Abstract
The procedure described below is useful for extracting proteins, nucleic acids, and glycosaminoglycans from 5–40 mg of cartilage or tissue-engineered cartilage samples. This extraction method will generate samples compatible with Western blot, RNase protection, dimethyl methylene blue (DMB) assay for glycosaminoglycan, Hoechst DNA assay, and hydroxyproline assay. Most soluble matrix molecules can be extracted from pulverized samples using 4 M guanidine HCl, during a 30-min period of vortex agitation at 4°C. Shorter agitation times can give inadequate solubilization. The guanidine HCl-insoluble pellet must be re-extracted with guanidine thiocyanate buffer, to solubilize RNA additionally. The final insoluble pellet can be rinsed with ethanol and digested with papain, to quantify collagen content as well as other insoluble or crosslinked material. Samples between 1 and 5 mg may be directly digested with a small volume of papain buffer for DMB, hydroxyproline, and Hoechst DNA assays.
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References
Farndale, R. W., Buttle, D. J., and Barrett, A. J. (1986) Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochem. Biophys. Acta 883, 173–177.
Kim Y._ J., Sah, R. L., Doong, J.-Y. H., and Grodzinsky, A. J. (1988) Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal. Biochem. 174, 168–176.
Farndale, R. W., Sayers, C. A., and Barrett, A. J. (1982) A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect. Tissue Res. 9, 247–248.
Chandrasekhar, S., Esterman, M. A., and Hoffman, H. A. (1987) Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal. Biochem. 161, 103–108.
Stegemann, H. and Stalder, K. (1967) Determination of hydroxyproline. Clin. Chim. Acta 18, 267–273.
Woessner, J. F. (1961) The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophys. 93, 440–447.
Burleigh, M. C., Barrett, A. J., and Lazarus, G. S. (1974) A lysosomal enzyme that degrades native collagen. Biochem. J. 137, 387–398.
Chomczynski, P. and Mackey, K. (1995) Modification of the tri reagent procedure for isolation of RNA from polysaccharide-and proteoglycan-rich sources. Biotechniques 19, 942–945.
Haines, D. S. and Gillespie, D. H. (1992) RNA abundance measured by a lysate RNase protection assay. Biotechniques 12, 736–741.
Binette, F., McQuaid, D. P., Haudenschild, D. R., Yaeger, P. C., McPherson, J. M., and Tubo, R. (1998) Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro. J. Orthop. Res. 16L, 207–216.
Hoemann, C. H., Sun, J., Chrzanowski, V., and Buschmann, M. D. (2002) A multivalent assay to detect DNA, RNA, glycosaminoglycan, protein, and collagen content of milligram samples of cartilage or chondrocytes grown in chitosan hydrogel. Anal. Biochem. 300, 1–10.
Gehrsitz, A., McKenna, L. A., Soder S., Kirchner, T., and Aigner T. (2002) Isolation of RNA from small human articular cartilage specimens allows quantification of mRNA expression levels in local articular cartilage defects. J. Orthop. Res. 19, 478–481.
Matyas, J. R., Huang, D., Chung, M., and Adams, M. E. (2002) Regional quantification of cartilage type II collagen and aggrecan mRNA in joints with early experimental osteoarthritis. Arthritis Rheum. 46, 1536–1543.
Bluteau, G., Gouttenoire, J., Conrozier, T., et al. (2002) Differential gene expression analysis in a rabbit model of osteoarthritis induced by anterior cruciate ligament (ACL) section. Biorheology 39, 247–258.
Langelier, A., Suetterlin, R., Hoemann, C. D., Aebi, U., and Buschmann, M. D. (2000) The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin and vimentin filaments. J. Histochem. Cytochem. 48, 1307–1320.
Sajdera, S. W. and Hascall, V. C. (1969) Proteinpolysaccharide complex from bovine nasal cartilage. J. Biol. Chem. 244, 77–87.
Heinegard, D. and Sommarin, Y. (1987) Isolation and characterization of proteoglycans. Methods Enzymol. 144, 319–372.
Mankin, H. J. and Lippiello, L. (1970) Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. J. Bone Joint Surg. Am. 52, 424–434.
Kempson, G. E., Muir, H., Swanson, S. A. V., and Freeman, M. A. R. (1970) Correlations between stiffness and the chemical constituents of cartilage on the human femoral head. Biochem. Biophys. Acta. 215, 70–77.
Venn, M. and Maroudas, A. (1977) Chemical composition and swelling of normal and osteoarthritic femoral head cartilage. Ann. Rheum. Dis. 36, 121–129.
Amiel, D., Coutts, R. D., Harwood, F. L., Ishizue, K. K., and Kleiner, J. B. (1988) The chondrogenesis of rib perichondrial grafts for repair of full thickness articular cartilage defects in a rabbit model: a one year postoperative assessment. Connect. Tissue Res. 18, 27–39.
Richardson, D. W. and Clark, C. C. (1990) Biochemical changes in articular cartilage opposing full-and partial-thickness cartilage lesions in horses. Am. J. Vet. Res. 51, 118–122.
Vachon, A. M., McIlwraith, C. W., and Keeley, F. W. (1991) Biochemical study of repair of induced osteochondral defects of the distal portion of the radial carpal bone in horses by use of periosteal autografts. Am. J. Vet. Res. 52, 328–332.
Brama, P. A., Tekoppele, J. M., Bank, R. A., Barneveld, A., and VanWeeren, P. R. (2000) Functional adaptation of equine articular cartilage: the formation of regional biochemical characteristics up to age one year. Equine Vet. J. 32, 217–221.
Burkhardt, D., Hwa, S. Y., and Ghosh, P. (2001) A novel microassay for the quantitation of the sulfated glycosaminoglycan content of histological sections: its application to determine the effects of diacerhein on cartilage in an ovine model of osteoarthritis. Osteoarthritis Cartilage 9, 238–247.
Murray, R. C., Birch, H. L., Lakhani, K., and Goodship, A. E. (2001) Subchondral bone thickness, hardness and remodelling are influenced by short-term exercise in a site-specific manner. J. Orthop. Res. 19, 1035–1042.
Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., Kimura, J. H., and Hunziker, E. B. (1992) Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J. Orthop. Res. 10, 745–758.
Eggli, P. S., Hunziker, E. B., and Schenk, R. K. (1988) Quantitation of structural features characterizing weight-and less-weight-bearing regions in articular cartilage: a stereological analysis of medial femoral condyles in young adult rabbits. Anat. Rec. 222, 217–227.
Sah, R. L., Yang, A. S., Chen, A. C., et al. (1997) Physical properties of rabbit articular cartilage after transection of the anterior cruciate ligament. J. Orthop. Res. 15, 197–203.
Lee, C. R., Grodzinsky, A. J., Hsu, H. P., Martin, S. D., and Spector, M. (2000) Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee. J. Orthop. Res. 18, 790–799.
Treppo, S., Koepp, H., Quan, E. C., Cole, A. A., Kuettner, K. E., and Grodzinsky, A. J. (2000) Comparison of biomechanical and biochemical properties of cartilage from human knee and ankle pairs. J. Orthop. Res. 18, 739–748.
Dumont, J., Ionescu, M, Reiner A., et al. (1999) Mature full-thickness articular cartilage explants attached to bone are physiologically stable over long-term culture in serum-free media. Connect. Tissue Res. 40, 259–272.
Ameer, G. A., Mahmood, T. A., and Langer, R. (2002) A biodegradable composite scaffold for cell transplantation. J. Orthop. Res. 20, 16–19.
Hunziker, E. B., Quinn, T. M., and Hauselmann, H. J. (2002) Quantitative structural organization of normal adult human articular cartilage. Osteoarthritis Cartilage 10, 564–572.
Lewis, R. J., MacFarland, A. K., Anandavijayan, S., and Aspden, R. M. (1988) Material properties and biosynthetic activity of articular cartilage from the bovine carpo-metacarpal joint. Osteoarthritis Cartilage 6, 383–392.
Mankin, H. J. (1974) The reaction of articular cartilage to injury and osteoarthritis (Second of Two Parts). N. Engl. J. Med. 291, 1335–1340.
Mow, V. C., Ratcliffe, A., and Poole A. R. (1992) Cartilage and diarthrodial joints as paradigms for hierarchical materials and structures. Biomaterials 13, 67–97.
Maroudas, A. (1990) Different ways of expressing concentration of cartilage constituents with special reference to the tissue’s organization and functional properties, in Methods in Cartilage Research (Maroudas, A. and Kuettner, K. E., eds.), Academic, London, pp. 211–219.
Eyre, D. (2001) Collagen of articular cartilage. Arthritis Res. 4, 30–35.
Page Thomas, D. P., King B., Stephens, T., and Dingle, J. T. (1991) In vivo studies of cartilage regeneration after damage induced by catabolin/interleukin-1. Ann. Rheum. Dis. 50, 75–80.
Richardson, D. W. and Clark, C. C. (1990) Biochemical changes in articular cartilage opposing full-and partial-thickness cartilage lesions in horses. Am. J. Vet. Res. 51, 118–122.
Verbruggen, G., Cornelissen, M., Almqvist, K. F., et al. (2000) Influence of aging on the synthesis and morphology of the aggrecans synthesized by differentiated human articular chondrocytes. Osteoarthritis Cartilage 8, 170–179.
Front, P. Aprile, F., Mitrovic, D. R., and Swann, D. A. (1989) Age-related changes in the synthesis of matrix macromolecules by bovine articular cartilage. Connect. Tissue Res. 19, 121–133.
Sims, C. D., Butler, P. E. M., Cao, Y. L., et al. (1998) Tissue engineered neocartilage using plasma derived polymer substrates and chondrocytes. Plast. Reconst. Surg. 101, 1580–1585.
Riesle, J., Hollander, A. P., Langer, R., Freed, L. E., and Vunjak-Novakovic, G. (1998) Collagen in tissue-engineered cartilage: types, structure, and crosslinks J. Cell. Biochem. 71, 313–327.
Hollander, A. P., Heathfield, T. F., Webber, C., et al. (1994) Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay. J. Clin. Invest. 93, 1722–1732.
Stone, J., Akhtar, H., Botchway, S., and Pennock, C. A. (1994) Interaction of 1,9-dimethylmethylene blue with glycosaminoglycans. Ann. Clin. Biochem. 31, 147–152.
Vunjak-Novakovic, G., Martin, I., Obradovic, B., et al. (1999) Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J. Orthop. Res. 17, 130–138.
Hoemann, C. D., Sun, J., Légaré, A., McKee, M. D., and Buschmann, M. D. Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle. Submitted.
Obradovic, B., Carrier, R. L., Vunjak-Novakovic, G., and Freed, L. E. (1999) Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage. Biotechnol. Bioeng. 63, 197–205.
Wu, F., Dunkelman, N., Peterson, A., Davisson, T., De La Torre, R., and Jain, D. (1999) Bioreactor development for tissue-engineered cartilage. Ann. NY Acad. Sci. 875, 405–411.
Grande, D. A., Halberstadt, C., Naughton, G., Schwartz, R., and Manji, R. (1997) Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts. J. Biomed. Mater. Res. 34, 211–220.
Yu, H., Grynpas, M., and Kandel, R. A. (1997) Composition of cartilagenous tissue with mineralized and non-mineralized zones formed in vitro. Biomaterials 18, 1425–1431.
Sun, Y., Hurtig, M., Pilliar, R. M., Grynpas, M., and Kandel, R. A. (2001) Characterization of nucleus pulposus-like tissue formed in vitro. J. Orthop. Res. 19, 1078–1084.
Nehrer, S., Breinan, H. A., Ramappa, A., et al. (1997) Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro. J. Biomed. Mater. Res. 38, 95–104.
Toolan, B. C., Frenkel, S. R., Pachence, J. M., Yalowitz, L., and Alexander, H. (1996) Effects of growth-factor enhanced culture on a chondrocyte-collagen implant for cartilage repair. J. Biomed. Mater. Res. 31, 273–280.
Bryant, S. J. and Anseth, K. S. (2001) Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. J. Biomed. Mater. Res. 59, 63–72.
Kisiday, J., Jin, M., Kurz, B., Hung, H., Semino, C., Zhang, S., and Grodzinsky, A. J. (2002) Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair. Proc. Natl. Acad. Sci. USA 99, 9996–10001.
Wong, M., Siegrist, M., Wang, X., and Hunziker, E. (2001) Development of mechanically stable alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis. J. Orthop. Res. 19, 493–499.
Passaretti, D., Silverman, R. P., Huang, W., et al. (2001) Cultured chondrocytes produce injectable tissue-engineered cartilage in hydrogel polymer. Tissue Eng. 7, 805–815.
Elisseeff, J., Anseth, K., Sims, D., et al. (1999) Transdermal photopolymerization of poly(ethylene oxide)-based injectable hydrogels for tissue-engineered cartilage. Plast. Reconstr. Surg. 104, 1014–1022.
Oegema, T. R., Carpenter, B. J., and Thompson, R. C. (1984) Fluorometric determination of DNA in cartilage of various species. J. Orthop. Res. 1, 345–351.
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Hoemann, C.D. (2004). Molecular and Biochemical Assays of Cartilage Components. In: De Ceuninck, F., Sabatini, M., Pastoureau, P. (eds) Cartilage and Osteoarthritis. Methods in Molecular Medicine, vol 101. Humana Press. https://doi.org/10.1385/1-59259-821-8:127
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DOI: https://doi.org/10.1385/1-59259-821-8:127
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