Real-time enzymatic biodegradation of collagen fibrils monitored by atomic force microscopy

The enzymatic degradation of type I collagen fibrils by the collagenase from Clostridium histolyticum has been investigated using real-time atomic force microscope (AFM) imaging under liquid. Using a commercial AFM with fluid cell attachment, we have observed the degradation of fibrils from their mature form down to fragments whose size reaches the limits of resolution of the instrument. Upon exposure to collagenase solutions, collagen fibrils become both shorter and thinner as a function of the enzymatic incubation time, suggesting that collagenase degrades the entire fibrillar structure in a non-specific manner. The results are discussed in terms of the postulated mechanism of C. histolyticum collagenase activity, along with the possibility of determining enzymatic rate constants for biodegradation processes using the AFM. Experimental advantages and shortcomings of this technique are compared with other methods of following enzymatic degradation and related biological processes, and suggestions are made in order to overcome some of the technical difficulties encountered with these studies.