Baseline Values of Gene Expression Levels for Cultured Chondrocytes Should be Obtained from Chondrocytes In Situ
K.M. Bagnall, J. Bater, C. Secretan, R. Barley, N. Jomha
Abstract
Introduction: Implantation of cells to repair spinal articular cartilage (AC) requires these cells to have a specific chondrocyte genotype. Current baseline values for chondrocyte genotype currently available in the literature have been determined after their release from the cartilage matrix. Molecular characteristics change extremely rapidly depending on the microenvironment of the cell and are likely not the same as those exhibited by chondrocytes in situ.
Objective: To determine chondrocyte genotype while still embedded within the cartilage matrix.
Materials and Methods: Samples of disease-free, donor, human AC were harvested and divided into two groups. One group was immediately frozen and ground using a freezer mill. In the other group, the chondrocytes were released from the cartilage matrix using collagenase. mRNA was extracted from both groups and gene expression levels for collagen types I and II, aggrecan and GAPDH were measured using RT-PCR.
Results: The gene expression levels for collagen types I and II for the cells released from the matrix were significantly higher than those obtained from cells in situ. Gene expression levels for aggrecan were not significantly different.
Conclusion: Gene expression levels for chondrocytes in situ differ significantly from chondrocytes released from the cartilage matrix.
Significance: If chondrocytes are to be cultured prior to implantation to repair spinal AC then the targeted baseline values should be obtained from cells in situ. Gene expression levels change very rapidly and values obtained from cells released from their matrix are significantly different and inaccurate.