Analysis of change in gait in the ovine stifle: normal, injured, and anterior cruciate ligament reconstructed.

BACKGROUND: Many patients who undergo anterior cruciate ligament (ACL) reconstructive surgery develop post-traumatic osteoarthritis (PTOA). ACL reconstructive surgery may not fully restore pre-injury joint biomechanics, thereby resulting in further joint damage and contributing to the development of PTOA. In an ovine model of idealized ACL reconstruction (ACL-R), it has been shown that signs of PTOA develop within surgical joints by 20 weeks post-surgery. The aim of the present study was to investigate whether altered kinematics contribute to early PTOA development within ACL-R joints of the ovine injury model by comparing the gait of these surgical animals to the gait of a stable normal control group, and an unstable injury group in which the ACL and medial collateral ligament (MCL) had been transected. METHODS: Fifteen skeletally mature female sheep were allocated evenly into 3 treatment groups: normal control, ACL-R, and ACL/MCL Tx (each group n = 5). Each animal's gait was recorded at baseline, 4 weeks post injury, and 20 weeks post injury. Principal component analysis (PCA) was used to identify the kinematic patterns that may be discriminant between treatment groups. Results from previous studies were referenced to present the amount of gross PTOA-like changes that occurred in the joints. RESULTS: ACL-R and ACL/MCL transected (Tx) animals developed a similar amount of early PTOA-like changes within the surgical joints, but differed significantly in the amount of kinematic change present at 20 weeks post-surgery. We showed that the stifle joint kinematics of ACL/MCL Tx differed significantly from those of CTRL and the majority of ACL-R animals, while no significant differences in joint kinematic changes were found between ACL-R and CTRL animals. CONCLUSIONS: These results suggest that the early PTOA-like changes reported in the ACL-R model cannot be attributed exclusively to post-surgical kinematic changes, and therefore biologic components in the post-injury environment must be contributing significantly to PTOA development.

Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study.

OBJECTIVE: To assess ovine synovial fluid (oSF) from different post-injury time points for (1) proteoglycan-4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, (3) cartilage boundary lubrication function, and (4) lubricant composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes after injury was also examined. METHODS: oSF was collected 2, 4, 10, and 20 weeks post anterior cruciate ligament (ACL) transection in five skeletally mature sheep. PRG4 and HA concentrations were measured using sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubrication of oSF was assessed using a cartilage-cartilage friction test. Gross damage to articular cartilage was also quantified at 20 weeks using modified Drez scoring protocol. RESULTS: Early (2-4 weeks) after ACL injury, PRG4 concentrations were significantly higher (P = 0.045, P = 0.037), and HA concentrations were substantially lower (P = 0.005, P = 0.005) compared to 20 weeks. The HA MW distribution also shifted towards lower ranges in the early post-injury stage. The kinetic friction coefficients were significantly higher 2-4 weeks post injury (P = 0.008 and P = 0.049) compared to 20 weeks. Poor cartilage boundary lubricating ability early after injury was associated with cartilage damage at 20 weeks. CONCLUSION: Altered composition and diminished boundary lubrication of oSF early after ACL transection may pre-dispose the articular cartilage to degenerative changes and initiate osteoarthritis (OA). These observations also provide potential motivation for biotherapeutic interventions at earlier time points post injury.

Changes of early post-traumatic osteoarthritis in an ovine model of simulated ACL reconstruction are associated with transient acute post-injury synovial inflammation and tissue catabolism.

The study described here tested the hypothesis that early intra-articular inflammation is associated with the development of post-traumatic osteoarthritis (PTOA) in a sheep model. We extended previously published work in which we investigated joint gross morphology and synovial mRNA expression of inflammatory and catabolic molecules 2 weeks after anatomic Anterior cruciate ligament (ACL) autograft reconstructive surgery (ACL-R). The same variables have been analyzed at 20 weeks post surgery together with new experimental variables at both time points. Animals were sacrificed at 20 weeks post ACL-R surgery and their joints graded for signs of PTOA. Synovial samples were harvested for histological grading plus mRNA and protein analysis for a panel of inflammatory and catabolic molecules. The mRNA expression levels for this panel plus connective tissue matrix turnover molecules were also investigated in cartilage samples. Results of gross morphological assessments at 20 weeks post surgery showed some changes consistent with early OA, but indicated little progression of damage from the 2 week time point. While significant alterations in mRNA levels for synovial inflammatory and catabolic molecules were detected at 2 weeks, values had normalized by 20 weeks. Similarly, all mRNA expression levels for inflammatory and catabolic molecules in articular cartilage had returned to normal levels by 20 weeks post ACL-R surgery. We conclude that synovial inflammatory processes are initiated very early after ACL-R surgery and may instigate events that lead to the gross cartilage and joint abnormalities observed as early as 2 weeks. However, the absence of sustained inflammation and joint instability may prevent OA progression.

Synovial and cartilage responsiveness to peri-operative hyaluronic acid ± dexamethasone administration following a limited injury to the rabbit stifle joint.

Posttraumatic osteoarthritis (PTOA) can develop after an injury to the knee. Previous studies have indicated that an intra-articular (IA) injection of the potent glucocorticoid dexamethasone (DEX) may significantly prevent induction of PTOA. The aim of the present study was to investigate the effectiveness of a single IA injection of hyaluronic acid (HA), alone and in combination with DEX following a localized intra-articular injury as a PTOA-preventing treatment option. An established rabbit model of surgical injury consisting of dual intra-articular (IA) drill holes in a non-cartilaginous area of the femoral notch near the origin of the anterior cruciate ligament (ACL) to allow for bleeding into the joint space was used. Immediately following surgery, subjects were treated with HA, HA + DEX, or received no treatment. An uninjured control group was used for comparison (N = 5/group). Rabbits were sacrificed and investigated at 9 weeks post-injury. At 9 weeks post-injury, there was a significant protective capacity of the single IA treatment of DEX + HA on the histological grade of the synovial tissue, and some variable location-specific effects of HA alone and HA + DEX interactions on cartilage damage. Thus, it is possible that co-treatment with HA may interfere with the effectiveness of the DEX. In vitro friction testing indicated that DEX did not interfere with the lubricating ability of HA or synovial fluid on cartilage. These results suggest that a single IA administration of HA in combination with DEX following an IA injury is not recommended for inhibition of PTOA progression in this model.

New understanding of the complex structure of knee menisci: implications for injury risk and repair potential for athletes.

Menisci help maintain the structural integrity of the knee. However, the poor healing potential of the meniscus following a knee injury can not only end a career in sports but lead to osteoarthritis later in life. Complete understanding of meniscal structure is essential for evaluating its risk for injury and subsequent successful repair. This study used novel approaches to elucidate meniscal architecture. The radial and circumferential collagen fibrils in the meniscus were investigated using novel tissue-preparative techniques for light and electron microscopic studies. The results demonstrate a unique architecture based on differences in the packaging of the fundamental collagen fibrils. For radial arrays, the collagen fibrils are arranged in parallel into ∼10 µm bundles, which associate laterally to form flat sheets of varying dimensions that bifurcate and come together to form a honeycomb network within the body of the meniscus. In contrast, the circumferential arrays display a complex network of collagen fibrils arranged into ∼5 µm bundles. Interestingly, both types of architectural organization of collagen fibrils in meniscus are conserved across mammalian species and are age and sex independent. These findings imply that disruptions in meniscal architecture following an injury contribute to poor prognosis for functional repair.

Technical issues in using robots to reproduce joint specific gait.

Reproduction of the in vivo motions of joints has become possible with improvements in robot technology and in vivo measuring techniques. A motion analysis system has been used to measure the motions of the tibia and femur of the ovine stifle joint during normal gait. These in vivo motions are then reproduced with a parallel robot. To ensure that the motion of the joint is accurately reproduced and that the resulting data are reliable, the testing frame, the data acquisition system, and the effects of limitations of the testing platform need to be considered. Of the latter, the stiffness of the robot and the ability of the control system to process sequential points on the path of motion in a timely fashion for repeatable path accuracy are of particular importance. Use of the system developed will lead to a better understanding of the mechanical environment of joints and ligaments in vivo.

Strength of medial structures of the knee joint are decreased by isolated injury to the medial collateral ligament and subsequent joint immobilization.

Past studies of the healing of the medial collateral ligament (MCL) in animal models have been conducted over a variety of healing intervals, some as early as 1 week. One concern with testing at early healing intervals is the difficulty in identifying and isolating the tissues that carry load. The purpose of this study was to determine if isolation of the MCL and healing time are critical factors in the assessment of structural strength in this model. Furthermore, the effect of immobilization on these critical factors was investigated. Our approach was to calculate the load-sharing ratio between the MCL and the MCL plus capsule. A 4 mm gap was created in the midsubstance of both hindlimb MCLs of 52 female New Zealand White rabbits (n=104). Of these, 29 rabbits had their right hindlimb pin immobilized (immobilized group), leaving the left hindlimb non-immobilized. Testing was performed at 3 (n=12), 6 (n=22), and 14 (n=24) weeks. The remaining 23 rabbits, which had both limbs non-immobilized (non-immobilized group), were tested at 3 (n=10), 6 (n=12), 14 (n=12), and 40 (n=12) weeks. For both groups, half of the specimens at each healing interval were used to test the MCL alone and half to test the MCL plus capsule, except for 3 week immobilized joints where only the MCL plus capsule was tested. Additionally, MCL (n=12), MCL plus capsule (n=6), and capsule alone (n=5) were tested from normal animals. The load-sharing ratio at MCL failure for the normal joint was 89%, suggesting an MCL-dominated response. For the non-immobilized group, the load-sharing ratio was 24% at 3 weeks of healing, suggesting a capsule-dominated response. At and after 6 weeks of healing, an MCL-dominated response was observed, with the ratio being 68% or greater. Thus, at less than 6 weeks of healing, the structural strength capabilities of the joint may be better represented by the medial structures rather than the isolated MCL. Immobilization delayed the transition from a capsule-dominated response to an MCL-dominated response in this model.

Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure.

Osteoblasts can be derived from embryonic stem cells (ESCs) by a 30 day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and respond to exogenously added 1,25α(OH)2 vitamin D3 with enhanced matrix mineralization. However, bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, a capacity that is executed by osteocytes. At present, it is unclear whether ESC-derived osteogenic cultures contain osteocytes and whether these are capable of responding to a relevant cyclic hydrostatic compression stimulus. Here, we show that ESC-osteoblastogenesis is followed by the generation of osteocytes and then mechanically load ESC-derived osteogenic cultures in a compression chamber using a cyclic loading protocol. Following mechanical loading of the cells, iNOS mRNA was upregulated 31-fold, which was consistent with a role for iNOS as an immediate early mechanoresponsive gene. Further analysis of matrix and bone-specific genes suggested a cellular response in favor of matrix remodeling. Immediate iNOS upregulation also correlated with a concomitant increase in Ctnnb1 and Tcf7l2 mRNAs along with increased nuclear TCF transcriptional activity, while the mRNA for the repressive Tcf7l1 was downregulated, providing a possible mechanistic explanation for the noted matrix remodeling. We conclude that ESC-derived osteocytes are capable of responding to relevant mechanical cues, at least such that mimic oscillatory compression stress, which not only provides new basic understanding, but also information that likely will be important for their use in cell-based regenerative therapies.

Efficient transfer of intact oligonucleotides into the nucleus of ligament scar fibroblasts by HVJ-cationic liposomes is correlated with effective antisense gene inhibition.

The efficacy of two different cationic liposomes, Lipofectin and hemagglutinating virus of Japan (HVJ)-cationic liposomes, on nuclear uptake of fluorescence-labeled phosphorothioate oligodeoxyribonucleotide (S-ODN) by ligament scar fibroblasts and suppression of decorin mRNA expression when antisense decorin S-ODN was transferred was investigated. There was no significant difference in nuclear uptake of fluorescent ODN between the two methods. However, only HVJ-cationic liposomes had a significant effect on suppression of decorin mRNA expression levels. To address the discrepancy, the molecular integrity of the transferred ODN in the cells was assessed by analysis of fluorescence resonance energy transfer (FRET) within double-fluorescence-labeled S-ODN. More than 70% of the ODN transfected by HVJ-cationic liposomes remained intact within the nucleus at 20 h after transfection, while the majority of the ODN transferred by Lipofectin was degraded at this point. These results suggest a strong relationship between the nuclear integrity of transfected antisense ODN and its suppression of target mRNA expression.

Pregnancy affects cellular activity, but not tissue mechanical properties, in the healing rabbit medial collateral ligament.

Recently, evidence has been accumulating that ligament and joint laxity is altered in women and rabbits during pregnancy. Furthermore, many female adolescents injure ligaments through participation in athletics and other activities. Therefore, to determine whether pregnancy has different effects on the injured and uninjured medial collateral ligament of the rabbit knee, we investigated cellular changes (mRNA levels) and alterations in tissue properties (biomechanics) accompanying pregnancy in animals with the medial collateral ligament injured during adolescence and bred for their primigravid pregnancy as young adults. Assessment of mRNA levels for matrix molecules, matrix metalloproteinases and tissue inhibitor of metalloproteinase-1, growth factors and sex hormone receptors, inflammatory cytokines, inducible nitric oxide synthase, and cyclooxygenase-2 by semiquantitative reverse transcription-polymerase chain reaction revealed that pregnancy had different impacts on scar and uninjured tissue for six of 15 genes assessed. A pregnancy-associated increase in laxity of the medial collateral ligament was observed for rabbits in the uninjured primigravida group; however, no increase was observed for injured rabbits during pregnancy. The injured ligament was already significantly more lax than the normal counterpart, and pregnancy did not lead to additional laxity or prevent the normal decline in laxity as the scar matured in nonpregnant animals. These results indicate that the impact of pregnancy on laxity and cell activity of the medial collateral ligament is dependent on whether the ligament is uninjured or injured. Pregnancy had no significant effect on structural (stiffness and failure load), material (stress at failure and Young's modulus), or viscoelastic (cyclic and static relaxation) properties of tissue from uninjured or injured medial collateral ligament. Therefore, the properties of the healing ligament were not adversely affected during pregnancy in this experimental model. However, it remains to be determined if these results with an injured medial collateral ligament can be extrapolated to the injured anterior cruciate ligament.

Ligament creep cannot be predicted from stress relaxation at low stress: a biomechanical study of the rabbit medial collateral ligament.

In normal daily activity, ligaments are probably subjected to repeated loading rather than to repeated deformation. The viscoelastic response to repeated loading is creep; this effect has significance for ligament reconstructions, which potentially "stretch out" over time. However, most experimental studies have examined the viscoelastic response to repeated deformation, stress relaxation. We hypothesized that the creep of a ligament could be predicted from its stress-relaxation behaviour. Left and right medial collateral ligaments of eight skeletally mature rabbits were subjected to either creep or stress-relaxation testing under comparable conditions. The time-dependent increase in strain (creep) and reduction in load (relaxation) from the tests were modelled with use of the quasilinear viscoelastic theory and generalized standard linear solid modelling. Ligaments were found to creep distinctly less than would be predicted from relaxation tests. Although the reason for this behaviour remains unknown, we speculate that it is due to the progressive recruitment of collagen fibres during creep.

Immobilization increases the vulnerability of rabbit medial collateral ligament autografts to creep.

Rehabilitation after soft-tissue autograft reconstructions is controversial because there is indirect evidence that some grafts fail by creeping over time. The vulnerability of soft-tissue grafts to creep over healing time and the effects of the load environment during healing on this vulnerability have never been studied specifically. We hypothesized that immobilization would decrease the magnitude of the vulnerability of ligament grafts to creep. Thirty-nine skeletally mature New Zealand White rabbits underwent a standardized medial collateral ligament autograft procedure to the right hindlimb, and 19 of the rabbits also had the limb rigidly pinned into flexion. Subgroups were killed at 3 or 8 weeks, and all isolated tibia/medial collateral ligament/femur complexes were tested for creep at 4.1 MPa under a standardized protocol. Eight normal medial collateral ligament controls were tested similarly. Results showed that all grafts were quantitatively more susceptible to cyclic and static creep than were normal medial collateral ligament controls (p < 0.05). By 3 weeks of healing, immobilization significantly increased the magnitude of the vulnerability of the grafts to cyclic, static, and total creep (all: p < 0.05). Furthermore, the grafts had more unrecovered creep strain than did the controls following a 20-minute recovery period. Contrary to our hypothesis, immobilization resulted in increased vulnerability of these ligament autografts to creep even with this relatively nonprovocative test of short duration and low stress. We postulate that following immobilization, this increase in the magnitude of susceptibility of the grafts to creep will result in functionally significant elongation of the graft if it is exposed to higher loads and over longer periods of time in vivo.

Altering ligament water content affects ligament pre-stress and creep behaviour.

The water content of a ligament can be altered by injury and surgical intervention in vivo, and inadvertently or purposely during in vitro tests. We investigated how altering the water content of the rabbit medial collateral ligament (MCL) affected its resulting creep behaviour (defined as an increase in strain from sequential cyclic and static creep tests). The water content of normal MCLs 4) was compared to that of MCLs soaked for 1 h in a sucrose solution (n = 4) or phosphate buffered saline (PBS; n = 8). Sucrose exposure decreased hydration and PBS exposure increased hydration. In addition, soaking in PBS caused a shift in ligament zero (the position where there was 0.1 N of tension on the ligament). Following the same single solution treatment, additional MCLs were creep tested at 4.1 MPa using a load based on the ligament cross-sectional area measured before solution treatment: sucrose (n = 4), PBS new "ligament zero" (n = 5). and PBS old "ligament zero" (n = 6). Normal MCLs were also tested at 4.1 MPa (n = 7) in a humidity chamber that maintained normal ligament water content. Additional MCLs were treated with both solutions in series (n = 12) to examine the reversibility of the mechanical changes caused by single solution treatment. This was the first investigation to show that ligament creep behaviour was clearly affected by the initial state of hydration: creep decreased with decreased hydration and creep increased with increased hydration. Another unique finding was that ligaments with increased hydration had decreased ligament functional length and increased ligament pre-stress. The creep behaviour of these ligaments was decreased if they were loaded from the pre-stressed state compared to the unloaded state. These results suggest that maintenance of physiological water content is important for in vitro mechanical testing of ligaments and controlling the low-load stress state of ligaments in situ.

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.

Ligament creep recruits fibres at low stresses and can lead to modulus-reducing fibre damage at higher creep stresses: a study in rabbit medial collateral ligament model.

Ligaments are subjected to a range of loads during different activities in vivo, suggesting that they must resist creep at various stresses. Cyclic and static creep tests of rabbit medial collateral ligament were used as a model to examine creep over a range of stresses in the toe- and linear-regions of the stress-strain curve: 4.1 MPa (n = 7), 7.1 MPa (n = 6), 14 MPa (n = 9) and 28 MPa (n = 6). We quantified ligament creep behaviour to determine if, at low stresses, modulus would increase in a cyclic creep test and collagen fibres would be recruited in a static creep test. At higher creep stresses, a decrease in measured modulus was expected to be a potential marker of damage. The increase in modulus during cyclic creep and the increase in strain during static creep were similar between the three toe-region stresses (4.1, 7.1, 14 MPa). However, at the linear-region stress (28 MPa), both these parameters increased significantly compared to the increases at the three toe-region stresses. A concurrent crimp analysis revealed that collagen fibres were recruited during creep, evidenced by decreased area of crimped fibres at the end of the static creep test. Interestingly, a predominance of straightened fibres was observed at the end of the 28 MPa creep test, suggesting a limited potential for fibre recruitment at higher, linear-region stresses. An additional 28 MPa (n = 6) group had mechanically detectable discontinuities in their stress-strain curves during creep that were related to reductions in modulus and suggested fibre damage. These data support the concept that collagen fibre recruitment is a mechanism by which ligaments resist creep at low stresses. At a higher creep stress, which was still only about a third of the failure capacity, damage to some ligaments occurred and was marked by a sudden reduction in modulus. In the cyclic tests, with continued cycling, the modulus increased back to original values obtained before the discontinuity suggesting that other fibres were being recruited to bear load. These results have important implications for our understanding of how fibre recruitment and stress redistribution act in normal ligament to minimize creep and restore modulus after fibre damage.

Compressive compared with tensile loading of medial collateral ligament scar in vitro uniquely influences mRNA levels for aggrecan, collagen type II, and collagenase.

To test the hypothesis that loading conditions can be used to engineer early ligament scar behaviors, we used an in vitro system to examine the effect that cyclic hydrostatic compression and cyclic tension applied to 6-week rabbit medial collateral ligament scars had on mRNA levels for matrix molecules, collagenase, and the proto-oncogenes c-fos and c-jun. Our specific hypothesis was that tensile stress would promote more normal mRNA expression in ligament whereas compression would lead to higher levels of mRNA for cartilage-like molecules. Femur (injured medial collateral ligament)-tibia complexes were subjected to a hydrostatic pressure of 1 MPa or a tensile stress of 1 MPa of 0.5 Hz for 1 minute followed by 14 minutes of rest. On the basis of a preliminary optimization experiment, this 15-minute testing cycle was repeated for 4 hours. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed for mechanically treated medial collateral ligament scars with use of rabbit specific primer sets for types I, II, and III collagen, decorin, biglycan, fibromodulin, versican, aggrecan, collagenase, c-fos, c-jun, and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. Cyclic hydrostatic compression resulted in a statistically significant increase in mRNA levels of type-II collagen (171% of nonloaded values) and aggrecan (313% of nonloaded values) but statistically significant decreases in collagenase mRNA levels (35% of nonloaded values). Cyclic tension also resulted in a statistically significant decrease in collagenase mRNA levels (66% of nonloaded values) and an increase in aggrecan mRNA levels (458% of nonloaded values) but no significant change in the mRNA levels for the other molecules. The results show that it is possible to alter mRNA levels for a subset of genes in scar tissue by supplying unique mechanical stimuli in vitro and thus that further investigation of scar engineering for potential reimplantation appears feasible.

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.

Intraoperative graft tensioning alters viscoelastic but not failure behaviours of rabbit medial collateral ligament autografts.

The effects of three different degrees of intraoperative graft tensioning on measures of ex vivo laxity, viscoelastic behaviour, and structural and material failure of isolated healing medial collateral ligament autografts were investigated in a rabbit model. The grafts were orthotopically replaced at one of three different loads (too tight, anatomic, or too loose) and were mechanically evaluated after 0, 12, 24, and 48 weeks of healing. Laxity of the ligament was influenced by intraoperative graft tensioning at time zero. However, after 12 weeks of healing, values for laxity were indistinguishable among the experimental groups. Cyclic load relaxation, a measure of viscoelastic behaviour, was significantly influenced by intraoperative graft tensioning, and this effect persisted even after 48 weeks of healing. Grafts placed under excessive tension relaxed one-third less than grafts placed under abnormally low in situ tension. The relevance of these differences remains to be determined. Intraoperative tensioning had no significant influence on characteristics of structural or material failure of the graft during the first year of healing. These results suggest that, in this model, control of graft tension at the time of placement and fixation does not improve the failure characteristics of the medial collateral ligament. The structural strength of the grafts collectively improved to nearly normal values after 48 weeks; however, material recovery was less complete. Failure loads averaged 89% of control values, whereas failure stress averaged only 52% after 48 weeks of healing.

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.

Method to assess in vivo knee stability longitudinally in an animal model of ligament injury.

The purpose of this study was to develop a method to prospectively quantify passive knee stability in an animal model of joint injury over time. Knee stability is defined here as the amount of translation or rotation of the tibia relative to the femur for a given application of force or moment, respectively. Five animals that had undergone transection of the anterior cruciate ligament and three control animals that had undergone a sham operation were anaesthetized and positioned in a stereotaxic frame. Motion of the tibia relative to the femur was quantified with use of reflective markers secured to modified bone pins and a three-dimensional motion analysis system. External forces and moments in the transverse plane of the tibia were measured with use of force transducers based on a strain-gauge design. Longitudinal measurements of knee stability were made before either sham surgery (control animals) or transection of the ligament (experimental animals), immediately after surgery, and at 2 and 4 months after transection. The results showed that the animals tolerated the procedures well and that systematic measurements could be obtained. The method described here has the practical advantage over cross-sectional experimental designs in that the number of subjects can be decreased while maintaining statistical power and has the further conceptual advantage that individual changes can be accounted for over time.