Matrix metalloproteinase-13 expression in rabbit knee joint connective tissues: influence of maturation and response to injury.

The hypothesis of the present work was that expression of matrix metalloproteinase-13 (MMP-13, collagenase-3) would be induced during conditions involving important matrix remodeling such as ligament maturation, scar healing and joint instability. Therefore, MMP-13 expression in the medial collateral ligament (MCL) during the variable situations of tissue maturation and healing was assessed. MMP-13 expression in three intra-articular connective tissues of the knee (i.e. articular cartilage, menisci and synovium) following the transection of the anterior cruciate ligament of the knee was evaluated at 3 and 8 weeks post-injury. MMP-13 mRNA (semi-quantitative RT-PCR) and protein (immunohistochemistry and Western blotting) were detected in all of the tissues studied. Significantly higher MCL mRNA levels for MMP-13 were detected during the early phases of tissue maturation (i.e. 29 days in utero and 2-month-old rabbits) compared to later phases (5- and 12-month-old rabbits). This pattern of expression was recapitulated following MCL injury, with very high levels of expression in scar tissue at 3 weeks post-injury and then a decline to levels not significantly different from control values by 14 weeks. Elevated mRNA levels correlated with increased protein levels for MMP-13 in both menisci and synovium following the transection of the anterior cruciate ligament and during medial collateral ligament healing. These results indicate that MMP-13 expression is regulated by a number of variables and that high levels of expression occur in situations when connective tissue remodeling is very active.     

3D finite element model of meniscectomy: changes in joint contact behavior

The goal of this study is to quantify changes in knee joint contact behavior following varying degrees of the medial partial meniscectomy. A previously validated 3D finite element model was used to simulate 11 different meniscectomies. The accompanying changes in the contact pressure on the superior surface of the menisci and tibial plateau were quantified as was the axial strain in the menisci and articular cartilage. The percentage of medial meniscus removed was linearly correlated with maximum contact pressure, mean contact pressure, and contact area. The lateral hemi-joint was minimally affected by the simulated medial meniscectomies. The location of maximum strain and location of maximum contact pressure did not change with varying degrees of partial medial meniscectomy. When 60% of the medial meniscus was removed, contact pressures increased 65% on the remaining medial meniscus and 55% on the medial tibial plateau. These data will be helpful for assessing potential complications with the surgical treatment of meniscal tears. Additionally, these data provide insight into the role of mechanical loading in the etiology of post-meniscectomy osteoarthritis.     

Ethylene glycol and glycerol loading and unloading in porcine meniscal tissue

The development of a long-term storage method for meniscus, a complex tissue of the knee prone to injury, would improve the procedure and outcomes of meniscus transplantation. Cryopreservation uses cryoprotective agents (CPAs) including ethylene glycol (EG) and glycerol to preserve a variety of live tissues, and understanding of the CPA permeation kinetics will be critical in designing a vitrification protocol for meniscus.The purpose of this preliminary study was to understand the loading and unloading behaviours of EG and glycerol in meniscus by observing their efflux. For the main experiment, lateral and medial porcine menisci were incubated with CPA for 24 h at three temperatures (i.e., 4, 22, and 37 °C). Then, the menisci were immersed in 25 ml of X-VIVO™10 and CPA efflux was recorded by monitoring the molality of two consecutive washout solutions at different time points. In a subsequent experiment, menisci were incubated in the CPA solutions for 48 h at 22 °C, and the results were compared to those obtained at 22 °C in the main experiment.Results showed a rapid efflux of CPA from meniscus at the beginning of each wash. With increasing temperature, the amount of CPA efflux (and hence loading) increased. Using 24 h incubation, EG loaded the menisci more completely than glycerol. But after 48 h of incubation, both EG and glycerol achieved approximately the same degree of meniscus loading.This study provides preliminary data that will facilitate future design of experiments aimed at development of meniscus permeation studies.     

Differential expression of VEGF isoforms and receptors in knee joint menisci under systemic hypoxia

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. We examined the expression levels of VEGF isoforms and their receptors in the medial and lateral meniscus of rabbits under normal physiologic conditions as well their expression levels after 8 and 24 h of systemic normobaric hypoxia (13%). VEGF121 is the most abundant VEGF isoform in the medial and lateral meniscus, followed by VEGF165, VEGF189, and VEGF183. While the soluble VEGF121 and VEGF165 are only upregulated at 8 h of hypoxia, the membrane-bound VEGF183 and VEGF189 are further increased at 24 h. VEGFR-2 is expressed at a much higher level than VEGFR-1 under normal conditions, and both receptors are upregulated under hypoxia. Differential expression levels under normoxia as well as a differential response to hypoxia may indicate different functions of VEGF isoforms in the meniscus.     

The effect of kinematic and kinetic changes on meniscal strains during gait

The menisci play an important role in load distribution, load bearing, joint stability, lubrication, and proprioception. Partial meniscectomy has been shown to result in changes in the kinematics and kinetics at the knee during gait that can lead to progressive meniscal degeneration. This study examined changes in the strains within the menisci associated with kinematic and kinetic changes during the gait cycle. The gait changes considered were a 5 deg shift toward external rotation of the tibia with respect to the femur and an increased medial-lateral load ratio representing an increased adduction moment. A finite element model of the knee was developed and tested using a cadaveric specimen. The cadaver was placed in positions representing heel-strike and midstance of the normal gait, and magnetic resonance images were taken. Comparisons of the model predictions to boundaries digitized from images acquired in the loaded states were within the errors produced by a 1 pixel shift of either meniscus. The finite element model predicted that an increased adduction moment caused increased strains of both the anterior and posterior horns of the medial meniscus. The lateral meniscus exhibited much lower strains and had minimal changes under the various loading conditions. The external tibial rotational change resulted in a 20% decrease in the strains in the posterior medial horn and increased strains in the anterior medial horn. The results of this study suggest that the shift toward external tibial rotation seen clinically after partial medial meniscectomy is not likely to cause subsequent degenerative medial meniscal damage, but the consequence of this kinematic shift on the pathogenesis of osteoarthritis following meniscectomy requires further consideration.     

Is the meniscus of the knee joint a fibrocartilage?

A histological analysis of the structure of intact knee joint menisci was carried out in adult dogs. By means of specific histochemical methods for the connective tissue and cartilage, it was found that the meniscus as a whole does not have a unique structure. The anterior and posterior horns are populated by round chondroid cells encircled by abundant interstitial substance and branched wavy connective fibers; blood vessels are present. The outer third of the meniscus is constituted of cross bundles of connective fibers, fibrocytes and spindle-like areas of loose connective tissue with blood vessels. The inner avascular two thirds of the meniscus are filled with parallel circumferentially oriented fascicles of connective fibers, ovally elongated chondroid cells, and a small quantity of chondroid interstitial substance. In some menisci, in the inner two thirds of the body, there are isles of typical cartilage, which show metachromasia of the beta type and rarely of the gamma type. The occurrence and way of the manifestation of cartilage are of an individual character. The structural duality of the knee meniscus is accounted for by its functional duality manifested in offering resistance to the forces of traction and pressure, the latter ones favoring the process of evolution of tissue from connective, through chondroid, to cartilaginous.     

Meniscus maturation in the swine model: changes occurring along with anterior to posterior and medial to lateral aspect during growth

The meniscus plays important roles in knee function and mechanics and is characterized by a heterogeneous matrix composition. The changes in meniscus vascularization observed during growth suggest that the tissue‐specific composition may be the result of a maturation process. This study has the aim to characterize the structural and biochemical variations that occur in the swine meniscus with age. To this purpose, menisci were collected from young and adult pigs and divided into different zones. In study 1, both lateral and medial menisci were divided into the anterior horn, the body and the posterior horn for the evaluation of glycosaminoglycans (GAGs), collagen 1 and 2 content. In study 2, the menisci were sectioned into the inner, the intermediate and the outer zones to determine the variations in the cell phenotype along with the inner–outer direction, through gene expression analysis. According to the results, the swine meniscus is characterized by an increasing enrichment in the cartilaginous component with age, with an increasing deposition in the anterior horn (GAGs and collagen 2; P < 0.01 both); moreover, this cartilaginous matrix strongly increases in the inner avascular and intermediate zone, as a consequence of a specific differentiation of meniscal cells towards a cartilaginous phenotype (collagen 2, P < 0.01). The obtained data add new information on the changes that accompany meniscus maturation, suggesting a specific response of meniscal cells to the regional mechanical stimuli in the knee joint.     

Tensile properties, collagen content, and crosslinks in connective tissues of the immature knee joint

Background

The major connective tissues of the knee joint act in concert during locomotion to provide joint stability, smooth articulation, shock absorption, and distribution of mechanical stresses. These functions are largely conferred by the intrinsic material properties of the tissues, which are in turn determined by biochemical composition. A thorough understanding of the structure-function relationships of the connective tissues of the knee joint is needed to provide design parameters for efforts in tissue engineering.

Methodology/Principal Findings

The objective of this study was to perform a comprehensive characterization of the tensile properties, collagen content, and pyridinoline crosslink abundance of condylar cartilage, patellar cartilage, medial and lateral menisci, cranial and caudal cruciate ligaments (analogous to anterior and posterior cruciate ligaments in humans, respectively), medial and lateral collateral ligaments, and patellar ligament from immature bovine calves. Tensile stiffness and strength were greatest in the menisci and patellar ligament, and lowest in the hyaline cartilages and cruciate ligaments; these tensile results reflected trends in collagen content. Pyridinoline crosslinks were found in every tissue despite the relative immaturity of the joints, and significant differences were observed among tissues. Notably, for the cruciate ligaments and patellar ligament, crosslink density appeared more important in determining tensile stiffness than collagen content.

Conclusions/Significance

To our knowledge, this study is the first to examine tensile properties, collagen content, and pyridinoline crosslink abundance in a direct head-to-head comparison among all of the major connective tissues of the knee. This is also the first study to report results for pyridinoline crosslink density that suggest its preferential role over collagen in determining tensile stiffness for certain tissues.     

Age-related changes of element contents in the human meniscus

The relative contents (RCs) of elements in the human menisci from 23 subjects in the age range between 65 and 93 yr were analyzed by inductively coupled plasma atomic emission spectrometry. The RCs of sulfur, calcium, and phosphorus in menisci increased progressively until the 80s, being the highest in the 80s, and thereafter decreased. The RCs of magnesium in menisci increased progressively until the 90s. Regarding the medial and lateral menisci, higher RCs of magnesium and iron, and a lower RC of phosphorus were found in lateral menisci in comparison with those in medial menisci. There were sexual differences in the RCs of calcium and phosphorus of medial and lateral menisci. The RCs of calcium and phosphorus were about 50% higher in women’s menisci than in men’s. Histological examinations showed that structureless mucoid masses were observed in the menisci, with very high RCs of calcium and phosphorus being detected.     

Chronic changes in the rabbit tibial plateau following blunt trauma to the tibiofemoral joint

The knee is often a site of injury that can often lead to a chronic disease known as osteoarthritis (OA). The disease may be initiated, in part, by acute injuries to joint cartilage and its cells. In a recent study by this laboratory, using Flemish Giant rabbits, an impact compressive load on the tibial femoral joint was shown to cause significant levels of acute damage to chondrocytes in cartilage of the medial and lateral tibial plateaus. In the current study, using the same model, histological and mechanical data from the plateaus were documented at 6 and 12 months post impact, and compared to the unimpacted control limbs and a limb from unimpacted, control animals. The mechanical properties of cartilage were measured with indentation relaxation tests on the medial and lateral plateaus in regions covered and uncovered by the meniscus. The histological studies on impacted limbs showed surface lesions on both plateaus, thickening of the underlying subchondral bone at 12 months and numerous occult microcracks at the calcified cartilage–subchondral bone interface at 6 and 12 months, without significant changes in cartilage thickness or its mechanical properties versus controls. Yet, there was an increase in both the matrix and fiber moduli and a decrease in the permeability of uncovered, medial plateau cartilage in both limbs of impacted animals between 6 and 12 months post impact that was not documented in control animals.     

Relationships among element contents in the medial meniscus

To elucidate compositional changes of elements in the meniscus with aging, the authors investigated the relationships among elements in the medial meniscus by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 16 men and 7 women, ranging in age from 65 to 93 yr. In the left medial meniscus, extremely significant correlations were found both between calcium and phosphorus contents and between sulfur and magnesium contents, whereas significant correlations were found between calcium and either iron or zinc contents, between phosphorus and either iron or zinc contents, and between iron and zinc contents. On the other hand, in the right medial meniscus, there were only an extremely significant correlation between calcium and phosphorus contents and a significant correlation between sulfur and magnesium contents. The common finding between the left and right medial menisci was an extremely significant correlation between calcium and phosphorus contents and a significant correlation between sulfur and magnesium contents.     

A new in vivo technique for 3-dimensional analysis of the translation of femoral condyles and the menisci responding to antagonistic muscle forces]

The aim of our study was to develop a 3-D MR-based technique for the analysis of meniscal and femoral translations during flexion of the knee, and under the influence of antagonistic muscle forces in healthy subjects. In an open MR system, 5 knees were examined at 30 degrees and 90 degrees flexion using a T1-weighted 3-D gradient echo sequence. A force of 30 Newtons, first in the extending and then in the flexing direction, was applied to the distal lower leg. After three-dimensional reconstruction, the minimal distances between the centre of the tibial plateau and the posterior edge of the menisci and femoral condyles were determined. At 30 degrees flexion, the minimum distance for the meniscus was larger medially than laterally (23.2 +/- 1.8 mm vs. 16.2 +/- 3.3 mm), and this also applied to the condyles (25.1 +/- 1.5 vs. 19.0 +/- 3.0 mm). During flexion to 90 degrees, a posterior translation of 0.5 +/- 0.2 mm was observed for the lateral, and of 3.4 +/- 1.2 mm for the medial, meniscus. The condyles demonstrated a different posterior translation (lateral 2.2 +/- 0.56 mm; medial 1.8 +/- 1.9 mm). No obvious differences were found between extension and flexion muscle activity for the different positions of the knee. In the present study, a new 3-D technique is presented for the analysis of the femoral and meniscal translation at various positions of the knee, and under muscle activity. The results suggest different translation for the menisci and condyles.     

Changes in the content of the fibrillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing

Summary The menisci are first seen as triangular aggregations of cells in the 20-day rabbit fetus. At 25-days, a matrix that contains types I, III and V collagens has formed. These collagens are also found in the 1-week neonatal meniscus, but by 3 weeks, type II collagen is present in some regions. By 12 to 14 weeks, typically cartilaginous areas with large cells in lacunae are found and by 2 years, these occupy the central regions of the inner two-thirds of the meniscus. The surface layers of the meniscus contain predominantly type I collagen. From 12 to 14 weeks onwards, there is little overlap between the regions with types I or II collagens, that is, these are discrete regions of type I-containing fibrocartilage and type II-containing cartilage. Types III and V collagens are found throughout the menisci, particularly in the pericellular regions. All the cells in the fetal and early neonatal menisci express the mRNA for type I collagen. At 3 weeks postnatal, cells that express type I collagen mRNA are found throughout the meniscus, but type II collagen mRNA is expressed only in the regions of developing cartilage. At 12- to 14-weeks, only type II collagen mRNA is expressed, except at the periphery next to the ligament where a few cells still express type I collagen mRNA. Rabbit menisci, therefore, undergo profound changes in their content and arrangement of collagens during postnatal development.     

The effect of meniscal tears and resultant partial meniscectomies on the knee contact stresses: a finite element analysis

Knee osteoarthritis (OA) is believed to result from high levels of contact stresses on the articular cartilage and meniscus after meniscal damage. This study investigated the effect of meniscal tears and partial meniscectomies on the peak compressive and shear stresses in the human knee joint. An elaborate three-dimensional finite element model of knee joint including bones, articular cartilages, menisci and main ligaments was developed from computed tomography and magnetic resonance imaging images. This model was used to model four types of meniscal tears and their resultant partial meniscectomies and analysed under an axial 1150 N load at 0° flexion. Three different conditions were compared: a healthy knee joint, a knee joint with medial meniscal tears and a knee joint following partial meniscectomies. The numerical results showed that each meniscal tear and its resultant partial meniscectomy led to an increase in the peak compressive and shear stresses on the articular cartilages and meniscus in the medial knee compartment, especially for partial meniscectomy. Among the four types of meniscal tears, the oblique tear resulted in the highest values of the peak compressive and shear stresses. For the four partial meniscectomies, longitudinal meniscectomy led to the largest increase in these two stresses. The lateral compartment was minimally affected by all the simulations. The results of this study demonstrate meniscal tear and its resultant partial meniscectomy has a positive impact on the maintenance of high levels of contact stresses, which may improve the progression of knee OA, especially for partial meniscectomy. Surgeons should adopt a prudent strategy to preserve the greatest amount of meniscus possible.     

Radial fibre proportions in human knee joint menisci. Measurement by scanning optical microscopy

The proportions of medial and lateral knee joint menisci represented by radially orientated collagen (COL) were measured in 42 specimens from 24 hospital patients examined post-mortem. Images of the fibre bundles were obtained by the 488-nm laser confocal scanning of hydrated, fixed radial blocks taken from the anterior, middle and posterior regions of the menisci after staining with picro-Sirius red. Measurements of the percentage of each image occupied by fluorescent, doubly refractile COL were made by means of a Kontron IBAS image analyser, after interactive segmentation. In areas adjoining the outer, lateral parts of both the medial and lateral menisci, the proportion of all samples identified as radial COL was 7.56 +/- 0.28%. The corresponding figure for areas near the inner, medial edges of the menisci was 17.80 +/- 0.80%. However, no relationship was demonstrable between age and sex and meniscal radial fibre optical density, and there was no difference between the proportion of radial fibres in the anterior, middle or posterior regions.     

Dynamic contact stress patterns on the tibial plateaus during simulated gait: A novel application of normalized cross correlation

The spatial distribution and pattern of local contact stresses within the knee joint during activities of daily living have not been fully investigated. The objective of this study was to determine if common contact stress patterns exist on the tibial plateaus of human knees during simulated gait. To test this hypothesis, we developed a novel normalized cross-correlation (NCC) algorithm and applied it to the contact stresses on the tibial plateaus of 12 human cadaveric knees subjected to multi-directional loads mimicking gait. The contact stress profiles at different locations on the tibial plateaus were compared, where regions with similar contact stress patterns were identified across specimens. Three consistent regional patterns were found, among them two most prominent contact stress patterns were shared by 9–12 of all the knees and the third pattern was shared by 6–8 knees. The first pattern was located at the posterior aspect of the medial tibial plateau and had a single peak stress that occurred during the early stance phase. The second pattern was located at the central-posterior aspects of the lateral plateau and consisted of two peak stresses coincident with the timing of peak axial force at early and late stance. The third pattern was found on the anterior aspect of cartilage-to-cartilage contact region on the medial plateau consisted of double peak stresses. The differences in the location and profile of the contact stress patterns suggest that the medial and lateral menisci function to carry load at different points in the gait cycle: with the posterior aspect of the medial meniscus consistently distributing load only during the early phase of stance, and the posterior aspect of the lateral meniscus consistently distributing load during both the early and late phases of stance. This novel approach can help identify abnormalities in knee contact mechanics and provide a better understanding of the mechanical pathways leading to post-traumatic osteoarthritis.     

Association of increased estrogen receptor beta2 expression with parity-induced alterations in the rat mammary gland

In this study, we investigated the cellular and molecular events involved in parity-related alterations in mammary gland (MG) proliferation and differentiation. Rat MGs were removed on day 9 of either first (nulliparous), second (primiparous) or third (multiparous) pregnancy. Expression of steroid hormone receptors along with cellular biomarkers of proliferation and differentiation were quantified in all MG tissue compartments by immunohistochemistry. Wnt-4 (a Wingless-like morphogenic gene involved in MG development), ERbeta and ERbeta2 mRNA were evaluated by RT-PCR analysis. Serum levels of mammotrophic hormones were measured. In comparison to nulliparous and primiparous rats, multiparous animals exhibited decreased luminal cell proliferation and PR levels, whereas alpha-lactalbumin, ERalpha, ERbeta and ERbeta2 expression were increased. In myoepithelial cells, while parity induced a decrease in proliferative activity, subsequent pregnancies and lactations lead to an increased state of differentiation. Our results showed that at least two periods of pregnancy and lactation were necessary to modify the studied parameters. The lower proliferative activity and higher differentiation state of the multiparous MG are associated with both a decreased PR expression and increased ERalpha and ERbeta expression. Since ERbeta and/or ERbeta2 isoform expression was related to parity history, results suggest that the decreased proliferative activity and PR expression observed in the MG of multiparous animals may be associated with overexpression of ERbeta and/or the ERbeta2 isoform, thereby antagonizing the proliferative effects associated with ERalpha.     

Age‐related modulation of angiogenesis‐regulating factors in the swine meniscus

An in‐depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti‐angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve‐CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro‐cartilaginous differentiation. Expression of VEGF and Ve‐CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.