sepharose and Osteoarthritis

Articular cartilage exhibits reduced self-healing following degeneration. This research evaluated the effects of hydrogels derived from various polysaccharides-gellan gum (GG), alginate, and agarose-on cartilage regeneration compared with that of hyaluronic acid (HA), which is commonly used in cartilage tissue engineering. Chondrocytes were isolated from the articular cartilage of New Zealand White (NZW) rabbits and stimulated with IL-1β followed by incubation with polysaccharides. The expressions of NF-κB and Cox-2 were decreased and those of IκBα, Sox-9, aggrecan, and type II collagen were increased in HA, GG, and Alginate groups. Osteochondral defects in NZW rabbits were treated with intra-articular polysaccharide injections; all except alginate resulted in tissue regeneration. Significant improvements were observed in cartilage regeneration in the GG and agarose groups. These results show that GG and agarose improve cartilage regeneration by suppressing inflammatory mediators and inducing cartilage formation and autophagy-related gene expression, indicating their potential for cartilage tissue engineering.

Topics: Alginates; Animals; Autophagy; Biomarkers; Cartilage, Articular; Cell Survival; Cells, Cultured; Chondrocytes; Chondrogenesis; Cross-Linking Reagents; Disease Models, Animal; Gene Expression Regulation; Hyaluronic Acid; Hydrogels; Inflammation; Male; Osteoarthritis; Polysaccharides; Polysaccharides, Bacterial; Rabbits; Regeneration; Rheology; RNA, Messenger; Sepharose; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Cell-based therapies have made an impact on the treatment of osteoarthritis; however, the repair and regeneration of thick cartilage defects is an important and growing clinical problem. Next-generation therapies that combine cells with biomaterials may provide improved outcomes. We have developed modular microenvironments that mimic the composition of articular cartilage as a delivery system for consistently differentiated cells.. Human bone marrow-derived mesenchymal stem cells (MSC) were embedded in modular microbeads consisting of agarose (AG) supplemented with 0%, 10% and 20% collagen Type II (COL-II) using a water-in-oil emulsion technique. AG and AG/COL-II microbeads were characterized in terms of their structural integrity, size distribution and protein content. The viability of embedded MSC and their ability to differentiate into osteogenic, adipogenic and chondrogenic lineages over 3 weeks in culture were also assessed.. Microbeads made with <20% COL-II were robust, generally spheroidal in shape and 80 ± 10 µm in diameter. MSC viability in microbeads was consistently high over a week in culture, whereas viability in corresponding bulk hydrogels decreased with increasing COL-II content. Osteogenic differentiation of MSC was modestly supported in both AG and AG/COL-II microbeads, whereas adipogenic differentiation was strongly inhibited in COL-II containing microbeads. Chondrogenic differentiation of MSC was clearly promoted in microbeads containing COL-II, compared with pure AG matrices.. Inclusion of collagen Type II in agarose matrices in microbead format can potentiate chondrogenic differentiation of human MSC. Such compositionally tailored microtissues may find utility for cell delivery in next-generation cartilage repair therapies.

Topics: Biocompatible Materials; Cartilage, Articular; Cell Differentiation; Cell- and Tissue-Based Therapy; Cells, Cultured; Chondrogenesis; Collagen Type II; Humans; Mesenchymal Stem Cells; Microspheres; Osteoarthritis; Osteogenesis; Sepharose; Wound Healing

In articular cartilage, chondrocytes reside within a gel-like pericellular matrix (PCM). This matrix provides a mechanical link through which joint loads are transmitted to chondrocytes. The stiffness of the PCM decreases in the most common degenerative joint disease, osteoarthritis. To develop a system for modeling the stiffness of both the healthy and osteoarthritic PCM, we determined the concentration-stiffness relationships for agarose. We extended these results to encapsulate chondrocytes in agarose of physiological stiffness. Finally, we assessed the relevance of stiffness for chondrocyte mechanotransduction by examining the biological response to mechanical loading for cells encapsulated in low- and high-stiffness gels. We achieved agarose equilibrium stiffness values as large as 51.3 kPa. At 4.0% agarose, we found equilibrium moduli of 34.3 ± 1.65 kPa, and at 4.5% agarose, we found equilibrium moduli of 35.7 ± 0.95 kPa. Cyclical tests found complex moduli of ~100-300 kPa. Viability was >96% for all studies. We observed distinct metabolomic responses in >500 functional small molecules describing changes in cell physiology, between primary human chondrocytes encapsulated in 2.0 and 4.5% agarose indicating that the gel stiffness affects cellular mechanotransduction. These data demonstrate both the feasibility of modeling the chondrocyte pericellular matrix stiffness and the importance of the physiological pericellular stiffness for understanding chondrocyte mechanotransduction.

Topics: Biomechanical Phenomena; Chondrocytes; Extracellular Matrix; Humans; Mechanotransduction, Cellular; Metabolomics; Models, Biological; Osteoarthritis; Sepharose

Limited blood supply and the avascular nature of articular cartilage restricts its self repair capacity, frequently leading to osteoarthritis. This work focuses on scaffolds for tissue repair from natural polymers, for example gelatin, chitosan, and agarose in the form of composite. A novel way of fabrication, known as cryogelation, is presented, in which matrices are synthesized at sub-zero temperature. Cell seeded scaffolds incubated under appropriate conditions result in the accumulation of matrix components on the surface of the gel in the form of neo-cartilage. Neo-cartilage exhibits similarity to native cartilage with respect to its physical, mechanical and biochemical properties. Based on the similarities of neo-cartilage to the native cartilage, it can provide a new approach for the treatment of localised joint injuries.

Topics: Cartilage; Chitosan; Chondrocytes; Cryogels; Extracellular Matrix; Gelatin; Humans; Joints; Osteoarthritis; Polymers; Sepharose; Tissue Engineering; Tissue Scaffolds

The goal of this study was to assess the incorporation of exudates of human platelet-rich fibrin (hPRF) that is abundant in platelet cytokines and growth factors into biodegradable fibrin (FB) scaffolds as a regeneration matrix for promoting chondrocyte proliferation and re-differentiation. hPRF was obtained from human blood by centrifugation without an anticoagulant, and the exudate of hPRF was collected and mixed with bovine fibrinogen, and then thrombin was added to form the FB scaffold. Proliferation and differentiation of human primary chondrocytes and a human chondrosarcoma cell line, the SW-1353, embedded in the three-dimensional (3D) scaffolds and on the two-dimensional (2D) surface of the FB scaffolds so produced were evaluated in comparison with an agarose (AG) scaffold serving as the control. Results demonstrated that the amounts of these cytokines and growth factors in hPRF exudates were higher than those in the blood-derived products except for TGF-β1. Chondrocytes and SW1353 cells on the 2D and 3D FB scaffolds with the addition of the exudates of PRF exhibited more-available proliferation and differentiation than cells on 2D and 3D FB and AG scaffolds. It was concluded that FB scaffolds can provide an appropriate environment for chondrocyte proliferation and re-differentiation, and it could be improved by adding exudates of hPRF. These 3D scaffolds have great promise for cartilage tissue engineering.

Topics: Animals; Biocompatible Materials; Blood Platelets; Cartilage; Cattle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chondrocytes; Collagen; Enzyme-Linked Immunosorbent Assay; Exudates and Transudates; Fibrin; Humans; Intercellular Signaling Peptides and Proteins; Osteoarthritis; Sepharose; Tissue Engineering

Mechanical loading and the fibronectin fragments (FN-fs) are known to stimulate the anabolic and catabolic processes in articular cartilage, possible through pathways mediated by *NO. This study examined the combined effects of dynamic compression and the NH(2)-hep I or COOH-hep II FN-fs on the expression levels of iNOS and COX-2 and production of *NO and PGE(2) release. Both types of fragments induced iNOS and COX-2 expression and stimulated the production of *NO release. This response was inhibited by dynamic compression. Inhibitor experiments indicated that both dynamic compression and the iNOS inhibitor were important in restoring cell proliferation and proteoglycan synthesis in the presence of the FN-fs. This is the first study which demonstrates a downregulation of the FN-f-induced iNOS and COX-2 expression by dynamic compression. The combination of mechanical and pharmacological interventions makes this study a powerful tool to examine further the interactions of biomechanics and cell signalling in osteoarthritis.

Topics: Animals; Biomechanical Phenomena; Cartilage, Articular; Cattle; Cell Proliferation; Chondrocytes; Cyclooxygenase 2; Down-Regulation; Fibronectins; Models, Biological; Nitric Oxide Synthase Type II; Osteoarthritis; Sepharose; Signal Transduction; Stress, Mechanical

The aim of this study was to assess whether enzymatically isolated chondrons from normal adult articular cartilage could be used as a model for the onset of osteoarthritis, by comparison with mechanically extracted chondrons from osteoarthritic cartilage. Enzymatically isolated chondrons (EC) were cultured for 4 weeks in alginate beads and agarose gel constructs. Samples were collected at days 1 and 2, and weekly thereafter. Samples were immunolabelled for types II and VI collagen, keratan sulphate and fibronectin and imaged using confocal microscopy. Mechanically extracted chondrons (MC) were isolated, immunohistochemically stained for type VI collagen and examined by confocal microscopy. In culture, EC showed the following characteristics: swelling of the chondron capsule, cell division within the capsule and remodelling of the pericellular microenvironment. This was followed by chondrocyte migration through gaps in the chondron capsule. Four types of cell clusters formed over time in both alginate beads and agarose constructs. Cells within clusters exhibited quite distinct morphologies and also differed in their patterns of matrix deposition. These differences in behaviour may be due to the origin of the chondrocytes in the intact tissue. The behaviour of EC in culture paralleled the range of morphologies observed in MC, which presented as single and double chondrons and large chondron clusters. This preliminary study indicates that EC in culture share similar structural characteristics with MC isolated from osteoarthritic cartilage, confirming that some processes that occur in osteoarthritis, such as pericellular remodelling, take place in EC cultures. The study of EC in culture may therefore provide an additional tool to investigate the mechanisms operating during the initial stages of osteoarthritis. Further investigation of specific osteoarthritic phenotype markers will, however, be required in order to validate the value of this model.

Topics: Alginates; Animals; Cartilage, Articular; Cell Movement; Chondrocytes; Collagen Type II; Collagen Type IV; Dogs; Fibronectins; Gels; Immunohistochemistry; Keratan Sulfate; Microscopy, Confocal; Microspheres; Models, Animal; Osteoarthritis; Sepharose; Tissue Culture Techniques

Understanding altered gene expression in osteoarthritic cartilage can lead to new targets for drug intervention. We established a functional assay based on chondrocyte cluster formation, a phenotype associated with osteoarthritis (OA), to screen an OA cartilage gene library. Previous reports have demonstrated that normal chondrocytes grown in suspension culture maintain their chondrocytic phenotype, however, certain growth factors such as basic fibroblast growth factor (bFGF) will induce the cells to proliferate in tight clusters similar to those seen in osteoarthritic cartilage. In this study we validate that overexpression of bFGF by retrovirally transduced normal chondrocytes would similarly induce the proliferation of tight cell clusters. We then used this approach as a basis to set up a functional screen where an entire OA cartilage cDNA library was tranduced into normal chondrocytes to search for other genes that would also induce cluster formation. Seven potential genes were isolated from the OA gene library, including BPOZ, IL-17 receptor C, NADH ubiquinone oxidoreductase, COMP, Soluble carrier 16 (MCT 3), C1r, and bFGF itself. None of the identified genes were upregulated by bFGF, however, all of them upregulated the expression of bFGF suggesting a common pathway. Although cluster formation is not considered to be destructive in OA cartilage, it is consistent with the disease and could yield answers to the altered phenotype. Further studies are needed to elucidate how these genes are linked to the disease state.

Topics: Cartilage, Articular; Cell Aggregation; Cell Division; Cells, Cultured; Chondrocytes; Cytological Techniques; Fibroblast Growth Factor 2; Gene Expression; Gene Expression Profiling; Gene Library; Genetic Vectors; Humans; Osteoarthritis; Retroviridae; Sepharose; Transduction, Genetic; Up-Regulation

The chondron represents the chondrocyte and its pericellular microenvironment and plays an important role in the progression of osteoarthritis. Type VI collagen is preferentially localized in the pericellular microenvironment of adult articular cartilage and increases during osteoarthritis. In this study, we characterized the pericellular sequestration of type VI collagen in long-term chondrocyte-agarose cultures, and assessed the action of interleukin-1 on type VI collagen deposition and assembly. Immunohistochemical and biochemical analysis showed that cultured chondrocytes initiate type VI collagen sequestration immediately upon plating and continue pericellular matrix sequestration in a time dependent manner. Confocal microscopy confirmed the cell surface localization and pericellular accumulation of type VI collagen, while image analysis identified a 'cargo-net like' organization of type VI collagen around each chondrocyte. Quantitative analysis revealed a primary phase of rapid cell division and low levels of type VI collagen sequestration, followed by a secondary phase of relative growth stability and high levels of type VI collagen deposition. Interleukin-1 treated cultures showed increased sequestration and retention of type VI collagen in an expanded microenvironment surrounding the chondrocytes. The data suggests a role for type VI collagen in the differentiation of the pericellular microenvironment in vitro. The increased type VI collagen sequestration promoted by interleukin-1 was consistent with previous studies on osteoarthritic cartilage, and implies a functional role for type VI collagen in the chondron remodeling associated with cartilage degradation.

Topics: Animals; Cartilage, Articular; Cell Count; Cell Division; Cells, Cultured; Chondrocytes; Collagen; Culture Media; Dogs; Gels; Interleukin-1; Microscopy, Confocal; Osteoarthritis; Sepharose