Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of Fetal Calf Serum

Abstract

Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood, however, pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology, and clustering of cultured bovine articular chondrocytes within stiff (2% w/v) and soft (0.2% w/v) three-dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; fetal calf serum (FCS) 1-10%;380 mOsm) for up to 7 days. Chondrocytes were fluorescently labeled after 1, 3, and 7 days with 5-chloromethylfluorescein-diacetate (CMFDA) and propidium iodide (PI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology, and clustering were quantified using Volocity™ software. In stiff gels after 7 d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However, in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P < 0.0001) and enhanced clustering (P < 0.05), compared to stiff gels. FCS exacerbated changes to density (P < 0.01), abnormal morphology (P < 0.001) and clustering (P < 0.01) compared to lower concentrations at the same gel strength. Reduced gel stiffness and/or increased FCS concentrations promoted chondrocyte proliferation and clustering, increased cell volume, and stimulated abnormal morphology, producing similar changes to those occurring in OA. The increased penetration of factors in FCS into soft gels may be important in the development of these abnormal chondrocyte properties. J. Cell. Physiol. 232: 1041-1052, 2017. © 2016 Wiley Periodicals, Inc.

The clustering and morphology of chondrocytes in normal and mildly degenerate human femoral head cartilage studied by confocal laser scanning microscopy

“…), as its depletion around chondrocytes could provide a weak area into which process(es) could develop. For example, chondrocytes cultured in soft, as opposed to stiff, agarose gels produce cytoplasmic processes with similar structures to those described in the present study (Karim & Hall, ). Alternatively, the development of processes could result from mechanically injured chondrocytes leading to an ‘active’ metabolic response and resulting in the release of cytokines/degradative enzymes and breakdown of the pericellular matrix.…”

The Role of Chondrocyte Morphology and Volume in Controlling Phenotype—Implications for Osteoarthritis, Cartilage Repair, and Cartilage Engineering

“…CLSM studies on fluorescently-labelled chondrocytes show similar processes also occasionally present after 7 days in stiff (2%) 3D agarose cultures containing FCS. However, their incidence rises markedly in soft (0.2%) agarose gels [ 42 ]. It is possible that the penetration of the growth/mitogenic factors in FCS into cartilage and stiff gels is severely restricted, but when the matrix or gel is weakened (by scalpel injury or culture in soft agarose gel) these factors can penetrate and stimulate chondrocyte morphological changes.…”

Investigating materials and orientation parameters for the creation of a 3D musculoskeletal interface co-culture model

“…Importantly, as viability is expressed as a percentage, this does not necessarily indicate an increase in cell number, rather a change in the ratio of live: dead cells present. A potential reason for this could be the presence of enzymes in the foetal bovine serum present in supplemented growth medium that can act to digest dead cellular DNA and therefore alter the dead cell ratio found through the quantitative assessment [ 36 , 37 ]. This was investigated over a period of 4 days with no significant effect (data not shown) but remains to be a potential factor that was influencing these cell populations.…”

“…9B ). Agarose has been used as a 3D scaffold before [ 36 ] and other studies have reported conflicting reports about cell viability in agarose. Ise et al reported an increase in hepatocytes proliferation and viability after 21 days of culture when encapsulate in 3% agarose [ 38 ].…”