Human Knee Articular Cartilage Research Articles

Unveiling inflammatory and prehypertrophic cell populations as key contributors to knee cartilage degeneration in osteoarthritis using multi-omics data integration

ObjectivesSingle-cell and spatial transcriptomics analysis of human knee articular cartilage tissue to present a comprehensive transcriptome landscape and osteoarthritis (OA)-critical cell populations.MethodsSingle-cell RNA sequencing and spatially resolved transcriptomic technology have...

An ultra-sensitive SPR immunosensor for quantitative determination of human cartilage oligomeric matrix protein biomarker

This paper reports the development of a novel surface plasmon resonance (SPR) immunosensor for ultra-sensitive quantitative determination of human articular cartilage oligomeric matrix protein (COMP), a major component of the extracellular matrix and an exploratory biomarker. Capture antibodies against human COMP (anti-COMP16F12) were covalently immobilized on an 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayer (SAM)-coated SPR sensor disk and a dual sandwich-type signal amplification strategy using biotinylated detection antibodies against COMP (anti-COMP17C10-biot) and streptavidin-conjugated quantum dots (SAv‒QDs) were used for the development of an immunosensor. The binding of high-mass SAv‒QDs via biotin–streptavidin interaction to the surface of the immunosensor resulted in a drastic increase in the sensitivity. The developed immunosensor was able to detect concentrations of COMP in a range from 2.80 to 680.54 fM with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.15 and 0.50 fM, respectively. The immunosensor exhibited good repeatability (relative standard deviation (RSD) 8.05%) and reproducibility (RSD 9.88%) as well as excellent operational stability (2.14 % decrease in SPR signal after 13 days). In addition, the analysis of secretomes of human knee articular cartilage explants from patients with osteoarthritis revealed that the immunosensor has good accuracy (analytical error less than 5 %). These results indicate that the immunosensor developed may be suitable for quantitative determination of COMP derived from articular cartilage and other synovial joint tissues in clinical studies.

Senescent cell population with ZEB1 transcription factor as its main regulator promotes osteoarthritis in cartilage and meniscus

ObjectivesSingle-cell level analysis of articular cartilage and meniscus tissues from human healthy and osteoarthritis (OA) knees.MethodsSingle-cell RNA sequencing (scRNA-seq) analyses were performed on articular cartilage and meniscus tissues from healthy...

Differential regulation of the water channel protein aquaporins in chondrocytes of human knee articular cartilage by aging

Knee cartilage is in an aqueous environment filled with synovial fluid consisting of water, various nutrients, and ions to maintain chondrocyte homeostasis. Aquaporins (AQPs) are water channel proteins that play an important role in water exchange in cells, and AQP1, -3, and -4 are known to be expressed predominantly in cartilage. We evaluated the changes in AQP expression in chondrocytes from human knee articular cartilage in patients of different ages and identified the key factor(s) that mediate age-induced alteration in AQP expression. The mRNA and protein expression of AQP1, -3 and -4 were significantly decreased in fibrocartilage compared to hyaline cartilage and in articular cartilage from older osteoarthritis patients compared to that from young patients. Gene and protein expression of AQP1, -3 and -4 were altered during the chondrogenic differentiation of C3H10T1/2 cells. The causative factors for age-associated decrease in AQP included H2O2, TNFα, and HMGB1 for AQP1, -3, and -4, respectively. In particular, the protective effect of AQP4 reduction following HMGB1 neutralization was noteworthy. The identification of other potent molecules that regulate AQP expression represents a promising therapeutic approach to suppress cartilage degeneration during aging.

Effect of Degradation and Osteoarthritis on the Viscoelastic Properties of Human Knee Articular Cartilage: An Experimental Study and Constitutive Modeling

Articular cartilage, as a hydrated soft tissue which covers diarthrodial joints, has a pivotal role in the musculoskeletal system. Osteoarthritis is the most common degenerative disease that affects most individuals over the age of 55. This disease affects the elasticity, lubrication mechanism, damping function, and energy absorption capability of articular cartilage. In order to investigate the effect of osteoarthritis on the performance of articular cartilage, the mechanical behavior of human knee articular cartilage was experimentally investigated. Progressive cyclic deformation was applied beyond the physiological range to facilitate degradation of the tissue. The relaxation response of the damaged tissue was modeled by means of a fractional-order nonlinear viscoelastic model in the framework of finite deformations. It is shown that the proposed fractional model well reproduces the tissue’s mechanical behavior using a low number of parameters. Alteration of the model parameters was also investigated throughout the progression of tissue damage. This helps predict the mechanical behavior of the osteoarthritic tissue based on the level of previous damage. It is concluded that, with progression of osteoarthritis, the articular cartilage loses its viscoelastic properties such as damping and energy absorption capacity. This is also accompanied by a loss of stiffness which deteriorates more rapidly than viscosity does throughout the evolution of tissue damage. These results are thought to be significant in better understanding the degradation of articular cartilage and the progression of OA, as well as in the design of artificial articular cartilages.

Estrogen receptor Alpha in human knee articular cartilage of healthy and osteoarthritic females

ObjectiveEstrogen and osteoarthritis association has previously been proposed. This study evaluates the presence of estrogen receptors (ER)-α in articular cartilage, and its association. MethodsA prospective cohort study of women undergoing anterior cruciate ligament reconstruction (controls) or total knee arthroplasty (cases) was performed. Cartilage samples were processed and ER-α expression was quantified. ResultsTwenty patients were included: 12 cases and 8 controls. ER-α expression was higher in the case subjects. ConclusionIncreased ER-α expression is associated with age, and degeneration. This suggests estrogen deficiency is a risk for osteoarthritis and is inversely related to proliferative looking chondrocytes.

Stimulation of \u03b17-nAChRs coordinates autophagy and apoptosis signaling in experimental knee osteoarthritis

Osteoarthritis (OA) is the most common chronic joint disease in the elderly population. Growing evidence indicates that a balance between autophagy and apoptosis in chondrocytes plays a key role in OA’s cartilage degradation. Thus, drugs targeting the balance between apoptosis and autophagy are potential therapeutic approaches for OA treatment. In previous studies, we found that the activation of α7 nicotinic acetylcholine receptors (α7-nAChRs) alleviated monosodium iodoacetate (MIA)-induced joint degradation and osteoarthritis pain. To explore the potential functions of α7-nAChRs in autophagy and apoptosis signaling in knee OA, we compared the expression of α7-nAChRs in human knee articular cartilage tissues from normal humans and OA patients. We found that knee joint cartilage tissues of OA patients showed decreased α7-nAChRs and an imbalance between autophagy and apoptosis. Next, we observed that α7-nAChRs deficiency did not affect cartilage degradation in OA development but reversed the beneficial effects of nicotine on mechanical allodynia, cartilage degradation, and an MIA-induced switch from autophagy to apoptosis. Unlike in vivo studies, we found that primary chondrocytes from α7-nAChRs knockout (KO) mice showed decreased LC3 levels under normal conditions and were more sensitive toward MIA-induced apoptosis. Finally, we found that α7-nAChRs deficiency increased the phosphorylation of mTOR after MIA treatment, which can also be observed in OA patients’ tissues. Thus, our findings not only confirmed that nicotine alleviated MIA-induced pain behavior and cartilage degradation via stimulating the α7-nAChRs/mTOR signal pathway but found the potential role of α7-nAChRs in mediating the balance between apoptosis and autophagy.

Assessment of Native Human Articular Cartilage: A Biomechanical Protocol.

Recapitulating the mechanical properties of articular cartilage (AC) is vital to facilitate the clinical translation of cartilage tissue engineering. Prior to evaluation of tissue-engineered constructs, it is fundamental to investigate the biomechanical properties of native AC under sudden, prolonged, and cyclic loads in a practical manner. However, previous studies have typically reported only the response of native AC to one or other of these loading regimes. We therefore developed a streamlined testing protocol to characterize the elastic and viscoelastic properties of human knee AC, generating values for several important parameters from the same sample. Human AC was harvested from macroscopically normal regions of distal femoral condyles of patients (n = 3) undergoing total knee arthroplasty. Indentation and unconfined compression tests were conducted under physiological conditions (temperature 37 °C and pH 7.4) and testing parameters (strain rates and loading frequency) to assess elastic and viscoelastic parameters. The biomechanical properties obtained were as follows: Poisson ratio (0.4 ± 0.1), instantaneous modulus (52.14 ± 9.47 MPa) at a loading rate of 1 mm/s, Young's modulus (1.03 ± 0.48 MPa), equilibrium modulus (7.48 ± 4.42 MPa), compressive modulus (10.60 ± 3.62 MPa), dynamic modulus (7.71 ± 4.62 MPa) at 1 Hz and loss factor (0.11 ± 0.02). The measurements fell within the range of reported values for human knee AC biomechanics. To the authors' knowledge this study is the first to report such a range of biomechanical properties for human distal femoral AC. This protocol may facilitate the assessment of tissue-engineered composites for their functionality and biomechanical similarity to native AC prior to clinical trials.

Progressive deformation-induced degradation of knee articular cartilage and osteoarthritis

Articular cartilage (AC) is a dense connective tissue in the diarthrodial joints. Osteoarthritis is a disease associated with progressive changes in the tissue structure which affects mechanical properties of the tissue. Experimental studies show that abnormal/excessive mechanical loading or changes in biochemical composition can alter the structure which leads to degradation and loss of stiffness. In this study, deformation-induced degradation of human knee articular cartilage is investigated by conducting excessive deformations and inspecting progressive changes in mechanical properties of the tissue. Evolution of the properties is formulated to predict the amount of damage and consequently to connect the loss of stiffness to the grade of osteoarthritis. It is shown that, for tensile strains up to almost 15%, the tissue may recover its initial properties without permanent damage but for higher deformations degradation is expected. The proposed model is not confined to tension as the evolution laws are expressed in a 3D framework. The model predicts higher thresholds for compression and shear (25–30%) which is in good correlation with experimental investigations. It is concluded that, modelling the alterations of mechanical properties can be an effective method for predicting degradation of the AC under excessive loading and its connection to the associated diseases.

Effects of solute size and tissue composition on molecular and macromolecular diffusivity in human knee cartilage

Articular cartilage is an avascular tissue. Accordingly, diffusivity represents a fundamental transport mechanism for nutrients and other molecular signals regulating its cell metabolism and maintenance of the extracellular matrix. Understanding how solutes spread into articular cartilage is crucial to elucidating its pathologies, and to designing treatments for repair and restoration of its extracellular matrix. As in other connective tissues, diffusivity in articular cartilage may vary depending both its composition and the specific diffusing solute. Hence, this study investigated the roles of solute size and tissue composition on molecular diffusion in knee articular cartilage. FRAP tests were conducted to measure diffusivity of five molecular probes, with size ranging from ~332Da to 70,000Da, in human knee articular cartilage. The measured diffusion coefficients were related to molecular size, as well as water and glycosaminoglycan (GAG) content of femoral and tibial condyle cartilage. Diffusivity was affected by molecular size, with the magnitude of the diffusion coefficients decreasing as the Stokes radius of the probe increased. The values of diffusion coefficients in tibial and femoral samples were not significantly different from one another, despite the fact that tibial samples exhibited significantly higher water content and lower GAG content of the femoral specimens. Water content did not affect diffusivity. In contrast, diffusivities of large molecules were sensitive to GAG content. This study provides new knowledge on the mechanisms of diffusion in articular cartilage. Our findings can be leveraged to further investigate osteoarthritis and to design treatments for cartilage restoration or replacement.

Molecular phenotyping of the surfaceome of migratory chondroprogenitors and mesenchymal stem cells using biotinylation, glycocapture and quantitative LC-MS/MS proteomic analysis

The complement of cell surface proteins, collectively referred to as the surfaceome, is a useful indicator of normal differentiation processes, and the development of pathologies such as osteoarthritis (OA). We employed biochemical and proteomic tools to explore the surfaceome and to define biomarkers in chondrogenic progenitor cells (CPC) derived from human OA knee articular cartilage. These cells have great therapeutic potential, but their unexplored biology limits their clinical application. We performed biotinylation combined with glycocapture and high throughput shotgun proteomics to define the surface proteome of human bone marrow mesenchymal stem cells (MSCs) and human CPCs. We prepared cell surface protein-enriched fractions from MSCs and CPCs, and then a proteomic approach was used to compare and evaluate protein changes between undifferentiated MSCs and CPCs. 1256 proteins were identified in the study, of which 791 (63%) were plasma membrane, cell surface or extracellular matrix proteins. Proteins constituting the surfaceome were annotated and categorized. Our results provide, for the first time, a repository of quantitative proteomic data on the surfaceome of two closely related cell types relevant to cartilage biology and OA. These results may provide novel insights into the transformation of the surfaceome during chondrogenic differentiation and phenotypic changes during OA development.

RHEB gene therapy maintains the chondrogenic characteristics and protects cartilage tissue from degenerative damage during experimental murine osteoarthritis

Osteoarthritis (OA) is characterized by cartilage degeneration resulting from hypertrophic changes in chondrocytes caused by altered gene expression. The involvement of Ras homolog enriched in brain (RHEB) in OA regulation is unclear. Human knee articular cartilage samples - were analyzed for structural and biological changes by histology, immunohistochemistry, real time PCR and western blotting. OA-mouse model developed by surgical destabilization of the medial meniscus (DMM) were treated with adenovirus harboring Rheb gene to analyze onset and progression of OA. Histological scoring, immunohistochemistry, and TUNEL assay was performed to assess cartilage damage across the entire joint. Human and mouse OA cartilage is degenerated and has markedly reduced levels of RHEB. Human OA-degenerated chondrocytes (DC) exhibited a fibroblastic phenotype and 80 % of degenerative cartilage were senescent, with higher levels of reactive oxygen species (ROS). Gene expression analysis of DC revealed almost no COL2A1 expression and reduced SOX9 and RHEB expression. Transient transfection of RHEB rescued the DC phenotype and reduced senescence and ROS levels markedly. RHEB overexpression also increased COL2A1 and SOX9 expression. In an OA-mouse model, the Rheb protein level decreased as the severity of OA increased. Ectopic expression of Rheb using adenovirus in mouse-OA cartilage suppressed surgically-induced OA pathogenesis accompanied by modulation of Adamts5, Mmp 13, Col 10, and Col2a1 expression. Rheb induction significantly reduced apoptosis in OA-cartilage. RHEB plays an important role in maintaining the chondrogenic characteristics of chondrocytes, and has potential in preventing progression of OA in the destabilize the medial meniscus (DMM) mouse model of OA.

Biologic Characteristics of Shoulder Articular Cartilage in Comparison to Knee and Ankle Articular Cartilage From Individual Donors.

To describe histological and metabolic characteristics of glenohumeral joint (GHJ) articular cartilage and compare to knee and ankle joints. Macroscopically healthy human humeral head, glenoid, knee, and ankle articular cartilage were obtained from tissue donors (N = 16, 9 males, 7 females; age 45-78 years), within 24 hours of death. Gross morphology of each joint was assessed using Collins grading. Cartilage explants were removed from the entire surface of each joint, cultured for 48 hours with or without interleukin-1β and processed for histology with Safranin O, proteoglycan (PG) synthesis/content, and polymerase chain reaction for collagen II, aggrecan, and SOX9. Results were compared between uncultured and cultured controls and across all 3 joints. Structural differences were seen on histology between GHJ cartilage and knee and ankle cartilage of the same Collins grade, specifically, depletion of Safranin O staining in the extracellular matrix. Treatment of glenoid and humerus specimens with IL-1β demonstrated a trend toward decreased PG synthesis in each explant but this decrease did not reach significance. There was no significant difference in PG synthesis between humerus, glenoid, knee, and ankle samples at baseline, day-0 control, 48-hour control, and 48 hours after treatment with 0.1 ng or 10 ng of IL-1β. There were no significant increases in collagen II, SOX9, and aggrecan expression in glenoid and humeral head cartilage samples treated with IL-1β compared to baseline controls. GHJ articular cartilage did not significantly differ from ankle or knee cartilage with regard to PG synthesis and gene expression. However, it did differ in its histological appearance in normal state.

Chondrotoxic Effects of Local Anesthetics on Human Knee Articular Cartilage: A Systematic Review.

To review the current literature on the effects of intraarticular local anesthetics on human knee articular cartilage. PubMed; MEDLINE; SCOPUS; PEDro; CINAHL databases (1/1990-06/1/2018) were searched for local anesthetic effects on human knee articular cartilage. Sixteen studies met the inclusion criteria, with outcome measures assessing chondrocyte viability, morphology, and histology. A systematic review was performed using PRISMA guidelines. Seven studies were identified evaluating lidocaine, with five of them demonstrating statistically significant chondrotoxic effects. Fourteen studies examined bupivacaine, with all but one study demonstrating a chondrotoxic effect. Eight studies examined ropivacaine and found a dose-dependent chondrotoxicity starting at 0.75%. Two studies evaluated levobupivacaine showing chondrotoxicity, with one study showing it to be more chondrotoxic than bupivacaine. One study looked at mepivacaine and showed it to have more chondrotoxicity than ropivacaine. When studied the chondrotoxicity was found to be both dose and time dependent. Also, the addition of corticosteroids appeared to worsen the chondrotoxic effects. Lidocaine, bupivacaine, ropivacaine, levobupivacaine, and mepivacaine were reported to have dose- and time-dependent deleterious effects on chondrocytes that appeared to be made worse by the coadministration of corticosteroids. Ropivacaine at concentrations of 0.5% or less was found to be the least chondrotoxic anesthetic. I.

Differential protein expression in human knee articular cartilage and medial meniscus using two different proteomic methods: a pilot analysis

BackgroundProteomics is an emerging field in the study of joint disease. Our two aims with this pilot analysis were to compare healthy human knee articular cartilage with meniscus, two tissues both known to become affected in the osteoarthritic disease process, and to compare two mass spectrometry (MS)-based methods: data-dependent acquisition (DDA) and data-independent acquisition (DIA).MethodsHealthy knee articular cartilage taken from the medial tibial condyle and medial meniscus samples taken from the body region were obtained from three adult forensic medicine cases. Proteins were extracted from tissue pieces and prepared for MS analysis. Each sample was subjected to liquid chromatography (LC)-MS/MS analysis using an Orbitrap mass spectrometer, and run in both DDA and DIA mode. Linear mixed effects models were used for statistical analysis.ResultsA total of 653 proteins were identified in the DDA analysis, of which the majority was present in both tissue types. Only proteins with quantitation information in both tissues (n = 90) were selected for more detailed analysis, of which the majority did not statistically significantly differ in abundance between the two tissue types, in either of the MS analyses. However, 21 proteins were statistically significantly different (p < 0.05) between meniscus and cartilage in the DIA analysis. Out of these, 11 proteins were also significantly different in the DDA analysis. Aggrecan core protein was the most abundant protein in articular cartilage and significantly differed between the two tissues in both methods. The corresponding protein in meniscus was serum albumin. Dermatopontin exhibited the highest meniscus vs articular cartilage ratio among the statistically significant proteins. The DIA method led to narrower confidence intervals for the abundance differences between the two tissue types than DDA.ConclusionsAlthough articular cartilage and meniscus had similar proteomic composition, we detected several differences by MS. Between the two analyses, DIA yielded more precise estimates and more statistically significant different proteins than DDA, and had no missing values, which makes it preferable for future LC-MS/MS analyses.

Isotropic morphometry and multicomponent T1 ρ mapping of human knee articular cartilage in vivo at 3T.

The progressive loss of hyaline articular cartilage due to osteoarthritis (OA) changes the functional and biochemical properties of cartilage. Measuring the T1 ρ along with the morphological assessment can potentially be used as noninvasive biomarkers in detecting early-stage OA. To correlate the biochemical and morphological data, submillimeter isotropic resolution for both studies is required. To implement a high spatial resolution 3D-isotropic-MRI sequence for simultaneous assessment of morphological and biexponential T1 ρ relaxometry of human knee cartilage in vivo. Prospective. Ten healthy volunteers with no known inflammation, trauma, or pain in the knee. Standard FLASH sequence and customized Turbo-FLASH sequence to acquire 3D-isotropic-T1 ρ-weighted images on a 3T MRI scanner. The mean volume and thickness along with mono- and biexponential T1 ρ relaxations were assessed in the articular cartilage of 10 healthy volunteers. Nonparametric rank-sum tests. Bland-Altman analysis and coefficient of variation. The mean monoexponential T1 ρ relaxation was 40.7 ± 4.8 msec, while the long and short components were 58.2 ± 3.9 msec and 6.5 ± 0.6 msec, respectively. The mean fractions of long and short T1 ρ relaxation components were 63.7 ± 5.9% and 36.3 ± 5.9%, respectively. Statistically significant (P ≤ 0.03) differences were observed in the monoexponential and long components between some of the regions of interest (ROIs). No gender differences between biexponential components were observed (P > 0.05). Mean cartilage volume and thickness were 25.9 ± 6.4 cm3 and 2.2 ± 0.7 mm, respectively. Cartilage volume (P = 0.01) and thickness (P = 0.03) were significantly higher in male than female participants across all ROIs. Bland-Altman analysis showed agreement between two morphological methods with limits of agreement between -1000 mm3 and +1100 mm3 for volume, and -0.78 mm and +0.46 mm for thickness, respectively. Simultaneous assessment of morphological and multicomponent T1 ρ relaxation of knee joint with 0.7 × 0.7 × 0.7 mm isotropic spatial resolution is demonstrated in vivo. Comparison with a standard method showed that the proposed technique is suitable for assessing the volume and thickness of articular cartilage. 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;48:1707-1716.

Erratum to: Development of an Electromechanical Grade to Assess Human Knee Articular Cartilage Quality.

Erratum to: Development of an Electromechanical Grade to Assess Human Knee Articular Cartilage Quality.

Development of an Electromechanical Grade to Assess Human Knee Articular Cartilage Quality.

Quantitative assessments of articular cartilage function are needed to aid clinical decision making. Our objectives were to develop a new electromechanical grade to assess quantitatively cartilage quality and test its reliability. Electromechanical properties were measured using a hand-held electromechanical probe on 200 human articular surfaces from cadaveric donors and osteoarthritic patients. These data were used to create a reference electromechanical property database and to compare with visual arthroscopic International Cartilage Repair Society (ICRS) grading of cartilage degradation. The effect of patient-specific and location-specific characteristics on electromechanical properties was investigated to construct a continuous and quantitative electromechanical grade analogous to ICRS grade. The reliability of this novel grade was assessed by comparing it with ICRS grades on 37 human articular surfaces. Electromechanical properties were not affected by patient-specific characteristics for each ICRS grade, but were significantly different across the articular surface. Electromechanical properties varied linearly with ICRS grade, leading to a simple linear transformation from one scale to the other. The electromechanical grade correlated strongly with ICRS grade (r=0.92, p<0.0001). Additionally, the electromechanical grade detected lesions that were not found visually. This novel grade can assist the surgeon in assessing human knee cartilage by providing a quantitative and reliable grading system.

Differences in protein expression using data-dependent and data-independent acquisition mass spectrometry in the analysis of human knee articular cartilage and menisci: a pilot analysis

Differences in protein expression using data-dependent and data-independent acquisition mass spectrometry in the analysis of human knee articular cartilage and menisci: a pilot analysis

The effect of additive compounds on glycerol-induced damage to human chondrocytes

High concentrations of cryoprotective agents are required for cryopreservation techniques such as vitrification. Glycerol is a common cryoprotective agent used in cryopreservation protocols but this agent is toxic at high concentrations. This work is an attempt to mitigate the toxic effects of high concentrations of glycerol on intact chondrocytes in human knee articular cartilage from total knee arthroplasty patients by simultaneous exposure to glycerol and a variety of additive compounds. The resulting cell viability in the cartilage samples as measured by membrane integrity staining showed that, in at least one concentration or in combination, all of the tested additive compounds (tetramethylpyrazine, ascorbic acid, chondroitin sulphate, glucosamine sulphate) were able to reduce the deleterious effects of glycerol exposure when examination of membrane integrity took place on a delayed time frame. The use of additive compounds to reduce cryoprotectant toxicity in articular cartilage may help improve cell recovery after cryopreservation.