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High-density cell culture is pivotal for the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Two high-density cell culture systems, micromass and pellet culture, have been used to induce chondrogenic differentiation of hMSCs. In micromass culture, the induced-cartilage tissues were larger, more homogenous and enriched in cartilage-specific collagen II but the fibrocartilage-like feature, collagen I, and hypertrophic chondrocyte feature, collagen X, were markedly decreased compared to those in pellet culture. Furthermore, real time RT-PCR analysis demonstrated that collagen II and aggrecan mRNA were up-regulated while collagen X and collagen I mRNA were down-regulated in micromass culture. Thus, the micromass culture system is a promising tool for in vitro chondrogenic studies.

Glucosamine (GlcN) fulfills many of the requirements as an ideal component in scaffolds used in cartilage tissue engineering. The incorporation of GlcN in a gelatin/hyaluronic acid (GH) cryogel scaffold could provide biological cues in maintaining the phenotype of chondrocytes. Nonetheless, substituting gelatin with GlcN may also decrease the crosslinking density and modulate the mechanical properties of the cryogel scaffold, which may be beneficial as physical cues for chondrocytes in the scaffold. Thus, we prepared cryogel scaffolds containing 9% GlcN (GH-GlcN9) and 16% GlcN (GH-GlcN16) by carbodiimide-mediated crosslinking reactions at −16 °C. The crosslinking density and the mechanical properties of the cryogel matrix could be tuned by adjusting the content of GlcN used during cryogel preparation. In general, incorporation of GlcN did not influence scaffold pore size and ultimate compressive strain but increased porosity. The GH-GlcN16 cryogel showed the highest swelling ratio and degradation rate in hyaluronidase and collagenase solutions. On the contrary, the Young’s modulus, storage modulus, ultimate compressive stress, energy dissipation level, and rate of stress relaxation decreased by increasing the GlcN content in the cryogel. The release of GlcN from the scaffolds in the culture medium of chondrocytes could be sustained for 21 days for GH-GlcN16 in contrast to only 7 days for GH-GlcN9. In vitro cell culture experiments using rabbit articular chondrocytes revealed that GlcN incorporation affected cell proliferation, morphology, and maintenance of chondrogenic phenotype. Overall, GH-GlcN16 showed the best performance in maintaining chondrogenic phenotype with reduced cell proliferation rate but enhanced glycosaminoglycans (GAGs) and type II collagen (COL II) secretion. Quantitative real-time polymerase chain reaction also showed time-dependent up-regulation of cartilage-specific marker genes (COL II, aggrecan and Sox9) for GH-GlcN16. Implantation of chondrocytes/GH-GlcN16 constructs into full-thickness articular cartilage defects of rabbits could regenerate neocartilage with positive staining for GAGs and COL II. The GH-GlcN16 cryogel will be suitable as a scaffold for the treatment of articular cartilage defects.

Intervertebral disc degeneration (IDD) is characterized by disordered extracellular matrix (ECM) metabolism, implicating subdued anabolism and enhanced catabolic activities in the nucleus pulposus (NP) of discs. Pro-inflammatory cytokines such as interleukin-1β (IL-1β) are considered to be potent mediators of ECM breakdown. Hemeoxygenase-1 (HO-1) has been reported to participate in cellular anti-inflammatory processes. The purpose of this study was to investigate HO-1 modulation of ECM metabolism in human NP cells under IL-1β stimulation. Our results revealed that expression of HO-1 decreased considerably during IDD progression. Induction of HO-1 by cobalt protoporphyrin IX attenuated the inhibition of sulfate glycosaminoglycan and collagen type II (COL-II) synthesis and ameliorated the reduced expressions of aggrecan, COL-II, SOX-6 and SOX-9 mediated by IL-1β. Induction of HO-1 also reversed the effect of IL-1β on expression of the catabolic markers matrix metalloproteinases-1, 3, 9 and 13. This was combined with inhibition of the activation of mitogen-activated protein kinase signaling. These findings suggest that HO-1 might play a pivotal role in IDD and that manipulating HO-1 expression might mitigate the impairment of ECM metabolism in NP, thus potentially offering a novel therapeutic approach to the treatment of IDD.

MicroRNAs have been shown to play a role in cartilage development, homeostasis and breakdown during osteoarthritis. We previously identified miR-3085 in humans as a chondrocyte-selective microRNA, however it could not be detected by Northern blot. The aim of the current study was to prove that miR-3085 is a microRNA and to investigate the function of miR-3085 in signaling pathways relevant to cartilage homeostasis and osteoarthritis. Here, we confirm that miR-3085 is a microRNA and not another class of small RNA using (1) a pre-miR hairpin maturation assay, (2) expression levels in a Dicer null cell line, and (3) Ago2 pulldown. MicroRNA-3085-3p is expressed more highly in micromass than monolayer cultured chondrocytes. Transfection of miR-3085-3p into chondrocytes decreases expression of COL2A1 and ACAN , both of which are validated as direct targets of miR-3085-3p. Interleukin-1 induces the expression of miR-3085-3p, at least in part via NFκB. In a feed-forward mechanism, miR-3085-3p then potentiates NFκB signaling. However, at early time points after transfection, its action appears to be inhibitory. MyD88 has been shown to be a direct target of miR-3085-3p and may be responsible for the early inhibition of NFκB signaling. However, at later time points, MyD88 knockdown remains inhibitory and so other functions of miR-3085-3p are clearly dominant. TGFβ1 also induces the expression of miR-3085-3p, but in this instance, it exerts a feedback inhibition on signaling with SMAD3 and SMAD4 shown to be direct targets. This in vitro analysis shows that miR-3085-3p functions in chondrocytes to induce IL-1-signaling, reduce TGFβ1 signaling, and inhibit expression of matrix genes. These data suggest that miR-3085-3p has a role in chondrocyte function and could contribute to the process of osteoarthritis.

The objective of this study is to investigate the expression of enzymes involved in the sulfation of articular cartilage from proximal metacarpophalangeal (PMC) joint cartilage and distal metacarpophalangeal (DMC) joint cartilage in children with Kashin–Beck disease (KBD). The finger cartilage samples of PMC and DMC were collected from KBD and normal children aged 5–14 years old. Hematoxylin and eosin staining as well as immunohistochemical staining were used to observe the morphology and quantitate the expression of carbohydrate sulfotransferase 3 (CHST-3), carbohydrate sulfotransferase 12 (CHST-12), carbohydrate sulfotransferase 13 (CHST-13), uronyl 2-O-sulfotransferase (UST), and aggrecan. In the results, the numbers of chondrocyte decreased in all three zones of PMC and DMC in the KBD group. Less positive staining cells for CHST-3, CHST-12, CHST-13, UST, and aggrecan were observed in almost all three zones of PMC and DMC in KBD. The positive staining cell rates of CHST-12 were higher in superficial and middle zones of PMC and DMC in KBD, and a significantly higher rate of CHST-13 was observed only in superficial zone of PMC in KBD. In conclusion, the abnormal expression of chondroitin sulfate sulfotransferases in chondrocytes of KBD children may provide an explanation for the cartilage damage, and provide therapeutic targets for the treatment.

Objectives:To investigate changes in gene expression in fibrillated and intact human osteoarthritis (OA) cartilage for evidence of an altered chondrocyte phenotype and hypertrophy.Methods:Paired osteochondral samples were taken from a high-load site and a low-load site from 25 OA joints and were compared with eight similar paired samples from age-matched controls. Gene expression of key matrix and regulatory genes was analysed by quantitative real-time reverse transcription-polymerase chain reaction on total RNA extracted from the cartilage.Results:There was a major change in chondrocyte gene expression in OA cartilage. SOX9 (38-fold) and aggrecan (4-fold) gene expression were both lower in OA (p<0.001), and collagen I (17-fold) and II (2.5-fold) gene expression were each increased in a subset of OA samples. The major changes in gene expression were similar at the fibrillated high-loaded site and the intact low-loaded site. There was no evidence of a generalised change in OA to proliferative or hypertrophic phenotype as seen in the growth plate, as genes associated with either stage of differentiation were unchanged (PTHrPR), or significantly downregulated (collagen X (14-fold, p<0.002), VEGF (23-fold, p<0.02), BCL-2 (5.6-fold, p<0.001), matrilin-1 (6.5-fold, p<0.001)). In contrast MMP-13 was significantly upregulated in the OA cartilage samples (5.3-fold, p<0.003).Conclusions:The expression of key chondrocyte genes, including aggrecan and SOX9, was decreased in OA cartilage and the changes were similar in both fibrillated high-loaded and intact low-loaded cartilage on the same joint. However, there was no significant upregulation of type X collagen, and other genes associated with chondrocyte further differentiation and hypertrophy.

This study tested whether activation of adrenoreceptors in chondrocytes has roles in degenerative remodelling of temporomandibular joint (TMJ) and to determine associated mechanisms. Unilateral anterior crossbite (UAC) was established to induce TMJ degeneration in rats. Saline vehicle, α2- and β-adrenoreceptor antagonists or agonists were injected locally into the TMJ area of UAC rats. Cartilage degeneration, subchondral bone microarchitecture and the expression of adrenoreceptors, aggrecans, matrix metalloproteinases (MMPs) and RANKL by chondrocytes were evaluated. Chondrocytes were stimulated by norepinephrine to investigate signal transduction of adrenoreceptors. Increased α2A-adrenoreceptor expression was observed in condylar cartilage of UAC rats, together with cartilage degeneration and subchondral bone loss. Norepinephrine depresses aggrecans expression but stimulates MMP-3, MMP-13 and RANKL production by chondrocytes through ERK1/2 and PKA pathway; these effects were abolished by an α2A-adrenoreceptor antagonist. Furthermore, inhibition of α2A-adrenoreceptor attenuated degenerative remodelling in the condylar cartilage and subchondral bone, as revealed by increased cartilage thickness, proteoglycans and aggrecan expression and decreased MMP-3, MMP-13 and RANKL expressions in cartilage, increased BMD, BV/TV and decreased Tb.Sp in subchondral bone. Conversely, activation of α2A-adrenoreceptor intensified aforementioned degenerative changes in UAC rats. It is concluded that activation of α2A-adrenergic signal in chondrocytes promotes TMJ degenerative remodelling by chondrocyte-mediated pro-catabolic activities.

Mesenchymal stem cells (MSCs) usually lose their stemness during in vitro expansion as they are deprived of their niche environment. Cell–extracellular matrix (ECM) interaction is known to play important roles in preserving the stemness of the cells in their stem cell niche environment. Previously, coating with bone marrow MSC (BMSC)-derived ECM was found able to maintain the differentiation potential of in vitro cultured MSCs. This study aimed to determine if this ECM coating could also maintain the stemness of cultured murine adipose-derived stem cells (ASCs) using a regular culture flask as a control. Cells were expanded in ECM-coated and ECM-noncoated flasks for two and four passages and then harvested for various analyses. The results showed that ASCs exhibited fibroblast-like spindle morphology in ECM-coated flasks, whereas ASCs gradually spread and enlarged in the ECM-noncoated flasks. After three and five passages, both groups of cells exhibited similar cytokinetics in the MSC culture medium (MesenPRO RS™ Medium). However, when cultured in Dulbecco's modified Eagles medium (DMEM) plus 10% fetal bovine serum, coating group cells exhibited more potent proliferation than control group cells with a significant difference in both passages 3 and 5 (p<0.01). When seeded at low density (500 cells/10-cm dish), coating group cells formed significantly more and larger sized cell colonies than control group cells with significant difference in cell colony numbers between two groups (p<0.05). In addition, coated colony cells were much smaller and more compactly arranged compared to control colony cells. Furthermore, ASCs expanded in coated flasks exhibited greater potentials for adipogenic, osteogenic, and chondrogenic differentiations than the cells expanded in regular flasks. Quantitatively, the Oil Red O staining area, Alizarin staining area, and Toluidine Blue staining area were all significantly larger than the respective staining areas of control cells (p<0.05). Real-time polymerase chain reaction also revealed significantly higher gene expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein (C/EBP), Rux2, osteocalcin, Sox9, collagen II, and aggrecan in ECM-coated group cells than in control group cells (p<0.05). Collectively, these results suggest that human BMSC decellular ECM coating helps to preserve the stemness of cultured murine ASCs.

Objective. To investigate the effects of Danshen on the imaging and histological parameters, expression levels of ECM-associated proteins and inflammatory factors, and antioxidative activity in the degenerated intervertebral disc (IVD) of SD rats. Methods. Sixty male rats were randomly divided into three groups (control, IDD, and Danshen IDD). Percutaneous needle puncture in Co8-9 intervertebral disc was conducted in all rats of the IDD and Danshen IDD groups to induce intervertebral disc degeneration (IDD). After operation, animals of the Danshen IDD group were administrated with Danshen granules (3 g/kg body weight ) by gavage once a day. Four weeks later, the coccygeal vertebrae were harvested and used for imaging (disc height and MR signal), histological, immunohistochemical, and biochemical [water content, glycosaminoglycans (GAG), superoxide dismutase (SOD2), glutathione (GSH), and malondialdehyde (MDA)] analyses. Results. The puncture induced significant decreased IVD space and MR T2 signal at both 2 and 4 weeks, which were attenuated by Danshen treatment. The disc degeneration in the IDD group (HE and Safranin O-Fast Green histological staining was markedly more serious compared with that in the control group. Four weeks of Danshen treatment significantly alleviated this degeneration compared with the IDD group. Needle puncture resulted in the upregulation of IL-1β and TNF-α, MMP-3, and downregulation of COL2 and aggrecan in the IDD group. However, this change was significantly weakened by Danshen treatment. Significantly lower water and GAG content, as well as the SOD2 and GSH levels, in the IDD group were found compared with those in the control group. However, the above parameters of the Danshen IDD group were significantly higher than those of the IDD group. Danshen treatment significantly decreased the content of MDA which was increased by needle puncture in the IDD group. Conclusion. Danshen can attenuate intervertebral disc degeneration in SD rats by suppressing the oxidation reaction.

Indian hedgehog (Ihh) is essential for embryonic mandibular condylar growth and disc primordium formation. To determine whether it regulates those processes during post-natal life, we ablated Ihh in cartilage of neonatal mice and assessed the consequences on temporomandibular joint (TMJ) growth and organization over age. Ihh deficiency caused condylar disorganization and growth retardation and reduced polymorphic cell layer proliferation. Expression of Sox9, Runx2, and Osterix was low, as was that of collagen II, collagen I, and aggrecan, thus altering the fibrocartilaginous nature of the condyle. Though a disc formed, it exhibited morphological defects, partial fusion with the glenoid bone surface, reduced synovial cavity space, and, unexpectedly, higher lubricin expression. Analysis of the data shows, for the first time, that continuous Ihh action is required for completion of post-natal TMJ growth and organization. Lubricin overexpression in mutants may represent a compensatory response to sustain TMJ movement and function.