In Situ Assessment of Porcine Osteochondral Repair Tissue in the Visible-Near Infrared Spectral Region

Abstract

Standard assessment of cartilage repair progression by visual arthroscopy can be subjective and may result in suboptimal evaluation. Visible-near infrared (Vis-NIR) fiber optic spectroscopy of joint tissues, including articular cartilage and subchondral bone, provides an objective approach for quantitative assessment of tissue composition. Here, we applied this technique in the 350-2,500 nm spectral region to identify spectral markers of osteochondral tissue during repair with the overarching goal of developing a new approach to monitor repair of cartilage defects in vivo. Full thickness chondral defects were created in Yucatan minipigs using a 5-mm biopsy punch, and microfracture (MFx) was performed as a standard technique to facilitate repair. Tissues were evaluated at 1 month (in adult pigs) and 3 months (in juvenile pigs) post-surgery by spectroscopy and histology. After euthanasia, Vis-NIR spectra were collected in situ from the defect region. Additional spectroscopy experiments were carried out in vitro to aid in spectral interpretation. Osteochondral tissues were dissected from the joint and evaluated using the conventional International Cartilage Repair Society (ICRS) II histological scoring system, which showed lower scores for the 1-month than the 3-month repair tissues. In the visible spectral region, hemoglobin absorbances at 540 and 570 nm were significantly higher in spectra from 1-month repair tissue than 3-month repair tissue, indicating a reduction of blood in the more mature repair tissue. In the NIR region, we observed qualitative differences between the two groups in spectra taken from the defect, but differences did not reach significance. Furthermore, spectral data also indicated that the hydrated environment of the joint tissue may interfere with evaluation of tissue water absorbances in the NIR region. Together, these data provide support for further investigation of the visible spectral region for assessment of longitudinal repair of cartilage defects, which would enable assessment during routine arthroscopy, particularly in a hydrated environment.

Keywords: cartilage repair; fiber optic spectroscopy; in situ optical spectroscopy; microfracture; visible–near infrared (Vis-NIR).

FIGURE 1

Schematic of light scattering through cartilage tissue and a semi-infinite reflecting surface.

FIGURE 2

(A) Second-derivative spectra of harvested cartilage tissue collected on top of a semi-infinite PET block or on Spectralon standard (without PET). The depth of detection of PET signal at higher and lower frequencies was monitored by assessment of the PET signal at 8865 cm⁻¹ and 6000 cm⁻¹, respectively. (B) 8865 cm⁻¹ signal intensity can be detected with a cartilage thickness of up to 4 mm. (C) 6000 cm⁻¹ PET signal intensity can be detected with a cartilage thickness of up to 3 mm only. ∗∗p < 0.01.

FIGURE 3

(A) Second-derivative spectra of cartilage tissue (1.6 mm) with and without a semi-infinite PET polymer (25 mm) and the polymer alone showing the PET peak at 375 nm and cartilage peak at 420 nm. (B) Second-derivative peak intensity with and without PET underneath cartilage tissue.

FIGURE 4

(A) Saf-O- and fast green-stained images from 1-month (n = 9) and 3-month (n = 6) animals with the defined repair tissue region. (B) Mean and standard deviation of ICRS II component scores (comparisons by the Wilcoxon rank sum test, significance at p < 05).

FIGURE 5

(A) FTIR images based on the 1338 cm^−1/Amide II areas after 1 month of repair in a mature animal (n = 9) and 3 months of repair in a juvenile animal (n = 6). The measurement lines of defect thickness are shown, including both cartilage repair and subchondral remodeling regions. (B) Defect thickness is significantly greater in the 3-month animals (Student’s t-test).

FIGURE 6

Average (A) raw and (B) second-derivative inverted Vis-NIR spectra from 1-month (n = 9) and 3-month (n = 6) defects in situ. The visible spectra clearly depict the differences in hemoglobin absorbance (540 and 570 nm) between 1-month and 3-month repair. Quantified second-derivative peak intensities of hemoglobin peaks at (C) 540 nm and (D) 570 nm showing greater hemoglobin detection in the 1-month tissue (Student’s t-test).

FIGURE 7

Average (A) raw and (B) second-derivative inverted NIR spectra from 1-month (n = 9) and 3-month (n = 6) defects in situ. The spectra highlight qualitative differences in water absorbances at 5,200 cm⁻¹ and 7,000 cm⁻¹ between 1 month and 3 months of repair. Quantified second-derivative peak intensities of (C) water absorbances and (D) matrix absorbances in 1- and 3-month defects. Although trends are present, no significant differences were found in intensities between 1- and 3-month defects (Student’s t-test).

FIGURE 8

(A) Schematic of data collection from the defect tissue (in situ) and individual components, repair tissue, and subchondral bone, ex vivo. (B) Second-derivative spectra from the defect in situ and isolated repair tissue and subchondral bone ex vivo in the visible range demonstrate the hemoglobin peak intensities at 540 and 570 nm in the defect tissue are more similar to those in the repair tissue than to those in subchondral bone. (C) Second-derivative spectra in the NIR region demonstrate the differences in the water absorbances in the repair tissue in ex vivo compared to in situ spectra. At 8250 cm⁻¹, the influence of subchondral bone on the in situ defect spectrum is apparent while absent in the isolated repair tissue spectrum.