A Method to Cleave Target Molecules in a Neocartilage

Abstract

The mechanical function of many matrix molecules is unknown. A common method to determine whether a molecule is a load-carrying structural molecule is to measure the mechanical properties of a tissue, digest the tissue with an enzyme specific for cleaving that molecule, and then remeasure the mechanical properties. A limitation of this technique is that there are no specific lytic enzymes for most molecules of interest. This article introduces a method that may allow evaluation of a large number of candidate structural molecules. A translated thrombin proteolytic recognition and cleavage site is inserted in the cDNA of a target molecule, and the target molecule then expressed in a cell that produces a tissue. After growing the tissue with cells expressing the engineered target molecule, the traditional procedure of mechanical testing, digesting, and retesting is performed. This method was demonstrated using decorin and its dermatan sulfate (DS) glycosaminoglycan chain in a neocartilage. A tissue was generated with cells expressing a genetically engineered decorin with a thrombin cleavage site. The tissue was then tested in tension and compression, digested with thrombin, and mechanically retested. The decorin protein was found in the tissue, the DS glycosaminoglycan chain was removed with thrombin digestion, and there was no change in the mechanical properties of the tissue due to the thrombin digestion relative to controls. These findings were in agreement with previously reported tests on decorin, collectively supporting the proposed method. All methods involving animals were reviewed and approved by our Institutional Review Board.