Meniscal Explants Research Articles

Selenium Reduces Early Signs of Tumor Necrosis Factor Alpha-Induced Meniscal Tissue Degradation.

Meniscal integrity is a prerequisite for sustained knee joint health and prevention of meniscal degeneration is a main research goal. Cartilage-protective effects of selenium have been described, but little is known about the impact on the meniscus. We therefore investigated the influence of sodium selenite on meniscal explants under tumor necrosis factor-alpha (TNFα)-stimulated proinflammatory conditions. Meniscal explant disks (3mm diameter×1mm thickness) were isolated from 2-year-old cattle and incubated with TNFα (10ng/ml) and sodium selenite (low dose, LoD 6.7ng/ml as being found in Insulin-Transferrin-Selenium medium supplements, ITS; medium-dose, MeD 40ng/ml described as physiological synovial concentration; high dose, HiD 100ng/ml described as optimal serum concentration). After 3days of culture glycosaminoglycan (GAG) release (DMMB assay), nitric oxide (NO) production (Griess assay), gene expression of matrix-degrading enzymes (quantitative RT-PCR), and apoptosis rate were determined. TNFα led to a significant raise of GAG release and NO production. LoD and MeD selenite significantly reduced the TNFα-induced GAG release (by 83, 55%, respectively), NO production (by 59, 40%, respectively), and apoptosis (by 68, 39%, respectively). LoD and MeD selenite showed a tendency to reduce the TNFα-mediated increase of inducible NO-synthase (iNOS) levels, LoD selenite furthermore matrix metalloproteinase (MMP)-3 transcription levels and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 levels. LoD and less pronounced MeD selenite show a substantial impact on the early meniscal inflammatory response. To our knowledge this is the first study showing the protective influence of selenium on meniscal tissue maintenance. To understand the superior potency of low-dose selenium on molecular level future studies are needed.

Augmented repair of radial meniscus tear with biomimetic electrospun scaffold: an in vitro mechanical analysis.

BackgroundLarge radial tears that disrupt the circumferential fibers of the meniscus are associated with reduced meniscal function and increased risk of joint degeneration. Electrospun fibrous scaffolds can mimic the topography and mechanics of fibrocartilaginous tissues and simultaneously serve as carriers of cells and growth factors, yet their incorporation into clinically relevant suture repair techniques for radial meniscus tears is unexplored. The purposes of this study were to (1) evaluate the effect of fiber orientation on the tensile properties and suture-retention strength of multilayered electrospun scaffolds and (2) determine the mechanical effects of scaffold inclusion within a surgical repair of a simulated radial meniscal tear. The experimental hypothesis was that augmentation with a multilayered scaffold would not compromise the strength of the repair.MethodsThree multilayered electrospun scaffolds with different fiber orientations were fabricated–aligned, random, and biomimetic. The biomimetic scaffold was comprised of four layers in the following order (deep to superficial)–aligned longitudinal, aligned transverse, aligned longitudinal, and random–respectively corresponding to circumferential, radial, circumferential, and superficial collagen fibers of the native meniscus. Material properties (i.e., ultimate stress, modulus, etc.) of the scaffolds were determined in the parallel and perpendicular directions, as was suture retention strength. Complete radial tears of lateral bovine meniscus explants were repaired with a double horizontal mattress suture technique, with or without inclusion of the biomimetic scaffold sheath. Both repair groups, as well as native controls, were cyclically loaded between 5 and 20 N for 500 cycles and then loaded to failure. Clamp-to-clamp distance (i.e., residual elongation) was measured following various cycles. Ultimate load, ultimate elongation, and stiffness, were also determined. Group differences were evaluated by one-way ANOVA or Student’s t-test where appropriate.ResultsAligned scaffolds possessed the most anisotropic mechanical properties, whereas random scaffolds showed uniform properties in the parallel and perpendicular directions. In comparison, the biomimetic scaffold possessed moduli in the parallel (68.7 ± 14.7 MPa) and perpendicular (39.4 ± 11.6 MPa) directions that respectively approximate the reported circumferential and radial tensile properties of native menisci. The ultimate suture retention load of the biomimetic scaffold in the parallel direction (7.2 ± 1.6 N) was significantly higher than all other conditions (p < 0.001). Biomimetic scaffold augmentation did not compromise mechanical properties when compared against suture repair in terms of residual elongation after 500 cycles (scaffold: 5.05 ± 0.89 mm vs. repair: 4.78 ± 1.24 mm), ultimate failure load (137.1 ± 31.0 N vs. 124.4 ± 21.4 N), ultimate elongation (12.09 ± 5.89 mm vs. 10.14 ± 4.61 mm), and stiffness (20.8 ± 3.6 vs. 18.4 ± 4.7 N/mm).ConclusionsWhile multilayered scaffold sheets were successfully fabricated to mimic the ultrastructure and anisotropic tensile properties of native menisci, improvements in suture retention strength or adoption of superior surgical techniques will be needed to further enhance the mechanical strength of repairs of radial meniscal tears.

Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo

Background: Although patients with an anterior cruciate ligament (ACL) injury have a high risk of developing posttraumatic osteoarthritis (PTOA), the role of meniscus hypertrophy and mineralization in PTOA after an ACL injury remains unknown. Purpose/Hypothesis: The purpose of this study was to determine if menisci respond to abnormal loading and if an ACL injury results in meniscus hypertrophy and calcification. The hypotheses were that (1) abnormal mechanical loading after an ACL injury induces meniscus hypertrophy and mineralization, which correlates to articular cartilage damage in vivo, and (2) abnormal mechanical loading on bovine meniscus explants induces the overexpression of hypertrophic and mineralization markers in vitro. Study Design: Controlled laboratory study. Methods: In vivo guinea pig study (hypothesis 1): Three-month-old male Hartley guinea pigs (n = 9) underwent ACL transection (ACLT) on the right knee; the left knee served as the control. Calcification in the menisci was evaluated by calcein labeling 1 and 5 days before knee harvesting at 5.5 months. Cartilage and meniscus damage and mineralization were quantified by the Osteoarthritis Research Society International score and meniscus grade, respectively. Indian hedgehog (Ihh), matrix metalloproteinase–13 (MMP-13), collagen type X (Col X), progressive ankylosis homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase–1 (ENPP1), alkaline phosphatase (ALP), inorganic pyrophosphate (PPi), and inorganic phosphate (Pi) concentrations were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. In vitro bovine meniscus explant study (hypothesis 2): Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 1, 2, and 3 hours. Cell viability was determined using live/dead staining. The levels of mRNA expression and protein levels were measured using real-time quantitative reverse transcription polymerase chain reaction and Western blot after 24, 48, and 72 hours in culture. The conditioned medium was collected for sulfated glycosaminoglycan (GAG) release and Pi/PPi assay. Results: In vivo guinea pig study: Meniscus size and area as well as intensity of meniscus calcification were significantly increased in the ACLT group compared with the control group. Both calcified area and intensity were correlated with cartilage damage in the ACLT group (meniscus calcified area: r = 0.925, P < .0001; meniscus calcified intensity: r = 0.944, P < .0001). Ihh, MMP-13, Col X, ANKH, ENPP1, and ALP expression were increased in the ACLT group compared with the control group. The Pi level and Pi/PPi ratio increased by 63% and 42%, respectively, in the ACLT group compared with the control group. In vitro bovine meniscus explant study: Cell death was found in the superficial zone of the bovine meniscus explants after loading for 3 hours. The mRNA expression and protein levels of MMP-13, ANKH, ENPP1, and ALP were up-regulated in all 3-hour loaded samples. The Pi/PPi ratio and sulfated GAG content in the culture medium were increased in the 3-hour loaded group. Conclusion: Meniscus hypertrophy and mineralization correlated to cartilage degeneration after ACL injuries. Clinical Relevance: The study data suggest that the suppression of meniscus hypertrophy and calcification may decrease the risk of PTOA after ACL injuries.

Osteoarthritic changes in vervet monkey knees correlate with meniscus degradation and increased matrix metalloproteinase and cytokine secretion

Meniscus injury increases osteoarthritis risk but its pathobiology in osteoarthritis is unclear. We hypothesized that older adult vervet monkeys would exhibit knee osteoarthritic changes and the degenerative menisci from these animals would secrete matrix metalloproteinases (MMPs) and pro-inflammatory cytokines that contribute to the development of osteoarthritis. In a cross sectional analysis of healthy young adult (9-12 years) and old (19-26 years) adult female vervet monkeys, knees were evaluated in vivo with computed tomography (CT) imaging, and joint tissues were morphologically graded at necropsy. Meniscus explants were subsequently cultured to evaluate meniscal MMP and cytokine secretion. CT images revealed significant bony osteoarthritic changes in 80% of older monkeys which included increases in osteophyte number and meniscal calcification. Meniscus and cartilage degradation scores were greater in the older monkeys and were positively correlated (r > 0.7). Menisci from older animals exhibiting osteoarthritic changes secreted significantly more MMP-1, MMP-3, and MMP-8 than healthy menisci from younger monkeys. Older menisci without significant osteoarthritic changes secreted more IL-7 than healthy young menisci while older osteoarthritic menisci secreted more IL-7 and granulocyte-macrophage colony-stimulating factor than healthy older menisci. Aged vervets develop naturally occurring knee osteoarthritis that includes involvement of the meniscus. Degenerative menisci secreted markedly increased amounts of matrix-degrading enzymes and inflammatory cytokines. These factors would be expected to act on the meniscus tissue and local joint tissues and may ultimately promote osteoarthritis development. These finding also suggest vervet monkeys are a useful animal model for studying the progression of osteoarthritis.

Meniscus is more susceptible than cartilage to catabolic and anti-anabolic effects of adipokines

This study compared the effects on cartilage and meniscus matrix catabolism and biosynthesis of several adipokines implicated in osteoarthritis (OA). Bovine cartilage and meniscus explants were cultured for 1 or 9 days in serum-free medium alone or with 0.02, 0.2, or 2 μg/ml of leptin, visfatin, adiponectin, or resistin. Media were supplemented with (3)H-proline or (35)S-sodium sulfate to evaluate protein and sulfated glycosaminoglycan (sGAG) accumulation on the last day of culture. Explants were assayed for radiolabel, sGAG, and DNA contents. Cultured media were assayed for sGAG, nitrite and lactate dehydrogenase. Cartilage tissue was minimally affected by adipokines, with only the highest resistin dose increasing sGAG release and nitrite production compared to controls. In sharp contrast, meniscus tissue was responsive to several adipokines, with elevated sGAG and nitrite release following treatment with resistin, leptin, or visfatin. Cartilage sGAG content was unaltered by adipokine treatment whereas meniscal sGAG content significantly decreased with resistin dosage. Protein ((3)H) incorporation was unaffected by adipokine treatment in both tissues. sGAG ((35)S) incorporation did not significantly vary with adipokine treatment in cartilage but was inhibited by treatment with leptin, visfatin, and resistin in meniscus. Our results indicate that meniscal tissue is more susceptible to adipokine-stimulated catabolism than is cartilage. Resistin had the strongest effect of the adipokines tested, inducing sGAG release in both tissues and depleting sGAG content in meniscus. These results suggest that increased adipokine levels due to obesity or joint injury may alter the mechanical integrity of the knee joint through biological pathways.

Overexpression of TGF-β via rAAV-Mediated Gene Transfer Promotes the Healing of Human Meniscal Lesions Ex Vivo on Explanted Menisci

Background: Direct application of therapeutic gene vectors derived from the adeno-associated virus (AAV) might be beneficial to improve the healing of meniscal tears. Purpose: To test the ability of recombinant AAV (rAAV) to overexpress the potent transforming growth factor–β (TGF-β) in primary cultures of human meniscal fibrochondrocytes, in human meniscal explants, and in experimental human meniscal lesions as a new tool to enhance meniscal repair. Study Design: Controlled laboratory study. Methods: The effects of the candidate treatment on the proliferative and metabolic activities of meniscal cells were monitored in vitro for up to 21 days and in situ in intact and injured human menisci for up to 15 days using biochemical, immunohistochemical, histological, and histomorphometric analyses. Results: Efficient production of TGF-β via rAAV was achieved in vitro and in situ, both in the intact and injured meniscus. Application of the rAAV TGF-β vector stimulated the levels of cell proliferation and matrix synthesis (type I collagen) compared with control gene transfer in all systems tested, especially in situ in regions of poor healing capacity and in sites of meniscal injury. No adverse effects of the candidate treatment were observed at the level of osseous differentiation, as tested by immunodetection of type X collagen. Most remarkably, a significant reduction of the amplitude of meniscal tears was noted after TGF-β treatment, an effect that was associated with increased expression levels of the α–smooth muscle actin contractile marker. Conclusion: Overexpression of TGF-β via rAAV gene transfer is capable of modulating the reparative activities of human meniscal cells, allowing for the healing of meniscal lesions by convenient injection in sites of injury. Clinical Relevance: Direct gene-based approaches using rAAV have strong potential to develop new therapeutic options that aim at treating human meniscal defects.

Bonding and Fusion of Meniscus Fibrocartilage Using a Novel Chondroitin Sulfate Bone Marrow Tissue Adhesive

The weak intrinsic meniscus healing response and technical challenges associated with meniscus repair contribute to a high rate of repair failures and meniscectomies. Given this limited healing response, the development of biologically active adjuncts to meniscal repair may hold the key to improving meniscal repair success rates. This study demonstrates the development of a bone marrow (BM) adhesive that binds, stabilizes, and stimulates fusion at the interface of meniscus tissues. Hydrogels containing several chondroitin sulfate (CS) adhesive levels (30, 50, and 70 mg/mL) and BM levels (30%, 50%, and 70%) were formed to investigate the effects of these components on hydrogel mechanics, bovine meniscal fibrochondrocyte viability, proliferation, matrix production, and migration ability in vitro. The BM content positively and significantly affected fibrochondrocyte viability, proliferation, and migration, while the CS content positively and significantly affected adhesive strength (ranged from 60±17 kPa to 335±88 kPa) and matrix production. Selected material formulations were translated to a subcutaneous model of meniscal fusion using adhered bovine meniscus explants implanted in athymic rats and evaluated over a 3-month time course. Fusion of adhered meniscus occurred in only the material containing the highest BM content. The technology can serve to mechanically stabilize the tissue repair interface and stimulate tissue regeneration across the injury site.

Potential Healing of Meniscal Injuries in the Avascular Zone after Radiofrequency Stimulation (SS-34)

Meniscal repair in the white-white zone has a high incidence of failure due to poor vascularity. Radiofrequency application has been shown to increase the angiogenic capabilities of fibroblasts in chronic tendinopathies. The purpose of this study was to evaluate the effect of radiofrequency (RF) stimulation, in conjunction with suture repair, on the healing of tears in the white-white zone of the meniscus. 54 New Zealand white rabbits underwent surgically induced meniscal injuries within the avascular (white-white) region. Rabbits were then divided into three treatment groups: no repair (N=6), suture repair only (N=15) and suture repair in conjunction with RF treatment (N=33). Radiofrequency was applied using a 0.8 mm radiofrequency wand at level 4 for 500 milliseconds. Rabbits were then sacrificed at 9, 28 and 84 days for gross, histological and biochemical examination by two blinded observers. Biochemical analyses included evaluation of cell proliferation (3H-thymidine) as well as mitogenic (IGF-1, bFGF) and angiogenic (VEGF, αV) factors. Of specimens repaired with radiofrequency combined with suture, 19 (58%) showed increased gross morphologic and histological healing compared to controls. Two meniscal explants treated with RF had complete histological healing. No significant healing was seen in specimens that had either no repair or repair with suture alone. Biochemically, at nine days, the rate of 3H-thymidine for meniscal tears treated with suture repair + RF was 590±80 cpm/mg dry tissue. This value was approximately 40% greater than the menisci treated with suture repair only, which had a value of 380±30 cpm/mg (p < 0.05). Normal, unrepaired meniscal tissue had a 3H-thymidine incorporation rate of 250±35 cpm/mg. Semiquantitative RT-PCR analysis showed that at nine days post-repair, the RF-treated menisci had significantly increased mR.N.A expression of IGF-1, bFGF, VEGF and αV relative to untreated repairs. By day 28 and 84, the levels appeared equivalent between all treatment groups (p > 0.05). This study demonstrated that in our animal model, radiofrequency application in adjunct to suture repair of white-white meniscal tears led to a significantly increased healing response both in vitro and in vivo. Although the mechanism by which RF stimulation improves the healing response is not completely understood, our biochemical analyses suggest that it may enhance cellular proliferation as well as the mitogenic and angiogenic capability of the meniscal fibroblast.

Functional characterization of normal and degraded bovine meniscus: Rate-dependent indentation and friction studies

The menisci are known to play important roles in normal joint function and the development of diseases such as osteoarthritis. However, our understanding of meniscus' load bearing and lubrication properties at the tissue level remains limited. The objective of this investigation was to characterize the site- and rate-dependency of the compressive and frictional responses of the meniscus under a spherical contact load. Using a custom testing device, indentation tests with rates of 1, 10, 25, 50, and 100μm/s were performed on bovine medial meniscus explants, which were harvested from five locations including the femoral apposing surface at the anterior, central, and posterior locations and the central portion at the deep layer and at the tibial apposing surface (n=5 per location). Sliding tests with rates of 0.05, 0.25, 1, and 5mm/s were performed on the central femoral aspect and central tibial aspect superficial samples (n=6 per location). A separate set of superficial samples were subjected to papain digestion and tested prior to and post treatment. Our findings are: i) the Hertz contact model can be used to fit the force responses of meniscus under the conditions tested; ii) the anterior region is significantly stiffer than the posterior region and tissue modulus does not vary with tissue depth at the central region; iii) the friction coefficient of the meniscus is on the order of 0.02 under migratory contacts and the femoral apposing surface tends to show lower friction than the tibial apposing surface; iv) the meniscus exhibits increased modulus and lubrication with increased indentation and sliding rates; v) matrix degradation impedes the functional load support and lubrication properties of the tissue. The site- and rate-dependent properties of the meniscus may be attributed to spatial variations of the tissue's biphasic structure. These properties substantiate the role of the meniscus as one of the important bearing surfaces of the knee. These data contribute to an improved understanding of meniscus function, and its role in degenerative joint diseases. In addition, the results provide functional metrics for developing engineered tissue replacements. This article is part of a Special Issue entitled Osteoarthritis.

Effects of Platelet-Rich Plasma Composition on Anabolic and Catabolic Activities in Equine Cartilage and Meniscal Explants.

Objective:To evaluate the effects of single- and double-spin preparations of platelet-rich plasma (PRP) on anabolic and catabolic activities of cartilage and meniscal explants in vitro.Methods:Single- and double-spin PRP was prepared using laboratory processing or commercial kits. The cellular contents were quantified, and each PRP was mixed in equal quantities with cell culture medium and added to cartilage or meniscus explant cultures, with or without interleukin 1 β (IL-1β). Extracellular matrix synthesis was quantified over 24 hours via 35S-sulfate and 3H-proline incorporation, while gene expression of catabolic enzymes was evaluated using real-time PCR.Results:The platelet concentration in single-spin laboratory PRP was 59% higher than blood. Platelet and white blood cell concentrations in single-spin laboratory and kit PRP were not significantly different, while the double-spin kit resulted in approximately 2.5-fold higher platelet and approximately 400-fold higher white blood cell concentrations. In cartilage cultures without IL-1β, radiolabel incorporation in single-spin PRP cultures was significantly higher than in double-spin cultures. Similar results were obtained for 35S-sulfate incorporation in meniscus cultures without IL-1β. In IL-1β, radiolabel incorporation was largely similar among all PRPs. After 24 hours of culture, ADAMTS-4 gene expression in cartilage was lowest for single-spin PRP, while expression in the double-spin kit was not significantly different from double-spin laboratory PRP in which platelets were concentrated 6-fold.ConclusionsThis study suggests that single-spin PRP preparations may be the most advantageous for intra-articular applications and that double-spin systems should be considered with caution.

Regional and fiber orientation dependent shear properties and anisotropy of bovine meniscus

Imaging of meniscal tissue reveals an extracellular matrix comprised of collagen fibrils arranged in circumferential bundles and radially aligned tie fibers, implicating structural material anisotropy. Biochemical analyses demonstrate regional disparities of proteoglycan content throughout the meniscal body, a constituent known to affect the shearing response of fibrocartilagenous tissue. Despite this phenomenological evidence and previous mechanical testing implicating otherwise, the meniscus if often modeled as a homogeneous, transversely isotropic material with little regard for regional specificity and material properties. The aim of this investigation was to determine if shear stress response homogeneity and directionality exists in and between bovine menisci with respect to anatomical location (medial and lateral), region (anterior, central, and posterior) and fiber orientation (parallel and perpendicular). Meniscus explants were subjected to lap shear strain at 0.002 s−1 with the circumferential collagen fibers oriented parallel or perpendicular to the loading axis. Comparisons were made using a piecewise linear elastic analysis. The toe region shear modulus was calculated from the first observed linear region, between 3% and 13% strain and the extended shear modulus was established after 80% of the maximum shear strain. The posterior region was significantly different than the central for the extended shear modulus, correlating with known proteoglycan distribution. Observed shearing anisotropy led to the use of an anisotropic hyperelastic model based on a two-fiber family composite, previously used for arterial walls. The chosen model provided an excellent fit to the sample population for each region. These data can be utilized in the advancement of finite element modeling as well as biomimetic tissue engineered constructs.

In vitro inhibition of compression-induced catabolic gene expression in meniscal explants following treatment with IL-1 receptor antagonist

BackgroundDamage to the knee meniscus may result in tears that are difficult or unable to heal, and are often treated by partial removal of the damaged tissue. In vitro, 20% dynamic compressive strains on meniscal tissue explants have resulted in an increase in the release of sulfated glycosaminoglycans (GAG) and nitric oxide (NO) from the tissue explants and increased expression of matrix metalloproteinases (MMP) and interleukin-1α (IL-1α). The objective of this study was to explore the efficacy of IL-1 blockade on the expression of a wide range of genes, as well as NO and GAG release, following dynamic compression of porcine meniscal explants. MethodsExplants were dynamically compressed for 2 h at 1 Hz to 0, 10, or 20% strain with and without a pretreatment of 500 ng/ml interleukin-1 receptor antagonist (IL-1RA). Relative changes in gene expression of IL-1α, MMP-1, -3, -13, A Disintegrin and Metalloproteinase with ThromboSpondin 4 (ADAMTS-4), ADAMTS-5, iNOS, aggrecan, and COX-2, as well as changes in NO and GAG release, were measured with standard biochemical assays. ResultsExpression of IL-1α, MMP-3, MMP-13, and ADAMTS-4 in superficial explants was significantly downregulated at 20% dynamic strain compared to 10% strain following treatment with IL-1RA. GAG and NO release were not significantly influenced by IL-1RA treatment. ConclusionsTreatment of meniscal explants with IL-1RA inhibited the expression of many catabolic genes following a single bout of high dynamic strain. IL-1RA may therefore be a potential therapy option during the acute phase of meniscal tear or meniscectomy treatment.

Meniscus and cartilage exhibit distinct intra-tissue strain distributions under unconfined compression

To examine the functional behavior of the surface layer of the meniscus by investigating depth-varying compressive strains during unconfined compression. Pairs of meniscus and articular cartilage explants (n=12) site-matched at the tibial surfaces were subjected to equilibrium unconfined compression at 5, 10, 15, and 20% compression under fluorescence imaging. Two-dimensional (2D) deformations were tracked using digital image correlation (DIC). For each specimen, local compressive engineering strains were determined in 200 μm layers through the depth of the tissue. In samples with sharp strain transitions, bilinear regressions were used to characterize the surface and interior tissue compressive responses. Meniscus and cartilage exhibited distinct depth-dependent strain profiles during unconfined compression. All cartilage explants had elevated compressive engineering strains near the surface, consistent with previous reports. In contrast, half of the meniscus explants tested had substantially stiffer surface layers, as indicated by surface engineering strains that were ∼20% of the applied compression. In the remaining samples, surface and interior engineering strains were comparable. 2D Green's strain maps revealed highly heterogeneous compressive and shear strains throughout the meniscus explants. In cartilage, the maximum shear strain appeared to be localized at 100-250 μm beneath the articular surface. Meniscus was characterized by highly heterogeneous strains during compression. In contrast to cartilage, which consistently had a compliant surface region, meniscal explants were either substantially stiffer near the surface or had comparable compressive stiffness through the depth. The relatively compliant interior may allow the meniscus to maintain a consistent surface contour while deforming during physiologic loading.

Interleukin-1α treatment of meniscal explants stimulates the production and release of aggrecanase-generated, GAG-substituted aggrecan products and also the release of pre-formed, aggrecanase-generated G1 and m-calpain-generated G1-G2

Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1alpha)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter x 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1alpha-treatment, GAG loss (DMMB assay), biosynthetic activity ([(35)SO(4)]-sulfate and [(3)H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.

Mechanical injury of explants from the articulating surface of the inner meniscus

Knee osteoarthritis is accelerated by damage to the meniscus, a fibrocartilage tissue that assists in load transmission. However, little is known about the mechanical or cellular response of the meniscus to injurious overloading. Here, in vitro studies explored injury to meniscal explants using a compressive overloading protocol that has been well characterized for articular cartilage. Cartilage samples were processed in parallel as a reference to the extensive literature on cartilage injury. Injured meniscal explants showed extensive cell death at the articulating surface but no gross tissue damage, while similar conditions of peak stress and strain resulted in cartilage surface fissures and cell death consistent with moderate overloading. Post-injury gene expression in meniscal explants indicated a decrease in seven of the nine catabolic and pro-inflammatory molecules surveyed, while cartilage experienced a downregulation in ADAMTS-5 and TNF-α only. These data demonstrated a resiliency of the meniscus to injury, and that an acute increase in catabolic activities is not necessarily a consequence of mechanical overloading.

RAAV-mediated overexpression of FGF-2 promotes cell proliferation, survival, and α-SMA expression in human meniscal lesions

Meniscal tears are a common problem in sports medicine. Direct application of therapeutic vectors derived from the adeno-associated virus might be beneficial to enhance meniscal repair. We tested the hypothesis that overexpression of fibroblast growth factor 2 (FGF-2) through recombinant adeno-associated virus (rAAV) vectors leads to detectable metabolic changes in human meniscal fibrochondrocytes and in human meniscal defects. rAAV-mediated gene transfer was investigated for its ability to promote FGF-2 secretion in human meniscal fibrochondrocytes in vitro, in intact human meniscal explants in situ, and in experimentally created human meniscal lesions. Effects of the treatment on cell proliferation and survival, extracellular matrix synthesis, and expression of the alpha-smooth muscle actin (alpha-SMA) contractile marker were monitored using biochemical, immunohistochemical, histological, and histomorphometric analyses. Efficient production of FGF-2 through rAAV could be achieved in vitro and in situ, both in the intact and injured meniscus. Application of the candidate FGF-2 vector allowed for enhanced cell proliferation and survival compared with control transduction, in particular in areas with poor healing capacity and in sites of injury, consistent with the mitogenic activities of the growth factor. Remarkably, a significant reduction of the amplitude of meniscal tears was noted after FGF-2 treatment, with increased levels of alpha-SMA expression. In contrast, there was no significant stimulation of synthesis of the major extracellular matrix components when the candidate vector was applied and instead, a decrease in the matrix/DNA contents was reported, in good agreement with the properties of FGF-2. Such a direct gene-based approach may have value in options aiming at treating human meniscal defects.

Meniscal tissue explants response depends on level of dynamic compressive strain

Following partial meniscectomy, the remaining meniscus is exposed to an altered loading environment. In vitro 20% dynamic compressive strains on meniscal tissue explants has been shown to lead to an increase in release of glycosaminoglycans from the tissue and increased expression of interleukin-1alpha (IL-1alpha). The goal of this study was to determine if compressive loading which induces endogenously expressed IL-1 results in downstream changes in gene expression of anabolic and catabolic molecules in meniscal tissue, such as MMP expression. Relative changes in gene expression of MMP-1, MMP-3, MMP-9, MMP-13, A Disintegrin and Metalloproteinase with ThromboSpondin 4 (ADAMTS4), ADAMTS5, TNFalpha, TGFbeta, COX-2, Type I collagen (COL-1) and aggrecan and subsequent changes in the concentration of prostaglandin E(2) released by meniscal tissue in response to varying levels of dynamic compression (0%, 10%, and 20%) were measured. Porcine meniscal explants were dynamically compressed for 2h at 1Hz. 20% dynamic compressive strains upregulated MMP-1, MMP-3, MMP-13 and ADAMTS4 compared to no dynamic loading. Aggrecan, COX-2, and ADAMTS5 gene expression were upregulated under 10% strain compared to no dynamic loading while COL-1, TIMP-1, and TGFbeta gene expression were not dependent on the magnitude of loading. This data suggests that changes in mechanical loading of the knee joint meniscus from 10% to 20% dynamic strain can increase the catabolic activity of the meniscus.

NRG1 RISK GENE MODULATES NEURONAL CORRELATES OF SPEECH PRODUCTION

Interest in non-invasive injectable therapies has rapidly risen due to their excellent safety profile and ease of use in clinical settings. Injectable hydrogels can be derived from the extracellular matrix (ECM) of specific tissues to provide a biomimetic environment for cell delivery and enable seamless regeneration of tissue defects. We investigated the in situ delivery of human mesenchymal stem cells (hMSCs) in decellularized meniscus ECM hydrogel to a meniscal defect in a nude rat model. First, decellularized meniscus ECM hydrogel retained tissue-specific proteoglycans and collagens, and significantly upregulated expression of fibrochondrogenic markers by hMSCs versus collagen hydrogel alone in vitro. The meniscus ECM hydrogel in turn supported delivery of hMSCs for integrative repair of a full-thickness defect model in meniscal explants after in vitro culture and in vivo subcutaneous implantation. When applied to an orthotopic model of meniscal injury in nude rat, hMSCs in meniscus ECM hydrogel were retained out to eight weeks post-injection, contributing to tissue regeneration and protection from joint space narrowing, pathologic mineralization, and osteoarthritis development, as evidenced by macroscopic and microscopic image analysis. Based on these findings, we propose the use of tissue-specific meniscus ECM-derived hydrogel for the delivery of therapeutic hMSCs to treat meniscal injury.

IL-1 and iNOS gene expression and NO synthesis in the superior region of meniscal explants are dependent on the magnitude of compressive strains

Partial meniscectomy is known to cause osteoarthritis (OA) of the underlying cartilage as well as alter the load on the remaining meniscus. Removal of 30-60% of the medial meniscus increases compressive strains from a maximum of approximately 10% to almost 20%. The goal of this study is to determine if meniscal cells produce catabolic molecules in response to the altered loading that results from a partial meniscectomy. Relative changes in gene expression of interleukin-1 (IL-1), inducible nitric oxide synthase (iNOS) and subsequent changes in the concentration of nitric oxide (NO) released by meniscal tissue in response to compression were measured. Porcine meniscal explants were dynamically compressed for 2 h at 1 Hz to simulate physiological stimulation at either 10% strain or 0.05 MPa stress. Additional explants were pathologically stimulated to either 0% strain, 20% strain or, 0.1 MPa stress. iNOS and IL-1 gene expression and NO release into the surrounding media were increased at 20% compressive strain compared to other conditions. Pathological unloading (0% compressive strain) of meniscal explants did not significantly change expression of IL-1 or iNOS genes, but did result in an increased amount of NO released compared to physiological strain of 10%. These data suggest that meniscectomies which reduce the surface area of the meniscus by 30-60% will increase the catabolic activity of the meniscus which may contribute to the progression of OA.

IL-1 and iNOS gene expression and NO synthesis in the superior region of meniscal explants are dependent on the magnitude of compressive strains

IL-1 and iNOS gene expression and NO synthesis in the superior region of meniscal explants are dependent on the magnitude of compressive strains