Arash Hanifi

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

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.

Infrared Fiber Optic Probe Evaluation of Degenerative Cartilage Correlates to Histological Grading

Background: Osteoarthritis (OA), a degenerative cartilage disease, results in alterations of the chemical and structural properties of tissue. Arthroscopic evaluation of full-depth tissue composition is limited and would require tissue harvesting, which is inappropriate in daily routine. Fourier transform infrared (FT-IR) spectroscopy is a modality based on molecular vibrations of matrix components that can be used in conjunction with fiber optics to acquire quantitative compositional data from the cartilage matrix. Purpose: To develop a model based on infrared spectra of articular cartilage to predict the histological Mankin score as an indicator of tissue quality. Study Design: Comparative laboratory study. Methods: Infrared fiber optic probe (IFOP) spectra were collected from nearly normal and more degraded regions of tibial plateau articular cartilage harvested during knee arthroplasty (N = 61). Each region was graded using a modified Mankin score. A multivariate partial least squares algorithm using second-derivative spectra was developed to predict the histological modified Mankin score. Results: The partial least squares model derived from IFOP spectra predicted the modified Mankin score with a prediction error of approximately 1.4, which resulted in approximately 72% of the Mankin-scored tissues being predicted correctly and 96% being predicted within 1 grade of their true score. Conclusion: These data demonstrate that IFOP spectral parameters correlate with histological tissue grade and can be used to provide information on tissue composition. Clinical Relevance: Infrared fiber optic probe studies have significant potential for the evaluation of cartilage tissue quality without the need for tissue harvest. Combined with arthroscopy, IFOP analysis could facilitate the definition of tissue margins in debridement procedures.

Clinical outcome of autologous chondrocyte implantation is correlated with infrared spectroscopic imaging-derived parameters

OBJECTIVE To investigate whether Fourier transform infrared imaging spectroscopy (FT-IRIS), a modality based on molecular vibrations, is a viable alternative to histology and immunohistochemistry (IHC) for assessment of tissue quality and patient clinical outcome. METHODS Osteochondral biopsies were obtained from patients (9-65 months post-surgery) who underwent an autologous chondrocyte implantation (ACI) procedure to repair a cartilage defect (N = 14). The repair tissue was evaluated histologically by OsScore grading, for the presence of types I and II collagen by IHC, and for proteoglycan (PG) distribution and collagen quality parameters by FT-IRIS. Patient clinical outcome was assessed by the Lysholm score. RESULTS Improvement in Lysholm score occurred in 79% of patients. IHC staining showed the presence of types I and II collagen in all samples, with a greater amount of collagen type II in the deep zone. The amount and location of immunostaining for type II collagen correlated to the FT-IRIS-derived parameters of relative PG content and collagen helical integrity. In addition, the improvement in Lysholm score post-ACI correlated positively with the OsScore, type II collagen (IHC score) and FT-IRIS-determined parameters. Regression models for the relation between improvement in Lysholm score and either OsScore, IHC area score or the FT-IRIS parameters all reached significance (p < 0.01). However, the FT-IRIS model was not significantly improved with inclusion of the OsScore and IHC score parameters. CONCLUSION Demonstration of the correlation between FT-IRIS-derived molecular parameters of cartilage repair tissue and patient clinical outcome lays the groundwork for translation of this methodology to the clinical environment to aid in the management of cartilage disorders and their treatment.

Compositional Assessment of Human Tracheal Cartilage by Infrared Spectroscopy

Objectives To assess the potential of infrared fiber-optic spectroscopy to evaluate the compositional properties of human tracheal cartilage. Study Design Laboratory-based study. Methods Twenty human cadaveric distal tracheas were harvested (age range 20-78 years; 6 females, 14 males) for compositional analysis. Histologic staining, Fourier transform infrared imaging spectroscopy data on collagen and proteoglycan (PG) content, and near-infrared (NIR) fiber-optic probe spectroscopic data that reflect protein and water content were evaluated. NIR fiber-optic probe data were also obtained from the proximal trachea in 4 human cadavers (age range 51-65 years; 2 females, 2 males) in situ for comparison to distal trachea spectral data. Results In the distal trachea cohort, the spectroscopic-determined ratio of PG/amide I, indicative of the relative amount of PG, was significantly higher in the tissues from the younger group compared to the older group (0.37 ± 0.08 vs 0.32 ± 0.05, P = .05). A principal component analysis of the NIR spectral data enabled separation of spectra based on tracheal location, likely due to differences in both protein and water content. The NIR-determined water content based on the 5200-cm−1 peak was significantly higher in the distal trachea compared to the proximal trachea (P < .001). Conclusions Establishment of normative compositional values and further elucidating differences between the segments of trachea will enable more directed research toward appropriate compositional end points in regenerative medicine for tracheal repair.

Spectroscopic Analysis of Human Tracheal Tissue during Decellularization

Objective To use mid-infrared (IR) spectroscopy to assess changes in the cartilaginous framework of human trachea during decellularization. Study Design Laboratory-based study. Setting Research laboratory. Methods Six cadaveric human tracheas were decellularized using a detergent enzymatic method (DEM). Tissue samples were obtained from each specimen after 0, 1, 10, and 25 DEM cycles for histologic and spectroscopic analysis. Decellularization was confirmed using hematoxylin and eosin (H&E) and 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) staining. Changes in cartilaginous framework were examined using Fourier transform infrared imaging spectroscopy (FT-IRIS) and an attenuated total reflectance (ATR) probe in the mid-IR frequencies. Results were statistically analyzed using 1-way analysis of variance (ANOVA) and principal component analysis (PCA). Results Six decellularized tracheal scaffolds were successfully created using a DEM protocol. Histologic examination showed near-complete nuclear loss following 25 DEM cycles. As observed with FT-IRIS analysis, the collagen absorbance signal (1336 cm−1) was predominantly in the perichondria and remained stable after 25 DEM cycles (P = .132), while the absorbance from sugar rings in proteoglycans and nucleic acids in hyaline cartilage (1080 cm−1) showed a significant decrease after 1 DEM cycle (P = .0007). Examination of the luminal surface of the trachea with an ATR probe showed raw mid-IR spectra consistent with cartilage. PCA showed significant separation of spectra corresponding to treatment cycle along the principal components 1 and 2. Conclusion Mid-IR spectroscopy is a viable method of monitoring changes in extracellular matrix components during the decellularization of human trachea.