Collagen fibrils from functionally distinct tendons have differing susceptibility to degradation by MMP-1

Abstract

Bovine forelimb flexor and extensor tendons are functionally distinct tendons that are commonly used as a model to examine high-stress, energy-storing and low-stress, positional tendons, respectively. These tendons are known to have structural differences at the nanoscale level of the collagen fibril which is the primary structural unit of tendon and other connective tissues. Collagen fibrils of energy-storing flexor tendons are smaller in size, more heavily crosslinked, and respond differently to mechanical loading. Meanwhile, these same tendons undergo less collagen turnover compared to positional extensor tendons and are more commonly injured. These observations raise the question of whether collagen fibril structure influences the collagen degradation processes necessary for remodeling.

At only tens of nanometers in diameter, collagen fibrils require specialized microscopy to be visualized. Atomic force microscopy (AFM) was used to image collagen fibrils before and after 5 hr exposure to matrix metalloproteinase-1 (MMP-1) to detect changes in fibril size upon degradation. Extensor fibrils were smaller following incubation while flexor fibrils were not. Additionally, a size-dependence to degradation where larger fibrils were degraded more than smaller ones was found, prompting a follow-up study at the population level.

Three potential models of degradation were created using the size decreases measured by AFM. This provided statistical requirements and reference distributions to guide a second in vitro experiment. Scanning electron microscopy (SEM) was used to image tissue sections from both tendons after 24 hr incubation with buffer or MMP-1. This required the development of a novel experimental protocol and custom semi-automated analysis pipeline to accurately measure fibril diameter from SEM images.

Fibrils in the extensor tendon showed clear evidence of degradation with an average 41% decrease in diameter in all fibrils measured. Upon comparison to the degradation model, size-dependence was confirmed. Flexor fibril degradation was limited to only large diameter fibrils that showed an average diameter decrease of 15%. These findings suggest that structural differences like collagen crosslinking inhibit MMP-1 degradation. This has implications for tissue remodeling and future studies: careful selection of tissue sources and fibril diameter are required until methods to resolve individual molecular crosslinking within a fibril are developed.