Resolving the Near-Infrared Spectrum of Articular Cartilage

Abstract

Objective: Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by (a) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and (b) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state.

Design: The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identified bands, spectra of the model compounds were compared with those of native cartilage. Since water obscures some bands in the NIR range, spectral measurements of the native cartilage were conducted under conditions of decreasing water content to amplify features of the solid matrix components. The identified spectral bands were then compared and examined in the resulting spectra of the intact cartilage samples.

Results: As water was progressively eliminated from cartilage, the specific contribution of the different matrix components was observed to correspond with those identified from the proxy cartilage component models.

Conclusion: Spectral peaks in the regions 5500 to 6250 cm^-1 and 8100 to 8600 cm^-1 were identified to be effective for characterizing cartilage proteoglycan and collagen contents, respectively.

Keywords: articular cartilage; collagen; matrix component models; near-infrared spectroscopy; proteoglycans.

Figure 1.

Typical near-infrared (NIR) absorbance spectra of cartilage matrix models (proteoglycan [PG] and collagen [COL]) showing distinct spectral peaks associated with the matrix components. The highlighted sections show regions that are useful for evaluation of articular cartilage in its physiological state. The specific band assignment of each peak identified is defined in Table 1 .

Figure 2.

Near infrared spectra of proteoglycan (PG) model gels at varying mass concentrations in saline.

Figure 3.

Near-infrared (NIR) spectra of proteoglycan (PG) and collagen (COL) models, with their mixtures at 83.3%, 80%, 75%, 66.7%, and 50% concentrations, respectively, in the direction of the arrow.

Figure 4.

Typical near-infrared (NIR) spectrum of articular cartilage showing the absorption spectrum and raw sample spectrum (inset).

Figure 5.

Principal component analysis of the spectra of native articular cartilage (AC) and its matrix component models in dry (COL, PG, and MIX = PG + COL) and different concentrations of wet (PG and MIX) states. (A) Plot of the first 2 principal component (PC) scores (AC = blue, dry = black markers, different concentrations of wet PG = red markers, and MIX = green markers), (B) the second and third principal component scores, and their corresponding loadings (C). Variance explained by PC1, PC2, and PC3 are 97%, 1.7%, and 0.7%, respectively. Shaded spectral regions show areas that are useful for evaluation of articular cartilage in its physiological state. COL, collagen; PG, proteoglycan.

Figure 6.

(A) Unprocessed near-infrared (NIR) spectra of articular cartilage showing systematic decrease in baseline as matrix free water is gradually removed by drying at room temperature for 16 hours (blue = 0-4 hours; red = 4-8 hours; green = 8-12 hours; black = 12-16 hours). (B) Baseline-corrected spectra show slight resolution of peaks (inset), specifically between 5500 and 6 250 cm⁻¹.

Figure 7.

Spectra of articular cartilage showing changes in spectral peaks as bound water is gradually dissociated from the solid matrix components using ethanol. Inset plots show the most noticeable peak spectral changes. Blue = first 15 minutes of treatment; red = next 2 hours of treatment; pink = next 12 hours of treatment. Note: For clarity, not all spectra are displayed.