Clinical Outcome 3 Years After Autologous Chondrocyte Implantation Does Not Correlate With the Expression of a Predefined Gene Marker Set in Chondrocytes Prior to Implantation but Is Associated With Critical Signaling Pathways

Abstract

Background: There is a need for tools to predict the chondrogenic potency of autologous cells for cartilage repair.

Purpose: To evaluate previously proposed chondrogenic biomarkers and to identify new biomarkers in the chondrocyte transcriptome capable of predicting clinical success or failure after autologous chondrocyte implantation.

Study design: Controlled laboratory study and case-control study; Level of evidence, 3.

Methods: Five patients with clinical improvement after autologous chondrocyte implantation and 5 patients with graft failures 3 years after implantation were included. Surplus chondrocytes from the transplantation were frozen for each patient. Each chondrocyte sample was subsequently thawed at the same time point and cultured for 1 cell doubling, prior to RNA purification and global microarray analysis. The expression profiles of a set of predefined marker genes (ie, collagen type II α1 [COL2A1], bone morphogenic protein 2 [BMP2], fibroblast growth factor receptor 3 [FGFR3], aggrecan [ACAN], CD44, and activin receptor-like kinase receptor 1 [ACVRL1]) were also evaluated.

Results: No significant difference in expression of the predefined marker set was observed between the success and failure groups. Thirty-nine genes were found to be induced, and 38 genes were found to be repressed between the 2 groups prior to autologous chondrocyte implantation, which have implications for cell-regulating pathways (eg, apoptosis, interleukin signaling, and β-catenin regulation).

Conclusion: No expressional differences that predict clinical outcome could be found in the present study, which may have implications for quality control assessments of autologous chondrocyte implantation. The subtle difference in gene expression regulation found between the 2 groups may strengthen the basis for further research, aiming at reliable biomarkers and quality control for tissue engineering in cartilage repair.

Clinical relevance: The present study shows the possible limitations of using gene expression before transplantation to predict the chondrogenic and thus clinical potency of the cells. This result is especially important as the chondrogenic potential of the chondrocytes is currently part of quality control measures according to European and American legislations regarding advanced therapies.

Keywords: articular cartilage; articular cartilage resurfacing; biology of cartilage; knee; tissue engineering.

Figure 1.

Dendogram showing no global clustering between the groups.

Figure 2.

Multivariate data analysis of the centered and normalized array data. (A) Score plot of principal component analysis (PCA) of the centered and normalized data from the arrays including all 10 patients. (B) Loading plot of discriminant analysis using orthogonal partial least square analysis (OPLS-DA) of the data from the array including all 10 patients. Dots indicate the set of transcripts that participate most to the separation of the groups; black dots, the set of 1443 transcripts; gray dots, the cloud of excluded transcripts. (C) Coomans plot showing the prediction PCA models for the 5 success patients and the 5 failure patients where the 5% of the variables that participated the most to the OPLS-DA were used to perform the PCA. Dotted line, 5% confidence limit of the model. (D) Coomans plot showing the prediction for all 10 patients. In this analysis, 3 randomly selected patients from the success and failure groups were included. The 5% of the variables that participated the most in this OPLS-DA were used as data in the PCA models. The excluded patients failed to fit their respective model. Dotted line, 5% confidence limit of the model.

Figure 3.

Heatmap of (A) chondrogenic genes and (B) the predefined set of gene markers showing no clustering of the success and failure groups. (Continued)

Figure 4.

Quantitative polymerase chain reaction validation of the results from the microarray analysis. Each value is plotted, and the mean is marked with a line. P < .05 was considered significant.