Matched frozen cartilage tissue from patients used for proteomic analysis was dissected transversely from the cartilage surface. Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) following the manufacturer’s instruction with modifications. Reverse transcription was conducted with miScript II RT Kit (Qiagen) following kit instructions. Quantification of blastema miRNAs (miR-21, miR-31, and miR-181c) and internal control miRNAs (miR-423, miR-191, and miR-U6) was assessed by quantitative real-time polymerase chain reaction (PCR) using the miScript SYBR Green PCR Kit (Qiagen). The ΔCt values for blastema miRNAs, representing the miRNA normalized to the mean of the internal control miRNAs, were used to evaluate the correlation of miRNA and cartilage ECM protein expression; these ΔCt values were computed as the Ct of the blastema miRNA minus the mean Ct of the internal control miRNAs.

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