Gene Expression Profile Is Different between Intact and Enzymatically Digested Equine Articular Cartilage

Abstract

Objectives: RNA isolation is necessary for the evaluation of gene expression. Due to the nature of its extracellular matrix, RNA isolation from articular hyaline cartilage is difficult and thus the tissue is commonly enzymatically digested in order to extract RNA from the obtained chondrocytes. We hypothesized that the digestion process affects the expression levels of common cartilage-associated genes. Design: Expression of cartilage-associated genes was compared between intact cartilage and digested chondrocytes from weight bearing and non-weight bearing regions of the equine fetlock joint. Results: The gene expression of SOX9, COL1A2, COL2A1, ACAN, and COLX were analyzed. Digested cartilage showed a significant decrease in the expression of COL1A2, COL2A1, and ACAN compared to intact cartilage in both joint regions, and an increase in COLX expression in non-weight bearing cartilage only. Conclusions: Enzymatic digestion of cartilage significantly impacts gene expression profile. We conclude that while RNA isolation from intact cartilage is more technically difficult, determination of gene expression should be conducted on intact cartilage if true representation of the in vivo processes is sought.

RNA Extraction from Cartilage: Issues, Methods, Tips

“…The majority of the studies that performed RNA extraction from fresh cartilage samples, straightaway frozen the tissue in liquid nitrogen before performing the extraction procedure immediately after [8,[30][31][32][33][34][35][36][37][38][39][40][41][42][43], or stored them at −80 • C after freezing, until subsequent processing [1,7,[9][10][11]22,[44][45][46][47][48][49][50][51][52]. Alternatively, when immediate tissue freezing is not possible, the use of an RNA-stabilization solution should be considered.…”

“…Particular instruments such as a freezer mill, a microdismembrator or, simply, a frozen mortar and pestle can be used for this purpose [39]. The most frequently used system is the freezer mill, which is an impact grinder using a steel impactor moved back and forth between the metal ends of a vial containing the sample by strong magnetic fields, while the vials are immersed in liquid nitrogen [8,9,11,19,33,34,37,38,45,48,66]. A different kind of mill provides for frozen cartilage samples in a precooled stainless-steel canister with stainless-steel grinding ball and immersed in liquid nitrogen for 2 min [43].…”

3D Printing for Bone-Cartilage Interface Regeneration

“…However, chondrocytes are usually digested with collagenase to facilitate complete isolation, which can be detrimental to the cells. That is, too much or too little collagenase can lead to failure or low yield ( Lepage et al, 2019 ). Hence, in a study conducted by Muhammad et al (2019) , a protocol for chondrocyte isolation was optimized using trypsin-ethylenediaminetetraacetic acid (EDTA), collagenase II in Hank’s balanced salt solution (HBSS), and collagenase II in Dulbecco’s modified Eagle medium/Nutrient Mixture F-12 (DMEM/F-12) for chondrocyte isolatio.…”

Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine

“…NC isolation is usually achieved through collagenase digestion; this step may play an important role in the overall success of the procedure, affecting both the yield and viability of isolated cells. In fact, collagenase treatment which is too intensive may often result in chondrocyte isolation failure [11]; conversely, collagenase treatment which is too mild may provide inadequate cell yield. In this respect, it is not to be excluded that the isolation outcome may be also affected by donor tissue composition, presumably because of individual variations resulting in unique collagen patterns in each donor, as observed in the case of pancreas tissue [12].…”