Quantitative cell and matrix changes in human humeral head osteoarthritic cartilage

APMIS 108: 64219. 2000 Printed in Denmark. All righfs reserved

Copvrighl 0 A P M I S 2000

kPMU8 ISSN 0903-4641

Quantitative cell and matrix changes in human humeral head osteoarthritic cartilage

RYUJI MORI, MITSUO OCHI, YASUO SAKAI, SOKICHI MANIWA and KENZO KAWASAKI

Orthopaedic Department, Shimane Medical University, Enya-cho, Izumo-shi, Shimane, Japan

Mori R, Ochi M, Sakai Y, Maniwa S & Kawasaki K. Quantitative cell and matrix changes in human humeral head osteoarthritic cartilage. APMIS 2000;108:642-8.

A new quantitative method was devised both to establish an objective standard for morphometric diagnosis and to determine the extent of degeneration in osteoarthritic cartilage. Eight normal and forty-eight osteoarthritic humeral heads, subsequently confirmed by light microscopy, were obtained at necropsy. The articular cartilage was observed in situ with a laser scanning confocal microscope (LSCM) and morphometric measurements determined cell density (celldmm), cell volume fraction (%) and mean cell volume (pm3). The osteoarthritic cartilages were classified according to the following four characteristics: increase in thickness, increase in cell volume fraction, decrease in cell volume fraction, and fibrous pannus. Deviations in cell density and cell volume fraction from normal means were calculated as extent of degeneration. Our present approach aims to provide valuable clues, such as objective stereological information and a unique reference for biochemical and traditional morphological analyses, that clinicians will be able to use in combination with other methods in order to establish a reliable diagnosis.

Key words: Osteoarthritis; morphology; confocal microscopy; quantitative analysis.

Ryuji Mori, Orthopaedic Department, Shimane Medical University, 89-1, Enya-cho, Izumo-shi, Shi- mane, Japan 693-8501. e-mail: [email protected]

A normal articular cartilage is highly inhomo- geneous (l), and pathological patterns of articular cartilage are further modified by produc- tion of new cartilage either adjacent to or within the sites of erosion (2). Thus, biochemical studies are of limited value as it is difficult to assess a process that is not only focal in nature but also occurs in heterogeneous tissues. Con- ventional light and electron microscopy are not optimal methods for quantifying structural information, although they can reveal structural details. The number of cell profiles in a two- dimensional section, for example, is a biased es- timate of the cell number because objects with longer caliper lengths are preferentially selected

Received June 28, 1999. Accepted July 2 1, 2000.

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(3, 4). A new methodology (1, 5 ) has been de- veloped to measure spatial variations in chondrocyte/matrix structural parameters in normal articular cartilage using laser scanning confocal microscopy (LSCM). Minimizing dis- turbances from artifacts such as cell shrinkage caused by conventional methods (l), this approach provides an ideal way to optically section thick slices of semitransparent biological tissues such as cartilage without disturbing the tissue physically. However, little quantitative information is currently available on the stereology of degenerated cartilage. In the present study, we applied LSCM for the first time to evaluate the status of osteoarthritic cartilage in an attempt to present a novel, informative and reliable diagnostic approach. The superiority and usefulness of the new unbiased methodology

QUANTITATIVE CHANGES OF OSTEOARTHRITIS

compared to the traditional histological approach is discussed.

MATERIALS A N D METHODS

Of the 58 humeral heads obtained from 29 subjects (65 to 85 years of age) at necropsy, 48 showed classic osteoarthritis while 8 displayed a normal cartilage histology (confirmed by conventional light microscopy). A pair of heads from a subject with swelling and deformities of the bilateral proximal interphalan- geal, metacarpophalangeal and wrist joints indicating rheumatoid arthritis (6) were not included in the study. Subjects with soft tissue swelling of three or more joint areas or rheumatoid nodules were likewise not included, though formal serological tests and clinical history, taking into consideration presence of morning stiffness, for example, were not used. Four of the osteoarthritic heads with cartilage thickness registering less than 1.0 mm coupled with deep defects were also excluded from the study. The 8 normal samples (66-80 years, average 74 years) were age- matched with 44 osteoarthritic cartilages (65-85 years, average 77 years). Two samples in the normal group made a pair from the same individual. In- formed consent was obtained from all subjects.

Because of normal variations in the intra-articular area, full-thickness cartilage samples measuring 5 X 5 mm of the surface area were exclusively excised from the exact center of the articular surface of the humeral head. This region was the central weight-bearing area at approximately 45” abducted and 30” flexed position of the shoulder. Cartilage fixation and iso- lation were conducted within 3 h after death. In the literature (7), cartilage samples prepared within 3 h of the postmortal period are recognized as fresh con-

trols because of the supreme viability of the cells throughout all layers of the articular cartilage.

A procedure designed by Wong et al. (1) was used with modifications for confocal and conventional light microscopy. The whole subchondral bone, except for a thin layer, was removed using a dental saw. The cartilage was cut coronally into approximately 1 mm full- thickness sections using an oscillating tissue slicer (DTK-1000; Douhan EM, Kyoto, Japan). After 48 h fixation with 5% glutaraldehyde (autofluorescent) and 0.02% Mayer’s hematoxylin, the sections were briefly washed with phosphate-buffered saline before mount- ing on standard microscopic slides for observation with an imaging system based on LSCM (Leica, Tokyo, Japan) where an oil immersion objective (x40) with a numerical aperture of 1 .O, a pinhole size of 100- 150 pm, and an argon laser of 488 nm wavelength was used. The cartilage thickness was measured optically, and zooming of the microscope was adjusted such that the height of the confocal image was set at one eighth the thickness (Fig. 1). Each of the eight fields was sec- tioned optically at 1 pm intervals to a depth of 30 pm (Fig. 2) . To compensate for the problem of light at- tenuation (8), the gain of each optical section was manually adjusted to maximize the contrast and the signal-to-noise ratio. The images were photographed and viewed using a computer (Power Macintosh 7600/ 200) before analysis with NIH image 1.57 software. An average of 60 cells was counted per zone to measure the cell density (cells/mm’) and cell volume fraction by point counting the area occupied by cells (“A). By taking the ratio of these parameters, estimates of so-called secondary parameters, including the mean cell volume (pm3) and mean matrix volume/cell (pm3), were calculated according to the method of Wong et al. (1). The abnormal stereology of osteoarthritic cartilage was quantified by deviation from normal values as follows:

Fig. 1. The thickness of an articular cartilage from the surface to the tidemark was divided into eight equal sites for morphometric analysis in normal cartilage (A) and osteoarthritis with fibrous pannus formation (B).

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Fig. 2. Cytoplasm fluorescence with intensity higher than the nucleus and surrounding matrix appears white in a plane at 25 pm (A) and 30 pm (B) depth in an osteoarthritic cartilage. Cells were recognized up to a depth of at least 30 pm, although the fluorescence attenuated with depth. The cell density was calculated as the total number of cells observed in a plane divided by the volume of the plane. In this example, two cells (arrowheads) were counted.

i= 1

where Dc represents the deviation index of the cell density; ci, the mean cell density of the sitei of normal cartilage; Ci, the cell density of the sitei, and

R

i= 1

where Dv represents the deviation index of the cell volume fraction; vi, the mean cell volume fraction of the sitei of the normal cartilage; Vi, the cell volume fraction of the sitei.

Standard paraffin sections of 5 pm thickness were prepared from a slice (1 mm thick) adjacent to the tissue used for confocal imaging. The sections stained for conventional light microscopy were scored using the Mankin’s histological-histochemical (H-H) grading system (9) by the same examiners (RM, YS), who were blind to the confocal findings. A maximum score of 14 was used to indicate the most severe con- dition, while the minimum score of 0 was considered normal (Fig. 3).

ANOVA and Dunnett were used to compare parameters. Correlations were examined using the Spearman rank correlation coefficient test.

RESULTS

To detect initial degenerative changes, the normal cartilage in this study was strictly limited

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to a zero score without surface irregularity, hyper- or hypo-cellularity besides indicating in- tense staining of safranin-0 with an intact tidemark (9). The number of cells per unit volume in the superficial site (4.15 X 1 04cells/mm3) was twice that in the lower sites. The cells occupied an average of 3.65% of the tissue, ranging between 3.15% in the superficial site and 3.86% in the deepest site. The mean cell volume increased by a factor of 2.6 from the surface (0.74X103 pm3) to the tidemark (1.9XlO’ pm’). The mean matrix volume per cell was least in the superficial site (2.41X104 pm3) and increased to 4.90X lo4 pm’ in the deepest site.

The stereological changes of osteoarthritic cartilages were grouped according to four characteristics: increase in thickness (IT), increase in cell volume fraction (IC), decrease in cell volume fraction (DC), and fibrous pannus (FP). Group IT (n= 12) was characterized by increased thickness (1.57-1.98 mm) surpassing the normal average value (1.54 mm, Fig. 3). While the cell density decreased (21.9-26.OX lo3 vs the normal average of 27.2X 103cells/mm3), the matrix volume per cell increased (34.5- 37.2X lo3 vs normal average of 36.7X lo3 pm’), and the number of cells within a unit surface area (1 mm2) was maintained at 81-105‘Yn of the normal average. Group IC (n=10) was characterized by increases in the mean cell volume


Cell densitv 0

E E 40 \ u) /

a

Site number Fig. 3. Histology and cell density distribution of a typical sample of osteoarthritic cartilage (0) compared with normal mean values (-). According to the histological-histochemical grading system (A), this cartilage (a paraffin section stained with safranin-0 and fast green; bar= 100 pm) scored 8 points (surface irregularity, hypocellul- arity. severe reduction of safranin-0 staining, and tidemark crossed by blood vessels were observed). The objective stereological system (B) classified the sample as belonging to the decrease-in-cell-volume fraction (DC) group and quantified the severity as 46 on the deviation index of cell density. Deviation was observed only near the surface in (B). Site l=tidemark, site 8=surface.

fraction (23.65%) compared with the normal average. This is basically due to an increase in the cell volume (1.44-1.56X102 pm3 vs the normal average of 1.44X lo2 pm3) because the overall cell density of this group was lower than the normal average except in three pieces. Group DC (n= 16) was characterized by decreases in the cell volume fraction (<3.65%), showing an apparent decrease in the cell density (1 5.0-26.8 vs 27.2X lo3 cells/mm3). Both increases and decreases in cell density and cell volume fraction were observed mostly at the surface and middle sites. Only minor changes were noticed near the tidemark (Tables 1 & 2). Group FP (n=6) was characterized by fibrous pannus formation, showing a distinct layer from the underlying cartilage tissues. This was analyzed separately

as site 9 in stereological measurements, regard- less of its thickness (Fig. 1). The cell density increased to a great extent at the fibrous pannus/ cartilage border. Three-dimensional observations showed the spindle shape of fibroblasts instead of the discshape of chondrocytes. The underlying cartilage revealed various patterns, ranging from increases to decreases in the cell volume fraction. In the shoulder and the hip, pannus formation is a relatively common find- ing even in osteoarthritis. However, the extent and the aggressiveness of this pannus with respect to underlying cartilage destruction is much less marked in osteoarthritis than in rheumatoid arthritis (10).

The Mankin's H-H system for scoring stereological patterns showed a decisive ten-

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dency. Normality, increase in thickness, increase and decrease in cell volume fraction, and fibrous pannus in Groups N, IT, IC, DC and FP were registered as 0, 1 4 , 3-9, 6-12, and 3-8, respec- tively. According to the Spearman rank correlation coefficient, the correlation between deviations in the cell density and H-H scores was 0.7 in Group IT (p=O.O11), -0.61 in IC (p= 0.069), 0.78 in DC (p=0.0057), and 0.67 in FP (p=0.055). The correlation between deviations in cell volume fraction and scores was 0.74 in Group IT ( ~ ~ 0 . 0 4 9 ) ~ -0.61 in IC (p=0.042), 0.81 in DC (p=0.035), and 0.71 in FP (p= 0.065). The correlation between deviations in cell density and the cartilage thickness was 0.014 in Group IT, -0.22 in IC, 0.16 in DC, and 0.34 in FP. The correlation between deviations in cell volume fraction and thickness was 0.023 in Group IT, -0.35 in IC, 0.22 in DC, and 0.19 in FP. Overall, the correlation between H-H scores and thickness was 0.55.

DISCUSSION

Previous histological and histochemical studies have indicated that cartilage is remodeled dur- ing degenerative osteoarthritis. This remodeling includes increases in cartilage thickness or dis- tension of the microenvironment following initiation of chondrocyte division and clonal proliferation to cope with the expanding microenvironment (1 1). In this study, confocal quanti- fication detected the initial change in osteoarthritis, including increases in the mean matrix volume per cell, while maintaining the number of cells per unit surface area (1 mm2), showing that increases in the cell volume fraction re- flected cell enlargement. The total cell number per unit surface area (1 mm2) decreased except for three pieces of cartilage Group IC (n=10). This demonstrates the limited chondrocyte proliferation and intrinsic repair of articular cartilage. The IC stage is thought to be followed by the DC phase. A previous isotopic study showed that at a specific stage of osteoarthritis, the cartilage was so badly damaged that it was in- capable of maintaining the repair responses involving cell proliferation and matrix synthesis (9). Although there was an overlap between the IT and DC groups with respect to overall cell

density, the former maintained a constant number of cells within a unit surface area, whereas the latter did not. Interestingly, both hypo- and hypercellularity were usually observed within the upper sites of the cartilage. FP formation is also considered to be one of the reparative activities in terms of cell profile. The structure, however, is completely different from native cartilage, even involving components of extracarti- lagenous origin such as the synovium (12). Thus, the parameters of the FP pattern must be interpreted with caution.

Group DC may represent not only cellular loss but also loss of the surface layer, since a normal surface zone has about twice the numerical cell density of deep zones. Therefore, there will probably be some criticism of direct graphical comparison by level of intact and eroded cartilage as being inappropriate. Readers must be cautioned that this system should not be applied in the case of late pathological stages of cartilage which manifest deep defects in the cartilage layer. At late stages, comparison of the thickness and proteoglycan contents, etc. could be useful since the 3-D structure has already been extensively de- stroyed. However, in this study, care was taken to ensure that cellular parameters would be sen- sitive to evaluation in early stages of osteoarthritis in humeral head which maintained more than 1 mm thickness, because of the wide-ranging variety of morphometric changes. In contrast, there was no correlation between the thickness of the cartilage layer in the present system and traditional pathological grading.

The present technique provides useful stereological information and serves as a unique reference for samples subjected to biochemical and traditional morphological analyses. Al- though minor swelling or shrinkage of the matrix even in fixative or culture fluid may have to be considered as well, only fixed samples were used in this study since we intended to develop a conventional system using readily available samples from the clinic. Conventional microscopy can also be used as an alternative to evaluate cellular parameters ( 13), while confocal microscopy can be used to make efficient measurements of cell density and volume fraction. Further studies using this methodology with more extensive sampling within a joint, including the less-weight-bearing portion, are war-

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ranted to promote a fuller understanding of the pathogenesis of osteoarthritis.

Our quantitative approach was devised to meet clinical needs. An arthroscopic biopsy of a small osteochondral specimen followed by histological examination has been performed to determine the efficacy of treatment (14, 15). However, classical pathological diagnoses using Mankin’s scoring system are, in part, empiric and are based on the qualitative observations and experience gained from examination of many specimens - a subjective technique. The scoring system has previously been shown to be unreliable (16). Therefore, it was not surprising that the present approach did not show a strong correlation with the Mankin’s scoring system. In addition, recent developments in surgical and pharmacological methodology for the treatment and prevention of chondral degeneration and injury (14, 15, 17) have led to heated controversy, emphasizing the need to establish an objective and reliable system for evaluating tissue quality (18). An objective analysis such as in our present approach would therefore provide many useful clues and could be used by clinicians in combination with other methods to establish a reliable diagnosis.

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Quantitative cell and matrix changes in human humeral head osteoarthritic cartilage

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