Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen and Hoechst 33258.

OBJECTIVE: To compare two fluorometric assays, utilizing (1) the bisbenzimidazole Hoechst 33258 and (2) PicoGreen, for determining DNA content in human cartilage. METHODS: Human articular and nasal septal cartilage explants were digested using proteinase K. Portions of sample digest were analysed for intrinsic and dye-enhanced fluorescence with either Hoechst 33258 or PicoGreen. RESULTS: Intrinsic tissue fluorescence in both articular and septal cartilage increased with age and was prominent at wavelengths used for Hoechst 33258 but relatively low at wavelengths used for PicoGreen. The relative contribution of intrinsic fluorescence to total dye-enhanced fluorescence of human cartilage was markedly greater for Hoechst 33258 (19-57%) than for PicoGreen (2-7%). Thus, in many situations, DNA in human cartilage can be assayed using PicoGreen without the need to correct for intrinsic cartilage fluorescence. The enhancement of fluorescence by each dye was found to be specific for DNA, as shown by fluorescence spectra, >90% sensitivity to DNase, and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in the absence or presence of proteinase K digested human cartilage, once intrinsic cartilage fluorescence was subtracted. PicoGreen was more sensitive for assaying DNA (0.9ng DNA/ml) than Hoechst 33258 (6ng DNA/ml) and can also be used in a microplate reader. CONCLUSION: PicoGreen can be used in a rapid and sensitive assay to quantify DNA in small samples of human cartilage.

Mechanisms of chondrocyte adhesion to cartilage: role of beta1-integrins, CD44, and annexin V.

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 beta1-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 degrees C. Under these conditions, flow cytometry analysis showed that chondrocytes expressed beta1-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 beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls. Similarly, results from a parallel-plate shear flow adhesion assay showed that blocking beta1-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 beta1-integrins also increased chondrocyte detachment, while blocking CD44 and annexin V also had no detectable effect. Under the conditions studied here, beta1-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.

Adhesive force of chondrocytes to cartilage. Effects of chondroitinase ABC.

Chondrocyte transplantation is a clinical procedure for cartilage repair. Transplanted cells may have difficulty attaching to the surface of chondral lesions because of the anti-adhesive properties of the proteoglycan rich matrix. This study used micromanipulation methods to determine if pretreatment of cartilage with chondroitinase ABC affects chondrocyte adhesion to cartilage and if chondrocytes adhere preferentially to the superficial, middle, or deep layers of cartilage. Bovine chondrocytes were transplanted in vitro on articular cartilage sections cut perpendicular to the articular surface. At various times between 15 and 75 minutes after seeding, a micropipette micromanipulation system was used to measure the adhesion force of individual chondrocytes to cartilage. The chondrocyte adhesion force increased with chondroitinase ABC treatment and seeding time but generally was similar for the different regions of articular cartilage (superficial, middle, deep layer) to which the cells were attached. For normal cartilage, the adhesion force increased from 1.29 +/- 0.24 mdyne after 15 to 30 minutes seeding to 5.29 +/- 0.25 mdyne after 60 to 75 minutes. Treatment with chondroitinase ABC at certain concentrations and durations (1.0 U/mL for 5 minutes or 0.5 or 1 U/mL for 15 minutes) led to an increase in adhesion force, whereas relatively low concentration or treatment time (0.25 U/mL for 15 minutes or 0.5 U/mL for 5 minutes) had little or no detectable effect. The increase in adhesion attributable to chondroitinase ABC treatment appeared most marked (+144% to +292%) for short (15 to 30 minutes) seeding durations but was still significant (+46%) for the longest seeding period (60 to 75 minutes) studied after the 1 U/mL for 15 minute treatment condition. These results provide direct biomechanical evidence that enzymatic treatment of a cartilage surface can enhance chondrocyte adhesion.

Effect of seeding duration on the strength of chondrocyte adhesion to articular cartilage.

Chondrocyte adhesion to cartilage may play an important role in the repair of articular defects by maintaining cells in positions where their biosynthetic products can contribute to the repair process. The objective of this in vitro study was to determine the effect of the duration of seeding time on the ability of chondrocytes to resist detachment from cartilage when subjected to mechanical perturbation (fluid-induced shear stress). Suspensions of adult bovine articular chondrocytes were prepared from primary, high-density monolayer cultures and infused into a parallel-plate shear-flow chamber where they settled onto 50-microm-thick sections of bovine articular cartilage at a density of approximately 20,000 cells/cm2. The chondrocytes were seeded and allowed to attach to the cartilage surface for specific durations (5-40 minutes) in medium including 10% serum at 22 degrees C, after which the cells were exposed to fluid flow-induced shear stresses (6-90 Pa). The fraction of detached cells at each shear stress was calculated from microscopic images. Shear stress was applied for 1 minute because this length of time was sufficient to induce steady-state cell detachment. Increasing the duration of cell seeding led to a more firm attachment of chondrocytes to cartilage. After 9 minutes of seeding, 50% cell detachment was induced by gravitational force alone. After 40 minutes of seeding, 50% detachment required 26 Pa of shear stress. Extrapolation of the data to account for the effect of repeated applications of cell suspensions to an individual cartilage substrate indicated that for a freshly prepared cartilage section, 50% detachment was induced by gravity after 25 minutes of seeding and by 2.3 Pa of shear stress after 40 minutes of seeding. The increase in resistance to shear stress-induced cell detachment with increasing seeding duration suggests that it may be beneficial to allow chondrocytes to stabilize in the absence of applied load for some time after chondrocyte transplantation for cartilage repair in vivo.