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Updated: Jun 12, 2026
Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice
Published on: September 29, 2023
Detection and evaluation of matrix metalloproteinases involved in cruciate ligament disease in dogs using multiplex
Lee A Breshears1, James L Cook, Aaron M Stoker
1Comparative Orthopaedic Laboratory, University of Missouri-Columbia, Columbia, MO, USA. lbreshears@aescparker.com
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.
Study Design:
In vitro experimental study.
Animals:
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.
Clinical Relevance:
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.
