Near infrared spectroscopic imaging assessment of cartilage composition: Validation with mid infrared imaging spectroscopy

Abstract

Disease or injury to articular cartilage results in loss of extracellular matrix components which can lead to the development of osteoarthritis (OA). To better understand the process of disease development, there is a need for evaluation of changes in cartilage composition without the requirement of extensive sample preparation. Near infrared (NIR) spectroscopy is a chemical investigative technique based on molecular vibrations that is increasingly used as an assessment tool for studying cartilage composition. However, the assignment of specific molecular vibrations to absorbance bands in the NIR spectrum of cartilage, which arise from overtones and combinations of primary absorbances in the mid infrared (MIR) spectral region, has been challenging. In contrast, MIR spectroscopic assessment of cartilage is well-established, with many studies validating the assignment of specific bands present in MIR spectra to specific molecular vibrations. In the current study, NIR imaging spectroscopic data were obtained for compositional analysis of tissues that served as an in vitro model of OA. MIR spectroscopic data obtained from the identical tissue regions were used as the gold-standard for collagen and proteoglycan (PG) content. MIR spectroscopy in transmittance mode typically requires a much shorter pathlength through the sample (≤10 microns thick) compared to NIR spectroscopy (millimeters). Thus, this study first addressed the linearity of small absorbance bands in the MIR region with increasing tissue thickness, suitable for obtaining a signal in both the MIR and NIR regions. It was found that the linearity of specific, small MIR absorbance bands attributable to the collagen and PG components of cartilage (at 1336 and 856 cm−1, respectively) are maintained through a thickness of 60 μm, which was also suitable for NIR data collection. MIR and NIR spectral data were then collected from 60 μm thick samples of cartilage degraded with chondroitinase ABC as a model of OA. Partial least squares (PLS) regression using NIR spectra as input predicted the MIR-determined compositional parameters of PG/collagen within 6% of actual values. These results indicate that NIR spectral data can be used to assess molecular changes that occur with cartilage degradation, and further, the data provide a foundation for future clinical studies where NIR fiber optic probes can be used to assess the progression of cartilage degradation.

Spatial correlation of native and engineered cartilage components at micron resolution

“…Utilization of 80 μm sections is unconventional in the MIR range, because the depth of penetration of the IR radiation is typically only several micrometers. However, this is also dependent on the specific absorptivity coefficient of the vibration being studied, as shown by a previous study in our laboratory where the effects of various sample thicknesses on the signal‐to‐noise ratio and linearity of absorbance was assessed . Data showed that the absorbance of amide I and II bands reached saturation due to attenuation of the signal, but the MIR bands at 856 and 1340 cm –1 , which have substantially smaller absorptivity coefficients, and all of the NIR bands did not reach saturation.…”

“…The absorbance seen in the MIR spectra at ∼856 cm –1 arises from the C–O–S vibrations of the sGAG chains. The intensity of this peak is directly correlated with the content of PG in cartilage, and it has been used previously to quantify the spatial distribution of PG in engineered cartilage . sGAGs are glycosylated proteins and make up 10–15% of the wet weight of articular cartilage .…”

“…This resulted in evaporation of the free water, and thus the majority of the spectral data was attributable to bound water. Imaging parameters were previously optimized by our laboratory with similar samples …”

“…FT‐IRIS images were first processed using the ISys chemical imaging analysis software (version 5.0.0.14; Malvern Instruments, Columbia, MD) to obtain compositional distributions based on the intensity of specific MIR and NIR peaks. The MIR peaks of interest were at ∼856 cm –1 (PG, C–O–S vibration) and 1340 cm –1 (collagen, side chain vibration), and the NIR peaks were at ∼5200 cm –1 (water, OH vibrations) and ∼4400, 4600, and 4890 cm –1 (matrix peaks, resulting from C–H and N–H vibrations) . The second derivative of the spectral images were obtained using the Savitzky‐Golay filter, applying 5 smoothing points for the MIR region (∼1800–750 cm –1 ) and 31 smoothing points for the NIR region (∼5500–4000 cm –1 ).…”

Vibrational spectroscopy and imaging: applications for tissue engineering

“…The absorption bands in the NIR region are overtones and combinations of the MIR fundamental vibrations of C–H, C–O, O–H and N–H bonds and are therefore appropriate for monitoring water, lipids, proteins and sugars. The main components of the cartilage ECM— water, collagen, and chondroitin sulfate (from proteoglycan) — have been identified and quantified using NIR spectroscopy with strong correlations to gold standard methods 73–75 . Palukuru et al 74 ascribed the peaks at 4050, 4260, 4610 and 4890 cm −1 to collagen, the peaks at 4020 and 4310 cm −1 to the sugar component of proteoglycans, and the peak at 4350–4375 cm −1 to chondroitin sulfate.…”