Mechanical load inhibits IL-1 induced matrix degradation in articular cartilage.

OBJECTIVE: Osteoarthritis is a disease process of cellular degradation of articular cartilage caused by mechanical loads and inflammatory cytokines. We studied the cellular response in native cartilage subjected to a mechanical load administered simultaneously with an inflammatory cytokine interleukin-1 (IL-1), hypothesizing that the combination of load and cytokine would result in accelerated extracellular matrix (ECM) degradation. METHODS: Mature bovine articular cartilage was loaded for 3 days (stimulation) with 0.2 and 0.5 MPa stresses, with and without IL-1 (IL-1alpha, 10 ng/ml), followed by 3 days of no stimulation (recovery). Aggrecan and collagen loss were measured as well as aggrecan cleavage using monoclonal antibodies AF-28 and BC-3 for cleavage by aggrecanases (ADAMTS) and matrix metalloproteinases (MMPs), respectively. RESULTS: Incubation with IL-1 caused aggrecan cleavage by aggrecanases and MMPs during the 3 days of stimulation. A load of 0.5 MPa inhibited the IL-1-induced aggrecan loss while no inhibition was found for the 0.2 MPa stress. There was no collagen loss during the treatments but upon load and IL-1 removal proteoglycan and collagen loss increased. Load itself under these conditions was found to have no effect when compared to the unloaded controls. CONCLUSIONS: A mechanical load of sufficient magnitude can inhibit ECM degradation by chondrocytes when stimulated by IL-1. The molecular mechanisms involved in this process are not clear but probably involve altered mechanochemical signal transduction between the ECM and chondrocyte.

Nondegradable hydrogels for the treatment of focal cartilage defects.

Nondegradable materials have long been suggested for the treatment of articular cartilage defects; however, the mechanics of the implant/tissue system necessary to ensure long-term function are unknown. The objective of this study was to explore the performance of nondegradable hydrogel implants in cartilage defects. Our hypothesis was that the structural integrity of the implant and surrounding tissue would be influenced by the compressive modulus of the material used, and that superior results would be obtained with the implantation of a more compliant material. Poly(vinyl alcohol)-poly(vinyl pyrrolidone) hydrogel implants of two different moduli were implanted into osteochondral defects in a rabbit model. Six-month postoperative histological and mechanical data were used to assess the wear and fixation of the implants. The compliant implants remained well fixed and a thin layer of soft tissue grew over the surface of the implants. However, gross deformation of the compliant implants occurred and debris was evident in surrounding bone. The stiffer implants were dislocated from their implantation site, but with no accompanying evidence of debris or implant deformation. Our hypothesis that superior results would be obtained with implantation of a more compliant material was rejected; a compromise between the wear and fixation properties dependent on modulus was found.

Effect of compressive strain on cell viability in statically loaded articular cartilage.

Physiological loading of articulating joints is necessary for normal cartilage function. However, conditions of excessive overloading or trauma can cause cartilage injury resulting in matrix damage and cell death. The objective of this study was to evaluate chondrocyte viability within mechanically compressed articular cartilage removed from immature and mature bovine knees. Twenty-three mature and 68 immature cartilage specimens were subjected to static uniaxial confined-creep compressions of 0-70% and the extent of cell death was measured using fluorescent microscopic imaging. In both age groups, cell death was always initiated at the articular surface and increased linearly in depth with increasing strain magnitude. However, most of the cell death was localized within the superficial zone (SZ) of the cartilage matrix with the depth never greater than approximately 500 microm or 25% of the thickness of the test specimen. The immature cartilage was found to have a significantly greater (> 2 times) amount (depth) of cell death compared to the mature cartilage, especially at the higher strains. This finding was attributed to the lower compressive modulus of the immature cartilage in the SZ compared to that of the mature cartilage, resulting in a greater local matrix strain and concomitant cell surface membrane strain in this zone when the matrix was compressed. These results provide further insight into the capacity of articular cartilage in different age groups to resist the severity of traumatic injury from compressive loads.

Intracellular calcium response of ACL and MCL ligament fibroblasts to fluid-induced shear stress.

This study examines the real-time intracellular calcium concentration, [Ca2+]i, response of canine medial collateral ligament (MCL) and anterior cruciate ligament (ACL) fibroblasts subjected to a fluid-induced shear stress of 25 dynes/cm2. In experiments using a modified Hanks' Balanced Salt Solution (HBSS) perfusate, both cell types demonstrated a significant increase in peak [Ca2+]i compared to respective no-flow controls, the response of MCL fibroblasts being nearly 2-fold greater than that of ACL fibroblasts. In studies where the cells were bathed in a medium of HBSS supplemented with 2% newborn bovine serum (NBS) and then introduced to flow with the same medium, ACL fibroblasts responded nearly 3-fold greater than MCL fibroblasts. Neomycin (10 mM), thapsigarigin (1 microM) and Ca(2+)-free media supplemented with EGTA (1 mM) were able to inhibit significantly the [Ca2+]i response to flow with HBSS in both fibroblasts. Thapsigargin also blocked the NBS flow response in both cell types, while neomycin and Ca(2+)-free media significantly inhibited the ACL response. Our findings demonstrate that ACL and MCL cells are not the same. These differences may be related to the disparate healing capacity of the ACL and MCL observed clinically.

Fourier transform infrared imaging spectroscopy analysis of collagenase-induced cartilage degradation.

Collagenase treatment of cartilage serves as an in vitro model of the pathological collagen degradation that occurs in the disease osteoarthritis (OA). Fourier transform infrared imaging spectroscopic (FT-IRIS) analysis of collagenase-treated cartilage is performed to elucidate the molecular origin of the spectral changes previously found at the articular surface of human OA cartilage. Bovine cartilage explants are treated with 0.1% collagenase for 0, 15, or 30 min. In situ collagen cleavage is assessed using immunofluorescent staining with an antibody specific for broken type II collagen. The FT-IRIS analysis of the control and treated specimens mirrors the differences previously found between normal and OA cartilage using an infrared fiber optic probe (IFOP). With collagenase treatment, the amide II/1338 cm(-1) area ratio increases while the 1238 cm(-1)/1227 cm(-1) peak ratio decreases. In addition, polarized FT-IRIS demonstrates a more random orientation of the collagen fibrils that correlate spatially with the immunofluorescent-determined regions of broken type II collagen. We can therefore conclude that the spectral changes observed in the collagenase-treated cartilage, and similarly in OA cartilage, arise from changes in collagen structure. These findings support the use of mid-infrared spectral analysis, in particular the minimally invasive IFOP, as potential techniques for the diagnosis and management of degenerative joint diseases such as osteoarthritis.

Proton spin-spin relaxation study of molecular dynamics and proteoglycan hydration in articular cartilage.

Spin-spin relaxation of proton magnetization in natural and deuterated articular cartilage is reported over a range of hydration. Information about macromolecular dynamics is deduced and a hydration stabilized macromolecular regime identified. There is good correspondence between NMR results and cartilage stoichiometry. A new measure for hydration of proteoglycans is found.

Effect of serum and platelet-derived growth factor on chondrocytes grown in collagen gels.

In this in vitro study, cell proliferation, viability, and morphology; proteoglycan (PG) synthesis; and gel contraction were assessed over a 15-day period (on days 3, 6, 9, 12, and 15) for mature bovine chondrocytes cultured in collagen gels. The environment within the gel was varied by changing the concentration of fetal bovine serum (1% and 10%) and platelet-derived growth factor-BB (PDGF; 0, 10, 50, 100 ng/ml) within the gel and incubation media. Our results showed that the amount of serum or PDGF added to the gels had no effect on cell viability, with >95% of cells remaining alive throughout the experiment. There was a significant increase in cell number over time in all groups, with a higher rate of cell proliferation in gels containing 10% serum and higher concentrations of PDGF. In addition, the amount of serum significantly affected gel contraction with or without PDGF. Gels containing 10% serum contracted on day 10-12, while none of the gels containing 1% serum contracted over the course of the experiment. The PG content within each gel increased with incubation time only for the gels containing 1% serum, and 10 or 100 ng/ml of PDGF. However, on a per cell basis, there was no change in the PG content with time when only serum was used and a significant decrease in the rate of PG production with the addition of PDGF (9.1-27.8 pgPG/cell/day). Cell morphology was also affected by PDGF, with the cells becoming more spindle shaped. Cell alignment within the gels appeared to be most affected by gel contraction. Collagen gels can act as cell carriers for the purpose of tissue engineering. These gels provide a three-dimensional environment in which chondrocytes can proliferate and produce matrix. We have shown how this environment can be controlled to affect gel contraction, rates of cell growth and PG production, and cellular morphology while maintaining cell viability. This information will be useful in determining the conditions in which chondrocytes can be grown within collagen gels and combined with cytokines to create an ideal tissue construct.

Water content and equilibrium water partition in immature cartilage.

The water content, equilibrium water partition, and total thickness for immature bovine articular cartilage were determined and compared to previously determined values for mature tissue. The bulk equilibrium water partition and thickness were significantly greater in immature tissue as compared to mature tissue, while the bulk water content was similar. Spatial differences were noted in each physical parameter.

Influence of cartilage conformation on its equilibrium water partition.

The spatial and bulk water equilibrium partition and fluid content were determined for normal adult bovine articular cartilage as a function of pH, temperature, and geometric confinement. Water partition averaged 60 +/- 7% at neutral pH and 37 degrees C and increased with decreasing pH and increasing temperature without a concomitant change in fluid content. The variation in water partition appeared to be a result of local conformation changes in the collagen fibril ultrastructure causing a transfer between free and trapped water volume. Removal of the lateral and subchondral bone geometric constraints caused an increase in both the water partition and fluid content. However, this partition variation could be accounted for solely from a change in free fluid volume without a change in the trapped fluid volume. These results suggest that in articular cartilage the proteoglycan-collagen interaction may be an important mechanism for controlling the partition of water between a freely exchangeable space and a space allowing no fluid exchange.

Effect of stress deprivation and cyclic tensile loading on the material and morphologic properties of canine flexor digitorum profundus tendon: an in vitro study.

The effect of stress deprivation and cyclic tensile loading on the mechanical and histologic properties of the canine flexor digitorum profundus tendon was examined using an in vitro system. Stress deprivation resulted in a progressive and statistically significant decrease in the tensile modulus over an 8-week period. Histologically, stress-deprived tendons demonstrated quantitative changes in the morphology and number of cells and in the alignment of collagen. The change in tensile properties was not associated with an alteration in the water content of the tissue, but the change appeared to be dependent on the presence of a viable cell population. Dead (acellular) tendons did not undergo any alteration in tensile modulus in this in vitro system. In vitro cyclic tensile loading of tendons over a 4-week time period resulted in a significant increase in the tensile modulus (93% of the control) compared with that of the stress-deprived tendons (68% of the control). This loading regimen also maintained the normal histologic pattern of the tendons. The results of this study are similar to those previously reported for in vivo studies and suggest that this in vitro model may represent a valid system with which to test the effects of various stress conditions on the tensile properties of tissues.

Measurement reproducibility of two commercial knee test devices.

Objective evaluation of patients' knee motion using mechanical devices, whether for diagnostic purposes or for assessing rehabilitative procedures, requires that these devices be reproducible, in order to avoid errors independent of the patients' condition. This study prospectively evaluates the reproducibility of two commercial knee test systems, the KT 1000 Knee Ligament Arthrometer and the Genucom Knee Analysis System, by performing repeated measurements on twenty normal men. Average knee motion, between-subject variance, and within-subject variance were determined by performing repeat tests on the same day and one week later. No significant difference was found between measurements taken on the two different test days, but the within-subject variation was high. We found that larger applied forces or moments resulted in a larger variation in mean displacements. However, the percent variation about the mean (coefficient of variation) decreased with increasing applied loads. The coefficient of variation for the KT 1000 varied from 8 to 33% and for the Genucom, from 13 to 87%. On an individual basis, large variations were found in repeated measures for both devices. To minimize errors, we recommend that repeated tests be performed, higher forces and moments utilized, and specific flexion angles be used for each device and test. Even under these conditions, caution must be exercised when evaluating individual subjects.

Prevention of ligament and meniscus atrophy by active joint motion in a non-weight-bearing model.

This study describes the effect of active joint motion on the maintenance of ligament and meniscus mass in a non-weight-bearing model of disuse. Denervation and fixation models of immobilization have shown that resorption of isotope and atrophy of mass occurred for hard tissue (bone) and soft tissues (ligament, tendon, or meniscus). A unilateral ankle disarticulation model of disuse that maintains active knee motion without weight bearing was studied for 8 weeks in dogs that were chronically prelabeled with three different isotopes. The effects of non-weight-bearing without denervation or fixation were analyzed for the resorption of isotopes, and net atrophy of bone mass (femur or tibia) and soft-tissue mass (collateral or cruciate ligaments, menisci). A large and similar loss of all three isotopes, as well as collagen and calcium mass occurred for whole femur and tibia; this indicated that mass loss was equivalent to bone resorption and suggests little replacement with new bone. No loss of isotope or mass per whole tissue occurred for the collateral and cruciate ligaments or menisci. The strength of the femur-anterior cruciate ligament-tibia complex was analyzed by a tensile failure test when a fast rate of deformation was applied; the results did not differ qualitatively or quantitatively between control and experimental limbs. The absence of weight bearing for 8 weeks resulted in marked bone atrophy without resorption or atrophy of soft tissues, or decrease of the mechanical strength for the femur-ligament-tibia complex.

Diminished material properties and altered bone structure in rat femora during pregnancy.

Pregnancy and lactation are known to cause structural and mechanical changes in bone, but the effects of pregnancy alone have not been evaluated thoroughly. This study used radiographic measurements, torsion testing, mineral analyses, and histological evaluation to determine whether there are changes in bone material and geometric properties during pregnancy in the growing rat, as implied by earlier biochemical and histological studies. The bones of pregnant 9 to 12-week-old rats and controls that were not pregnant and were matched by age (but not weight) were evaluated at times corresponding to 5, 10, 15, and 20 days of the 23-day gestation period to address the following questions: (a) How is the growth of whole bone affected by pregnancy in the growing rat (as determined by radiographic analyses)? (b) How are the mechanical properties (structural and material) of whole bone affected by pregnancy (as assessed by torsion testing)? (c) Are there changes in the characteristics of bone mineral during pregnancy (as determined by measurement of mineral content and x-ray diffraction analyses)? and (d) Are there detectable morphological or ultrastructural differences between the bones of pregnant and control rats (as assessed by analyses based on histology and back-scattered electron imaging)? The presence of statistically significant differences in this study was determined initially on the basis of a two-factor analysis of variance. In general, significant differences were noted only at late gestation (day 20), when the bones were longer and had a greater outer radius and cortical thickness; this indicates that more growth occurred during pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the posterolateral and cruciate ligaments in the stability of the human knee. A biomechanical study.

Injury to the posterolateral structures of the knee, including the popliteus tendon and arcuate complex, frequently results in poorly understood patterns of instability. To evaluate the static function of these tissues, we used a mechanical testing apparatus that allowed five degrees of freedom to test seventeen specimens from human cadavera at angles of flexion that ranged from zero to 90 degrees. Selective section of the lateral collateral ligament, popliteus-arcuate (deep) ligament complex, anterior cruciate ligament, and posterior cruciate ligament was performed. At all angles of flexion, the lateral collateral ligament and deep ligament complex functioned together as the principal structures preventing varus rotation and external rotation of the tibia, while the posterior cruciate ligament was the principal structure preventing posterior translation. However, at angles of flexion of 30 degrees or less, the amount of posterior translation after section of only the lateral collateral ligament and the deep structures was similar to that noted after isolated section of the posterior cruciate ligament. Isolated section of the posterior cruciate ligament did not affect varus or external rotation of the tibia at any position of flexion of the knee. When the posterior cruciate ligament was sectioned after the lateral collateral ligament and deep ligament complex had been cut, a large increase in posterior translation and varus rotation resulted at all angles of flexion. In addition, at angles of flexion of more than 30 degrees, external rotation of the tibia also increased. The application of internal tibial torque resulted in no increase in tibial rotation after isolated section of the anterior cruciate ligament or combined section of the lateral collateral ligament and deep ligament complex. However, combined section of all three structures increased internal rotation at 30 and 60 degrees of flexion. The increases in external rotation that were produced by section of the lateral collateral ligament and deep ligament complex were not changed by the addition of the section of the anterior cruciate ligament.

An in vitro biomechanical evaluation of anterior-posterior motion of the knee. Tibial displacement, rotation, and torque.

We tested the anterior-posterior motion of nine normal cadaver knees in zero to 90 degrees of flexion using a specially designed apparatus. This apparatus applied a dynamic anterior-posterior force to each knee and measured the resulting tibial displacement, rotation, and torque. In the intact knee, an anterior force produced an internal tibial torque and internal tibial rotation, while a posterior force produced an external torque and external rotation. Anterior-posterior displacement increased by 30 per cent when the tibia was allowed to rotate freely about its neutral rotation position. Isolated section of the anterior cruciate ligament produced more than double the amount of anterior displacement without affecting posterior displacement. Isolated section of the posterior cruciate ligament produced almost triple the amount of posterior displacement without affecting anterior displacement. After cutting either the anterior or the posterior cruciate ligament, the resulting internal or external secondary tibial rotation disappeared. It appears, therefore, that the anterior and posterior cruciate ligaments are the primary restraints to motion in the anterior and posterior directions as well as the causes of internal and external tibial rotation during anterior and posterior motion.

The effect of medial meniscectomy on anterior-posterior motion of the knee.

We used an in vitro knee-testing apparatus to measure anterior-posterior displacement of the tibia on the femur and the accompanying tibial rotation in response to an applied anterior-posterior force. Testing was performed on nine intact knees, on five knees after medial meniscectomy, on three knees after isolated section of the anterior cruciate ligament, and on eight knees after both excision of the medial meniscus and section of the anterior cruciate ligament. The induced anterior-posterior displacement and the coupled rotation were unaffected by meniscectomy. Isolated section of the anterior cruciate ligament allowed a significant (p less than 0.05) increase in anterior displacement but had no effect on posterior displacement. The coupled internal rotation associated with anterior displacement was lost after section of the anterior cruciate ligament. Excision of the medial meniscus and section of the anterior cruciate ligament allowed significantly (p less than 0.05) greater increases in anterior displacement than those already increased by isolated section of the anterior cruciate ligament.

Biomechanical evaluation of a simulated Bankart lesion.

The purpose of this study was to determine the effect of sectioning of the anterior part of the inferior glenohumeral ligament (a simulated Bankart lesion) on load-induced multidirectional glenohumeral motion. Nine fresh, intact cadaveric shoulders were tested on a special apparatus that constrained three rotations but allowed simultaneous measurement of anterior-posterior, superior-inferior, and medial-lateral translation. Coupled anterior-posterior and superior-posterior translations were recorded while anterior, posterior, superior, and inferior forces of fifty newtons were applied sequentially. Testing was done in three positions of humeral elevation in the scapular plane, in three positions of humeral rotation, and with an externally applied joint-compression load of twenty-two newtons. A liquid-metal strain-gauge was placed on the posterior band of the inferior glenohumeral ligament to assess concomitant posterior capsular strain during the various test conditions. All shoulders were tested intact and again after the inferior glenohumeral ligament and the labrum had been detached from the glenoid from just superior to the anterior band of the inferior glenohumeral ligament to a point just posterior to the infraglenoid tubercle. The simulated Bankart lesion resulted in selected increases in anterior translation at all positions of elevation, in posterior translation at 90 degrees of elevation, and in inferior translation at all positions of elevation. However, these increases were very small; the maximum mean increase in translation seen over-all was only 3.4 millimeters, which occurred during inferior translation at 45 degrees of elevation.(ABSTRACT TRUNCATED AT 250 WORDS)

Tendon-healing in a bone tunnel. A biomechanical and histological study in the dog.

Our study evaluated tendon-to-bone healing in a dog model. Twenty adult mongrel dogs had a transplantation of the long digital extensor tendon into a 4.8-millimeter drill-hole in the proximal tibial metaphysis. Four dogs were killed at each of five time-periods (two, four, eight, twelve, and twenty-six weeks after the transplantation), and the histological and biomechanical characteristics of the tendon-bone interface were evaluated. Serial histological analysis revealed progressive reestablishment of collagen-fiber continuity between the bone and the tendon. A layer of cellular, fibrous tissue was noted between the tendon and the bone, along the length of the bone tunnel; this layer progressively matured and reorganized during the healing process. The collagen fibers that attached the tendon to the bone resembled Sharpey fibers. High-resolution radiographs showed remodeling of the trabecular bone that surrounded the tendon. At the two, four, and eight-week time-periods, all specimens had failed by pull-out of the tendon from the bone tunnel. The strength of the interface was noted to have significantly and progressively increased between the second and the twelfth week after the transplantation. At the twelve and twenty-six-week time-periods, all specimens had failed by pull-out of the tendon from the clamp or by mid-substance rupture of the tendon. The progressive increase in strength was correlated with the degree of bone ingrowth, mineralization, and maturation of the healing tissue, noted histologically.

Effect of lesions of the superior portion of the glenoid labrum on glenohumeral translation.

Lesions of the superior portion of the glenoid labrum were created in seven cadaveric shoulders. The shoulders were mounted on a special apparatus attached to a servocontrolled hydraulic materials-testing device. Sequential fifty-newton anterior, posterior, superior, and inferior forces and a twenty-two-newton joint compressive load were applied to the shoulders. In addition, a fifty-five-newton force was applied to the tendon of the long head of the biceps brachii. The shoulders were tested in seven positions of glenohumeral elevation and rotation. An isolated lesion of the anterosuperior portion of the labrum, which did not involve the supraglenoid insertion of the biceps brachii, had no significant effect on anteroposterior or superoinferior glenohumeral translation, either with or without application of the fifty-five-newton force to the biceps brachii tendon. In contrast, a complete lesion of the superior portion of the labrum that destabilized the insertion of the biceps resulted in significant increases in anteroposterior and superoinferior glenohumeral translations. At 45 degrees of glenohumeral elevation, the complete lesion led to a 6.0-millimeter increase in anterior translation when the arm was in neutral rotation and to a 6.3-millimeter increase when the arm was in internal rotation; inferior translation also increased, by 1.9 to 2.5 millimeters. The increases in translation persisted despite application of a fifty-five-newton force to the long head of the biceps.

A method for the postmortem evaluation of an in situ total hip replacement.

A method was developed for the post-mortem evaluation of a total hip replacement retrieved in situ. The hip replacement had been implanted for six years. The evaluation procedure employed existing techniques in a logical sequence such that earlier tests would not compromise results from subsequent ones. These techniques included the measurement of range of motion, aspiration of the joint for analysis of debris, radionuclide arthrograms for determination of looseness of the components, gross and histological inspection of the capsule and synovial tissue, serial sectioning of the femoral component, determination of some mechanical properties of the bone and femoral stem, and scanning electron microscopy of the articulating surfaces of the prosthetic components. Positive findings in the specimen studied were: excessive wear of the articulating surface of the acetabular component, and associated polyethylene debris in the aspirate and surrounding synovial and granulation tissue.