The periodontal ligament is a dense connective tissue between the root cementum and the alveolar bone that anchors the tooth and maintains the structural integrity of these mineralized tissues. Fibroblasts in the periodontal ligament, the major cell type, are regarded as multipotential, or form a heterogeneous population that can differentiate into either cementoblasts or osteoblasts, depending on needs and conditions. Thus, the ligament has been regarded as the source of cementoblasts and osteoblasts. Indeed, cultured fibroblasts from the ligament exhibit bone cell-like properties in vitro, including a relatively large amount of alkaline phosphatase activity, and the expression of bone-matrix proteins such as osteopontin and secreted protein, acidic and rich in cysteine (SPARC). Similar to bone-forming cultures, cells of the periodontal ligament undergo osteoblastic differentiation in response to dexamethasone, a synthetic glucocorticoid widely used to induce differentiation of osteoblasts, as characterized by elevated alkaline phosphatase activity, increased expression of osteopontin and bone sialoprotein, and the formation of mineralized nodules.

The mechanisms by which cells of the periodontal ligament maintain their phenotype and differentiate into mineralized tissue-forming cells remain unclear. In attempts to examine the involvement of polypeptide growth factors, we have earlier investigated the role of EGF and its receptor in dexamethasone-induced in vitro differentiation of rat periodontal-ligament cells. We found that EGF antagonized differentiation and up-regulated EGF-R expression, whereas EGF-R was down-regulated in the course of differentiation. Therefore, the EGF/EGF-R system appears to be important as a phenotype stabilizer by functioning as a negative regulator of osteoblastic differentiation in these cells. Although in vivo observations on the rat supported these in vitro results, a detailed study on the role of EGF/EGF-R in cells of the human periodontal ligament cultured in conditions closer to the physiological, without dexamethasone, is still needed.

Physiologically, the periodontal ligament is continuously subjected to mechanical stress caused by occlusal forces. Furthermore, remodelling of the ligament and alveolar bone occurs in response to orthodontic forces. These facts led us to speculate that responses of the ligament to mechanical stress are involved in its cell proliferation and differentiation. In fact, it has been shown that a variety of cells respond to mechanical stress, such as tension force, compression and fluid shear stress, by demonstrating significant changes in their structure and function. In bone, Raab-Cullen et al. (1994)have reported, using the rat tibia 4-point bending model, that external mechanical loading induces a rapid and transient increase in mRNA expression for c-fos, a gene associated with proliferation and/or differentiation in bone development and fracture repair. Several in vitro studies on cultured osteoblastic cells have also demonstrated elevated amounts of bone-related molecules, such as alkaline phosphatase, osteopontin and osteocalcin, in response to mechanical stretching. Therefore, it appears that essential functions of osteoblasts in bone remodelling are affected by experimentally loaded mechanical stress and that cellular responses to mechanical stress are crucial in homeostasis, adaptation to the environment, and regeneration of bone. In contrast, only a small number of studies have addressed the responsiveness of cells of the periodontal ligament to mechanical stress, in which the ability of cultured cells to proliferate in response to tension force was demonstrated. As we have shown that such cells maintain their phenotype and differentiate into osteoblastic cells through mechanisms involving the EGF/EGF-R system, it was of interest to investigate how they and the EGF/EGF-R system respond to mechanical stress. Furthermore, the interaction between mechanical stress and EGF/EGF-R has not, we believe, been reported elsewhere.

Our aim now was to elucidate the role of EGF and EGF-R in the proliferation and differentiation of cells of the human periodontal ligament in mechanical stress-loaded conditions in vitro. For this purpose, cultured ligament cells were loaded with cyclic stretch using flexible-bottomed culture plates and their responses were monitored. Then the effect of EGF on these responses was examined. Finally, we evaluated changes in the amount and autophosphorylation of EGF-R in response to cyclic stretching.

Fibroblastic cells were obtained from explant cultures of human healthy periodontal ligament taken from a third molar that had been extracted for orthodontic reasons, as described by Matsuda et al. (1996b). The tissues were minced, put in culture dishes and incubated in DMEM (Gibco Laboratories, Grand Island, NY) supplemented with 10% FBS (Intergen Company, Purchase, NY), non-essential amino acids, 10 mM sodium pyruvate, vitamins (Gibco) and an antibiotic mixture (1 U/ml penicillin, 1 U/ml streptomycin, 1 U/ml gentamycin; Sigma Chemical Co., St. Louis, MO) at 37°C in a humidified atmosphere of 5% CO₂–95% air. When the outgrowing cells reached confluency, they were trypsinized with 0.05% trypsin (1:250)–0.53 mM EDTA 4 Na (Gibco) in PBS for secondary culture. Cultures were maintained until confluency and passed at a 1:4 split ratio. All the experiments were done on cells of between three and seven passages.

As an experimental model of mechanical stretch, tension force was loaded on to cells cultured on a flexible substratum (25 mm dia., Flex I culture plate; Flexcell International Corporation, McKeesport, PA) by applying vacuum-operated negative pressure using the Flexercell Strain Unit Model FX-2000, which is capable of controlling the magnitude as well as the frequency of cell deformation. Although this system has been used in many investigations, one problem with the apparatus is that the substrate of the strain well is strained in a non-uniform manner; deformation is greatest at its periphery and least at its centre. Therefore, we aimed to examine the net response of the total number of cells to mechanical strain. Cells were subjected to 9 or 18% of maximum strain for 5 s followed by 5-s relaxation (6 cycles/min). According to the manufacturer, in these conditions the strains are distributed inhomogeneously such as ?5–9% or ?4–18%.

Cells were plated on to a Flex I culture plate at concentrations of 2.5×10⁴, 5.0×10⁴, or 1.0×10⁵ per well and incubated in DMEM/10% FBS for 24 h. After replacement of the medium with fresh DMEM/10% FBS, Flex I plates were placed on the Flexercell strain unit to apply cyclic stretch to the cells. At 1, 3, or 5 days of incubation under cyclic stretching, cells were harves ted by trypsinization and counted in a haemocytometer.

After 5 days of culture with or without stretch, cells were rinsed with PBS twice and fixed by incubation with 10% neutral-buffered formalin for 30 min. The flexible substrate of 25 mm dia. was then removed from the plate and cut to 20×20 mm square. Actin stress fibres were stained with 33 nM of rhodamine phalloidin (Molecular Probes, Inc., Eugene, OR) for 30 min. Stained cells were viewed on an LSM410 invert laser-scan microscope (Carl Zeiss, Jena, Germany). For rhodamine visualization, fluorescence was excited at 543 nm and emitted light was detected at 590 nm.

As one of the differentiation markers of the ligament cells, their alkaline phosphatase activity was examined. Ten thousand cells were plated on Flex I culture plates in DMEM/10% FBS and incubated until they reached confluency. After further incubation for 24 h in DMEM/1% FBS containing 50 mg/ml ascorbic acid and 10 mM ?-glycerophosphate (mineralizing medium) with or without 10 nM dexamethasone (Wako Pure Chemical Industries, Osaka, Japan), cells were then subjected to cyclic stretching for 2, 4 or 6 days. Alkaline phosphate activity of cell lysates was determined by using p-nitrophenyl phosphate (Wako) as a substrate. The enzyme activity was expressed as U/mg protein. For enzyme staining, cells were washed with PBS and fixed in 10% cold neutral-buffered formalin for 15 min. After washing with distilled water, cells were incubated for 45 min at room temperature in alkaline-phosphatase substrate solution consisting of 0.1 mg/ml naphthol AS MX-PO₄ (Dojin Chemical, Kumamoto, Japan), 0.4% N,N-dimethyl-formamide (Wako) and 0.6 mg/ml Fast red-violet LB salt (Sigma) in 0.1 M Tris–HCl, pH 8.3. Cells were then washed with distilled water and with 0.5 M HCl. Finally, they were stained with 0.25% alcian blue 8GX (Sigma) in 0.5 M HCl.