Compromised autophagy precedes meniscus degeneration and cartilage damage in mice.

OBJECTIVE: Autophagy is a cellular homeostasis mechanism that facilitates normal cell function and survival. Objectives of this study were to determine associations between autophagic responses with meniscus injury, joint aging, and osteoarthritis (OA), and to establish the temporal relationship with structural changes in menisci and cartilage. METHODS: Constitutive activation of autophagy during aging was measured in GFP-LC3 transgenic reporter mice between 6 and 30 months. Meniscus injury was created by surgically destabilizing the medial meniscus (DMM) to induce posttraumatic OA in C57BL/6J mice. Levels of autophagy proteins and activation were analyzed by confocal microscopy and immunohistochemistry. Associated histopathological changes, such as cellularity, matrix staining, and structural damage, were graded in the meniscus and compared to changes in articular cartilage. RESULTS: In C57BL/6J mice, basal autophagy was lower in the meniscus than in articular cartilage. With increasing age, expression of the autophagy proteins ATG5 and LC3 was significantly reduced by 24 months. Age-related changes included abnormal Safranin-O staining and reduced cellularity, which preceded structural damage in the meniscus and articular cartilage. In mice with DMM, autophagy was induced in the meniscus while it was suppressed in cartilage. Articular cartilage exhibited the most profound changes in autophagy and structure that preceded meniscus degeneration. Systemic administration of rapamycin to mice with DMM induced autophagy activation in cartilage and reduced degenerative changes in both meniscus and cartilage. CONCLUSION: Autophagy is significantly affected in the meniscus during aging and injury and precedes structural damage. Maintenance of autophagic activity appears critical for meniscus and cartilage integrity.

Histopathological analyses of murine menisci: implications for joint aging and osteoarthritis.

OBJECTIVE: To establish a standardized protocol for histopathological assessment of murine menisci that can be applied to evaluate transgenic, knock-out/in, and surgically induced OA models. METHODS: Knee joints from C57BL/6J mice (6-36 months) as well as from mice with surgically-induced OA were processed and cut into sagittal sections. All sections included the anterior and posterior horns of the menisci and were graded for (1) surface integrity, (2) cellularity, (3) Safranin-O staining distribution and intensity. Articular cartilage in the knee joints was also scored. RESULTS: The new histopathological grading system showed good inter- and intra-class correlation coefficients. The major age-related changes in murine menisci in the absence of OA included decreased Safranin O staining intensity, abnormal cell distribution and the appearance of acellular areas. Menisci from mice with surgically-induced OA showed severe fibrillations, partial/total loss of tissue, and calcifications. Abnormal cell arrangements included both regional hypercellularity and hypocellularity along with hypertrophy and cell clusters. In general, the posterior horns were less affected by age and OA. CONCLUSION: A new standardized protocol and histopathological grading system has been developed and validated to allow for a comprehensive, systematic evaluation of changes in aging and OA-affected murine menisci. This system was developed to serve as a standardized technique and tool for further studies in murine meniscal pathophysiology models.

Higher strains in the inner region of the meniscus indicate a potential source for degeneration.

Complex structural properties of menisci can be characterized in part by their inhomogeneous strain response under compression. This pilot study explored the feasibility to quantify characteristic strain distributions on meniscus cross-sections subjected to static compression using electronic speckle pattern interferometry (ESPI). Cross-sectional specimens of 5-mm thickness were harvested from eight human menisci. After application of 20% pre-strain, strain maps in response to 10µm compression were captured with ESPI. The 10µm compression induced an aggregate strain of nominally 0.14% and resulted in highly non-uniform strain distributions. Local compressive strain captured by ESPI ranged from 0.03% to 0.7%. The highest strain was in the central region of meniscus cross-sections, and the lowest magnitude of strain was at the femoral surface of the meniscus. After stratifying for age, peak compressive strain in older menisci (71±6 years, n=4) was 0.33%±0.09, compared to 0.25%±0.06 in younger menisci (34±9 years, n=4). In conclusion, this study captured for the first time continuous strain distribution maps over entire meniscus cross-sections. The non-uniform strain distributions demonstrated inhomogeneous structural properties. Age-related differences in characteristic strain distributions likely represent degenerative changes. As such, ESPI provides a novel strategy of further characterize meniscal function and degeneration.

Comparison of cartilage histopathology assessment systems on human knee joints at all stages of osteoarthritis development.

OBJECTIVE: To compare the MANKIN and OARSI cartilage histopathology assessment systems using human articular cartilage from a large number of donors across the adult age spectrum representing all levels of cartilage degradation. DESIGN: Human knees (n=125 from 65 donors; age range 23-92) were obtained from tissue banks. All cartilage surfaces were macroscopically graded. Osteochondral slabs representing the entire central regions of both femoral condyles, tibial plateaus, and the patella were processed for histology and Safranin O - Fast Green staining. Slides representing normal, aged, and osteoarthritis (OA) tissue were scanned and electronic images were scored online by five observers. Statistical analysis was performed for inter- and intra-observer variability, reproducibility and reliability. RESULTS: The inter-observer variability among five observers for the MANKIN system showed a similar good Intra-class correlation coefficient (ICC>0.81) as for the OARSI system (ICC>0.78). Repeat scoring by three of the five readers showed very good agreement (ICC>0.94). Both systems showed a high reproducibility among four of the five readers as indicated by the Spearman's rho value. For the MANKIN system, the surface represented by lesion depth was the parameter where all readers showed an excellent agreement. Other parameters such as cellularity, Safranin O staining intensity and tidemark had greater inter-reader disagreement. CONCLUSION: Both scoring systems were reliable but appeared too complex and time consuming for assessment of lesion severity, the major parameter determined in standardized scoring systems. To rapidly and reproducibly assess severity of cartilage degradation, we propose to develop a simplified system for lesion volume.

Macroscopic and histopathologic analysis of human knee menisci in aging and osteoarthritis.

OBJECTIVE: Meniscus lesions following trauma or associated with osteoarthritis (OA) have been described, yet meniscus aging has not been systematically analyzed. The objectives of this study were to (1) establish standardized protocols for representative macroscopic and microscopic analysis, (2) improve existing scoring systems, and (3) apply these techniques to a large number of human menisci. DESIGN: Medial and lateral menisci from 107 human knees were obtained and cut in two different planes (triangle/cross section and transverse/horizontal section as well) in three separate locations (middle portion, anterior and posterior horns). All sections included vascular and avascular regions and were graded for (1) surface integrity, (2) cellularity, (3) matrix/fiber organization and collagen alignment, and (4) Safranin-O staining intensity. The cartilage in all knee compartments was also scored. RESULTS: The new macroscopic and microscopic grading systems showed high inter-reader and intra-reader intraclass correlation coefficients. The major age-related changes in menisci in joints with no or minimal OA included increased Safranin-O staining intensity, decreased cell density, the appearance of acellular zones, and evidence of mucoid degeneration with some loss of collagen fiber organization. The earliest meniscus changes occurred predominantly along the inner rim. Menisci from OA joints showed severe fibrocartilaginous separation of the matrix, extensive fraying, tears and calcification. Abnormal cell arrangements included decreased cellularity, diffuse hypercellularity along with cellular hypertrophy and abnormal cell clusters. In general, the anterior horns of both medial and lateral menisci were less affected by age and OA. CONCLUSIONS: New standardized protocols and new validated grading systems allowed us to conduct a more systematic evaluation of changes in aging and OA menisci at a macroscopic and microscopic level. Several meniscus abnormalities appear to be specific to aging in the absence of significant OA. With aging the meniscal surface can be intact but abnormal matrix organization and cellularity were observed within the meniscal substance. The increased Safranin-O staining appears to represent a shift from fibroblastic to chondrocytic phenotype during aging and early degeneration.

Effect of meniscus replacement fixation technique on restoration of knee contact mechanics and stability.

The menisci are important biomechanical components of the knee. We developed and validated a finite element model of meniscal replacement to assess the effect of surgical fixation technique on contact behavior and knee stability. The geometry of femoral and tibial articular cartilage and menisci was segmented from magnetic resonance images of a normal cadaver knee using MIMICS (Materialise, Leuven, Belgium). A finite element mesh was generated using HyperWorks (Altair Inc, Santa Ana, CA). A finite element solver (Abaqus v6.9, Simulia, Providence, RI) was used to compute contact area and stresses under axial loading and to assess stability (reaction force generated during anteroposterior translation of the femur). The natural and surgical attachments of the meniscal horns and peripheral rim were simulated using springs. After total meniscectomy, femoral contact area decreased by 26% with a concomitant increase in average contact stresses (36%) and peak contact stresses (33%). Replacing the meniscus without suturing the horns did little to restore femoral contact area. Suturing the horns increased contact area and reduced peak contact stresses. Increasing suture stiffness correlated with increased meniscal contact stresses as a greater proportion of tibiofemoral load was transferred to the meniscus. A small incremental benefit was seen of simulated bone plug fixation over the suture construct with the highest stiffness (50 N/mm). Suturing the rim did little to change contact conditions. The nominal anteroposterior stiffness reduced by 3.1 N/mm after meniscectomy. In contrast to contact area and stress, stiffness of the horn fixation sutures had a smaller effect on anteroposterior stability. On the other hand suturing the rim of the meniscus affected anteroposterior stability to a much larger degree. This model emphasizes the importance of the meniscus in knee biomechanics. Appropriate meniscal replacement fixation techniques are likely to be critical to the clinical success of meniscal replacement. While contact conditions are mainly sensitive to meniscus horn fixation, the stability of the knee under anteroposterior shear loads appeared to be more sensitive to meniscal rim fixation. This model may also be useful in predicting the effect of biomaterial mechanical properties and meniscal replacement shape on knee contact conditions.

A nonlinear viscoelastic finite element model of polyethylene.

A nonlinear viscoelastic finite element model of ultra-high molecular weight polyethylene (UHMWPE) was developed in this study. Eight cylindrical specimens were machined from ram extruded UHMWPE bar stock (GUR 1020) and tested under constant compression at 7% strain for 100 sec. The stress strain data during the initial ramp up to 7% strain was utilized to model the "instantaneous" stress-strain response using a Mooney-Rivlin material model. The viscoelastic behavior was modeled using the time-dependent relaxation in stress seen after the initial maximum stress was achieved using a stored energy formulation. A cylindrical model of similar dimensions was created using a finite element analysis software program. The cylinder was made up of hexahedral elements, which were given the material properties utilizing the "instantaneous" stress-strain curve and the energy-relaxation curve obtained from the experimental data. The cylinder was compressed between two flat rigid bodies that simulated the fixtures of the testing machine. Experimental stress-relaxation, creep and dynamic testing data were then used to validate the model. The mean error for predicted versus experimental data for stress relaxation at different strain levels was 4.2%. The mean error for the creep test was 7% and for dynamic test was 5.4%. Finally, dynamic loading in a hip arthroplasty was modeled and validated experimentally with an error of 8%. This study establishes a working finite element material model of UHMWPE that can be utilized to simulate a variety of postoperative arthroplasty conditions.

Dynamic compression of chondrocytes induces a Rho kinase-dependent reorganization of the actin cytoskeleton.

The signal transduction mechanisms in chondrocytes that recognize applied forces and elicit the appropriate biochemical cellular responses are not well characterized. A current theory is that the actin cytoskeleton provides an intracellular framework onto which mechanosensation mechanisms are assembled. The actin cytoskeleton is linked to the extracellular matrix at multi-protein complexes called focal adhesions, and evidence exists that focal adhesions mediate the conversion of external physical forces into appropriate biochemical signal transduction events. The Rho GTPases affect the arrangement of actin cytoskeletal structures, and enhance the formation of focal adhesions, which link the cytoskeleton to the extracellular matrix. A major effector pathway downstream of Rho is the activation of Rho kinase (ROCK), which phosphorylates and activates Lim kinase, which in turn phosphorylates and inhibits the actin-depolymerizing protein cofilin. The objectives of this study were threefold: first, to quantify the actin reorganization in response to dynamic compression of agarose-embedded chondrocytes. Second, to test whether Rho kinase is required for the actin cytoskeletal reorganization induced by dynamic compression. Third, to test whether dynamic compression alters the intracellular localization of Rho kinase and actin remodeling proteins in chondrocytes. Dynamic compression of agarose-embedded chondrocytes induced actin cytoskeletal remodeling causing a significant increase in punctate F-actin structures. Rho kinase activity was required for these cytoskeletal changes. Dynamic compression increased the amount of phosphorylated Rho kinase. The chemokine CCL20 and inducible nitric oxide synthase (iNOS) were the most highly upregulated genes by dynamic compression and this response was reduced by the Rho kinase inhibitors. In conclusion, we show that dynamic compression induces changes in the actin cytoskeleton of agarose-embedded chondrocytes, and we establish methodology to quantify these changes. Furthermore, we show that Rho kinase activity is required for this actin reorganization and gene expression induced by dynamic compression.

Polyethylene wear and variations in knee kinematics.

A six-station knee wear simulator was used to test a posterior cruciate-retaining total knee arthroplasty design. Six implants each were tested in three groups; low intensity, high intensity, and malalignment using kinematic inputs from normal gait data, more severe loading conditions, and 3 degrees varus malalignment, respectively. For each group, gravimetric wear of the polyethylene inserts was measured for 5,000,000 cycles. Knee wear testing showed significantly different results for the three groups. Low intensity group inserts had mean wear rates of 3.1 (+/- 1.2) mg per million cycles. High intensity group inserts had significantly higher mean wear rates of 7.4 (+/- 2.7) mg per million cycles. Malalignment group inserts had the highest wear rates of 9.2 (+/- 3.3) mg per million cycles. The wear generated in the knee simulator seems to be dependent on the relative motions and loads at the articulating surface. The high intensity groups were subjected to motions that included reciprocating anteroposterior translations and a higher peak axial load than the low intensity group. This resulted in increasing the amount of wear. Varus malalignment also increased the total wear significantly. These results may explain some of the wide variations in wear seen in retrieved knee implants.

Quadriceps moment arm and quadriceps forces after total knee arthroplasty.

Knee prosthetic designs that increase quadriceps moment arm can reduce quadriceps tension and patellofemoral compressive forces. Six knees from cadavers were tested on the Oxford knee rig, which simulates closed chain knee extension under load. Three conditions were tested sequentially for each knee: Normal, Control (implanted with the Osteonics 7000 knee design), and Scorpio (implanted with the Osteonics Scorpio design). The center of flexion-extension of the Scorpio design was 10 mm posterior to that of Control that served to lengthen the quadriceps moment arm. An electromagnetic tracking system measured dynamic knee kinematics, and a uniaxial load cell measured quadriceps tension. The Scorpio design reduced quadriceps tension when compared with the Normal or Control knee ranging from 5% to 20%. This was statistically significant at flexion angles greater than 50 degrees. In three knees, the patellar component was instrumented with a triaxial load cell that measured patellofemoral forces. Patellofemoral forces were lower with the Scorpio design compared with the Control. Increasing quadriceps lever arm reduces quadriceps forces and can facilitate activities of daily living and enhance patient rehabilitation. Reduced quadriceps forces may result in reduced patellofemoral forces that can have a beneficial effect on anterior knee pain, patellar component wear, and loosening.

Polyethylene contact stresses, articular congruity, and knee alignment.

Increased conformity at the tibiofemoral articulation increases contact area and reduces contact stresses in total knee arthroplasty. Malalignment, however, can increase polyethylene contact stresses. The effect of knee alignment and articular conformity on contact stresses was evaluated in a finite element model. The polyethylene insert and femoral component were modeled in high- and low-conformity conditions. An axial tibial load of 3000 N was applied across the tibiofemoral articulation at different knee positions ranging from 0 degrees, to 90 degrees, flexion, 0 to 10 mm anteroposterior translation, 0 degrees to 10 degrees axial rotation, and coronal plane angulation (liftoff). Increased conformity significantly reduced contact stresses in neutral alignment (by 44% at 0 degrees flexion and 36% at 60 degrees and 90 degrees flexion). Liftoff significantly increased contact stresses in low- and high-conformity conditions, but to a lesser degree in the high-conformity condition. Malalignment in rotation was most detrimental especially with the high-conformity insert design. Overall, increasing articular conformity reduced stresses when the knee was well-aligned. However, malalignment in axial rotation was detrimental. Mobile-bearing knee designs with increased articular congruity may result in lower contact stresses, especially the rotating-bearing designs that theoretically minimize rotational malalignment.

In vivo changes after mechanical injury.

Chondrocytes undergo apoptosis in response to mechanical injury in vitro. The current clinical study correlates arthroscopic and magnetic resonance imaging results with biopsy specimens of cartilage from patients with knee injury. Twenty patients were evaluated at a mean 2.7 months after acute knee injury. The mean age of the patients was 32 years and the mean weight was 83 kg. Cartilage lesions were graded separately on magnetic resonance images and arthroscopy in a blinded manner. During arthroscopy, a 1.8 mm diameter biopsy specimen was obtained from the edge of cartilage lesion. The biopsy specimen underwent histologic examination by safranin O staining and detection of chondrocyte apoptosis by the presence of deoxyribonucleic acid fragmentation. There was a positive correlation in 50% (10 of 20) when the presence or absence of cartilage lesions by magnetic resonance imaging was correlated with arthroscopy. All cases of partial thickness or full-thickness cartilage loss that were seen by arthroscopy also were detected by magnetic resonance images. Apoptotic cells were significantly more numerous in biopsy specimens from lesions compared with control biopsy specimens. The findings of reduced cell viability attributable to apoptosis may have profound implications for cartilage repair. This opens potential therapeutic avenues for the treatment of posttraumatic cartilage lesions through apoptosis prevention.