Outer Meniscus Research Articles

Interleukin-1α induces focal degradation of biglycan and tissue degeneration in an in-vitro ovine meniscal model

We have developed an ovine meniscal explant model where the focal degradative events leading to characteristic fragmentation patterns of biglycan in human OA of the knee and hip, and evident in animal models of knee OA and IVD degeneration are reproduced in culture. Lateral and medial menisci were dissected into outer, mid and inner zones and established in explant culture±IL-1 (10ng/ml). The biglycan species present in conditioned media samples and in GuHCl extracts of tissues were examined by Western blotting using two C-terminal antibodies PR-85 and EF-Bgn. Clear differences were evident in the biglycan species in each meniscal tissue zone with the medial outer meniscus having lower biglycan levels and major fragments of 20, 28, 33 and 36, 39kDa. Similar fragmentation was detected in articular cartilage samples, 42-45kDa core protein species were also detected. Biglycan fragmentation was not as extensive in the IL-1 stimulated meniscal cultures with 36, 39, 42 and 45kDa biglycan species evident. Thus the medial meniscus outer zone displayed the highest levels of biglycan processing in this model and correlated with a major zone of meniscal remodelling in OA in man. Significantly, enzymatic digests of meniscal tissues with MMP-13, ADAMTS-4 and ADAMTS-5 have also generated similar biglycan species in-vitro. Zymography confirmed that the medial outer zone was the region of maximal MMP activity. This model represents a convenient system to recapitulate matrix remodelling events driven by IL-1 in pathological cartilages and in animal models of joint degeneration.

Histological and biological comparisons between complete and incomplete discoid lateral meniscus

ABSTRACTThe discoid lateral meniscus (DLM) is an anatomically abnormal meniscus that covers a greater area of the tibial plateau than the normal meniscus. The DLM is classified into two types: complete (CDLM) and incomplete (ICDLM) types. In this study, we investigated the histological and cell biological characteristics of CDLM and ICDLM. The number of blood vessels, proteoglycan deposition, and collagen distribution were assessed using meniscal tissues. Collagen production was also investigated in CDLM and ICDLM cells. The intercondylar region of the CDLM had a higher number of blood vessels than the inner region of the ICDLM. Safranin O staining density and type II collagen deposition in ICDLM were higher than those in CDLM. Type II collagen-positive cells were higher in ICLDM than in CDLM. CDLM cells showed slender fibroblastic morphology, while ICDLM cells were triangular chondrocytic in shape. This study demonstrated that the intercondylar region of the CDLM showed similar properties to the outer region of the meniscus. The inner region of the ICDLM, on the other hand, differed from the intercondylar region of the CDLM. Our results suggest that the intercondylar region of the CDLM may have a high healing potential like the outer meniscus.

ROCK inhibition stimulates SOX9/Smad3-dependent COL2A1 expression in inner meniscus cells.

Proper functioning of the meniscus depends on the composition and organization of its fibrocartilaginous extracellular matrix. We previously demonstrated that the avascular inner meniscus has a more chondrocytic phenotype compared with the outer meniscus. Inhibition of the Rho family GTPase ROCK, the major regulator of the actin cytoskeleton, stimulates the chondrogenic transcription factor Sry-type HMG box (SOX) 9-dependent α1(II) collagen (COL2A1) expression in inner meniscus cells. However, the crosstalk between ROCK inhibition, SOX9, and other transcription modulators on COL2A1 upregulation remains unclear in meniscus cells. The aim of this study was to investigate the role of SOX9-related transcriptional complex on COL2A1 expression under the inhibition of ROCK in human meniscus cells. Human inner and outer meniscus cells were prepared from macroscopically intact lateral menisci. Cells were cultured in the presence or absence of ROCK inhibitor (ROCKi, Y27632). Gene expression, collagen synthesis, and nuclear translocation of SOX9 and Smad2/3 were analyzed. Treatment of ROCKi increased the ratio of type I/II collagen double positive cells derived from the inner meniscus. In real-time PCR analyses, expression of SOX9 and COL2A1 genes was stimulated by ROCKi treatment in inner meniscus cells. ROCKi treatment also induced nuclear translocation of SOX9 and phosphorylated Smad2/3 in immunohistological analyses. Complex formation between SOX9 and Smad3 was increased by ROCKi treatment in inner meniscus cells. Chromatin immunoprecipitation analyses revealed that association between SOX9/Smad3 transcriptional complex with the COL2A1 enhancer region was increased by ROCKi treatment. This study demonstrated that ROCK inhibition stimulated SOX9/Smad3-dependent COL2A1 expression through the immediate nuclear translocation of Smad3 in inner meniscus cells. Our results suggest that ROCK inhibition can stimulates type II collagen synthesis through the cooperative activation of Smad3 in inner meniscus cells. ROCKi treatment may be useful to promote the fibrochondrocytic healing of the injured inner meniscus.

An apparatus with a horizontal capillary tube intended for measurement of the surface tension of supercooled liquids

New experimental apparatus for measurement of the surface tension of liquids under the metastable supercooled state has been designed and assembled in the study. The measuring technique is similar to the method employed by P.T. Hacker [NACA TN 2510] in 1951. A short liquid thread of the liquid sample was sucked inside a horizontal capillary tube partly placed in a temperature-controlled glass chamber. One end of the capillary tube was connected to a setup with inert gas which allowed for precise tuning of the gas overpressure in order of hundreds of Pa. The open end of the capillary tube was precisely grinded and polished before the measurement in order to assure planarity and perpendicularity of the outer surface. The liquid meniscus at the open end was illuminated by a laser beam and observed by a digital camera. Application of an increasing overpressure of the inert gas at the inner meniscus of the liquid thread caused variation of the outer meniscus such that it gradually changed from concave to flat and subsequently convex shape. The surface tension at the temperature of the inner meniscus could be evaluated from the overpressure corresponding to exactly planar outer meniscus. Detailed description of the new setup together with results of the preliminary tests is provided in the study.

Building an Anisotropic Meniscus with Zonal Variations

Toward addressing the difficult problems of knee meniscus regeneration, a self-assembling process has been used to re-create the native morphology and matrix properties. A significant problem in such attempts is the recapitulation of the distinct zones of the meniscus, the inner, more cartilaginous and the outer, more fibrocartilaginous zones. In this study, an anisotropic and zonally variant meniscus was produced by self-assembly of the inner meniscus (100% chondrocytes) followed by cell seeding the outer meniscus (coculture of chondrocytes and meniscus cells). After 4 weeks in culture, the engineered, inner meniscus exhibited a 42% increase in both instantaneous and relaxation moduli and a 62% increase in GAG/DW, as compared to the outer meniscus. In contrast, the circumferential tensile modulus and collagen/DW of the outer zone was 101% and 129% higher, respectively, than the values measured for the inner zone. Furthermore, there was no difference in the radial tensile modulus between the control and zonal engineered menisci, suggesting that the inner and outer zones of the engineered zonal menisci successfully integrated. These data demonstrate that not only can biomechanical and biochemical properties be engineered to differ by the zone, but they can also recapitulate the anisotropic behavior of the knee meniscus.

Gene signature of the embryonic meniscus

The meniscus is a fibrocartilagenous disc in the knee that protects the joint from damage. Meniscal injuries are common, however repair efforts are largely unsuccessful and are not able to prevent the degenerative changes that result in development of osteoarthritis. Tissue regeneration in adults often recapitulates events of embryonic development, suggesting the regulatory pathways controlling morphogenesis are candidate repair signals. Here we use laser capture microdissection to collect mouse embryonic day 16 (E16) meniscus, articular cartilage, and cruciate ligaments. RNA isolated from these tissues was then used to perform genome-wide microarray analysis. We found 38 genes were differentially expressed between E16 meniscus and articular cartilage and 43 genes were differentially expressed between E16 meniscus and cruciate ligaments. Included in our data set were extracellular matrix proteins, transcription factors, and growth factors, including TGF-β modulators (Lox, Dpt) and IGF-1 pathway members (Igf-1, Igfbp2, Igfbp3, Igfbp5). Ingenuity Pathway Analysis revealed that IGF-1 signaling was enriched in the meniscus compared to the other joint structures, while qPCR showed that Igf-1, Igfbp2, and Igfbp3 expression declined with age. We also found that several meniscus-enriched genes were expressed either in the inner or outer meniscus, establishing that regionalization of the meniscus occurs early in development.

The Knee Joint Tissues Differ Significantly in TGFβ1 Expression and Its Sensitivity

The knee joint is the largest and most complex joint in the human body. In this study, we investigated TGFβ1 expression in the outer meniscus, inner meniscus and articular cartilage of rabbit and human knee tissue (outer and inner menisci) in order to determine the potential role of this factor in normal meniscal function. We also examined the potential of TGF-β1 stimulation to promote tissue regeneration in the two different regions of rabbit knee meniscus tissue. Immunohistochemical investigations of TGF-β1 were performed on rabbit and human knee tissue. The rabbit outer, inner and articular cartilage cells were culture and stimulated with TGF-β1 followed by cell proliferation assay and extracellular matrix analysis. Regulatory studies were performed using TGF-β1 inhibitors SB-431542 and PD98059. Gene expression was analyzed by quantitative polymerase chain reaction. We found marked regional variation in the expression of TGF-β1 in rabbit and human knee. TGF-β1 expressions are relatively greater in the outer meniscus than inner meniscus. Furthermore, we found that exogenous TGF-β1 stimulation increased cell proliferation and aggrecan synthesis more so in the outer than in the inner meniscus. Articular cartilage tissue shows moderate levels of cell proliferation and ECM synthesis when compared with outer and inner meniscus. These findings suggest that growth factors used to enhance the repair and regeneration of meniscal tissue should be tailored to enhance region-specific variation in cell proliferation and extracellular matrix synthesis.

Chondromodulin‐I derived from the inner meniscus prevents endothelial cell proliferation

The meniscus is a fibrocartilaginous tissue that plays an important role in controlling complex biomechanics of the knee. A perimeniscal capillary plexus supplies the outer meniscus, whereas the inner meniscus is composed of avascular tissue. Anti-angiogenic molecules, such as chondromodulin-I (ChM-I) and endostatin, have pivotal roles in preserving the avascularity of cartilage. However, the anti-angiogenic role of ChM-I is unclear in the meniscus. We hypothesized that the inner meniscus might maintain its avascular feature by expressing ChM-I. Immunohistochemical analyses revealed that ChM-I was mainly detected in the inner and superficial zones of the meniscus. On the other hand, endostatin distribution was similar between the inner and outer meniscus. In Western blot, ChM-I was detected only in the inner meniscus, whereas endostatin was equally observed in both inner and outer menisci. In addition, ChM-I concentration of the inner meniscus-derived conditioned medium was higher than that of the outer meniscus-derived medium. ChM-I removal from the inner meniscus-derived medium and functional blocking of ChM-I significantly increased endothelial cell proliferation. In this study, we demonstrated that the inner meniscus contained larger amounts of ChM-I, and that the inner meniscus-derived ChM-I inhibited endothelial cell proliferation. Our results suggest that ChM-I may be a key anti-angiogenic factor for maintaining the avascularity of the inner meniscus.

Mechanical stretch increases Smad3‐dependent CCN2 expression in inner meniscus cells

The intrinsic zone-specific properties of the menisci are determined by biomechanical environments. In this study, we examined mechanical stretch-dependent expression of multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, and investigated the role of CCN2 in meniscus cells. Uni-axial cyclic tensile strain (CTS) was applied using a STB-140 system. CTS-induced expression of CCN2 and α1(I) collagen (COL1A1) was assessed by quantitative real-time PCR analysis. The distribution of CCN2 and Smad2/3 in stretched cells was investigated by immunohistochemical analysis. Smad2/3-dependent CCN2 transactivation was measured by luciferase reporter assay. The relationship between Smad2/3 and CTS-induced CCN2 transcription was investigated by chromatin immunoprecipitation. CTS stimulated gene expression of CCN2 and COL1A1 in inner meniscus cells, but not in outer meniscus cells. Recombinant CCN2 increased COL1A1 expression only in inner meniscus cells. CCN2 synthesis and nuclear translocalization of phosphorylated Smad2/3 in inner meniscus cells were stimulated by CTS. The CCN2 promoter activity was synergistically enhanced by overexpressed Smad3 in stretched inner meniscus cells, but was not by Smad2. Chromatin immunoprecipitation revealed that CTS increased the association between Smad3 and the Smad-binding element on the CCN2 proximal promoter in inner meniscus cells. Our results suggest that stretch-induced CCN2 may have a crucial role in regulating COL1A1 expression in the inner meniscus.

Discrimination of meniscal cell phenotypes using gene expression profiles

The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus). The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

An Overview of the Different Approaches Used in the Development of Meniscal Tissue Engineering

The menisci are important fibrocartilaginous structures which give lubrication, shock absorption, nutrition and stabilisation to the knee joint, and also help transfer load. The meniscus' extracellular matrix possesses a complex architecture which is not uniform throughout the tissue. The inner third of the meniscus is composed of hyaline cartilage and the outer meniscus is composed of fibrocartilage. In a mature meniscus only the outer 10-25% is vascularised. There are various types of pathology associated with the meniscus. Previously, surgical techniques used to be considered as conventional treatment for meniscal lesions. However lesions in the avascular regions of the meniscus would rarely heal appropriately. It has been found that total menisectomies in patients may increase their chance of suffering from osteoarthritis in the future. Meniscal tissue engineering has been developed in an attempt to help improve the healing potential of avascular meniscal regions. Many different concepts and approaches have been tried and tested, such as the application of natural and synthetic scaffolds, mesenchymal stem cells, growth factors, fibrin glue and more. The objective of this review is to summarise the different approaches that have been used in the development of meniscal tissue engineering. The focus of this review is to evaluate the strengths and weaknesses of the studies that have been carried out, and from there determine what we have learnt from them in order to further the development in meniscal tissue engineering.

Biomechanics of meniscus cells: regional variation and comparison to articular chondrocytes and ligament cells

Central to understanding mechanotransduction in the knee meniscus is the characterization of meniscus cell mechanics. In addition to biochemical and geometric differences, the inner and outer regions of the meniscus contain cells that are distinct in morphology and phenotype. This study investigated the regional variation in meniscus cell mechanics in comparison with articular chondrocytes and ligament cells. It was found that the meniscus contains two biomechanically distinct cell populations, with outer meniscus cells being stiffer (1.59 ± 0.19 kPa) than inner meniscus cells (1.07 ± 0.14 kPa). Additionally, it was found that both outer and inner meniscus cell stiffnesses were similar to ligament cells (1.32 ± 0.20 kPa), and articular chondrocytes showed the highest stiffness overall (2.51 ± 0.20 kPa). Comparison of compressibility characteristics of the cells showed similarities between articular chondrocytes and inner meniscus cells, as well as between outer meniscus cells and ligament cells. These results show that cellular biomechanics vary regionally in the knee meniscus and that meniscus cells are biomechanically similar to ligament cells. The mechanical properties of musculoskeletal cells determined in this study may be useful for the development of mathematical models or the design of experiments studying mechanotransduction in a variety of soft tissues.

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

IntroductionThe main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.MethodsHuman menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.ResultsCo-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.ConclusionsCo-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.

Regional Effects of Enzymatic Digestion on Knee Meniscus Cell Yield and Phenotype for Tissue Engineering

An abundant cell source is the cornerstone of most tissue engineering strategies, but extracting cells from the knee meniscus is hindered by its dense fibrocartilaginous matrix. Identifying a method to efficiently isolate meniscus cells is important, as it can reduce the cost and effort required to perform meniscus engineering research. In this study, six enzymatic digestion regimens used for cartilaginous cell isolation were used to isolate cells from the outer, middle, and inner regions of the bovine knee meniscus. Each regimen in each region was assessed in terms of cell yield, impact on cell phenotype, and cytotoxicity. All digestion regimens caused an overall upregulation of cartilage-specific genes Sox9, collagen type I (Col 1), collagen type II (Col 2), cartilage oligomeric matrix protein, and aggrecan (AGC) in cells from all meniscus regions, but was highest for cells isolated using 1075 U/mL of collagenase for 3 h (high collagenase). In response to isolation, outer meniscus cells showed highest upregulation of Sox9 and Col 1 genes, whereas greatest upregulation for middle meniscus cells was seen in Col 1 expression, and Col 2 expression for inner cells. Cell yield was highest in all regions when subjected to 45 min of 61 U/mL pronase followed by 3 h of 1075 U/mL collagenase (pronase/collagenase [P/C]) digestion regimen (outer: 6.57±0.37, middle: 12.77±1.41, inner: 22.17±1.47×10(6) cells/g tissue). The second highest cell yield was achieved using the low collagenase (LC) digestion regimen that applied 433 U/mL of collagenase for 18 h (outer: 1.95±0.54, middle: 3.3±4.4, inner: 6.06±2.44×10(6) cells/g tissue). Cytotoxicity analysis showed higher cell death in the LC group compared with the P/C group. Self-assembled constructs formed from LC-isolated cells were less dense than constructs formed from P/C-isolated cells, and P/C constructs showed higher glycosaminoglycan content and compressive moduli than LC constructs. All isolation methods tested resulted in similar phenotypic changes in meniscus cells from each region. These results indicate that, compared with other common isolation protocols, the P/C isolation method is able to more efficiently isolate meniscus cells from all regions that can produce tissue engineered constructs.

Regional variation in the mechanical role of knee meniscus glycosaminoglycans

High compressive properties of cartilaginous tissues are commonly attributed to the sulfated glycosaminoglycan (GAG) fraction of the extracellular matrix (ECM), but this relationship has not been directly measured in the knee meniscus, which shows regional variation in GAG content. In this study, biopsies from each meniscus region (outer, middle, and inner) were either subjected to chondroitinase ABC (CABC) to remove all sulfated GAGs or not. Compressive testing revealed that GAG depletion in the inner and middle meniscus regions caused a significant decrease in modulus of relaxation (58% and 41% decreases, respectively, at 20% strain), and all regions exhibited a significant decrease in viscosity (outer: 29%; middle: 58%; inner: 62% decrease). Tensile properties following CABC treatment were unaffected for outer and middle meniscus specimens, but the inner meniscus displayed significant increases in Young's modulus (41% increase) and ultimate tensile stress (40% increase) following GAG depletion. These findings suggest that, in the outer meniscus, GAGs contribute to increasing tissue viscosity, whereas in the middle and inner meniscus, where GAGs are most abundant, these molecules also enhance the tissue's ability to withstand compressive loads. GAGs in the inner meniscus also contribute to reducing the circumferential tensile properties of the tissue, perhaps due to the pre-stress on the collagen network from increased hydration of the ECM. Understanding the mechanical role of GAGs in each region of the knee meniscus is important for understanding meniscus structure-function relationships and creating design criteria for functional meniscus tissue engineering efforts.

Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells

The meniscus plays an important role in controlling the biomechanics of the knee. However, the mechanical stress-related response in meniscus cells remains unclear. We investigated mechanical stretch-regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry-type HMG box (SOX) 9 and α1(II) collagen (COL2A1) were assessed by real-time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch-related protein-DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA-binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretch-induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression.

Migration responses of outer and inner meniscus cells to applied direct current electric fields

Injuries to the inner regions of the knee meniscus do not heal and can result in degenerative changes to the articular surface, ultimately leading to osteoarthritis. A possible stimulus to enhance meniscus healing is to use electric fields that induce galvanotaxis. In this study, a novel characterization of the effects of direct current electric fields on migration characteristics of meniscus cells was performed. Primary and passaged inner and outer meniscus cells were exposed to varying electric field strengths from 0 to 6 V/cm. Cell migration was tracked using time lapse digital photography, and cell displacement and cathodal direct velocity were quantified. Cytoskeletal staining was performed to examine actin distribution and nuclear content. Cell adhesion strength was quantified as a function of wall shear stress. Meniscus cells exhibited cathodal migration and cell elongation perpendicular to the applied electric field accompanied by actin reorganization. Outer meniscus cells migrated quicker and exhibited lower adhesion strengths when compared to inner meniscus cells. Passaged cells exhibited higher migration characteristics when compared to primary cells. Overall, this study demonstrated that electric fields can significantly enhance and direct meniscus cell migration and suggests the potential for their incorporation in strategies of meniscus repair and tissue engineering.

Inner Meniscus Cells Maintain Higher Chondrogenic Phenotype Compared with Outer Meniscus Cells

Meniscus cells have several distinct properties in cellular morphology and extracellular matrix production. Inner meniscus cells are considered to have more chondrocytic phenotype compared with outer meniscus cells. However, the chondrogenic property of each meniscus cell has not been elucidated in detail. In this study, we investigated the difference between human inner and outer meniscus-derived cells in extracellular matrix deposition and chondrogenic potential. Monolayer-cultured inner meniscus cells showed small and ovoid shapes though slender and fibroblastic cells were obtained from outer half of human meniscus. The syntheses of type II collagen and safranin O-stained proteoglycans were increased in chondrogenic pellets derived from inner meniscus cells, rather than in outer meniscus cell-derived pellets. On the other hand, adipogenic lipid vacuoles were equally accumulated in both inner and outer meniscus cells after adipogenic treatment. Chondrogenic treatments also enhanced the expression of chondrogenic marker genes, such as Sry-type HMG box (SOX) 9, Scleraxis, and α1(II) collagen, in inner meniscus cells. However, SOX9 expression was not increased in outer meniscus cells even after chondrogenic treatment. This study demonstrated that inner meniscus cells maintained higher chondrogenic potential compared with outer meniscus cells. Our results suggest that the difference between inner and outer meniscus cells in chondrogenic property might have an essential role in preserving a zone-specific meniscal feature.

033 EFFECTS OF FEEDING A HIGH OMEGA-3 FATTY ACID DIET ON THE PAIN-RELATED DISABILITY IN DOGS WITH NATURALLY OCCURRING OSTEOARTHRITIS

expression of all MMPs was greater in meniscus than AC. In general MMP expression in the outer meniscus was more responsive to TNFα. TIMP-1 and -3 were generally decreased in all tissues by IL-1α and TNFα. There was more pro and active MMP-2, and pro-MMP-13 in control cultures of outer meniscus than AC or inner meniscus. Unlike AC, both meniscal zones released active MMP-13 after IL-1α and TNFα treatment, and active MMP-2 after IL-1α (inner) or both cytokines (outer). Conclusions: Aggrecan processing differed between outer versus inner meniscus, with the later being similar to AC. Aggrecan proteolysis in the outer meniscus rather than AC, may be responsible for MMP-generated neoepitopes detected in OA human knee synovial fluid. The meniscus was generally more responsive to TNFα than AC, particularly the outer zone. Expression of MMPs and ADAMTS was higher in meniscus than AC, and unlike AC active MMPs were released from meniscus. The meniscus may contribute significantly to metalloproteinase levels in the joint, and therefore to cartilage breakdown in OA. Differential regulation of MMPs and ADAMTS in the knee joint meniscus compared with AC, and between inner and outer meniscal zones has important implications in OA therapy.

The Knee Meniscus: A Complex Tissue of Diverse Cells

This review describes the knee meniscus and its diverse cell populations. Situated between the femur and tibia, the meniscus acts to transmit loads within the knee while maintaining joint stability. Not only does this tissue display complex geometry and anatomy, its cellular profile ranges from fibroblast-like to chondrocyte-like. When the tissue first begins to develop in the body, its cells are similar in shape and morphology, but as it matures, these cells take on distinct characteristics. The spindle-shaped cells of the outer meniscus are well-suited to maintaining a fibrous extracellular matrix rich in collagen type I. The round, inner meniscus cells produce both collagen types I and II, and glycosaminoglycans, giving rise to a hyaline-like inner portion of the tissue. Cells intermediately located display characteristics of both cell types. Fibrochondrocytes are also known to be highly dependent on mechanical stimulation to maintain healthy tissue, and display regional variation in response to different biomolecular cues. Investigating this cell population under a variety of conditions can lead to a better understanding of the pathophysiology and regenerative processes of the meniscus.