A versatile assay for gelatinases using succinylated gelatin.

A spectrophotometric assay using succinylated gelatin as substrate is described for measuring the catalytic activity of gelatinases. The assay is based on measurement of primary amines exposed as a result of hydrolysis of the substrate by gelatinases. Comparison of hydrolysis by matrix metalloproteinase (MMP) 1, 2, 3, 7, 9 indicated that succinylated gelatin was primarily digested by MMP-2 and -9. The assay is rapid (<60 min), specific, suitable for measuring gelatinolytic activity of enzymes and high volume screening of MMP-2 and -9 inhibitors. Sensitivity of the assay is comparable to that of gelatin zymography, under similar experimental conditions. Thus, the assay combines ease and rapidity of assays based on synthetic peptide substrates with specificity of the gelatin zymography technique.

Transplantation of adenovirally transduced allogeneic chondrocytes into articular cartilage defects in vivo.

Gene transfer to chondrocytes followed by intra-articular transplantation may allow for functional modulation of chondrocyte biology and enhanced repair of damaged articular cartilage. We chose to examine the loss of chondrocytes transduced with a recombinant adenovirus containing the gene for Escherichia coli beta-galactosidase (Ad.RSVntlacZ), followed by transplantation into deep and shallow articular cartilage defects using New Zealand White rabbits as an animal model. A type I collagen matrix was used as a carrier for the growth of the transduced chondrocytes and to retain the cells within the surgically created articular defects. Histochemical analysis of matrices recovered from the animals 1, 3 and 10 days after implantation showed the continued loss of lacZ positive chondrocytes. The number of cells recovered from the matrices was also compared with the initial innoculum of transduced cells present within the matrices at the time of implantation. The greatest loss of transduced cells was observed in the first 24 h after implantation. The numbers of transduced cells present within the matrices were relatively constant between 1 and 3 days postimplantation, but had progressively declined by 10 days postimplantation. These results suggest that transduction of chondrocytes followed by intra-articular transplantation in this rabbit model may enable us to examine the biological effects of focal transgenic overexpression of proteins involved in cartilage homeostasis and repair.

Transplantation of transduced chondrocytes protects articular cartilage from interleukin 1-induced extracellular matrix degradation.

Gene therapy used in the context of delivering a therapeutic gene(s) to chondrocytes offers a new approach for treating chondrocyte-mediated cartilage degradation associated with various human arthropathies including osteoarthritis. In this study, gene delivery to human osteoarthritis chondrocytes in monolayer culture was demonstrated using two adenoviral vectors (Ad.CMVlacZ and Ad.RSVntlacZ) carrying the Escherichia coli beta-galactosidase marker gene, and a third vector (Ad.RSV hIL-1ra) containing the cDNA for human interleukin-1 receptor antagonist. At an moi of 10(3) plaque-forming units/chondrocyte, > 90% of the infected cells stained positive for E. coli beta-galactosidase activity, indicating a high efficiency of transduction. Genetically modified chondrocytes were then transplanted onto the articular surface of osteoarthritic cartilage organ cultures with and without the underlying subchondral bone. Both in situ staining of the cartilage organ cultures for E. coli beta-galactosidase activity and examination by scanning electron microscopy indicated that the transplanted chondrocytes adhered and integrated into the articular surface and continued to express transgenic protein. Chondrocytes transduced with Ad.RSV hIL-1ra and seeded onto the surface of osteoarthritic cartilage secreted high levels of biologically active IL-1 receptor antagonist. The Ad.RSV hIL-1ra-treated cartilage samples were resistant to IL1-induced proteoglycan degradation over 10 d of sustained organ culture. These data demonstrate that transplantation of transduced chondrocytes onto the articular surface protects cartilage from IL-1-induced extracellular matrix degradation.

A protocol for rapid screening of proteoglycan-degrading metalloproteinase inhibitors.

A procedure is described for rapid, high-volume screening of proteoglycan-degrading metalloproteinase inhibitors. The procedure was developed by modifying the proteoglycan-polyacrylamide particle assay for proteases, polysaccharidases (Nagase and Woessner, 1980), and the 1,9-dimethylmethylene blue (DMB) assay for sulfated glycosaminoglycans (Farndale et al., 1982). The following modifications have made the new protocol suitable for evaluating a large number of compounds simultaneously: 1) Pipetting steps were automated using a pipetting robot. 2) Speed and sensitivity of the 1,9-dimethylmethylene assay were increased by scaling down the assay and measuring absorbance using an automatic multiwell plate reader. 3) Data capture and analysis were simplified using computer programs. The modified procedure is rapid, sensitive, reliable, and requires a small sample size. Above all, the procedure is suitable for high-volume screening: approximately 100 compounds can be evaluated in 1 day.

Preparation and high-performance size-exclusion chromatographic (HPSEC) analysis of fluorescein isothiocyanate-hydroxyethyl starch: macromolecular probes of the blood-lymph barrier.

A procedure is described for the preparation of fluorescein isothiocyanate-labeled hydroxyethyl starch (FITC-HES). Chromatographic techniques for the purification and analysis of FITC-HES that include the development of a high-performance size-exclusion chromatographic (HPSEC) technique with a fluorescence spectrophotometer-computer detection system are described. FITC-HES macromolecules have a wide range of hydrodynamic radii (ae greater than 120- less than 10 A) and the substitution ratio of FITC to the size-selected HES macromolecules remains constant throughout the chromatographic range. The predominant isoelectric point (pI) of the multiple acidic isomers of FITC-HES is 4.6. In vitro, the very large FITC-HES macromolecules (greater than 100 A) are rapidly degraded (15 sec) by alpha-amylase in control dog plasma. While most of the large molecules (100-20 A) remain intact for greater than 24 hr, this degradation is not associated with the loss of FITC from HES. In vivo, the rate of this reaction appeared to be accelerated and the degradation of FITC-HES between 0.1 and 12 hr was small. Illustration of the HPSEC quantitation of the spectrum of FITC-HES macromolecules in "near steady-state" lymph (L) and plasma (P) samples and the L/P ratio show that this technique can be used to describe the size selectivity of the blood-lymph barrier under conditions of unaltered capillary pressure. We propose that the size-selected solvent-drag reflection coefficient (sigma f) curves for the anionic FITC-HES under conditions of elevated capillary pressure is a measure of macromolecular convective permeability of the blood-lymph barrier.