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Chondrocytes are the first skeleton-specific cell type to appear during development and are required for the longitudinal growth of the skeleton. Chondrocytes are also needed in joints to allow mobility of various skeletal elements. Cartilage matrix is composed of collagens and proteoglycans, with sparse populations of chondrocytes. Chondrocytes are the growth plate forming cells in the long bones and thus, act in the bone formation processes. Chondrogenesis and endochondral ossification are the cartilage differentiation processes that lead to the skeletogenesis during growth and development of vertebrates as well as skeletal repair in the adults (Karesenty at al., 2009). Chondrocytes are highly sensitive to mechanical, biochemical and other stressful stimuli. Cartilage injury leads to an irreversible cartilage loss as differentiated chondrocytes do not divide and hence do not compensate for the defects. Cartilage in the limbs is most susceptible to the normal and pathological stresses such as osteoarthritis and inflammatory arthritis. In joint pathologies, cytokines are the direct or indirect players in the regulation of inflammation that leads to the irreversible destruction of extracellular matrix of cartilage and bone. Disturbance in the homeostatic balance of anabolic and catabolic, anti- and pro-inflammatory cytokines induces cartilage and bone destructions.
Interleukin 3 (IL-3), a cytokine secreted by activated T lymphocytes stimulates the proliferation, differentiation and survival of pluripotent haematopoietic stem cells (HSC). It is a broadly acting haematopoietic-regulatory protein with activities on a number of cell lineages including macrophages, mast cells, neutrophils, eosinophils and megakaryocytes. Previously we have shown that IL-3 potently and irreversibly inhibits osteoclast differentiation induced by receptor activator of NF-κB ligand (RANKL) and TNF-α (Khapli et al., 2003; Yogesha et al., 2005). IL-3 is a potent inhibitor of bone resorption induced by TNF-¡ and inhibits bone resorption even in the presence of other proinflammatory cytokines such as IL-1a, TGF-b1, TGF-b3, IL-6 and PGE2 (Yogesh et al., 2009). Recently we have observed that IL-3 increases osteoblasts differentiation and bone matrix synthesis in human mesenchymal stem cells derived from bone marrow. IL-3 increases osteoblast differentiation through secretion of bone morphogenetic protein 2 (manuscript under review). IL-3 also showed in vivo anti-inflammatory effect and indirectly protects cartilage and bone damage in inflammatory arthritis (Yogesha et al., 2009). However, how IL-3 protects the cartilage damage is presently not known. Thus, in the present study we propose to investigate the role of IL-3 and other cytokines in differentiation of chondrocytes.
Survey of the work done in the research area and the need for more research
Chondrocytes differentiate from mesenchymal stem cells. Two families of transcription factors play central roles in chondrogenesis and in all aspects of skeletogenesis. These are the Sox and Runx proteins. The role of Sox9, a high mobility group box- containing protein with homology to Sry, in chondrocytes differentiation was first revealed through the molecular elucidation of a rare chondrodysplasia, the Campomelic dysplasia, which is caused by inactivating mutations in this protein (Foster et al., 1994; Wagner et al., 1994). Sox9 is expressed in cells of the mesenchymal condensations and in proliferating chondrocytes, but not in hypertrophic chondrocytes. Subsequently, it was shown through the generation of chimeric mice, Sox9 haplo-insufficient mice, and ultimately mesenchymal cell-specific and chondrocyte- specific Sox 9- deficient mice that Sox9 is necessary for the differentiation of mesenchymal cells into proliferating chondrocytes and for the expression in chondrocytes of the α1 (II) collagen, α (XI) collagen, and aggrecan genes, all markers of nonhypertrophic chondrocytes (Bi et al., 1999; Akiyama et al., 2002). As the mesenchyme differentiates into chondrocytes, the cells begin to produce an extra cellular matrix rich in type II collagen and aggrecan. During development, following early chondrocyte differentiation, the cells rapidly proliferate, enlarging the cartilage templates that preform individual skeletal elements.
Runx2 (runt-related transcription factor 2), a transcription factor important for osteoblast differentiation, also plays important role in chondrocyte hypertrophy. Runx2 expression initially occurs in mesenchymal cells just before their differentiation into chondrocytes, and its expression is maintained in chondrocytes until the cells complete terminal differentiation. Runx2 expression level varies with the differentiation stages of chondrocytes suggesting that Runx2 plays role in the regulation of chondrocyte differentiation in addition to promoting chondrocyte maturation. Overexpression of Runx2 in chondrocytes accelerates chondrocyte maturation and matrix mineralization in vitro and endochondral bone formation in vivo [Enomoto et al., 2000; Takeda et al., 2001; Ueta et al., 2001]. Chondrocyte maturation is disturbed in Runx2-/- mice [Inada et al., 1999; Kim et al., 1999]. Runx2-/- chondrocytes differentiate into adipocytes, accompanied with the loss of chondrocyte phenotype, thus indicating that Runx2 is also involved in maintenance of the chondrocyte phenotype and inhibition of adipogenesis [Enomoto et al., 2003].
Chondrogenic differentiation has been shown to occur when MSCs are cultured in 3D culture format and in serum-free medium supplemented with one or more members of the TGF-β superfamily [Barry et al., 2001]. Under these conditions, cells lose their fibroblastic morphology and begin to express cartilage-specific matrix components. In vitro chondrogenesis is typically carried out in a micromass pellet culture system, which allows cell-cell interactions similar to those occurring in prechondrogenic condensations during embryonic development. Bone morphogenetic proteins (BMPs) form a subgroup of TGF-β superfamily of growth factors. They were originally described by their capacity to induce ectopic bone and cartilage formation in vivo [Peister et al., 2004], and are known to initiate, promote and maintain osteogenesis and chondrogenesis [Nochi et al., 2004; Pizette and Niswander., 2000].
IL-3, a cytokine secreted by Th cells, stimulates proliferation, differentiation, and survival of pluripotent hematopoietic stem cells. The receptor for IL-3 consists of a heterodimer of the IL-3 specific α chain and a common β chain that is shared with GM-CSF and IL-5 [de Groot et al., 1998]. We have previously demonstrated that IL-3 inhibits RANKL [Khapli et al., 2003] and TNF- α -induced osteoclast differentiation in purified mouse osteoclast precursors [Yogesha et al., 2005]. Recently, we have also demonstrated that IL-3 potently and irreversibly inhibits TNF-α induced bone resorption in vitro and prevents development of inflammatory arthritis as well as cartilage and bone loss in mice [Yogesha et al., 2009]. These results indicated the potent inhibitory nature of IL-3 on osteoclast differentiation and bone resorption. IL-3 also enhances osteoblast differentiation and bone formation from mesenchymal stem cells. However, the role of IL-3 on differentiation of chondrocytes is not known. In the proposed work we aim at investigating the role of IL-3 in regulation of chondrogenesis.
Aims and objectives
To standardize the in vitro method of chondrocyte differentiation, and optimal culture conditions for maintenance of functional chondrocytes.
To evaluate the role of IL-3 and other cytokines on regulation of chondrocyte development.
Plan of research and methodologies to be used
Isolation of chondrocytes
Chondrocytes will be isolated from the rib cage and the knee balls of 2-4 days old female mice pups using collagenase enzyme treatment. The cells obtained will be cultured in high glucose DMEM with 100 units/ml penicillin, 100 μg/ml streptomycin and 2mM L-glutamine. Chondrocytes adhere to the plastic surface of the culture vessel and proliferate. The cells will be fed every 2 days and passaged after 80% confluency.
Samples collection, isolation and propagation of MSCs
We will also use MSC for differentiation of chondrocytes. MSCs will be isolated from bone marrow. Bone marrow will be harvested from 6-8 weeks old mice. Nucleated cells will be isolated with a density gradient (Ficoll-paque) and resuspended in culture medium containing αMEM supplemented with 10% FCS, 100 units /ml penicillin, 100μg /ml streptomycin and 2mM L-glutamine. Cells will be incubated for 48-72 hours, and non-adherent cells will be discarded and adherent cells will be washed thoroughly. MSCs proliferate in culture with an attached well-spread morphology. The cells are incubated for 7-10 days and passaged after 80% confluency. For the clonal expansion of MSCs cells will be cloned by limiting dilution. MSCs will also be isolated by sorting of the labeled cells.
Characterization of multipotent MSCs
MSCs will be assessed for the presence of cell surface markers CD29, CD44, CD73, CD90 and CD105; and absence of haemopoietic cell markers CD11b, CD34, and CD45 using FACS and confocal analysis.
Chondrocyte differentiation and characterization
Chondrogenic differentiation has been shown to occur when MSCs are cultured in 3D culture format and in serum-free media (αMEM) containing L- proline and growth factors as ITS and TGF-β3 etc. In vitro chondrogenesis is typically carried out in a micromass pellet culture system, which allows cell-cell interactions similar to those occurring in prechondrogenic condensations during embryonic development. The chondrocyte pellets will be assessed for the specific markers as Sox 9, aggrecan, collagen type II, chondroadherin, collagen type X, BMP-2 and also for the presence of IL-3R alpha.
Effect of IL-3 on chondrocytes differentiation
The approach towards assessing the effect of IL-3 on chondrocytes would be through proliferation and toxicity assays as well as the functional assays. It would also include the expression studies at the gene and protein levels. Finally, the same studies will be done in the respective mice models.
Other techniques to be used
Standard techniques such as cell isolation and culture, RNA isolation and RT-PCR, immunoblotting, confocal microscopy, flow cytometry, differential gene expression analysis by semi-quantitative gene assay by real-time RT-PCR, proliferation and toxicity assays, histochemical assays, in vivo experiments etc will be used in proposed studies.
The kinds of conclusions expected and their possible value
This proposed study will help to understand the mechanisms of differentiation of mesenchymal stem cells into chondrocytes, and may help in developing the therapeutic agents to treat cartilage damage.