$3.00+ 0.00 OOIl3-9969/90 Copyright 0 1990Pergamon Press plc
Archs oral Biol. Vol. 35, No. 8, pp. 597401, 1990 Printed in Great Britain. All rights reserved
MEASUREMENTS OF THE ISOMETRIC CONTRACTILE FORCES GENERATED BY DOG PERIODONTAL LIGAMENT FIBROBLASTS IN VITRO S. KASUGAI,’S. SUZUKI,*S. SHIBATA,~S. YASUI,~ H. AMANO’ and H. OGURA’ Departments of ‘Pharmacology, *Orthodontics II, ‘Anatomy II and 4Periodontology, Faculty of Dentistry, Tokyo Medical and Dental University, Yushima l-5-45, Bunkyo-ku, Tokyo, 113, Japan (Accepted 2 March 1990) Summary--One hypothesis for the mechanism of tooth eruption is that the periodontal ligament fibroblasts generate the eruptive force. To assess the force generated, these fibroblasts were obtained by explant culture of ligament from mandibular premolars of a dog and were cultured in collagen gel matrices. The forces generated by them under isometric conditions were continuously measured for 120 h with a strain gauge. At the same time the number of cells in the gel was counted and the force measured was calculattd as the force generated by lo4 cells. Shortly after the start of culture, the force per lo4 cells increased rapidly; it reached 5.2 x lO-4 N at 8 h, and then remained at the same level for about 48 h. Our findings suggest that fibroblasts of the periodontal ligament may generate sufficient force for tooth eruption. Key words: libroblast contraction, periodontal ligament, tooth eruption.
INTRODUCIION
MATERIALS AND METHODS
The mechanism of tooth eruption is not clearly understood although several hypotheses have been proposed and discussed for many years. From experiments with root resection and root transection, it can be concluded that the periodontal ligament is the source of the eruptive force (Berkovitz and Thomas, 1969; Berkovitz, 1,971). Ness (1967) suggested that periodontal ligament fibroblasts might generate this force. In the rodent incisor these cells migrate in an occlusal direction (Chiba, 1968; Zajicek, 1974; Beertsen, 1975; Perera and Tonge, 1981). Beertsen, Everts and van den Hooff (1974) have proposed that while migrating they pull the tooth forward into the mouth. Fibroblasts in culture can generate tensional forces (James and Taylor, 1969). After fibroblasts have been seeded into a three-dimensional collagen gel matrix, the gel gradually contracts (Bell, Ivarsson and Merrill, 1979). This experimental system has been widely used to study the contraction of fibroblasts and experiments with fibroblasts from periodontal ligament sh,ow that these are also contractile (Bellows, Melcher and Aubin, 1981, 1982a). An in vitro model of tooth eruption using ligament fibroblasts and collagen gel matrix has been described (Bellows, Melcher and Aubin, 1983). These in vitro findings endorse the possibility that such fibroblasts may play some part in tooth eruption. However, the forces generated by them have not been measured and it is not clear whether these are sufficient to effect tooth eruption. We have now sought to measure the contractile force of periodontal ligament fibroblasts.
A male dog (14 kg) was premeditated with 0.5 mg atropine sulphate (Tanabe Pharmaceutical Co. Ltd, Tokyo, Japan) and anaesthetized with 20 mg xylazin (Celactar, Bayer Japan Co. Ltd, Tokyo, Japan) and 50 mg ketamine chloride (Ketalar, Sankyo Co. Ltd, Tokyo, Japan). Lidocaine (Fujisawa Pharmaceutical Co. Ltd, Osaka, Japan) was injected for local anaesthesia. Lower premolars were extracted and the periodontal ligament was scraped from the root surface with a scalpel. The explants of the ligament were soaked in phosphate-buffered saline (Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) containing 200 U/ml penicillin, 200 pg/ml streptomycin and 0.5 pg/ml fungizone (Antibiotics Solution, Whittake M.A. Bioproducts Inc., MD, U.S.A.). The explants were transferred to a 60 mm culture dish (Corning, New York, U.S.A.), fixed in place with a cover glass, and culture medium gently added [Eagle’s minimal essential medium (Nissui) containing 60 pg/ml kanamycin and supplemented with 15% (v/v) fetal bovine serum (Filtron Pty Ltd, Victoria, Australia)]. The medium was maintained at pH 7.4 with NaHCO, under an atmosphere of 95% air, 5% CO,, at 37°C. After 14 days, the cells were subcultured in phosphate-buffered saline containing 0.5% trypsin (1: 250, Difco Laboratories, Detroit, MI, U.S.A.). At the third passage, the fibroblasts were stored in the liquid nitrogen. They were then cultured again and used in our study.
AOB 35/bB
Experimental
apparatus
We constructed an apparatus from strain-gauge chips (KFC-03-Cl-16, Kyowa Electronic Instruments Co. Ltd, Tokyo, Japan) and an amplifier IC (AD522,
597
598
S.
KAsUGAIet al. Strain WJW
Fig. 1, Schematic illustration of the experimental apparatus: it consists of two stainless-steel meshes: one mesh (1) was connected to the edge of the culture dish; the other (2) was floating with the aid of fishing floats (3) and was connected by wire to a strain gauge (4). The collagen gel (5) was placed between the two meshes and cultured in the medium (6). Devices, MA, U.S.A.) by following the instructions of Analog Devices. The amplifier was connected to a recorder (2210, LKB, Bromma, Sweden). Before starting the experiments, the strain gauge was placed in a COZ incubator at 37°C and calibrated.
Analog
Preparation of collagen gel and measurement of force
Bovine acid-soluble type I collagen (Cell Matrix I, Nitta Gelatin Co., Ltd, Osaka, Japan) was neutralized by alkaline buffer and minimal essential medium according to the instructions of the supplier. The final concentration of the collagen solution was 2.4 mg/ml. We made square moulds from 1% agar medium and placed two stainless-steel meshes opposite each other in each mould. Then, 5 x lo4 or 2.5 x lo4 fibroblasts were incorporated into 250 ~1 of neutralized collagen solution, poured into the mould and incubated at 37°C for 15 min. The gel and meshes were then transferred to a 60 mm culture dish filled with culture medium. As shown in Fig. 1, one mesh was connected to the edge of the culture dish and the other to a fishing float. The culture dish was placed in a CO* incubator and the float was connected to the strain gauge. To prevent contamination, both culture dish and strain gauge were covered with a large plastic box. The force generated by the gel was recorded continuously over 120 h. Control experiments used gel without cells. Cell counting
To determine the number of cells within gels at various times, gels containing 5 x lo4 fibroblasts were prepared as described above and cultured for 8, 24, 48, 72 and 120 h. Gels between the two meshes were cut out and transferred to a centrifuge tube, where 1 ml of Hank’s balanced salt solution (Nissui) containing 0.04% collagenase (Type I, Sigma Chemical Co., St Louis, MO, U.S.A.) was added. The tubes were stirred at 37°C for 15 min and cetrifuged (400 g, 5 min). The supernatants were discarded and 100 ~1 of crystal violet solution (0.1 M citric acid and 0.1% crystal violet) was added. The number of cells present was then counted in a Barker-Turk counter. Morphological observation
Gels containing fibroblasts were inspected by phase-contrast microscopy to find any changes in cell morphology.
RESULTS
Figure 2 shows the force changes generated by the gel over 120 h. The force generated by gels containing periodontal ligament fibroblasts increased rapidly during the first 8 h, and more slowly thereafter. The time and force curves were constant when we inoculated the gel with the same number of cells. When half that number of cells was inoculated, the force was approximately halved throughout the experimental period. Gels without cells did not generate a measurable force. The change in the number of fibroblasts in the gels is shown in Fig. 3. This did not change during the first 8 h of culture and then increased linearly. From these findings, the force generated by lo4 cells over 120 h was calculated and is shown in Fig. 4. The force per lo4 cells reached 5.2 x 10e4 N at 8 h and remained at almost the same level for about 48 h, after which it decreased gradually. The force per lo4 cells was 3.2 x 10m4N at 120 h. At the start of culture within the gel, the fibroblasts were spherical. Numerous filopodia were soon seen; pseudopodia began to extend from the cells after 6 h in culture, and the fibroblasts then gradually elongated. After 12 h, they were much elongated and had long pseudopodia. Proliferation of some of the fibroblasts was seen after 16 h. Cell to cell contact was also seen as the cell density increased. Between 16 and 120 h, cell shape did not change remarkably.
0
24
48
72
96
120
nme (h) Fig. 2. Force generated by collagen gel over 120 h: collagen gel initially containing either 5 x 10’ (a) or 2.5 x 10’ periodontal ligament fibroblasts (b) or collagen gel without fibroblasts (c).
Contractile force of periodontal fibroblasts
Tlme (h)
Fig. 3. The increase is. the number of fibroblasts in collagen gels over 120h. Each point and bar is the mean value and f 1 SD of 4 samples respectively. DISCUSSION
In previous studies, fibroblasts and other cell types were seeded into collagen gel matrices and shrinkage of the gel resulting from cell contraction was measured (Bell et al., 1979; Steinberg et al., 1980; Bellows et al., 1981; Buttle and Ehrlich, 1983; Gillery, Maquart and Borel, 1986; Ehrlich, 1988; Hughes and Issbemer, 1988; Kasugai and Ogura, 1988; Montesano and Orci, 1988; Nishiyama et al., 1988). In our experiments, fibroblasts were also seeded in collagen gel, but the gel could not shrink freely because one side of it was fixed and the other side was connected to the strain gauge. The maximal change in length of the gel was small (approx. 100 pm or 1% of the initial gel length) throughout the experiment; thus we could measure the isometric force generated within it. Gels without fibroblasts generated no force (Fig. 2) and thus the forces measured were generated directly by those cells. Soon after the start of culture, the ligament fibroblasts generated a force that increased linearly up to 8 h (Fig. 2). Nishiyama et al. (1988) found that several hours elapsed before the start of contraction; this difference between findings might be due to their lower collagen concentration (0.5 mg/ml) and cell density (1.0 x 104/ml). In this initial period (O-8 h in culture), fibroblasts of the periodontal ligament
lime (h) Fig. 4. Force generated by lti periodontal ligament fibroblasts. Each point was calculated from the value measured by the strain gauge (Fig. 2) and the number of fibroblasts (Fig. 3). Each point and bar is the mean value and + 1 SD of 4 samples (O-72 h) or 2 samples (120 h).
599
develop filopodia containing many microfilaments (Bellows et al., 1982b; Tomasek, Hay and Fujiwara, 1982); microtubules align along the long axis of the cell body (Tomasek and Hay, 1984). The contraction of collagen gels containing fibroblasts is inhibited by cytoskeletal inhibitors, such as Colcemid, colchicine and cytochalasins (Bell et al., 1979; Bellows et al., 1982a, 1983; Kasugai and Ogura, 1988; Nishiyama et al., 1988). The force generated by fibroblasts is thus considered to be related to their attachment to the collagen and their development of cell processes; the cytoskeletal systems play an important part in these. One of the factors that affects gel contraction is the number of cells in the gel (Bell et al., 1979). We counted this number, and the forces measured by the strain gauge was calculated as the force generated by lo4 cells. We did not check other factors that affect gel contraction, such as collagen concentration (Gillery et al., 1986), collagen type (Ehrlich, 1988), serum concentration (Gillery et al., 1986) and growth factors (Montesano and Orci, 1988). Between 8 and 72 h in our culture system, lo4 fibroblasts could generate a force of about 5 x 10e4 N (Fig. 4). James and Taylor (1969) used the bending of calibrated microneedles to measure the contractility of whole sheets of fibroblasts stretched between two bone fragments. They estimated the stress per cell as 1.65 x lo-‘N. Our value (5 x lo-* N per cell) was approximately one-third of theirs, but the difference might be due to differences of cells and methods. Harris, Wild and Stopak (1980) cultured chick-heart fibroblasts on a thin silicone membrane and found cell movement and distortion of the membrane. Although they estimated the traction force as 1 x lo-* N/pm of the advancing margin of the cell, they did not state the traction force of an individual cell. A single smooth-muscle cell has been shown to generate a force of l-2 x 10m6N (Yagi, Becker and Fay, 1988); the force generated by the periodontal fibroblasts was therefore approx. 1 or 2% of that generated by the smooth-muscle cell. Changing the medium interferes with continuous force measurement, so we did not do this throughout the experiment. The gradual decline in the force generated by lti fibroblasts after 72 h (Fig. 4) might be due to depletion of nutrients within the medium, but this is unlikely because the number of cells continued to increase (Fig. 3). Remodelling of the collagen matrix by the fibroblasts, or a phenotypic change might explain this decrease. Bellows et al. (1981) showed that fibroblasts from monkey periodontal ligament contracted collagen gels significantly faster than did other cell types, such as human gingival fibroblasts, fetal rat calvaria cells, porcine periodontal ligament epithelial cells and rat osteosarcoma cells. By measuring gel contraction, we earlier found that the fibroblasts of dog periodontal ligament contracted collagen gels more rapidly than did dog gingival fibroblasts (Kasugai et al., 1988). In contrast, Hughes and Issbemer (1988) found that fibroblasts from human periodontal ligament did not contract collagen gels more than fibroblasts from other oral tissues. Gel contraction is also dependent upon the number of cell passages (Steinberg et al., 1980; Buttle and Ehrlich, 1983), and ligament
Berkovitz B. K. B. (1971) The effect of root transection and fibroblasts derived from various species might have partial root resection on the unimpeded eruption rate of different characteristics. the rat incisor. Archs oral Biol. 16, 1033-1043. The force needed to prevent eruption of rat Berkovitz B. K. B. and Thomas N. R. (1969) Unimpeded mandibular incisors was between 2 and 5 g (approx. eruption in the root-resected lower incisor of the rat with 2-5 x 10e2 N) (Taylor and Butcher, 1951) and 7 g a preliminary note on root transection. Archs oral Biol. (approx. 7 x 10e2 N) in rabbit maxillary incisors 14, 771-780. (Miura and Ito, 1968). Burn-Murdoch (1981) found Burn-Murdoch R. A. (1981) Evidence that the response that forces (2.5 x 10m3-1.5 x 10e2N) applied in a to applied forces of continuously erupting rat incisors contains more than one component. Archs oral Biol. 26, direction that opposed the eruption of rat maxillary 989-993. incisors made these teeth sink into their sockets. Buttle D. J. and Ehrlich H. P. (1983) Comparative studies Michaeli, Steigman and Weinreb (1987) estimated of collagen lattice contraction utilizing a normal and a that there are more than IO6fibrobfasts in the toothtransformal cell line. J. Cell Physiol. 116, 159-166. related part of the periodontal ligament of a rat Chiba M. (1968) Movement, during unimpeded eruption, incisor. We calculated that the force generated by 1O4 of the position of cells, and of material incorporating fibroblasts was 5 x low4 N. Therefore, our findings tritiated proline, in the lingual periodontal membrane of suggest that periodontal ligament fibroblasts may be the mandibular incisors of adult male mice. J. dent. Res. able to generate enough force for tooth eruption. We 47, 986. Ehrlich H. P. (1988) The modulation of contraction of have measured the force generated in vitro by fibrofibroblast populated collagen lattices by type I, II and III blasts from teeth of limited eruption, and compared collagen. Tiss. Cell 20, 47-50. this with the eruptive force of continuous erupting Gillery P., Maquart F. X. and Bore1 J. P. (1986) Fibronectin teeth. There seems to be no evidence either that the dependence of the contraction of collagen lattices by eruptive mechanism of continuous erupting teeth is human skin fibroblasts. Expl Cell Res. 167, 29-37. similar to that of teeth of limited eruption or that the Harris A. K., Wild P. and Stopak D. (1980) Silicone rubber forces generated by their ligament cells are different. substrata: a new wrinkle in the study of cell locomotion. However, our findings suggest that the tooth-eruptive Science 208, 177-179. force. may come from fibroblasts of the periodontal Hughes F. J. and Issberner J. P. (1988) Phenotypic variations in contraction of fibroblasts derived from the perioligament. Acknowledgements-We
are very grateful to Dr K. Toda, Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental University, for his kind technical advice. We also greatly appreciate Dr C. G. Bellows, MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, for his critical reading and correcting of English. This study was supported by Grant-in-Aid for scientific research (No. 63570867), from the Ministry of Education, Science and Culture, Japan. REFERENCES Beertsen W. (1975) Migration of fibroblasts in the periodontal ligament of the mouse incisor as revealed by autoradiography. Archs oral Biol. 20, 659-666. Beertsen W., Everts V. and Hooff A. van den (1974) Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. Archs oral Biol. 19, 1087-1098. Bell E., Ivarsson B. and Merrill C. (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. nam. Acad. Sci. U.S.A. 76, 127+1278.
Bellows C. G., Melcher A. H. and Aubin J. E. (1981) Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types. J. Cell Sci. SO, 299-314. Bellows C. G., Melcher A. H. and Aubin J. E. (1982a) Association between tension and orientation of periodontal ligament fibroblasts and exogenous collagen fibres in collagen gels in viao. J. Cell. Sci. Ss, 125-138. Bellows C. G., Melcher A. H., Bhargava U. and Aubin J. E. (1982b) Fibroblasts contracting three-dimensional collagen gels exhibit ultrastructure consistent with either contraction or protein secretion. J. Ultrastrucf. Res. 78, 178-192.
Bellows C. G., Melcher A. H. and Aubin J. E. (1983) An in-vitro model for tooth eruption utilizing periodontal ligament fibroblasts and collagen lattices. Archs oral Biol. 28, 715-722.
dontal tissue. J. dent. Res. 67, 646. James D. W. and Taylor J. F. (1969) The stress developed by sheets of chick fibroblasts in vitro. Expl Cell Res. 54, 107-l IO.
Kasugai S. and Ogura H. (1988) Effects of cytoskeletal inhibitors on the contraction of fibroblasts. Jap. J. Pharmac. Suppl. 46, 233P. Kasugai S., Amano H., Ogura H., Yasui S., Suzuki S., Susami T. and Shibata S. (1988) Characteristics of cultured cells derived from gingiva and periodontal ligament. J. Stomatol. Sot. Jap. 55, 253.
Michaeli Y., Steigman S. and Weinreb M. Jr (1987) Longterm effect of loading on the fibroblast population of the periodontal ligament in the rat lower incisor. Archs oral biol. 32, 355-361.
Miura F. and Ito G. (1968) Eruntive force of rabbits uaner incisors. Trans. E&. Orrhodokr, Sot. 121-126. _a Montesano R. and Orci L. (1988) Transforming growth factor /I stimulates collagen-matrix contraction by fibroblasts: implications for wound healing. Proc. natn. Acad. Sci. U.S.A. 85, 48944897. Ness A. R. (1967) Eruption. In The Mechanism of Tooth Support (Edited by Anderson D. J., Eastoe J. E., Melcher
A. H. and Picton D. C. A.) pp. 8488. Wright, Bristol. Nishiyama T., Tominaga N., Nakajima K. and Hayashi T. (1988) Quantitative evaluation of the factors affecting the process of fibroblast-mediated collagen gel contraction by separating the process into three phases. Collagen Rel. Res. 8, 259-273.
Perera K. A. S. and Tonge C. H. (1981) Fibroblast cell proliferation in the mouse molar periodontal ligament. J. Anat. 133, 77-90.
Steinberg B. M., Smith K., Colozzo M. and Pollack R. (1980) Establishment and transformation diminish the ability of fibroblasts to contract a native collagen gel. J. Cell Biol. 87, 304-308.
Taylor A. C. and Butcher E. 0. (1951) The regulation of eruption rate in the incisor teeth of the white rat. J. exp. Zool. 117, 165-188. Tomasek J. J. and Hay E. D. (1984) Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels. J. Cell Biol. 99, 536-549.
Contractile force of periodontal fibroblasts Tomasek J. J., Hay E. D. and Fujiwara K. (1982) Collagen modulates cell shape and cytoskelton of embryonic cornea1 and fibroma fibroblasts: distribution of actin, a-actinin, and myosin. Devl Biol. 92, 107-122. Yagi S., Becker P. L and Fay F. S. (1988) Relationship
601
between force and Ca*+ concentration in smooth muscle cells as revealed by measurements on single cells. Proc. natn. Acad. Sci. U.S.A. 85, 41094113. Zajicek G. (1974) Fibroblast cell kinetics in the periodontal ligament of the mouse. Cell Tim. Kinet. 7, 479-492.
Archs oral Biol. Vol. 35, No. 8, pp. 597401, 1990 Printed in Great Britain. All rights reserved
MEASUREMENTS OF THE ISOMETRIC CONTRACTILE FORCES GENERATED BY DOG PERIODONTAL LIGAMENT FIBROBLASTS IN VITRO S. KASUGAI,’S. SUZUKI,*S. SHIBATA,~S. YASUI,~ H. AMANO’ and H. OGURA’ Departments of ‘Pharmacology, *Orthodontics II, ‘Anatomy II and 4Periodontology, Faculty of Dentistry, Tokyo Medical and Dental University, Yushima l-5-45, Bunkyo-ku, Tokyo, 113, Japan (Accepted 2 March 1990) Summary--One hypothesis for the mechanism of tooth eruption is that the periodontal ligament fibroblasts generate the eruptive force. To assess the force generated, these fibroblasts were obtained by explant culture of ligament from mandibular premolars of a dog and were cultured in collagen gel matrices. The forces generated by them under isometric conditions were continuously measured for 120 h with a strain gauge. At the same time the number of cells in the gel was counted and the force measured was calculattd as the force generated by lo4 cells. Shortly after the start of culture, the force per lo4 cells increased rapidly; it reached 5.2 x lO-4 N at 8 h, and then remained at the same level for about 48 h. Our findings suggest that fibroblasts of the periodontal ligament may generate sufficient force for tooth eruption. Key words: libroblast contraction, periodontal ligament, tooth eruption.
INTRODUCIION
MATERIALS AND METHODS
The mechanism of tooth eruption is not clearly understood although several hypotheses have been proposed and discussed for many years. From experiments with root resection and root transection, it can be concluded that the periodontal ligament is the source of the eruptive force (Berkovitz and Thomas, 1969; Berkovitz, 1,971). Ness (1967) suggested that periodontal ligament fibroblasts might generate this force. In the rodent incisor these cells migrate in an occlusal direction (Chiba, 1968; Zajicek, 1974; Beertsen, 1975; Perera and Tonge, 1981). Beertsen, Everts and van den Hooff (1974) have proposed that while migrating they pull the tooth forward into the mouth. Fibroblasts in culture can generate tensional forces (James and Taylor, 1969). After fibroblasts have been seeded into a three-dimensional collagen gel matrix, the gel gradually contracts (Bell, Ivarsson and Merrill, 1979). This experimental system has been widely used to study the contraction of fibroblasts and experiments with fibroblasts from periodontal ligament sh,ow that these are also contractile (Bellows, Melcher and Aubin, 1981, 1982a). An in vitro model of tooth eruption using ligament fibroblasts and collagen gel matrix has been described (Bellows, Melcher and Aubin, 1983). These in vitro findings endorse the possibility that such fibroblasts may play some part in tooth eruption. However, the forces generated by them have not been measured and it is not clear whether these are sufficient to effect tooth eruption. We have now sought to measure the contractile force of periodontal ligament fibroblasts.
A male dog (14 kg) was premeditated with 0.5 mg atropine sulphate (Tanabe Pharmaceutical Co. Ltd, Tokyo, Japan) and anaesthetized with 20 mg xylazin (Celactar, Bayer Japan Co. Ltd, Tokyo, Japan) and 50 mg ketamine chloride (Ketalar, Sankyo Co. Ltd, Tokyo, Japan). Lidocaine (Fujisawa Pharmaceutical Co. Ltd, Osaka, Japan) was injected for local anaesthesia. Lower premolars were extracted and the periodontal ligament was scraped from the root surface with a scalpel. The explants of the ligament were soaked in phosphate-buffered saline (Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) containing 200 U/ml penicillin, 200 pg/ml streptomycin and 0.5 pg/ml fungizone (Antibiotics Solution, Whittake M.A. Bioproducts Inc., MD, U.S.A.). The explants were transferred to a 60 mm culture dish (Corning, New York, U.S.A.), fixed in place with a cover glass, and culture medium gently added [Eagle’s minimal essential medium (Nissui) containing 60 pg/ml kanamycin and supplemented with 15% (v/v) fetal bovine serum (Filtron Pty Ltd, Victoria, Australia)]. The medium was maintained at pH 7.4 with NaHCO, under an atmosphere of 95% air, 5% CO,, at 37°C. After 14 days, the cells were subcultured in phosphate-buffered saline containing 0.5% trypsin (1: 250, Difco Laboratories, Detroit, MI, U.S.A.). At the third passage, the fibroblasts were stored in the liquid nitrogen. They were then cultured again and used in our study.
AOB 35/bB
Experimental
apparatus
We constructed an apparatus from strain-gauge chips (KFC-03-Cl-16, Kyowa Electronic Instruments Co. Ltd, Tokyo, Japan) and an amplifier IC (AD522,
597
598
S.
KAsUGAIet al. Strain WJW
Fig. 1, Schematic illustration of the experimental apparatus: it consists of two stainless-steel meshes: one mesh (1) was connected to the edge of the culture dish; the other (2) was floating with the aid of fishing floats (3) and was connected by wire to a strain gauge (4). The collagen gel (5) was placed between the two meshes and cultured in the medium (6). Devices, MA, U.S.A.) by following the instructions of Analog Devices. The amplifier was connected to a recorder (2210, LKB, Bromma, Sweden). Before starting the experiments, the strain gauge was placed in a COZ incubator at 37°C and calibrated.
Analog
Preparation of collagen gel and measurement of force
Bovine acid-soluble type I collagen (Cell Matrix I, Nitta Gelatin Co., Ltd, Osaka, Japan) was neutralized by alkaline buffer and minimal essential medium according to the instructions of the supplier. The final concentration of the collagen solution was 2.4 mg/ml. We made square moulds from 1% agar medium and placed two stainless-steel meshes opposite each other in each mould. Then, 5 x lo4 or 2.5 x lo4 fibroblasts were incorporated into 250 ~1 of neutralized collagen solution, poured into the mould and incubated at 37°C for 15 min. The gel and meshes were then transferred to a 60 mm culture dish filled with culture medium. As shown in Fig. 1, one mesh was connected to the edge of the culture dish and the other to a fishing float. The culture dish was placed in a CO* incubator and the float was connected to the strain gauge. To prevent contamination, both culture dish and strain gauge were covered with a large plastic box. The force generated by the gel was recorded continuously over 120 h. Control experiments used gel without cells. Cell counting
To determine the number of cells within gels at various times, gels containing 5 x lo4 fibroblasts were prepared as described above and cultured for 8, 24, 48, 72 and 120 h. Gels between the two meshes were cut out and transferred to a centrifuge tube, where 1 ml of Hank’s balanced salt solution (Nissui) containing 0.04% collagenase (Type I, Sigma Chemical Co., St Louis, MO, U.S.A.) was added. The tubes were stirred at 37°C for 15 min and cetrifuged (400 g, 5 min). The supernatants were discarded and 100 ~1 of crystal violet solution (0.1 M citric acid and 0.1% crystal violet) was added. The number of cells present was then counted in a Barker-Turk counter. Morphological observation
Gels containing fibroblasts were inspected by phase-contrast microscopy to find any changes in cell morphology.
RESULTS
Figure 2 shows the force changes generated by the gel over 120 h. The force generated by gels containing periodontal ligament fibroblasts increased rapidly during the first 8 h, and more slowly thereafter. The time and force curves were constant when we inoculated the gel with the same number of cells. When half that number of cells was inoculated, the force was approximately halved throughout the experimental period. Gels without cells did not generate a measurable force. The change in the number of fibroblasts in the gels is shown in Fig. 3. This did not change during the first 8 h of culture and then increased linearly. From these findings, the force generated by lo4 cells over 120 h was calculated and is shown in Fig. 4. The force per lo4 cells reached 5.2 x 10e4 N at 8 h and remained at almost the same level for about 48 h, after which it decreased gradually. The force per lo4 cells was 3.2 x 10m4N at 120 h. At the start of culture within the gel, the fibroblasts were spherical. Numerous filopodia were soon seen; pseudopodia began to extend from the cells after 6 h in culture, and the fibroblasts then gradually elongated. After 12 h, they were much elongated and had long pseudopodia. Proliferation of some of the fibroblasts was seen after 16 h. Cell to cell contact was also seen as the cell density increased. Between 16 and 120 h, cell shape did not change remarkably.
0
24
48
72
96
120
nme (h) Fig. 2. Force generated by collagen gel over 120 h: collagen gel initially containing either 5 x 10’ (a) or 2.5 x 10’ periodontal ligament fibroblasts (b) or collagen gel without fibroblasts (c).
Contractile force of periodontal fibroblasts
Tlme (h)
Fig. 3. The increase is. the number of fibroblasts in collagen gels over 120h. Each point and bar is the mean value and f 1 SD of 4 samples respectively. DISCUSSION
In previous studies, fibroblasts and other cell types were seeded into collagen gel matrices and shrinkage of the gel resulting from cell contraction was measured (Bell et al., 1979; Steinberg et al., 1980; Bellows et al., 1981; Buttle and Ehrlich, 1983; Gillery, Maquart and Borel, 1986; Ehrlich, 1988; Hughes and Issbemer, 1988; Kasugai and Ogura, 1988; Montesano and Orci, 1988; Nishiyama et al., 1988). In our experiments, fibroblasts were also seeded in collagen gel, but the gel could not shrink freely because one side of it was fixed and the other side was connected to the strain gauge. The maximal change in length of the gel was small (approx. 100 pm or 1% of the initial gel length) throughout the experiment; thus we could measure the isometric force generated within it. Gels without fibroblasts generated no force (Fig. 2) and thus the forces measured were generated directly by those cells. Soon after the start of culture, the ligament fibroblasts generated a force that increased linearly up to 8 h (Fig. 2). Nishiyama et al. (1988) found that several hours elapsed before the start of contraction; this difference between findings might be due to their lower collagen concentration (0.5 mg/ml) and cell density (1.0 x 104/ml). In this initial period (O-8 h in culture), fibroblasts of the periodontal ligament
lime (h) Fig. 4. Force generated by lti periodontal ligament fibroblasts. Each point was calculated from the value measured by the strain gauge (Fig. 2) and the number of fibroblasts (Fig. 3). Each point and bar is the mean value and + 1 SD of 4 samples (O-72 h) or 2 samples (120 h).
599
develop filopodia containing many microfilaments (Bellows et al., 1982b; Tomasek, Hay and Fujiwara, 1982); microtubules align along the long axis of the cell body (Tomasek and Hay, 1984). The contraction of collagen gels containing fibroblasts is inhibited by cytoskeletal inhibitors, such as Colcemid, colchicine and cytochalasins (Bell et al., 1979; Bellows et al., 1982a, 1983; Kasugai and Ogura, 1988; Nishiyama et al., 1988). The force generated by fibroblasts is thus considered to be related to their attachment to the collagen and their development of cell processes; the cytoskeletal systems play an important part in these. One of the factors that affects gel contraction is the number of cells in the gel (Bell et al., 1979). We counted this number, and the forces measured by the strain gauge was calculated as the force generated by lo4 cells. We did not check other factors that affect gel contraction, such as collagen concentration (Gillery et al., 1986), collagen type (Ehrlich, 1988), serum concentration (Gillery et al., 1986) and growth factors (Montesano and Orci, 1988). Between 8 and 72 h in our culture system, lo4 fibroblasts could generate a force of about 5 x 10e4 N (Fig. 4). James and Taylor (1969) used the bending of calibrated microneedles to measure the contractility of whole sheets of fibroblasts stretched between two bone fragments. They estimated the stress per cell as 1.65 x lo-‘N. Our value (5 x lo-* N per cell) was approximately one-third of theirs, but the difference might be due to differences of cells and methods. Harris, Wild and Stopak (1980) cultured chick-heart fibroblasts on a thin silicone membrane and found cell movement and distortion of the membrane. Although they estimated the traction force as 1 x lo-* N/pm of the advancing margin of the cell, they did not state the traction force of an individual cell. A single smooth-muscle cell has been shown to generate a force of l-2 x 10m6N (Yagi, Becker and Fay, 1988); the force generated by the periodontal fibroblasts was therefore approx. 1 or 2% of that generated by the smooth-muscle cell. Changing the medium interferes with continuous force measurement, so we did not do this throughout the experiment. The gradual decline in the force generated by lti fibroblasts after 72 h (Fig. 4) might be due to depletion of nutrients within the medium, but this is unlikely because the number of cells continued to increase (Fig. 3). Remodelling of the collagen matrix by the fibroblasts, or a phenotypic change might explain this decrease. Bellows et al. (1981) showed that fibroblasts from monkey periodontal ligament contracted collagen gels significantly faster than did other cell types, such as human gingival fibroblasts, fetal rat calvaria cells, porcine periodontal ligament epithelial cells and rat osteosarcoma cells. By measuring gel contraction, we earlier found that the fibroblasts of dog periodontal ligament contracted collagen gels more rapidly than did dog gingival fibroblasts (Kasugai et al., 1988). In contrast, Hughes and Issbemer (1988) found that fibroblasts from human periodontal ligament did not contract collagen gels more than fibroblasts from other oral tissues. Gel contraction is also dependent upon the number of cell passages (Steinberg et al., 1980; Buttle and Ehrlich, 1983), and ligament
Berkovitz B. K. B. (1971) The effect of root transection and fibroblasts derived from various species might have partial root resection on the unimpeded eruption rate of different characteristics. the rat incisor. Archs oral Biol. 16, 1033-1043. The force needed to prevent eruption of rat Berkovitz B. K. B. and Thomas N. R. (1969) Unimpeded mandibular incisors was between 2 and 5 g (approx. eruption in the root-resected lower incisor of the rat with 2-5 x 10e2 N) (Taylor and Butcher, 1951) and 7 g a preliminary note on root transection. Archs oral Biol. (approx. 7 x 10e2 N) in rabbit maxillary incisors 14, 771-780. (Miura and Ito, 1968). Burn-Murdoch (1981) found Burn-Murdoch R. A. (1981) Evidence that the response that forces (2.5 x 10m3-1.5 x 10e2N) applied in a to applied forces of continuously erupting rat incisors contains more than one component. Archs oral Biol. 26, direction that opposed the eruption of rat maxillary 989-993. incisors made these teeth sink into their sockets. Buttle D. J. and Ehrlich H. P. (1983) Comparative studies Michaeli, Steigman and Weinreb (1987) estimated of collagen lattice contraction utilizing a normal and a that there are more than IO6fibrobfasts in the toothtransformal cell line. J. Cell Physiol. 116, 159-166. related part of the periodontal ligament of a rat Chiba M. (1968) Movement, during unimpeded eruption, incisor. We calculated that the force generated by 1O4 of the position of cells, and of material incorporating fibroblasts was 5 x low4 N. Therefore, our findings tritiated proline, in the lingual periodontal membrane of suggest that periodontal ligament fibroblasts may be the mandibular incisors of adult male mice. J. dent. Res. able to generate enough force for tooth eruption. We 47, 986. Ehrlich H. P. (1988) The modulation of contraction of have measured the force generated in vitro by fibrofibroblast populated collagen lattices by type I, II and III blasts from teeth of limited eruption, and compared collagen. Tiss. Cell 20, 47-50. this with the eruptive force of continuous erupting Gillery P., Maquart F. X. and Bore1 J. P. (1986) Fibronectin teeth. There seems to be no evidence either that the dependence of the contraction of collagen lattices by eruptive mechanism of continuous erupting teeth is human skin fibroblasts. Expl Cell Res. 167, 29-37. similar to that of teeth of limited eruption or that the Harris A. K., Wild P. and Stopak D. (1980) Silicone rubber forces generated by their ligament cells are different. substrata: a new wrinkle in the study of cell locomotion. However, our findings suggest that the tooth-eruptive Science 208, 177-179. force. may come from fibroblasts of the periodontal Hughes F. J. and Issberner J. P. (1988) Phenotypic variations in contraction of fibroblasts derived from the perioligament. Acknowledgements-We
are very grateful to Dr K. Toda, Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental University, for his kind technical advice. We also greatly appreciate Dr C. G. Bellows, MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, for his critical reading and correcting of English. This study was supported by Grant-in-Aid for scientific research (No. 63570867), from the Ministry of Education, Science and Culture, Japan. REFERENCES Beertsen W. (1975) Migration of fibroblasts in the periodontal ligament of the mouse incisor as revealed by autoradiography. Archs oral Biol. 20, 659-666. Beertsen W., Everts V. and Hooff A. van den (1974) Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. Archs oral Biol. 19, 1087-1098. Bell E., Ivarsson B. and Merrill C. (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. nam. Acad. Sci. U.S.A. 76, 127+1278.
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