Archs
oral
Bid.
Vol.
31. No.
6, pp.
471178.
00034969
1992
92 $5.00
+ 0.00
Copyright 0 1992Pergamon Press Ltd
Pnnted in Great Britain. All rights reserved
EFFECTS OF TRANSFORMING GROWTH FACTOR+, ON CULTURED DENTAL FOLLICLE CELLS FROM RAT MANDIBULAR MOLARS G. E. WISE, F. LIN and W. FAN Department of Anatomy and Cell Biology, Texas College of Osteopathic Medicine/University North Texas, 3500 Camp Bowie Blvd, Fort Worth, TX 76107-2690, U.S.A.
of
(Accepted 9 January 1992)
Summary-Analysis of the total proteins secreted by cultured dental follicle cells revealed that transforming growth factor-fl, (TGF-P,) stimulated them to secrete more extracellular matrix proteins into a serum-free medium than did follicle cells not exposed to the growth factor. Electrophoresis and scanning densitometry showed that secretion of all the major proteins was increased by exposure to the growth factor but the amounts ranged from a 66% increase for one of the procollagen chains to a 7% increase for fibronectin. Immunofluorescence using anti-type I collagen and anti-fibronectin showed that the intracellular concentration and intracellular localization of the antibodies was not changed by incubating the cells with the growth factor. The growth factor did not cause an increase in cell number but did modify the association of the cells in the culture, causing them to aggregate into clusters whereas the control cells formed a confluent monolayer. These results suggest that TGF-8, may signal the fibroblasts of the dental follicle to secrete the extracellular matrix needed for its development into a periodontal ligament. Key words: dental follicle. tissue culture, TGF-/3,, secretion, collagen, fibronectin.
INTRODLCTIOY
In teeth of limited eruption, the presence of a dental follicle is required in order for the tooth to erupt (Cahill and Marks, 1980; Marks and Cahill, 1984). The follicle probably regulates eruption by signalling for the influx of mononuclear cells (monocytes) into the follicle observed at the onset of eruption (Marks, Cahill and Wise, 1983; Wise, Marks and Cahill, 1985; Wise and Fan, 1989) and these monocytes, in turn, probably fuse to produce the increase in osteoclasts also seen at this time (Marks et al., 1983, Wise et al., 1985). The increased numbers of osteoclasts are necessary to resorb the alveolar bone to form an eruption pathway for the tooth. In rat molars we have shown that TGF-fi, is present in the stellate reticulum adjacent to the dental follicle on the days immediatelv prior to the maximal influx of monocytes and then is not observed in the stellate reticulum after that time in the postnatal rats (iVise and Fan, 1991). Because TGF-/I, has been shown to be chemotactic for monocytes (Wahl et al., 1987; Wiseman et nl., 1988), we hypothesized that TGF-/3, may leave the stellate reticulum and that at least one of its actions may be to enter the fenestrated capillaries of the dental follicle and attract the monocytes to the follicle to initiate tooth eruption. In addition to these cellular changes in the dental follicle, there are extracellular changes as well. In Abbreviations
minimal essential medium; : MEM, SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel ekctrophoresis; TGF-8,. transforming growth factor-&. 471
particular, the follicle transforms from a loose connective tissue sac that is primarily cellular (fibroblasts) to a denser connective tissue with a greatly increased extracellular matrix (Wise et al., 1988; Gorski et al., 1988). This transition reflects the fact that the follicle is the precursor of the periodontal ligament. Thus, an additional effect of TGF-/?, may be to stimulate the cells of the dental follicle to synthesize the molecules of the extracellular matrix. In short, TGF-fi, may initiate the development of the periodontal ligament. Cultures of rat dental follicle cells have been established and have shown that these cells secrete extracellular matrix proteins such as fibronectin and type I procollagens (Wise, Lin and Fan, 1991). Hence, the purpose of our study was to determine the effects of TGF-8, on the secretory abilities of these cells in vitro, as well as its effect on cell number and orientation. MATERIALS AND METHODS Cell culture
First and second mandibular molars were surgically removed from 6 to7-day-old postnatal rats and their dental follicles were microdissected, followed by trypsinization and culture of the dental follicle cells (fibroblasts) as described by Wise et al. (1991). For these experiments, cultured cells of passages 4-6 were used. Passages this late were used to ensure that there were no epithelial cells present from the primary culture. The phenotype of these later passaged cells, however, appeared the same as that of the cells of earlier passages.
G.
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WISE er al.
Immunof?uorescence
Rabbit anti-rat type I collagen (Chemicon, Temecula, CA. U.S.A.) and rabbit anti-human fibronectin (ICN Biomedicals. Costa ?vlesa, CA. U.S.A.) were used as previously detailed (Wise er al., 1991) to detect collagen and fibronectin in the permeabilized cultured cells. Fluorescein-conjugated anti-rabbit IgG (ICN Biomedicals, Costa Mesa, CA. U.S.A.) was used to detect the anti-collagen or anti-fibronectin and the cells were observed with a Zeiss photomicroscope III equipped with epifluorescence. For assessing the effect of TGF-/?, on collagen and fibronectin staining within the cells, the cells were incubated in 5 ng of TGF-/?, (R&D Systems, Minneapolis, MN, U.S.A.) in serum-free Eagle modified MEM (ICN Biomedicals, Costa Mesa, CA, U.S.A.) for 24 h before incubation with the appropriate antibody. Controls to determine the specificity of the antibody involved either omission of the primary antibody or its substitution by rabbit non-immune serum.
for 20 min to separate the cell debris. The cells in each flask were then rinsed with MEM and counted. The samples of media were then lyophilized and dialysed as described by Wise et al. (1991). Each lyophilized sample was then dissolved in 100 ~1 of distilled water and the protein concentrations of the samples were determined by the Bradford assay (Bradford, (1976). SDS-PAGE
An equal volume of each above sample (10 ~1) and a sample of 2 pg each of reference-weight proteins were loaded on to separate lanes of a 6% SDS-polyacrylamide gel. The gel was then electrophoresed with a constant current of 10 mA/slab until the bromophenol tracking-dye front was about 1 cm from the bottom of the gel. Next, the gel was stained with Coomassie blue R-250 for 1 h and a Quick Scan R&D scanning densitometer was used to semiquantitate the relative amounts of protein in the stained bands. RESULTS
Protein assay
Cells grown on circular IO-cm dia plastic dishes were trypsinized and counted with a ZM Coulter counter (Hialeah. FL, U.S.A.). Next. 7 x IO’ cells were plated into each 150 cm’ flask that contained 20 ml of tissue culture medium (MELI) containing 15% newborn calf serum (ICN Biomedicals, Costa Mesa, CA, U.S.A.). After 24 h the nearly confluent cells of each flask were rinsed thrice with 10ml of serum-free medium. The cells in each flask were then incubated with 15 ml of serum-free LfEM either in the presence of TGF-/?, (1 ng TGF-/?, ml of MEM, 5 ng/ml or 10 ng ml) or in its absence for 24 h in a 37’C/5% CO: incubator. After incubation, the medium of each flask was collected separately and 1 mM concentrations of phenylmethyl sulphonyl fluoride, EDTA and B-mercaptoethanol were added to each. The media were then centrifuged at 15OOg
By immunofluorescence microscopy the cultured cells were stained heavily for fibronectin (Fig. 1) and type I collagen (Fig. 2) after they had been permeabilized and incubated with the appropriate antibody. When the cells were incubated in TGF-pi for 24 h before immunofluorescence, there was no change in the staining for fibronectin (Fig. 3) or for collagen when compared with cells not incubated in TGF-fl,. Controls did not fluoresce. Placing the cultured cells in a serum-free medium for 24 h and analysing the medium revealed that proteins had been secreted into it. As seen in an SDS-gel profile in Fig. 4, some of the major proteins secreted included those of molecular weights 220 (fibronectin), 180 and I56 (procollagens) and 66 kDa. No proteins were present in the serum-free medium before the cells were put in it.
Plate I Fig. I. Cultured dental follicle cells that had been permeabilized and then incubated with anti-fibronectin followed by fluorescein-conjugated IgG. Note that the cells fluoresce strongly, especially in the perinuclear cytoplasm. The nucleus does not stain. x 330 Fig. 2. Localization of collagen in cultured follicle cells using anti-type I collagen is almost identical to the fluorescent pattern of anti-tibronectin. x 580 Fig. 3. Cultured dental follicle cells that had been incubated in TGF-fi, for 24 h before detection of fibronectin with anti-fibronectin. The cells still fluoresce as heavily and have the same staining pattern as do the cells not incubated with TGF-P, (see Fig. 1). x 330 Plate 2 Fig. 4. SDS-polyacrylamide gel of proteins secreted into serum-free medium by cultured dental follicle cells comparing effects of TGF-1, with control. Lane (a), molecular-weight standards in kDa; lane (b), control in which cells had not been incubated in TGF-&; lane (c), cells had been incubated with 1 ng TGF-/I,/ml of serum-free medium; lane (d), cells had been incubated with 5 ng YGF-p, and lane (e), cells had been incubated with 10 ng TGF-8,. No unique proteins are secreted under the influence of TGF-8, but an increase in proteins normally secreted is seen-see Tables 1 and 2. Plate 3 Fig. 5. Phase-contrast view of fourth-passage cultured dental follicle cells that have reached confluency and form a monolayer. x 33 Fig. 6. Fourth-passage cultured dental follicle cells that had been exposed to TGF-B, for 72 h. Cells are arranged in clusters (arrows) with spaces almost devoid of cells between the aggregates. x 33 Fig. 7. High-power view of one of the clusters of cells that form in response to incubation with TGF-8,. Note the ordered arrangement of cells within this large mound. x I65
Effects of TGF-& on dental follicle
Plate 1
473
G. E. WISE et al
474
a F
b .a-
c
-mq’---
d
e
y?-T
=-w-7
#jf##milu) 200
220
m 180
_uaQInw
156
116
a
Plate 2
Effects of TGF-b, on dental follicle
Plate 3
475
416
G. E. WISE et al.
Table I. Effects of different concentrations of TGF-/?, on cell number and total protein secretion of cultured dental follicle cells Concentration TGF-8, Control (no TGF-B,) I ng/ml 5 ng/ml 10 ng/ml
Cells
Cells
Proteins
Increase
(before)
(after)
proteins (JL,~)
(ngiceh)
(%)
700,000
569,600
83.829
0.147
-
700,000 700,000 700,000
595,200 558,400 576,000
117.351 136.224 100.905
0.197 0.244 0.175
34 66 19
Total
Measurements of total proteins secreted into the serum-free medium were conducted after the cells had been in the medium for 24 h.
Adding
TGF-/I,
to the cultured
cells increased
form a confluent monolayer. cells that had been incubated
In contrast,
DISCCSSION
the
total amount of protein secreted. As seen in Table 1, concentrations of TGF-8, of 1, 5 or 10 ng/ml of serum-free medium were added to the cells. All concentrations increased the amount of total proteins secreted but 5 ng was the optimal amount for maximal secretion. Because there is a slight decrease in cell number when the cells are incubated in serum-free medium, the amount of protein secreted per cell was calculated. This was determined to be 0.147 ng/cell in the controls as compared to 0.244 ng/ceIl after the optimal amount of TGF-B, (5 ng/ml) had been added. This represents a 66% increase in the amount of proteins secreted after incubation in TGF-p, for 24 h. To determine if TGF-/I, preferentially increased the secretion of a given protein, the cells were incubated in the different concentrations of TGF-/I, and equal volumes of the secreted proteins of each experiment were run on SDS-PAGE (Fig. 4). Scanning the Coomassie blue-stained gels with a densitometer revealed that the amount of the 156 kDa protein was increased the most (Table 2). Each major protein of the experimental group increased in amount over its counterpart in the controls (Table 2), although only a 7% increase was seen for the 220 kDa protein after incubation with 5 ng of TGF-/?,/ml. TGF-/I, also modifies the association of the cells in culture. As seen in Fig. 5, fourth-passage controls similar
in TGF-pi for 72 h with the medium changed every 48 h associated closely into large clusters with spaces essentially devoid of ceils between the clusters (Fig. 6). A higher-power view of one of these aggregates showed that the cells in a cluster formed a mound that appeared to be arranged, to a degree, in an ordered pattern (Fig. 7).
The ability of TGF-fi, to stimulate the synthesis and secretion of collagen and fibronectin has previously been demonstrated in chick embryo fibroblasts (Ignotz and Massague. 1986). Moreover, this growth factor causes an increase in the level of mRNAs for these proteins (Ignotz, Indo and Massague, 1987). As shown here, TGF-fi, can increase the secretion of extracellular matrix proteins of cultured dental follicle cells. The nature of these proteins (fibronectin, procollagens) has been documented by Wise et al. (1991). In both rat molars and dog premolars. electron microscopy has shown that there is a dramatic increase in the number and size of collagen fibrils in the extracellular matrix of the dental follicle from the time before eruption to its near conclusion (Wise et al., 1988; Gorski et al., 1988). This was confirmed biochemically in the dog premolar, where there was a 2.5-fold increase in collagen over this g-week span (Gorski et al., 1988). In conjunction with this, small fibrils of approx. 12.7 nm, which may be elastic fibres, appear for the first time in the matrix near the end of eruption (Wise et al., 1988; Gorski er nl., 1988). These changes in the extracellular matrix of the dental follicle may well be initiated by TGF-/?,. In an earlier study, we showed that TGF-/I, is present in the stellate reticulum of the 1st mandibular molar of the rat on days 1 and 2 postnatally (Wise and Fan, 1991). Because the dental follicle is immediately adjacent to the stellate reticulum, it is possible that some of the TGF-/I, in the stellate reticulum could have a paracrine effect on the dental follicle cells, stimulating them to secrete more extracellular matrix proteins. This, in turn, would actually be a signal for the formation of the periodontal ligament, which is derived from the dental follicle. Signalling for the
Table 2. Comparison of the effect of different concentrations of TGF-8, on secretion of individual proteins by cultured dental follicle cells Molecular weight @Da) 66 156 180 220
Control W) 100 100 100 100
1 ng TGF-&/ml W) 125 246 116 104
5 ng TGF$, ‘ml W) 170 273 123 107
10 ng TGF#,/ml W) 114 162 138 101
Comparisons made based on scanning densitometry of Coomassie blue-stained 6% SDS-gels. Controls were the base line and were set at 100%. Proteins were collected from the serum-free medium after the cells had been in the medium for 24 h.
477
Effects of TGF-8, on dental follicle
formation of the periodontal ligament is a major developmental event, not only because it is the onset of the formation of the structure that anchors the tooth to its bony socket but also because the ligament may play a role in postfunctional eruption. Cahill and Marks (1982) have shown that not until after the tooth pierces the gingiva does the periodontal ligament become attached to the alveolar bone. Thus, at that point in tooth eruption the ligament may help in moving the tooth to its final destination. This seems likely in view of the fact that, in teeth of continuous eruption, Berkovitz and Thomas (1969) have shown that severance of the periodontal ligament prevents eruption. In teeth of limited (e.g. dog premolars or rat molars) and continuous eruption (rodent or lagomorph incisors), the periodontal ligament might serve to propel the tooth once it has escaped its bony crypt. In short, the ligament may play a part in postfunctional but not prefunctional eruption. We used 6-7-day-old postnatal rats as the source of the follicles in order to provide enough of this tissue for surgical removal, trypsinization and culture. TGF-/?,, as discussed above, is present in the stellate reticulum on days 1 and 2 (Wise and Fan, 1991) and perhaps is exerting its effect earlier than day 6. However, that does not preclude the possibility that dental follicle cells could respond to TGF-8, at times other than day I or 2. Moreover, regardless of the age of the rats from which the dental follicles were isolated. the possibility remains that the follicle cells will dedifferentiate to some extent in culture. In addition to promoting secretion of collagen and fibronectin, it is probable that TGF-/I, is stimulating the synthesis of these proteins. Incubating the cells in TGF-/I, does not alter the fluorescent staining for these proteins within the cells, despite the fact that more protein is being secreted than in controls. Thus, new proteins are probably being synthesized, as reflected in the continued staining for them within the cells; i.e. if no new extracellular matrix proteins were being synthesized, the intracellular staining for them should decrease after they have been secreted. The appearance of putative elastic fibrils in the developing, periodontal ligament may also be related to the action of TGF-/I,. Studies with cultures of smooth muscle cells have shown that TGF-P, stimulates the synthesis of elastin (Liu and Davidson, 1988). Thus, TGF-p, may be stimulating the synthesis and/or secretion of all of the major fibrils of the periodontal ligament. Experiments involving the injection of TGF-/I, in ciw are in progress to determine if it does indeed stimulate the synthesis and secretion of the ligament components. Concomitant with the stimulation of changes in the extracellular matrix, TGF-/I, may be modifying the orientation of the dental follicle cells themselves. As eruption begins the fibroblasts of the follicle appear to become oriented more in parallel to each other (e.g. see Wise et al., 1985) and this ordered array may result from a TGF-/I, signal. This speculation is based on the fact that in &To the cultures of dental follicle cells treated with TGF-/I, clustered together instead of forming a monolayer and, within these clusters, there was a suggestion of a parallel orientation.
In addition to TGF-/I, stimulating the secretion of major extracellular proteins (type I collagen and fibronectin), the secretion of a smaller protein of 66 kDa was also increased after incubation with TGF+I,. This protein might be. the extracellular matrix protein that has been shown to be a substrate for urokinase (Keski-Oja and Vaheri, 1982). This protein appears to be associated with fibronectin and perhaps helps in the association of the fibronectin with the matrix (Keski-Oja and Vaheri, 1982). A protein of this molecular weight has been shown to be part of the extracellular matrix in human fibroblast cultures. Finally, the fact that TGF-j, may initiate the formation of the periodontal ligament does not preclude the possibility that it may also act as a chemoattractant for the influx of monocytes into the dental follicle to initiate tooth eruption. In fact, because TGF-/I, does not stimulate the dental follicle cells in culture to secrete proteins different from the extracellular matrix proteins they normally secrete, it may be that TGF-/I, itself is the chemoattractant rather than it stimulating the follicle cells to secrete a chemoattractant for the monocytes. Concomitant with this, the effect of TGF-/I, on the follicle cells themselves might be to stimulate them to secrete the extracellular matrix proteins of the periodontal ligament. Acknowledgemenls-This work was supported by NIDR Grant ROI DE08911 to G.E.W. The authors thank MS Carolyn Bannon for typing this manuscript.
REFERENCES Berkovitz B. K. B. and Thomas N. R. (1969) Unimpeded eruption in the root-resected lower incisor of the rat with a preliminary note on root transection. Archs oral Biol. 14, 771-780. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyf. Biochem. 72, 248-254.
Cahill D. R. and Marks S. C. Jr (1980) Tooth eruption: evidence for the central role of the dental follicle. J. oral Path. 9, 189-200.
Cahill D. R. and Marks S. C. Jr (1982) Chronology and histology of exfoliation and eruption of mandibular premolars in dogs. J. Morph. 171, 213-218. Gorski J. P., Marks S. C. Jr, Cahill D. R. and Wise G. E. (1988) Developmental changes in the extracellular matrix of the dental follicle during tooth eruption. Conn. Tiss. Res. 18, 175-190.
Ignotz R. A. and Massague J. (1986) Transforming growth factor-b stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. biol. Chem. 261, 4337-4345. Ignotz R. A., Endo T. and Massague J. (1987) Regulation of fibronectin and type I collagen on RSA levels by transforming growth factor-/?. J. biol. Chem. 262, 6443-6446.
Keski-Oja J. and Vaheri A. (1982) The cellular target for the plasminogen activator, urokinase, in human fibroblasts66000-dalton protein. Biochem. biophys. Acta 720, 141-146.
Liu J. M. and Davidson J. M. (1988) The elastogenic effect of recombinant transforming growth factor-beta on porcine aortic smooth muscle cells. Biochem. biophys. Res. Commun. 154, 895-961.
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G. E. WISE et al.
Marks S. C. Jr and Cahill D. R. (1984) Experimental study in the dog of the non-active role of the tooth in the eruptive process, Archs oral Biol. 29, 3 I l-322. Marks S. C. Jr, Cahill D. R. and Wise G. E. (1983) The cytology of the dental follicle and adjacent alveolar bone during tooth eruption in the dog. Am. J. Anat. 168, 277-289.
Wahl S. M., Hunt D. A., Wakefield L. M., McCartneyFrancis N., Wahl L. M.. Roberts A. B. and Spom M. B. (1987) Transforming growth factor-beta (TTGF-beta) induces monocyte chemotaxis and growth factor production. Proc. natn. Acad. Sci., U.S.A. &J, 5788-5792. Wise G. E. and Fan W. (1989) Changes in the tartrateresistant acid phosphatase cell population in dental follicles and bony crypts of rat molars during tooth eruption. J. denr. Res. 68, 150-156. Wise G. E. and Fan W. (1991) lmmunolocalization of transforming growth factor beta in rat molars. J. oral Path. Med. 20, 74-80.
Wise G. E., Marks S. C. Jr and Cahill D. R. (1985) Ultrastructural features of the dental follicle associated with formation of the tooth eruption pathway in the dog. 1. oral Path. 14, 15-26. Wise G. E., Marks S. C. Jr, Cahill D. R. and Gorski J. P. (1988) Ultrastructural features of the dental follicle and enamel organ prior to and during tooth eruption. In The Biological Mechanisms of Tooth Eruption and Rool Resorption (Ed. Davidovitch 2.). pp. 243-249. EBSCO
Media, Birmingham, AL. Wise G. E., Lin F. and Fan W. (1992) Culture and characterization of dental follicle cells from rat molars. Cell Tiss. Res. 267, 483-492. Wiseman D. M., Polverine P. J., Kamp D. W. and Liebovich S. J. (1988) Transforming growth factor-beta (TGF-beta) is chemotactic for human monocytes and induces their expression of angiogenie activity. Biochem. biophys. Res. Commun. 157, 793-800.
oral
Bid.
Vol.
31. No.
6, pp.
471178.
00034969
1992
92 $5.00
+ 0.00
Copyright 0 1992Pergamon Press Ltd
Pnnted in Great Britain. All rights reserved
EFFECTS OF TRANSFORMING GROWTH FACTOR+, ON CULTURED DENTAL FOLLICLE CELLS FROM RAT MANDIBULAR MOLARS G. E. WISE, F. LIN and W. FAN Department of Anatomy and Cell Biology, Texas College of Osteopathic Medicine/University North Texas, 3500 Camp Bowie Blvd, Fort Worth, TX 76107-2690, U.S.A.
of
(Accepted 9 January 1992)
Summary-Analysis of the total proteins secreted by cultured dental follicle cells revealed that transforming growth factor-fl, (TGF-P,) stimulated them to secrete more extracellular matrix proteins into a serum-free medium than did follicle cells not exposed to the growth factor. Electrophoresis and scanning densitometry showed that secretion of all the major proteins was increased by exposure to the growth factor but the amounts ranged from a 66% increase for one of the procollagen chains to a 7% increase for fibronectin. Immunofluorescence using anti-type I collagen and anti-fibronectin showed that the intracellular concentration and intracellular localization of the antibodies was not changed by incubating the cells with the growth factor. The growth factor did not cause an increase in cell number but did modify the association of the cells in the culture, causing them to aggregate into clusters whereas the control cells formed a confluent monolayer. These results suggest that TGF-8, may signal the fibroblasts of the dental follicle to secrete the extracellular matrix needed for its development into a periodontal ligament. Key words: dental follicle. tissue culture, TGF-/3,, secretion, collagen, fibronectin.
INTRODLCTIOY
In teeth of limited eruption, the presence of a dental follicle is required in order for the tooth to erupt (Cahill and Marks, 1980; Marks and Cahill, 1984). The follicle probably regulates eruption by signalling for the influx of mononuclear cells (monocytes) into the follicle observed at the onset of eruption (Marks, Cahill and Wise, 1983; Wise, Marks and Cahill, 1985; Wise and Fan, 1989) and these monocytes, in turn, probably fuse to produce the increase in osteoclasts also seen at this time (Marks et al., 1983, Wise et al., 1985). The increased numbers of osteoclasts are necessary to resorb the alveolar bone to form an eruption pathway for the tooth. In rat molars we have shown that TGF-fi, is present in the stellate reticulum adjacent to the dental follicle on the days immediatelv prior to the maximal influx of monocytes and then is not observed in the stellate reticulum after that time in the postnatal rats (iVise and Fan, 1991). Because TGF-/I, has been shown to be chemotactic for monocytes (Wahl et al., 1987; Wiseman et nl., 1988), we hypothesized that TGF-/3, may leave the stellate reticulum and that at least one of its actions may be to enter the fenestrated capillaries of the dental follicle and attract the monocytes to the follicle to initiate tooth eruption. In addition to these cellular changes in the dental follicle, there are extracellular changes as well. In Abbreviations
minimal essential medium; : MEM, SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel ekctrophoresis; TGF-8,. transforming growth factor-&. 471
particular, the follicle transforms from a loose connective tissue sac that is primarily cellular (fibroblasts) to a denser connective tissue with a greatly increased extracellular matrix (Wise et al., 1988; Gorski et al., 1988). This transition reflects the fact that the follicle is the precursor of the periodontal ligament. Thus, an additional effect of TGF-/?, may be to stimulate the cells of the dental follicle to synthesize the molecules of the extracellular matrix. In short, TGF-fi, may initiate the development of the periodontal ligament. Cultures of rat dental follicle cells have been established and have shown that these cells secrete extracellular matrix proteins such as fibronectin and type I procollagens (Wise, Lin and Fan, 1991). Hence, the purpose of our study was to determine the effects of TGF-8, on the secretory abilities of these cells in vitro, as well as its effect on cell number and orientation. MATERIALS AND METHODS Cell culture
First and second mandibular molars were surgically removed from 6 to7-day-old postnatal rats and their dental follicles were microdissected, followed by trypsinization and culture of the dental follicle cells (fibroblasts) as described by Wise et al. (1991). For these experiments, cultured cells of passages 4-6 were used. Passages this late were used to ensure that there were no epithelial cells present from the primary culture. The phenotype of these later passaged cells, however, appeared the same as that of the cells of earlier passages.
G.
472
E.
WISE er al.
Immunof?uorescence
Rabbit anti-rat type I collagen (Chemicon, Temecula, CA. U.S.A.) and rabbit anti-human fibronectin (ICN Biomedicals. Costa ?vlesa, CA. U.S.A.) were used as previously detailed (Wise er al., 1991) to detect collagen and fibronectin in the permeabilized cultured cells. Fluorescein-conjugated anti-rabbit IgG (ICN Biomedicals, Costa Mesa, CA. U.S.A.) was used to detect the anti-collagen or anti-fibronectin and the cells were observed with a Zeiss photomicroscope III equipped with epifluorescence. For assessing the effect of TGF-/?, on collagen and fibronectin staining within the cells, the cells were incubated in 5 ng of TGF-/?, (R&D Systems, Minneapolis, MN, U.S.A.) in serum-free Eagle modified MEM (ICN Biomedicals, Costa Mesa, CA, U.S.A.) for 24 h before incubation with the appropriate antibody. Controls to determine the specificity of the antibody involved either omission of the primary antibody or its substitution by rabbit non-immune serum.
for 20 min to separate the cell debris. The cells in each flask were then rinsed with MEM and counted. The samples of media were then lyophilized and dialysed as described by Wise et al. (1991). Each lyophilized sample was then dissolved in 100 ~1 of distilled water and the protein concentrations of the samples were determined by the Bradford assay (Bradford, (1976). SDS-PAGE
An equal volume of each above sample (10 ~1) and a sample of 2 pg each of reference-weight proteins were loaded on to separate lanes of a 6% SDS-polyacrylamide gel. The gel was then electrophoresed with a constant current of 10 mA/slab until the bromophenol tracking-dye front was about 1 cm from the bottom of the gel. Next, the gel was stained with Coomassie blue R-250 for 1 h and a Quick Scan R&D scanning densitometer was used to semiquantitate the relative amounts of protein in the stained bands. RESULTS
Protein assay
Cells grown on circular IO-cm dia plastic dishes were trypsinized and counted with a ZM Coulter counter (Hialeah. FL, U.S.A.). Next. 7 x IO’ cells were plated into each 150 cm’ flask that contained 20 ml of tissue culture medium (MELI) containing 15% newborn calf serum (ICN Biomedicals, Costa Mesa, CA, U.S.A.). After 24 h the nearly confluent cells of each flask were rinsed thrice with 10ml of serum-free medium. The cells in each flask were then incubated with 15 ml of serum-free LfEM either in the presence of TGF-/?, (1 ng TGF-/?, ml of MEM, 5 ng/ml or 10 ng ml) or in its absence for 24 h in a 37’C/5% CO: incubator. After incubation, the medium of each flask was collected separately and 1 mM concentrations of phenylmethyl sulphonyl fluoride, EDTA and B-mercaptoethanol were added to each. The media were then centrifuged at 15OOg
By immunofluorescence microscopy the cultured cells were stained heavily for fibronectin (Fig. 1) and type I collagen (Fig. 2) after they had been permeabilized and incubated with the appropriate antibody. When the cells were incubated in TGF-pi for 24 h before immunofluorescence, there was no change in the staining for fibronectin (Fig. 3) or for collagen when compared with cells not incubated in TGF-fl,. Controls did not fluoresce. Placing the cultured cells in a serum-free medium for 24 h and analysing the medium revealed that proteins had been secreted into it. As seen in an SDS-gel profile in Fig. 4, some of the major proteins secreted included those of molecular weights 220 (fibronectin), 180 and I56 (procollagens) and 66 kDa. No proteins were present in the serum-free medium before the cells were put in it.
Plate I Fig. I. Cultured dental follicle cells that had been permeabilized and then incubated with anti-fibronectin followed by fluorescein-conjugated IgG. Note that the cells fluoresce strongly, especially in the perinuclear cytoplasm. The nucleus does not stain. x 330 Fig. 2. Localization of collagen in cultured follicle cells using anti-type I collagen is almost identical to the fluorescent pattern of anti-tibronectin. x 580 Fig. 3. Cultured dental follicle cells that had been incubated in TGF-fi, for 24 h before detection of fibronectin with anti-fibronectin. The cells still fluoresce as heavily and have the same staining pattern as do the cells not incubated with TGF-P, (see Fig. 1). x 330 Plate 2 Fig. 4. SDS-polyacrylamide gel of proteins secreted into serum-free medium by cultured dental follicle cells comparing effects of TGF-1, with control. Lane (a), molecular-weight standards in kDa; lane (b), control in which cells had not been incubated in TGF-&; lane (c), cells had been incubated with 1 ng TGF-/I,/ml of serum-free medium; lane (d), cells had been incubated with 5 ng YGF-p, and lane (e), cells had been incubated with 10 ng TGF-8,. No unique proteins are secreted under the influence of TGF-8, but an increase in proteins normally secreted is seen-see Tables 1 and 2. Plate 3 Fig. 5. Phase-contrast view of fourth-passage cultured dental follicle cells that have reached confluency and form a monolayer. x 33 Fig. 6. Fourth-passage cultured dental follicle cells that had been exposed to TGF-B, for 72 h. Cells are arranged in clusters (arrows) with spaces almost devoid of cells between the aggregates. x 33 Fig. 7. High-power view of one of the clusters of cells that form in response to incubation with TGF-8,. Note the ordered arrangement of cells within this large mound. x I65
Effects of TGF-& on dental follicle
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Effects of TGF-b, on dental follicle
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Table I. Effects of different concentrations of TGF-/?, on cell number and total protein secretion of cultured dental follicle cells Concentration TGF-8, Control (no TGF-B,) I ng/ml 5 ng/ml 10 ng/ml
Cells
Cells
Proteins
Increase
(before)
(after)
proteins (JL,~)
(ngiceh)
(%)
700,000
569,600
83.829
0.147
-
700,000 700,000 700,000
595,200 558,400 576,000
117.351 136.224 100.905
0.197 0.244 0.175
34 66 19
Total
Measurements of total proteins secreted into the serum-free medium were conducted after the cells had been in the medium for 24 h.
Adding
TGF-/I,
to the cultured
cells increased
form a confluent monolayer. cells that had been incubated
In contrast,
DISCCSSION
the
total amount of protein secreted. As seen in Table 1, concentrations of TGF-8, of 1, 5 or 10 ng/ml of serum-free medium were added to the cells. All concentrations increased the amount of total proteins secreted but 5 ng was the optimal amount for maximal secretion. Because there is a slight decrease in cell number when the cells are incubated in serum-free medium, the amount of protein secreted per cell was calculated. This was determined to be 0.147 ng/cell in the controls as compared to 0.244 ng/ceIl after the optimal amount of TGF-B, (5 ng/ml) had been added. This represents a 66% increase in the amount of proteins secreted after incubation in TGF-p, for 24 h. To determine if TGF-/I, preferentially increased the secretion of a given protein, the cells were incubated in the different concentrations of TGF-/I, and equal volumes of the secreted proteins of each experiment were run on SDS-PAGE (Fig. 4). Scanning the Coomassie blue-stained gels with a densitometer revealed that the amount of the 156 kDa protein was increased the most (Table 2). Each major protein of the experimental group increased in amount over its counterpart in the controls (Table 2), although only a 7% increase was seen for the 220 kDa protein after incubation with 5 ng of TGF-/?,/ml. TGF-/I, also modifies the association of the cells in culture. As seen in Fig. 5, fourth-passage controls similar
in TGF-pi for 72 h with the medium changed every 48 h associated closely into large clusters with spaces essentially devoid of ceils between the clusters (Fig. 6). A higher-power view of one of these aggregates showed that the cells in a cluster formed a mound that appeared to be arranged, to a degree, in an ordered pattern (Fig. 7).
The ability of TGF-fi, to stimulate the synthesis and secretion of collagen and fibronectin has previously been demonstrated in chick embryo fibroblasts (Ignotz and Massague. 1986). Moreover, this growth factor causes an increase in the level of mRNAs for these proteins (Ignotz, Indo and Massague, 1987). As shown here, TGF-fi, can increase the secretion of extracellular matrix proteins of cultured dental follicle cells. The nature of these proteins (fibronectin, procollagens) has been documented by Wise et al. (1991). In both rat molars and dog premolars. electron microscopy has shown that there is a dramatic increase in the number and size of collagen fibrils in the extracellular matrix of the dental follicle from the time before eruption to its near conclusion (Wise et al., 1988; Gorski et al., 1988). This was confirmed biochemically in the dog premolar, where there was a 2.5-fold increase in collagen over this g-week span (Gorski et al., 1988). In conjunction with this, small fibrils of approx. 12.7 nm, which may be elastic fibres, appear for the first time in the matrix near the end of eruption (Wise et al., 1988; Gorski er nl., 1988). These changes in the extracellular matrix of the dental follicle may well be initiated by TGF-/?,. In an earlier study, we showed that TGF-/I, is present in the stellate reticulum of the 1st mandibular molar of the rat on days 1 and 2 postnatally (Wise and Fan, 1991). Because the dental follicle is immediately adjacent to the stellate reticulum, it is possible that some of the TGF-/I, in the stellate reticulum could have a paracrine effect on the dental follicle cells, stimulating them to secrete more extracellular matrix proteins. This, in turn, would actually be a signal for the formation of the periodontal ligament, which is derived from the dental follicle. Signalling for the
Table 2. Comparison of the effect of different concentrations of TGF-8, on secretion of individual proteins by cultured dental follicle cells Molecular weight @Da) 66 156 180 220
Control W) 100 100 100 100
1 ng TGF-&/ml W) 125 246 116 104
5 ng TGF$, ‘ml W) 170 273 123 107
10 ng TGF#,/ml W) 114 162 138 101
Comparisons made based on scanning densitometry of Coomassie blue-stained 6% SDS-gels. Controls were the base line and were set at 100%. Proteins were collected from the serum-free medium after the cells had been in the medium for 24 h.
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Effects of TGF-8, on dental follicle
formation of the periodontal ligament is a major developmental event, not only because it is the onset of the formation of the structure that anchors the tooth to its bony socket but also because the ligament may play a role in postfunctional eruption. Cahill and Marks (1982) have shown that not until after the tooth pierces the gingiva does the periodontal ligament become attached to the alveolar bone. Thus, at that point in tooth eruption the ligament may help in moving the tooth to its final destination. This seems likely in view of the fact that, in teeth of continuous eruption, Berkovitz and Thomas (1969) have shown that severance of the periodontal ligament prevents eruption. In teeth of limited (e.g. dog premolars or rat molars) and continuous eruption (rodent or lagomorph incisors), the periodontal ligament might serve to propel the tooth once it has escaped its bony crypt. In short, the ligament may play a part in postfunctional but not prefunctional eruption. We used 6-7-day-old postnatal rats as the source of the follicles in order to provide enough of this tissue for surgical removal, trypsinization and culture. TGF-/?,, as discussed above, is present in the stellate reticulum on days 1 and 2 (Wise and Fan, 1991) and perhaps is exerting its effect earlier than day 6. However, that does not preclude the possibility that dental follicle cells could respond to TGF-8, at times other than day I or 2. Moreover, regardless of the age of the rats from which the dental follicles were isolated. the possibility remains that the follicle cells will dedifferentiate to some extent in culture. In addition to promoting secretion of collagen and fibronectin, it is probable that TGF-/I, is stimulating the synthesis of these proteins. Incubating the cells in TGF-/I, does not alter the fluorescent staining for these proteins within the cells, despite the fact that more protein is being secreted than in controls. Thus, new proteins are probably being synthesized, as reflected in the continued staining for them within the cells; i.e. if no new extracellular matrix proteins were being synthesized, the intracellular staining for them should decrease after they have been secreted. The appearance of putative elastic fibrils in the developing, periodontal ligament may also be related to the action of TGF-/I,. Studies with cultures of smooth muscle cells have shown that TGF-P, stimulates the synthesis of elastin (Liu and Davidson, 1988). Thus, TGF-p, may be stimulating the synthesis and/or secretion of all of the major fibrils of the periodontal ligament. Experiments involving the injection of TGF-/I, in ciw are in progress to determine if it does indeed stimulate the synthesis and secretion of the ligament components. Concomitant with the stimulation of changes in the extracellular matrix, TGF-/I, may be modifying the orientation of the dental follicle cells themselves. As eruption begins the fibroblasts of the follicle appear to become oriented more in parallel to each other (e.g. see Wise et al., 1985) and this ordered array may result from a TGF-/I, signal. This speculation is based on the fact that in &To the cultures of dental follicle cells treated with TGF-/I, clustered together instead of forming a monolayer and, within these clusters, there was a suggestion of a parallel orientation.
In addition to TGF-/I, stimulating the secretion of major extracellular proteins (type I collagen and fibronectin), the secretion of a smaller protein of 66 kDa was also increased after incubation with TGF+I,. This protein might be. the extracellular matrix protein that has been shown to be a substrate for urokinase (Keski-Oja and Vaheri, 1982). This protein appears to be associated with fibronectin and perhaps helps in the association of the fibronectin with the matrix (Keski-Oja and Vaheri, 1982). A protein of this molecular weight has been shown to be part of the extracellular matrix in human fibroblast cultures. Finally, the fact that TGF-j, may initiate the formation of the periodontal ligament does not preclude the possibility that it may also act as a chemoattractant for the influx of monocytes into the dental follicle to initiate tooth eruption. In fact, because TGF-/I, does not stimulate the dental follicle cells in culture to secrete proteins different from the extracellular matrix proteins they normally secrete, it may be that TGF-/I, itself is the chemoattractant rather than it stimulating the follicle cells to secrete a chemoattractant for the monocytes. Concomitant with this, the effect of TGF-/I, on the follicle cells themselves might be to stimulate them to secrete the extracellular matrix proteins of the periodontal ligament. Acknowledgemenls-This work was supported by NIDR Grant ROI DE08911 to G.E.W. The authors thank MS Carolyn Bannon for typing this manuscript.
REFERENCES Berkovitz B. K. B. and Thomas N. R. (1969) Unimpeded eruption in the root-resected lower incisor of the rat with a preliminary note on root transection. Archs oral Biol. 14, 771-780. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyf. Biochem. 72, 248-254.
Cahill D. R. and Marks S. C. Jr (1980) Tooth eruption: evidence for the central role of the dental follicle. J. oral Path. 9, 189-200.
Cahill D. R. and Marks S. C. Jr (1982) Chronology and histology of exfoliation and eruption of mandibular premolars in dogs. J. Morph. 171, 213-218. Gorski J. P., Marks S. C. Jr, Cahill D. R. and Wise G. E. (1988) Developmental changes in the extracellular matrix of the dental follicle during tooth eruption. Conn. Tiss. Res. 18, 175-190.
Ignotz R. A. and Massague J. (1986) Transforming growth factor-b stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. biol. Chem. 261, 4337-4345. Ignotz R. A., Endo T. and Massague J. (1987) Regulation of fibronectin and type I collagen on RSA levels by transforming growth factor-/?. J. biol. Chem. 262, 6443-6446.
Keski-Oja J. and Vaheri A. (1982) The cellular target for the plasminogen activator, urokinase, in human fibroblasts66000-dalton protein. Biochem. biophys. Acta 720, 141-146.
Liu J. M. and Davidson J. M. (1988) The elastogenic effect of recombinant transforming growth factor-beta on porcine aortic smooth muscle cells. Biochem. biophys. Res. Commun. 154, 895-961.
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Marks S. C. Jr and Cahill D. R. (1984) Experimental study in the dog of the non-active role of the tooth in the eruptive process, Archs oral Biol. 29, 3 I l-322. Marks S. C. Jr, Cahill D. R. and Wise G. E. (1983) The cytology of the dental follicle and adjacent alveolar bone during tooth eruption in the dog. Am. J. Anat. 168, 277-289.
Wahl S. M., Hunt D. A., Wakefield L. M., McCartneyFrancis N., Wahl L. M.. Roberts A. B. and Spom M. B. (1987) Transforming growth factor-beta (TTGF-beta) induces monocyte chemotaxis and growth factor production. Proc. natn. Acad. Sci., U.S.A. &J, 5788-5792. Wise G. E. and Fan W. (1989) Changes in the tartrateresistant acid phosphatase cell population in dental follicles and bony crypts of rat molars during tooth eruption. J. denr. Res. 68, 150-156. Wise G. E. and Fan W. (1991) lmmunolocalization of transforming growth factor beta in rat molars. J. oral Path. Med. 20, 74-80.
Wise G. E., Marks S. C. Jr and Cahill D. R. (1985) Ultrastructural features of the dental follicle associated with formation of the tooth eruption pathway in the dog. 1. oral Path. 14, 15-26. Wise G. E., Marks S. C. Jr, Cahill D. R. and Gorski J. P. (1988) Ultrastructural features of the dental follicle and enamel organ prior to and during tooth eruption. In The Biological Mechanisms of Tooth Eruption and Rool Resorption (Ed. Davidovitch 2.). pp. 243-249. EBSCO
Media, Birmingham, AL. Wise G. E., Lin F. and Fan W. (1992) Culture and characterization of dental follicle cells from rat molars. Cell Tiss. Res. 267, 483-492. Wiseman D. M., Polverine P. J., Kamp D. W. and Liebovich S. J. (1988) Transforming growth factor-beta (TGF-beta) is chemotactic for human monocytes and induces their expression of angiogenie activity. Biochem. biophys. Res. Commun. 157, 793-800.
