Fourier Transform Infrared Imaging and Infrared Fiber Optic Probe Spectroscopy Identify Collagen Type in Connective Tissues

Abstract

Hyaline cartilage and mechanically inferior fibrocartilage consisting of mixed collagen types are frequently found together in repairing articular cartilage. The present study seeks to develop methodology to identify collagen type and other tissue components using Fourier transform infrared (FTIR) spectral evaluation of matrix composition in combination with multivariate analyses. FTIR spectra of the primary molecular components of repair cartilage, types I and II collagen, and aggrecan, were used to develop multivariate spectral models for discrimination of the matrix components of the tissues of interest. Infrared imaging data were collected from bovine bone, tendon, normal cartilage, meniscus and human repair cartilage tissues, and composition predicted using partial least squares analyses. Histology and immunohistochemistry results were used as standards for validation. Infrared fiber optic probe spectral data were also obtained from meniscus (a tissue with mixed collagen types) to evaluate the potential of this method for identification of collagen type in a minimally-invasive clinical application. Concentration profiles of the tissue components obtained from multivariate analysis were in excellent agreement with histology and immunohistochemistry results. Bone and tendon showed a uniform distribution of predominantly type I collagen through the tissue. Normal cartilage showed a distribution of type II collagen and proteoglycan similar to the known composition, while in repair cartilage, the spectral distribution of both types I and II collagen were similar to that observed via immunohistochemistry. Using the probe, the outer and inner regions of the meniscus were shown to be primarily composed of type I and II collagen, respectively, in accordance with immunohistochemistry data. In summary, multivariate analysis of infrared spectra can indeed be used to differentiate collagen type I and type II, even in the presence of proteoglycan, in connective tissues, using both imaging and fiber optic methodology. This has great potential for clinical in situ applications for monitoring tissue repair.

Comparison of Engineered Peptide-Glycosaminoglycan Microfibrous Hybrid Scaffolds for Potential Applications in Cartilage Tissue Regeneration

“…The FVC-CP spectra demonstrated a broad peak in the amide I region with split peaks at 1637 cm . Such split peaks are characteristic of Col-II and thus confirms the incorporation of Col-II to FVC-CP composites [78]. Upon incorporation of Ch, once again a broadening of the hydroxyl region peak was observed as expected.…”

Vibrational spectroscopy of articular cartilage

“…In addition, a PLS regression model for detecting the relative amounts of type I and type II collagens was built using pure compound mixtures. The model was shown to give reliable results in bone, tendon, normal and repair cartilage, and meniscus (46). The relationship between the FTIR spectra of bovine AC and its compressive stiffness was studied using PLS regression models combined with genetic algorithm for variable selection.…”

“…Furthermore, the clinical outcome of autologous chondrocyte implantation in human AC was shown to correlate with the proteoglycan content and the collagen integrity determined using infrared fiber optic probe (65). Recently, the feasibility of identifying collagen type based on infrared fiber optic measurements was demonstrated, which could be beneficial for monitoring tissue repair (46).…”

Infrared microspectroscopic determination of collagen cross-links in articular cartilage

“…FTIR microspectroscopy, a combination of spectroscopy and microscopy, is an intriguing method, as it enables determination of collagen and proteoglycan distributions from unstained histological articular cartilage sections. This is conducted either by calculating integrated areas of absorption peaks 4 or by calibrating multivariate models, e.g., principal component 5 or partial least squares (PLS) 6,7 regression models, against reference information about the investigated compound. The capability of FTIR microspectroscopy to determine collagen and proteoglycan contents has been demonstrated and validated against multiple reference methods.…”

“…The capability of FTIR microspectroscopy to determine collagen and proteoglycan contents has been demonstrated and validated against multiple reference methods. [5][6][7][8][9] Currently, the standard method to determine the cross-link concentrations in tissues is high-performance liquid chromatography (HPLC). As it is a destructive method, dedicated sample blocks are needed for the analysis.…”