Preservation of osteochondral allografts used for transplantation is critical to ensure favorable outcomes for patients after surgical treatment of cartilage defects. To study the biological effects of protocols currently used for cartilage storage, we investigated differences in gene expression between stored allograft cartilage and fresh cartilage from living donors using high throughput molecular screening strategies. We applied next generation RNA sequencing (RNA-seq) and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) to assess genome-wide differences in mRNA expression between stored allograft cartilage and fresh cartilage tissue from living donors. Gene ontology analysis was used to characterize biological pathways associated with differentially expressed genes. Our studies establish reduced levels of mRNAs encoding cartilage related extracellular matrix (ECM) proteins (i.e., COL1A1, COL2A1, COL10A1, ACAN, DCN, HAPLN1, TNC, and COMP) in stored cartilage. These changes occur concomitantly with increased expression of “early response genes” that encode transcription factors mediating stress/cytoprotective responses (i.e., EGR1, EGR2, EGR3, MYC, FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND). The elevated expression of “early response genes” and reduced levels of ECM-related mRNAs in stored cartilage allografts suggests that tissue viability may be maintained by a cytoprotective program that reduces cell metabolic activity. These findings have potential implications for future studies focused on quality assessment and clinical optimization of osteochondral allografts used for cartilage transplantation.
Bibliographical noteFunding Information:
This work was supported, in whole or in part, by National Institutes of Health grants R01 AR049069 (to Andre J. van Wijnen), R03 AR066342-02 (to Annalise N. Larson), T32 AR56950 (to Eric A. Lewallen), and F32 AR066508 (to Amel Dudakovic). We also appreciate the generous philanthropic support of William and Karen Eby, and the charitable foundation in their names. We also thank Susan Puffer and Renae Olson for assistance with handling surgical specimens and the members of our laboratory, as well as our colleagues David Deyle and David Lewallen for sharing reagents and/or stimulating discussions.
- RNA sequencing