Spatial correlation of native and engineered cartilage components at micron resolution

Abstract

Tissue engineering (TE) approaches are being widely investigated for repair of focal defects in articular cartilage. However, the amount and/or type of extracellular matrix (ECM) produced in engineered constructs does not always correlate with the resultant mechanical properties. This could be related to the specifics of ECM distribution throughout the construct. Here, we present data on the amount and distribution of the primary components of native and engineered cartilage (i.e., collagen, proteoglycan (PG), and water) using Fourier transform infrared imaging spectroscopy (FT-IRIS). These data permit visualization of matrix and water at 25 μm resolution throughout the tissues, and subsequent colocalization of these components using image processing methods. Native and engineered cartilage were cryosectioned at 80 μm for evaluation by FT-IRIS in the mid-infrared (MIR) and near-infrared (NIR) regions. PG distribution correlated strongly with water in native and engineered cartilage, supporting the binding of water to PG in both tissues. In addition, NIR-derived matrix peaks correlated significantly with MIR-derived collagen peaks, confirming the interpretation that these absorbances arise primarily from collagen and not PG. The combined use of MIR and NIR permits assessment of ECM and water spatial distribution at the micron level, which may aid in improved development of TE techniques.

Near infrared spectroscopic assessment of engineered cartilage for implantation in a pre-clinical model

“…Analysis of specific second derivative NIR peak intensities and ratios allows quantification of several parameters directly associated with water and protein content 40 . Spectral absorbance peaks of water are observed at 5300, 7100 and 8600 cm -1 , with each peak related to water molecules present in different tissue environments, such as free and hydrogen-bound tissue water 20,22,[40][41][42][43][44][45][46] . Their relative intensities reflect differences in tissue type and composition 22 , and thus, the empirical evaluation of the water peak ratios may inform changes in molecular environments within the TEC, and correlations to TEC construct quality and related in vivo repair tissue quality.…”

Applications of Vibrational Spectroscopy for Analysis of Connective Tissues

“…Further yet, considering that NIR light is non-ionizing, NIR spectroscopy may be applied for the analysis of living tissues and organisms, including in humans [ 51 , 52 , 53 , 54 , 55 , 56 ]. Considerable advances have been made in these applications in the last few years, including in functional NIR imaging [ 57 , 58 ], and tissue imaging [ 42 , 46 , 49 , 59 , 60 ], which shows the potential to provide valuable data on compositional properties associated with tissue quality and health in situ and in vivo.…”

“…Two examples of major advances in vibrational spectroscopy are the coupling of spectrometers to imaging systems and fiber optic probes, which enables an important expansion of the biomedical application of these methods. When coupled to imaging systems [ 1 , 2 , 27 , 31 , 34 , 42 , 46 , 49 , 59 , 60 , 61 , 63 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 ], it is possible to obtain hyperspectral images of tissue sections, in which each micron-sized pixel corresponds to a spatially-defined spectrum. Each hyperspectral image can be comprised of arrays of hundreds to thousands of spectra and allow the analysis of multiple individual components based on the selection of specific intensities or absorbances present in the spectra, presenting an excellent source of information on the amount and distribution of tissue components.…”

“…Additionally, polarized infrared light can be used with FTIR imaging to reveal the zonal distribution of differently oriented collagen fibers based on the ratio of the amide I to amide II absorbances [ 69 ]. The information in these foundational studies of native cartilage has subsequently been applied in many areas of cartilage research, including towards understanding of degraded osteoarthritic tissues [ 102 , 114 , 115 , 116 ], and characterization of developing engineered constructs [ 60 , 117 , 118 , 119 ].…”

Nondestructive assessment of tissue engineered cartilage based on biochemical markers in cell culture media: application of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy

“…IR approaches in the mid IR (MIR) and near IR (NIR) spectral regions can be employed in the evaluation of construct composition, although NIR has somewhat lower molecular specificity. 8,11,12 Fourier transform infrared (FTIR) in the MIR range has been widely used for engineered tissue evaluation, 8,9,[13][14][15] but is often destructive to the constructs, due to the requirement to thin section the tissues.…”