Osteoarthritic Human Articular Cartilage Research Articles

Site-specific elastic and viscoelastic biomechanical properties of healthy and osteoarthritic human knee joint articular cartilage

Articular cartilage exhibits site-specific biomechanical properties. However, no study has comprehensively characterized site-specific cartilage properties from the same knee joints at different stages of osteoarthritis (OA).Cylindrical osteochondral explants (n = 381) were harvested from donor-matched lateral and medial tibia, lateral and medial femur, patella, and trochlea of cadaveric knees (N = 17). Indentation test was used to measure the elastic and viscoelastic mechanical properties of the samples, and Osteoarthritis Research Society International (OARSI) grading system was used to categorize the samples into normal (OARSI 0–1), early OA (OARSI 2–3), and advanced OA (OARSI 4–5) groups.OA-related changes in cartilage mechanical properties were site-specific. In the lateral and medial tibia and trochlea sites, equilibrium, instantaneous and dynamic moduli were higher (p < 0.001) in normal tissue than in early and advanced OA tissue. In lateral and medial femur, equilibrium, instantaneous and dynamic moduli were smaller in advanced OA, but not in early OA, than in normal tissue. The phase difference (0.1–0.25 Hz) between stress and strain was significantly smaller (p < 0.05) in advanced OA than in normal tissue across all sites except medial tibia.Our results indicated that in contrast to femoral and patellar cartilage, equilibrium, instantaneous and dynamic moduli of the tibia and trochlear cartilage decreased in early OA. These may suggest that the tibia and trochlear cartilage degrades faster than the femoral and patellar cartilage. The information is relevant for developing site-specific computational models and engineered cartilage constructs.

Inhibition of HOXD11 promotes cartilage degradation and induces osteoarthritis development

The 5′-HOXD genes are important for chondrogenesis in vertebrates, but their roles in osteoarthritis (OA) are still ambiguous. In our study, 5′-HOXD genes involvement contributing to cartilage degradation and OA was investigated. In bioinformatics analysis of 5′-HOXD genes, we obtained the GSE169077 data set related to OA in the GEO and analyzed DEGs using the GEO2R tool attached to the GEO. Then, we screened the mRNA levels of 5′-HOXD genes by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). We discovered that OA chondrocyte proliferation was inhibited, and apoptosis was increased. Moreover, it was discovered that SOX9 and COL2A1 were downregulated at mRNA and protein levels, while matrix metalloproteinases (MMPs) and a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTSs) were upregulated. According to the results of differentially expressed genes (DEGs) and qRT-PCR, we evaluated the protein level of HOXD11 and found that the expression of HOXD11 was downregulated, reversed to MMPs and ADAMTSs but consistent with the cartilage-specific factors, SOX9 and COL2A1. In the lentivirus transfection experiments, HOXD11 overexpression reversed the effects in OA chondrocytes. In human OA articular cartilage, aberrant subchondral bone was formed in hematoxylin–eosin (H&E) and Safranin O and fast green (SOFG) staining results. Furthermore, according to immunohistochemistry findings, SOX9 and HOXD11 expression was inhibited. The results of this study established that HOXD11 was downregulated in OA cartilage and that overexpression of HOXD11 could prevent cartilage degradation in OA.

Visible and Near-Infrared Spectroscopy Enables Differentiation of Normal and Early Osteoarthritic Human Knee Joint Articular Cartilage

Osteoarthritis degenerates cartilage and impairs joint function. Early intervention opportunities are missed as current diagnostic methods are insensitive to early tissue degeneration. We investigated the capability of visible light-near-infrared spectroscopy (Vis-NIRS) to differentiate normal human cartilage from early osteoarthritic one. Vis-NIRS spectra, biomechanical properties and the state of osteoarthritis (OARSI grade) were quantified from osteochondral samples harvested from different anatomical sites of human cadaver knees. Two support vector machines (SVM) classifiers were developed based on the Vis-NIRS spectra and OARSI scores. The first classifier was designed to distinguish normal (OARSI: 0–1) from general osteoarthritic cartilage (OARSI: 2–5) to check the general suitability of the approach yielding an average accuracy of 75% (AUC = 0.77). Then, the second classifier was designed to distinguish normal from early osteoarthritic cartilage (OARSI: 2–3) yielding an average accuracy of 71% (AUC = 0.73). Important wavelength regions for differentiating normal from early osteoarthritic cartilage were related to collagen organization (wavelength region: 400–600 nm), collagen content (1000–1300 nm) and proteoglycan content (1600–1850 nm). The findings suggest that Vis-NIRS allows objective differentiation of normal and early osteoarthritic tissue, e.g., during arthroscopic repair surgeries.

Label-free, multi-parametric assessments of cell metabolism and matrix remodeling within human and early-stage murine osteoarthritic articular cartilage

Osteoarthritis (OA) is characterized by the progressive deterioration of articular cartilage, involving complicated cell-matrix interactions. Systematic investigations of dynamic cellular and matrix changes during OA progression are lacking. In this study, we use label-free two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to assess cellular and extracellular matrix features of murine articular cartilage during several time points at early stages of OA development following destabilization of medial meniscus surgery. We detect significant changes in the organization of collagen fibers and crosslink-associated fluorescence of the superficial zone as early as one week following surgery. Such changes become significant within the deeper transitional and radial zones at later time-points, highlighting the importance of high spatial resolution. Cellular metabolic changes exhibit a highly dynamic behavior, and indicate metabolic reprogramming from enhanced oxidative phosphorylation to enhanced glycolysis or fatty acid oxidation over the ten-week observation period. The optical metabolic and matrix changes detected within this mouse model are consistent with differences identified in excised human cartilage specimens from OA and healthy cartilage specimens. Thus, our studies reveal important cell-matrix interactions at the onset of OA that may enable improved understanding of OA development and identification of new potential treatment targets.

Analysis of RNA Polyadenylation in Healthy and Osteoarthritic Human Articular Cartilage.

Polyadenylation (polyA) defines the 3' boundary of a transcript's genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist for a gene. This causes variance in 3' regulatory domains and can affect coding sequence if intronic events occur. The distribution of polyA sites on articular chondrocyte transcripts has not been studied so we aimed to define their transcriptome-wide location in age-matched healthy and osteoarthritic knee articular cartilage. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeq-Reverse 3' RNA sequencing approach, where each read runs 3' to 5' from within the polyA tail into the transcript and contains a distinct polyA site. Differential expression of transcripts was significant altered between healthy and osteoarthritic samples with enrichment for functionalities that were strongly associated with joint pathology. Subsequent examination of polyA site data allowed us to define the extent of site usage across all the samples. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, in the genes affected, there was potential for the APA to have functional relevance. We have characterised the polyadenylation landscape of human knee articular chondrocytes and conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed.

Inhibition of KDM7A/B histone demethylases restores H3K79 methylation and protects against osteoarthritis

ObjectivesIn osteoarthritis, methylation of lysine 79 on histone H3 (H3K79me), a protective epigenetic mechanism, is reduced. Histone methylation levels are dynamically regulated by histone methyltransferases and demethylases. Here, we aimed...

SCD1 deficiency exacerbates osteoarthritis by inducing ferroptosis in chondrocytes.

Osteoarthritis (OA) is a severe joint disease that causes cartilage destruction and mobility loss. Abnormal fatty acid metabolism of chondrocytes plays a role in OA development. Stearoyl-CoA desaturase (SCD1) is a rate-limiting enzyme in the anabolism of unsaturated fatty acids. This study aimed to investigate the role of the SCD1 protein in the degenerative process of OA. The GSE176199 gene expression profile dataset was analyzed by Gene Set Enrichment Analysis (GSEA). An animal model of OA was established using C57BL/6J wild-type (WT) (n=40) and SCD1 knockout (SCD1-KO) (n=20) mice. The histological scoring method of the Osteoarthritis Research Society International (OARSI) was used to quantify the degree of cartilage degeneration. The expression of SCD1 protein and relevant ferroptosis indicators were evaluated. The GSEA analysis showed that unsaturated fatty acid synthesis was inhibited in human OA chondrocytes. Meanwhile, the expression of SCD1 protein was significantly reduced in human OA articular cartilage. SCD1-KO mice exhibited early OA and accelerated cartilage loss after destabilization of medial meniscus (DMM)-induced OA. Furthermore, we found that the SCD1-PPARG axis regulates articular cartilage homeostasis via a mechanism involving the induction of ferroptosis-related gene expression in ATDC5 chondrocytes. SCD1 deficiency exacerbates OA by inducing ferroptosis in chondrocytes.

Progenitor Cells in Healthy and Osteoarthritic Human Cartilage Have Extensive Culture Expansion Capacity while Retaining Chondrogenic Properties.

Articular cartilage-derived progenitor cells (ACPCs) are a potential new cell source for cartilage repair. This study aims to characterize endogenous ACPCs from healthy and osteoarthritic (OA) cartilage, evaluate their potential for cartilage regeneration, and compare this to cartilage formation by chondrocytes. ACPCs were isolated from full-thickness healthy and OA human cartilage and separated from the total cell population by clonal growth after differential adhesion to fibronectin. ACPCs were characterized by growth kinetics, multilineage differentiation, and surface marker expression. Chondrogenic redifferentiation of ACPCs was compared with chondrocytes in pellet cultures. Pellets were assessed for cartilage-like matrix production by (immuno)histochemistry, quantitative analyses for glycosaminoglycans and DNA content, and expression of chondrogenic and hypertrophic genes. Healthy and OA ACPCs were successfully differentiated toward the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Both ACPC populations met the criteria for cell surface marker expression of mesenchymal stromal cells (MSCs). Healthy ACPCs cultured in pellets deposited extracellular matrix containing proteoglycans and type II collagen, devoid of type I collagen. Gene expression of hypertrophic marker type X collagen was lower in healthy ACPC pellets compared with OA pellets. This study provides further insight into the ACPC population in healthy and OA human articular cartilage. ACPCs show similarities to MSCs, yet do not produce calcified matrix under well-established osteogenic culture conditions. Due to extensive proliferative potential and chondrogenic capacity, ACPCs show potential for cartilage regeneration and possibly for clinical application, as a promising alternative to MSCs or chondrocytes.

Comparative characterization of mesenchymal progenitor cells from osteoarthritic and rheumatoid arthritic human articular cartilage

Comparative characterization of mesenchymal progenitor cells from osteoarthritic and rheumatoid arthritic human articular cartilage

Endoglin Level Is Critical for Cartilage Tissue Formation In Vitro by Passaged Human Chondrocytes.

Transforming growth factor beta (TGFβ) signaling is required for in vitro chondrogenesis. In animal models of osteoarthritis (OA), TGFβ receptor alterations are detected in chondrocytes in severe OA cartilage. It is not known whether such changes are dependent on the grade of human OA and if they affect chondrogenesis. Thus, the purpose of this study was to determine if human OA chondrocytes obtained from low-grade or high-grade disease could form cartilage tissue and to assess the role of the co-receptors, endoglin (ENG) and TGFβ receptor 3 (TGFBRIII), in the regulation of this tissue generation in vitro. We hypothesized that the grade of OA disease would not affect the ability of cells to form cartilage tissue and that the TGFβ co-receptor, ENG, would be critical to regulating tissue formation. Chondrocytes isolated from low-grade OA or high-grade OA human articular cartilage (AC) were analyzed directly (P0) or passaged in monolayer to P2. Expression of the primary TGFβ receptor ALK5, and the co-receptors ENG and TGFβRIII, was assessed by image flow cytometry. To assess the ability to form cartilaginous tissue, cells were placed in three-dimensional culture at high density and cultured in chondrogenic media containing TGFβ3. ENG knockdown was used to determine its role in regulating tissue formation. Overall, grade-specific differences in expression of ALK5, ENG, and TGFβRIII in primary or passaged chondrocytes were not detected; however, ENG expression increased significantly after passaging. Despite the presence of ALK5, P0 cells did not form cartilaginous tissue. In contrast, P2 cells derived from low-grade and high-grade OA AC formed hyaline-like cartilaginous tissues of similar quality. Knockdown of ENG in P2 cells inhibited cartilaginous tissue formation compared to controls indicating that the level of ENG protein expression is critical for in vitro chondrogenesis by passaged articular chondrocytes. This study demonstrates that it is not the grade of OA, but the levels of ENG in the presence of ALK5 that influences the ability of human passaged articular chondrocytes to form cartilaginous tissue in vitro in 3D culture. This has implications for cartilage repair therapies. Impact statement These findings are important clinically, given the limited availability of osteoarthritis (OA) cartilage tissue. Being able to use cells from all grades of OA will increase our ability to obtain sufficient cells for cartilage repair. In addition, it is possible that endoglin (ENG) levels, in the presence of ALK5 expression, may be suitable to use as biomarkers to identify cells able to produce cartilage.

Protocol for the Isolation of Intact Chondrons from Healthy and Osteoarthritic Human Articular Cartilage.

Chondrons are the main functional microanatomical units in cartilage, consisting of chondrocytes and the directly surrounding pericellular matrix (PCM). They have attracted attention as a more physiological and biomimetic in vitro model for evaluating chondrocyte function and metabolism as compared to single chondrocytes. Chondrons may be more suitable for in vitro studies than primary chondrocytes that have been isolated without PCM since their in situ and in vivo states remain intact: chondrocytes within their PCM do not undergo the rapid dedifferentiation that proliferating single chondrocytes undergo in culture. Therefore, chondrons may be a better model for studying chondrocyte biology and responses to pro-inflammatory and anti-inflammatory cytokines, growth factors and novel therapeutics. In this chapter, we present a concise and unified protocol for enzymatic isolation of intact chondrons from human articular cartilage and determination of their viability.

RNA Isolation from Articular Cartilage Tissue.

Isolation of high-quality RNA directly from tissues is desirable to obtain precise information of in vivo gene expression profiles in cells embedded within their extracellular matrix (ECM). It is well known that purification of RNA from cartilage tissues is particularly challenging due to low cell (chondrocyte) content and its dense ECM rich in large negatively charged proteoglycans that can copurify with RNA. Older methodologies to purify RNA from cartilage involved the use of concentrated denaturing solutions containing guanidinium isothiocyanate followed by ultracentrifugation in cesium trifluoroacetate. Such ultracentrifugation approaches are rarely used now since the emergence of more user-friendly mini spin column chromatography kits. For this chapter, we tested and compared three methods to isolate RNA from immature murine articular (femoral head) cartilage and found that the combination of TRIzol® reagent and spin column chromatography (Norgen Total RNA Purification Kit) was the best approach to generate higher quality RNA. Here, the average RNA Integrity Number (RIN), as determined by Bioanalyzer technology, was 7.1. We then applied this method to attempt to isolate RNA directly from human articular cartilage harvested from three osteoarthritic (OA) knee joint specimens. As expected, the concentration and quality of RNA obtained differed between samples. However, from one specimen, we were able to isolate approximately 3μg of total RNA (including small noncoding RNAs) from 100mg of human OA cartilage with a RIN=7.9. Despite the patient-to-patient variabilities that are known to exist between cartilage specimens from OA joints, we have demonstrated that it is possible to obtain reasonably high levels of RNA from human OA articular cartilage at a quality suitable for downstream analyses including microarray and RNA-Seq. A detailed description of our preferred RNA purification methodology, which can be used to isolate RNA from human, bovine, or rodent cartilage tissue, is provided in this chapter.

Modulation of Hedgehog Signaling by Kappa Opioids to Attenuate Osteoarthritis.

Inhibition of hedgehog (HH) signaling prevents cartilage degeneration and promotes repair in animal models of osteoarthritis (OA). This study, undertaken in OA models and in human OA articular cartilage, was designed to explore whether kappa opioid receptor (KOR) modulation via the inhibition of HH signaling may have therapeutic potential for achieving disease-modifying activity in OA. Primary human articular cartilage and synovial tissue samples from patients with knee OA undergoing total joint replacement and from healthy human subjects were obtained from the National Disease Research Interchange. For in vivo animal studies, a partial medial meniscectomy (PMM) model of knee OA in rats was used. A novel automated 3-dimensional indentation tester (Mach-1) was used to quantify the thickness and stiffness properties of the articular cartilage. Inhibition of HH signaling through KOR activation was achieved with a selective peptide agonist, JT09, which reduced HH signaling via the cAMP/CREB pathway in OA human articular chondrocytes (P = 0.002 for treated versus untreated OA chondrocytes). Moreover, JT09 markedly decreased matrix degeneration induced by an HH agonist, SAG, in pig articular chondrocytes and cartilage explants (P = 0.026 versus untreated controls). In vivo application of JT09 via intraarticular injection into the rat knee joint after PMM surgery significantly attenuated articular cartilage degeneration (60% improvement in the tibial plateau; P = 0.021 versus vehicle-treated controls). In JT09-treated rats, cartilage content, structure, and functional properties were largely maintained, and osteophyte formation was reduced by 70% (P = 0.005 versus vehicle-treated controls). The results of this study define a novel mechanism for the role of KOR in articular cartilage homeostasis and disease, providing a potential unifying mechanistic basis for the overlap in disease processes and features involving opioid and HH signaling. Moreover, this study identifies a potential novel therapeutic strategy in which KOR modulation can improve outcomes in patients with OA.

Sprifermin (rhFGF18) versus vehicle induces a biphasic process of extracellular matrix remodeling in human knee OA articular cartilage ex vivo

Sprifermin, recombinant human fibroblast growth factor 18 (rhFGF18), induces cartilage regeneration in knees of patients with osteoarthritis (OA). We hypothesized that a temporal multiphasic process of extracellular matrix (ECM) degradation and formation underlie this effect. We aimed to characterize the temporal ECM remodeling of human knee OA articular cartilage in response to sprifermin treatment. Articular cartilage explants from patients with knee OA (npatients = 14) were cultured for 70 days, with permanent exposure to sprifermin (900, 450, 225 ng/mL), FGF18 (450 ng/mL), insulin-like growth factor-1 (100 ng/mL, positive control) or vehicle (nreplicates/treatment/patient = 2). Metabolic activity (AlamarBlue) and biomarkers of type IIB collagen (PIIBNP) formation (Pro-C2 enzyme-linked immunosorbent assay [ELISA]) and aggrecanase-mediated aggrecan neo-epitope NITEGE (AGNx1 ELISA) were quantified once a week. At end of culture (day 70), gene expression (quantitative reverse transcription polymerase chain reaction) and proteoglycan content (Safranin O/Fast green staining) were quantified. The cartilage had continuously increased metabolic activity, when treated with sprifermin/FGF18 compared to vehicle. During days 7–28 PIIBNP was decreased and NITEGE was increased, and during days 35–70 PIIBNP was increased. At end of culture, the cartilage had sustained proteoglycan content and relative expression of ACAN < COL2A1 < SOX9 < COL1A1, indicating that functional chondrocytes remained in the explants. Sprifermin induces a temporal biphasic cartilage remodeling in human knee OA articular cartilage explants, with early-phase increased aggrecanase activity and late-phase increased type II collagen formation.

Progenitor cells from healthy and Osteoarthritic human cartilage show potential for one-stage repair treatment

Purpose: This study aims to isolate and characterize articular cartilage-derived progenitor cells (ACPCs) from human cartilage. ACPCs from healthy and osteoarthritic cartilage were compared. In addition, the potential to use these ACPCs in addition to primary chondrocytes for one-stage procedures to treat cartilage defects is assessed. Methods: Cells were isolated from full-thickness healthy (n=6, age 46-49, mean age 48) and osteoarthritic (n=6, age 41-82, mean age 62) human cartilage. Subsequently, ACPCs were isolated from the total cell population by clonal growth after a differential fibronectin adhesion assay. Mesenchymal stromal cells (MSCs) were isolated from human bone marrow. Healthy and osteoarthritic ACPCs were characterized and compared to MSCs by multilineage differentiation and flow cytometry analysis to assess cell surface marker expression. Full-depth chondrocytes of the same donors were assessed as well. Next, ACPCs were cocultured with osteoarthritic chondrocytes in 3D pellet cultures and compared to cocultures of MSCs and chondrocytes. Pellets were harvested after 28 days and assessed for cartilage-like matrix production using quantitative biochemical analyses for glycosaminoglycans and collagen. (Immuno)histochemistry was performed to visualize proteoglycan and collagen production. Results: Healthy and osteoarthritic ACPCs were successfully isolated and differentiated into the adipogenic and chondrogenic lineage (Figure 1), but failed to produce calcified matrix when exposed to osteogenic induction media. Full-depth chondrocytes derived from the same donors were able to produce calcified matrix upon induction of osteogenic differentiation. Both ACPC populations met the criteria for cell surface marker expression to identify mesenchymal stromal cells (MSCs) as determined by flow cytometry. Cartilage-like matrix production was observed in ACPC pellet cultures, as well as pellets consisting of a coculture of ACPCs and chondrocytes. Conclusions: In conclusion, this study provides further insight into a progenitor cell population which is present in both healthy and osteoarthritic human articular cartilage. These populations show similarities to MSCs. Furthermore, ACPCs show potential for use complementary to osteoarthritic chondrocytes in one-stage cartilage repair treatments, as a potential alternative to MSCs.

NOV/CCN3 induces cartilage protection by inhibiting PI3K/AKT/mTOR pathway.

Osteoarthritis (OA), an age‐related degenerative joint disease, is pathologically characterized by articular cartilage degeneration and synovial inflammation. Nephroblastoma overexpressed (NOV or CCN3), a matricellular protein, is a primary member of the CCN family (Cyr61, Ctgf, NOV) of proteins and is involved in various inflammatory disorders. Previous studies reported that CCN3 might play a therapeutic role in OA. However, the underlying mechanism remains unclear. In this study, we confirmed the expression of CCN3 was decreased in human and rat OA articular cartilage. Recombinant CCN3 ameliorated the IL‐1β‐induced matrix catabolism, as demonstrated by MMP1, MMP3, MMP13, ADAMTS5 and iNOS expression, in vitro. In addition, the degradation of cartilage matrix such as collagen 2 and aggrecan could be reversed by CCN3. Furthermore, we found CCN3 promoted autophagy as Atg5, Beclin1 and LC3‐II expression were increased. High‐mobility group box 1 was negatively correlated with CCN3 in IL‐1β‐induced osteoarthritis responses, and HMGB1 is involved in the protective effect of CCN3 in OA. Moreover, CCN3 overexpression decreased the expression of HMGB1 and reversed the IL‐1β induced MMPs production. Additionally, recombinant CCN3 or CCN3 overexpression attenuated the activation of PI3K/AKT/mTOR pathway induced by IL‐1β. Our study presents new mechanisms of CCN3 in osteoarthritis and indicates that CCN3 can serve as a novel potential therapeutic target for osteoarthritis.

Through-thickness patterns of shear strain evolve in early osteoarthritis

Given the structural changes associated with the progression of Osteoarthritis (OA), we hypothesized that patterns of through-thickness, large-strain shear evolve with early-stage OA. We therefore aimed to determine whether and how patterns of shear strains change during early-stage OA to 1) gain insight into the progression of OA by quantifying changes in local deformations; 2) gauge the potential of patterns in shear strain to serve as image-based biomarkers of early-stage OA; and 3) provide high-resolution, through-thickness data for proposing, fitting, and validating constitutive models for cartilage. We completed displacement-driven, large-strain shear tests (5, 10, 15%) on 44 specimens of variably advanced osteoarthritic human articular cartilage as determined by both Osteoarthritis Research Society International (OARSI) grade and PLM-CO score. We recorded the through-thickness deformations with a stereo-camera system and processed these data using digital image correlation (DIC) to determine full-thickness patterns of strains and relative zonal recruitments, i.e., the average shear strain in a through-thickness zone weighted by its relative thickness and normalized by the applied strain. We observed three general shapes for the curves of averaged through-thickness, Green-Lagrange shear strains during progression of OA. We also observed that during the progression of OA only the deep zone is recruited differently under shear in a statistically significant way. We propose that changes in through-thickness patterns of shear strain could provide sensitive biomarkers for early clinical detection of OA. The relative zonal recruitment of the deep zone decreases with progressing OA (OARSI grade) and microstructural remodeling (PLM-CO score), which do not consistently affect recruitment of the superficial and middle zones.

Human Diseased Articular Cartilage Contains a Mesenchymal Stem Cell-Like Population of Chondroprogenitors with Strong Immunomodulatory Responses.

Background: osteoarthritic human articular cartilage (AC)-derived cartilage cells (CCs) with same-donor bone marrow (BMSCs) and adipose tissue (ASCs)-derived mesenchymal stem cells were compared, in terms of stemness features, and secretory and immunomodulatory responses to inflammation. Methods: proteoglycan 4 (PRG4) presence was evaluated in AC and CCs. MSCs and CCs (n = 8) were cultured (P1 to P4) and characterized for clonogenicity, nanog homeobox (NANOG), and POU class 5 homeobox 1 (POU5F1) expression, immunotypification, and tri-lineage differentiation. Their basal and interleukin-1β (IL-1β)-stimulated expression of matrix metalloproteases (MMPs), tissue inhibitors (TIMPs), release of growth factors, and cytokines were analyzed, along with the immunomodulatory ability of CCs. Results: PRG4 was mainly expressed in the intact AC surface, whereas shifted to the intermediate zone in damaged cartilage and increased its expression in CCs upon culture. All cells exhibited a similar phenotype and stemness maintenance over passages. CCs showed highest chondrogenic ability, no adipogenic potential, a superior basal secretion of growth factors and cytokines, the latter further increased after inflammatory stimulation, and an immunomodulatory behavior. All stimulated cells shared an increased MMP expression without a corresponding TIMP production. Conclusion: based on the observed features, CCs obtained from pathological joints may constitute a potential tissue-specific therapeutic target or agent to improve damaged cartilage healing, especially damage caused by inflammatory/immune mediated conditions.

Non-Invasive Monitoring of Functional State of Articular Cartilage Tissue with Label-Free Unsupervised Hyperspectral Imaging

Damage and degradation of articular cartilage leads to severe pain and loss of mobility. The development of new therapies for cartilage regeneration for monitoring their effect requires further study of cartilage, ideally at a molecular level and in a minimally invasive way. Hyperspectral microscopy is a novel technology which utilises endogenous fluorophores to non-invasively assess the molecular composition of cells and tissue. In this study, we applied hyperspectral microscopy to healthy bovine articular cartilage and osteoarthritic human articular cartilage to investigate its capacity to generate informative molecular data and characterise disease state and treatment effects. We successfully demonstrated label-free fluorescence identification of collagen type I and II – isolated in cartilage here for the first time and the co-enzymes free NADH and FAD which together give the optical redox ratio that is an important measure of metabolic activity. The intracellular composition of chondrocytes was also examined. Differences were observed in the molecular ratios within the superficial and transitional zones of the articular cartilage which appeared to be influenced by disease state and treatment. These findings show that hyperspectral microscopy could be useful for investigating the molecular underpinnings of articular cartilage degradation and repair. As it is non-invasive and non-destructive, samples can be repeatedly assessed over time, enabling true time-course experiments with in-depth molecular data. Additionally, there is potential for the hyperspectral approach to be adapted for patient examination to allow the investigation of cartilage state. This could be of advantage for assessment and diagnosis as well as treatment monitoring.

Increased WISP1 expression in human osteoarthritic articular cartilage is epigenetically regulated and decreases cartilage matrix production.

Previously, we have shown the involvement of Wnt-activated protein Wnt-1-induced signaling protein 1 (WISP1) in the development of OA in mice. Here, we aimed to characterize the relation between WISP1 expression and human OA and its regulatory epigenetic determinants. Preserved and lesioned articular cartilage from end-stage OA patients and non-OA-diagnosed individuals was collected. WISP1 expression was determined using immunohistochemistry and damage was classified using Mankin scoring. RNA expression and DNA methylation were assessed in silico from genome-wide datasets (microarray analysis and RNA sequencing, and 450 k-methylationarrays, respectively). Effects of WISP1 were tested in pellet cultures of primary human chondrocytes. WISP1 expression in cartilage of OA patients was increased compared with non-OA-diagnosed controls and, within OA patients, WISP1 was even higher in lesioned compared with preserved regions, with expression strongly correlating with Mankin score. In early symptomatic OA patients with disease progression, higher synovial WISP1 expression was observed as compared with non-progressors. Notably, increased WISP1 expression was inversely correlated with methylation levels of a positional CpG-dinucleotide (cg10191240), with lesioned areas showing strong hypomethylation for this CpG as compared with preserved cartilage. Additionally, we observed that methylation levels were allele-dependent for an intronic single-nucleotide polymorphism nearby cg10191240. Finally, addition of recombinant WISP1 to pellets of primary chondrocytes strongly inhibited deposition of extracellular matrix as reflected by decreased pellet circumference, proteoglycan content and decreased expression of matrix components. Increased WISP1 expression is found in lesioned human articular cartilage, and appears epigenetically regulated via DNA methylation. In vitro assays suggest that increased WISP1 is detrimental for cartilage integrity.