FTIR IMAGING OF THE COMPOSITION OF TERRITORIAL AND INTERTERRITORIAL MATRIX OF HUMAN
ARTICULAR CARTILAGE
+*Rieppo, J; **Ruotsalainen, H; ***Töyräs, J; **Kurkijärvi, J; ****Kiviranta, I; *Hyttinen, M M; **Jurvelin, J S; *Helminen, H J
+University of Kuopio, Department of Anatomy, Kuopio, Finland
[email protected]
METHODS
Cartilage samples were prepared from femur, tibia and patellar groove of
human cadaver knee joints (n=13). Full-thickness cartilage layer was
detached from the subchodral bone with a razor blade. Cartilage was
embedded into OCT embedding media with two pieces of nitrocellulose
membrane. Nitrocellulose was used to normalize differences in section
thickness as previously described [1]. Subsequently, 10 m-thick
sections were cut with cryomicrotome and sections were directly placed
onto ZnSe-windows. After air-drying (minimum 24 h) samples were
measured using PerkinElmer Spectrum Spotlight 300-imaging system.
IR-images were acquired with 8 cm-1 spectral and 6.25 m-pixel
resolution and two repeated scans were averaged for each pixel.
Collagen content was analyzed using the average background corrected
absorption of amide region (1710-1510 cm-1) and PGs with carbon
region absorption (1075-975 cm-1).
RESULTS
FT-IRIS measurement showed high collagen contents in the superficial
and deep cartilage whereas PG concentration was monotonically
increasing (Fig. 1). Highest collagen and PG levels were reached in the
deep cartilage. Especially in the deep cartilage, PG concentration around
cells increased (Fig. 1 and 2b). Territorial matrix surrounding the cell
showed distinctively different composition, as compared to
interterritorial matrix between the cells. Territorial matrix revealed
higher PG concentration and lower collagen content compared to
interterritorial matrix (Fig. 2a and b).
DISCUSSION
Present study demonstrated the suitability of FT-IRIS for
characterization of highly inhomogeneous ECM composition. Chemical
images showed complex distribution patterns of collagen and PGs
around the cells. PG distribution, even its high concentration around the
cells, can also be seen using light microscopy with staining techniques,
whereas the methods for quantitation of spatial collagen distribution
have been lacking. Both collagen and PG distributions were effectively
measured with a singe imaging session. This quantitative information on
the main matrix constituents, and especially their distribution around the
cells, allows us to develop more realistic theoretical models to describe
mechanical interactions, the fluid flow and stresses, between the cell and
its matrix environment. Detailed information on the physiological
conditions at the chondrocyte-ECM interface is essential to improve
understanding of mechanotransduction in cartilage.
High
Col/PG
content
INTRODUCTION:
Extracellural matrix (ECM) of articular cartilage consists mainly of
fibrillar collagen (type II) and proteoglycans (PGs). Composition and
properties of ECM generate unique biomechanical properties of articular
cartilage. ECM composition varies in different layers of the tissue.
Collagen content is high in the superficial and deep layers of the tissue
whereas PGs show nearly monotonously increasing concentration from
superficial to deep tissue. Solid matrix constitution directly controls
water distribution within tissue. Detailed description of the structural
characteristics of the matrix is essential in order to understand complex
mechanical features of articular cartilage. As the traditional biochemical
methods fail to preserve spatial compositional information, these
techniques are often limited to the determination to average (bulk)
content information of collagen or PGs only. Therefore, compositional
differences of territorial and interterritorial ECM are not well
characterized. The present study introduces Fourier transform infrared
imaging spectroscopy (FT-IRIS) for the characterization of ECM
composition, revealing detailed compositional differences between the
territorial and interterritorial matrix.
A
B
Low
Figure 1. Spatial distribution of collagen (A) and PGs (B) in human
articular cartilage. Images are from the same sample and formed by
using amide region (collagen) and carbon region (PGs). Interterritorial
matrix shows high collagen content and low PG content whereas
territorial matrix around the cells has high PG content, often visualized
as a ring surrounding the cell, and low collagen content.
A
B
Figure 2. A single cell and its territorial/interterritoraila matrix in deep
cartilage. Chemical images of collagen (A) and proteoglycans (B) are
produced with FT-IRIS. Collagen shows low/high concentrations in
territorial/interterritorial matrix, respectively (A). PGs show high
concentration in territorial matrix and lower concentrations in
interterritorial matrix (B). Length of the scalebar is 20 m.
**University of Kuopio, Dept. of Applied Physics, Kuopio, Finland.
*** Kuopio University Hospital, Dept. of Clinical Physiology and
Nuclear Medicine, Kuopio, Finland.
**** Jyväskylä Central Hospital, Dept. of Surgery, Jyväskylä, Finland.
REFERENCES
1. Rieppo J et al. 2004 Appl Spectrosc. 58(1):137-40.
51st Annual Meeting of the Orthopaedic Research Society
Poster No: 1500