A method to cleave target molecules in a neocartilage

David A. Krawczak, Jack L. Lewis

Research output: Contribution to journalReview articlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)430-436
Number of pages7
JournalConnective Tissue Research
Volume53
Issue number5
DOIs
StatePublished - Oct 2012

Bibliographical note

Funding Information:
This study was made possible by Grant Number AR060196 from NIAMS/NIH and by support from the Minnesota Supercomputer Institute of the University of Minnesota. The authors have no financial or personal relationships with other individuals or organizations that could bias this work and serve as a potential conflict of interest.

Keywords

  • Cartilage
  • Matrix
  • Mechanical properties
  • Microstructure
  • Structural molecules

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