Pathological calcification in osteoarthritis: an outcome or a disease initiator?

In the progression of osteoarthritis, pathological calcification in the affected joint is an important feature. The role of these crystallites in the pathogenesis and progression of osteoarthritis is controversial; it remains unclear whether they act as a disease initiator or are present as a result of joint damage. Recent studies reported that the molecular mechanisms regulating physiological calcification of skeletal tissues are similar to those regulating pathological or ectopic calcification of soft tissues. Pathological calcification takes place when the equilibrium is disrupted. Calcium phosphate crystallites are identified in most affected joints and the presence of these crystallites is closely correlated with the extent of joint destruction. These observations suggest that pathological calcification is most likely to be a disease initiator instead of an outcome of osteoarthritis progression. Inhibiting pathological crystallite deposition within joint tissues therefore represents a potential therapeutic target in the management of osteoarthritis.     

Evidence for a low-molecular-weight plasma peptide which stimulates chick chondrocyte metabolism

Plasma contains a number of insulin-like activities (ILA) of molecular weights 7,000 to 90,000 (somatomedins and insulin-like proteins) which stimulate cellular metabolism and may function as growth factors. We have found evidence for the presence of an 800 Dalton peptide in human plasma which markedly stimulates the metabolism of chick chondrocytes. This peptide was extracted from human Cohn fraction IV-1 by procedures similar to those used for somatomedin isolations. At the Sephadex G-50 column separation step, the fraction with molecular weights of 300–1,000 was found to markedly stimulate chick chondrocyte metabolism. Rechromatography on Sephadex G-25 concentrated activity in peptides of molecular weight of about 800. An HPLC separation on a silica C-18 reverse phase column gave elution of the active peptide at 18% acetonitrile in water. This bioactivity appears to be a peptide which is free of lipids, carbohydrates, nucleic acids, metal ions, and immunoreactive insulin. This factor markedly increased the metabolism of cultured chick chondrocytes, but had only marginal activity on rat chondrocytes. When added at 1 μg/ml to chick chondrocytes cultured in F-12 medium plus 1.5% fetal calf serum, the HPLC-purified activity increased DNA synthesis 7.3-fold, lipid synthesis 10.2-fold, and lactate production 2.9-fold after 48 h incubation. However, unlike somatomedins A and C, this factor did not displace insulin from placental membranes. These results suggest that low-molecular-weight peptides, which are smaller than the somatomedins, may contribute to the total ILA of human plasma.     

Small animal models to understand pathogenesis of osteoarthritis and use of stem cell in cartilage regeneration

Osteoarthritis (OA) is one of the most common diseases, which affect the correct functionality of synovial joints and is characterized by articular cartilage degradation. Limitation in the treatment of OA is mostly due to the very limited regenerative characteristic of articular cartilage once is damaged. Small animal models are of particular importance for mechanistic analysis to understand the processes that affect cartilage degradation. Combination of joint injury techniques with the use of stem cells has been shown to be an important tool for understanding the processes of cartilage degradation and regeneration. Implementation of stem cells and small animal models are important tools to help researchers to find a solution that could ameliorate and prevent the symptoms of OA.     

Integrated Study of Globally Expressed microRNAs in IL-1β-stimulated Human Osteoarthritis Chondrocytes and Osteoarthritis Relevant Genes: A Microarray and Bioinformatics Analysis

This study was undertaken to identify and characterize the globally expressed microRNAs (miRNAs) involved in interleukin-1β (IL-1β)-induced joint damage and to predict whether miRNAs can regulate the catabolic effects in osteoarthritis (OA) chondrocytes. Out of 1347 miRNAs analyzed by microarrays in IL-1β-stimulated OA chondrocytes, 35 miRNAs were down-regulated, 1 miRNA was up-regulated, and the expression of 1311 miRNAs remained unchanged. Bioinformatics analysis showed the key inflammatory mediators and key molecular pathways are targeted by differentially expressed miRNAs. Novel miRNAs identified could have important diagnostic and therapeutic potentials in the development of novel therapeutic strategies for pain managements in OA.     

Development of a serum-free system to study the effect of growth hormone and insulinlike growth factor-I on cultured postembryonic growth plate chondrocytes

Summary We have developed a serum-free system to culture postembryonic growth plate chondrocytes while maintaining some important phenotypic characteristics of their tissue of origin. This serum-free medium was as effective as medium containing 10% newborn bovine serum (NBS) for recovering the cells from enzymatic isolation. Surface secretory activity of chondrocytes cultured in monolayer, assessed through scanning electron microscopy, was also comparable to cells grown in medium containing serum. The effects of growth hormone (GH) and insulinlike growth factor-I (IGF-I) were also studied using the serum-free medium. GH had no effect on cell density and morphology of the cells compared to the control without the hormone. In contrast, chondrocytes grown in medium containing IGF-I had a marked increase in cell density after 3 days and presented similar morphologic characteristics to cells grown in the presence of NBS. The growth factors required for proliferation of chondrocytes cultured in the serum-free medium are IGF-I and fibroblast growth factor (100 ng/ml, respectively). Addition of ascorbic acid to the serum-free medium (0 to 50 μg/ml) produced a dose-dependant decrease in cell proliferation. This medium should provide a useful tool for studying the effects of different growth factors/hormones in the regulation of longitudinal bone growth and their interactions.     

Comparison of three multi-cryoprotectant loading protocols for vitrification of porcine articular cartilage

Vitrification is a cryopreservation technique for the long-term storage of viable tissue, but the success of this technique relies on multiple factors. In 2012, our group published a working vitrification protocol for intact human articular cartilage and reported promising chondrocyte recovery after using a four-step multi-cryoprotectant (CPA) loading method that required 570 min. However, this protocol requires further optimization for clinical practice. Herein, we compared three multi-step CPA loading protocols to investigate their impact on chondrocyte recovery after vitrification of porcine articular cartilage on a bone base, including our previous four-step protocol (original: 570 min), and two shorter three-step protocols (optimized: 420 min, and minimally vitrifiable: 310 min). Four different CPAs were used including glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol. As vitrification containers, two conical tubes (50 ml and 15 ml) were evaluated for their heat transfer impact on chondrocyte recovery after vitrification. Osteochondral dowels were cored into two diameters of 10.0 mm and 6.9 mm with an approximately 10-mm thick bone base, and then allocated into the twelve experimental groups based on CPA loading protocol, osteochondral dowel size, and vitrification container size. After vitrification at −196 °C and tissue warming and CPA removal, samples in all groups were assessed for both chondrocyte viability and metabolic activity. The optimized protocol proposed based on mathematical modelling resulted in similar chondrocyte recovery to our original protocol and it was 150 min shorter. Furthermore, this study illustrated the role of CPA permeation (dowel size) and heat transfer (container size) on vitrification protocol outcome.     

CTGF facilitates cell‐cell communication in chondrocytes via PI3K/Akt signalling pathway

PurposesGap junction intercellular communication (GJIC) is essential for articular cartilage to respond appropriately to physical or biological stimuli and maintain homeostasis. Connective tissue growth factor (CTGF), identified as an endochondral ossification genetic factor, plays a vital role in cell proliferation, migration and adhesion. However, how CTGF regulates GJIC in chondrocytes is still unknown. This study aims to explore the effects of CTGF on GJIC in chondrocytes and its potential biomechanism.Materials and methodsqPCR was performed to determine the expression of gene profile in the CCN family in chondrocytes. After CTGF treatment, CCK‐8 assay and scratch assay were performed to explore cell proliferation and migration. A scrape loading/dye transfer assay was adopted to visualize GJIC in living chondrocytes. Western blot analysis was done to detect the expression of Cx43 and PI3K/Akt signalling. Immunofluorescence staining was used to show protein distribution. siRNA targeting CTGF was used to detect the influence on cell‐cell communication.ResultsThe CTGF (CCN2) was shown to be the highest expressed member of the CCN family in chondrocytes. CTGF facilitated functional gap junction intercellular communication in chondrocytes through up‐regulation of Cx43 expressions. CTGF activated PI3K/Akt signalling to promote Akt phosphorylation and translocation. Suppressing CTGF also reduced the expression of Cx43. The inhibition of PI3K/Akt signalling decreased the expressions of Cx43 and thus impaired gap junction intercellular communication enhanced by CTGF.ConclusionsFor the first time, we provide evidence to show CTGF facilitates cell communication in chondrocytes via PI3K/Akt signalling pathway.