Modeling the Effect of Annulus Fibrosus Stiffness on the Stressed State of a Vertebral L1 Body and Nucleus Pulposus.

The investigation examines the transference of stiffness from intervertebral discs (IVDs) to the lumbar body of the L1 vertebra and the interactions among adjacent tissues. A computational model of the vertebra was developed, considering parameters such as cortical bone thickness, trabecular bone elasticity, and the nonlinear response of the nucleus pulposus to external loading. A nonlinear dynamic analysis was performed, revealing certain trends: a heightened stiffness of the annulus fibrosus correlates with a significant reduction in the vertebral body's ability to withstand external loading. At a supplied displacement of 6 mm, the vertebra with a degenerative disc reached its yielding point, whereas the vertebrae with a healthy annulus fibrosus exhibited a strength capacity exceeding 20%. The obtained findings and proposed methodology are potentially useful for biomedical engineers and clinical specialists in evaluating the condition of the annulus fibrosus and predicting its influence on the bone components of the spinal system.

Menstrual Blood-Derived Stem Cell Paracrine Factors Possess Stimulatory Effects on Chondrogenesis In Vitro and Diminish the Degradation of Articular Cartilage during Osteoarthritis.

Articular cartilage is an avascular tissue with a limited capacity for self-regeneration, leading the tissue to osteoarthritis (OA). Mesenchymal stem cells (MSCs) are promising for cartilage tissue engineering, as they are capable of differentiating into chondrocyte-like cells and secreting a number of active molecules that are important for cartilage extracellular matrix (ECM) synthesis. The aim of this study was to evaluate the potential of easily accessible menstrual blood-derived MSC (MenSC) paracrine factors in stimulating bone marrow MSC (BMMSCs) chondrogenic differentiation and to investigate their role in protecting cartilage from degradation in vitro. MenSCs and BMMSCs chondrogenic differentiation was induced using four different growth factors: TGF-ß3, activin A, BMP-2, and IGF-1. The chondrogenic differentiation of BMMSCs was stimulated in co-cultures with MenSCs and cartilage explants co-cultured with MenSCs for 21 days. The chondrogenic capacity of BMMSCs was analyzed by the secretion of four growth factors and cartilage oligomeric matrix protein, as well as the release and synthesis of cartilage ECM proteins, and chondrogenic gene expression in cartilage explants. Our results suggest that MenSCs stimulate chondrogenic response in BMMSCs by secreting activin A and TGF-ß3 and may have protective effects on cartilage tissue ECM by decreasing the release of GAGs, most likely through the modulation of activin A related molecular pathway. In conclusion, paracrine factors secreted by MenSCs may turn out to be a promising therapeutical approach for cartilage tissue protection and repair.

Evaluation of Cartilage Integrity Following Administration of Oral and Intraarticular Nifedipine in a Murine Model of Osteoarthritis.

Osteoarthritis (OA) ranks as the prevailing type of arthritis on a global scale, for which no effective treatments are currently available. Arterial hypertension is a common comorbidity in OA patients, and antihypertensive drugs, such as nifedipine (NIF), may affect the course of OA progression. The aim of this preclinical study was to determine the effect of nifedipine on healthy and OA cartilage, depending on its route of administration. In this study, we used the destabilization of medial meniscus to develop a mouse model of OA. Nifedipine was applied per os or intraarticularly (i.a.) for 8 weeks to both mice with OA and healthy animals. Serum biomarker concentrations were evaluated using the Luminex platform and alterations in the knee cartilage were graded according to OARSI histological scores and investigated immunohistochemically. Nifedipine treatment per os and i.a. exerted protective effects, as assessed by the OARSI histological scores. However, long-term nifedipine i.a. injections induced the deterioration of healthy cartilage. Lubricin, cartilage intermediate layer matrix protein (CILP), collagen type VI (COLVI), CILP, and Ki67 were upregulated by the nifedipine treatment. Serum biomarkers MMP-3, thrombospondin-4, and leptin were upregulated in the healthy groups treated with nifedipine, while only the levels of MMP-3 were significantly higher in the OA group treated with nifedipine per os compared to the untreated group. In conclusion, this study highlights the differential effects of nifedipine on cartilage integrity, depending on the route of administration and cartilage condition.

Stimulation of Chondrocyte and Bone Marrow Mesenchymal Stem Cell Chondrogenic Response by Polypyrrole and Polypyrrole/Gold Nanoparticles.

Bone marrow mesenchymal stem cells (BMMSCs) possess a strong ability to differentiate into the chondrogenic lineage, which is important for cartilage regeneration. External stimuli, such as electrical stimulation (ES), are frequently studied for chondrogenic differentiation of BMMSCs; however, the application of conductive polymers such as polypyrrole (Ppy), has never been used for stimulating BMMSCs chondrogenesis in vitro before. Thus, the aim of this study was to evaluate the chondrogenic potential of human BMMSCs after stimulation with Ppy nanoparticles (Ppy NPs) and compare them to cartilage-derived chondrocytes. In this study, we tested Ppy NPs without and with 13 nm gold NPs (Ppy/Au) for BMMSCs and chondrocyte proliferation, viability, and chondrogenic differentiation for 21 days, without the use of ES. The results demonstrated significantly higher amounts of cartilage oligomeric matrix protein (COMP) in BMMSCs stimulated with Ppy and Ppy/Au NPs, as compared to the control. The expression of chondrogenic genes (SOX9, ACAN, COL2A1) in BMMSCs and chondrocytes were upregulated by Ppy and Ppy/Au NPs, as compared to controls. Histological staining with safranin-O indicated higher extracellular matrix production in Ppy and Ppy/Au NPs stimulated samples, as compared to controls. In conclusion, Ppy and Ppy/Au NPs stimulate BMMSC chondrogenic differentiation; however, BMMSCs were more responsive to Ppy, while chondrocytes possessed a stronger chondrogenic response to Ppy/Au NPs.

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

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

The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

Electrical Stimulation in Cartilage Tissue Engineering.

Electrical stimulation (ES) has been frequently used in different biomedical applications both in vitro and in vivo. Numerous studies have demonstrated positive effects of ES on cellular functions, including metabolism, proliferation, and differentiation. The application of ES to cartilage tissue for increasing extracellular matrix formation is of interest, as cartilage is not able to restore its lesions owing to its avascular nature and lack of cells. Various ES approaches have been used to stimulate chondrogenic differentiation in chondrocytes and stem cells; however, there is a huge gap in systematizing ES protocols used for chondrogenic differentiation of cells. This review focuses on the application of ES for chondrocyte and mesenchymal stem cell chondrogenesis for cartilage tissue regeneration. The effects of different types of ES on cellular functions and chondrogenic differentiation are reviewed, systematically providing ES protocols and their advantageous effects. Moreover, cartilage 3D modeling using cells in scaffolds/hydrogels under ES are observed, and recommendations on reporting about the use of ES in different studies are provided to ensure adequate consolidation of knowledge in the area of ES. This review brings novel insights into the further application of ES in in vitro studies, which are promising for further cartilage repair techniques.

Modeling the Impact of Meniscal Tears on von Mises Stress of Knee Cartilage Tissue.

The present study aims to explore the stressed state of cartilage using various meniscal tear models. To perform this research, the anatomical model of the knee joint was developed and the nonlinear mechanical properties of the cartilage and meniscus were verified. The stress-strain curve of the meniscus was obtained by testing fresh tissue specimens of the human meniscus using a compression machine. The results showed that the more deteriorated meniscus had greater stiffness, but its integrity had the greatest impact on the growth of cartilage stresses. To confirm this, cases of radial, longitudinal, and complex tears were examined. The methodology and results of the study can assist in medical diagnostics for meniscus treatment and replacement.

The Effects of Mechanical Load on Chondrogenic Responses of Bone Marrow Mesenchymal Stem Cells and Chondrocytes Encapsulated in Chondroitin Sulfate-Based Hydrogel.

Articular cartilage is vulnerable to mechanical overload and has limited ability to restore lesions, which leads to the development of chronic diseases such as osteoarthritis (OA). In this study, the chondrogenic responses of human bone marrow mesenchymal stem cells (BMMSCs) and OA cartilage-derived chondrocytes in 3D chondroitin sulfate-tyramine/gelatin (CS-Tyr)/Gel) hydrogels with or without experimental mechanical load have been investigated. Chondrocytes were smaller in size, had slower proliferation rate and higher level of intracellular calcium (iCa2+) compared to BMMSCs. Under 3D chondrogenic conditions in CS-Tyr/Gel with or without TGF-ß3, chondrocytes more intensively secreted cartilage oligomeric matrix protein (COMP) and expressed collagen type II (COL2A1) and aggrecan (ACAN) genes but were more susceptible to mechanical load compared to BMMSCs. ICa2+ was more stably controlled in CS-Tyr/Gel/BMMSCs than in CS-Tyr/Gel/chondrocytes ones, through the expression of L-type channel subunit CaV1.2 (CACNA1C) and Serca2 pump (ATP2A2) genes, and their balance was kept more stable. Due to the lower susceptibility to mechanical load, BMMSCs in CS-Tyr/Gel hydrogel may have an advantage over chondrocytes in application for cartilage regeneration purposes. The mechanical overload related cartilage damage in vivo and the vague regenerative processes of OA chondrocytes might be associated to the inefficient control of iCa2+ regulating channels.

Chondroitin Sulfate-Tyramine-Based Hydrogels for Cartilage Tissue Repair.

The degradation of cartilage, due to trauma, mechanical load or diseases, results in abundant loss of extracellular matrix (ECM) integrity and development of osteoarthritis (OA). Chondroitin sulfate (CS) is a member of the highly sulfated glycosaminoglycans (GAGs) and a primary component of cartilage tissue ECM. In this study, we aimed to investigate the effect of mechanical load on the chondrogenic differentiation of bone marrow mesenchymal stem cells (BM-MCSs) encapsulated into CS-tyramine-gelatin (CS-Tyr/Gel) hydrogel in order to evaluate the suitability of this composite for OA cartilage regeneration studies in vitro. The CS-Tyr/Gel/BM-MSCs composite showed excellent biointegration on cartilage explants. The applied mild mechanical load stimulated the chondrogenic differentiation of BM-MSCs in CS-Tyr/Gel hydrogel (immunohistochemical collagen II staining). However, the stronger mechanical load had a negative effect on the human OA cartilage explants evaluated by the higher release of ECM components, such as the cartilage oligomeric matrix protein (COMP) and GAGs, compared to the not-compressed explants. Finally, the application of the CS-Tyr/Gel/BM-MSCs composite on the top of the OA cartilage explants decreased the release of COMP and GAGs from the cartilage explants. Data suggest that the CS-Tyr/Gel/BM-MSCs composite can protect the OA cartilage explants from the damaging effects of external mechanical stimuli. Therefore, it can be used for investigation of OA cartilage regenerative potential and mechanisms under the mechanical load in vitro with further perspectives of therapeutic application in vivo.

Experimental Evaluation of Quantum Dots and Antibodies Conjugation by Surface Plasmon Resonance Spectroscopy.

The application of antibody-functionalized quantum dots (QDs) in different areas has been widely described in the literature. However, a standard routine method for obtaining information on the conjugation efficiency of QDs with antibodies in terms of the interaction of the functionalized QDs with a specific antigen is still lacking. Herein, surface plasmon resonance (SPR) spectroscopy is proposed for this purpose. Gold-coated SPR sensor disks were modified with a self-assembled monolayer of 11-mercaptoundecanoic acid, and carbodiimide cross-linker chemistry was used to covalently immobilize the CD44 biomarker on the premodified surface (Au/CD44). Meanwhile, QDs functionalized with amine-derivatized polyethylene glycol (PEG) (QDs-NH2) were chosen for conjugation with antibodies because of their low non-specific adsorption on the Au/CD44 surface. Prior to conjugation, the surface binding capacity (Bmax) and equilibrium dissociation constant (KD) of the specific antibodies against CD44 (anti-CD44) were found to be 263.32 ± 2.44 m° and 1.00 × 10-7 ± 2.29 × 10-9 M, respectively. QDs-NH2 and anti-CD44 were conjugated at their initial molar ratios of 1:3, 1:5, 1:10 and 1:12. SPR measurements showed that the conjugates (QDs-anti-CD44) prepared using 1:10 and 1:12 molar ratios interacted comparably with immobilized CD44 biomarkers. The equilibrium angles in the case of 10- and 12-fold concentrations of anti-CD44 were calculated to be 60.43 ± 4.51 and 61.36 ± 4.40 m°, respectively. This could be explained by the QDs-NH2 and anti-CD44 having a similar surface loading (about four molecules per QDs-NH2) and similar hydrodynamic diameters, which were 46.63 ± 3.86 and 42.42 ± 0.80 nm for the 1:10 and 1:12 ratios, respectively. An initial QDs-NH2: anti-CD44 molar ratio of 1:10 was chosen as being optimal. SPR spectroscopy proved to be the right choice for QDs-anti-CD44 conjugation optimization, and can be used for the evaluation of conjugation efficiency for other nanostructures with various bio-recognition molecules.

Carbon Dioxide Sensing by Immune Cells Occurs through Carbonic Anhydrase 2-Dependent Changes in Intracellular pH.

CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.

Magneto-Immunoassay for the Detection and Quantification of Human Growth Hormone.

Physiological and endocrine maintenance of a normal human growth hormone (hGH) concentration is crucial for growth, development, and a number of essential biological processes. In this study, we describe the preparation and characterization of magnetic nanoparticles coated with a gold shell (MNPs-Au). The optimal surface concentration of monoclonal anti-hGH antibodies (m-anti-hGH) on magnetic nanoparticles, as well as conditions that decrease non-specific interactions during the magneto-immunoassay, were elaborated. After the selective recognition, separation, and pre-concentration of hGH by MNPs-Au/m-anti-hGH and the hGH interaction with specific polyclonal biotin-labeled antibodies (p-anti-hHG-B) and streptavidin modified horseradish peroxidase (S-HRP), the MNPs-Au/m-anti-hGH/hGH/p-anti-hGH-B/S-HRP immunoconjugate was formed. The concentration of hGH was determined after the addition of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide substrate solution for HRP; the absorbance at 450 nm was registered after the addition of STOP solution. The developed sandwich-type colorimetric magneto-immunoassay is characterized by a clinically relevant linear range (from 0.1 to 5.0 nmol L-1, R2 0.9831), low limit of detection (0.082 nmol L-1), and negligible non-specific binding of other antibodies or S-HRP. The obtained results demonstrate the applicability of the developed magneto-immunoassay for the concentration and determination of hGH in the serum. Additionally, important technical solutions for the development of the sandwich-type colorimetric magneto-immunoassay are discussed.

Exploring the translational potential of clusterin as a biomarker of early osteoarthritis.

BACKGROUND: Clusterin (CLU; also known as apolipoprotein J) is an ATP-independent holdase chaperone that prevents proteotoxicity as a consequence of protein aggregation. It is a ∼60 kDa disulfide-linked heterodimeric protein involved in the clearance of cellular debris and the regulation of apoptosis. CLU has been proposed to protect cells from cytolysis by complement components and has been implicated in Alzheimer's disease due to its ability to bind amyloid-ß peptides and prevent aggregate formation in the brain. Recent studies suggest that CLU performs moonlighting functions. CLU exists in two major forms: an intracellular form and a secreted extracellular form. The intracellular form of CLU may suppress stress-induced apoptosis by forming complexes with misfolded proteins and facilitates their degradation. The secreted form of CLU functions as an extracellular chaperone that prevents protein aggregation. METHODS: In this review, we discuss the published literature on the biology of CLU in cartilage, chondrocytes, and other synovial joint tissues. We also review clinical studies that have examined the potential for using this protein as a biomarker in synovial and systemic fluids of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). RESULTS: Since CLU functions as an extracellular chaperone, we propose that it may be involved in cytoprotective functions in osteoarticular tissues. The secreted form of CLU can be measured in synovial and systemic fluids and may have translational potential as a biomarker of early repair responses in OA. CONCLUSION: There is significant potential for investigating synovial and systemic CLU as biomarkers of OA. Future translational and clinical orthopaedic studies should carefully consider the diverse roles of this protein and its involvement in other comorbidities. Therefore, future biomarker studies should not correlate circulating CLU levels exclusively to the process of OA pathogenesis and progression. Special attention should be paid to CLU levels in synovial fluid. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: There is significant potential for investigating synovial and systemic CLU as a predictive biomarker of osteoarthritis (OA) progression and response to novel treatments and interventions. Given that CLU plays diverse roles in other comorbidities such as rheumatoid arthritis (RA) and obesity, future translational and clinical orthopaedic biomarker studies should not directly correlate circulating CLU levels to the process of OA pathogenesis and progression. However, special attention should be paid to CLU levels in synovial fluid. The cytoprotective properties of CLU may support the implementation of regenerative strategies and new approaches for developing targeted therapeutics for OA.

Cardiovascular Drugs and Osteoarthritis: Effects of Targeting Ion Channels.

Osteoarthritis (OA) and cardiovascular diseases (CVD) share many similar features, including similar risk factors and molecular mechanisms. A great number of cardiovascular drugs act via different ion channels and change ion balance, thus modulating cell metabolism, osmotic responses, turnover of cartilage extracellular matrix and inflammation. These drugs are consumed by patients with CVD for many years; however, information about their effects on the joint tissues has not been fully clarified. Nevertheless, it is becoming increasingly likely that different cardiovascular drugs may have an impact on articular tissues in OA. Here, we discuss the potential effects of direct and indirect ion channel modulating drugs, including inhibitors of voltage gated calcium and sodium channels, hyperpolarization-activated cyclic nucleotide-gated channels, ß-adrenoreceptor inhibitors and angiotensin-aldosterone system affecting drugs. The aim of this review was to summarize the information about activities of cardiovascular drugs on cartilage and subchondral bone and to discuss their possible consequences on the progression of OA, focusing on the modulation of ion channels in chondrocytes and other joint cells, pain control and regulation of inflammation. The implication of cardiovascular drug consumption in aetiopathogenesis of OA should be considered when prescribing ion channel modulators, particularly in long-term therapy protocols.

Mechanotransducive Biomimetic Systems for Chondrogenic Differentiation In Vitro.

Osteoarthritis (OA) is a long-term chronic joint disease characterized by the deterioration of bones and cartilage, which results in rubbing of bones which causes joint stiffness, pain, and restriction of movement. Tissue engineering strategies for repairing damaged and diseased cartilage tissue have been widely studied with various types of stem cells, chondrocytes, and extracellular matrices being on the lead of new discoveries. The application of natural or synthetic compound-based scaffolds for the improvement of chondrogenic differentiation efficiency and cartilage tissue engineering is of great interest in regenerative medicine. However, the properties of such constructs under conditions of mechanical load, which is one of the most important factors for the successful cartilage regeneration and functioning in vivo is poorly understood. In this review, we have primarily focused on natural compounds, particularly extracellular matrix macromolecule-based scaffolds and their combinations for the chondrogenic differentiation of stem cells and chondrocytes. We also discuss different mechanical forces and compression models that are used for In Vitro studies to improve chondrogenic differentiation. Summary of provided mechanical stimulation models In Vitro reviews the current state of the cartilage tissue regeneration technologies and to the potential for more efficient application of cell- and scaffold-based technologies for osteoarthritis or other cartilage disorders.

Different phenotypes and chondrogenic responses of human menstrual blood and bone marrow mesenchymal stem cells to activin A and TGF-ß3.

BACKGROUND: Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor ß (TGF-ß3) and activin A. METHODS: MenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-ß3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture. RESULTS: MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-ß3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-ß3 alone were more pronounced in BMMSCs in vitro. CONCLUSION: These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders.

The Expression of Inflammasomes NLRP1 and NLRP3, Toll-Like Receptors, and Vitamin D Receptor in Synovial Fibroblasts From Patients With Different Types of Knee Arthritis.

Activated rheumatoid arthritis (RA) synovial fibroblasts (SFs) are among the most important cells promoting RA pathogenesis. They are considered active contributors to the initiation, progression, and perpetuation of the disease; therefore, early detection of RASF activation could advance contemporary diagnosis and adequate treatment of undifferentiated early inflammatory arthritis (EA). In this study, we investigated the expression of nucleotide-binding, oligomerization domain (NOD)-like receptor family, pyrin domain containing (NLRP)1, NLRP3 inflammasomes, Toll-like receptor (TLR)1, TLR2, TLR4, vitamin D receptor (VDR), and secretion of matrix metalloproteinases (MMPs) in SFs isolated from patients with RA, osteoarthritis (OA), EA, and control individuals (CN) after knee surgical intervention. C-reactive protein, general blood test, anticyclic citrullinated peptide (anti-CCP), rheumatoid factor (RF), and vitamin D (vitD) in patients' sera were performed. Cells were stimulated or not with 100 ng/ml tumor necrosis factor alpha (TNF-α) or/and 1 nM or/and 0.01 nM vitamin D3 for 72 h. The expression levels of NLRP1, NLRP3, TLR1, TLR2, TLR4, and VDR in all examined SFs were analyzed by quantitative real-time PCR (RT-qPCR). Additionally, the secretion of IL-1ß by SFs and MMPs were determined by ELISA and Luminex technology. The expression of NLRP3 was correlated with the levels of CRP, RF, and anti-CCP, suggesting its implication in SF inflammatory activation. In the TNF-α-stimulated SFs, a significantly lower expression of NLRP3 and TLR4 was observed in the RA group, compared with the other tested forms of arthritis. Moreover, upregulation of NLRP3 expression by TNF-α alone or in combination with vitD3 was observed, further indicating involvement of NLRP3 in the inflammatory responses of SFs. Secretion of IL-1ß was not detected in any sample, while TNF-α upregulated the levels of secreted MMP-1, MMP-7, MMP-8, MMP-12, and MMP-13 in all patient groups. Attenuating effects of vitD on the expression of NLRP3, TLR1, and TLR4 suggest potential protective effects of vitD on the inflammatory responses in SFs. However, longer studies may be needed to confirm or fully rule out the potential implication of vitD in SF activation in inflammatory arthritis. Both VDR and NLRP3 in the TNF-α-stimulated SFs negatively correlated with the age of patients, suggesting potential age-related changes in the local inflammatory responses.

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.

Swelling-activated ClC-3 activity regulates prostaglandin E2 release in human OUMS-27 chondrocytes.

Articular chondrocytes are exposed to dynamic osmotic environments during normal joint loading, and thus, require effective volume regulatory mechanisms. A regulatory volume decrease (RVD) is one of the mechanisms for protecting chondrocytes from swelling and damage. Swelling-activated Cl- currents (ICl,swell) are responsible for the RVD, but the molecular identity in chondrocytes is largely unknown. In this study, we reveal that in human OUMS-27 chondrocytes, ICl,swell can be elicited by hypoosmotic stimulation (180 mOsm) and be inhibited by classical Cl- channel blockers, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and be attenuated by siRNA knockdown of ClC-3. Our molecular analyses revealed that ClC-3A is expressed as a major splice variant in both human articular chondrocytes and OUMS-27 cells. The onset and early phase of RVD following hypoosmotic stress in OUMS-27 cells were affected by DIDS and ClC-3 knockdown. Hypoosmotic stimulation caused Ca2+ influx and subsequent release of prostaglandin E2 (PGE2) in OUMS-27 cells, and both of these responses were reduced by DIDS and ClC-3 knockdown. These results strongly suggest that ClC-3 is responsible for ICl,swell and RVD under the hypoosmotic environments. It is likely that ClC-3 is associated with the pathogenesis of cartilage degenerative diseases including osteoarthritis via PGE2 release.