A Multivalent Assay to Detect Glycosaminoglycan, Protein, Collagen, RNA, and DNA Content in Milligram Samples of Cartilage or Hydrogel-Based Repair Cartilage (1)
Mature cartilage tissue is composed mainly of insoluble collagen and large aggregating proteoglycan, with only 1 to 5% of the tissue volume being occupied by cells. Since the ability of cartilage to withstand compressive forces is directly proportional to its GAG and collagen content, the levels of these two matrix components are a most crucial determinant of repair tissue properties.
All soluble cell and matrix proteins appeared to become solubilized by a single extraction in buffered 4M GuCl. In all samples tested, most of the RNA remained insoluble in 4 M GuCl, but could be subsequently extracted in 4 M GITC. Most of the GAG and DNA that could be extracted by guanidine salt solutions was extracted in the first GuCl extraction, with a 30-min vortex period showing more complete extraction than a 5-min vortexing time.
Papain digests of cells in chitosan, at the recommended pH 6.5, carried no detectable DNA. Increasing the pH to 7.5, where chitosan should bear little charge, rendered the cellular DNA quantifiable with Hoechst 33258. Papain-digested chitosan and agarose samples gave a lower apparent DNA content than expected, based on the number of cells cast in the gel. For cartilage samples, the primary GuCl extract had incomplete DNA recovery compared with papain-digested cartilage. The GuCl extracts of pulverized cartilage consistently harbored around 60% of the total cellular DNA detected in papain digests; however, the remaining DNA was largely recovered in the papain-digested guanidine-insoluble pellet.
As was observed for the DNA assay, papain digests of chitosan-encapsulated chondrocytes at pH 6.5 failed to yield any detectable GAG, presumably due to an inhibitory complex formed between negatively charged GAG and chitosan which carries a net positive charge at pH 6.5. Raising the papain digestion to pH 7.5 yielded soluble GAG that was detected in the DMMB assay. A single GuCl extraction of adult articular cartilage contained 90% of those GAG levels detected in the combined levels in GuCl, GITC, and papain-digested insoluble pellets. Re-extraction of the primary GuCl-insoluble pellet in either GuCl or GITC yielded at most 9%. more GAG. The combined GAG content of guanidine-soluble and guanidine-insoluble GAG gave similar values to those obtained from papain-digested cartilage. soluble GAG levels could be determined from a single 4 M GuCl extract of hydrogel-cultured chondrocytes or cartilage. Additionally, insoluble and most probably cross-linked GAG could be determined from papain digests of guanidine-insoluble pellets. The collagen present in a 3-week culture is around 50% cross-linked. In calf and adult cartilage, guanidine-insoluble collagen comprised around 80% of the collagen detected in papain digests of parallel samples. The reduced collagen yield in the guanidine-insoluble pellets relative to the papain-digested samples could also be partly due to guanidine solubilization of other collagen types (types IX and XI).
Similar type II collagen RNA expression levels in all samples, whereas aggrecan RNA expression was slightly augmented in chitosan-cultured chondrocytes in vitro. Proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, was only detected in cultured chondrocytes and calf cartilage.
For SDS–PAGE, samples must first be precipitated with ethanol to remove guanidium salts, to prevent precipitation of SDS detergent in the sample buffer. The Hoescht DNA and DMMB GAG assays, and GITC was found to interact colorimetrically with the DMMB reagent in the GAG assay, prohibiting the direct analysis of GITC extracts by DMMB assay. The chitosan polymer was highly soluble in 4 M GITC than in 4 M GuCl, with several negative consequences of GITC solubilization of chitosan. EDTA present in the Hoechst assay caused precipitation of soluble chitosan, causing an aberrant fluorescent signal. Storing papain digests at 280°C caused a large amount of precipitation upon thawing and loss of up to 50% of the original DNA fluorescence. If fresh cultured cells were lysed in 4 M GuCl, up to 50% of the DNA failed to become solubilized, when comparing the DNA yield with that obtained using papain-digested cells or frozen lysed cells. Freezing the cells prior to extraction in 4 M GuCl resulted in full DNA recovery.