Human Chondrocyte Cultures as Models of Cartilage-Specific Gene Regulation

Abstract

The human adult articular chondrocyte is a unique cell type that has reached a fully differentiated state as an end point of development. Within the cartilage matrix, chondrocytes are normally quiescent and maintain the matrix constituents in a low-turnover state of equilibrium. Isolated chondrocytes in culture have provided useful models to study cellular responses to alterations in the environment such as those occurring in different forms of arthritis. However, expansion of primary chondrocytes in monolayer culture results in the loss of phenotype, particularly if high cell density is not maintained. This chapter describes strategies for maintaining or restoring differentiated phenotype by culture in suspension, gels, or scaffolds. Techniques for assessing phenotype involving primarily the analysis of synthesis of cartilage-specific matrix proteins as well as the corresponding mRNAs are also described. Approaches for studying gene regulation, including transfection of promoter-driven reporter genes with expression vectors for transcriptional and signaling regulators, chromatin immunoprecipitation, and DNA methylation are also described.

Human Osteoarthritic Cartilage Shows Reduced In Vivo Expression of IL-4, a Chondroprotective Cytokine that Differentially Modulates IL-1β-Stimulated Production of Chemokines and Matrix-Degrading Enzymes In Vitro

“…Little is currently known, however, regarding the ability of IL-4 to provide a protective, anti-inflammatory action in human cartilage. To undertake this section of the study we adopted a high-density culture model of primary chondrocytes, used within the first passages as a universally accepted method to regain a differentiated phenotype [36], [43]. This was confirmed by SOX-9 protein expression and expression of all the IL-4 receptor subunits, particularly the IL-2R γ chain at both RNA and protein levels.…”

Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA)

“…Consequently, further expansion and consecutive passages are needed, which on the other hand can lead to dedifferentiation (Mirando et al, 2014; Shortkroff & Spector, 1999; Thirion & Berenbaum, 2004). The latter is evident by morphological changes of the cells from polygonal to more elongated, as well as through a reduction in the growth rate (Cetinkaya et al, 2011; Haudenschild et al, 2001; Otero et al, 2012). For example, development of novel scaffolds for cartilage tissue engineering often requires a million cells per sample scaffold (the number depends on the size of the scaffold to be tested), exposing the high demand for cells and at the same time one of the major bottlenecks in development of novel tissue engineering solutions.…”

“…Therefore, it comes to no surprise that novel approaches for chondrocyte isolation are highly desired, especially considering the high prices of ordered cells. Optimisation of isolation yields, abundant cell sources and efficient culturing procedures that lead to preparation of desired, reproducible and relatively affordable cell cultures or/and material-cell constructs with good durability are therefore highly rated novelties in recent research (Dehne et al, 2009; Naranda et al, 2016; Otero et al, 2012). …”

“…More specifically, this analysis should include the evaluation expression of genes related to cartilage specific markers (e.g., collagen type 2 and aggrecan). To follow-up possible dedifferentiation towards the fibroblastic phenotype (Duan et al, 2015; Goldring, Tsuchimochi & Ijiri, 2006; Haudenschild et al, 2001; Makris et al, 2015; Otero et al, 2012), we propose simultaneous measurement of up-regulation of collagen type 1.…”

An update on the pathophysiology of osteoarthritis

“…Its main function is to provide a smooth, lubricated surface for articulation and to facilitate the transmission of loads with a low frictional coefficient (19). Throughout life, cartilage is continually remodeled as chondrocytes replace the degraded matrix macromolecules with newly synthesized components, although it is recognized that this is an exceptionally slow process in adults; proteoglycan turnover can take up to 2 decades whereas the half-life of collagen is estimated to range from several decades to more than 100 years (20)(21)(22). Although articular cartilage can tolerate a tremendous amount of intensive and repetitive physical stress, it manifests a striking inability to heal even a minor injury (2 (23), it has inflammatory and metabolic components (1,16,(24)(25)(26)(27).…”