Noninvasive assessment of engineered cartilage properties would enable better control of the developing tissue towards the desired structural and compositional endpoints through optimization of the biochemical environment in real time. The objective of this study is to assess the matrix constituents of cartilage using near-infrared spectroscopy (NIRS), a technique that permits full-depth assessment of developing engineered tissue constructs. Mid-infrared (mid-IR) and NIR data were acquired from full-thickness cartilage constructs that were grown up to 4 weeks with and without mechanical stimulation. Correlations were assessed between established mid-IR peak areas that reflect the relative amount of collagen (amide I, amide II, and 1338 cm<sup>−1</sup>) and proteoglycan (PG), (850 cm<sup>−1</sup>), and the integrated area of the NIR water absorbance at 5190 cm<sup>−1</sup>. This analysis was performed to evaluate whether simple assessment of the NIR water absorbance could yield information about matrix development. It was found that an increase in the mid-IR PG absorbance at 850 cm<sup>−1</sup> correlated with the area of the NIR water peak (Spearman's rho = 0.95, <i>p</i> < 0.0001). In the second analysis, a partial least squares method (PLS1) was used to assess whether an extended NIR spectral range (5400–3800 cm<sup>−1</sup>) could be utilized to predict collagen and proteoglycan content of the constructs based on mid-IR absorbances. A subset of spectra was randomly selected as an independent prediction set in this analysis. Average of the normalized root mean square errors of prediction of first-derivative NIR spectral models were 7% for 850 cm<sup>−1</sup> (PG), 11% for 1338 cm<sup>−1</sup> (collagen), 8% for amide II (collagen), and 8% for amide I (collagen). These results demonstrate the ability of NIRS to monitor macromolecular content of cartilage constructs and is the first step towards employing NIR to assess engineered cartilage in situ.

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