PicoGreen can be used in a rapid and sensitive assay to quantify DNA in small samples of human cartilage.

Objective. To determine 1) the kinetics and strength of adhesion of human articular chondrocytes to a cut cartilage surface, and 2) the role of specific integrins in mediating such adhesion, using an in vitro model.Methods. Human articular chondrocytes isolated from cadaveric donors (mean ؎ SD age 38 ؎ 13 years) were cultured in high-density or low-density monolayer. Following release from culture with trypsin and a 2-2.5-hour recovery period, chondrocytes were analyzed either for adhesion to cartilage or for integrin expression by flow cytometry.Results. Following culture in monolayer, adhesion of chondrocytes to cartilage increased with time, from 6-16% at 10 minutes to a maximum of 59-82% at 80-320 minutes. After 80 minutes of adhesion, the resistance of cells to flow-induced shear stress (50% detachment) was ϳ21 Pa. Chondrocyte adhesion to cartilage decreased with pretreatment of cells with monoclonal antibodies that bound to and blocked certain integrins. After an 80-minute incubation time, adhesion of chondrocytes cultured in high-density monolayer decreased from the value of IgG1-treated controls (55%) with blocking of the ␤1 integrin subunit (to 23%) or with blocking of ␣5␤1 (to 36%). Following expansion of chondrocytes in low-density monolayer, the mechanisms of adhesion to cartilage were generally similar. After an 80-minute incubation time, adhesion of chondrocytes cultured in low-density monolayer decreased from the value of IgG1-treated controls (62%) with blocking of the ␤1 integrin subunit (to 30%) or with blocking of ␣5␤1 (to 44%). Additionally, adhesion of these cells decreased to 46% by blocking of ␣v␤5, with a similar trend in effect for chondrocytes cultured in high-density monolayer. Blocking of the ␣1 or ␣3 integrin subunits or ␣v␤3 had no detectable effect on adhesion, even though these receptors were detected by flow cytometry.Conclusion. Under the culture and seeding conditions studied, ␤1, ␣5␤1, and ␣v␤5 integrins mediate human chondrocyte adhesion to cartilage. These chondrocyte integrins have a potential role in the initial adhesion and retention of chondrocytes at a cartilage defect site following clinical procedures of chondrocyte transplantation.

The initial adhesion of transplanted chondrocytes to surrounding host cartilage may be important in the repair of articular defects. Adhesion may position cells to secrete molecules that fill the defect and integrate repair tissue with host tissue. While chondrocytes are known to become increasingly adherent to cartilage with time, the molecular basis for this is unknown. The objective of this study was to investigate the role of (31-integrin, CD44, and annexin V receptors in chondrocyte adhesion to cartilage. Chondrocytes were cultured in high density monolayer, released with trypsin, and allowed to recover in suspension for 2 h at 37°C. Under these conditions, flow cytometry analysis showed that chondrocytes expressed (31-integrins, CD44, and annexin V. In a rapid screening assay to assess chondrocyte adhesion to cartilage, cell detachment decreased from 79% at 10 min following transplantation to 10% at 320 min. Treatment of cells with a monoclonal antibody to block p1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls. Similarly, results from a parallel-plate shear flow adhesion assay showed that blocking Dl -integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls at each level of applied shear (0-70 Pa). In both assays, treatment of cells with reagents that block CD44 (hyaluronan oligosaccharides or monoclonal Ab IM7) or annexin V (polyclonal Ab #8958) had no detectable effect on adhesion. With cartilage treated with chondroitinase ABC, blocking pl -integrins also increased chondrocyte detachment, while blocking CD44 and annexin V also had no detectable effect. Under the conditions studied here, 01-integrins appear to mediate chondrocyte adhesion to a cut cartilage surface. Delineation of the mechanisms of adhesion may have clinical implications by allowing cell manipulations or matrix treatments to enhance chondrocyte adhesion and retention at a defect site.

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