Composition of the Chondrocyte Microenvironment in Healthy and Osteoarthritic Cartilage:
Fourier Transform Infrared Spectroscopy Analysis
+1Saarakkala, S; 1Jurvelin, J S; 1Mäkitalo J; 1Korhonen, R K
+1Department of Physics, University of Kuopio, Kuopio, Finland
Corresponding author: simo.saarakkala@uku.fi
INTRODUCTION:
Cartilage cells, chondrocytes, are essential for the maintenance and
mechanical behavior of articular cartilage. In osteoarthritis (OA), the
structure, composition and mechanical properties of the cell
microenvironment alter [1]. This may change the mechanical signals
experienced by cells [1,2], alter tissue biosynthetic activity, and affect
possible cell death.
Fourier transform infrared imaging (FTIRI) has been used for
determination of the composition of cartilage tissue (collagen,
proteoglycans) in a single imaging session. The aim of this study was to
test the capability of FTIR to detect changes in the proteoglycan (PG)
and collagen contents in the vicinity of chondrocytes of normal and
osteoarthritic (OA) human articular cartilage. Further, we compared the
microenvironment of the superficial and deep zone cells.
METHODS:
Fourteen patellae from right knees of cadaveric human donors (12 males
and two females, age 55 ± 18 years) were collected as described earlier
[3]. The material included both intact and spontaneously degenerated
samples (n=84). Cartilage layer was detached from the subchondral bone
and prepared for histological and FTIRI analyses.
For the histological evaluation, 3 µm thick microscopic sections were
prepared and stained with Safranin-O. Mankin scoring was used for
grading OA of the samples [4].
Unstained microscopic sections were prepared for FTIRI
(PerkinElmer Spectrum 300 instrument, PerkinElmer Inc., Shelton, CT,
USA). Pixel size of the FTIRI was 6.25 µm. Spatial collagen and PG
content of the samples were determined using the Amide I (1720-1585
cm-1) and carbon (1140-985 cm-1) peak, respectively [5]. Subsequently,
obtained spatial collagen and PG maps were analyzed in the vicinity of
chondrocytes for the following sample groups: healthy (n=4, Mankin
score = 0-1, mild OA (n=5, Mankin score = 5) and severe OA (n=5,
Mankin score = 10). Collagen and PG contents in a cell
microenvironment were analyzed by manually drawing a line profiles
through the individual cells (10 cells from each sample; 5 cells from the
superficial and deep zones). In the superficial and deep zones, the
profiles were drawn perpendicular and parallel to the sample surface,
respectively, i.e. perpendicular to the primary collagen fibril orientation.
Total length of the line profile was ~69 µm, including a cell in the center
of the line. In each sample, the collagen and PG profiles were averaged
for the surface and deep zones. The profiles were normalized by the
parameter value at the edge of each profile.
RESULTS:
At the similar tissue depths, collagen and PG content decreased as
Mankin score increased (data not shown). The normalized collagen
content around the deep zone chondrocytes was similar in all sample
groups (Fig. 1). In the superficial zone of the samples with severe OA,
the collagen content of the pericellular matrix decreased with respect to
that of the extracellular matrix, as compared to the normal samples or
samples with mild OA.
In the deep zone, PG content in the cell microenvironment, as
compared to that of the extracellular matrix, increased as OA progressed
(Fig. 2). Relative values for the PG content of intact and severe OA
cartilage were similar both in the superficial and deep zones, however,
mild OA cartilage showed an increase in the normalized PG content
around cells in the superficial zone.
DISCUSSION:
In the present study, composition in the microenvironment of
chondrocytes of normal and osteoarthritic human cartilage was
characterized using FTIRI. The method provides novel information on
compositional changes in the cell environment during OA progression.
Our results suggest that OA may change the collagen and PG content
around cells differently in the deep and superficial zones of cartilage.
In mild OA, an increased relative PG content in the pericellular
matrix of the superficial zone may suggest that cells have increased the
production of aggrecan. However, the cells were not able to maintain the
PG content further away from the cells. In severe OA, the production of
aggrecan may have been reduced, indicated by the decreased relative PG
content in the pericellular matrix.
Figure 1. Collagen content (area of Amide I peak) in the
microenvironment of chondrocytes (cell locates in the middle of the
profile) in the deep (left) and superficial (right) zones of cartilage.
Figure 2. PG content (area of the carbon peak) in the microenvironment
of chondrocytes (cell locates in the middle of the profile) in the deep
(left) and superficial (right) zones of cartilage.
In the superficial zone, but not in the deep zone, the relative collagen
content of the pericellular matrix was low in severely osteoarthritic
cartilage with broken collagen network. This is consistent with earlier
studies (see review article [1]). Obviously, the superficial zone is then
subjected to high mechanical loads and, possibly, impaired mechanical
conditions alter the cell microenvironment in the superficial tissue
differently than in the deep tissue.
In an earlier study, FTIRI was used to investigate the cell
microenvironment of human cartilage in the deep zone [6]. Although
that study included only three samples, similar results were obtained for
Amide I absorption as we found here. In addition to the deep zone, the
present study focused on investigating normal and OA cartilage in
several stages, and showed differences in the superficial and deep zones.
However, the results are still preliminary and the changes in the cell
microenvironment during OA progression should be confirmed with
greater amount of samples, enabling statistical testing.
To conclude, despite the limited resolution for highly detailed
investigations, FTIRI provides a potential method to clarify
compositional changes in the cell microenvironment in healthy and
pathological cartilage. By implementing this information into a
theoretical model, more realistic estimates on the cell-matrix interactions
may be obtained [2]. The OA changes in cell microenvironment are still
poorly understood and more experimental and theoretical studies are
warranted. By far, FTIRI is the only experimental technique enabling
simultaneous measurement of PG and collagen spatial distribution even
from unprocessed cryosections.
REFERENCES:
[1] Guilak et al., Ann N Y Acad Sci 1068:498-512, 2006.
[2] Korhonen et el., J Biomech Eng 130:021003, 2008.
[3] Kiviranta et al., Osteoarthritis Cartilage 16:796-804, 2008.
[4] Mankin et al., J Bone Joint Surg Am 53:523-37, 1971.
[5] Boskey et al., Biomaterials 28:2465-78, 2007.
[6] Bi et al., Biochim Biophys Acta 1758:934-41, 2006.
Poster No. 1049 • 55th Annual Meeting of the Orthopaedic Research Society