A comparative evaluation of the mechanical properties of the rabbit medial collateral and anterior cruciate ligaments

J. Biomechonics Vol. 25, No Printed in Great Britain

4. pp. 377-386,

1992. 0

0021-9290/92 $5.00+.00 1992 Pergamon Press plc

A COMPARATIVE EVALUATION OF THE MECHANICAL PROPERTIES OF THE RABBIT MEDIAL COLLATERAL AND ANTERIOR CRUCIATE LIGAMENTS SAVIOL-Y. Woo, PETER 0. NEWTON, DEIDRE A. MACKENNA and ROGER M. LYON Orthopaedic Bioengineering Laboratory, University of California, San Diego and Veterans Affairs Medical Center. San Diego, California, U.S.A. and Musculoskeletal Research Laboratories, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A.

Abstract-The biomechanical properties of the medial collateral and anterior cruciate ligaments from 30 New Zealand White rabbits were measured. Because ofits complex geometry, the ACL was divided into two portions (medial and lateral) to provide uniform loading. This allowed an examination of the intraligamentous properties. A laser micrometer system was used to measure the cross-sectional area for tensile stress and a video dimension analyzer was used to measure the strain. The mechanical properties (stress-strain curves) of the MCL and ACL were different, with the modulus (determined between 4 and 7% strain) in the MCL (1120+ 153 MPa) more than twice that of either portion of the ACL (516+64 and 516 + 69 MPa for the medial and lateral portions, respectively). This higher modulus correlated with the more uniform and dense appearance of the collagen fibrils examined with scanning electron microscopy (SEM).

INTRODUCTION

The medial collateral (MCL) and anterior cruciate ligaments (ACL) are two commonly injured knee ligaments that have vastly different intrinsic healing responses. Clinical studies have demonstrated that the MCL has a reparative capacity which generally results in normal joint function within months of rupture (Fetto and Marshall, 1978; Jones et nl., 1986). This observation has been substantiated quantitatively in experimental studies using dogs and rabbits (Woo et al., 1987b; Frank et ul., 1983). On the other hand, the ruptured ACL is much more problematic. Reconstitution into a functional unit after a complete midsubstance tear of the ACL is rare (Girgis et al., 1975; Hawkins et a/., 1986; Noyes et al., 1983; G’Donoghue et al., 1966). These marked differences have prompted substantial interest in characterizing the differences in the biomechanical, biochemical and histological properties of these two ligaments. Distinguishing histological characteristics of the MCL and ACL at the light microscopic level have also been demonstrated. Fibroblasts of the MCL are spindle-shaped and are randomly distributed amongst the collagen, while those of the ACL are more often oval and arranged in columns between collagen fibers (Frank et al., 1983; Noyes et al., 1983; Vasseur et al., 1985). Unique ultrastructural features of the fibroblasts and pericellular region of the MCL and ACL have been documented using transmission electron microscopy (TEM) (Lyon et al., 1989). Biochemical analysis of these two ligaments have revealed similar Type I collagen content; however, differences in the

6 August 1991. Address correspondence to S. L-Y. Woo, Department of Orthopaedic Surgery, M268 Scaife Hall, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. Received in final form

Type III collagen content, glycosaminoglycan content and collagen cross-links have been noted (Amiel et al., 1984). In light of these histological and biochemical differences between the MCL and ACL, a comparative evaluation of the biomechanical properties of these ligaments would be of interest. Kennedy et al. (1976) evaluated the tensile behavior of the isolated human MCL and ACL and found similar structural properties (load-elongation curves) for these ligaments. Butler er af. (1986) compared the mechanical properties (stress-strain relationships) of ligaments around the human knee with those of patellar tendon and noted differences between the ligaments and patellar tendon. However, the ACL, posterior cruciate ligament, and lateral collateral ligament were found to behave similarly. More recently, Butler et al. (1991) have made intra-articular comparisons of the ACL comparing the anteromedial, anterolateral and posterior bundles of human specimens. They found that the anteromedial and anterolateral bundles had similar mechanical properties. The posterior bundle had a similar strain at failure, but had a lower maximum stress and strain-energy density than the other two bundles. The mean modulus of the posterior bundle was approximately 50% lower than the anterior bundles, but it was not statistically different. In human studies, however, it is difficult to control many variables such as age, sex, and activity levels, which have been shown to affect the mechanical properties of ligaments (Woo et al., 1990b, 1991). This study offers a controlled animal model in which the mechanical properties of the rabbit MCL and ACL were compared in sex- and age-matched animals. In addition, to alleviate the complex geometry, the ACL was divided into two portions, allowing intra-ligamentous properties to be examined. Finally, in order to interpret the results, the ligament morphology was

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examined using scanning electron microscopy (SEM) and the ultrastructural appearance was correlated with the mechanical properties. MATERIALS

AND METHODS

Specimen preparation

From each of the 30 skeletally mature New Zealand White rabbits (4.8 + 0.2 kg, mean body mass + S.E.) one hind limb was randomly chosen, disarticulated at the hip, wrapped in two airtight plastic bags and stored at -20°C prior to tensile testing. This storage method has been demonstrated to have no effect on the mechanical properties of the ligaments (Woo et al., 1986). On the day of testing, the limbs were thawed and the skin and muscles removed. For 10 legs, the soft tissues of the knee were removed except for the MCL, yielding a femur-MCL-tibia complex (FMTC). The distal 3-4 mm of the femur and the proximal 3-4 mm of the tibia were resected using a bone saw [Fig. l(A)] to allow for the measurement of the MCL crosssectional shape and area. Care was taken not to disturb the MCL insertion sites. Two thin black parallel lines (using VerhoetT’s elastin stain) were placed across the medial aspect of the ligament, 1 cm apart, centered at the joint line, to define a gauge length for strain determination. For the remaining 20 rabbit knees, the preparation was performed in a similar fashion, except that the ACL was left intact, creating a femur-ACL-tibia complex. Because the ACL is arranged in an anatomically complex fashion and various portions of the

MCL

COMPLEX

WOO

et al.

ACL are of different lengths (Fig. 2), it is impossible to simultaneously and uniformly load all portions of the ACL during a simple tensile test of the femur-ACL-tibia complex. Therefore, in 10 knees, the lateral portion of the ACL was transected and removed as demonstrated in Fig. 3, leaving the medial portion intact, creating a femur-ACL/medial portion-tibia complex (FATC,). This preparation reduced the geometric complexity and allowed a more uniform stress distribution in the ligament during tensile testing. For the remaining 10 knees, the medial portion was transected and removed, leaving a femur-ACL/lateral portion-tibia complex (FATCL). Similar to the FMTC, a portion of the lateral femoral condyle distal to the ACL insertion was resected along with the entire medial femoral condyle for area measurement and for visualization during strain determination with the VDA [Fig. l(B)]. Care was taken so that the insertion sites of the portion of the ACL being tested were not disturbed. Again, two stain lines were placed on the surface of the ligament (0.5 cm apart) as a gauge length for strain measurement. Specimens were kept moist with physiologic saline solution throughout the preparation. Cross-sectional shape and area determination

The cross-sectional shape and area in the region between the gauge length markers of the MCL as well as of the medial and lateral portions of the ACL were measured. Each specimen was mounted in a specially designed jig to be placed on the laser micrometer system (Lee and Woo, 1988), with the ligament held

ACL COMPLEX

90*

-

ROTATION

ACL

MCL-

A

B

Fig. 1. Schematicsof the (A)femur-MCL-tibia complex (FMTC) and (B)femur-ACL/medial portion-tibia complex (FATC,). Note that portions of the condyles were resectedfor cross-sectional shape determination. The ligament insertion sites were not disturbed.

Fig. 2. Posterior view of the rabbit ACL. The sulcus is used as a landmark to divide the medial and lateral portions of the ACL (see arrow).

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Fig. 8. Scanning electron photomicrographs (900 x ) of ligament cross sections (prepared by L. Kitabayashi and A. Sisk). (A) MCL: showing densely packed collagen fibers distribmed uniformly. (B) ACL: showing collagen fibers arranged in a fascicular pattern, with a large interfasicular region (arrows).

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An evaluation of rabbit ligament mechanical properties

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al

Fia. 3. Schematic diagrams of the ACL beinn dissected into a medial Dortion suecimen. A similar division was performed for the lateral port&n. _

CUT EDGE OF FEMUR

Fig. 4. Schematic diagram of the laser micrometer system used to determine the cross-sectional shape and area of the ligament substance of an FMTC specimen. The specimen is placed in the path of the laser beam, creating a shadow equal to the ligament profile width. The specimen is rotated through 180”, with measurements taken at 3” increments.

perpendicular to the collimated laser beam. As the laser beam traversed the ligament, the width of the ligament shadow (profile width) and its distance from the top of the beam was detected by the laser beam receiver (accuracy 0.001 mm). The ligament was rotated within the path of the laser beam, at 3” angular increments through 180”, and the corresponding profile widths of the ligament were recorded (Fig. 4). The cross-sectional shape of the ligament was reconstructed using a computer algorithm described by L?, and Woo (1988), and the cross-sectional area was mte.grated using Simpson’s rule. The measurement process requires l-2 min per specimen and has been shown to be accurate and reproducible (Woo et al., 1990a). BM

25:4-D

These cross-sectional area data were used for the determination of tensile stress in the ligament. Tensile testing Medial collateral ligament. Techniques used in our laboratory to measure the stress-strain relationship of the FMTC have been previously described in detail (Woo et al., 1983). The femur and tibia were rigidly fixed in specially designed clamps using 3.2 mm diameter pins and mounted on an Instron testing machine (Canton, MA), with the knee in 90” of flexion. The clamps were adjusted so that the applied tensile load was in line with the longitudinal axis of the MCL [Fig. 5(A)]. A small tare load of 0.5 N was applied to

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ACL

Fig. 5. The clamps used for tensile testing of the bone-ligament-bone complexes. Note that the tensile load is applied in line and along the length of the ligament. Thin parallel black lines are placed on the ligament substance to serve as gauge length markers for strain measurement. (A) FMTC clamp. (B) FATC clamp: the specimen is mounted such that the medial or lateral portion of the ACL is untwisted and, thus, can be loaded uniformly in tension. the FMTC to establish a uniform position from which to begin tensile loading. Cyclic preconditioning between 0.0 and 0.5 mm extension (approximately 0 and 3% strain of the MCL substance, respectively) at an extension rate of 10 mm min- ’ was performed for 10 cycles. The FMTC was then loaded to failure at the same rate, and the load-elongation curve was obtained. The test was recorded on videotape for strain analysis using the video dimension analyzer (VDA) system (see description below). The mode of failure of each bone-ligament-bone complex was noted. Anterior cruciate ligament. The FATCu and FATC, were tested in a fashion similar to the FMTC. The tibia and femur were fixed in clamps with 2.0 mm diameter Steinmann pins and methylmethacrylate bone cement. The clamp allowed for untwisting of the ligament specimen and adjustments to obtain the alignment of tensile load along the ligament [Fig. 5(B)]. Again, a 0.5 N tare load was applied, and cyclic preconditioning between 0.0 and 0.3 mm of extension (approximately 0 and 3% strain of the midsubstance of the ligament) at a rate of 10 mm min- ’ was performed for 10 cycles. Tensile loading to failure was then performed at 10 mmmin-’ and a load-elongation curve was obtained. The tests were

for strain determination using the VDA system and the failure modes of the boneligament-bone complexes were noted. videotaped

Tensile-strain determination Strain of the ligament substance was determined using the VDA system (Woo et al., 1983). The two windows in the VDA system automatically triggered on the gauge length markers (the two dark Verhoeff stain lines) placed on the ligament. A voltage output from the VDA represented the initial distance between the gauge length markers (I,). This initial distance corresponds to the ligament under the tare load of 50 g to provide a reproducible gauge length. During tensile stretch, the distance between the markers (I) increased, resulting in a corresponding increase in the output voltage. The strain of the ligament substance was defined as [(f-I,)/&,]. The frequency response of the VDA system is 120 Hz. This method has previously been demonstrated to be accurate to within 0.5% (Woo et al., 1983). Statistical analysis Using the cross-sectional area of the ligament and the strain determinations, the load-elongation curves

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An evaluation of rabbit ligament mechanical properties were transformed into stress-strain curves to represent the mechanical behavior of the ligament substance. The properties were compared for statistical significance using a one-way analysis of variance (ANOVA). The individual differences between groups were further evaluated using multiple t-tests, utilizing the Bonferroni procedure to evaluate the statistical significance. A confidence level of 95% was chosen to define significance. Ultrastructure

The cross-sectional shape of the MCL was found to be generally oblong, with a width to thickness ratio of approximately 3. The individual portions of the ACL were found to be more ovoid in cross section (Fig. 6). Table 1 shows the cross-sectional areas of the MCL and the two portions of the ACL and demonstrates that the two portions of the ACL are similar to each other. Cyclic stress relaxation

The cyclic stress relaxation data for the MCL, medial and lateral portions of the ACL were obtained and compared. The normalized stress (peak stress for each cycle/peak stress of the first cycle) was found to gradually decrease during the 10 cycles of loading and unloading. The normalized stress at the 10th cycle for both the MCL and the two portions of the ACL (Table 1) was not statistically different (p>O.OS). Table 1. Cross-sectional area and cyclic relaxation data for each bone-ligament-bone complex. Statistical significance was not demonstrated in either case

MCL ACL: medial ACL: lateral

-

60

MCI_

70

--c-

ACL -- Medial Portion

60

-

ACL -- Lateral Portion

I

50 40 30 20 10 0

0

I

2

3

4

5

6

7

Fig. 7. Mechanical properties of the mid-substance of the MCL and ACL as represented by the stress-strain curves.

Cross-sectional shape and area

Ligament

Fig. 6. Typical cross-sectional shape for the MCL and the two portions of the ACL. The reconstructed area and shape are obtained from the laser micrometer system (see Fig. 4).

Strain (%)

RESULTS

(mm2)

Cyclic relaxation at 10th cycle (% of first cycle)

3.4kO.2 3.6kO.4 3.5kO.3

87&l 88&l 90*1

Cross-sectional area

Medial ACL

Lateral ACL

MCL

Two additional skeletally mature animals of similar body mass were used to evaluate the ultrastructural differences between the MCL and ACL. From each animal, one limb was utilized to examine the MCL and the contralateral limb was used to examine the ACL. Cross sections from the mid-joint region of the ligaments were examined using scanning electron microscopy (SEM). Fresh specimens were immediately fixed in 2.5% glutaraldehyde in phosphate buffer for 12-24 h. The specimens were then post-fixed in 2% osmic acid for 224 h, dehydrated in alcohol and Freon, critical-point-dried, and carbon-coated. A Cambridge 360 scanning electron microscope was utilized to view the specimens at magnifications between 900 and 2500 times.

Mechanical properties of ligament substance

The mechanical properties of the MCL and the medial and lateral portions of the ACL were found to be different (Fig. 7). The modulus (slope of the stress-strain curve measured between 4 and 7% strain) was significantly greater for the MCL (1120 f 153 MPa) than either the medial (516+64 MPa) or lateral (516f69 MPa) portions of the ACL (pO.5). The failure modes for the three different structures also varied (Table 2). The failure of the ligament was defined as the point where the bone-ligament-bone complex could no longer bear load. The type or location of the failure was characterized as defined below. A ‘substance’ failure was defined as the one in which the ligament substance ruptured without evidence of failure of the insertion sites. An ‘avulsion’ failure was defined as the one where the ligament substance appeared intact and the specimen failed at an insertion to bone. Finally, a ‘combined’ failure was

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384

Table 2. Distribution of failure modes for the boneligament-bone complexes Failure mode Substance Avulsion Combined (substance and avulsion)

FMTC

FATCM

FATC,

10 0 0

4 1 5

1 9 0

defined as the one where both ligament substance rupture and avulsion occurred. All FMTC specimens failed in the mid-substance of the MCL, while the FATCu specimens had four substance failures and only one FATC, specimen had a substance failure. The tensile strength and the ultimate strain for the MCL substance were 110f 19 MPa and 14.5 f l.O%, respectively. These data agreed well with the previous data from our laboratory (Woo et al., 1987a). Due to the varied failure modes, the tensile strength and the ultimate strain for both portions of the ACL could not be determined or compared statistically. However, the MCL specimens were 77% higher in tensile strength than the four FATC, that failed in the ligament substance. Note, however, that the stress and strain at failure values of the combined failure specimens (5/10 FATCu specimens) were only slightly lower than the tensile strength and the ultimate strain of the specimens that failed in the ligament substance (4/10 specimens), i.e. 51.7k5.9 MPa vs 62.3k5.2 MPa for the stress at failure (p> 0.2) and 12.0+0.5% vs 12.5 + 1.5% for strain at failure (p>O.5), respectively. Morphology

Examination of the MCL and ACL under SEM showed marked differences between the tissues (Fig. 8). The ACL has a distinct fascicular structure with large amounts of interfascicular space and loose connective tissue (shown with arrow) that appears to be absent in the MCL. Additionally, the collagen fibrils aligned with the long axis in the MCL appear to be more densely packed and more uniformly distributed than in the ACL. DISCUSSION

This study demonstrates the differences in the mechanical properties of the MCL and ACL by tensile testing of the bone-ligament-bone complexes. Noncontact testing methods were employed to measure the cross-sectional shape and area (using the laser micrometer system) and ligament substance strain without the contribution of the ligament insertion sites (using the video dimension analyzer). As a result, the stress-strain curves of the ligament substance were determined. Furthermore, in this study the tensile loads were applied in line with the longitudinal axis of the ligament substance. In the case of the MCL, this is rather straightforward as it has a relatively uniform

cross section. On the other hand, the complex anatomy of the ACL presents difficulties which make simultaneous loading of all the collagen fibers of the entire ligament nearly impossible. Butler et al. (1986, 1991) as well as our research group (Hollis et al., 1988) have dealt with this problem in the human ACL by dividing the ligament into several bundles, and testing each bundle individually in order to achieve a more uniform tensile-stress distribution in the tissue. This concept was employed in this study. In the rabbit, two portions of the ACL can often be identified. Posteriorly, a sulcus frequently exists that can be used to separate the medial and lateral portions of the ACL (Fig. 2). The portions can be divided and untwisted providing a relatively uniform specimen. As a result, the mechanical properties of the two portions of the ACL during uniaxial testing can be evaluated accurately. These portions are not intended to represent a functional division of the rabbit ACL; instead, the division is made to allow uniform loading. Any variation that is introduced during dissection of the ACL portions is accounted for by determination of the mechanical properties (i.e. the stress represents the load normalized by the area of the sample). Butler er al. (1986) reported the human ACL, posterior cruciate ligament and lateral collateral ligament to have similar modulus, tensile strength and strainenergy density, but these mechanical properties were significantly different from those of the patellar tendon. In this study, the mechanical properties of the MCL substance of skeletally mature rabbits were found to be different from those of the medial and lateral portions of the ACL. The modulus and the stress at failure for the MCL were found to be significantly greater than for either portion of the ACL. Additionally, the cross-sectional area and shape of these ligaments varied. The whole MCL had approximately the same area as one half of the ACL. This, combined with the difference observed in the modulus, might suggest that the structural properties of these two ligaments may be similar. More recently, Butler et al. (1991) have studied the intra-ligamentous properties of the ACL. The human ACL was divided into three bundles: anteromedial (AMB), anterolateral (ALB), and posterior (PB). They found that the properties were similar for the AMB and ALB. However, the PB specimens had a significantly lower maximum stress and strain-energy density than the ABM and ALB. The modulus of the PB was almost half that of the AMB and ALB (154.9 f 119.5 MPa vs 283.1+ 114.4 and 284.9& 140.6 MPa, respectively), but statistical significance could not be shown-presumably due to high specimen variation. In the rabbit, the existence of a posterior bundle is not readily apparent. However, the similarity of the AMB and ALB in the human is paralleled by the similarity of the medial and lateral portions of the rabbit ACL shown in this study. The differences in the failure mode indicate differences in the relative strength of the ligament substance

An evaluation of rabbit ligament mechanical properties and the insertions to bone for the MCL and ACL. In the skeletally mature rabbit, the MCL insertion to bone is stronger than the ligament substance as all failures occurred in the MCL substance. In contrast, the medial and lateral portions of the rabbit ACL failed often as a combination of the insertion sites and ligament substance, suggesting that they have relatively equal strengths. This is further supported by noting that the stresses and strains at failure for the medial ACL specimens were similar in the cases of substance and combined failure modes A knowledge of the differences in the microscopic and ultrastructural appearance of the MCL and ACL may help in understanding their differences in the mechanical properties. Danyichuk et af. (1978) have clearly demonstrated in the human ACL, regions of ‘endotenon’, which appear to bind longitudinal collagen fibers into subfascicles and fascicles. Yahia and Drouin (1989) have recently demonstrated a similar arrangement in the canine ACL. This interfascicular region of endotenon is present in the rabbit ACL as well. The collagen fibers of the MCL, on the other hand, are packed closely together without a prominent interfascicular region of loose connective tissue. Cross-sectional scanning electron micrographs demonstrate this dramatic difference in collagen fiber arrangement (Fig. 8). The cross-sectional area of longitudinally oriented collagen fibrils per unit of ligament cross-sectional area appears less in the ACL than in the MCL. Recent data from our laboratory show that the amount of longitudinally oriented collagen in the ligament is in fact higher in the rabbit MCL than the ACL by approximately 15%. We have also shown that the distribution of the fibril diameters is different in the two ligaments (Hart et al., 1991). These differences may explain, in part, the lower modulus and stress at failure for the ACL in comparison with the MCL (Newton et al., 1990). Biochemical differences between these ligaments have been demonstrated previously. The rabbit cruciate ligaments have a higher percentage of Type III collagen (12%) compared to the MCL (9%). Also, the cruciate ligaments have twice the amount of glycosaminoglycans. In addition, the reducible collagen cross-link, dihydroxylysinonorleucine, is present in greater amounts in the cruciate ligaments compared to the MCL (Amiel et al., 1984). The role of parallel collagen fibers in resisting tensile force is well recognized (Danielsen, 1981; Minns and Soden, 1973; Vogel, 1974). However, the role of proteoglycan-collagen (Minns and Soden, 1973) and collagen-collagen (Lapiere et al., 1977) interactions are less well understood. The differences in the biochemical composition in addition to the cellular and ultrastructural characteristics may help to explain the differences in the stress--strain relationships of the MCL and ACL. Additional investigations involving the cellular and molecular basis for the differences in these tissues are of interest. If these basic properties can be better characterized, then it may be possible to understand

385

the different healing responses of these ligaments and to alter the treatment in the hope of yielding improved results. Acknowledgements-Supported by VA RR&D Grant No. A188-3RA, an NSF Graduate Fellowship, and NIH Grants AR01484 and AR34264 The technical assistance of Mr J. Xerogeanes and Mr J. Weiss is gratefully acknowledged.

REFERENCES

Amiel, D., Frank, C., Harwood, F., Fronek, J. and Akeson, W. (1984) Tendons and ligaments: Morphological and biochemical comparison. J. orthop. Res. 1, 257-265. Butler, D. L., Guan, Y., Kay, M. D., Feder, S. M. and Cummings, J. F. (1991) Location-dependent variations in the material properties of anterior cruciate ligament subunits. Trans. orthop. Res. Sot. 16, 234. Butler, D. L., Kay, M. D. and Stouffer, D. C. (1986) Comparison of material properties in fascicle-bone units from human patellar tendon and knee ligaments. J. Biomechanits 19, 425-432. Danielsen, C. C. (1981) Mechanical properties of reconstituted collagen fibrils. Conn. Tissue kes: 9, 52-V. Danvlchuk. K. D.. Finlav. J. B. and Kraek. J. P. (1978) M&rostructural organ&ion of human and bovine cruciate ligaments. Clin. Orthop. 131, 294298. Fetto, J. F. and Marshall, J. L. (1978) Medial collateral ligament injuries of the knee: a rationale for treatment. Clin. Orthop. 132, 206-218. Frank, C. B., Woo, S. L-Y., Amiel, D., Harwood, F. L., Gomez M. A. and Akeson. W. H. (1983) Medial collateral ligament healing: A multidisciplinary assessment in rabbits. Am. J Sports Med. 11, 379-389. Girgis, F. G., Marshall, J. L. and Monajem, A. R. S. (1975) The cruciate ligaments of the knee joint. Clin. Orthop. 106, 216231. Hart, R. A., Newton, P. 0. and Woo, S. L-Y. (1991) Quantitative morphology of the anterior cruciate and medial collateral ligaments. Trans. orthop. Res. Sot. 16, 181.

Hawkins, R. J., Misamore, G. W. and Merritt, T. R. (1986) Follow-up of the acute nonoperated isolated anterior cruciate ligament tear. Am. .I. Sports Med. 14, 205-210. Hollis, J. M., Marcin, J. P., Horibe, S. and Woo, S. L-Y. (1988) Load determination in ACL fiber bundles under knee loading. Trans orthop. Res. Sot. 13, 58. Jones, R. E. E., Bradford, H. and Francis, P. (1986) Nonoperative management of isolated Grade III collateral ligament injury in high school football players. Clin. Orthop. 213, 137-140. Kennedy, J. C., Weinberg, H. W. and Wilson, A. S. (1974) The anatomy and function of the anterior cruciate ligament. J. Bone Jt Surg. 56A, 223-236. Lapiere, C. M., Nusgens, B. and Pierard, G. E. (1977) Interaction between collagen Type I and Type III in conditioning bundles organization. Corm. Tissue Res. 5, 21-29.

Lee, T. Q. and Woo, S. L-Y. (1988) A new method for determining cross-sectional shape and area of soft tissues. .I. biomech. Engng 110, 110-l 14. Lyon, R. M., Billings, E., Woo, S. L-Y., Newton, P. O., Kitabayashi, L., Amiel, D. and Akeson, W. H. (1989) The ACL: A fibrocartilaginous structure. Trans. orthop. Res. sot. 14, 189.

Minns, R. J., and Soden, P. D. (1973) The role of the fibrous components and ground substance and the mechanical properties of biological tissues: A preliminary investigation. J. Biomechanics 6, 153-165. Newton, P. O., MacKenna,D. A., Danto, M. I., Kitabayashi, L. R., Akeson, W. H. and Woo, S. L-Y. (1990) Improved

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methodologies to differentiate the mechanical properties of the rabbit anterior cruciate ligament (ACL) and medial collateral ligament (MCL). Truns. orrhop. Res. Sot. 15,509. Noyes, F. R., Mooar, P. A., Matthews, D. S. and Butler, D. L. (i983) The symptomatic anterior cruciate-deficient’knee. J. Bone Jt Surg. 65A, 154-162. O’Donoghue, D. H., Rockwood, C. A., Frank, G. R., Jack, S. C. and Kenyon, R. (1966) Repair of the anterior cruciate ligament in dogs. .I. Bone Jt Surg. 48A, 503-519. Vasseur, P. B., Pool, R. R., Arnoczky, S. P. and Lau, R. E. (1985) Correlative biomechanical and histological study of the cranial cruciate ligament in dogs. Am. J. Vet. Res. 46, 1842-1854. Vogel, H. G. (1974) Correlation between tensile strength and collagen content in rat skin. Effect of age and cortical treatment. Conn. Tissue Res. 2, 177-182. Woo, S. L-Y., Danto, M. I., Ohland, K. J., Lee, T. Q. and Newton, P. 0. (1990a) The use of a laser micrometer system to determ&e thd cross-sectional shape and area of ligaments: A comparative study with two existing methods. J. biomech.Engng 112, 426-431. Woo, S. L-Y., Gomez, M. A., Seguchi, Y., Endo, C. M. and Akeson, W. H. (1983) Measurement of mechanical properties of ligament substance from a bone-ligament-bone preparation. J. orthop. Res. 1, 22-29.

Woo, S. L-Y., Gomez, M. A., Sites, T. J., Newton, P. O., Orlando, C. A. and Akeson, W. H. (1987a) The biomechani&aI and morphological changes in the medial collateral ligament of the rabbit after immobilization and remobilizatyon. J. Bone Jt Surg. 69A, 1200-1211. Woo, S. L-Y., Hollis, J. M., Adams, D. J., Lyon, R. M. and Takai, S. (1991) Tensile properties of the human femur-anterior cruciate ligament-tibia complex: the effects of specimen age and orientation. Am. .I. Sports Med. 19, 217-225. Woo, S. L-Y., Inoue, M., McGurk-Burleson, E. and Gomez, M. A. (1987b) Treatment of the medial collateral ligament injury.‘dm. i. Sports Med. 15, 22-29. Woo, S. L-Y., Ohland, K. J. and Weiss, J. A. (1990b) Aging and sex-related changes in the biomechanical properties of the rabbit medial collateral ligament. Mech. Ageing Deuelop. 15, 129-142. Woo, S. L-Y., Orlando, C. A., Camp, J. F. and Akeson, W. H. (1986) Effects of postmortem storage by freezing on ligament tensile behavior. J. Biomechanics 19. 399404. Yahia, L.-H. and Drouin, G. (1989) Micros&pica1 investigation of canine anterior cruciate linament and-1 natellar -~-~ tendon: collagen fascicle morphology and architecture. J. orthop. Res. 7, 243-251.