THE ANATOMICAL RECORD zyxw 2103375-682 zy (1984) Morphometry of Articular Cartilage: A Stereological Method Using Light Microscopy KARI PAUKKONEN, KALEVI SELKAINAHO, JUKKA JURVELIN, AND HEIKKI J. HELMINEN zyxwvut Departments ofAnatomy zyxwvut (K.P, J.J., H. J. H.) and Applied Mathematics zyx (K.S.), University ofKuopw, SF-70211 Kuopio 21. Finland ABSTRACT A zyxwvut stereological method is introduced for studying the light microscopic morphometry of articular cartilage. New parameters are presented for defining the three-dimensional structure of the articular cartilage. These include volume density of the lacunae and nuclei, numerical density of cells in 3-dimensional space, total number of cells, total volume of the lacunae in cartilage, volume and diameter of the lacuna and nucleus, the nucleusAacuna volume ratio, and the mean volume of the matrix per cell. Using the lateral tibial condyle of the knee joint of eight rabbits, we investigated tissue prepa- ration, sampling, morphometry, and the conditions necessary for validity and precision of the estimators. The method proved easy to use, and the estimators were precise. Previous light microscopic studies of the morphometry of articular cartilage have been confined to two-dimensional examination of tissue sections. In such studies the main pa- rameters have been cartilage thickness and cell (profile) density (Stockwell, 1967, 1971, 1979; Vignon et al., 1974,1976). The relation- ship of the two-dimensional measurements to three-dimensional parameters (which would define the structure more precisely) and the validity and precision of the mea- surements have not been taken into consid- eration. In other words, true stereological methods have not been used to study the morphometry of articular cartilage. Saaf (1950) used “prestereological” morphometri- cal methods in his study of the articular car- tilage of guinea pigs. Hamerman and Schubert (1962) computed the volume den- sity of the cartilage cells as the ratio of the cell mass to the total mass of the organic tissue. During recent developments in stereology, much attention has been paid to statistical aspects, sampling, and construction of un- biased as well as efficient and accurate ster- eological estimators (Cruz-Orive and Weibel, 1981; Gundersen and Qsterby, 1981; Mathieu et al., 1981; Miles, 1978; Miles and Davy, 1976, 1977; Nicholson, 1978; Shay, 1975; see also Weibel, 1979, 1980).The aim of our study was to apply this knowledge and thereby de- sign a stereological method for studying ar- ticular cartilage. MATERIALS Articular cartilage from eight male New Zealand white (NZW)rabbits 4.5 to 5 months of age was examined. The mean weight of the rabbits was 3,240 gm (range = 2,895- 3,730 gm). When an animal had been killed, the tibia of the right leg was dissected free and immediately placed in 2% glutaralde- hyde (see below) at room temperature for 1 hour. Subsequently, the articular cartilage of the lateral tibial condyle was sawed into slices using a Leitz 1600 saw microtome. Blocks with surface dimensions of 0.3 x 1.5 mm were prepared the saw cuts were per- pendicular to the articular surface and reached the subchondral bone (Fig. la). Un- der a stereomicroscope, the articular carti- lage was separated from the subchondral bone; and the tissue was kept moist with 0.9% NaC1. From each condyle, 30-40 tissue blocks were fixed for 2 hours at 4°C in 2% purified glutaraldehyde in 0.15 M cacodylate buffer, pH 7.3, containing 0.2% ruthenium red. The tissue blocks were then treated ac- cording to the method of Shepard and Mitch- ell (1977). Treatment of the blocks included staining for 30 min with 1% p-phenylenedi- Received November 4, 1983; accepted July 16, 1984 zyx 0 1984 ALAN R. LISS. INC