Effect of physical crosslinking methods on collagen-fiber durability in proteolytic solutions.
We previously demonstrated that ultraviolet (UV) or dehydrothermal (DHT) crosslinking partially denatured fibers extruded from an insoluble type I collagen dispersion. In this study denaturation effects were evaluated by measuring collagen-fiber sensitivity to trypsin. Shrinkage-temperature measurements and sensitivity to collagenase served as indices of crosslinking. UV or DHT crosslinking increased the collagen-fiber shrinkage temperature, resistance to degradation in collagenase, and durability under load in collagenase. However, in trypsin solutions, solubility was significantly increased for UV (approximately 11%) or DHT (approximately 15%) crosslinked fibers compared with uncrosslinked fibers (approximately 4%). Size-exclusion chromatography indicated that no intact collagen alpha-chains were present in the soluble fraction of fibers exposed to trypsin (MW < 1 kD). Interestingly, UV-crosslinked collagen fibers remained intact an order of magnitude longer (4840 +/- 739 min) than DHT-crosslinked (473 +/- 39 min) or uncrosslinked (108 +/- 53 min) fibers when placed under load in trypsin solutions. These data indicate that mechanical loading during incubation in a trypsin solution measures denaturation effects not detected by the trypsin-solubility assay. Our results suggest that DHT-crosslinked collagen fibers should not be used as load-bearing implants. UV-crosslinked fibers may retain more native structure and should exhibit greater resistance to nonspecific proteases in vivo.
Weadock
K S
KS
Orthopaedic Research Laboratory, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick 08903, USA.
Miller
E J
EJ
Keuffel
E L
EL
Dunn
M G
MG
D000818:Animals; D002417:Cattle; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D003432:Cross-Linking Reagents; D010450:Endopeptidases / Q000378:metabolism*; D011489:Protein Denaturation; D012995:Solubility; D012996:Solutions; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D012996:Solutions; D003094:Collagen; D010450:Endopeptidases; D014357:Trypsin; D017364:Collagenases