tensile modulus of the canine patellar tendon with age. On an average the maximum stress for substance failures of the PT was 190% of the CCL at less than 1 yr and approximately 220% at 12 yr. The ratio of strain energy density to failure for the PT to that of the
171
CCL was, on an average, 1.0 at less than 1 yr and 5.0 at 12 yr. The above data generally indicate that the PT gets progressively stiffer and stronger than the CCL with age. It is interesting to note, from limited data, that no significant changes with age have been documented in the human PT (Haut et al., 1988). For a similar test condition (moistened tissues at room temperature), the tensile modulus and maximum stress of the human PT is 191+ 16 MPa (n=6) and 30.6k2.6 MPa (n=6), respectively. Grood et al. (1977) documented that the tensile mechanical properties of the human ACL decrease significantly with age, as did those of the canine CCL. The tensile modulus, maximum stress and strain energy density of the human ACL decrease approximately 111 MPa, 38 MPa and from 10 J cnm3, respectively, at 16-26 yr of age to approximately 65 MPa, 13 MPa and 3 J cmm3, respectively, at 48-86 yr of age. On an average, the ratio of tensile modulus for the human PT and ACL is approximately 1.7 for younger humans and approximately 2.9 for older. The ratios of maximum stress between the two tissues is approximately 0.8 at the younger ages and 2.4 at the older ages. The above data suggest that differences in the tensile mechapical characteristics of the canine PT and CCL as a function of age are largely similar to the data collected to date on humans. Hubbard and Soutas-Little (1984) also indicate no significant decrease in the tensile strength and modulus of human palmaris longus and extensor hallucis longus tendons with age. Yamada (1970) documents no changes in the ultimate tensile strengths of the calcaneal tendon between 10 and 60 yr of age. The reasons for differential human aging effects in various tissue structures are largely unknown. Vasseur et al. (1985) discussed differences in the histological appearance. of the ACL and other ligaments. They suggest the effects may be due to different physiological functions, indirectly related to the levels of stress in each tissue structure. While the actual levels of physiological load in the PT compared to the ACL can only be speculated, we hypothesize that the patellar tendon may, in fact, carry greater loads more regularly than the ACL. Physiologically, the PT may respond by continually repairing microdamage (Vasseur et al., 1985) more effectively than the ACL to maintain a level of tensile quality. This may be related to differing blood supplies in the CCL and PT (Alm and Stromberg, 1974), or other unknown reasons. Earlier studies, primarily with the rat, correlate the content of collagen insoluble in acetic acid to tensile strength. In studies on skin and tail tendon of the rat a gradual decrease in the content of acetic acid insoluble collagen during senescence is correlated with a gradual decrease in strength (Vogel, 1978). In the current study on canine PT, only about 1% of collagen was soluble in acetic acid. This compares to approximately 8% at 1 month of age in the rat tail tendon and 3% at 24 months of age (Vogel, 1978). The content of acetic acid soluble collagen for rat skin varies from approx-
172
R. C. HAUTet al.
imately 11% at 1 month of age to 3% at 24 months. The lower percentage of acetic acid soluble collagen in the canine patellar tendon may be due to a species difference, or possibly higher levels of physiological stress in this tissue compared to skin or tail tendon of the rat. Resistance of collagen to the enzyme pepsin, which attacks the n-terminal region of the macromolecule, was a better method of grading strength of crosslink densities in canine tendon collagen. The actual mechanisms of strength in connective tissues at the ultrastructural level are still largely unknown, but the types and degrees of crosslinking are suspected to play a major role (Nimni and Harkness, 1988). A slight downward trend (P~0.05) was noted for the maximum stress in the canine PT with age for substance failures only. This was qualitatively similar to a decrease in total collagen content of the tendon with age (Fig. 6). On the other hand, the fraction of collagen insoluble in pepsin increased with age (Fig. 7). The tensile modulus was shown to positively correlate with the content of collagen insoluble in pepsin. These data reflected our early impressions that aged collagen becomes more crosslinked and, therefore, more insoluble, which correlates with the properties of the tendon substance, per se. The failure properties of the bonetendon-bone preparation may involve some aspects of the bone insertion. The content of total collagen in the tendon may be, for some unknown reasons, a better correlate of this aspect of the tendon preparation. If the results of this study on the dog are generally relevant to the human, they may suggest that reconstruction of a torn ACL with a portion of the PT is not a problem, based solely on the strength of the whole PT as age progresses. We assume here that the properties of the graft are largely reflected in those of the whole tendon. In humans, some variations have been measured between properties of the medial and central thirds of the PT (Noyes et al., 1984). This oftenquoted study dealing with the relative strengths of the central third PT graft and the ACL is based on human tissues aged 26 k6 yr. The study indicates, on the average, that a central (14 mm wide) portion of the human patellar tendon has a strength 168% and structural stiffness 380% that of the ACL. The ratios would change slightly for the medial portion of the PT, which is sometimes used for reconstruction of the ACL. The use of approximately one-third of the patellar tendon would seem to allow extra strength and stiffness needed to account for any postsurgical remodeling and resorption of the graft (Butler, 1989). The above comments suggesting that the PT may be an even more superior graft tissue with increasing age, because it does not degenerate as rapidly as the ACL, must be tempered by questions about the dog model and whether a proportionately higher stiffness in a one-third PT graft would negatively jeopardize the final outcome of the reconstruction because of a greater mismatch in tensile properties between the graft and the damaged ACL. We also must qualify this
hypothesis since whole tendons were examined in the current study. Alterations due to age may, in fact, vary across the width of the patellar tendon. Additional experimental research using the dog model is needed to answer this question. The current study also suggests that clinical studies are needed on the more aged human patient. We hope this reseach, using a dog model, will provide some impetus for these studies. Acknowledgements-This research was partially supported by the Companion Animal Fund, College of Veterinary Medicine, Michigan State University. The authors acknowledge the help and valuable discussions with Dr Gregory Fink, Department of Pharmacology and Toxicology, on the statistical interpretation of these data. We also thank Aimee Farquhar, Kristina Haut, and David J. Licciardello for their valuable technical contributions to the research project.
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Ahn, A. and Stromberg, B. (1974) Vascular anatomy of the patellar and cruciate ligaments: a micrographic and histologic investigation in the dog. Acta Chir. stand. (Suppl.) 445,25-35. Amocxky, S. P. (1990) Animal models for knee ligament research. In Knee Ligaments: Structure, Functiorr~lnjury and Renair (Edited bv Daniel. D.. Akeson. W. and O’Connor, J.i pp. 401-417. Raven Press, New’York. Burks, R. T., Haut, R. C. and Lancaster, R. L. (1990) Biomechanical and histological observations on the dog patellar tendon after removal of its central one-third. Am. J. Sports Med. 18(2), 146-152. Butler, D. L. (1989) Anterior cruciate ligament: its normal response and replacement. J. orthop. Res. 7, 910-921. Butler, D. L., Hulse, D. A., Kay, M. D., Grood, E. S., Shires, P. K., D’Ambroska, R. and Shoji, H. (1983) Biomechanics of cranial cruciate ligament reconstruction in the dog. IImechanical properties. Vet. Surg. U(3), 113-118. Grood, E. S., Noyes, F. R. and Butler, D. L. (1977) Age related changes in the mechanical properties of knee ligaments. ASME Biomech. Symp. 23,213-216. Guan, Y. and Butler, D. (1990) Location-dependent variations in the material properties of anterior cruciate ligament subunits. ASME Adv. in Bioengng 17, 5-7. Hamlin, C. R. and Kohn, R. R. (1971) Evidence for progressive, age-related structural changes in post-mature human collagen. Biochem Biophys. Acta 236,458-467. Haut, R. C., Powlison, A., Rutherford, G. N. and Kateley, J. R. (1988) Some effects of donor age and sex on the mechanical properties of patellar tendon graft tissues. ASME Adv. in Bioengng 15, 75-78. Hubbard, R. P. and Soutas-Little, R. W. (1984) Mechanical properties of human tendon and their age dependence. J. biomech. Engng 106, 144-150. Johnson, S. G., Hulse, D. A., Hogan, H. A., Nelson, J. K. and Boothe, H. W. (1989) System behavior of commonly used cranial cruciate ligament reconstruction autografts. Vet. Surg. 18(6), 459-465. Mitchell, T. W. and Rigby, B. J. (1975) In vivo and in vitro aging of collagen examined using isometric melting technique. Biochem Biophys. Acta 393,531-541. Nikolaou, P. K., Seaber, A. V., Glisson, R. R., Ribbeck, B. M. and Bassett, F. H. (1986) Anterior cruciate ligament allograft transplantation. Am. .I. Sports Med. 14(5), 348-360. Nimni, M. E. and Harkness, R. D. (1988) Molecular structure and functions of collagen. In Collagen (Edited by Nimni M.), pp. l-77. CRC Press, Boca Raton, Florida. Nordin, M. and FrankeL V. H. (19801 Biomechanics of collagenous tissues. In &sic Biomech&cs of the Skeletal
Mechanical properties of the canine patellar tendon System (Edited by Frankel, V. H. and Nordin, M.), pp. 87- 110. Lea and Febiger, Philadelphia. Noyes, F. R., Butler, D. L., Grood, E. S., Zemicke, R. F. and Hefty, M. S. (1984) Biomechanical analysis of human ligament grafts used in knee ligament repairs and reconstructions. J. Bone Jr Surg. 66A, 344-352. Noyes, F. R. and Grood, E. S. (1976) The strength of the anterior cruciate ligament: age and species-related changes. J. Bone Jt Surg. 58, 1074-1082. Schnider, S. L. and Kohn, R. R. (1981) Effects of age and diabetes mellitis on the solubility and nonenzymatic glycosylation of human skin collagen. J. Clin. Invest. 67, 1630-1635. Shino, K., Kawasaki, T., Horose, H., Gotoh, I., Inone, M. and Ono, K. (1984) Replacement of the anterior cruciate ligament by an allogenic tendon graft. J. Bone Jt Surg. 66B, 672-681. Stegemann, H. (1958) Mikro vestimmung von hydroxyprolin mit chloramin-tund -dimethylamino benzaldehyd. Physiol. c/rem. 311(l), 41-45. Stouffer, D. C., Butler, D. L. and Hosney, D. (1985) The relationship between crimp pattern and mechanical response of human patellar tendon-bone units. J. biomech. Engng 17, 158-165. Vasseur, P. B., Pool, R. R., Amoczky, S. P. and Lau, R. E.
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(1985) Correlative biomechanical and histological study of the cranial cruciate ligament in dogs. Am. J. Vet. Res. 46, 1842-1854. Vasseur, P. B., Rod&o, J. J., Stevenson, S., Clark, G. and Sharkey, N. (1987) Replacement of the anterior cruciate ligament with a bone-ligament-bone anterior cruciate ligament allograft in dogs. Clin. Orthop. 219, 268-277. Viidik, A. (1972) Aging of collagen in complex tissues: a micro-methodical study of the thermal reaction. Experientia 15(6), 641-642. Vogel, H. B. (1978) Influence of maturation and age on mechanical and biochemical parameters of connective tissue of various organs in the rat. Corm. Tissue Res. 6, 83-94. Woo, S. L.-Y., Orlando, C. A., Camp, J. R. and Akeson, W. H. (1986) Effects of postmortem storage by freezing on ligament tensile behavior. J. Biomechanics 19(5), 399404. Woo, S. L.-Y., Gomez, M. A., Sites, T. J., Newton, P. W., Orlando, C. A. and Akeson, W. H. (1987) The biomechanical and morphological changes in the medial collateral ligament of the rabbit after immobilization and remobilization. .I. Bone Jt Surg. 69A, 1200-1211. Yamada, H. (1970) Strength ofBiological Materials (Edited by Evans, F. G.), p. 273. Williams and Wilkins, Baltimore, MD.
Vol. 25, No. 2, pp, 163-173.
1992. (0
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MECHANICAL PROPERTIES OF THE CANINE PATELLAR TENDON: SOME CORRELATIONS WITH AGE AND THE CONTENT OF COLLAGEN ROGERC. HAUT,* RONALD L. LANcAsTERt and CHARLESE. DECAMPS *Department of Biom~h~i~, College of Osteopathic Medicine, A414 East Fee Hall, Michigan State University, East Lansing, MI 48824, U.S.A., ?Johnson & Johnson Orthopaedics, 325 Paramount Drive, Raynham, MA 02767-0350, U.S.A. and $Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, U.S.A. Abstract-Portions of the patellar tendon (PT) are currently used for autogenous and allogeneic reconstruction of a torn or damaged anterior cruciate ligament (ACL). Age-related changes in the mechanical properties of the PT may influence its use in this reco~t~ction procedure. Age-dependent changes in the PT were determined in the dog, which is often used to experimentally study this reconstruction. Tensile failure experiments were performed at 100% s-l on patella-patellar tendon-tibia preparations from dogs aged 0.5-15 yr. The contents of collagen soluble and insoluble in pepsin were also measured at each age. Fifty-nine percent (16/27) of the preparations failed by avulsion at the patella, but neither the failure load nor the mode of failure were a function of age. Failure load and energy were higher for tendon substance failures compared to avulsions of bone from the patella. While a positive, linear correiation was measured between tensile modulus of the PT and age, the slope of regression was not si~ifi~ntly different from zero. The content of total collagen in the PT decreased significantly with age. The content of collagen insoluble in pepsin, however, increased with age and positively correlated with tensile modulus of the tendon. These results are different from those reported for the canine CCL, by others, which degenerates withage. Age-related changes in the mechanical properties of the canine PT are qualitatively similar to earlier, limited data on human patellar tendons. These data from the dog model are discussed in terms of their possible relevance to some questions regarding the outcome of experimental ACL reconstruction using the dog model and its potential relevance to the more aged human patient.
The mechanical
INTRODUCTION
In recent years there has been a significant amount of information documented on the mechanical properties of ligaments and tendons surrounding the knee. Much of this literature has been generated to investigate various methods and consequences of reconstructive surgery to repair a torn or otherwise damaged anterior cruciate ligament (ACL). The use of a portion of an autogenous or allogeneic patellar tendon (PT) is a popular method (Butler, 1989). Frequent rupture of the cranial cruciate ligament (CCL) in the dog has increased its importance as an animal model for human knee ligament research. This research has been conducted to develop surgical techniques, prosthetic implants, and post-operative management methods (Butler et al., 1983; Johnson et al., 1989; Shino et al., 1984; Nikolaou et al., 1986;Vasseur et al., 1987). It is commonly assumed that these data are applicable to the human (Amoczky, 1990). The outcome of experimental reconstructive surgery, however, can depend on graft placement, pretension, and the initial tensile qualities of the graft (Butler, 1989). Normal joint function, and probably the outcome of a reconstruction procedure, may depend on the properties of the graft with respect to the original ACL. Received in jinal form 26 April 1991.
properties
of the canine
CCL de-
pend on age (Vasseur et al., 1985). This structure is analogous to the human ACL. Tensile loading experiments on the canine CCL indicate a significant decrease in material properties (modulus, maximum stress, and failure strain energy density) from 1 to 15 yr of age. Yet animal age is not always controlled in these research projects. Histological studies suggest that degenerative changes in the canine caudal cruciate and collateral ligaments are not as severe; hence, these changes are tissue specific. Vasseur et al. (1985) speculate that CCL degeneration may be more rapid because of a higher physiological state of stress. Guan and Butler (1990) suggest that individual fascicles of the ACL under the greatest tension physiologically have the largest material strength and modulus. Other authors indicate that disease and/or immobilization degrade strength parameters (Woo et al., 1987). With these somewhat contradictory results and the unknown state of stress in the PT compared to the CCL, or ACL, it is difficult to predict age-related changes in the PT based on the above studies. The tensile mechanical properties of soft connective tissues have been correlated with the content, organization and physical properties of collagen (Nordin and Frankel, 1980). The physical properties of collagen are largely dependent on intermolecular and intramol~uiar crosslinkages. During the process of 163
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aging the molecular stability, entropy, and crosslinking of collagen increase (Viidik, 1972; Hamlin and Kohn, 1971; Mitchell and Rigby, 1975). Studies have shown that various mechanical parameters, such as tensile strength, ultimate load and modulus, change with age and correlate with the content of collagen soluble in buffered saline and weak acids (Vogel, 1978). The primary purpose of this study was to document the mechanical properties of the canine patellar tendon at various ages, in part because animal age is not always a tightly controlled parameter in studies of reconstructive surgery. Since most of the previous studies correlating mechanical parameters with contents of soluble collagen were conducted on rats, these biochemical data were also gathered and correlated with various tensile parameters for the canine PT. This information provides new correlations between biochemical and biomechanical parameters on tissues used more often in orthopaedic research. The data also provide important information on trends in the age-related tensile properties of the PT and CCL of the dog model. These relative properties may, in part, dictate the outcome of animal experimentation and its applicability to reconstructive surgery in the human patient. MATERIALS
AND METHODS
Twenty-seven dogs (26.7k8.4 kg) of medium and large breeds were obtained at necropsy. Fourteen animals were male. Medical records were screened to eliminate animals having known histories of musculoskeletal disorders, knee surgery or trauma. The animals were of 18 different ages ranging from 6 to 180 months. After necropsy the hind limbs were disarticulated and frozen at -20°C for an unspecified time (~9 months). Storage at this temperature has been shown not to influence the mechanical properties of ligaments and tendons (Woo et al., 1986). One limb was used per animal for experiments reported here. Prior to tensile tests the limbs were thawed overnight at room temperature (27°C). During isolation of the patella-patellar tendon-tibia complex (PPT), the preparation was kept moistened with a continuous drip of 37°C phosphate-buffered saline (pH 7.2). All soft tissue was removed from the bones, except the patellar tendon. The dimensions of the tendon were measured with vernier calipers by a single investigator (Burks et al., 1990). The initial length was determined from the inferior pole of the patella to the proximal-posterior aspect of the tibia1 attachment. The width and thickness were measured at three locations down the length of the tendon and averaged. A rectangular cross section was assumed. The cross-sectional areas varied by approximately 5% down the lengths. All measurements were made under a slight pretension, sufficient to merely straighten the tendon. For the biomechanical failure experiments, the patella was potted with room temperature curing epoxy in a specially designed box-type fixture using methods
et al.
previously documented (Burks et al., 1990). Care was taken that the epoxy did not contact the tendon. The tibia was potted in a PVC tube which was clamped into a restraint fixture, so that the limb’s flexion angle was approximately 70” (Fig. 1). This configuration oriented the patellar tendon vertically, so it could be stretched in pure tension in a servo-controlled hydraulic testing machine. The specimen was kept moistened with a room temperature, phosphate-buffered saline solution continuously sprayed on the tendon from an overhead drip device during mechanical experiments. Each preparation was cyclically loaded (preconditioned) between 90 and 180 N at 1 Hz for 20 cycles. This range of loading was assumed to be within a physiological range. This was immediately followed by a failure experiment at a nominal strain rate of 100% of the tendon’s length per second. Specimen elongation was monitored as grip-to-grip deformation. This deformation was from the insertion into the patella to the insertion of the PT into the tibia. In earlier studies (Burks et al., 1990) no slippage was observed during experiments filmed with an 8 mm video camera. Resistive loads were recorded continuously. In the cyclic experiments, creep was defined as the change in peak strain (normalized stretch) between the first and fortieth cycle (Fig. 2). A physiological stiffness was determined as the overall average slope of the loading segment in the fortieth cycle. Failure mechanisms were determined from direct post-test examination of the tissue preparation. The elongation and load data were used to determine the failure load and failure energy. Structural stiffness was evaluated by the slope of the response prior to failure in the mid-range, where a linear response was observed between approximately 2 and 8 mm of deformation (Fig. 3). This range was slightly adjusted for each specimen to measure slope in the most representative linear range of response. This corresponded to a range of strains from approximately 5 to 25%. This method of analysis does not take into account nonuniform strains along the length of tendon, shown to exist in the human PT preparation (Stouffer et al., 1985). The data were normalized by dividing load by average cross-sectional area and elongation by initial length to yield stress and strain, respectively. The slope of the stress-strain curve in the linear range was termed the tensile modulus. In cases of mid-substance failure the area under the stress-strain energy curve to the maximum stress was termed the failure strain energy density of the tendon. Immediately after the mechanical tests, the tissues were wrapped in saline moistened gauze for biochemical analyses. Tissue collagen was measured in six separate aliquots (15-29 mg) of pulverized tendon. Tissue was collected away from bone insertions within the middle third of the tendon. Three aliquots were used to measure fractions of collagen soluble in acetic acid and pepsin according to Schnider and Kohn (1981). Following removal of lipids, collagen was extracted overnight three times in 0.5 M acetic acid at
Fig. 1. The tensile loading fixture used to extend the patella-patellar tendon-tibia preparation to structural failure. The limb was flexed so that the patellar tendon was aligned in the servo-hydraulic testing machine.
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Mechanical properties of the canine patellar tendon
TIME
(seconds)
Fig. 2. This typical creep experiment at 1 Hz indicated that the PT continuously extended with each load cycle between 90 and 180 N. The slope of the force4ongation curve in the fortieth cycle was used to determine the physiological stiffness of the PT. No age dependencies were noted in creep compliance or stiffness.
FAILURE LOAD
r
FAILURE ENERGY
\.
T
0
2
4
a
6
DEFORMATION
10
12
(mm)
Fig. 3. A typical plot of the tensile force4ongation response of the canine PT at 100%/s. The failure energy and failure load were computed from these data. Structural stiffness was measured as the slope in the linear region. The data were normalized to compute failure stress, strain and strain energy density.
4°C. Supematants were pooled. The pellet was then digested in 2 ml of pepsin (P7012, Sigma Chemical) at a concentration of 1 mg ml- ’ for 18 h at 4°C. Salt was used to help precipitate the pepsin-soluble fraction. The remaining pellet was the insoluble fraction. Hydroxyproline was measured using the method of Stegemann (1958). Collagen content was quantified as
micrograms hydroxyproline per milligram dry weight of tissue. Regression analyses were used to determine whether parameters varied linearly with animal age. A statistically significant change in any parameter was indicated when the slope of linear regression differed statistically from zero, using the Student’s t-test at a
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level PcO.05. When the analysis of linear regression indicated a slope not statistically different from zero, the experimental data were documented as mean f one standard deviation. A Student’s t-test was used for statistical comparisons of means between any two groups of parameters.
energy for substance failures on an average (26 f 9 J) exceeded that for avulsion types of failure (lo+ 6 J), but the results were not significantly different (P > 0.05). The tensile modulus was computed from the slope of the stress-strain curve, analogous to the computation of structural stiffness from force-elongation responses. While a positive trend was noted in the tensile modulus of the patellar tendon with age, no significance could be attached to this effect (Fig. 5). The tensile modulus of the canine patellar tendon averaged 474_+ 101 MPa between the ages of 6 and 180 months. The tensile failure data were analyzed separately for cases exhibiting substance failures of the tendon. For the eleven experiments in this category maximum stress, strain and failure strain energy density did not vary with animal age, being 122f25.6 MPa, 32.3 + 7.8% and 25.7 + 8.7 J cmw3, respectively. Significant variations were recorded in the contents of collagen in the canine patellar tendon with age of the animal. The concentration of total collagen (measured in three separate aliquots and averaged) decreased significantly with age (Fig. 6). The concentration of collagen soluble in acetic acid was less than 1% for all age groups. The content of collagen insoluble in pepsin showed a significant rise with age (Fig. 7). At six months of age approximately 60% of the collagen was soluble in pepsin, while at 180 months nearly all the collagen was insoluble. Regression analyses were performed to test the dependence of tensile modulus on the total content and the insoluble fraction of collagen. The tensile modulus was positively correlated (P < 0.05) with the content of insoluble collagen in the tendon (Fig. 8). No linear dependence was indicated between total collagen content and tensile modulus. In cases where
RESULTS
Regression analyses indicated that the overall geometry (or size) of the canine patellar tendon in this population of medium and large breeds did not vary with age. The average length, cross-sectional area and volume of the canine patellar tendon was 3.41 kO.65 cm, 0.24kO.07 cm’, 0.82kO.32 cm3, respectively. Low level, preconditioning experiments were conducted to estimate the tensile response under an approximated physiological level of loading. Cyclic creep elongation developed continuously over the entire 20 cycles for all ages. The stiffness of the patellar tendon preparation for the physiological range of loading did not vary with age, being on an average 308 _+66 kN m- I. The corresponding tensile modulus of the tendon within this range of loading was 457 + 98 MPa. Under cyclic loading the tendon elongated 1.4f0.68% for all age groups. In this investigation we also studied the tensile failure properties of the patella-patellar tendon-tibia complex. Fifty-nine percent (16/17) of the preparations failed by an avulsion fracture of bone from the patella (Fig. 4). Neither the failure loads nor the mode of failure was a function of animal age. Failure load, however, was a function of the mode of failure. Avulsion fractures occurred at 208Ok 620 N, while substance failures of the tendon occurred at a significantly higher level of 3250+ 700 N. In a similar fashion,
0
.
0
Avulsion
0
Substance
. .
0 .
0
0
l
.
0
0
Age (months) Fig. 4. Tensile failure loads for the canine PT preparation were not a function of age. Avulsion-type fractures occurred in 50% of the experiments irrespective of age and yielded lower loads on an average than substance failures.
Mechanical properties of the canine patellar tendon
‘
6004
l
l
l
.
.
.
. 500..
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l
b
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Y :
169
. 300.7
8
. l
zoo-y -0.56x+ 426.45 loo-
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P > 0.05
30
so
60
120
210
165
Age (months)
Fig. 5. The tensile modulus of the canine PT increased slightly with age, but not a statistically significant variation. This contrasts with earlier work of Vasseur et al. (1989, showing that the modulus of the canine ACL decreases significantly with age.
y --0.16x+116.62 P < 0.05
Age (months)
Fig. 6. The total content of collagen in the canine PT decreased significantly with age. The content was measured as micrograms of hydroxyproiine per dry tissue weight. This correlated qualitatively with tensile strength of the patellar tendon. substance failures occurred, the maximum tensile stress did not significantly depend on the total content of collagen or the insoluble fraction. All parameters (geometrical, biomechanical and biochemical) were separately anaIyzed for gender differences. No significant gender dependencies were uncovered in this study. BM 25:2-D
DKSCUSSION The major objective of this research was to document alterations in the tensile mechanical properties of the canine PT due to age. Earlier studies suggest these changes may be tissue-specific and dependent on
R. C. HAUTet of.
170
.
“I
0
CiO
60
90
.
120
150
Iilo
210
Age ~months) Fig. 7. The concentration of insoluble (crosslinked) collagen in the canine PT significantly increased from nearly 40% at 6 months to nearly 100% at 15 yr of age. The collagen concentration was measured as micrograms of hydroxyproline per dry weight. This biochemical parameter correlated with tensile modulus of the canine patellar tendon.
.
. .
I
y * l.69~ + 356.17
I
20
40
60
insoluble
60
Collagen
100
120
140
Content
Fig. 8. The study indicated that a significant, positive linear correlation existed in the canine PT between the content of insoluble (crosslinked) collagen and the tensile modulus of the tendon. The concentration of collagen was measured as micrograms of hydroxyproline per dry weight.
the state of physiology stress in the tissue (Vasseur et al., 1985). The content, organization, and physical properties of collagen have been shown to change with age and correlate with some of the tensile properties of other collagenous tissues (Vogel, 1978). Since the biochemistry of collagen is known to depend on age,
we felt that additions correlations were warranted on tissues more often used in current orthopaedic research. Numerous studies have been and will be conducted dealing with questions of the surgical reconstruction of the ACL with portions of the PT using a canine. model. Relative differences between the ACL
Mechanical properties of the canine patellar tendon
and PT may influence the outcome of these studies. Specimen age could play a significant role. The results of this study on the tensile mechanical properties of the canine PT were quite different from those reported earlier by Vasseur et al. (1985) on the CCL. For a similar population of animals, namely, medium and large breeds from necropsy, the degenerative changes in the CCL were greater than those measured in the PT. Qualitatively the variation in tensile mechanical properties of the canine PT and CCL around a linear regression line were comparable between the two studies. Some variability was expected because the activity level of the animals was not controlled in either study. Immobilization (Woo et al., 1987) and extended cage activity (Noyes et al., 1984) have been shown to significantly influence the strength and modulus of the ACL, MCL and other collagenous structures. The effects of any undetected systemic diseases on the properties of ligaments and tendons are largely unknown (Noyes and Grood, 1976), and may also contribute to variability in the data reported in this study. While the animals were screened, some minor errors in animal age can be expected, since we had to depend on client information on their pets. The covariant type of statistical analysis conducted here helped to handle this by employing numerous ages in the study. In Vasseur et al. (1985) degeneration in mechanical properties of the CCL with age exceeded the variation in test data about the regression line. This was not the case for the canine PT. In the CCL the tensile modulus decreased from approximately 250 MPa at less than 1 yr of age to approximately 200 MPa at 15 yr. The current study indicated that the tensile modulus of the canine PT averaged 474+ 101 MPa for all ages, and even tended to increase with age. The ratio of tensile modulus for the PT to that of the CCL varied from approximately 1.9 at 1 yr to 2.4 at 15 yr, using data from Vasseur et al. (1985) and the current study. The failure mode for the patella-patellar tendon-tibia was also quite different from that documented for the canine CCL. While 41% of the cases failed in the substance for the PT preparation, Vasseur et al. (1985) indicate all substance failures for the CCL. Other studies indicate that for ligaments a higher incidence of avulsion failures is associated with extended periods of immobilization and cage activity (Woo et al., 1987; Noyes and Grood, 1976). Some of the spread in data could, admittedly, be caused by our lack of control in the activity level of these animals. However, it would be very difficult and costly to maintain control of this parameter over a 15 yr span. On the other hand, analysis of only cases in which substance failures occurred (11 specimens aged 0.5-12 yr) also did not suggest age-dependent changes in the maximum stress or tensile modulus of the canine patellar tendon with age. On an average the maximum stress for substance failures of the PT was 190% of the CCL at less than 1 yr and approximately 220% at 12 yr. The ratio of strain energy density to failure for the PT to that of the
171
CCL was, on an average, 1.0 at less than 1 yr and 5.0 at 12 yr. The above data generally indicate that the PT gets progressively stiffer and stronger than the CCL with age. It is interesting to note, from limited data, that no significant changes with age have been documented in the human PT (Haut et al., 1988). For a similar test condition (moistened tissues at room temperature), the tensile modulus and maximum stress of the human PT is 191+ 16 MPa (n=6) and 30.6k2.6 MPa (n=6), respectively. Grood et al. (1977) documented that the tensile mechanical properties of the human ACL decrease significantly with age, as did those of the canine CCL. The tensile modulus, maximum stress and strain energy density of the human ACL decrease approximately 111 MPa, 38 MPa and from 10 J cnm3, respectively, at 16-26 yr of age to approximately 65 MPa, 13 MPa and 3 J cmm3, respectively, at 48-86 yr of age. On an average, the ratio of tensile modulus for the human PT and ACL is approximately 1.7 for younger humans and approximately 2.9 for older. The ratios of maximum stress between the two tissues is approximately 0.8 at the younger ages and 2.4 at the older ages. The above data suggest that differences in the tensile mechapical characteristics of the canine PT and CCL as a function of age are largely similar to the data collected to date on humans. Hubbard and Soutas-Little (1984) also indicate no significant decrease in the tensile strength and modulus of human palmaris longus and extensor hallucis longus tendons with age. Yamada (1970) documents no changes in the ultimate tensile strengths of the calcaneal tendon between 10 and 60 yr of age. The reasons for differential human aging effects in various tissue structures are largely unknown. Vasseur et al. (1985) discussed differences in the histological appearance. of the ACL and other ligaments. They suggest the effects may be due to different physiological functions, indirectly related to the levels of stress in each tissue structure. While the actual levels of physiological load in the PT compared to the ACL can only be speculated, we hypothesize that the patellar tendon may, in fact, carry greater loads more regularly than the ACL. Physiologically, the PT may respond by continually repairing microdamage (Vasseur et al., 1985) more effectively than the ACL to maintain a level of tensile quality. This may be related to differing blood supplies in the CCL and PT (Alm and Stromberg, 1974), or other unknown reasons. Earlier studies, primarily with the rat, correlate the content of collagen insoluble in acetic acid to tensile strength. In studies on skin and tail tendon of the rat a gradual decrease in the content of acetic acid insoluble collagen during senescence is correlated with a gradual decrease in strength (Vogel, 1978). In the current study on canine PT, only about 1% of collagen was soluble in acetic acid. This compares to approximately 8% at 1 month of age in the rat tail tendon and 3% at 24 months of age (Vogel, 1978). The content of acetic acid soluble collagen for rat skin varies from approx-
172
R. C. HAUTet al.
imately 11% at 1 month of age to 3% at 24 months. The lower percentage of acetic acid soluble collagen in the canine patellar tendon may be due to a species difference, or possibly higher levels of physiological stress in this tissue compared to skin or tail tendon of the rat. Resistance of collagen to the enzyme pepsin, which attacks the n-terminal region of the macromolecule, was a better method of grading strength of crosslink densities in canine tendon collagen. The actual mechanisms of strength in connective tissues at the ultrastructural level are still largely unknown, but the types and degrees of crosslinking are suspected to play a major role (Nimni and Harkness, 1988). A slight downward trend (P~0.05) was noted for the maximum stress in the canine PT with age for substance failures only. This was qualitatively similar to a decrease in total collagen content of the tendon with age (Fig. 6). On the other hand, the fraction of collagen insoluble in pepsin increased with age (Fig. 7). The tensile modulus was shown to positively correlate with the content of collagen insoluble in pepsin. These data reflected our early impressions that aged collagen becomes more crosslinked and, therefore, more insoluble, which correlates with the properties of the tendon substance, per se. The failure properties of the bonetendon-bone preparation may involve some aspects of the bone insertion. The content of total collagen in the tendon may be, for some unknown reasons, a better correlate of this aspect of the tendon preparation. If the results of this study on the dog are generally relevant to the human, they may suggest that reconstruction of a torn ACL with a portion of the PT is not a problem, based solely on the strength of the whole PT as age progresses. We assume here that the properties of the graft are largely reflected in those of the whole tendon. In humans, some variations have been measured between properties of the medial and central thirds of the PT (Noyes et al., 1984). This oftenquoted study dealing with the relative strengths of the central third PT graft and the ACL is based on human tissues aged 26 k6 yr. The study indicates, on the average, that a central (14 mm wide) portion of the human patellar tendon has a strength 168% and structural stiffness 380% that of the ACL. The ratios would change slightly for the medial portion of the PT, which is sometimes used for reconstruction of the ACL. The use of approximately one-third of the patellar tendon would seem to allow extra strength and stiffness needed to account for any postsurgical remodeling and resorption of the graft (Butler, 1989). The above comments suggesting that the PT may be an even more superior graft tissue with increasing age, because it does not degenerate as rapidly as the ACL, must be tempered by questions about the dog model and whether a proportionately higher stiffness in a one-third PT graft would negatively jeopardize the final outcome of the reconstruction because of a greater mismatch in tensile properties between the graft and the damaged ACL. We also must qualify this
hypothesis since whole tendons were examined in the current study. Alterations due to age may, in fact, vary across the width of the patellar tendon. Additional experimental research using the dog model is needed to answer this question. The current study also suggests that clinical studies are needed on the more aged human patient. We hope this reseach, using a dog model, will provide some impetus for these studies. Acknowledgements-This research was partially supported by the Companion Animal Fund, College of Veterinary Medicine, Michigan State University. The authors acknowledge the help and valuable discussions with Dr Gregory Fink, Department of Pharmacology and Toxicology, on the statistical interpretation of these data. We also thank Aimee Farquhar, Kristina Haut, and David J. Licciardello for their valuable technical contributions to the research project.
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Ahn, A. and Stromberg, B. (1974) Vascular anatomy of the patellar and cruciate ligaments: a micrographic and histologic investigation in the dog. Acta Chir. stand. (Suppl.) 445,25-35. Amocxky, S. P. (1990) Animal models for knee ligament research. In Knee Ligaments: Structure, Functiorr~lnjury and Renair (Edited bv Daniel. D.. Akeson. W. and O’Connor, J.i pp. 401-417. Raven Press, New’York. Burks, R. T., Haut, R. C. and Lancaster, R. L. (1990) Biomechanical and histological observations on the dog patellar tendon after removal of its central one-third. Am. J. Sports Med. 18(2), 146-152. Butler, D. L. (1989) Anterior cruciate ligament: its normal response and replacement. J. orthop. Res. 7, 910-921. Butler, D. L., Hulse, D. A., Kay, M. D., Grood, E. S., Shires, P. K., D’Ambroska, R. and Shoji, H. (1983) Biomechanics of cranial cruciate ligament reconstruction in the dog. IImechanical properties. Vet. Surg. U(3), 113-118. Grood, E. S., Noyes, F. R. and Butler, D. L. (1977) Age related changes in the mechanical properties of knee ligaments. ASME Biomech. Symp. 23,213-216. Guan, Y. and Butler, D. (1990) Location-dependent variations in the material properties of anterior cruciate ligament subunits. ASME Adv. in Bioengng 17, 5-7. Hamlin, C. R. and Kohn, R. R. (1971) Evidence for progressive, age-related structural changes in post-mature human collagen. Biochem Biophys. Acta 236,458-467. Haut, R. C., Powlison, A., Rutherford, G. N. and Kateley, J. R. (1988) Some effects of donor age and sex on the mechanical properties of patellar tendon graft tissues. ASME Adv. in Bioengng 15, 75-78. Hubbard, R. P. and Soutas-Little, R. W. (1984) Mechanical properties of human tendon and their age dependence. J. biomech. Engng 106, 144-150. Johnson, S. G., Hulse, D. A., Hogan, H. A., Nelson, J. K. and Boothe, H. W. (1989) System behavior of commonly used cranial cruciate ligament reconstruction autografts. Vet. Surg. 18(6), 459-465. Mitchell, T. W. and Rigby, B. J. (1975) In vivo and in vitro aging of collagen examined using isometric melting technique. Biochem Biophys. Acta 393,531-541. Nikolaou, P. K., Seaber, A. V., Glisson, R. R., Ribbeck, B. M. and Bassett, F. H. (1986) Anterior cruciate ligament allograft transplantation. Am. .I. Sports Med. 14(5), 348-360. Nimni, M. E. and Harkness, R. D. (1988) Molecular structure and functions of collagen. In Collagen (Edited by Nimni M.), pp. l-77. CRC Press, Boca Raton, Florida. Nordin, M. and FrankeL V. H. (19801 Biomechanics of collagenous tissues. In &sic Biomech&cs of the Skeletal
Mechanical properties of the canine patellar tendon System (Edited by Frankel, V. H. and Nordin, M.), pp. 87- 110. Lea and Febiger, Philadelphia. Noyes, F. R., Butler, D. L., Grood, E. S., Zemicke, R. F. and Hefty, M. S. (1984) Biomechanical analysis of human ligament grafts used in knee ligament repairs and reconstructions. J. Bone Jr Surg. 66A, 344-352. Noyes, F. R. and Grood, E. S. (1976) The strength of the anterior cruciate ligament: age and species-related changes. J. Bone Jt Surg. 58, 1074-1082. Schnider, S. L. and Kohn, R. R. (1981) Effects of age and diabetes mellitis on the solubility and nonenzymatic glycosylation of human skin collagen. J. Clin. Invest. 67, 1630-1635. Shino, K., Kawasaki, T., Horose, H., Gotoh, I., Inone, M. and Ono, K. (1984) Replacement of the anterior cruciate ligament by an allogenic tendon graft. J. Bone Jt Surg. 66B, 672-681. Stegemann, H. (1958) Mikro vestimmung von hydroxyprolin mit chloramin-tund -dimethylamino benzaldehyd. Physiol. c/rem. 311(l), 41-45. Stouffer, D. C., Butler, D. L. and Hosney, D. (1985) The relationship between crimp pattern and mechanical response of human patellar tendon-bone units. J. biomech. Engng 17, 158-165. Vasseur, P. B., Pool, R. R., Amoczky, S. P. and Lau, R. E.
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(1985) Correlative biomechanical and histological study of the cranial cruciate ligament in dogs. Am. J. Vet. Res. 46, 1842-1854. Vasseur, P. B., Rod&o, J. J., Stevenson, S., Clark, G. and Sharkey, N. (1987) Replacement of the anterior cruciate ligament with a bone-ligament-bone anterior cruciate ligament allograft in dogs. Clin. Orthop. 219, 268-277. Viidik, A. (1972) Aging of collagen in complex tissues: a micro-methodical study of the thermal reaction. Experientia 15(6), 641-642. Vogel, H. B. (1978) Influence of maturation and age on mechanical and biochemical parameters of connective tissue of various organs in the rat. Corm. Tissue Res. 6, 83-94. Woo, S. L.-Y., Orlando, C. A., Camp, J. R. and Akeson, W. H. (1986) Effects of postmortem storage by freezing on ligament tensile behavior. J. Biomechanics 19(5), 399404. Woo, S. L.-Y., Gomez, M. A., Sites, T. J., Newton, P. W., Orlando, C. A. and Akeson, W. H. (1987) The biomechanical and morphological changes in the medial collateral ligament of the rabbit after immobilization and remobilization. .I. Bone Jt Surg. 69A, 1200-1211. Yamada, H. (1970) Strength ofBiological Materials (Edited by Evans, F. G.), p. 273. Williams and Wilkins, Baltimore, MD.
