958

BRIEF REPORT

PERICELLULAR PROTEOGLYCAN CONCENTRATIONS IN EARLY DEGENERATIVE ARTHRITIS

NELSON MITCHELL and NORA SHEPARD

It is commonly agreed that the earliest biochemi- cal changes in degenerative arthritis are loss of pro- teoglycan as evidenced by diminished staining with cat- ionic dyes or direct biochemical analysis of diseased cartilage (1,2). There have been no reports of analysis for proteoglycan concentration in minute areas throughout the various zones of articular cartilage.

We have recently shown that a satisfactory anal- ysis for proteoglycan can be carried out by electron mi- croprobe using brominated toluidine blue in the fixa- tion process of cartilage (3). Toluidine blue combines mole for mole with glycosaminoglycan and a measure of bromine in BR-TBO sections is a measure of pro- teoglycan (4). This article reports the spot analyses for proteoglycan in the various zones of early degenerative arthritic cartilage.

MATERIALS AND METHODS

Samples of articular cartilage were removed from 4 patients undergoing arthroplasty of the knee. Al- though all the patients had advanced degenerative ar- thritis requiring knee arthroplasty, the samples removed

From the Electron Microscopic Unit of Shriners Hospital, Montreal, Canada.

Supported by the Shriners of North America and a grant from the Medical Research Council of Canada.

Address reprint requests to Electron Microscopy Unit, Shrin- ers Hospital, 1529 Cedar Avenue, Montreal, Quebec, Canada, H3G IA6.

Submitted for publication August 1 I , 1980; accepted in re- vised form January 29, I98 1.

Arthritis and Rheumatism, Vol. 24, No. 7 (July 1981)

were taken from those areas where a moderate degree of cartilage thickeness remained and the disease was thought to be at a relatively early stage in that region (Figure 1). Samples of normal cartilage for controls were removed from 3 patients undergoing amputation who had otherwise normal knee joints. The blocks, 10 to 16 in number, were fixed in 2% buffered glutaralde- hyde plus 0.1% brominated toluidine blue; they were then buffer rinsed, osmicated, alcohol dehydrated, and embedded in Spurr plastic.

Half-micron sections were placed on copper grids and studied in a Philips 400 transmission electron microscope with scanning transmission electron micros- copy attachment. After initial photography of the region of interest, the specimen was tilted 40" toward the x-ray detector and using a gun current of 40 microamperes, 80 KV and 100 nm spot size, regions of interest were ana- lyzed for 100 seconds. Three to six such 100-second counts were performed on the same spot and if the first 3 counts fell within 2 standard deviations of each other, these results were averaged. Counts falling outside 2 standard deviations were discarded on the assumption that these represented specimen contamination, elec- tronic instability, or beam shift.

All spectra underwent computerized background subtraction to remove Bremstralung radiation using the EDAX Edit and Nova 2 computer systems. The number of x-ray counts within a given spectra are indicated above the individual spectrum. This method permits quantitation of bromine and hence proteoglycan from area to area within the same section, though not neces- sarily when one compares one section with another be-

BRIEF REPORTS 959

RESULTS

Figure 1. A sample of articular cartilage removed at operation. Some fibrillation is seen at the surface, and peripheral rims are noted around deeper cells. The patient had early degenerative arthritis ( x 307).

cause of variations in section thickness and operating conditions of the microscope. Four to six specimens from each of the patients with arthritis were studied, and 2 to 3 specimens from the control were examined.

The concentration of bromine and, therefore, proteoglycan was not significantly different between the most superficial areas of the matrix and those somewhat deeper within the tangential zone (Figure 2). In the deeper zones of the same patient, chondrocytes were seen with very large pericellular spaces and rims about them. The proteoglycan content within these rims was 2 to 4 times greater than in the matrix, some distance from the cell (Figures 3 and 4).

Some patients had cartilage that was fibrillated and contained clefts. The proteoglycan concentration again did not vary from the surface adjacent to the joint space, to that somewhat deeper (Figure 5). In this sec- tion, however, a segment of cartilage that appeared to be encapsulated with joint debris and probably repre- sented a free fragment was found to have much more proteoglycan than the intact matrix. The deeper portion of the transitional zone in this patient again showed chondrocytes with pericellular rims containing much more proteoglycan than the adjacent matrix (Figures 6 and 7).

A gradient of proteoglycan concentration was noted as one moved from the cell membrane out through the rim, into the adjacent matrix (Figure 8). Clusters of chondrocytes or clones were seen in the deepest cartilage zones. There was a narrow rim around the clone almost devoid of proteoglycan. Although the matrix concentration of proteoglycan at some distance from the clone was similar to that of the other regions studied, the matrix proteoglycan inside the clone was 50% greater than on the outside. Cells in the clone showed 3 times as much proteoglycan in their peri- cellular space as in the adjacent intact matrix (Figure 9).

DISCUSSION

Although the earliest biochemical changes in de- generative arthritis are said to consist of a loss of pro- teoglycan from the cartilage as a whole (1,2), this study has shown that from area to area within the cartilage a great variety of proteoglycan concentrations may be seen, particularly when comparing the pericellular space with the rest of the matrix. The most striking find- ing was that of a large number of chondrocytes at all levels which had pericellular spaces bounded by a pe- ripheral rim made up of an amorphous material. These are not seen in normal cartilage but have been de- scribed in rheumatoid cartilage (5 ) . The proteoglycan concentration within this rim was considerably higher

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Figure 2. Counts for Br (proteoglycan) at surface of cartilage are similar to those of deeper areas ( x 1.600).

than the adjacent matrix. A gradient of concentrations was found in the pericellular space, becoming least ad- jacent to cell membranes and greater as the rim was reached, only to diminish as one moved into the adjoin- ing matrix. Clones were seen in the deeper zone lying

within a very narrow region almost devoid of pro- teoglycan. The proteoglycan concentration, however, inside the clonal matrix was considerably greater than in the adjacent matrix and the concentration was even higher in the pericellular spaces within the clone.

Figure 3. Chondrocyte from the transitional zone showing much greater amounts of proteoglycan in the pericellular space than in the distant matrix (X 3,900).

BRIEF REPORTS

Figure 4. A chondrocyte from a transitional zone in normal knee cartilage shows no such rim or proteoglycan disparity (X 6,400).

Figure 5. Cartilage with fibrillation cleft and free fragment (arrow) near joint surface. The proteoglycan content of the fragment is 2-3 times that of matrix (X 1,600).

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Figure 6. Chondrocyte from radial zone with peripheral rim. Proieoglycan content in rim (pr) is greater than distant ma- trix (X 10,600).

It is known that in degenerative arthritis overall synthesis rates in cartilage for collagen and pro- teoglycan are increased, but no net increased accumula- tion of proteoglycan has been recorded (1,6).

It seems reasonable to speculate that in the early diseased cartilage studied and described in this report, chondrocytes have a considerable capacity to produce proteoglycan. Although the evidence for this is circum- stantial, it is difficult to imagine why the proteoglycan concentration should be higher in the pericellular zone

than in the adjoining matrix, unless it has been recently produced by a stimulated cell. Alternatively, one might propose that the adjoining matrix has been rendered deficient in proteoglycan because of enzymatic degrada- tion sweeping down from the joint surface. However, no such gradient of proteoglycan concentration was seen in the intact matrix. Finally it is possible that the pro- teoglycan around the cells is resistent to enzymatic deg- radation since other studies have shown this pro- teoglycan to be least stable under normal preparative

BRIEF REPORTS

Figure 7. A similar cell from control cartilage analyzed for both bromine (proteoglycan) and sulfur shows variations between the matrix (M) and pencellular space @s) (X 4,925).

Figure 8. The amount of proteoglycan increases from cell membrane (cm) to peripheral rim @r) (X 12,500).

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Figure 9. A clone lying within a narrow rim with little proteoglycan (arrow). The PG content of the pericellular space and the clone itself is greater than in the distant matrix (X 1,800).

procedures. It is difficult to understand how it could bet- ter resist enzymatic degradation. We conclude that in- creased proteoglycan is seen around active cells and constitutes a net increase for that area, though not for the cartilage as a whole.

The function of the clone has always been an enigma. The analysis carried out suggested that the clone is found lying in a narrow zone of proteoglycan depleted matrix. The clonal matrix and the pericellular matrix in the clone were extremely rich in proteoglycan, thus suggesting that these cells have synthesized new proteoglycan or at least somehow prevented normal degradation. This substantiates semiquantitatively other observations made by light microscopy which suggested augmented proteoglycan in clones (7,8). The point to point analysis by microprobe has allowed us to identify these changes in very small areas of the clone.

This study reminds us that the chondrocyte in a diseased environment may still have considerable ca- pacity to synthesize new matrix. If this potential could be better understood and harnessed, it might open up new possibilities for repair. Proteoglycan concentration patterns should be studied in different stages of disease

and in animal models to determine if mechanical or chemical treatment can alter such conditions.

REFERENCES 1. Mankin HJ: The reaction of articular cartilage to injury

and osteoarthritis. N Engl J Med 29:1335-1340, 1974 2. Mankin HJ, Dorfman H, Lippiello L: Biochemical and

metabolic abnormalities in articular cartilage from osteo- arthritic human hips. J Bone Joint Surg 53A:523-537, 1971

3. Mitchell N, Shepard N, Harrod J: The use of brominated toluidine blue in the analysis for proteoglycan. Histochem

4. Rosenberg L: Chemical basis for the histological use of Safranin 0 in the study of articular cartilage. J Bone Joint Surg 53A:69-82, 1971

5. Mitchell NS, Shepard N: The ultrastructure of articular cartilage in rheumatoid arthritis. J Bone Joint Surg 52A:1405-1423, 1970

6. Mitchell NS, Cruess RL: A classification of degenerative arthritis. Can Med Assoc J 117:763-765, 1977

7. Collins PH, McElligott TF: Sulfate (35S0,) uptake by chondrocytes in relation to histological changes in osteo- arthritic human cartilage. Ann Rheum Dis 19:318, 1960

8. Meachim G: The effect of scarification on articular carti- lage in the rabbit. J Bone Joint Surg 45B:150-161, 1963

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