A simplified measurement of degraded collagen in tissues: application in healthy, fibrillated and osteoarthritic cartilage.
Intact triple helical collagen molecules are highly resistant to proteolytic enzymes, whereas degraded (unwound) collagen is easily digested. This fact was exploited to develop a simplified method for the quantification of the amount of degraded collagen in the collagen network of connective tissues. Essentially, the method involves extraction of proteoglycans with 4 M guanidinium chloride, selective digestion of degraded collagen by alpha-chymotrypsin, hydrolysis in 6 M HCl of the released fragments as well as the residual tissue, and then measurement of the amount of hydroxyproline in both pools. Since the digestion of degraded collagen by alpha-chymotrypsin and measurement of hydroxyproline is not restricted to a specific collagen type, this technique can be applied to a wide variety of connective tissues. The method was validated with articular cartilage. Levels of in situ degraded collagen were about four-fold higher in degenerated (fibrillated) cartilage than in its healthy counterpart derived from the same donor. More detailed investigations revealed that the collagen damage in degenerated cartilage is more extensive at the cartilage surface than in the region adjacent to bone. This was not the case in healthy cartilage; identical low values were obtained at the surface and close to the bone. An impaired collagen network has been hypothesized to be the reason for the swelling of cartilage in osteoarthritis (OA). The present paper presents the first experimental evidence to support this hypothesis: more damage to the collagen network (i.e., more degraded collagen molecules within fibrils) is linearly related to more extensive swelling of the OA tissue in hypotonic saline.
Bank
R A
RA
TNO Prevention and Health, Division of Vascular and Connective Tissue Research, Leiden, The Netherlands.
Krikken
M
M
Beekman
B
B
Stoop
R
R
Maroudas
A
A
Lafeber
F P
FP
te Koppele
J M
JM
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D003094:Collagen / Q000378:metabolism*; D005260:Female; D005449:Fluorenes / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D015203:Reproducibility of Results; D009764:o-Phthalaldehyde / Q000378:metabolism
D005449:Fluorenes; D009764:o-Phthalaldehyde; D003094:Collagen; C054007:1-(9-fluorenyl)methyl chloroformate; D002918:Chymotrypsin