Search for an endogenous collagenase in chicken endochondral bone matrix vesicles.

Abstract

While isolating matrix vesicles from avian endochondral bone, collagenase activity was discovered unexpectedly. The question was raised whether this collagenase activity was endogenous or if it was due to the bacterial collagenase used to release the matrix vesicles from bone. In related experiments done, collagen partially degraded by collagenase mineralized better than undegraded collagen. This study would then attempt to show if an endogenous collagenase is found, whether it facilitates collagen mineralization by allowing better access for matrix vesicles by the "nicking" of collagen. From this two-month study evidence was found that an endogenous collagenase as well as a gelatinase was present on matrix vesicles. SDS gel electrophoresis and zymography were used to determine the presence of collagenases in purified matrix vesicle fractions. Western blots with antibodies to bacterial collagenase, matrix metallo-proteinases 13 (MMP-13), an endogenous collagenase, and MMP-2, an endogenous gelatinase, were also done to determine the presence of an endogenous collagenase. Another facet was added to this study to determine if contamination by exogenous collagenase during matrix vesicle isolation could be removed. From the final Western blots done, it was verified that even after exhaustive washings of the column and centrifugation, purified matrix vesicles contained contaminating bacterial collagenase. Due to the research completed, the matrix vesicle isolation protocol was changed from using bacterial collagenase to a trypsin isolation method to prevent any exogenous collagenase contamination.