Journal of Orthopaedic Research 8:54%5554 Raven Press, Ltd., New York 0 1990 Orthopaedic Research Society
Acute Hemarthrosis: A Histological, Biochemical, and Biomechanical Correlation of Early Effects on the Anterior Cruciate Ligament in a Rabbit Model Kenneth K. Ishizue, Roger M. Lyon, David Amiel, and Savio L-Y. Woo Division of Orthopaedics and Rehabilitation, University of California at San Diego, La Jolla, California, U.S.A.
Summary: The early histological, biochemical, and biomechanical characteristics of the anterior cruciate ligament (ACL) were determined in a rabbit model of acute hemarthrosis. The ACLs of 19 rabbits were given seven consecutive daily knee injections of 2 ml of fresh autologous blood, and then compared to contralateral ACLs from control knees injected with 2 ml of lactated Ringer’s solution daily for 7 days. The rabbits were then sacrificed. Synovial proliferation with iron deposition within synoviocytes was observed; however, the architecture of the ACL was maintained. Additionally, the total collagen content, collagenase activity, and biomechanical properties of the ACL were unaltered. Key Words: Hemarthrosis-Anterior cruciate ligament (ACL)-Collagenase-Biomechanics of ACL.
environment, may contribute to the poor results reported with primary ACL repair. This is supported by earlier animal studies from our group in a model of ACL injury and by clinical and animal studies of chronic hemarthrosis . The presence of blood in the joint seems to promote changes in intra- and periarticular structures that can be detrimental to ligament healing. Longterm exposure to blood as seen in chronic hemarthrosis, such as cases of hemophilia, has been shown to produce a degenerative arthritis (8,17). Animal models of chronic hemarthrosis have confirmed the detrimental effects of intra-articular blood on articular cartilage although the exact mechanism by which blood elicits the observed changes remains unclear (2,7,13,18). Fabry in 1982 (3) proposed that proteoglycan degrading enzymes were responsible for the degenerative changes seen in hemophilic arthropathy. In Fabry’s study (3), the lysosomal enzyme cathepsin D was used as an indicator of this activity. Few investigators have studied the effect of blood on other articular structures such as the anterior
Recent advances in the understanding of knee biomechanics have shown the importance of the anterior cruciate ligament (ACL) in joint function and the importance of the preservation or restoration of this structure when possible. Acute injuries of the ACL are often associated with hemarthrosis and, unlike other ligamentous injuries such as the medial collateral ligament (MCL), are much less likely to heal. This may in part be due to certain anatomic features of the ACL. The ACL is an intra-articular structure covered with a synovial sheath. Once injured, the ACL has a tendency to retract, thus losing approximation of the ruptured ligament ends. Results of reapproximation of this structure with primary repair have been disappointing. This suggests that other factors are likely to be involved in failure, such as potential deleterious effects of the synovial environment. A break in the synovial sheath of the ACL, and exposure to the synovial Received March 10, 1989; accepted December 14, 1989. Address correspondence and reprint requests to Prof. D. Amiel at University of California at San Diego, M-030, La Jolla, CA 92093, U.S.A.
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EFFECTS OF ACUTE HEMARTHROSIS ON RABBIT ACL
cruciate ligament (ACL). In an animal model, Pforringer (14) has demonstrated small decreases in the biomechanical properties of the femur-ACGtibia complex (FATC) after hemarthrosis, as well as histological changes (15) in the ACL substance. Clinically, intra-articular structures are exposed to blood in association with acute traumatic knee injury. Noyes (12) has shown that 72% of patients presenting with an acute hemarthrosis of the knee have an acute tear of the ACL. Studies from our laboratories (10) demonstrated in a rabbit model of acute hemarthrosis that the menisci (lateral and medial), which lack a synovial covering, demonstrated an increase in degradative activity assessed by collagenase activity. Furthermore, in a rabbit model of acute injury in which the ACL was surgically sectioned, we observed rapid changes in the histologic features, total collagen content, and collagenase activity of the “injured” ACL (1). Since clinical ACL injuries are often associated with hemarthrosis, we sought to investigate the early effects of hemarthrosis over a time period similar to our previous study. We suspected that soft tissue structures with direct exposure to the articular environment (such as the menisci) would be more greatly affected by proteases than the ACL, which is protected from the articular environment by its synovial sheath. The purpose of this investigation was to determine the early effects of hemarthrosis on histological, biochemical, and biomechanical properties of the ACL. MATERIALS AND METHODS Animal Model The knees of 19 male New Zealand white rabbits (body weight of 3.0 0.2 kg) were used as a model of acute hemarthrosis. Two milliliters of autologous blood harvested fresh from ear veins were injected once daily into the left knee for seven consecutive days. Two milliliters of lactated Ringer’s solution was injected into right knees to serve as a sham control (16). Between injections, the animals were allowed cage activity. Animals were sacrificed on the 9th day. After the animals were killed, all knees were evaluated for the absence of sepsis. Two animals were evaluated histologically. Eight animals were used for biochemical evaluation. Nine animals were used for the biomechanical properties of the ACL.
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Histology The ACLs were harvested, placed in cassettes, and fixed in 10% buffered formalin. Specimens were mounted in paraffin, and 6 pm sections were stained with both hematoxylin and eosin (H&E) and Prussian Blue. These sections were evaluated for overall tissue organization, synovial proliferation, and iron deposition. Biochemistry Since collagen represents the major structural protein of the ACL and menisci and is responsible for most of the tensile strength of this tissue, assays for total collagen and collagenase were used. At the time of sacrifice, the ACL was aseptically harvested, rinsed with sterile phosphate-buffered saline, and placed individually in 1 ml of serum-free Earle’s MEM (Irvine Scientific Co., Irvine, CA) with the addition of fungibact, nonessential amino acids, L-glutamine, and vitamin E. Tissues were incubated in 35 mm diameter petri dishes (Costar, Cambridge, MA) and maintained in a humidified environment of 95% oxygen and 5% C 0 2 for 3 days. At the end of the incubation period, the media were harvested and assayed immediately for collagenase activity. Residual tissue was defatted with ether/ acetone, dried, and dry weights obtained. The total collagen content of the residual tissue was determined. Collagenase Assays
Acid-soluble type I collagen extracted from rabbit skin was acetylated with [‘4C]acetic anhydride by the method of Gisslow (5). This served as a substrate for collagenase determinations performed in a collagen film assay described by Johnson-Wint (9). The labeled substrate was suspended in 0.15 M phosphate buffer (pH 7.4 with 0.02% Na azide) and plated into a 96-well culture dish (Costar) to obtain 10,000 counts/min/well. Eighty microliters of media from each 3-day explant was assayed in duplicate. Ten millimolar CaCl,, 100 mM Tris buffer (pH 8. l), and fresh media were added to obtain a final reaction volume of 200 pl containing 1 mM Ca and 10 mM tris at neutral pH. At the end of incubation, the reaction volume (200 11.1) was mixed with 10 ml of betaphase scintillation cocktail (Westchem, Princeton, NJ) and counted for 1 min in a model LS6800 liquid scintillation spectrometer (Beckman, Ana-
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heim, CA). Sample counts were compared to an index group of controls of 0.1% clostridial collagenase and 0.01% TPCK-treated trypsin (Sigma, St. Louis, MO). Counts released with trypsin were assumed to represent the degradation of noncollagenous protein, or denatured collagen. Counts released above trypsin blanks were used as an indication that collagenase activity was present. Results were normalized by expressing counts per minute (cpm) per mg of dry weight of tissue from the original culture. The validity of the procedure for collagenase activity was confirmed with a separate set of incubations, with and without the presence of 2 mM EDTA (a calcium chelator and known inhibitor of collagenase activity). Products of degradation in the presence and absence of inhibitor were processed by acetone precipitation and SDS-PAGE chromatography. The characteristic primary degradation products of collagenase activity were identified, as well as their disappearance with the addition of EDTA. Seven animals were available to evaluate collagenase activity. Although connective tissue explants would be expected to release collagenase into media, it was assumed that by comparing experimental to control tissues, the in vivo effects of experimental hemarthrosis would be reflected by differences observed in vitro. The in vitro method of assaying media for collagenase activity was selected because of constraints of sample size and expected difficulties in attempting to purify collagenase from such small specimens. Total Collagen
Anterior cruciate ligaments remaining after incubation were dried, weighed, and ground in a Wiley mill. Aliquots weighing 3-5 mg were hydrolyzed in 6 N HC1 for 3 h at 130°C. The hydroxyproline content was determined as described by Woessner (21) and calculations made to express total collagen as a percentage of dry weight. Biomechanics The knees of nine animals underwent biomechanical analysis. Anterior-posterior (A-P) knee translation, as well as mechanical properties of the ACL substance, was determined. Immediately after sacrifice, the animals' hindlimbs were removed, wrapped in saline-soaked gauze, placed in plastic
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bags, and stored at -20°C for 1-3 weeks prior to testing. A-P Joint Translation
All muscle and soft tissues were carefully dissected from the knee, leaving only the knee ligaments and joint capsule intact. The femur and tibia were placed in a clamp used in previous studies (23), which allows the knee to be mounted on an Instron Universal Testing Machine at specifically chosen flexion angles. In this study, the joint was tested in both 45" and 90" of knee flexion. The tibia was aligned horizontally and the femur rotated in the sagittal plane to obtain either 45" or 90" of knee flexion. The loading axis was vertical, which provided an A-P load perpendicular with respect to the tibia. A cyclic load of +5 N was applied 10 times at a deformation rate of 10 mm/min. The loaddisplacement curve was recorded, and the total A-P translation was taken to be the maximum displacement between the peak loads. Tensile Testing
Following A-P translation test, the joint capsule and knee ligaments except for the ACL were removed. The lateral half of the ACL was removed, leaving the anteromedial (AM) bundle intact. This was done to allow more uniform loading of the ACL during tensile testing. To allow correct orientation of the ligament fibers, the femur and tibia were carefully trimmed using a rongeur until a small plug of bone (from 0.5-1.0 cm in diameter) remained at the proximal and distal ACL insertion sites. The femoral and tibia1 bone plugs were each mounted in 25 mm diameter spheres of polymethyl methacrylate (PMMA) bone cement. With this testing device, careful adjustments in the bone and ligament alignment allowed uniform loading of ligament fibers during mechanical testing. The cross-sectional area of the AM bundle of the ACL was measured using digital vernier calipers. Specimens were kept moist with 0.9% saline throughout the preparation. Dark elastin (Verhoff) stain lines approximately 7 mm apart were carefully applied to the ligament to act as gauge length markers for strain measurement by the Video Dimension Analyzer (VDA) system (22). The PMMA spheres and a bone-ligament-bone complex were mounted in the custom testing jig, which allowed adjustments in three translational
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EFFECTS OF ACUTE HEMARTHROSIS ON RABBIT ACL planes and around three rotational axes. Therefore, tensile loading could be applied uniformly along the long axis of the ligament. The bone-AM bundlebone complex was immersed in a 37°C physiological saline bath for 30 min. A preload of 0.5 N was applied, followed by cyclic preconditioning between 0 and 0.3 mm displacement for 10 cycles at a rate of 10 mm/min. The bone-ligament-bone specimen was then loaded to failure at a rate of 50 mdmin. The load vs. time curve was recorded on the Instron strip-chart recorder. The ligament strain vs. time response was measured by means of a VDA system. With this information, both structural properties of the femur-AM bundle-tibia complex, as well as mechanical properties of the AM bundle of the ACL, were determined (22). There was technical failure in tensile testing on one of the knees with hemarthrosis; therefore, it was not used in the analysis. Statistical Analysis Collagenase activity and total collagen values were compared between blood-treated and control tissues with a paired t test. A significance level of 0.05 was selected. A-P joint translation was compared using two-way analysis of variance (ANOVA), so that the effects of both hemarthrosis and knee flexion angle on A-P translation could be evaluated. Unpaired Student’s I tests were used to compare several parameters representing the mechanical properties, and a p value of c0.05 was considered to be significant.
RESULTS Gross
At the end of the treatment period, all bloodtreated knees exhibited a persistent bloody effusion. The ACL synovial sheath exhibited yellowbrown discoloration and appeared to be edematous. All knees were grossly free of infection. Random cultures of these knees confirmed that sterility had been maintained. Histology Hypertrophic synovium with synoviocytes laden with iron deposits were noted about the ACL from blood-treated knees (Fig. 1). In contrast, within the ligament substance, the overall cellular and matrix organization was unaltered in the experimental samples (Fig. 2A and B). Collagenase Collagenase activity in the media from the ACL showed no significant difference between bloodtreated (121.7 f 13 cpmlmg of dry weight) and control (114.1 k 25 c p d m g of dry weight) knees (p = 0.76) (Fig. 3). Total Collagen The ACL of the experimental knee showed a total collagen level of 82.4 2 1.3%, with a control of 80.6
FIG. 1. Blood-treated synoviurn (iron stain, ~ 1 0 ) .
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FIG. 2. (A) Blood-treated ACL, H&E stain ( ~ 4 0 )Note . the regular cellularity of the collagenous matrix. (B) Control ACL, H&E stain ( ~ 4 0 ) .
B
2 1.5%. These values were not significantly different (p = 0.33) (Fig. 4).
Biomechanics The results of a two-way ANOVA showed that the A-P joint translation in the setting of acute hemarthrosis was not significantly different (p > 0.05) from sham-injected knees at 90" flexion (0.7 2 0.1 and 0.8 k 0.1 mm, respectively), nor was there a difference (p > 0.05) at 45"flexion (1.2 ? 0.1 and 1.3 2 0.1 mm, respectively). There was, however, a significant effect (p < 0.05) of knee flexion angle, with greater A-P translation at 45" compared to 90" in both control and experimental knees. J Orthop Res, Vol. 8. No.4, 1990
The stress-strain curves obtained from the tensile tests of the AM bundle are shown in Fig. 5. The tangent modulus (defined between 4 and 7%), the tensile strength, and strain at failure are detailed in Table 1 using unpaired t tests. These parameters representing the mechanical properties of the bundles were not found to be significantly different (p > 0.4) between the hemarthrosis and control groups. During load to failure testing, six of eight hemarthrosis specimens (75%) failed within the ligament substance and, similarly, six of nine control specimens (67%) failed in the ligament substance. Thus, overall, 7 1% of the bone-ligament-bone preparations failed in the ligament substance, while 29% failed by either bony avulsion or pullout from the PMMA bone cement plug.
EFFECTS OF ACUTE HEMARTHROSIS ON RABBIT ACL
553
2ool
INJECTED
-
0 Ringers Q Blood
0
I
2
3
4
6
Strain (%)
"
ACL N=7 FIG. 3. Collagenase activity. Note no difference between blood-treated and control ACL (mean 2 SEM). Paired t test, p = 0.76.
All of the specimens were used for determination of tangent modulus; only those specimens that failed in the ligament substance were included for determinations of ligament ultimate strain and tensile strength. DISCUSSION
Most experimental models have studied the effects of chronic hemarthrosis on articular cartilage. The changes seen include intracellular deposition of iron in chondrocytes (4,8,17,18), an increase in protease activity from cartilage (3), a drop in glycosaminoglycan content of articular surfaces (2,17), decreased shear strength of cartilage (2), and associated early cartilage degeneration (3,7). It is apparent that the synovial lining is not a passive bystander in this process, as reports of synovial hyINJECTED
El Ringers Q Blood
r
8
FIG. 5. Rabbit ACL-AM bundle stress-strain curves.
pertrophy with intracellular deposits of iron (7,13,17) have noted. The synovitis thus elicited may promote secretion of proteases into the articular environment (6,20). Although this may in itself be responsible for the degenerative changes seen in hemophilic arthropathy, other postulated mechanisms include the introduction of enzymes from the granulocyte fraction of blood (19) and a direct effect of iron deposits on chondrocytes leading to detrimental alterations in cellular metabolism (8,18). Pforringer (14,15), in a two-part paper on the effects of acute hemarthrosis on mechanical and histologic properties of the ACL, noted a slight reduction in structural properties of the boneligament-bone complex with hemarthrosis. The differences increased with injury added to the synovial sheath of the ACL. The changes in the ultimate load for both cases were the most significant 4 weeks after surgery, with a 5N and 17 N decrease from a control load of 470 N for the hemarthrosis and synovectomy groups, respectively. At 1 week, the ultimate load values of the hemarthrosis group was 3 N less than the control value. Thus, the effect of hemarthrosis represented only a 1% decrease in the ultimate load. Statistical analysis was not presented. In our model of acute hemarthrosis, we have atTABLE 1. Mechanical properties of AM bundle of the ACL (mean L- SEM)
Group Control Hemarthrosis
N=8
FIG. 4. Total collagen. Note no difference between bloodtreated and control ACL (mean f SEM). Paired t test, p = 0.33.
p value (unpaired t test)
Tangent modulus (lo2 MPa)
Tensile strength" (MPa)
Strain at failure" (%)
6.0 f 0.5 (n = 9) 5.0 2 0.8 (n = 8)
51.2 f 4.3 (n = 6) 50.2 2 6.6 (n = 6 )
13.0 2 2.0 (n = 6 ) 13.0 1.5 (n = 6)
p > 0.4
p > 0.30
p > 0.50
Only samples that failed in ligament substance are used.
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tempted to correlate effects on the histological, biochemical, and biomechanical properties of an uninjured ACL. A proliferative synovitis was elicited. As noted by others, deposits of iron within the synovial cells were noted; however, the substance of the ACL showed no microscopic evidence of response to blood treatment. As well, no significant changes were noted in the total collagen content, collagenase activity, or biomechanical properties of the ACL. We have previously demonstrated an increase in meniscal collagenase in an identical model of acute hemarthrosis (10). Lindy et al. (11) have described the release of collagenase from human rheumatoid menisci and correlated higher levels of collagenase activity with increased severity of disease. They postulated that meniscal tissue has the ability to elaborate its own collagenase. It is possible that the collagenase activity observed from blood-treated menisci originated from the blood itself or an inflammatory response of synovial tissue to hemarthrosis. However, there is growing evidence that all connective tissues are metabolically active and have the ability to elaborate their own degradative enzymes. Although inconclusive, the elevated levels of collagenase may reflect the reaction of meniscus tissue to an altered and potentially hostile environment. In contrast, the ACL may be a privileged intra-articular structure, since it possesses a synovial covering that allows it to be protected from the articular environment. With trauma, synovial injury would be expected to occur, and this protective function of the ACL may be disturbed. Subsequent exposure of ligament substance to the intraarticular environment and hemarthrosis may produce changes in the ligament. Whether this mechanism may be responsible for the poor results reported with primary ACL repair is an important subject for further investigation. Acknowledgment: We would like to acknowledge financial support from NIH AR34264 and the Malcolm and Dorothy Coutts Institute for Joint Rehabilitation and Research. Also, the technical assistance of Ms. Deidre MacKenna, Dr. Peter Newton, Mr. Mike Furniss, and Ms. Linda Kitabayashi and the word processing skills of Ms. Fran Shepherd are gratefully acknowledged.
REFERENCES 1. Amiel D, Ishizue KK, Harwood FL, Kitabayashi L, Akeson WH: Injury of the anterior cruciate ligament: the role of
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collagenase in ligament degeneration. J Orthop Res 7:486, 1989 2. Convery FR, Woo SL-Y, Akeson WH, Amiel D, Malcom LL: Experimental hemarthrosis in the knee of the mature canine. Arthritis Rheum 1959, 1976 3. Fabry G: Early biochemical and histological findings in experimental haemarthrosis in dogs. Arch Orthop Traumat Surg 100:167, 1982 4. Ghadially FN, Oryschak AF, Ailsby RL, Mehta PN: Electron probe x-ray analysis of siderosomes in haemarthrotic articular cartilage. Virchows Arch [B] 16:43, 1974 5 . Gisslow MT, McBride BC: A rapid sensitive collagenase assay. Anal Biochem 68:70, 1975 6. Goldberg VM, Barstein A, Dawson M: The influence of a experimental immune synovitis on the failure mode and strength of the rabbit ACL. J Bone Joint Surg [Am] 64:900, 1982 7. Hoaglund FT: Experimental hemarthrosis. J Bone Joint Surg [Am] 49:285, 1967 8. Hough AJ, Barfield WG, Sokoloff L: Cartilage in hemophilic arthropathy; ultrastructural and microanalytical studies. Arch Pathol Lab Med 100:91, 1976 9. Johnson-Wint B: A quantitative collagen film collagenase assay for large numbers of samples. Anal Biochem 104:175, 1980 10. Ishizue KK, Lyon R, Amiel D, Woo SL-Y, Kitabayashi L, Harwood FL, Gomez M, Akeson WH: Hemarthrosis: a biochemical and mechanical evaluation of effects on the anterior cruciate ligament and menisci. Trans Orthop Res SOC 1 3 5 5 , 1988 11. Lindy S, Turto H, Sorsa T, Halme J, Lauhio A, Suomalainen K, Uitto V-J, Wegelius 0: Increased collagenase activity in human rheumatoid meniscus. Scand J Rheumatol 15:237, 1986 12. Noyes FR, Bassett RW, Grood ES, Butler DL: Arthroscopy in acute traumatic hemarthrosis of the knee. J Bone Joint Surg [Am] 62:687, 1980 13. Parsons JR, Zingler BM, McCeon JJ: Mechanical and histologic studies of acute joint hemorrhage. Orthopedics 10: 1019, 1987 14. Pforringer W: Hamarthros und kreuzbander-biomechanische untersuchangen teil 1 . Unfallchirugie 8:353, 1982 15. Pforringer W: Hamarthros und kreuzbander-morphologische untersuchungen teil 2. Unfallchirugie 8:368, 1982 16. Reagan FB, McInerny VK, Treadwell BV, Zarins B, Mankin HJ: Irrigating solutions for arthroscopy: a metabolic study. J Bone Joint Surg [Am] 65:629-631, 1983 17. Rippey JJ, Hill RR, Lurie A, Sweet M, Thonar E, Handelsman JE: Articular cartilage degradation and the pathology of haemophilic arthropathy. South Afr Med J 54:345, 1978 18. Roy S: Ultrastructure of articular cartilage in experimental hemarthrosis. Arch Pathol 86:69, 1968 19. Weissman G, Spilberg I, Krakauer K: Arthritis induced in rabbits by lysates of granulocyte lysosomes. Arthritis Rheum 12:103, 1969 20. Werb Z, Reynolds JJ: Stimulation by endocytosis of the secretion of collagenase and neutral proteinase from rabbit synovil fibroblasts. J Exp Med 140:1482, 1974 21. Woessner JF Jr: The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Siophys 93:440, 1961 22. Woo SL-Y, Gomez MA, Seguchi Y, Endo CM, Akeson WH: Measurement of mechanical properties of ligaments from a bone-ligament-bone preparation. J Orthop Res 1 :22-29, 1983 23. Woo SL-Y, Hollis JM, Roux RD, Gomez MA, Inoue M, Kleiner JB, Akeson WH: The effects of knee flexion on the structural properties of the femur-anterior cruciate ligament-tibia complex (FATC). J Biomech 20557-564, 1987
Acute Hemarthrosis: A Histological, Biochemical, and Biomechanical Correlation of Early Effects on the Anterior Cruciate Ligament in a Rabbit Model Kenneth K. Ishizue, Roger M. Lyon, David Amiel, and Savio L-Y. Woo Division of Orthopaedics and Rehabilitation, University of California at San Diego, La Jolla, California, U.S.A.
Summary: The early histological, biochemical, and biomechanical characteristics of the anterior cruciate ligament (ACL) were determined in a rabbit model of acute hemarthrosis. The ACLs of 19 rabbits were given seven consecutive daily knee injections of 2 ml of fresh autologous blood, and then compared to contralateral ACLs from control knees injected with 2 ml of lactated Ringer’s solution daily for 7 days. The rabbits were then sacrificed. Synovial proliferation with iron deposition within synoviocytes was observed; however, the architecture of the ACL was maintained. Additionally, the total collagen content, collagenase activity, and biomechanical properties of the ACL were unaltered. Key Words: Hemarthrosis-Anterior cruciate ligament (ACL)-Collagenase-Biomechanics of ACL.
environment, may contribute to the poor results reported with primary ACL repair. This is supported by earlier animal studies from our group in a model of ACL injury and by clinical and animal studies of chronic hemarthrosis . The presence of blood in the joint seems to promote changes in intra- and periarticular structures that can be detrimental to ligament healing. Longterm exposure to blood as seen in chronic hemarthrosis, such as cases of hemophilia, has been shown to produce a degenerative arthritis (8,17). Animal models of chronic hemarthrosis have confirmed the detrimental effects of intra-articular blood on articular cartilage although the exact mechanism by which blood elicits the observed changes remains unclear (2,7,13,18). Fabry in 1982 (3) proposed that proteoglycan degrading enzymes were responsible for the degenerative changes seen in hemophilic arthropathy. In Fabry’s study (3), the lysosomal enzyme cathepsin D was used as an indicator of this activity. Few investigators have studied the effect of blood on other articular structures such as the anterior
Recent advances in the understanding of knee biomechanics have shown the importance of the anterior cruciate ligament (ACL) in joint function and the importance of the preservation or restoration of this structure when possible. Acute injuries of the ACL are often associated with hemarthrosis and, unlike other ligamentous injuries such as the medial collateral ligament (MCL), are much less likely to heal. This may in part be due to certain anatomic features of the ACL. The ACL is an intra-articular structure covered with a synovial sheath. Once injured, the ACL has a tendency to retract, thus losing approximation of the ruptured ligament ends. Results of reapproximation of this structure with primary repair have been disappointing. This suggests that other factors are likely to be involved in failure, such as potential deleterious effects of the synovial environment. A break in the synovial sheath of the ACL, and exposure to the synovial Received March 10, 1989; accepted December 14, 1989. Address correspondence and reprint requests to Prof. D. Amiel at University of California at San Diego, M-030, La Jolla, CA 92093, U.S.A.
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EFFECTS OF ACUTE HEMARTHROSIS ON RABBIT ACL
cruciate ligament (ACL). In an animal model, Pforringer (14) has demonstrated small decreases in the biomechanical properties of the femur-ACGtibia complex (FATC) after hemarthrosis, as well as histological changes (15) in the ACL substance. Clinically, intra-articular structures are exposed to blood in association with acute traumatic knee injury. Noyes (12) has shown that 72% of patients presenting with an acute hemarthrosis of the knee have an acute tear of the ACL. Studies from our laboratories (10) demonstrated in a rabbit model of acute hemarthrosis that the menisci (lateral and medial), which lack a synovial covering, demonstrated an increase in degradative activity assessed by collagenase activity. Furthermore, in a rabbit model of acute injury in which the ACL was surgically sectioned, we observed rapid changes in the histologic features, total collagen content, and collagenase activity of the “injured” ACL (1). Since clinical ACL injuries are often associated with hemarthrosis, we sought to investigate the early effects of hemarthrosis over a time period similar to our previous study. We suspected that soft tissue structures with direct exposure to the articular environment (such as the menisci) would be more greatly affected by proteases than the ACL, which is protected from the articular environment by its synovial sheath. The purpose of this investigation was to determine the early effects of hemarthrosis on histological, biochemical, and biomechanical properties of the ACL. MATERIALS AND METHODS Animal Model The knees of 19 male New Zealand white rabbits (body weight of 3.0 0.2 kg) were used as a model of acute hemarthrosis. Two milliliters of autologous blood harvested fresh from ear veins were injected once daily into the left knee for seven consecutive days. Two milliliters of lactated Ringer’s solution was injected into right knees to serve as a sham control (16). Between injections, the animals were allowed cage activity. Animals were sacrificed on the 9th day. After the animals were killed, all knees were evaluated for the absence of sepsis. Two animals were evaluated histologically. Eight animals were used for biochemical evaluation. Nine animals were used for the biomechanical properties of the ACL.
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Histology The ACLs were harvested, placed in cassettes, and fixed in 10% buffered formalin. Specimens were mounted in paraffin, and 6 pm sections were stained with both hematoxylin and eosin (H&E) and Prussian Blue. These sections were evaluated for overall tissue organization, synovial proliferation, and iron deposition. Biochemistry Since collagen represents the major structural protein of the ACL and menisci and is responsible for most of the tensile strength of this tissue, assays for total collagen and collagenase were used. At the time of sacrifice, the ACL was aseptically harvested, rinsed with sterile phosphate-buffered saline, and placed individually in 1 ml of serum-free Earle’s MEM (Irvine Scientific Co., Irvine, CA) with the addition of fungibact, nonessential amino acids, L-glutamine, and vitamin E. Tissues were incubated in 35 mm diameter petri dishes (Costar, Cambridge, MA) and maintained in a humidified environment of 95% oxygen and 5% C 0 2 for 3 days. At the end of the incubation period, the media were harvested and assayed immediately for collagenase activity. Residual tissue was defatted with ether/ acetone, dried, and dry weights obtained. The total collagen content of the residual tissue was determined. Collagenase Assays
Acid-soluble type I collagen extracted from rabbit skin was acetylated with [‘4C]acetic anhydride by the method of Gisslow (5). This served as a substrate for collagenase determinations performed in a collagen film assay described by Johnson-Wint (9). The labeled substrate was suspended in 0.15 M phosphate buffer (pH 7.4 with 0.02% Na azide) and plated into a 96-well culture dish (Costar) to obtain 10,000 counts/min/well. Eighty microliters of media from each 3-day explant was assayed in duplicate. Ten millimolar CaCl,, 100 mM Tris buffer (pH 8. l), and fresh media were added to obtain a final reaction volume of 200 pl containing 1 mM Ca and 10 mM tris at neutral pH. At the end of incubation, the reaction volume (200 11.1) was mixed with 10 ml of betaphase scintillation cocktail (Westchem, Princeton, NJ) and counted for 1 min in a model LS6800 liquid scintillation spectrometer (Beckman, Ana-
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heim, CA). Sample counts were compared to an index group of controls of 0.1% clostridial collagenase and 0.01% TPCK-treated trypsin (Sigma, St. Louis, MO). Counts released with trypsin were assumed to represent the degradation of noncollagenous protein, or denatured collagen. Counts released above trypsin blanks were used as an indication that collagenase activity was present. Results were normalized by expressing counts per minute (cpm) per mg of dry weight of tissue from the original culture. The validity of the procedure for collagenase activity was confirmed with a separate set of incubations, with and without the presence of 2 mM EDTA (a calcium chelator and known inhibitor of collagenase activity). Products of degradation in the presence and absence of inhibitor were processed by acetone precipitation and SDS-PAGE chromatography. The characteristic primary degradation products of collagenase activity were identified, as well as their disappearance with the addition of EDTA. Seven animals were available to evaluate collagenase activity. Although connective tissue explants would be expected to release collagenase into media, it was assumed that by comparing experimental to control tissues, the in vivo effects of experimental hemarthrosis would be reflected by differences observed in vitro. The in vitro method of assaying media for collagenase activity was selected because of constraints of sample size and expected difficulties in attempting to purify collagenase from such small specimens. Total Collagen
Anterior cruciate ligaments remaining after incubation were dried, weighed, and ground in a Wiley mill. Aliquots weighing 3-5 mg were hydrolyzed in 6 N HC1 for 3 h at 130°C. The hydroxyproline content was determined as described by Woessner (21) and calculations made to express total collagen as a percentage of dry weight. Biomechanics The knees of nine animals underwent biomechanical analysis. Anterior-posterior (A-P) knee translation, as well as mechanical properties of the ACL substance, was determined. Immediately after sacrifice, the animals' hindlimbs were removed, wrapped in saline-soaked gauze, placed in plastic
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bags, and stored at -20°C for 1-3 weeks prior to testing. A-P Joint Translation
All muscle and soft tissues were carefully dissected from the knee, leaving only the knee ligaments and joint capsule intact. The femur and tibia were placed in a clamp used in previous studies (23), which allows the knee to be mounted on an Instron Universal Testing Machine at specifically chosen flexion angles. In this study, the joint was tested in both 45" and 90" of knee flexion. The tibia was aligned horizontally and the femur rotated in the sagittal plane to obtain either 45" or 90" of knee flexion. The loading axis was vertical, which provided an A-P load perpendicular with respect to the tibia. A cyclic load of +5 N was applied 10 times at a deformation rate of 10 mm/min. The loaddisplacement curve was recorded, and the total A-P translation was taken to be the maximum displacement between the peak loads. Tensile Testing
Following A-P translation test, the joint capsule and knee ligaments except for the ACL were removed. The lateral half of the ACL was removed, leaving the anteromedial (AM) bundle intact. This was done to allow more uniform loading of the ACL during tensile testing. To allow correct orientation of the ligament fibers, the femur and tibia were carefully trimmed using a rongeur until a small plug of bone (from 0.5-1.0 cm in diameter) remained at the proximal and distal ACL insertion sites. The femoral and tibia1 bone plugs were each mounted in 25 mm diameter spheres of polymethyl methacrylate (PMMA) bone cement. With this testing device, careful adjustments in the bone and ligament alignment allowed uniform loading of ligament fibers during mechanical testing. The cross-sectional area of the AM bundle of the ACL was measured using digital vernier calipers. Specimens were kept moist with 0.9% saline throughout the preparation. Dark elastin (Verhoff) stain lines approximately 7 mm apart were carefully applied to the ligament to act as gauge length markers for strain measurement by the Video Dimension Analyzer (VDA) system (22). The PMMA spheres and a bone-ligament-bone complex were mounted in the custom testing jig, which allowed adjustments in three translational
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EFFECTS OF ACUTE HEMARTHROSIS ON RABBIT ACL planes and around three rotational axes. Therefore, tensile loading could be applied uniformly along the long axis of the ligament. The bone-AM bundlebone complex was immersed in a 37°C physiological saline bath for 30 min. A preload of 0.5 N was applied, followed by cyclic preconditioning between 0 and 0.3 mm displacement for 10 cycles at a rate of 10 mm/min. The bone-ligament-bone specimen was then loaded to failure at a rate of 50 mdmin. The load vs. time curve was recorded on the Instron strip-chart recorder. The ligament strain vs. time response was measured by means of a VDA system. With this information, both structural properties of the femur-AM bundle-tibia complex, as well as mechanical properties of the AM bundle of the ACL, were determined (22). There was technical failure in tensile testing on one of the knees with hemarthrosis; therefore, it was not used in the analysis. Statistical Analysis Collagenase activity and total collagen values were compared between blood-treated and control tissues with a paired t test. A significance level of 0.05 was selected. A-P joint translation was compared using two-way analysis of variance (ANOVA), so that the effects of both hemarthrosis and knee flexion angle on A-P translation could be evaluated. Unpaired Student’s I tests were used to compare several parameters representing the mechanical properties, and a p value of c0.05 was considered to be significant.
RESULTS Gross
At the end of the treatment period, all bloodtreated knees exhibited a persistent bloody effusion. The ACL synovial sheath exhibited yellowbrown discoloration and appeared to be edematous. All knees were grossly free of infection. Random cultures of these knees confirmed that sterility had been maintained. Histology Hypertrophic synovium with synoviocytes laden with iron deposits were noted about the ACL from blood-treated knees (Fig. 1). In contrast, within the ligament substance, the overall cellular and matrix organization was unaltered in the experimental samples (Fig. 2A and B). Collagenase Collagenase activity in the media from the ACL showed no significant difference between bloodtreated (121.7 f 13 cpmlmg of dry weight) and control (114.1 k 25 c p d m g of dry weight) knees (p = 0.76) (Fig. 3). Total Collagen The ACL of the experimental knee showed a total collagen level of 82.4 2 1.3%, with a control of 80.6
FIG. 1. Blood-treated synoviurn (iron stain, ~ 1 0 ) .
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FIG. 2. (A) Blood-treated ACL, H&E stain ( ~ 4 0 )Note . the regular cellularity of the collagenous matrix. (B) Control ACL, H&E stain ( ~ 4 0 ) .
B
2 1.5%. These values were not significantly different (p = 0.33) (Fig. 4).
Biomechanics The results of a two-way ANOVA showed that the A-P joint translation in the setting of acute hemarthrosis was not significantly different (p > 0.05) from sham-injected knees at 90" flexion (0.7 2 0.1 and 0.8 k 0.1 mm, respectively), nor was there a difference (p > 0.05) at 45"flexion (1.2 ? 0.1 and 1.3 2 0.1 mm, respectively). There was, however, a significant effect (p < 0.05) of knee flexion angle, with greater A-P translation at 45" compared to 90" in both control and experimental knees. J Orthop Res, Vol. 8. No.4, 1990
The stress-strain curves obtained from the tensile tests of the AM bundle are shown in Fig. 5. The tangent modulus (defined between 4 and 7%), the tensile strength, and strain at failure are detailed in Table 1 using unpaired t tests. These parameters representing the mechanical properties of the bundles were not found to be significantly different (p > 0.4) between the hemarthrosis and control groups. During load to failure testing, six of eight hemarthrosis specimens (75%) failed within the ligament substance and, similarly, six of nine control specimens (67%) failed in the ligament substance. Thus, overall, 7 1% of the bone-ligament-bone preparations failed in the ligament substance, while 29% failed by either bony avulsion or pullout from the PMMA bone cement plug.
EFFECTS OF ACUTE HEMARTHROSIS ON RABBIT ACL
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2ool
INJECTED
-
0 Ringers Q Blood
0
I
2
3
4
6
Strain (%)
"
ACL N=7 FIG. 3. Collagenase activity. Note no difference between blood-treated and control ACL (mean 2 SEM). Paired t test, p = 0.76.
All of the specimens were used for determination of tangent modulus; only those specimens that failed in the ligament substance were included for determinations of ligament ultimate strain and tensile strength. DISCUSSION
Most experimental models have studied the effects of chronic hemarthrosis on articular cartilage. The changes seen include intracellular deposition of iron in chondrocytes (4,8,17,18), an increase in protease activity from cartilage (3), a drop in glycosaminoglycan content of articular surfaces (2,17), decreased shear strength of cartilage (2), and associated early cartilage degeneration (3,7). It is apparent that the synovial lining is not a passive bystander in this process, as reports of synovial hyINJECTED
El Ringers Q Blood
r
8
FIG. 5. Rabbit ACL-AM bundle stress-strain curves.
pertrophy with intracellular deposits of iron (7,13,17) have noted. The synovitis thus elicited may promote secretion of proteases into the articular environment (6,20). Although this may in itself be responsible for the degenerative changes seen in hemophilic arthropathy, other postulated mechanisms include the introduction of enzymes from the granulocyte fraction of blood (19) and a direct effect of iron deposits on chondrocytes leading to detrimental alterations in cellular metabolism (8,18). Pforringer (14,15), in a two-part paper on the effects of acute hemarthrosis on mechanical and histologic properties of the ACL, noted a slight reduction in structural properties of the boneligament-bone complex with hemarthrosis. The differences increased with injury added to the synovial sheath of the ACL. The changes in the ultimate load for both cases were the most significant 4 weeks after surgery, with a 5N and 17 N decrease from a control load of 470 N for the hemarthrosis and synovectomy groups, respectively. At 1 week, the ultimate load values of the hemarthrosis group was 3 N less than the control value. Thus, the effect of hemarthrosis represented only a 1% decrease in the ultimate load. Statistical analysis was not presented. In our model of acute hemarthrosis, we have atTABLE 1. Mechanical properties of AM bundle of the ACL (mean L- SEM)
Group Control Hemarthrosis
N=8
FIG. 4. Total collagen. Note no difference between bloodtreated and control ACL (mean f SEM). Paired t test, p = 0.33.
p value (unpaired t test)
Tangent modulus (lo2 MPa)
Tensile strength" (MPa)
Strain at failure" (%)
6.0 f 0.5 (n = 9) 5.0 2 0.8 (n = 8)
51.2 f 4.3 (n = 6) 50.2 2 6.6 (n = 6 )
13.0 2 2.0 (n = 6 ) 13.0 1.5 (n = 6)
p > 0.4
p > 0.30
p > 0.50
Only samples that failed in ligament substance are used.
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tempted to correlate effects on the histological, biochemical, and biomechanical properties of an uninjured ACL. A proliferative synovitis was elicited. As noted by others, deposits of iron within the synovial cells were noted; however, the substance of the ACL showed no microscopic evidence of response to blood treatment. As well, no significant changes were noted in the total collagen content, collagenase activity, or biomechanical properties of the ACL. We have previously demonstrated an increase in meniscal collagenase in an identical model of acute hemarthrosis (10). Lindy et al. (11) have described the release of collagenase from human rheumatoid menisci and correlated higher levels of collagenase activity with increased severity of disease. They postulated that meniscal tissue has the ability to elaborate its own collagenase. It is possible that the collagenase activity observed from blood-treated menisci originated from the blood itself or an inflammatory response of synovial tissue to hemarthrosis. However, there is growing evidence that all connective tissues are metabolically active and have the ability to elaborate their own degradative enzymes. Although inconclusive, the elevated levels of collagenase may reflect the reaction of meniscus tissue to an altered and potentially hostile environment. In contrast, the ACL may be a privileged intra-articular structure, since it possesses a synovial covering that allows it to be protected from the articular environment. With trauma, synovial injury would be expected to occur, and this protective function of the ACL may be disturbed. Subsequent exposure of ligament substance to the intraarticular environment and hemarthrosis may produce changes in the ligament. Whether this mechanism may be responsible for the poor results reported with primary ACL repair is an important subject for further investigation. Acknowledgment: We would like to acknowledge financial support from NIH AR34264 and the Malcolm and Dorothy Coutts Institute for Joint Rehabilitation and Research. Also, the technical assistance of Ms. Deidre MacKenna, Dr. Peter Newton, Mr. Mike Furniss, and Ms. Linda Kitabayashi and the word processing skills of Ms. Fran Shepherd are gratefully acknowledged.
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collagenase in ligament degeneration. J Orthop Res 7:486, 1989 2. Convery FR, Woo SL-Y, Akeson WH, Amiel D, Malcom LL: Experimental hemarthrosis in the knee of the mature canine. Arthritis Rheum 1959, 1976 3. Fabry G: Early biochemical and histological findings in experimental haemarthrosis in dogs. Arch Orthop Traumat Surg 100:167, 1982 4. Ghadially FN, Oryschak AF, Ailsby RL, Mehta PN: Electron probe x-ray analysis of siderosomes in haemarthrotic articular cartilage. Virchows Arch [B] 16:43, 1974 5 . Gisslow MT, McBride BC: A rapid sensitive collagenase assay. Anal Biochem 68:70, 1975 6. Goldberg VM, Barstein A, Dawson M: The influence of a experimental immune synovitis on the failure mode and strength of the rabbit ACL. J Bone Joint Surg [Am] 64:900, 1982 7. Hoaglund FT: Experimental hemarthrosis. J Bone Joint Surg [Am] 49:285, 1967 8. Hough AJ, Barfield WG, Sokoloff L: Cartilage in hemophilic arthropathy; ultrastructural and microanalytical studies. Arch Pathol Lab Med 100:91, 1976 9. Johnson-Wint B: A quantitative collagen film collagenase assay for large numbers of samples. Anal Biochem 104:175, 1980 10. Ishizue KK, Lyon R, Amiel D, Woo SL-Y, Kitabayashi L, Harwood FL, Gomez M, Akeson WH: Hemarthrosis: a biochemical and mechanical evaluation of effects on the anterior cruciate ligament and menisci. Trans Orthop Res SOC 1 3 5 5 , 1988 11. Lindy S, Turto H, Sorsa T, Halme J, Lauhio A, Suomalainen K, Uitto V-J, Wegelius 0: Increased collagenase activity in human rheumatoid meniscus. Scand J Rheumatol 15:237, 1986 12. Noyes FR, Bassett RW, Grood ES, Butler DL: Arthroscopy in acute traumatic hemarthrosis of the knee. J Bone Joint Surg [Am] 62:687, 1980 13. Parsons JR, Zingler BM, McCeon JJ: Mechanical and histologic studies of acute joint hemorrhage. Orthopedics 10: 1019, 1987 14. Pforringer W: Hamarthros und kreuzbander-biomechanische untersuchangen teil 1 . Unfallchirugie 8:353, 1982 15. Pforringer W: Hamarthros und kreuzbander-morphologische untersuchungen teil 2. Unfallchirugie 8:368, 1982 16. Reagan FB, McInerny VK, Treadwell BV, Zarins B, Mankin HJ: Irrigating solutions for arthroscopy: a metabolic study. J Bone Joint Surg [Am] 65:629-631, 1983 17. Rippey JJ, Hill RR, Lurie A, Sweet M, Thonar E, Handelsman JE: Articular cartilage degradation and the pathology of haemophilic arthropathy. South Afr Med J 54:345, 1978 18. Roy S: Ultrastructure of articular cartilage in experimental hemarthrosis. Arch Pathol 86:69, 1968 19. Weissman G, Spilberg I, Krakauer K: Arthritis induced in rabbits by lysates of granulocyte lysosomes. Arthritis Rheum 12:103, 1969 20. Werb Z, Reynolds JJ: Stimulation by endocytosis of the secretion of collagenase and neutral proteinase from rabbit synovil fibroblasts. J Exp Med 140:1482, 1974 21. Woessner JF Jr: The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Siophys 93:440, 1961 22. Woo SL-Y, Gomez MA, Seguchi Y, Endo CM, Akeson WH: Measurement of mechanical properties of ligaments from a bone-ligament-bone preparation. J Orthop Res 1 :22-29, 1983 23. Woo SL-Y, Hollis JM, Roux RD, Gomez MA, Inoue M, Kleiner JB, Akeson WH: The effects of knee flexion on the structural properties of the femur-anterior cruciate ligament-tibia complex (FATC). J Biomech 20557-564, 1987
