Phenotype Of Articular Chondrocytes Research Articles

Silk fibroin/carboxymethyl chitosan hydrogel with tunable biomechanical properties has application potential as cartilage scaffold

Tissue engineering is a promising strategy for cartilage repair and regeneration. However, an ideal scaffolding material that not only mimics the biomechanical properties of the native cartilage, but also supports the chondrogenic phenotype of the seeding cells is in need. In this study, we developed a silk fibroin (SF) and carboxymethyl chitosan (CMCS) composite hydrogel with enzymatic cross-links (horseradish peroxidase and hydrogen peroxide) and β-sheet cross-links (ethanol treatment). Results of Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA), and X-ray diffraction (XRD) verified that SF/CMCS composite hydrogels had a tunable β-sheet structure. Therefore, by increasing the time of ethanol treatment from 0 h to 8 h, a series of parameters including pore size (from 50 to 300 μm), equilibrium swelling (from 78.1 ± 2.6% to 91.9 ± 0.9%), degradation (from 100% to 9% reduction in mass over 56 days), rheological properties (storage modulus from 177 Pa to 88,904 Pa), and mechanical properties (compressive modulus from 13 to 829 kPa) of the hydrogels were adjusted. In particular, the material parameters of the hydrogels with 2 h ethanol treatment appeared most suitable for engineered cartilage. Furthermore, the in vitro cellular experiments showed that the hydrogels supported the adhesion, proliferation, glycosaminoglycan synthesis, and chondrogenic phenotype of rabbit articular chondrocytes. Finally, subcutaneous implantation of the hydrogels in mice showed no infections or local inflammatory responses, indicating a good biocompatibility in vivo. In conclusion, the chemical-physical cross-linking SF/CMCS composite hydrogels, with tunable material properties and degradation rate, good biocompatibility, are promising scaffolds for cartilage tissue engineering.

Adseverin, an actin binding protein, regulates articular chondrocyte phenotype.

Chondrocytes dedifferentiate as a result of monolayer culture for cell number expansion. This is associated with the development of an elongated shape, increased actin polymerization, development of stress fibres, and expression of contractile molecules. Given the changes in actin status with dedifferentiation, the hypothesis of this study was that adseverin, an actin severing and capping protein, plays a role in regulating chondrocyte phenotype and function. This study reports that serial passaging of articular chondrocytes in monolayer culture resulted in loss of adseverin protein expression as early as Day 14 of culture and remained repressed in Passage 2 (P2) cells. Knockdown of adseverin by siRNA in primary chondrocytes promoted an increase in cell size and an elongated shape, actin stress fibres, decreased G-/F-actin ratio, and increased number of actin-free barbed ends. The cells also showed increased expression of the contractile genes and proteins, vinculin and α-smooth muscle actin, and increased ability to contract collagen gels. These are all features of dedifferentiation. These effects were due to adseverin as adseverin overexpression following transfection of the green fluorescent protein-adseverin plasmid partially reversed all of these changes in P2 chondrocytes. Furthermore, sox9 and aggrecan chondrogenic gene expression was upregulated, and collagen type I genes expression was downregulated with adseverin overexpression. The change in aggrecan mRNA expression had functional consequence as these cells exhibited increased total proteoglycan synthesis. These findings demonstrate that adseverin regulates features indicative of redifferentiation in passaged articular chondrocytes through modulation of the actin cytoskeleton status and potentially may regulate the maintenance of phenotype in primary chondrocytes.

Effects of three potential anabolic disease-modifying osteoarthritis drugs – sprifermin, IGF1 and BMP7 – on matrix production and the phenotype of articular chondrocytes

Effects of three potential anabolic disease-modifying osteoarthritis drugs – sprifermin, IGF1 and BMP7 – on matrix production and the phenotype of articular chondrocytes

The Importance of Physioxia in Mesenchymal Stem Cell Chondrogenesis and the Mechanisms Controlling Its Response.

Articular cartilage covers the surface of synovial joints and enables joint movement. However, it is susceptible to progressive degeneration with age that can be accelerated by either previous joint injury or meniscectomy. This degenerative disease is known as osteoarthritis (OA) and it greatly affects the adult population. Cell-based tissue engineering provides a possible solution for treating OA at its earliest stages, particularly focal cartilage lesions. A candidate cell type for treating these focal defects are Mesenchymal Stem Cells (MSCs). However, present methods for differentiating these cells towards the chondrogenic lineage lead to hypertrophic chondrocytes and bone formation in vivo. Environmental stimuli that can stabilise the articular chondrocyte phenotype without compromising tissue formation have been extensively investigated. One factor that has generated intensive investigation in MSC chondrogenesis is low oxygen tension or physioxia (2–5% oxygen). In vivo articular cartilage resides at oxygen tensions between 1–4%, and in vitro results suggest that these conditions are beneficial for MSC expansion and chondrogenesis, particularly in suppressing the cartilage hypertrophy. This review will summarise the current literature regarding the effects of physioxia on MSC chondrogenesis with an emphasis on the pathways that control tissue formation and cartilage hypertrophy.

RGDSP functionalized carboxylated agarose as extrudable carriers for chondrocyte delivery

The limited potential of cartilage to regenerate itself has led to development of new strategies and biomaterials for cartilage tissue engineering and regenerative medicine. Although de novo strategies for cartilage repair have been realized, extrudable hydrogels that can be administered in minimally invasive manner while simultaneously supporting chondrogenic differentiation could lead to development of new systems to deliver cells to cartilage lesions. In this work, we explored the suitability of thermo-reversible, extrudable gels derived from carboxylated agarose for maintaining human articular chondrocyte (HAC) phenotype. Towards this objective, we have investigated the impact of hydrogel stiffness and presence of integrin-binding peptide sequence GGGGRGDSP on HAC differentiation potential. We discovered that stiffer hydrogels (5.8 kPa) are more efficient than softer counterparts (0.6 kPa) in promoting chondrogenesis. Interestingly, in GGGGRGDSP modified gels, a synergy between stiffness and RGD signaling led to enhanced expression of chondrogenic related genes (aggrecan, collagen type II and sox9). These findings were also supported by quantitative analysis of sulfated glycosaminoglycans. Since carboxylated agarose are highly suitable as bioink for 3D bioprinting, we propose that extrudable GGGGRGDSP-linked stiff carboxylated agarose as a medium for direct printing of chondrocyte into cartilage lesion.

Enpp1 inhibits ectopic joint calcification and maintains articular chondrocytes by repressing hedgehog signaling.

The differentiated phenotype of articular chondrocytes of synovial joints needs to be maintained throughout life. Disruption of the articular cartilage, frequently associated with chondrocyte hypertrophy and calcification, is a central feature in osteoarthritis (OA). However, the molecular mechanisms whereby phenotypes of articular chondrocytes are maintained and pathological calcification is inhibited remain poorly understood. Recently, the ecto-enzyme Enpp1, a suppressor of pathological calcification, was reported to be decreased in joint cartilage with OA in both human and mouse, and Enpp1 deficiency causes joint calcification. Here, we found that hedgehog (Hh) signaling activation contributes to ectopic joint calcification in the Enpp1-/- mice. In the Enpp1-/- joints, Hh signaling was upregulated. Further activation of Hh signaling by removing the patched 1 gene in the Enpp1-/- mice enhanced ectopic joint calcification, whereas removing Gli2 partially rescued the ectopic calcification phenotype. In addition, reduction of Gαs in the Enpp1-/- mice enhanced joint calcification, suggesting that Enpp1 inhibits Hh signaling and chondrocyte hypertrophy by activating Gαs-PKA signaling. Our findings provide new insights into the mechanisms underlying Enpp1 regulation of joint integrity.

Functional tissue-engineered microtissue derived from cartilage extracellular matrix for articular cartilage regeneration

We developed a promising cell carrier prepared from articular cartilage slices, designated cartilage extracellular matrix (ECM)-derived particles (CEDPs), through processes involving physical pulverization, size screening, and chemical decellularization. Rabbit articular chondrocytes (ACs) or adipose-derived stem cells (ASCs) rapidly attached to the surface of the CEDPs and proliferated with high cell viability under microgravity (MG) condition in a rotary cell culture system (RCCS) or static condition. Gene profiling results demonstrated that ACs expanded on CEDPs exhibited significantly enhanced chondrogenic phenotypes compared with monolayer culture, and that ASCs differentiated into a chondrogenic phenotype without the use of exogenous growth factors. Moreover, MG culture conditions in a RCCS bioreactor were superior to static culture conditions in terms of maintaining the chondrogenic phenotype of ACs and inducing ACS chondrogenesis. With prolonged expansion, functional microtissue aggregates of AC- or ASC-laden CEDPs were formed. Further, AC- or ASC-based microtissues were directly implanted in vivo to repair articular osteochondral defects in a rabbit model. Histological results, biomechanical evaluations, and radiographic assessments indicated that AC- and ASC-based microtissues displayed equal levels of superior hyaline cartilage repair, whereas the other two treatment groups, in which osteochondral defects were treated with CEDPs alone or fibrin glue, exhibited primarily fibrous tissue repair. These findings provide an alternative method for cell culture and stem cell differentiation and a promising strategy for constructing tissue-engineered cartilage microtissues for cartilage regeneration. Statement of SignificanceDespite the remarkable progress in cartilage tissue engineering, cartilage repair still remains elusive. In the present study, we developed a cell carrier, namely cartilage extracellular matrix-derived particles (CEDPs), for cell proliferation of articular chondrocytes (ACs) and adipose-derived stem cells (ASCs), which improved the maintenance of chondrogenic phenotype of ACs, and induced chondrogenesis of ASCs. Moreover, the functional microtissue aggregates of AC- or ASC-laden CEDPs induced equal levels of superior hyaline cartilage repair in a rabbit model. Therefore, our study demonstrated an alternative method for chondrocyte culture and stem cell differentiation, and a promising strategy for constructing tissue-engineered cartilage microtissues for in vivo articular cartilage repair and regeneration.

Human Articular Chondrocytes Retain Their Phenotype in Sustained Hypoxia While Normoxia Promotes Their Immunomodulatory Potential.

ObjectiveTo assess the phenotype of human articular chondrocytes cultured in normoxia (21% O2) or continuous hypoxia (2% O2).DesignChondrocytes were extracted from patients undergoing total knee replacement (n = 5) and cultured in ~21% (normoxic chondrocytes, NC) and 2% (hypoxic chondrocytes, HC) oxygen in both monolayer and 3-dimensional (3D) pellet culture and compared with freshly isolated chondrocytes (FC). Cells were assessed by flow cytometry for markers indicative of mesenchymal stromal cells (MSCs), chondrogenic-potency and dedifferentiation. Chondrogenic potency and immunomodulatory gene expression was assessed in NC and HC by reverse transcription quantitative polymerase chain reaction. Immunohistochemistry was used to assess collagen II production following 3D pellet culture.ResultsNC were positive (>97%, n = 5) for MSC markers, CD73, CD90, and CD105, while HC demonstrated <90% positivity (n = 4) and FC (n = 5) less again (CD73 and CD90 <20%; CD105 <40%). The markers CD166 and CD151, indicative of chondrogenic de-differentiation, were significantly higher on NC compared with HC and lowest on FC. NC also produced the highest levels of CD106 and showed the greatest levels of IDO expression, following interferon-γ stimulation, indicating immunomodulatory potential. NC produced the highest levels of CD49c (>60%) compared with HC and FC in which production was <2%. Hypoxic conditions upregulated expression of SOX9, frizzled-related protein (FRZB), fibroblast growth factor receptor 3 (FGFR3), and collagen type II (COL2A1) and downregulated activin receptor-like kinase 1 (ALK1) in 3 out of 4 patients compared with normoxic conditions for monolayer cells.ConclusionsHypoxic conditions encourage retention of a chondrogenic phenotype with some immunomodulatory potential, whereas normoxia promotes dedifferentiation of chondrocytes toward an MSC phenotype with loss of chondrogenic potency but enhanced immunomodulatory capacity.

Recent Insights into the Contribution of the Changing Hypertrophic Chondrocyte Phenotype in the Development and Progression of Osteoarthritis.

Osteoarthritis (OA) is an extremely prevalent age-related condition. The economic and societal burden due to the cost of symptomatic treatment, inability to work, joint replacement, and rehabilitation is huge and increasing. Currently, there are no effective medical therapies that delay or reverse the pathological manifestations of OA. Current treatment options are, without exception, focused on slowing down progression of the disease to postpone total joint replacement surgery for as long as possible and keeping the associated pain and joint immobility manageable. Alterations in the articular cartilage chondrocyte phenotype might be fundamental in the pathological mechanisms of OA development. In many ways, the changing chondrocyte phenotype in osteoarthritic cartilage resembles the process of endochondral ossification as seen, for instance, in developing growth plates. However, the relative contribution of endochondral ossification to the changing chondrocyte phenotype in the development and progression of OA remains poorly described. In this review, we will discuss the current knowledge regarding the cartilage endochondral phenotypic changes occurring during OA development and progression, as well as the molecular and environmental effectors driving these changes. Understanding how these molecular mechanisms determine the chondrocyte cell fate in OA will be essential in enabling cartilage regenerative approaches in future treatments of OA.

Non-contact Coculture Reveals a Comprehensive Response of Chondrocytes Induced by Mesenchymal Stem Cells Through Trophic Secretion.

Coculture between mesenchymal stem cells (MSCs) and chondrocytes has significant implications in cartilage regeneration. However, a conclusive understanding remains elusive. Previously, we reported that rabbit bone marrow-derived MSCs (rbBMSCs) could downregulate the differentiated phenotype of rabbit articular chondrocytes (rbACs) in a non-contact coculture system for the first time. In the present study, a systemic investigation was performed to understand the biological characteristics of chondrocytes in coculture with MSCs. Firstly, cells (MSCs and chondrocytes) from different origins were cocultured in transwell system. Different chondrocytes, when cocultured with different MSCs respectively, consistently demonstrated stimulated proliferation, transformed morphology and declined glycosaminoglycan secretion of chondrocytes. Next, cell surface molecules and the global gene expression of rbACs were characterized. It was found that cocultured rbACs showed a distinct surface molecule profile and global gene expression compared to both dedifferentiated rbACs and rbBMSCs. In the end, cocultured rbACs were passaged and induced to undergo the chondrogenic redifferentiation. Better growth and chondrogenesis ability were confirmed compared with control cells without coculture. Together, chondrocytes display comprehensive changes in coculture with MSCs and the cocultured rbACs are beneficial for cartilage repair.

The changing role of TGFβ in healthy, ageing and osteoarthritic joints.

Transforming growth factor-β (TGFβ) is a pleiotropic cytokine that is important in the regulation of joint homeostasis and disease. TGFβ signalling is induced by loading and has an important function in maintaining the differentiated phenotype of articular chondrocytes. Concentrations of active TGFβ differ greatly between healthy and osteoarthritic joints, being low in healthy joints and high in osteoarthritic joints, leading to the activation of different signalling pathways in joint cells. The characteristic pathology of osteoarthritic joints, such as cartilage damage, osteophyte formation and synovial fibrosis, seems to be stimulated or even caused by the high levels of active TGFβ, in combination with altered chondrocyte signalling pathways (which are also observed in ageing joints). In this Review, the changing role of TGFβ in normal joint homeostasis, ageing and osteoarthritis is discussed: TGFβ counteracts pathological changes in a young healthy joint, alters its signalling during ageing and is a driving force of pathology in osteoarthritic joints.

Generation of Immortalized Equine Chondrocytes With Inducible Sox9 Expression Allows Control of Hypertrophic Differentiation.

Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower expression of chondrocyte specific collagen II mRNA (P < 0.0001) and up regulation of the hypertrophic marker collagen X (P < 0.0001) in three dimensional cultures. A Col2a1 promoter driven GFP reporter was constructed for real time monitoring of chondrogenic differentiation and a significant increase in promoter activation was observed in cultures treated with the growth factor TGFβ-3 (P < 0.05). To recapitulate the native articular chondrocyte phenotype we further transduced large T antigen immortalized chondrocytes with lentiviral vectors allowing either constitutive or doxycycline inducible expression of Sox9. In 3D cultures, the Sox9 over-expressing chondrocytes secreted significantly higher levels of extracellular matrix polysaccharide glycosaminoglycan (P < 0.05), while up-regulating collagen II and Aggrecan mRNA (P < 0.05) in both expression systems with a similar patterns observed with imunohistochemical staining. High levels of collagen X mRNA and protein were maintained with constitutive sox9 reflecting hypetrophic differentiation but significantly lower expression could be achieved with inducible Sox9. In conclusion, immortalization of equine chondrocytes results in stable proliferation but a reduction of chondrogenic potential whilst modulation of sox9 expression enabled control of hypertrophic characteristics. J. Cell. Biochem. 118: 1201-1215, 2017. © 2016 Wiley Periodicals, Inc.

Synthesis, Biological Evaluation, and Docking Studies of a Novel Sulfonamido-Based Gallate as Pro-Chondrogenic Agent for the Treatment of Cartilage

Gallic acid (GA) and its derivatives are anti-inflammatory agents and are reported to have potent effects on Osteoarthritis (OA) treatment. Nonetheless, it is generally accepted that the therapeutic effect and biocompatibility of GA is much weaker than its esters due to the high hydrophilicity. The therapeutic effect of GA on OA could be improved if certain structural modifications were made to increase its hydrophobicity. In this study, a novel sulfonamido-based gallate was synthesized by bonding sulfonamide with GA, and its biological evaluations on OA were investigated. Results show that 5-[4-(Pyrimidin-2-ylsulfamoylphenyl)]-carbamoyl-benzene-1,2,3-triyl triacetate (HAMDC) was able to reverse the effects induced by Interleukin-1 (IL-1) stimulation, and it also had a great effect on chondro-protection via promoting cell proliferation and maintaining the phenotype of articular chondrocytes, as well as enhancing synthesis of cartilage specific markers such as aggrecan, collagen II and Sox9. Furthermore, a docking study showed that HAMDC fits into the core of the active site of a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), which provides an explanation for its activity and selectivity.

Context-specific protection of TGFα null mice from osteoarthritis.

Transforming growth factor alpha (TGFα) is a growth factor involved in osteoarthritis (OA). TGFα induces an OA-like phenotype in articular chondrocytes, by inhibiting matrix synthesis and promoting catabolic factor expression. To better understand TGFα’s potential as a therapeutic target, we employed two in vivo OA models: (1) post-traumatic and (2) aging related OA. Ten-week old and six-month old male Tgfa null mice and their heterozygous (control) littermates underwent destabilization of the medial meniscus (DMM) surgery. Disease progression was assessed histologically using the Osteoarthritis Research Society International (OARSI) scoring system. As well, spontaneous disease progression was analyzed in eighteen-month-old Tgfa null and heterozygous mice. Ten-week old Tgfa null mice were protected from OA progression at both seven and fourteen weeks post-surgery. No protection was seen however in six-month old null mice after DMM surgery, and no differences were observed between genotypes in the aging model. Thus, young Tgfa null mice are protected from OA progression in the DMM model, while older mice are not. In addition, Tgfa null mice are equally susceptible to spontaneous OA development during aging. Thus, TGFα might be a valuable therapeutic target in some post-traumatic forms of OA, however its role in idiopathic disease is less clear.

Unloading results in rapid loss of TGFβ signaling in articular cartilage: role of loading-induced TGFβ signaling in maintenance of articular chondrocyte phenotype?

Recently it was shown that loading of articular cartilage explants activates TGFβ signaling. Here we investigated if invivo chondrocytes express permanently high TGFβ signaling, and the consequence of the loss of compressive loading-mediated TGFβ signaling on chondrocyte function and phenotype. Bovine articular cartilage explants were collected within 10min post mortem and stained immediately and after 30, 60 and 360min for phosphorylated-Smad2, indicating active TGFβ signaling. Explants were unloaded for 48h and subsequently repeatedly loaded with a compressive load of 3MPa. In addition, explants were cultured unloaded for 2 weeks and the effect of loading or exogenous TGFβ on proteoglycan level and chondrocyte phenotype (Col10a1 mRNA expression) was analyzed. Unloading of articular cartilage results in rapid loss of TGFβ signaling while subsequent compressive loading swiftly restored this. Loading and exogenous TGFβ enhanced expression of TGFβ1 and ALK5. Unloading of explants for 2 weeks resulted in proteoglycan loss and increased Col10a1 expression. Both loading and exogenous TGFβ inhibited elevated Col10a1 expression but not proteoglycan loss. Our data might imply that invivo regular physiological loading of articular cartilage leads to enduring TGFβ signaling and TGFβ-induced gene expression. We propose a hypothetical model in which loading activates a self-perpetuating system that prevents hypertrophic differentiation of chondrocytes and is crucial for cartilage homeostasis.

Chondro-Protective and Antiarthritic Effects of Sulfonamido-Based Gallate-ZXHA-TC in Vitro and in Vivo.

The effects of gallic acid (GA) on arthritis are limited by weak antioxidant effects and inferior biological properties of GA. We recently described a new series of synthesized GA derivatives by coupling with sulfonamides. Among these analogs, a novel compound synthesized from GA and sulfadimoxine (SDM) named ZXHA-TC exhibited the most robust anti-inflammatory potential. In this current study, the chondro-protective and antiarthritic effects of ZXHA-TC were investigated both in vitro and in vivo. In the in vitro study, ZXHA-TC exerted chondro-protective effects as evidenced by promoting cell proliferation and the maintaining of the phenotype of articular chondrocytes treated with interleukin-1-beta (IL-1β). The potential of ZXHA-TC to slow the progress of osteoarthritis (OA) was suggested by a reduction in matrix metalloproteinases (MMPs) and the up-regulation of the tissue inhibitor of metalloproteinase-1 (TIMP-1). In a rabbit anterior cruciate ligament transaction (ACLT) model of OA, ZXHA-TC exerted a protective effect on arthritis as assessed by macroscopic scores, histological, qRT-PCR, and immunohistochemical analyses. The effects of ZXHA-TC on inhibiting the production of inflammatory mediators in OA may be mediated partly by the suppression of the PI3K/AKT pathway or MAPK cascades, leading to NF-κB inactivation. Thus, this study indicates that ZXHA-TC may be developed as a potential therapeutic agent for OA.

DKK1 AND FRZB are necessary for chondrocyte (RE)differentiation and prevention of cell hypertrophy in 3D cultures of human chondrocytes and human mesenchymal stem cells

Purpose: Autologous chondrocytes and mesenchymal stem cells (MSCs) in either monoculture or co-culture, for the enhancement of chondrogenesis, show great promise for treatment of cartilage disorders. However, maintaining the chondrogenic phenotype and avoiding hypertrophy of chondrogenic differentiating MSCs remain a big challenge in these cell-based strategies. Previously, we identified Dickkopf 1 homolog (DKK1) and frizzled-related protein (FRZB) as key factors in controlling the articular chondrocyte phenotype. DKK1 and FRZB are naturally occurring antagonists of the WNT signaling pathway. The expression of DKK1 and FRZB is low in cultured human mesenchymal stem cells (hMSCs) and high in cultured human articular chondrocytes (hChs). We have previously shown that addition of either DKK1 or FRZB blocks hypertrophy in chondrogenically differentiating hMSCs and that expression of DKK1 and FRZB is lost in OA. We hypothesized that DKK1 and FRZB are necessary for chondrogenesis and for preventing cell hypertrophy in chondrocyte terminal differentiation. Methods: To block DKK1 and FRZB the variable domain of single chain heavy chain only antibodies (VHH) was used to neutralize DKK1 and FRZB. We tested our hypothesis using 3D pellet cultures of three relevant human cell based systems: isolated hChs, isolated hMSCs and co-culture of hChs with hMSCs. The effects were analyzed using real-time quantitative polymerase chain reaction (qPCR), histology, and immunohistochemistry (IHC). Results: In the presence of DKK1 and FRZB neutralizing VHH, glycosaminoglycan (GAG) deposition and Collagen type II staining were significantly reduced in redifferentiating chondrocytes (Figure 1 A and B) and in chondrogenic differentiating hMSCs, indicating loss of chondrogenic potential. Upon neutralization of DKK1 and FRZB in co-cultures, the cells in pellets showed hypertrophic differentiation as indicated by matrix mineralization, apoptosis and significantly increased expression of the hypertrophic markers, COL10A1, MMP13 and RUNX2. This is indicative of terminal differentiation. Conclusions: DKK1 and FRZB are necessary for multiple steps during chondrogenesis: first DKK1 and FRZB are necessary for the initial steps of chondrogenic differentiation of hMSCs and in redifferentiation of cultured chondrocytes, and secondly in preventing chondrocyte hypertrophy in terminal differentiation of articular chondrocytes (Figure 2).Figure 2. DKK1 and FRZB are necessary for multiple steps during chondrogenesis to ensure chondrogenesis and to prevent cell hypertrophy in terminal differentiation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Heparin‐based self‐assembling peptide scaffold reestablish chondrogenic phenotype of expanded de‐differentiated human chondrocytes

The use of chondrocytes in cell-based therapies for cartilage lesions are limited by quantity and, therefore, require an in vitro expansion. As monolayer culture leads to de-differentiation, different culture techniques are currently under development to recover chondrocyte phenotype after cell expansion. In the present work, we studied the capacity of the bimolecular heparin-based self-assembling peptide scaffold (RAD16-I) as a three-dimensional (3D) culture system to foster reestablishment of chondrogenic phenotype of de-differentiated human Articular Chondrocytes (AC). The culture was performed in a serum-free medium under control and chondrogenic induction and good viability results were observed after 4 weeks of culture in both conditions. Cells changed their morphology to a more elongated shape and established a cellular network that induced the condensation of the constructs in the case of chondrogenic medium, leading to a compacted structure with improved mechanical properties. Specific extracellular matrix (ECM) proteins of mature cartilage, such as collagen type II and aggrecan were up-regulated under chondrogenic medium and significantly enhanced with the presence of heparin in the scaffold. 3D constructs became highly stained with toluidine blue dye after 4 weeks of culture, indicating the presence of synthetized proteoglycans (PGs) by the cells. Interestingly, the full viscoelastic behavior was closely related to that found in chicken native cartilage. Altogether, the results suggest that the 3D culture model described can help de-differentiated human chondrocytes to recover its cartilage phenotype. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1694-1706, 2016.

Derepression of MicroRNA-138 Contributes to Loss of the Human Articular Chondrocyte Phenotype.

To investigate the function of microRNA-138 (miR-138) in human articular chondrocytes (HACs). The expression of miR-138 in intact cartilage and cultured chondrocytes and the effects of miR-138 overexpression on chondrocyte marker genes were investigated. Targets of miR-138 relevant to chondrocytes were identified and verified by overexpression of synthetic miRNA mimics and inhibitors, luciferase assays, chromatin immunoprecipitation, and RNA immunoprecipitation of native argonaute 2, using quantitative polymerase chain reaction, Western blotting, and luciferase assays. Expression levels of miR-138 were maintained at relatively low levels in intact human cartilage but were greatly increased upon loss of the differentiated phenotype in culture, with a concomitant decrease in the major cartilage extracellular matrix component COL2A1. We showed that miR-138 is able to repress the expression of COL2A1 by directly targeting Sp-1 and hypoxia-inducible factor 2α (HIF-2α), 2 transcription factors that are essential for COL2A1 transcription. We further demonstrated a direct association of these targets with miR-138 in the RNA-induced silencing complex and confirmed binding of Sp-1 to the COL2A1 promoter region in HACs. We propose that an evolutionary pressure helps to suppress expression levels of miR-138 in human cartilage, thus enabling expression of appropriate tissue-specific matrix genes. Inhibition of miR-138 may serve as a potential therapeutic strategy to maintain the chondrocyte phenotype or reduce the progression of dedifferentiation in cultured HACs.

Effects of Cytoskeleton Modulation on the Phenotype of Articular Chondrocytes in Monolayer and Pellet Culture

Effects of Cytoskeleton Modulation on the Phenotype of Articular Chondrocytes in Monolayer and Pellet Culture