Detection and evaluation of matrix metalloproteinases involved in cruciate ligament disease in dogs using multiplex bead technology.
OBJECTIVE
To measure matrix metalloproteinases (MMPs) suspected to be involved in the initiation or progression of osteoarthritis (OA) in cranial cruciate ligament (CCL) explant culture media using multiplex bead technology.


METHODS
In vitro experimental study.


METHODS
Adult dogs with (n=10) and without (n=10) CCL deficiency.


METHODS
Based on clinical, radiographic, and gross evidence of CCL deficiency, excised CCL were classified as normal and intact (n=10) or partially torn (n=10). The ligament was excised and immediately placed in tissue culture. Culture media were sampled and replaced on days 3 and 6. MMP-1, 2, 3, 9, and 13 were quantified in explant media using a multiplexing machine that uses flow cytometry, microspheres, spectral dyes, lasers, digital signal processing, and traditional chemistry. MMP concentrations were determined using a standard curve constructed from the serial dilution of positive controls. Media MMP concentrations comparing the type of ligament and the time frame were analyzed using a Mann-Whitney rank sum test.


RESULTS
Media exposed to intact ligaments had >3 times the amount of MMP-2 than for partially torn ligaments on day 6 (P=.006). Media exposed to intact ligaments also had significantly higher levels of MMP-3 than for partially torn ligaments on day 3 (P=.035) and on day 6 (P=.05).


CONCLUSIONS
MMP multiplexing is a reliable, cost-effective, efficient, and sample-sparing method of MMP quantification. MMP-2, 3, 9, and 13 are released from CCL explants exposed to culture media and can be detected using multiplex bead technology.


CONCLUSIONS
CCL remnants exposed to the intra-articular environment may release degradative enzymes in vivo similar to that demonstrated in this in vitro study. Because MMPs are known to be involved in the initiation and progression of OA, debridement of these remnants as a component of treatment for cruciate disease in dogs deserves consideration.
Breshears
Lee A
LA
Comparative Orthopaedic Laboratory, University of Missouri-Columbia, Columbia, MO, USA. lbreshears@aescparker.com
Cook
James L
JL
Stoker
Aaron M
AM
Fox
Derek B
DB
D000818:Animals; D016118:Anterior Cruciate Ligament / Q000201:enzymology*; D004283:Dog Diseases / Q000201:enzymology*; D004285:Dogs; D005434:Flow Cytometry / Q000662:veterinary; D020781:Matrix Metalloproteinase 1 / Q000032:analysis; D053509:Matrix Metalloproteinase 13 / Q000032:analysis; D020778:Matrix Metalloproteinase 2 / Q000032:analysis; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D020780:Matrix Metalloproteinase 9 / Q000032:analysis; D020782:Matrix Metalloproteinases / Q000032:analysis*; D008863:Microspheres
D053509:Matrix Metalloproteinase 13; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1