Research Highlights

Objective

To assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage.

Method

Fresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination.

Results

NLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes.

Conclusion

Thin images of living articular cartilage using NLOM may be obtained with (sub-) cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.

Spectra of endogenous nonlinear optical signals in articular cartilage used for imaging. (A) Intensity image of chondrocytes within cartilage matrix. (B) Spectral filtering of SHG signal at 400 nm specifically imaging collagen. (C) Spectral filtering of TPF signal at 520 nm specifically imaging the chondron. Scale bar is 8 µm.

NLOM images (SHG and TPF) and SOFG stained histology of normal (A&B) and degenerative joint diseased (C) articular cartilage. Extracellular TPF corresponds with proteoglycan content (orange staining).

Verhoeff's stained histology suggesting the presence of elastin (grey layers) within the superficial layer. Depth-dependent intensity (SHG + TPF), TPF and SHG images showing the ability to segment elastin-like fibers (TPF) and collagen (SHG) near the articular surface and matrix compositional and morphological changes with depth.

Histology and NLOM images of (A) normal, (B) early fibrillar matrical degeneration and (C) fibrocartilage of advanced degenerative joint disease. Intensity (column 1) and SHG16 NLOM images using polarized incident laser light vertically (column 2) and horizontally (column 3) oriented are shown.

(In press, Osteoarthritis and Cartilage, 2005)

References

1. Muir H. The chondrocyte, architect of cartilage. Biomechanics, structure, function and molecular biology of cartilage matrix macromolecules. Bioessays 1995;17(12):1039-48.
2. Lai WM, Hou JS, Mow VC. A Triphasic Theory for the Swelling and Deformation Behaviors of Articular Cartilage. Journal of Biomechanical Engineering 1991;113:245-58.
3. Hardingham TE, Beardmore-Gray M, Dunham DG, Ratcliffe A. Cartilage proteoglycans. Ciba Found Symp. 1986;124:30-46.
4. Maroudas A, Bannon C. Measurement of swelling pressure in cartilage and comparison with the osmotic pressure of constituent proteoglycans. Biorheology 1981;18(3-6):619-32.
5. Urban JP, Maroudas A, Bayliss MT, Dillon J. Swelling pressures of proteoglycans at the concentrations found in cartilaginous tissues. Biorheology 1979;16(6):447-64.