OBJECTIVES: This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic cell line (ATDC5) cells and the maintenance of murine articular cartilage in vitro. METHODS: ATDC5 and articular cartilage tissue explant were cultured in the medium containing different sulfated glycosaminoglycans. Cell proliferation, differentiation, cartilage formation, and mechanism were observed using cell proliferation assay, Alcian blue staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively. RESULTS: Results showed that HEP and DS primarily activated the bone morphogenetic protein (BMP) signal pathway, while CS primarily activated the protein kinase B (AKT) signal pathway, further promoted ATDC5 cell proliferation and matrix production, and increased Sox9, Col2a1, and Aggrecan expression. CONCLUSIONS This study investigated the differences and mechanisms of different sulfated glycosaminoglycans in chondrogenesis and cartilage homeostasis maintenance. HEP promotes cartilage formation and maintains the normal state of cartilage tissue in vitro, while CS plays a more effective role in the regeneration of damaged cartilage tissue.
Animals ; Mice ; Cartilage/metabolism* ; Cell Differentiation ; Cells, Cultured ; Chondrocytes/metabolism* ; Chondrogenesis/physiology* ; Glycosaminoglycans/pharmacology*

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.
Animals ; Cartilage/cytology* ; Chondrocytes/drug effects* ; Chondrogenesis/drug effects* ; Extracellular Matrix/metabolism* ; Quercetin/pharmacology* ; Rats ; Signal Transduction/drug effects* ; Tissue Scaffolds

Articular cartilage lesions due to injury or other pathology are often difficult to heal, and the outcomes of the clinical treatment widely used today are far from satisfaction. Adipose-derived stem cells (ADSCs) are multipotent stem cells from adipose tissue. Tissue engineering based on the ability of ADSCs to differentiate into chondrocytes provides a new idea for the repair and regeneration of articular cartilage defects. The method for inducing the differentiation of ADSCs into chondrocytes in vitro who have been quite well established, which mainly include the use of growth factors and scaffolds to mimic the in vivo microenvironment, thereby promoting the differentiation of ADSCs into chondrocytes.
Adipocytes ; Adipose Tissue ; Cartilage, Articular ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; Chondrogenesis ; Stem Cells ; Tissue Engineering

Osteoarthritis is a musculoskeletal disease representative of an aging society. As medical conditions are usually complicated in an aging population, osteoarthritis becomes more frequently encountered in the physician's office. There is a growing need, therefore, for physicians to pay attention to this common orthopedic condition. Cartilage degeneration, arthritic pain, and joint dysfunction are major manifestations of osteoarthritis, and degenerated cartilage is difficult to repair with conventional treatment modalities. Scientists and physicians have developed various therapeutic strategies, including the use of stem cells. Here, we discuss previous and current progress in cartilage regenerative therapy against osteoarthritis.
Adult Stem Cells ; Aging ; Cartilage ; Chondrogenesis ; Induced Pluripotent Stem Cells ; Joints ; Musculoskeletal Diseases ; Orthopedics ; Osteoarthritis ; Physicians' Offices ; Stem Cells

BACKGROUND: Articular cartilage lesions occur frequently but unfortunately damaged cartilage has a very limited intrinsic repair capacity. Therefore, there is a high need to develop technology that makes cartilage repair possible. Since joint damage will lead to (sterile) inflammation, development of this technology has to take into account the effects of inflammation on cartilage repair. METHODS: A literature search has been performed including combinations of the following keywords; cartilage repair, fracture repair, chondrogenesis, (sterile) inflammation, inflammatory factors, macrophage, innate immunity, and a number of individual cytokines. Papers were selected that described how inflammation or inflammatory factors affect chondrogenesis and tissue repair. A narrative review is written based on these papers focusing on the role of inflammation in cartilage repair and what we can learn from findings in other organs, especially fracture repair. RESULTS: The relationship between inflammation and tissue repair is not straightforward. Acute, local inflammation stimulates fracture repair but appears to be deleterious for chondrogenesis and cartilage repair. Systemic inflammation has a negative effect on all sorts of tissue repair. CONCLUSION: Findings on the role of inflammation in fracture repair and cartilage repair are not in line. The currently widely used models of chondrogenesis, using high differentiation factor concentrations and corticosteroid levels, are not optimal. To make it possible to draw more valid conclusions about the role of inflammation and inflammatory factors on cartilage repair, model systems must be developed that better mimic the real conditions in a joint with damaged cartilage.
Cartilage ; Cartilage, Articular ; Chondrogenesis ; Cytokines ; Immunity, Innate ; Inflammation ; Joints ; Macrophages

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) become hypertrophic in long term despite chondrogenic differentiation following the pathway of growth plate chondrocytes. This terminal differentiation leads to phenotypically unstable cartilage and was mirrored in vitro by addition of hypertrophy inducing medium. We investigated how intrinsic TGF-β signaling is altered in pro-hypertrophic conditions. METHODS AND RESULTS: Human bone marrow derived MSC were chondrogenically differentiated in 3D culture. At day 14 medium conditions were changed to 1. pro-hypertrophic by addition of T3 and withdrawal of TGF-β and dexamethasone 2. pro-hypertrophic by addition of BMP 4 and withdrawal of TGF-β and dexamethasone and 3. kept in prochondrogenic medium conditions. All groups were treated with and without TGFβ-type-1-receptor inhibitor SB431542 from day 14 on. Aggregates were harvested for histo- and immunohistological analysis at d14 and d28, for gene expression analysis (rt-PCR) on d1, d3, d7, d14, d17, d21 and d28 and for Western blot analysis on d21 and d28. Induction of hypertrophy was achieved in the pro-hypertrophic groups while expression of TGFβ-type-1- and 2-receptor and Sox 9 were significantly downregulated compared to pro-chondrogenic conditions. Western blotting showed reduced phosphorylation of Smad 2 and 3 in hypertrophic samples, reduced TGF-β-1 receptor proteins and reduced SOX 9. Addition of SB431542 did not initiate hypertrophy under pro-chondrogenic conditions, but was capable of enhancing hypertrophy when applied simultaneously with BMP-4. CONCLUSIONS: Our results suggest that the enhancement of hypertrophy in this model is a result of both activation of pro-hypertrophic BMP signaling and reduction of anti-hypertrophic TGFβ signaling.
Blotting, Western ; Bone Marrow ; Cartilage ; Chondrocytes ; Chondrogenesis ; Dexamethasone ; Down-Regulation ; Gene Expression ; Growth Plate ; Humans ; Hypertrophy ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Phosphorylation

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy. Physiological cell environment not only connects cells to each other, but also connects cells to the extracellular matrix that provide mechanical support, thus exposing the entire cell surface and activating signaling pathways. Hydrogel is a polymeric material that swells in water and maintains a distinct 3-dimensional (3D) network structure by cross linking. In this study, we investigated the optimized cellular function for canine adipose tissue-derived MSCs (cAD-MSCs) using hydrogel. We observed that the expression levels of Ki67 and proliferating cell nuclear antigen, which are involved in cell proliferation and stemness, were increased in transwell-hydrogel (3D-TN) compared to the transwell-normal (TN). Also, transforming growth factor-β1 and SOX9, which are typical bone morphogenesis-inducing factors, were increased in 3D-TN compared to the TN. Collagen type II alpha 1, which is a chondrocyte-specific marker, was increased in 3D-TN compared to the TN. Osteocalcin, which is a osteocyte-specific marker, was increased in 3DTN compared to the TN. Collectively, preconditioning cAD-MSCs via 3D culture systems can enhance inherent secretory properties that may improve the potency and efficacy of MSCs-based therapies for bone regeneration process.
Bone Regeneration ; Cell Proliferation ; Cell- and Tissue-Based Therapy ; Chondrogenesis ; Collagen Type II ; Extracellular Matrix ; Hydrogel ; Hydrogels ; Mesenchymal Stromal Cells ; Osteocalcin ; Osteogenesis ; Polymers ; Proliferating Cell Nuclear Antigen ; Tissue Engineering ; Water

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

For the cartilage repair, the cell sources currently adopted are primarily chondrocytes or mesenchymal stem cells (MSCs). Due to the fact that chondrocytes dedifferentiate during 2-dimensional (2D) expansion, MSCs are generally more studied and considered to have higher potential for cartilage repair purposes. Here we question if the dedifferentiated chondrocytes can regain the chondrogenic potential, to find potential applications in cartilage repair. For this we chose chondrocytes at passage 12 (considered to have sufficiently dedifferentiated) and the expression of chondrogenic phenotypes and matrix syntheses were examined over 14 days. In particular, the chondrogenic potential of MSCs was also compared. Results showed that the dedifferentiated chondrocytes proliferated actively over 14 days with almost 2.5-fold increase relative to MSCs. Moreover, the chondrogenic ability of chondrocytes was significantly higher than that of MSCs, as confirmed by the expression of a series of mRNA levels and the production of cartilage extracellular matrix molecules in 2D-monolayer and 3-dimensional (3D)-spheroid cultures. Of note, the significance was higher in 3D-culture than in 2D-culture. Although more studies are needed such as the use of different cell passages and human cell source, and the chondrogenic confirmation under in vivo conditions, this study showing that the dedifferentiated chondrocytes can also be a suitable cell source for the cell-based cartilage repair, as a counterpart of MSCs, will encourage further studies regarding this issue.
Animals ; Cartilage ; Chondrocytes* ; Chondrogenesis ; Extracellular Matrix ; Humans ; Mesenchymal Stromal Cells ; Phenotype ; Rats* ; RNA, Messenger

The aim of this study was to prepare inclusion nanocomplexes of hyaluronic acid-β-cyclodextrin and simvastatin (HA-β-CD/SIM) and evaluate in vitro anti-inflammation effects on lipopolysaccharide (LPS)-activated synoviocytes and chondrogenic differentiation effects on rat adipose-derived stem cells (rADSCs). The β-CD moieties in HA-β-CD could incorporate SIM to form HA-β-CD/SIM nanocomplexes with diameters of 297–350 nm. HA-β-CD/SIM resulted in long-term release of SIM from the nanocomplexes for up to 63 days in a sustained manner. In vitro studies revealed that HA-β-CD/SIM nanocomplexes were able to effectively and dose-dependently suppress the mRNA expression levels of proinflammatory markers such as matrix metallopeptidase-3 (MMP-3), MMP-13, cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) in LPS-stimulated synoviocytes. HA-β-CD/SIM-treated rADSCs significantly and dose-dependently enhanced mRNA expressions of aggrecan, collagen type II (COL2A1), and collagen type X (COL10A1), implying that HA-β-CD/SIM greatly induced the chondrogenic differentiation of rADSCs. Conclusively, HA-β-CD/SIM nanocomplexes will be a promising therapeutic material to alleviate inflammation as well as promote chondrogenesis.
Aggrecans ; Animals ; Chondrogenesis ; Collagen Type II ; Collagen Type X ; Cyclooxygenase 2 ; In Vitro Techniques* ; Inflammation ; Interleukin-6 ; Rats ; RNA, Messenger ; Simvastatin* ; Stem Cells ; Thrombospondins ; Tumor Necrosis Factor-alpha