Anisotropic Chitosan Scaffolds Generated by Electrostatic Flocking Combined with Alginate Hydrogel Support Chondrogenic Differentiation

The replacement of damaged or degenerated articular cartilage tissue remains a challenge, as this non-vascularized tissue has a very limited self-healing capacity. Therefore, tissue engineering (TE) of cartilage is a promising treatment option. Although significant progress has been made in recent years, there is still a lack of scaffolds that ensure the formation of functional cartilage tissue while meeting the mechanical requirements for chondrogenic TE. In this article, we report the application of flock technology, a common process in the modern textile industry, to produce flock scaffolds made of chitosan (a biodegradable and biocompatible biopolymer) for chondrogenic TE. By combining an alginate hydrogel with a chitosan flock scaffold (CFS+ALG), a fiber-reinforced hydrogel with anisotropic properties was developed to support chondrogenic differentiation of embedded human chondrocytes. Pure alginate hydrogels (ALG) and pure chitosan flock scaffolds (CFS) were studied as controls. Morphology of primary human chondrocytes analyzed by cLSM and SEM showed a round, chondrogenic phenotype in CFS+ALG and ALG after 21 days of differentiation, whereas chondrocytes on CFS formed spheroids. The compressive strength of CFS+ALG was higher than the compressive strength of ALG and CFS alone. Chondrocytes embedded in CFS+ALG showed gene expression of chondrogenic markers (COL II, COMP, ACAN), the highest collagen II/I ratio, and production of the typical extracellular matrix such as sGAG and collagen II. The combination of alginate hydrogel with chitosan flock scaffolds resulted in a scaffold with anisotropic structure, good mechanical properties, elasticity, and porosity that supported chondrogenic differentiation of inserted human chondrocytes and expression of chondrogenic markers and typical extracellular matrix.     

Expression of Transient Receptor Potential Vanilloid (TRPV) Channels in Different Passages of Articular Chondrocytes

Ion channels play important roles in chondrocyte mechanotransduction. The transient receptor potential vanilloid (TRPV) subfamily of ion channels consists of six members. TRPV1-4 are temperature sensitive calcium-permeable, relatively non-selective cation channels whereas TRPV5 and TRPV6 show high selectivity for calcium over other cations. In this study we investigated the effect of time in culture and passage number on the expression of TRPV4, TRPV5 and TRPV6 in articular chondrocytes isolated from equine metacarpophalangeal joints. Polyclonal antibodies raised against TRPV4, TRPV5 and TRPV6 were used to compare the expression of these channels in lysates from first expansion chondrocytes (P0) and cells from passages 1-3 (P1, P2 and P3) by western blotting. TRPV4, TRPV5 and TRPV6 were expressed in all passages examined. Immunohistochemistry and immunofluorescence confirmed the presence of these channels in sections of formalin fixed articular cartilage and monolayer cultures of methanol fixed P2 chondrocytes. TRPV5 and TRPV6 were upregulated with time and passage in culture suggesting that a shift in the phenotype of the cells in monolayer culture alters the expression of these channels. In conclusion, several TRPV channels are likely to be involved in calcium signaling and homeostasis in chondrocytes.     

The Effects of Tgfb1 and Csf3 on Chondrogenic Differentiation of iPS Cells in 2D and 3D Culture Environment

Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor β1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.     

Notch信号通路相关分子在脊椎生长过程中的差异表达及其对软骨细胞分化的调控作用

目的探讨Notch信号通路相关分子在脊椎生长过程中的差异表达及其对软骨细胞分化的调控作用。方法采用RT-PCR检测各不同发育年龄段小鼠椎间盘Notch信号分子mRNA的表达情况,筛选调控脊椎正常生长发育的重要信号分子,并采用免疫组化法进行验证。用重组腺病毒介导Dll-1、Jag-1、骨形态发生蛋白-2(bone morphogeneticprotein-2,BMP-2)在髓核(nucleus pulposus,NP)细胞中过表达,RT-PCR检测软骨细胞特征性标志物蛋白聚糖(aggrecan,ACAN)和Ⅱ型胶原(collagen typeⅡ,COL2A1)的表达,甲苯胺蓝染色检测软骨细胞基质分泌情况。结果小鼠脊椎生长发育过程中,Notch信号分子mRNA的表达总体呈下降趋势,其中Dll-1、Dll-3、Jag-1下降趋势尤其显著;Dll-1和Jag-1广泛表达于软骨细胞胞膜,随着小鼠年龄的增大,二者在椎间盘中的表达显著下降;BMP-2过表达可促进NP细胞成软骨分化,ACAN和COL2A1表达升高,甲苯胺蓝染色增强;Dll-1、Jag-1过表达可显著抑制BMP-2诱导的成软骨分化效应。结论 Notch信号分子,尤其是Dll-1、Jag-1对正常脊椎生长及椎间盘软骨细胞的成熟分化具有明显的负性调节作用。     

Physosmotic Induction of Chondrogenic Maturation Is TGF-β Dependent and Enhanced by Calcineurin Inhibitor FK506

Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-β-inducible protein TGFBI, as well as chondrogenic (SOX9, Col2) and terminal differentiation markers (e.g., Col10) were affected by osmolarity. Particularly, the expression of minor collagens (e.g., Col9, Col11) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-β receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies.     

Role of Insulin-Transferrin-Selenium in Auricular Chondrocyte Proliferation and Engineered Cartilage Formation in Vitro

The goal of this study is to determine the effects of Insulin-Transferrin-Selenium (ITS) on proliferation of auricular chondrocytes and formation of engineered cartilage in vitro. Pig auricular monolayer chondrocytes and chondrocyte pellets were cultured in media containing 1% ITS at different concentrations of fetal bovine serum (FBS, 10%, 6%, 2%, 0%), or 10% FBS alone as a control for four weeks. Parameters including cell proliferation in monolayer, wet weight, collagen type I/II/X (Col I, II, X) and glycosaminoglycan (GAG) expression, GAG content of pellets and gene expression associated with cartilage formation/dedifferentiation (lost cartilage phenotype)/hypertrophy within the chondrocyte pellets were assessed. The results showed that chondrocytes proliferation rates increased when FBS concentrations increased (2%, 6%, 10% FBS) in ITS supplemented groups. In addition, 1% ITS plus 10% FBS significantly promoted cell proliferation than 10% FBS alone. No chondrocytes grew in ITS alone medium. 1% ITS plus 10% FBS enhanced cartilage formation in terms of size, wet weight, cartilage specific matrices, and homogeneity, compared to 10% FBS alone group. Furthermore, ITS prevented engineered cartilage from dedifferentiation (i.e., higher index of Col II/Col I mRNA expression and expression of aggrecan) and hypertrophy (i.e., lower mRNA expression of Col X and MMP13). In conclusion, our results indicated that ITS efficiently enhanced auricular chondrocytes proliferation, retained chondrogenic phenotypes, and promoted engineered cartilage formation when combined with FBS, which is potentially used as key supplementation in auricular chondrocytes and engineered cartilage culture.     

Caspase-1 Inhibition Impacts the Formation of Chondrogenic Nodules,and the Expression of Markers Related to Osteogenic Differentiation and Lipid Metabolism

Caspase-1, as the main pro-inflammatory cysteine protease, was investigated mostly with respect to inflammation-related processes. Interestingly, caspase-1 was identified as being involved in lipid metabolism, which is extremely important for the proper differentiation of chondrocytes. Based on a screening investigation, general caspase inhibition impacts the expression of Cd36 in chondrocytes, the fatty acid translocase with a significant impact on lipid metabolism. However, the engagement of individual caspases in the effect has not yet been identified. Therefore, the hypothesis that caspase-1 might be a candidate here appears challenging. The primary aim of this study thus was to find out whether the inhibition of caspase-1 activity would affect Cd36 expression in a chondrogenic micromass model. The expression of Pparg, a regulator Cd36, was examined as well. In the caspase-1 inhibited samples, both molecules were significantly downregulated. Notably, in the treated group, the formation of the chondrogenic nodules was apparently disrupted, and the subcellular deposition of lipids and polysaccharides showed an abnormal pattern. To further investigate this observation, the samples were subjected to an osteogenic PCR array containing selected markers related to cartilage/bone cell differentiation. Among affected molecules, Bmp7 and Gdf10 showed a significantly increased expression, while Itgam, Mmp9, Vdr, and Rankl decreased. Notably, Rankl is a key marker in bone remodeling/homeostasis and thus is a target in several treatment strategies, including a variety of fatty acids, and is balanced by its decoy receptor Opg (osteoprotegerin). To evaluate the effect of Cd36 downregulation on Rankl and Opg, Cd36 silencing was performed using micromass cultures. After Cd36 silencing, the expression of Rankl was downregulated and Opg upregulated, which was an inverse effect to caspase-1 inhibition (and Cd36 upregulation). These results demonstrate new functions of caspase-1 in chondrocyte differentiation and lipid metabolism-related pathways. The effect on the Rankl/Opg ratio, critical for bone maintenance and pathology, including osteoarthritis, is particularly important here as well.     

Establishment of a New Device for Electrical Stimulation of Non-Degenerative Cartilage Cells In Vitro

In cell-based therapies for cartilage lesions, the main problem is still the formation of fibrous cartilage, caused by underlying de-differentiation processes ex vivo. Biophysical stimulation is a promising approach to optimize cell-based procedures and to adapt them more closely to physiological conditions. The occurrence of mechano-electrical transduction phenomena within cartilage tissue is physiological and based on streaming and diffusion potentials. The application of exogenous electric fields can be used to mimic endogenous fields and, thus, support the differentiation of chondrocytes in vitro. For this purpose, we have developed a new device for electrical stimulation of chondrocytes, which operates on the basis of capacitive coupling of alternating electric fields. The reusable and sterilizable stimulation device allows the simultaneous use of 12 cavities with independently applicable fields using only one main supply. The first parameter settings for the stimulation of human non-degenerative chondrocytes, seeded on collagen type I elastin-based scaffolds, were derived from numerical electric field simulations. Our first results suggest that applied alternating electric fields induce chondrogenic re-differentiation at the gene and especially at the protein level of human de-differentiated chondrocytes in a frequency-dependent manner. In future studies, further parameter optimizations will be performed to improve the differentiation capacity of human cartilage cells.     

Silk Fiber-Reinforced Hyaluronic Acid-Based Hydrogel for Cartilage Tissue Engineering

A continuing challenge in cartilage tissue engineering for cartilage regeneration is the creation of a suitable synthetic microenvironment for chondrocytes and tissue regeneration. The aim of this study was to develop a highly tunable hybrid scaffold based on a silk fibroin matrix (SM) and a hyaluronic acid (HA) hydrogel. Human articular chondrocytes were embedded in a porous 3-dimensional SM, before infiltration with tyramine modified HA hydrogel. Scaffolds were cultured in chondropermissive medium with and without TGF-β1. Cell viability and cell distribution were assessed using CellTiter-Blue assay and Live/Dead staining. Chondrogenic marker expression was detected using qPCR. Biosynthesis of matrix compounds was analyzed by dimethylmethylene blue assay and immuno-histology. Differences in biomaterial stiffness and stress relaxation were characterized using a one-step unconfined compression test. Cell morphology was investigated by scanning electron microscopy. Hybrid scaffold revealed superior chondro-inductive and biomechanical properties compared to sole SM. The presence of HA and TGF-β1 increased chondrogenic marker gene expression and matrix deposition. Hybrid scaffolds offer cytocompatible and highly tunable properties as cell-carrier systems, as well as favorable biomechanical properties.     

Protective Effects of a Hyaluronan-Binding Peptide (P15-1) on Mesenchymal Stem Cells in an Inflammatory Environment

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1β)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.     

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.     

The Involvement of Mutual Inhibition of ERK and mTOR in PLCγ1-Mediated MMP-13 Expression in Human Osteoarthritis Chondrocytes

The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA) pathogenesis currently remains controversial. Our previous study shows that PLCγ1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLCγ1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLCγ1, p-PLCγ1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLCγ1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLCγ1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLCγ1 on MMP-13 may be partly attributed to the inhibition of the PLCγ1/mTOR axis on the PLCγ1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLCγ1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy.     

Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process

Monosodium urate (MSU) crystals, which are highly precipitated in the joint cartilage, increase the production of cartilage-degrading enzymes and pro-inflammatory mediators in cartilage, thereby leading to gouty inflammation and joint damage. In this study, we investigated the effect of MSU crystals on the viability of human articular chondrocytes and the mechanism of MSU crystal-induced chondrocyte death. MSU crystals significantly decreased the viability of primary chondrocytes in a time- and dose-dependent manner. DNA fragmentation was observed in a culture medium of MSU crystal-treated chondrocytes, but not in cell lysates. MSU crystals did not activate caspase-3, a marker of apoptosis, compared with actinomycin D and TNF-α-treated cells. MSU crystals did not directly affect the expression of endoplasmic reticulum (ER) stress markers at the mRNA and protein levels. However, MSU crystals significantly increased the LC3-II level in a time-dependent manner, indicating autophagy activation. Moreover, MSU crystal-induced autophagy and subsequent chondrocyte death were significantly inhibited by 3-methyladenine, a blocker of autophagosomes formation. MSU crystals activated autophagy via inhibition of phosporylation of the Akt/mTOR signaling pathway. These results demonstrate that MSU crystals may cause the death of chondrocytes through the activation of the autophagic process rather than apoptosis or ER stress.     

Urocortin-1 Is Chondroprotective in Response to Acute Cartilage Injury via Modulation of Piezo1

Post-traumatic OA (PTOA) is often triggered by injurious, high-impact loading events which result in rapid, excessive chondrocyte cell death and a phenotypic shift in residual cells toward a more catabolic state. As such, the identification of a disease-modifying OA drug (DMOAD) that can protect chondrocytes from death following impact injury, and thereby prevent cartilage degradation and progression to PTOA, would offer a novel intervention. We have previously shown that urocortin-1 (Ucn) is an essential endogenous pro-survival factor that protects chondrocytes from OA-associated pro-apoptotic stimuli. Here, using a drop tower PTOA-induction model, we demonstrate the extent of Ucn’s chondroprotective role in cartilage explants exposed to excessive impact load. Using pathway-specific agonists and antagonists, we show that Ucn acts to block load-induced intracellular calcium accumulation through blockade of the non-selective cation channel Piezo1 rather than TRPV4. This protective effect is mediated primarily through the Ucn receptor CRF-R1 rather than CRF-R2. Crucially, we demonstrate that the chondroprotective effect of Ucn is maintained whether it is applied pre-impact or post-impact, highlighting the potential of Ucn as a novel DMOAD for the prevention of injurious impact overload-induced PTOA.     

Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage

The composition and organisation of the extracellular matrix (ECM), particularly the pericellular matrix (PCM), in articular cartilage is critical to its biomechanical functionality; the presence of proteoglycans such as aggrecan, entrapped within a type II collagen fibrillar network, confers mechanical resilience underweight-bearing. Furthermore, components of the PCM including type VI collagen, perlecan, small leucine-rich proteoglycans—decorin and biglycan—and fibronectin facilitate the transduction of both biomechanical and biochemical signals to the residing chondrocytes, thereby regulating the process of mechanotransduction in cartilage. In this review, we summarise the literature reporting on the bidirectional reciprocity of the ECM in chondrocyte mechano-signalling and articular cartilage homeostasis. Specifically, we discuss studies that have characterised the response of articular cartilage to mechanical perturbations in the local tissue environment and how the magnitude or type of loading applied elicits cellular behaviours to effect change. In vivo, including transgenic approaches, and in vitro studies have illustrated how physiological loading maintains a homeostatic balance of anabolic and catabolic activities, involving the direct engagement of many PCM molecules in orchestrating this slow but consistent turnover of the cartilage matrix. Furthermore, we document studies characterising how abnormal, non-physiological loading including excessive loading or joint trauma negatively impacts matrix molecule biosynthesis and/or organisation, affecting PCM mechanical properties and reducing the tissue’s ability to withstand load. We present compelling evidence showing that reciprocal engagement of the cells with this altered ECM environment can thus impact tissue homeostasis and, if sustained, can result in cartilage degradation and onset of osteoarthritis pathology. Enhanced dysregulation of PCM/ECM turnover is partially driven by mechanically mediated proteolytic degradation of cartilage ECM components. This generates bioactive breakdown fragments such as fibronectin, biglycan and lumican fragments, which can subsequently activate or inhibit additional signalling pathways including those involved in inflammation. Finally, we discuss how bidirectionality within the ECM is critically important in enabling the chondrocytes to synthesise and release PCM/ECM molecules, growth factors, pro-inflammatory cytokines and proteolytic enzymes, under a specified load, to influence PCM/ECM composition and mechanical properties in cartilage health and disease.     

Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids Prevent Mesenchymal Stromal-Cell-Derived Chondrocyte Hypertrophy

Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-β, 1–14 days) and thyroid hormone hypertrophic medium (T3, 15–28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-β2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs’ therapeutic effect on cartilage regeneration and hypertrophy suppression.     

Biological and Chemical Removal of Primary Cilia Affects Mechanical Activation of Chondrogenesis Markers in Chondroprogenitors and Hypertrophic Chondrocytes

Chondroprogenitors and hypertrophic chondrocytes, which are the first and last stages of the chondrocyte differentiation process, respectively, are sensitive to mechanical signals. We hypothesize that the mechanical sensitivity of these cells depends on the cell surface primary cilia. To test this hypothesis, we removed the primary cilia by biological means with transfection with intraflagellar transport protein 88 (IFT88) siRNA or by chemical means with chloral hydrate treatment. Transfection of IFT88 siRNA significantly reduced the percentage of ciliated cells in both chondroprogenitor ATDC5 cells as well as primary hypertrophic chondrocytes. Cyclic loading (1 Hz, 10% matrix deformation) of ATDC5 cells in three-dimensional (3D) culture stimulates the mRNA levels of chondrogenesis marker Type II collagen (Col II), hypertrophic chondrocyte marker Type X collagen (Col X), and a molecular regulator of chondrogenesis and chondrocyte hypertrophy bone morphogenetic protein 2 (BMP-2). The reduction of ciliated chondroprogenitors abolishes mechanical stimulation of Col II, Col X, and BMP-2. In contrast, cyclic loading stimulates Col X mRNA levels in hypertrophic chondrocytes, but not those of Col II and BMP-2. Both biological and chemical reduction of ciliated hypertrophic chondrocytes reduced but failed to abolish mechanical stimulation of Col X mRNA levels. Thus, primary cilia play a major role in mechanical stimulation of chondrogenesis and chondrocyte hypertrophy in chondroprogenitor cells and at least a partial role in hypertrophic chondrocytes.     

Gremlin-1 and BMP-4 Overexpressed in Osteoarthritis Drive an Osteochondral-Remodeling Program in Osteoblasts and Hypertrophic Chondrocytes

Osteoarthritis (OA) is a whole joint disease characterized by an important remodeling of the osteochondral junction. It includes cartilage mineralization due to chondrocyte hypertrophic differentiation and bone sclerosis. Here, we investigated whether gremlin-1 (Grem-1) and its BMP partners could be involved in the remodeling events of the osteochondral junction in OA. We found that Grem-1, BMP-2, and BMP-4 immunostaining was detected in chondrocytes from the deep layer of cartilage and in subchondral bone of knee OA patients, and was positively correlated with cartilage damage. ELISA assays showed that bone released more Grem-1 and BMP-4 than cartilage, which released more BMP-2. In vitro experiments evidenced that compression stimulated the expression and the release of Grem-1 and BMP-4 by osteoblasts. Grem-1 was also overexpressed during the prehypertrophic to hypertrophic differentiation of murine articular chondrocytes. Recombinant Grem-1 stimulated Mmp-3 and Mmp-13 expression in murine chondrocytes and osteoblasts, whereas recombinant BMP-4 stimulated the expression of genes associated with angiogenesis (Angptl4 and osteoclastogenesis (Rankl and Ccl2). In conclusion, Grem-1 and BMP-4, whose expression at the osteochondral junction increased with OA progression, may favor the pathological remodeling of the osteochondral junction by inducing a catabolic and tissue remodeling program in hypertrophic chondrocytes and osteoblasts.     

Inhibition of Spinal TRPV1 Reduces NMDA Receptor 2B Phosphorylation and Produces Anti-Nociceptive Effects in Mice with Inflammatory Pain

Transient receptor potential vanilloid 1 (TRPV1) has been implicated in peripheral inflammation and is a mediator of the inflammatory response to various noxious stimuli. However, the interaction between TRPV1 and N-methyl-D-aspartate (NMDA) receptors in the regulation of inflammatory pain remains poorly understood. This study aimed to investigate the analgesic effects of intrathecal administration of capsazepine, a TRPV1 antagonist, on carrageenan-induced inflammatory pain in mice and to identify its interactions with NMDA receptors. Inflammatory pain was induced by intraplantar injection of 2% carrageenan in male ICR mice. To investigate the analgesic effects of capsazepine, pain-related behaviors were evaluated using von Frey filaments and a thermal stimulator placed on the hind paw. TRPV1 expression and NMDA receptor phosphorylation in the spinal cord and glutamate concentration in the spinal cord and serum were measured. Intrathecal treatment with capsazepine significantly attenuated carrageenan-induced mechanical allodynia and thermal hyperalgesia. Moreover, carrageenan-enhanced glutamate and phosphorylation of NMDA receptor subunit 2B in the spinal cord were suppressed by capsazepine administration. These results indicate that TRPV1 and NMDA receptors in the spinal cord are associated with inflammatory pain transmission, and inhibition of TRPV1 may reduce inflammatory pain via NMDA receptors.