The early effects of joint immobilization on medial collateral ligament healing in an ACL-deficient knee: a gross anatomic and biomechanical investigation in the adult rabbit model.

In this study, the short-term effects of immobilization on joint damage and medial collateral ligament (MCL) healing were investigated in unstable, anterior cruciate ligament (ACL)-deficient knees in rabbits. Forty-six 12-month-old female New Zealand white rabbits were separated into three groups. Animals from each group had surgery on their right knees: group I, sham controls (n = 9); group II, complete transection of the ACL and removal of a 4 mm segment (gap injury) of MCL midsubstance with no immobilization of the limb (n = 19); and group III, same injuries to the ACL and MCL (as group II) but with immobilization of the limb (n = 18). No surgical repair of disrupted ligaments was performed. Left knees served as unoperated contralateral controls. All animals were allowed unrestricted cage activity until sacrifice in subgroups at 3, 6, and 14 weeks of healing when biomechanical properties of all MCLs were measured. All knee joints were systematically examined for gross evidence of damage to articular cartilage, menisci, and periarticular soft tissues. To monitor relative in vivo loads on injured limbs during healing, hindlimb weight bearing was assessed at biweekly intervals. Results indicated that animals in both groups II and III bore relatively lower loads (compared to preinjury values) on their injured hindlimbs. Mechanical testing of MCLs showed only minor changes in sham controls, while group II and III healing MCLs demonstrated significantly lower force and stress at MCL complex failure compared to contralateral controls. In specific comparisons of group III to group II animals, we noted that immobilization prevented joint damage over the early intervals studied. In addition, immobilization resulted in MCL laxity similar to contralateral control values but inhibited development of structural strength and stiffness in healing MCLs. These results suggest that in the rabbit, short-term immobilization of an ACL-deficient knee offers some advantages to the joint and to certain low load behaviors of the healing MCL, but it also results in a smaller quantity of scar tissue that is less able to resist higher loads. Longer-term studies involving remobilization are necessary before the effects of brief immobilization on joint damage and MCL healing in this ACL-deficient model can be fully defined.     

Immobilization alters cell metabolism in an immature ligament.

This investigation examined the metabolic and cellular effects of immobilization on immature rabbit medial collateral ligaments (MCLs). Thirty-two three-month-old rabbits had their right knee pinned in flexion for intervals of two weeks, one month, two months, or three months before being killed for comparison with MCLs from nonpinned contralateral controls and MCLs from 16 normal age-matched controls. Twelve animals were studied by gait analysis after either one month or three months of immobilization and were killed with the others for in vitro analysis of ligament mass, collagen synthesis, plasminogen activator secretion, or DNA content. Immobilized hindlimbs carried less weight than did contralaterals, and MCLs from those hindlimbs synthesized less collagen and secreted less plasminogen activator than did controls at all intervals. Contralaterals also showed some signs of altered cell metabolism compared with normal controls, suggesting that they are not normal by these assessment criteria. With increasing periods of knee immobilization, immobilized MCLs switched progressively from an anabolic to a more catabolic state, apparently contributing to the failure of immobilized ligaments to increase normally in mass. Knee immobilization alters the metabolic behavior of MCLs in both knees in growing rabbits, and, based on these results, either knee joint motion or hindlimb load bearing could be necessary for normal rabbit medial collateral ligament growth and maturation.     

Healing ligaments have decreased cyclic modulus compared to normal ligaments and immobilization further compromises healing ligament response to cyclic loading.

Ligaments help maintain joint stability by resisting excessive strain during the repetitive loading experienced during daily activity. Healing ligaments may be less able to fulfill this role, straining more under equivalent loading than normal ligaments. We examined the cyclic stress-strain response of normal and healing ligaments to repetitive low loads (<10% of the normal ligament failure strength). Rabbit medial collateral ligaments (MCLs) were surgically gapped in either a unilateral (right MCL; n=23) or bilateral (right and left MCLs; n=17) fashion with immobilization of the right hindlimb in the bilateral group. These MCL scars were allowed to heal for 3, 6, and 14 weeks and were cyclic creep tested at 2.2, 4.1, and 7.1 MPa, respectively. Creep test stresses were a constant 30% of the failure strength of non-immobilized scars at the different healing intervals. Normal MCLs were creep tested at 4.1 and 7.1 MPa (n=13). The cyclic modulus of the non-immobilized scars was less than that of normal ligaments. The percent increase in modulus during cycling was greater for scars than for normal ligaments, likely related to increased viscous dissipation or material inferiorities in scars. Furthermore, immobilization significantly decreased the ability of scars to resist strain, with a majority of immobilized scars failing during repetitive loading. Such failures were preceded by a reduction in cyclic modulus indicating damage to the healing ligaments that was predictive of eventual total failure. The implications of this study are that joints with healing ligaments may have increased strain in joint structures while they are under stress, potentially leading to joint instability. Although immobilization could be used temporarily to maintain joint stability, remobilization would likely lead to total failure of the healing ligament.     

A quantitative analysis of matrix alignment in ligament scars: a comparison of movement versus immobilization in an immature rabbit model

This investigation quantified the alignment of fibrillar matrix in normal rabbit medial collateral ligaments (MCLs) and in healing MCLs from animals treated with or without knee immobilization. Twenty-four immature female rabbits were given complete midsubstance injuries to their right MCLs. Fifteen of them had that knee pin immobilized in flexion, while the remaining nine were allowed unrestricted cage activity. Animals were sacrificed in groups of three at intervals of 3, 6, or 14 weeks after injury, and both healing MCLs and unoperated contralateral controls were fixed in situ for subsequent removal, freeze-fracture, and preparation for scanning electron microscopy (SEM). A random sampling of SEM photographs followed by automated, statistically validated image processing was used to quantify alignment of matrix in all samples. Results showed that nonimmobilized MCL scars in this model do remodel over 14 weeks of healing, returning to normal alignment values in that time. Surprisingly, MCL scars in immobilized knees were even better, with mean matrix alignments falling statistically within normal MCL limits at all healing intervals studied. If not due to an unknown sampling or fixation artifact, these results suggest that gross knee flexion and extension is not a prerequisite for scar matrix alignment in this immature model of ligament healing.     

Cellularity and apoptosis after radiofrequency-induced shrinkage of collagenous tissue: assessment of postoperative immobilization using an in vivo rabbit model

Background Electrothermally-assisted capsular shrinkage has been gaining increased acceptance in the treatment of shoulder instability. Its indication in ACL-deficient knees has been discussed recently.

Methods We examined the influence of immobilization on cell homeostasis of healing collagenous tissue after radiofrequency energy was applied to the patellar tendon in 23 rabbits. The animals were killed immediately after surgery (n = 6) or 3 weeks after surgery (n = 17). 10 rabbits were allowed normal cage activity, whereas the treated hind limb of 7 animals was immobilized for 3 weeks in a cast. Feulgen staining was used to stain the DNA of cell nuclei. Cells undergoing apoptosis were identified by the TUNEL method. Quantitative histological assessment was performed using imaging analysis software.

Results Severe cellular damage in RF-treated collagenous tissue was partly induced by the immediate onset of apoptosis. At 3 weeks after surgery, non-immobilized tendon showed increased cellularity and apoptosis, whereas immobilization prevented the increase in cellularity and apoptosis significantly. The calculated ratio of apoptosis was not influenced by any postoperative treatment.

Interpretation Diminished cellularity and apoptosis during tissue remodeling, due to immobilization, may protect the shortened collagenous scaffold from stretching and further optimize the clinical outcome after radiofrequency shrinkage. To stabilize the shrunken tissue, proliferation during postoperative wound healing should be minimized by careful rehabilitation.     

New experimental procedures to evaluate the biomechanical properties of healing canine medial collateral ligaments

Both clinical and animal studies have indicated that early mobilization and exercise may improve the healing of injured medial collateral ligaments (MCLs). To investigate these effects, transected canine MCLs were subjected to three different treatment regimens: (a) no surgical repair with 6 weeks mobilization; (b) surgical repair with 3 weeks immobilization followed by 3 weeks remobilization; and (c) surgical repair with 6 weeks immobilization. After sacrifice, knee laxity was measured in a newly designed varus-valgus (V-V) laxity device. Each knee was tested subsequently in tension to determine the structural properties of the femur-MCL-tibia complex (FMTC) and the mechanical properties of the healing MCL substance. It was found that the V-V laxity increased for all experimental knees, but that early mobilization enhanced joint stability. The structural properties of the FMTC and the mechanical properties of the MCL substance were also lower than the contralateral controls. Again, the early mobilization groups had better results.     

Immobilization of the knee joint alters the mechanical and ultrastructural properties of the rabbit anterior cruciate ligament

The effects of immobilization of the knee joint on the mechanical and ultrastructural properties of the anterior cruciate ligament have not been well documented. Our goal was to determine these effects in a rabbit model and to assess the effect of knee flexion angle during immobilization. The knee joint was immobilized in either 170° or 105° of flexion, and new methodologies were utilized to determine the mechanical properties of the anterior cruciate ligament. In specimens from knees that had been immobilized, the cross-sectional area of the ligament was 74% of the control value. The stress-strain curve was altered slightly, and the strain at failure increased 32–40%. The modulus and stress at failure did not decrease significantly. There was no significant difference between the mechanical properties of the knees immobilized at 170° and 105° of flexion. Histological and ultrastructural evaluation demonstrated changes in the shape and intracellular makeup of the fibroblasts from the ligament after immobilization. This cellular response may account for the alterations in the mechanical properties of the anterior cruciate ligament.     

Adverse effects on rabbit anterior cruciate ligament after knee immobilization: changes in permeability of horseradish peroxidase

Changes in the permeability of horseradih peroxidase (HRP) and the histology of the rabbit anterior cruciate ligament (ACL) caused by knee immobilization were studied. The knees of adult rabbits were immobilized for 2–8 weeks by a long leg cast with the knee flexed at 120°, and some joints were allowed to be remobilized for 4 weeks after 6 or 8 weeks’ immobilization. After 1 ml of HRP was injected into the knee joint and allowed to permeate into the ACL, the degree of permeation at the mid-portion of the ACL between femur and tibia was visualized with diaminobenzidine tetrahydrochloride. The ACL was examined histologically by light and transmission electron microscopy. Although the HRP permeated into almost the entire area of the transverse section of the non-immobilized normal ACL, as the period of immobilization increased, the extent of permeation gradually decreased. The decrease was significant from week 4 to week 6 and from week 6 to week 8. Degenerative change with necrosis of cells at the central area of the ACL was noted in the 6-week immobilization group, and the 8-week period of immobilization increased the extent of this degeneration. Although the permeation recovered in both the 6-week and 8-week immobilization groups 4 weeks after remobilization, there were fewer cells with a normal morphology in the central area in the 8-week immobilization group than in the control. These results suggest that long-term immobilization adversely affects the ACL. Received: 4 April 1997     

Healing of the medial collateral ligament after a combined medial collateral and anterior cruciate ligament injury and reconstruction of the anterior cruciate ligament: Comparison of repair and nonrepair of medial collateral ligament tears in rabbits

The optimal treatment for a combined injury of the medial collateral and anterior cruciate ligaments is controversial, and the question remains as to whether repair of the medial collateral ligament and reconstruction of the anterior cruciate ligament improves healing of the medial collateral ligament. We compared reconstruction of the anterior cruciate ligament with and without repair of the medial collateral ligament in a rabbit model of a combined injury of these two ligaments. The anterior-posterior translation and varus-valgus rotation of the knee, the structural properties of the femur-medial collateral ligament-tibia complex, and the mechanical properties of the midsubstance of the medial collateral ligament were evaluated immediately after surgery and at 6 and 12 weeks postoperatively. Repair of the medial collateral ligament led to significantly less varus-valgus rotation of the knee than did no repair, but the anterior-posterior translation of the knees in the repair and nonrepair groups were not significantly different at any study time. At 12 weeks, the cross-sectional area and ultimate load in the repair group were 60 and 53% greater, respectively, than in the nonrepair group. Among 12 specimens that were repaired (six specimens at 6 weeks and six specimens at 12 weeks), failure occurred within the midsubstance in four (two at each time period); in all of the specimens that were not repaired, failure occurred at the tibial insertion site. There was no significant difference between the modulus of the midsubstance in the repaired and the nonrepaired medial collateral ligaments. Thus, the improved structural properties of the femur-medial collateral ligament-tibia complexes that were repaired resulted from an increase in cross-sectional area of the repaired medial collateral ligament and healing of the tibial insertion site. Postoperative healing time had little effect on the tensile properties. In this rabbit model, repair of the medial collateral ligament with reconstruction of the anterior cruciate ligament may lead to better healing of the medial collateral ligament in the early phase than does reconstruction of the anterior cruciate ligament alone.     

Biological and Biomechanical Evaluation of Autologous Tendon Combined with Ligament Advanced Reinforcement System Artificial Ligament in a Rabbit Model of Anterior Cruciate Ligament Reconstruction

Objective

To compare the biomechanical and histological changes in a rabbit model after reconstructing the anterior cruciate ligament (ACL) with solely autologous tendon and with autologous tendon combined with the ligament advanced reinforcement system (LARS) artificial ligament.

Methods

Anterior cruciate ligament reconstruction was performed in 72 knees from 36 healthy New Zealand white rabbits (bodyweight, 2500–3000 g). The Achilles tendons were harvested bilaterally. The left ACL were reconstructed solely with autografts (autologous tendon group), while the right ACL were reconstructed with autografts combined with LARS ligaments (combined ligaments group). The gross observation, histological determination, and the tension failure loads in both groups were evaluated at 12 weeks (n = 18) and 24 weeks (n = 18) postoperatively.

Results

Gross examination of the knee joints showed that all combined ligaments were obviously covered by a connective tissue layer at 12 weeks, and were completely covered at 24 weeks. Fibrous tissue ingrowth was observed between fascicles and individual fibers in the bone–artificial ligament interface at both time points; this fibrovascular tissue layer localized at the bone–artificial ligament interface tended to be denser in specimens obtained at 24 weeks compared with those obtained at 12 weeks. The tension failure loads of the knees were similar in the autologous tendon group and the combined ligaments group at 12 weeks (144.15 ± 3.92 N vs. 140.88 ± 2.75 N; P > 0.05), and at 24 weeks (184.15 ± 1.96 N vs. 180.88 ± 3.21 N; P > 0.05).

Conclusion

Reconstructing the ACL in rabbits using autologous tendon combined with the LARS artificial ligament results in satisfactory biointegration, with no obvious immunological rejection between the autologous tendon and the artificial ligament, and is, therefore, a promising ACL reconstruction method.

     

Treatment of ligament laxity by electrothermal shrinkage or surgical plication: a morphologic and mechanical comparison

Capsular plication or thermal shrinkage can be used to enhance surgical joint stabilization. We compared mechanical or morphologic properties of the medial collateral ligament of the rabbit knee treated by either bipolar radiofrequency electrothermal shrinkage or surgical plication. After 12 weeks, the medial collateral ligaments were procured from treated and contralateral knees to undergo viscoelastic (creep) testing, quantitative transmission electron microscopy, and immunohistochemistry. Creep strain in thermal (1.85% +/- 0.32%) and plicated (1.92% +/- 0.36%) ligaments was almost twice that of the control group (1.04% +/- 0.15%), although there was no difference between treatment modalities. The morphologic parameters of all 3 groups were significantly different (P < .001). The thermal ligaments demonstrated predominantly small fibrils, whereas the plicated group displayed an intermediate distribution of heterogeneous fibrils, suggesting a different pattern of remodeling. Viscoelastic properties are similar after thermal shrinkage or plication, though inferior to those of intact ligaments.     

Changes in compression and distances between tibial and femoral condyles during immobilization of rabbit knee

Summary The compression forces and the distances between tibial and femoral condyles were measured during the development of osteoarthritis produced by immobilizing rabbit knees. The compression was measured in both the immobilized and non-immobilized knees with a sub-miniature pressure sensor. Compression increased up to threefold during the four first weeks of immobilization and decreased thereafter to below the initial level. These results give one explanation why immobilization produced degenerative joint changes; they also support the hypothesis that capsular tissues can play a role in the pathogenesis of osteoarthritis.     

Early medial collateral ligament scars have inferior creep behaviour.

Recent evidence suggests that ligaments are subject to repetitive loads in vivo. Hence, the creep behaviour (increase in strain under constant or repetitive stress) of ligament scars is of significance, since healing ligaments may elongate permanently over time. A rabbit medial collateral ligament model was used to assess the creep behaviour of healing ligaments at stresses corresponding to 30% of the scar failure strength at 3 (n = 6), 6 (n = 6), and 14 (n = 5) weeks of healing. The stresses for the creep tests of scars (and contralateral controls) were 2.2, 4.1, and 7.1 MPa for the 3, 6, and 14-week healing intervals, respectively. Normal medial collateral ligaments from comparable rabbits were tested at two of the corresponding stresses: 4.1 (n = 7) and 7.1 (n = 6) MPa. Total creep strain-the cumulative increase in strain resulting from serial cyclic and static creep testing-was independent of the order of testing and was compared between scars and controls. Water contents after testing were also quantified. Water contents before testing were assessed for additional animals: six normal animals and three from each healing interval. At 3 weeks of healing, the total creep strain of medial collateral ligament scars was four times greater than that for contralateral controls tested at the same stress. Although there was improvement from 3 to 14 weeks, the total creep strain of scars remained more than two times greater than that of controls at 14 weeks. Scar water content decreased with healing from elevated initial values, possibly contributing to the marginally improved creep response. Comparisons of this deficiency in scar creep with previously published scar abnormalities in the same model suggest that collagen crosslink density, proteoglycan content, soft-tissue flaws, and the combined effect of collagen fibre changes may be mechanistic factors involved in scar creep.     

Changes in 35S-Sulphate Uptake in Different Tissues in the Knee and Hip Regions of Rabbits During Immobilization, Remobilization and the Development of Osteoarthritis

In order to observe phenomena accompanying the development of osteoarthritis of the rabbit knee, consistently appearing within 4—6 weeks as a consequence of immobilization of this joint, studies on ³⁵S-sulphate (Na₂³⁵SO₄) uptake were carried out. The investigation suggests that immobilization of the knee leads within 2-4 weeks to increased sulphate uptake in bone, cartilage and ligaments in the knee region and in the hip (not immobilized) of the same leg. The first marked changes in ssS-sulphate uptake were found in the ligaments and the articular cartilage as early as after 4 days of immobilization.     

Changes in 35S-Sulphate Uptake in Different Tissues in the Knee and Hip Regions of Rabbits During Immobilization,Remobilization and the Development of Osteoarthritis

In order to observe phenomena accompanying the development of osteoarthritis of the rabbit knee, consistently appearing within 4—6 weeks as a consequence of immobilization of this joint, studies on ³⁵S-sulphate (Na₂³⁵SO₄) uptake were carried out. The investigation suggests that immobilization of the knee leads within 2–4 weeks to increased sulphate uptake in bone, cartilage and ligaments in the knee region and in the hip (not immobilized) of the same leg. The first marked changes in ssS-sulphate uptake were found in the ligaments and the articular cartilage as early as after 4 days of immobilization.     

Increased expression of the β1, α5 and αv integrin adhesion receptor subunits occurs coincident with remodeling of stress-deprived rabbit anterior cruciate and medial collateral ligaments

The biomechanical, biochemical, and morphological properties of the anterior cruciate and medial collateral ligaments are dramatically altered in response to deprivation of normal physical forces and joint motion. Integrin adhesion receptors are known to play important roles in the tissue remodeling that occurs in the course of normal wound repair. We propose that integrins play a similar role in the remodeling of the extracellular matrix in stress-deprived periarticular ligaments. This study tests the hypothesis that altered expression of integrins on ligament fibroblasts accompanies this remodeling. The left knees of 15 New Zealand White rabbits were surgically immobilized in acute flexion and the right knees served as controls (no operation). The anterior cruciate and medial collateral ligaments were harvested at 1, 3, 5, 9, or 12 weeks after immobilization. Sections from the ligaments were immunostained with monoclonal antibodies specific for the integrin subunits β₁, α₅, α₆, and α_v, as well as with a negative control antibody. Fibroblasts within both the stress-deprived anterior cruciate and medial collateral ligaments demonstrated markedly increased staining for the β₁, α₅, and α_v subunits, as compared with the controls. The increased staining was greatest at 9 weeks in the anterior cruciate ligament and at 12 weeks in the medial collateral ligament. Western blot study of ligament proteins extracted with sodium dodecyl sulfate demonstrated an increased amount of β₁ subunit protein in both ligaments from knees that were stress deprived for 9 and 12 weeks, as compared with the control ligaments. These studies establish that stress deprivation in this animal model is accompanied by increased expression of integrin cell-surface receptors containing the β₁, α₅, and α_v subunits on anterior cruciate and medial collateral ligament fibroblasts, with a time course similar to that previously reported for indices of extracellular matrix remodeling induced by stress deprivation. This outcome suggests an important role for these integrins in the remodeling of stress-deprived ligaments.     

Healing of experimental ligament rupture: findings by scanning electron microscopy

The medial collateral ligaments of the knee joints of 16 rats were cut. Sixteen ligaments were allowed to heal spontaneously while the others were immediately sutured under the microscope. Four sham-operated immobilized rats served as controls. The ligamentous healing was registered after 4-5 weeks of immobilization under a scanning electron microscope (SEM). In the control ligaments, collagen bundles of variable thickness were observed as the main supporting elements. Numerous thin fibers were seen between the bundles. The surgically corrected ligaments showed a microstructural picture often difficult to distinguish from the controls. The unsutured ligaments, however, showed a much more variable picture. In some unsutured cases ligamentous regeneration was blocked by extensive scar formation. In many unsutured cases, however, signs of ligamentous regeneration were observed. It was noted that the SEM is a useful and sophisticated tool for studies on ligament healing.     

Injury location affects ligament healing: A morphologic and mechanical study of the healing rabbit medial collateral ligament

Based on the heterogeneity of the rabbit medial collateral ligament (MCL) along its length, we tested the hypothesis that injury location would affect its healing response. The right MCL of 80 skeletally mature New Zealand white rabbits was sectioned adjacent to bone at the femoral end (40 rabbits) or the tibial end (40 rabbits) and reapposed with sutures. Animals were killed after 3, 6, 14, or 40 weeks of healing to examine wounds histologically (2 rabbits per healing interval) and mechanically (8 rabbits per healing interval). Results of the mechanical tests were compared to midsubstance MCL repairs (24 rabbits) and to uninjured normal MCLs (20 rabbits). The morphology of the near-insertion repairs was characterized by abnormal callus-like formation and patchy bone resorption, particularly at the tibial insertion. Mechanically, insertional injuries remodeled towards normal MCL low-load, viscoelastic and failure properties more slowly than midsubstance injuries at the early healing intervals. After 40 weeks of healing, few injury-specific differences persisted. All injured ligaments had ultimate strengths 15-35 percent short of normal at 40 weeks and the femo-rally-injured ligaments were weaker than normal at this time. These results suggest that rabbit MCLs, injured near either end, heal more slowly that those injured in their midsubstance and develop abnormal insertion morphology.     

Influence of immobilization on autograft healing in the knee joint

Summary The influence of immobilization on patellar tendon autograft healing was investigated in 12 sheep with replaced posterior cruciate ligaments of the knee (PCLs). In one group achillotomy immobilized the operated knee joint. In another group initial reduced weightbearing was obtained by hanging the sheep in a special trapeze. In the third group no protection at all was given to the operated knee. The sheep in this group went within 8 weeks from partial use of the operated leg to unrestricted weightbearing and mobility. Biomechanical testing 16 weeks after the operation demonstrated a considerable decrease in the maximum force, ligament stiffness, and strain values in the achillotomy group, as well as an increase in posterior laxity. The best data were observed in the no-protection group. This study demonstrates the negative effect of immobilization on autograft healing in a sheep PCL model. Even early in the healing process tissue may be extremely sensitive to stress and strain.Supported by the Deutsche Forschungsgemeinschaft (OE 88 2-1)     

The effect of temporary exercising of a joint during an immobilization period: an experimental study on rabbits

The right hind limbs of adult rabbits were immobilized for 5 weeks. The rabbits were then allowed to move freely for 8 weeks. The rabbits of the control group were immobilized uninterruptedly. In a second group of rabbits the plaster was removed during the immobilization period 6 days a week for 5 minutes for careful exercise and in a third group of rabbits, for violent exercise. The temporary careful exercise was of no benefit and was sometimes harmful, and the violent exercise was usually injurious for the immobilized knees as regards the stiffening, thickening and the development of radiologically detectable degenerative changes.