A multivalent assay to detect glycosaminoglycan, protein, collagen, RNA, and DNA content in milligram samples of cartilage or hydrogel-based repair cartilage.
The biochemical measure of success in assisted cartilage repair is normally judged by repair tissue cell density, mRNA and protein expression, and accumulation of extracellular matrix molecules. Existing methods to solubilize cartilage matrix proteoglycans and cellular DNA for quantification, such as papain digestion, often destroy one or more species of the above-named parameters, in order to render others measurable. We have therefore developed a methodology to measure specific levels of mRNA, protein, DNA, glycosaminoglycan, and collagen content on single pulverized 10-mg samples of cartilage, or tissue-engineered cartilage, using successive extractions in concentrated guanidine hydrochloride (GuCl) and guanidine thiocyanate (GITC) solutions. Conditions were developed to solubilize most cellular proteins, DNA, proteoglycans, and some matrix proteins with an initial GuCl extraction step. A subsequent extraction with GITC was essential to solubilize the majority of the cellular RNA. Guanidine-insoluble material was rendered soluble by papain digestion, to enable quantification of collagen, residual glycosaminoglycan, and residual unextracted DNA in individual samples. In general, total collagen, GAG, and DNA content measured in multivalent-extracted samples was similar to that obtained with samples digested directly with papain. Moreover, we were able to reliably detect, in these same multivalent extracts, expressed mRNA as well as specific cellular and extracellular matrix proteins. This multivalent assay could be applied to a variety of cells cultured in biopolymers and to tissues from which biochemical components may be otherwise difficult to extract.
Hoemann
Caroline D
CD
BioSynTech, 475 Armand-Frappier, Laval, Quebec H7V 4B3, Canada. hoemann@grbb.polymtl.ca
Sun
Jun
J
Chrzanowski
Veronica
V
Buschmann
Michael D
MD
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002356:Cartilage / Q000737:chemistry*; D002417:Cattle; D019902:Chondrocytes / Q000737:chemistry; D003094:Collagen / Q000032:analysis*; D004247:DNA / Q000032:analysis*; D005470:Fluorometry / Q000379:methods; D006025:Glycosaminoglycans / Q000032:analysis*; D006146:Guanidines / Q000737:chemistry; D020100:Hydrogels / Q000737:chemistry; D006909:Hydroxyproline / Q000737:chemistry; D010206:Papain / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D012313:RNA / Q000032:analysis*; D015203:Reproducibility of Results; D012260:Ribonucleases / Q000737:chemistry; D012995:Solubility; D014020:Tissue Extracts / Q000737:chemistry
D006025:Glycosaminoglycans; D006146:Guanidines; D020100:Hydrogels; D011506:Proteins; D014020:Tissue Extracts; D012313:RNA; D003094:Collagen; D004247:DNA; D012260:Ribonucleases; D010206:Papain; D006909:Hydroxyproline