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mRNA expression profile differences before and after enzymatic digestion of equine articular cartilage

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Title: mRNA expression profile differences before and after enzymatic digestion of equine articular cartilage
Author: Sharma, Rishi
Department: Department of Biomedical Sciences
Program: Biomedical Sciences
Advisor: Koch, Thomas
Abstract: Focal cartilage defects of developmental or traumatic origin affect horses and humans in similar manners. Current treatments have poor long-term outcomes. Ex vivo tissue-engineered cartilage is a potential treatment option. Tissue engineering benchmarks and process checkpoints rely on accurate knowledge of the normal biological properties of cartilage. Assessment of cartilage gene expression is commonly compared to chondrocytes isolated from enzymatically digesting cartilage. However, studies have demonstrated that gene expression of cultured chondrocytes change over time making them poor proxies for gene expression of chondrocytes within normal native cartilage tissue. More accurate knowledge about normal cartilage biology is needed to inform future cartilage-engineering approaches. The overall hypothesis of work presented is that enzymatic digestion of articular cartilage causes rapid changes in the levels of specific chondrocyte RNAs. First an RNA isolation protocol was optimized to enhance yield and quality for mRNA analysis, next generation sequencing, and miRNA studies. Subsequently, the expression of SOX9, COL1A2, COL2A1, ACAN, and COLX was compared between native and enzymatically-digested cartilage from weight bearing and non-weight bearing regions 24 hours after initiation of digestion and without an in vitro chondrocyte culture period. Homogenization method significantly impacted RNA yield, with large ball homogenization producing greater yields of RNA. Digested cartilage showed a significant decrease in the expression of COL1A2, COL2A1, and ACAN compared to native cartilage and an increase in COLX expression in non-weight bearing digested cartilage. Thus, enzymatic digestion of cartilage rapidly and significantly impacts gene expression profiles. Determination of normal cartilage biology should be conducted on native untreated cartilage.
Date: 2017-09-07

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