Cartilage is important in the growth and development of bone structure. The mandibular cartilage exhibits a morphology and density well adapted to the functional demands placed upon it. However, the factors that initiate and control endochondral ossification in the mandibular condyle are poorly understood. By using cartilage from a long bone, there are reports that mechanical forces regulate skeletal growth and development, and are important factors in controlling local ossification. Use of the functionally isolated, externally loadable, avian ulna as an experimental model in vivo has confirmed the relation between dynamic strain and the change in the bone mass over a period of weeks.

In the mandible, changes in loading by feeding a soft diet of reducing the incisal function of young rats affect the number of chondrocytes and the thickness of the cell layers of mandibular condyle in vivo, but in those models it is difficult to exclude completely any other factors that might influence skeletal growth.

Organ cultures of fetal cartilage from small animals are well suited to clarifying endochondral growth processes at the tissue level. In this experimental system, the structure and function of extracellular-matrix components in the mandibular condyle can be explored. A few investigators have studied the effect of direct mechanical loading on the growth of skeletal tissues in vitro. In particular, a method of applying mechanical force by compressing gas phase above cultures in a closed chamber has been developed. Compressive force increases the deposition of mineral and proteoglycans in the matrix.

Our purpose now was to clarify the change in the mandibular condyle under compressive loading. For well-controlled conditions, an organ culture of the fetal rat mandibular condyle was used under intermittent compressive loading. The cultured condyles were then evaluated histomorphologically and immunohistochemically.

At 20 days’ gestation, 10 fetuses were removed from pregnant Wistar rats (Shizuoka Agricultural Cooperative Association for Laboratory Animals, Hamamatsu, Japan), and non-calcified mandibular condyles excised. After 1-h preincubation, each condyle was placed in 1 ml of Fitton–Jackson-modified BGJb medium (Gibco, Grand Island, NY), supplemented with 100 ?g/ml ascorbic acid (Sigma, St. Louis, MO, USA). The cultures were placed in a 37°C incubator with a 5% CO₂ atmosphere for 8 days. The medium was changed every 2 days.

Ten condyles were cultured with intermittent compressive loading as the experimental group, and another 10 were cultured without loading as the control group.

Intermittent compressive loading was as in Klein-Nulend’s study (1986). Loading was generated by compressing the gas phase within a closed culture chamber that contained the culture dishes. The pressure was 7 kPa above ambient, and applied at 0.3 Hz.

Fig. 1. Schematic diagram showing apparatus for application of mechanical loading generated by compressing the gas phase within a closed chamber.

At the end of the culture period, the maximum width of the sagittal plane of the condyle was measured. Then the condyles were fixed in periodate–lysine–paraformaldehyde, pH 6.2, for 24 h. Fixed specimens were mineralized in Tris-buffered 10% EDTA, pH 7.2, for a period ranging from 1 to 2 weeks, and then embedded in paraffin. They were sectioned at 4?m thickness and stained with haematoxylin and eosin. In the apical region of the sagittal plane of condyles, the thickness of the fibroblast layer, the chondroprogenitor cell layer and the maturing chondroblast layer was measured. The ratio of the thickness of respective layers and the total thickness of three layers to the maximum width of the condyle was calculated on all specimens from the experimental group, the control group and in 7-day-old rats (see below). The mean ratios of the respective groups were compared statistically by Wilcoxon’s signed-rank test.

Rabbit anti-type I collagen antibody was purchased from Advance Co. (Tokyo, Japan). Rabbit antirat fibronectin polyclonal antibody was purchased from Chemicon International Inc. (El Segundo, CA). Antitype I collagen antibody was diluted 1:1000 and antifibronectin antibody was diluted 1:100 was phosphate-buffered saline.

Condyles designated for immunohistochemical studies were mineralized, embedded and sectioned as described above. Endogenous peroxidase activity was quenched by incubating in 3% H₂O₂ for 10 min. Sections were treated with non-immunized goat serum for 10 min to block any non-specific binding of the antibody. They were then incubated with first antibodies at 4°C in a moist chamber overnight. After washing in chilled phosphate-buffered saline the sections were incubated with biotinylated anti-rabbit IgG antibody for 10 min at room temperature, and streptoavidin peroxidase solution was applied for 5 min at room temperature. These sections were then reacted with a solution of H₂O₂ containing 3,3-diaminobenzidine. Next, they were dehydrated in graded ethanols and xylene, and mounted. For reference, the specimens of mandibular condyles of 7-day-old rats, born at 21 days’ gestation, were sampled, and immunohistochemical staining for type I collagen and fibronectin were observed.

After 8 days of culture the mean width of the sagittal plane of the condyle was 1.14 mm in the experimental group and 1.07 mm in the control group. The mean width of the sagittal plane of 7-day-old rat condyles was 1.90 mm (n=6), significantly wider than the cultured condyles (p<0.05).

In the 20-day fetal rats, the condylar cartilage exhibited four distinct zones of cells based on morphological characteristics: fibroblasts (fibrous zone), chondroprogenitor cells (proliferative zone), maturing chondroblasts (first differentiated cartilage cells) and hypertrophic chondrocytes. At the end of the culture period, the thickness of the fibroblast layer was 3.46% of the maximum condylar width in the experimental group, 3.08% in the control group and 2.40% in the 7-day-old rats. The respective mean ratios for the thickness of the chondroprogenitor cell layers were 1.17% in the experimental group, 1.04% in the control group and 7.07% in the 7-day-old rats, and for the thickness of the maturing chondroblast layer 2.56% in the experimental group, 2.09% in the control group and 18.90% in the 7-day-old rats. In the fibroblast layer, there were no significant difference between groups. In the chondroprogenitor cell layer and the maturing chondroblast layer, there was no significant difference between the experimental and control groups, but in both groups these two layers were significantly thinner than in 7-day-old rats. The mean ratio of the total thickness of the three layers to the maximum width of the condyle was 7.19% in the experimental group, 6.21% in the control group and 28.01% in the 7-day-old rats. Hypertrophic chondrocytes made up a large proportion of the cultured condyle, and there was no bone matrix. On the other hand, the hypertrophic chondrocyte layer in 7-day-old rats was thinner than in the cultured condyle, and there was bone matrix adjacent to the layer.

Fig. 2. The ratio of (a) the thickness of the fibroblast layer, (b) the chondroprogenitor cell layer, (c) the maturing chondroblast layer and (d) the total thickness of three layers to the maximum width of condyles in the respective groups. NS, p?0.05, *p<0.05.

The number of cells and the size of each cell were similar in the control and the experimental groups.

Fig. 3. The upper surface of the mandibular condyle: (a) experimental group; (b) control group. In both sections, the chondroprogenitor cell layer and the maturing chondroblast layer are very thin. H&E, ×50.