Cartilage plays an essential part in vertebrate development and growth. The mandibular condyle is distinguished from the articular cartilages of long bones by several morphological, physiological and functional differences: it differs from most other articular cartilages in its cellular organization and populations, responses to biomechanical forces, and extracellular-matrix composition.

Much in vitro evidence suggests that many growth factors are important in the growth and differentiation of chondrocytes. Recently, basic fibroblast growth factor and its receptor genes were identified in vivo in cartilaginous tissues from fetal rats and mice and also in the growth plate and articular cartilages from rat, mouse, and chicken. Both local and systemic administration of basic fibroblast growth factor stimulated endochrondral ossification.

Basic fibroblast growth factor is a multipotential, 18-kDa polypeptide that stimulates mitogenesis, chemotaxis, and cell differentiation. It is thought to interact specifically with heparan sulphate, as its binding to heparan sulphate is necessary for the expression of its biological activity. In vitro experiments further indicate that basic fibroblast growth factor stimulates cell proliferation by preventing terminal differentiation of chondrocytes and that down-regulation of the expression of heparan sulphate can take place during this process.

Thus far we believe there has been no report on the in vivo location of basic fibroblast growth factor in combination with heparan sulphate in the mandibular condylar cartilage, which has a different origin, both spatially and temporally, from primary cartilage such as epiphyseal articular cartilage. We have now sought to determine the distribution of basic fibroblast growth factor and heparan sulphate in adult rat mandibular condylar cartilage by immunohistochemical labelling and to compare the findings with those on articular cartilage.

Mandibular condylar and epiphyseal articular cartilages from the proximal tibia of 12 Wistar rats, 8 weeks of age (150–170 g), were used. Animals were treated according to the principles of laboratory animal care described in the revised NIH guidelines (1985), and tissues were dissected during deep anaesthesia with diethyl ether. The specimens were cut into halves longitudinally with a sharp razor and fixed in Bouin’s solution for 6 h at 4°C without demineralization. After fixation, the tissue blocks were washed with 0.01 M PBS (pH 7.2), dehydrated by passage through graded concentrations of ethanol, cleared in xylene, and then embedded in paraffin wax. Longitudinal sections (5 ?m) were cut, deparaffinized, hydrated, dried, and then subjected to immunohistochemical staining in a humidified chamber at room temperature according to the following schedule.

The sections were immersed in 1% BSA in phosphate-buffered saline (PBS) for 1 h to prevent non-specific protein binding before the staining. First, the sections were incubated overnight at 4°C with monoclonal antibody against bovine basic fibroblast growth factor type II (UBI, New York, NY, U.S.A.) at a dilution of 1:200. After several rinses with PBS, incubation with the secondary antibody (rabbit antimouse IgG; 1:1000 dilution in PBS; E.Y. Lab., San Mateo, CA, U.S.A.) was done for 40 min at room temperature. The sections were rinsed with PBS and then submerged for 30 min in a 1:20 dilution of protein A–gold (10 nm dia.; Bio Cell, Cardiff, U.K.). After several rinses with PBS followed by distilled water, they were silver stained for 30 min with the R-Gent silver enhancement kit (Aurion, Wageningen, The Netherlands) without any counterstain. For staining heparan sulphate, the alkaline phosphatase/anti-alkaline phosphatase method was applied: the sections were treated with monoclonal antibody against heparan sulphate (Seikagaku Co., Tokyo, Japan) at a dilution of 1:100 overnight at 4°C. After several rinses with PBS, they were then incubated with the secondary antibody (biotinylated rabbit antimouse immunoglobulin; 1:200 dilution in PBS; Nichirei, Tokyo, Japan) for 40 min at room temperature. Thereafter, the sections were incubated in streptavidin–alkaline phosphatase solution (Nichirei, Tokyo, Japan) for 40 min at room temperature; and then the alkaline phosphatase reaction was carried out using naphthol phosphate/fast red as chromogen for visualization of heparan sulphate.

Controls for immunohistochemical procedures included the omission of primary and/or secondary antibodies as negative controls.

A schematic drawing of the tissue sections examined is shown in Fig. 1: four different cellular zones are recognized in rat mandibular condylar cartilage. Both basic fibroblast growth factor and heparan sulphate were present in chondrocytes in various morphological zones including the fibrous, proliferative, mature-cell and hypertrophic zones, but were not seen in calcifying cartilage. Immunogold–silver staining showed basic fibroblast growth factor in the nuclei of the chondrocytes in the proliferative and mature-cell zones, but rather weak perinculear staining was observed in hypertrophic cells. The alkaline phosphatase labelling reaction for heparan sulphate was prominent in the cytoplasm of cells in all chondrocyte layers, but an even more intense reaction was seen in the hypertrophic zone. The control sections showed absolutely negative results. In contrast, in epiphyseal articular cartilage, the chondrocytes beneath the superficial zone (intermediate zone) were immunoreactive for both basic fibroblast growth factor and heparan sulphate, and superficial cells with a flattened appearance revealed immunolabelling only for heparan sulphate. No staining for either of these molecules was found in calcifying cartilage.

Fig. 1. Schematic illustration of the topographical orientation of the mandibular condylar cartilage examined here. The middle portion of the cartilage [squared in (a)] was subjected to histochemical examination. The cartilage layer was divided into four cellular zones (b): F, fibrous zone; P, proliferative zone; M, mature-cell zone; H, hypertrophic zone.

Fig. 2. Immunohistochemistry for basic fibroblast growth factor (bFGF) (a, b, c) and heparan sulphate (HS) (d, e, f) in mandibular condylar cartilage. Positive staining for bFGF in the nuclei of all layers of chondrocytes (a), but a rather weak perinuclear reaction in hypertrophic chondrocytes (b). Cytoplasmic staining for HS in all layers of chondrocytes (d), but a rather intense staining in hypertrophic cells (e). Control sections show no immunostaining for either bFGF (c) or HS (f). Bar=100 ?m; (a, d)×50; (b, c, e, f)×100.

Fig. 3. Immunohistochemistry for basic fibroblast growth factor (bFGF) (a) and heparan sulphate (HS) (b) in epiphyseal articular cartilage. Only the chondrocytes beneath the superficial zone stain for both bFGF (a) and HS (b); the superficial cells label for HS only (b). No staining for either bFGF or HS in the deeper region consisting of calcifying cartilage (arrows). Insets: Control sections. Bar=100 ?m; (a, b)×50.

To the best of our knowledge, this is the first study to demonstrate both basic fibroblast growth factor and heparan sulphate immunohistochemically in the adult rat mandibular condylar cartilage in vivo. The distribution of basic fibroblast growth factor in condylar cartilage had a very different pattern from that in epiphyseal articular cartilage. An earlier histochemical study on basic fibroblast growth factor in the epiphyseal growth plate of young rats demonstrated localization in the resting and proliferative zones. In contrast, Twal et al. (1994) showed a different pattern of localization in chicken growth plate after enzyme treatment of frozen sections: they noted that whereas basic fibroblast growth factor acts as a chondrocyte mitogen in the proliferative zone, it may serve as a chemotatic signal for metaphyseal blood-cell proliferation when released from hypertrophic chondrocytes.