| Collagen is the structural molecule of choice in vertebrates and is the most abundant protein on earth. Its primary role is that of bearing and transmitting mechanical (principally tensile) loads. Though collagen degradation is a normal part of collagen homeostasis, excessive collagenolysis has been implicated in a number of human diseases such as arthritis, cancer, and atherosclerosis. Collagen is susceptible to cleavage via bacterial collagenase (Clostridium histolyticum) and members of Matrix Metalloproteinase (MMP) family. It has been hypothesized that mechanical loads can influence the rate of enzymatic cleavage of collagen molecules in native tissue. The purpose of the investigation is to study in vitro the behavior of reconstituted collagen fibrils in networks supporting mechanical strain and then exposed to enzymatic cleavage by bacterial collagenase and MMP. In a custom designed, environmentally-controlled, reaction micro-chamber, type I collagen solution was self assembled by neutralizing purified collagen (3.0 mg/mL), with PBS buffer for bacterial collagen experiments and with Tris buffer for MMP experiment. Collagen fibrillogenesis (induced by temperature elevation to 37°C) was observed by DIC (using Nikon TE2000-E). Mechanical strain was applied on the fibrillar network using micromanipulators and micropipettes. To determine if the load protected the cleavage of loaded collagen fibril, bacterial collagenase and in the separate experiments MMP-8 was added to the assembled matrix. The results demonstrated that unstrained fibrils were removed quickly while strained fibrils were degraded at a significantly slower rate. Using the edge detection image analysis tool, the degradation curve shows the statistical difference in the start and the end of degradation for loaded and unloaded fibrils. This indicates the strain-stabilization of collagen against enzymatic cleavage. Also, in another result, preliminary data suggest that mechanical signals direct the preferential incorporation of collagen monomers into fibrils. Using the above two conclusions, in vitro, we propose a novel method where organized collagenous tissue can be produced merely by application of appropriate strain in the presence of excess of monomers and an appropriate concentration of enzymes. |
