ooo3-9969 9’ 55.00+ 0.00
Archs oral Biol. Vol. 37. No. 3, pp. 231-236. 1992
Copyright c 1992 Pergamon Press pIc
Printed in Great Britain. All rights reserved
THE EFFECTS OF GROWTH FACTORS ON DNA SYNTHESIS, PROTEOGLYCAN SYNTHESIS AND ALKALINE PHOSPHATASE ACTIVITY IN BOVINE DENTAL PULP CELLS M. NAKASHIMA Department of Conservative Dentistry, Faculty of Dentistry. Kyushu University 61, Fukuoka, 812, Japan (Accepted
22
August
1991)
Summary-Platelet-derived growth factor (PDGF), insulin-like growth factor-l and -II (IGF-I and -II), acidic fibroblast growth factor (aFGF). basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated [“‘I]-deoxyuridine incorporation about 13-, 6.2-, 4.6-, 3.8, 3.l- and l.2-fold. respectively, above control values at a concentration of 50 ngiml. Transforming growth factor-p (TGF-8) decreased incorporation about 30% at the same dose. aFGF, IGF-I, IGF-II, bFGF and TGF-8 increased [“S]-sulphate incorporation 23 I, 7 I, 64,42 and 39%, respectively, in proliferating cells, while EGF, IGF-I. TGF-/I and PDGF decreased incorporation about 30%, and aFGF increased incorporation 80% in stationary-stage culture. TGF-/I, PDGF, aFGF and bFGF caused 6540% inhibition of alkaline phosphatase activity in proliferating and stationary cultures. These findings suggest that the proliferation of pulp cells may be stimulated mainly by PDGF and IGF-I. and the production of extracellular matrix proteoglycan may be enhanced by aFGF, IGF-I and IGF-II. Furthermore, TGF-/?, PDGF, aFGF and bFGF may regulate the differentiation of pulp cells into odontoblasts. Key words: growth factors, dental phosphatase
pulp, cell culture,
DNA
INTRODUCTION
Pulp repair and dental
synthesis,
proteoglycan
synthesis,
alkaline
activity.
after abrasion,
erosion,
caries,
1979; Childs et al., 1982; Assoian et al., 1983, 1984; Oka and Orth, 1983). bFGF has been detected in macrophages (Baird, Mormtde and Bohlen, 1985). TGF-fi, IGF-I, IGF-II and bone morphogenetic protein have been found in dentine matrix (Butler, Mikufski and Urist, 1977; Conover and Urist, 1982; Mera, 1988; Katz and Reddi, 1988; Kawai and Urist, 1989; Finkelman et al., 1990; Bessho et ol., 1991). It is possible that when pulp tissues are injured, some of these factors are released from platelets, damaged cells and tissues, and dentine matrix, thus stimulating the proliferation of pulp cells, the production of extracellular matrices and the differentiation of odontoblasts. The effects of these growth factors on pulp cells during repair in adult pulps have not been studied. An in vitro study of a rat pulp cell line RPC-C2A showed that EGF stimulates DNA synthesis and proliferation of the pulp cell, but TGF-8 had no effect (Liang et al., 1990). I have now examined the effects of PDGF, TGF-8, EGF, aFGF, bFGF, IGF-I and IGF-II on DNA and proteoglycan synthesis, and-alkaline phosphatase activity (a marker for mature dental pulp cells) in primary cell cultures of dental pulp.
fracture
procedures is thought to involve a sequence of cellular events similar to wound healing in soft tissue. These events include chemotaxis, proliferation of cells of mesenchymal origin at the site of injury and production of extracellular matrices. Calcified reparative dentine, the ultimate evidence of pulp repair, is analogous to scar tissue in other connective tissues (Stanley, 1984). Growth factors such as PDGF, TGF-8, EGF and aFGF and bFGF are thought to play important roles in the repair of various tissues (Buckley et al., 1985; Grotendorst et al., 198.5; Sporn and Roberts, 1986; Calvin and Antoniades, 1987; Lynch et al., 1987; Mustoe et al., 1987; Schultz et al., 1987; Sprugel et al., 1987; Rifkin and Moscatelli, 1989; Gospodarowicz, 1990; Habenicht et al., 1990; Lyons and Moses, 1990; Marie, Hott and Perheentupa, 1990; Ross, BowenPore and Raines, 1990). Platelets sequester PDGF, TGF-fi and EGF-like protein and release them upon degranulation after injury (Antoniades, Scher and Stiles, 1979; Heldin, Westermark and Wasteson, Abbreoiutions:
BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s minimal essential medium; EGF, epidermal growth factor; aFGF, bFGF, acidic and basic fibroblast growth factor; HBSS, Hank’s balanced salt solution; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; PNP, p-nitrophenol; TGF, transforming growth factor.
>lATERIALS
Cell
AND METHODS
culture
Primary cell cultures were obtained by my method (Nakashima, 1991). In brief, the mandibular permanent first incisors were extracted from about 231
232
M. NAKASHIWA
3-yr-old cows and cracked open. The pulp was pulled out and minced into pieces. The tissue was washed with calcium-free HBSS, pH 7.0, and incubated at 37’C in 0.2% trypsin (Difco Laboratories, Detroit, Ml, U.S.A., 1:2SO)-0.02% EDTA in calcium-free HBSS for 30 min. The solution was replaced with 0.2% collagenase (Wako Chemical Co., Japan) in HBSS containing 5 mM calcium. At 30 min intervals, dissociated cells were transferred to DMEM (Gibco Laboratories, Grand Island, NY, U.S.A.) containing 10% calf serum (Gibco Laboratories, Grand Island, NY, U.S.A.), penicillin and streptomycin (200 pg/ml each). Residual tissue was incubated in fresh collagenase solution. This process was repeated three times. The cell suspensions isolated in the first 30 min, second 30min and third 30 min incubations were collected together and inoculated in 24 multiwell dishes at a density of 5 x lo4 cells per ml. Cultures reached confluence on day 14 and were multilayered on day 28. Growth factors
Human PDGF (mol. wt. 30,000; gift from Dr T. Nakamura, Department of Biology, Faculty of Science, Kyushu University), recombinant human TGF-j (mol. wt. 25,000; Nakarahi Tesque, Japan), human EGF (mol. wt. 6100; Aase them Corp., Japan), bovine aFGF (mol. wt. 13,400; Toyobo Corp., Japan), bovine bFGF (mol. wt. 13,400; Toyobo Corp., Japan), recombinant human IGF-I (mol. wt. 7700; Collaborative Res. lncorp., U.S.A.) and recombinant human IGF-II (mol. wt. 8700; Genzyme Corp., U.S.A.) were used as mitogenic agents. DNA synthesis
On day 6 of incubation during the proliferating stage, the culture medium was changed to DMEM containing 0.2% BSA 24 h before stimulation. The growth factors at various concentrations were added to the fresh medium supplemented with 0.2% BSA or 10% calf serum, and cultivation was continued for 24 h. Cultures were labelled with [‘*‘I]-iodo2-deoxyuridine (2200 Ci/m mol, I mCi/2ml, New England Nuclear Corp., Boston, MA, U.S.A.) containing unlabelled deoxyuridine (1.6 pmol/mCi [‘*‘I]deoxyuridine) in a final concentration of 0.5 pCi/ml during the last 12 h of the cultivation. The media were washed out with PBS. The cells were fixed with 10% trichloroacetic acid at 4°C for 15 min and dissolved in I M NaOH at 37°C for 2 h. The radioactivity of the solution was measured by a gamma counter. Proteoglycan
synthesis
Proteoglycan synthesis was measured according to the methods of Suzuki, Yoneda and Shimomura (1976) and Saami and Tarnmi (1977). On day 6 during the proliferating stage and day 14 during the stationary stage of cultivation, the cultures were incubated for 24 h in DMEM with 0.2% BSA. Growth factors were added at a final concentration of 5 ng/ml. The cell layer was washed three times with serum-free, factor-free DMEM, and incubations were continued for an additional 20 h in media supplemented with 5 FCi of [“S]-sulphatelml (New England Nuclear Corp., Boston, MA, U.S.A.). After
labelling, the medium was removed and chilled. The cells and insoluble extra-cellular materials were solubilized in 0.5 M NaOH and then neutralized with 10 M HCl. Part of the neutralized materials was used for protein determination (Lowry et al., 1951). The remainder and the cold culture medium were digested with 1 mg/ml actinase (Kaken Kagaku Corp., Japan) in 0.05 M tris-HCl buffer, pH 7.8, containing 5 mM CaCl, for 12 h at 55’C. Then 50 ~1 of water containing 5pg of chondroitin sulphate (Wake Chemical Co., Japan) and 500 111of 2 mM MgSO, were added. Glycosaminoglycans were precipitated for 1 h with 500 ~1 of 1% cetylpyridinium chloride in 0.02 M NaCl. The precipitate was collected onto a filtration membrane (HA-Millipore, 0.45 itrn pore size, Millipore Corp., Bedford. MA, U.S.A.) held in a membrane holder (Millipore Corp., Bedford, MA, U.S.A.). The non-precipitable glycoproteins were washed through with 1% cetylpyridinium chloride in 0.02 M NaCl. The radioactivity was counted in 10 ml of scintillation cocktail (ACS Il. Amersham, Arlington Heights, IL, U.S.A.). The data were expressed as counts/min/pg protein. Alkaline phosphatase
assay
On day 7 during the proliferating stage, day 14 during the stationary stage and day 28 during multilayered stage, the cultures were stimulated by growth factors at a final concentration of 5 ngiml in DMEM in the presence and absence of 10% calf serum for 48 h. Cell layers were extracted in IO mM tris-HCl buffer, pH 7.4, containing 0.1% Triton X-100 and then sonicated. Enzyme activity was assayed (Lowry et al., 1954) using PNP phosphate (Sigma Chemical Co., St Louis, MO, U.S.A.) as the substrate incubating at 37°C for 30 min. The reaction was stopped by the addition of 1 ml of 0.05 N NaOH and the absorbances were measured at 410 nm. The data were expressed as pmol PNP/h/mg protein. Protein concentrations were determined (Lowry et al., 1951). RESULTS
DNA synthesis
PDGF and IGF-I stimulated [“‘I]-deoxyuridine incorporation at a concentration above 1 ng/ml, and IGF-II above 5ng/ml. Acidic FGF and bFGF showed a similar dose dependent curve, and [‘2Sl]deoxyuridine incorporation gradually increased at a concentration of 0.1-50 ng/ml. EGF had a little stimulatory effect on DNA synthesis at a concentration above 0.5 ng/ml. TGF-/l inhibited DNA synthesis above 5 ng/ml (Fig. 1). At a concentration of 50 ng/ml, PDGF, IGF-I, IGF-II, aFGF, bFGF and EGF stimulated [“‘I]-deoxyuridine incorporation about 13., 6.2-, 4.6-, 3.8-, 3.1- and 1.2.fold, respectively, above control value with a remarkable statistical significance (p < 0.001, except EGF, p < 0.01). Only TGF-fi decreased (‘2sl]-deoxyuridine incorporation about 30% (p < 0.001). Ten per cent calf serum stimulated about a 2300% increase in [‘*‘I]-deoxyuridine incorporation as compared with 0.2% BSA because it provided an enriched supplement of growth factors and nutrients. It was chosen, therefore, to examine the inhibitory effect of growth factors. In 10% calf
Growth factors on pulp cell
233
Table 1. Effects of the seven types of growth factor and 10% calf serum on [‘?+sulphate incorporation in the proliferating and stationary stage
Control aFGF bFGF PDGF TGF-fl IGFI IGFII EGF lO%CS
Proliferating stage
Stationary stage
2830 k 312 9356 k 797’ 4006 + 340* 3015 + 532 3925 L-431’ 4831 f 322. 4631 + 698. 2593 & 453 1824 * 107’
1687 + 197 3035 f 225* 1567 + 22 1221 * 790 1166+ 163’ 1127 + 154* 1624+78 1081 & 1IO’ 1229 f 90*
Results expressed in terms of counts/min/pg protein, mean *SD of six determinations from one of three experiments. *Significantly different from control value (p < 0.001).
0,
0
0.01 0.1
0.5
I
5
nglml Fig. I. Dose-response of primary pulp cells stimulated by aFGF (O-O), bFGF (0.. O), PDGF (A.-A), TGF-j (A---A), IGF-I (I--m), IGF-II (Cl--0) and EGF (+ --- +) in 0.2% BSA-supplemented culture. Symbols represent counts per min per well of [12JI]-deoxyuridine incorporation in DNA, mean f SD of four wells from one of three experiments. *p c 0.01, l p c 0.001 compared with control. serum-supplemented culture, TGF-j3 showed an inhibition of DNA synthesis at a concentration above 5 n&ml in two of three separate assays. Other factors had no effect (Fig. 2). Proteoglycan
synthesis
stage of culture, aFGF, IGF-I, IGF-II, bFGF, and TGF-/I increased [“S]sulphate incorporation 231, 71, 64, 42 and 39%, respectively, as compared with control (p < 0.001). Ten per cent calf serum significantly decreased the During
the proliferating
incorporation 37% (p < 0.001). PDGF and EGF had no significant effect (Table 1). In stationary-stage cultures, EGF, IGF-I, TGF-/?, 10% calf serum and PDGF decreased [“S]-sulphate incorporation 36, 33, 31, 28 and 27%. respectively (p c 0.001). Only aFGF significantly increased [“S]sulphate incorporation 80% (p < 0.001) (Table 1). Alkaline phosphatase
PDGF, aFGF and bFGF caused 6540% inhibition of alkaline phosphatase activity as compared with controls in both serum-free and 10% calf serumsupplemented culture of proliferating, stationery and multilayered stages except in serum-free culture of stationary stage (p < 0.001 and 0.01). TGF-fl significantly decreased alkaline phosphatase activity by 65 and 40%, respectively, in serum-free cultures of proliferating and stationary stages (p < 0.001 and 0.01). The other growth factors had no significant effect (Table 2). Table 2. Effects of the seven types of growth factor on alkaline phosphatase activity in the absence and the presence of 10% calf serum in proliferating, stationary and multilayered stages Proliferating stage Control aFGF bFGF PDGF TGF-8 IGF-I IGF-II EGF
nglml Fig. 2. Doscresponse of primary pulp cells stimulated aFGF (O-O), bFGF (0. * .O), PDGF (A.-A), TGF-B (A---A), IGF-I (W-B), IGF-II (O--o), and EGF (+ --- +) in 10% calf serum-supplemented culture. Symbols represent counts per min per well of [‘“Ij-deoxyuridine incorporation in DNA, mean f SD of four wells from one of three experiments. l p < 0.001 compared with control.
actioity
Control aFGF bFGF PDGF TGF-fl IGF-I IGF-II EGF
11.31 2 5.71 5 4.16 2 6.79 i 4.09 + 11.622 10.61 2 9.19 i
Stationary stage
Absence 2.31 3.70 & 0.52 0.97* 3.32 + 0.44 0.39t 3.28 + 0.48 0.62* 2.76 + 0.29 0.23t 2.57 rf:0.24’ 1.62 3.10 f 0.39 1.74 3.09 f 0.24 0.75 3.47 + 0.53
Multi-layered stage 5.97 k 1.32 3.01 + 0.59* 3.01 & 0.16* 2.37 + 0.21. 5.65 k 1.30 5.45 f 0.60 4.51 & 0.68 3.99 & 0.35
Presence 3.02 k 0.31 1.23& 0.23 1.86 5 0.22t 0.68 5 0.09’ 1.96 & 0.27* 0.54 + 0.13* 1.52 i 0.31t 0.51 * 0.10’ 3.05 k 0.39 0.78 k 0.22 2.42 & 0.14 1.34 kO.18 2.11 kO.40 1.18 kO.15 2.66 k 0.52 0.93 * 0.14
Results expressed in terms of pmol/h. mg protein, mean k SD of four determinations from one of two experiments. *Significantly different from control value (p < 0.01). tSignificantly different from control value (p < O.OOI).
bl.
234
NAKASHIMA
DISCLSSION
I had earlier characterized the primary pulp cells used here (Nakashima, 1991). The trypsin pretreatment/collagenase separation was chosen because a large enough number of primary cells can be obtained from one isolation, so enabling multiple analyses, Primary cell culture is considered to retain more of the phenotypic characteristics of the original pulp cells than with subcultured and cloned cells. The pulp cell likeness of these ceils has been shown by the presence of high alkaline phosphatase activity and parathyroid hormone-stimulated, CAMP production (Nakashima, 1991). In long-term culture, nodules are seen in which initial calcification occurs around preodontoblast-like cells, suggesting that the isolated pulp cell may be able to differentiate into an odontoblast in uifro. Therefore, one may speculate about the reaction of pulp cells to growth factors in wound healing by extrapolation from the effects of these factors on DNA synthesis, proteoglycan synthesis and alkaline phosphatase activity in the primary pulp cell culture. EGF stimulates in citro proliferation of dental mesenchymal cells isolated from tooth germs (Steidler and Reade, 1981; Thesleff, Ekblom and Keski-Oja, 1983; Partanen, Ekblom and Thesleff, 1985; Partanen and Thesleff, 1989), while FGF and PDGF have no effect (Partanen et al., 1985). In cloned pulp cells, EGF stimulated DNA synthesis; whereas TGF-8 did not (Liang et al., 1990). PDGF (Ross, Raines and Bowen-Pore, 1986), aFGF (Uhlrich et al., 1986), bFGF (Delli-Bovi et al., 1987, 1988), IGFs (Rechler ef al., 1974; Haselbacher and Humbel, 1976; Phillips and Vassilopoulou-Sellin, 1980a, b) and EGF (Canalis and Raisz, 1979) are known to stimulate proliferation of fibroblasts. My pulp cells were isolated from permanent bovine incisors whose root development was complete. After 6 days of culture the cells were predominantly stellate-like fibroblasts with a few short processes. PDGF, IGF-I, IGF-II, aFGF, bFGF and EGF were all potent mitogens for these pulp cells, showing the similarity between cultured pulp cells and fibroblasts. The difference in the effect of FGF and PDGF may be due to the developmental stage of the pulp, the culture system, or both. TGF-fl can act either as a growth inhibitory or a mitogenic factor, depending on cell source, cell population, cell density, presence or absence of serum, culture conditions and whether other growth factors are present (Bonewald and Mundy, 1990). TGF-j? inhibited DNA synthesis in cultured pulp cells in the absence of serum. Discrepancies were observed in cultures supplemented with 10% calf serum where TGF-fl inhibited DNA synthesis in two of three separate assays. TGF-/? and IGFs increase the expression of sulphate proteoglycan present in the extracellular matrix and culture medium of various cell types including fibroblasts (Phillips and Vassilopoulou-Sellin, 1980a, b; Chen, Hoshi and Mckeehan, 1987; Bassols and Massagtie, 1988). The effect of FGF on sulphate proteoglycan synthesis by chondrocytes varies depending upon the stage of differentiation of cells and culture conditions (Sachs et al., 1982; Kato and Gospodarowicz, 1985; Hamerman, Sasse and
Klagsbrun, 1986). Water-soluble dentine extracts increased proteoglycan synthesis in embryonic chick limb mesenchymal cells and stimulated chondrogenesis as detected by metachromatic staining with toluidine blue (Rabinowitz, Syftestad and Caplan, 1990). In my work, aFGF, IGF-I, IGF-II, bFGF and TGF-P stimulated proteoglycan synthesis during the proliferating stage, while EGF, IGF-I, TGF-/? and PDGF inhibited proteoglycan synthesis and only aFGF stimulated the synthesis during the stationary stage. Thus, the effects were dependent on the degree of differentiation of the cells. The acquisition of alkaline phosphatase activity may be a prerequisite for the differentiation and possible specialization of pulp ceils in cico (Miller, Everett and Cramer, 1976) because alkaline phosphatase activity in the subodontoblastic layer is higher than in the odontoblastic layer and highest in the pulp (Nuki and Bonting. 1961; Goggins and Fullmer, 1967; Yoshiki and Kurahashi, 1971). Alkaline phosphatase as a pulp-capping agent stimulates the pulp tissue to form dentine matrix (Seltzer, 1959; Seltzer, Bender and Kaufman, 1961). Therefore, I used alkaline phosphatase activity as a marker to indicate the effect of growth factors on the differentiation of pulp cells; changes in alkaline phosphatase activity may reflect their state of maturation. Inhibition of alkaline phosphatase activity by aFGF has been reported in calvarial cell cultures and organ cultures (Rodan et al., 1987; Shen et al., 1989; Nicolas et al., 1990) and by EGF (Nicolas et al., 1990). In an osteosarcoma cell line, bFGF similarly inhibits alkaline phosphatase activity (Miller and Puzas, 1988). There are discrepancies in the reported effects of TGF-fi on osteoblastic cell lines (Noda and Rodan, 1986; Pfeilschifter, D’Souza and Mundy, 1987). It has been suggested that changes in alkaline phosphatase activity may reflect the maturation state of the osteoblast (Bonewald and Mundy, 1990). TGF-P and EGF also show suppressive effects in cloned pulp cells (Liang et al., 1990). In my study, TGF-fi, bFGF, aFGF and PDGF inhibited alkaline phosphatase activity. These growth factors may control the expression of alkaline phosphatase in vitro by preventing differentiation during periods of active cell proliferation and regulating the expression of mature cell phenotype. These results suggest that in pulp wound healing, proliferation of mesenchymal pulp cells may be stimulated mainly by PDGF and IGF-I, and the production of extracellular matrix proteoglycan may be enhanced by aFGF, IGF-I and IGF-II. Furthermore, TGF-8, PDGF, aFGF and bFGF may be possible regulators of the differentiation of pulp cells into odontoblasts. Acknowledgements-1
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Noda M. and Rodan G. A. (1986) Type /I transforming growth factor inhibits proliferation and expression of alkaline phosphatase in murine osteoblast-like cells. Biochem. biophys. Res. Commun. 140, 56-65. Nuki K. and Bonting S. L. (1961) Quantitative histochemistry of the developing hamster tooth: alkaline phosphatase and lactic dehydrogenase. J. Histochem. Cytochem. 9, 117-125.
Oka Y. and Orth D. N. (1983) Human plasma epidermal growth factor//?-urogastrone is associated with blood platelets. J. clin. Invest. 72, 249-259. Partanen A.-M. and Thesleff I. (1989) Growth factors and tooth development. Devl Biol. 33, 165-172. Partanen A.-M., Ekblom P. and Thesleff I. (1985) Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth. Deal Biol. 111, 84-94. Pfeilschifter J., D’Souza S. M. and Mundy G. R. (1987) Effects of transforming growth factor-beta on osteoblastic osteosarcoma cells. Endocrinology 121, 212-218. Phillips L. S. and Vassilopoulou-Sellin R. (1980a) Somatomedins. New Engl. J. Med. 302, 371-380. Phillips L. S. and Vassilopoulou-Sellin R. (1980b) Somatomedins. New Engl. J. Med. 302, 438-446. Rabinowitz T., Syftestad G. T. and Caplan A. I. (1990) Chondrogenic stimulation of embryonic chick limb mesenchymal cells by factors in bovine and human dentine extracts. Archs oral Biol. 35, 49-54. Rechler M. M., Podskalny J. M.. Goldfine I. D. and Wells C. A. (1974) DNA synthesis in human fibroblasts: stimulation by insulin and by nonsuppressible insulin-like activity (NSILA). J. clin. Endocrinol. Merab. 39, 512-521. Rifkin D. B. and Moscatelli D. (1989) Recent developments in the cell biology of basic fibroblast growth factor. J. Cell Bio/. 109, l-6. Rodan S. B., Wesolowski G., Thomas K. and Rodan G. A. (1987) Growth stimulation of rat calvaria osteoblastic cells by acidic fibroblast growth factor. Endocrinology 121, 1917-1923. Ross R., Raines E. W. and Bowen-Pore D. F. (1986) The biology of platelet-derived growth factor. Cell 46, 155-169. Ross R., Bowen-Pore D. F. and Raines E. W. (1990) Platelet derived growth factor and its role in health and disease. Phil. Trans. R. Sot. Lond. 3274 155-169. Saami H. and Tammi M. (1977) A rapid method for separation and assay of radiolabeled mucopolysaccharides from cell culture medium. Analyr. Biochem. 81. 40-46.
Sachs B. L., Goldberg V. M., Moskowitz R. W. and Malemud C. J. (1982) Response of articular chondrocytes to pituitary fibroblast growth factor (FGF). J. cell. Physiol. 112, 51-59.
Schultz G. S., White M., Mitchell R., Brown G., Lynch J., Twardzik D. R. and Todaro G. J. (1987) Epithelial wound healing enhanced by transforming growth factor-a and Vaccinia growth factor. Science 235. 350-352. Seltzer S. (1959)-Reparative dentinogenesis: Oral Surg. 12, 595-602. Seltzer S., Bender I. B. and Kaufman I. J. (1961) Root canal dressings: their usefulness in endodontic therapy reconsidered. Oral Surg. 14, 603-610. Shen V., Kohler G., Huang J., Huang S. S. and Peck W. A. (1989) An acidic fibroblast growth factor stimulates DNA synthesis, inhibits collagen and alkaline phosphatase synthesis and induces resorption in bone. Bone Miner. 7, 205-219.
Spom M. B. and Roberts A. B. (1986) Peptide growth factors and inflammation, tissue repair, and cancer. J. clin. Incest. 78, 329-332.
Sprugel K. H., McPherson J. M., Clowes A. W. and Ross R. (1987) Effects of growth factors in vioo-I. Cell ingrowth into porous subcutaneous chambers. Am. J. Path. 129, 601-613.
Stanley H. R. (1984) Pulpal responses. In Pathway of the Pulp (Eds Cohen S. and Bums R. C.), 3rd edn, Chap. 14, pp. 465-489. C. V. Mosby Co., Toronto. Steidler N. E. and Reade P. C. (1981) Epidermal growth factor and proliferation of odontogenic cells in culture. J. dent. Res. 60, 1977-1982.
Suzuki F., Yoneda T. and Shimomura Y. (1976) Calcitonin and parathyroid-hormone stimulation of acid mucopolysaccharide synthesis in cultured chondrocytes isolated from growth cartilage. FEBS Len. 70, 155-158. Thesleff I., Ekblom P. and Keski-Oja J. (1983) Inhibition of morphogenesis and stimulation of vascular proliferation in embryonic tooth cultures by a sarcoma growth factor preparation. Cancer Res. 43, 5902-5909. Uhlrich S., Lagente O., Choay J., Courtois Y. and Lenfant M. (1986) Structure activity relationship in heparin: stimulation of non-vascular cell by a synthetic heparin pentasaccharide in cooperation with human acidic fibroblast growth factors. Biochem. biophys. Res Commun. 139, 728-732.
Yoshiki S. and Kurahashi Y. (1971) A light and electron microscopic study of alkaline phosphatase activity in the early stage of dentinogenesis in the young rat. Archs oral Biol. 16, 1143-l 154.
Archs oral Biol. Vol. 37. No. 3, pp. 231-236. 1992
Copyright c 1992 Pergamon Press pIc
Printed in Great Britain. All rights reserved
THE EFFECTS OF GROWTH FACTORS ON DNA SYNTHESIS, PROTEOGLYCAN SYNTHESIS AND ALKALINE PHOSPHATASE ACTIVITY IN BOVINE DENTAL PULP CELLS M. NAKASHIMA Department of Conservative Dentistry, Faculty of Dentistry. Kyushu University 61, Fukuoka, 812, Japan (Accepted
22
August
1991)
Summary-Platelet-derived growth factor (PDGF), insulin-like growth factor-l and -II (IGF-I and -II), acidic fibroblast growth factor (aFGF). basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated [“‘I]-deoxyuridine incorporation about 13-, 6.2-, 4.6-, 3.8, 3.l- and l.2-fold. respectively, above control values at a concentration of 50 ngiml. Transforming growth factor-p (TGF-8) decreased incorporation about 30% at the same dose. aFGF, IGF-I, IGF-II, bFGF and TGF-8 increased [“S]-sulphate incorporation 23 I, 7 I, 64,42 and 39%, respectively, in proliferating cells, while EGF, IGF-I. TGF-/I and PDGF decreased incorporation about 30%, and aFGF increased incorporation 80% in stationary-stage culture. TGF-/I, PDGF, aFGF and bFGF caused 6540% inhibition of alkaline phosphatase activity in proliferating and stationary cultures. These findings suggest that the proliferation of pulp cells may be stimulated mainly by PDGF and IGF-I. and the production of extracellular matrix proteoglycan may be enhanced by aFGF, IGF-I and IGF-II. Furthermore, TGF-/?, PDGF, aFGF and bFGF may regulate the differentiation of pulp cells into odontoblasts. Key words: growth factors, dental phosphatase
pulp, cell culture,
DNA
INTRODUCTION
Pulp repair and dental
synthesis,
proteoglycan
synthesis,
alkaline
activity.
after abrasion,
erosion,
caries,
1979; Childs et al., 1982; Assoian et al., 1983, 1984; Oka and Orth, 1983). bFGF has been detected in macrophages (Baird, Mormtde and Bohlen, 1985). TGF-fi, IGF-I, IGF-II and bone morphogenetic protein have been found in dentine matrix (Butler, Mikufski and Urist, 1977; Conover and Urist, 1982; Mera, 1988; Katz and Reddi, 1988; Kawai and Urist, 1989; Finkelman et al., 1990; Bessho et ol., 1991). It is possible that when pulp tissues are injured, some of these factors are released from platelets, damaged cells and tissues, and dentine matrix, thus stimulating the proliferation of pulp cells, the production of extracellular matrices and the differentiation of odontoblasts. The effects of these growth factors on pulp cells during repair in adult pulps have not been studied. An in vitro study of a rat pulp cell line RPC-C2A showed that EGF stimulates DNA synthesis and proliferation of the pulp cell, but TGF-8 had no effect (Liang et al., 1990). I have now examined the effects of PDGF, TGF-8, EGF, aFGF, bFGF, IGF-I and IGF-II on DNA and proteoglycan synthesis, and-alkaline phosphatase activity (a marker for mature dental pulp cells) in primary cell cultures of dental pulp.
fracture
procedures is thought to involve a sequence of cellular events similar to wound healing in soft tissue. These events include chemotaxis, proliferation of cells of mesenchymal origin at the site of injury and production of extracellular matrices. Calcified reparative dentine, the ultimate evidence of pulp repair, is analogous to scar tissue in other connective tissues (Stanley, 1984). Growth factors such as PDGF, TGF-8, EGF and aFGF and bFGF are thought to play important roles in the repair of various tissues (Buckley et al., 1985; Grotendorst et al., 198.5; Sporn and Roberts, 1986; Calvin and Antoniades, 1987; Lynch et al., 1987; Mustoe et al., 1987; Schultz et al., 1987; Sprugel et al., 1987; Rifkin and Moscatelli, 1989; Gospodarowicz, 1990; Habenicht et al., 1990; Lyons and Moses, 1990; Marie, Hott and Perheentupa, 1990; Ross, BowenPore and Raines, 1990). Platelets sequester PDGF, TGF-fi and EGF-like protein and release them upon degranulation after injury (Antoniades, Scher and Stiles, 1979; Heldin, Westermark and Wasteson, Abbreoiutions:
BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s minimal essential medium; EGF, epidermal growth factor; aFGF, bFGF, acidic and basic fibroblast growth factor; HBSS, Hank’s balanced salt solution; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; PNP, p-nitrophenol; TGF, transforming growth factor.
>lATERIALS
Cell
AND METHODS
culture
Primary cell cultures were obtained by my method (Nakashima, 1991). In brief, the mandibular permanent first incisors were extracted from about 231
232
M. NAKASHIWA
3-yr-old cows and cracked open. The pulp was pulled out and minced into pieces. The tissue was washed with calcium-free HBSS, pH 7.0, and incubated at 37’C in 0.2% trypsin (Difco Laboratories, Detroit, Ml, U.S.A., 1:2SO)-0.02% EDTA in calcium-free HBSS for 30 min. The solution was replaced with 0.2% collagenase (Wako Chemical Co., Japan) in HBSS containing 5 mM calcium. At 30 min intervals, dissociated cells were transferred to DMEM (Gibco Laboratories, Grand Island, NY, U.S.A.) containing 10% calf serum (Gibco Laboratories, Grand Island, NY, U.S.A.), penicillin and streptomycin (200 pg/ml each). Residual tissue was incubated in fresh collagenase solution. This process was repeated three times. The cell suspensions isolated in the first 30 min, second 30min and third 30 min incubations were collected together and inoculated in 24 multiwell dishes at a density of 5 x lo4 cells per ml. Cultures reached confluence on day 14 and were multilayered on day 28. Growth factors
Human PDGF (mol. wt. 30,000; gift from Dr T. Nakamura, Department of Biology, Faculty of Science, Kyushu University), recombinant human TGF-j (mol. wt. 25,000; Nakarahi Tesque, Japan), human EGF (mol. wt. 6100; Aase them Corp., Japan), bovine aFGF (mol. wt. 13,400; Toyobo Corp., Japan), bovine bFGF (mol. wt. 13,400; Toyobo Corp., Japan), recombinant human IGF-I (mol. wt. 7700; Collaborative Res. lncorp., U.S.A.) and recombinant human IGF-II (mol. wt. 8700; Genzyme Corp., U.S.A.) were used as mitogenic agents. DNA synthesis
On day 6 of incubation during the proliferating stage, the culture medium was changed to DMEM containing 0.2% BSA 24 h before stimulation. The growth factors at various concentrations were added to the fresh medium supplemented with 0.2% BSA or 10% calf serum, and cultivation was continued for 24 h. Cultures were labelled with [‘*‘I]-iodo2-deoxyuridine (2200 Ci/m mol, I mCi/2ml, New England Nuclear Corp., Boston, MA, U.S.A.) containing unlabelled deoxyuridine (1.6 pmol/mCi [‘*‘I]deoxyuridine) in a final concentration of 0.5 pCi/ml during the last 12 h of the cultivation. The media were washed out with PBS. The cells were fixed with 10% trichloroacetic acid at 4°C for 15 min and dissolved in I M NaOH at 37°C for 2 h. The radioactivity of the solution was measured by a gamma counter. Proteoglycan
synthesis
Proteoglycan synthesis was measured according to the methods of Suzuki, Yoneda and Shimomura (1976) and Saami and Tarnmi (1977). On day 6 during the proliferating stage and day 14 during the stationary stage of cultivation, the cultures were incubated for 24 h in DMEM with 0.2% BSA. Growth factors were added at a final concentration of 5 ng/ml. The cell layer was washed three times with serum-free, factor-free DMEM, and incubations were continued for an additional 20 h in media supplemented with 5 FCi of [“S]-sulphatelml (New England Nuclear Corp., Boston, MA, U.S.A.). After
labelling, the medium was removed and chilled. The cells and insoluble extra-cellular materials were solubilized in 0.5 M NaOH and then neutralized with 10 M HCl. Part of the neutralized materials was used for protein determination (Lowry et al., 1951). The remainder and the cold culture medium were digested with 1 mg/ml actinase (Kaken Kagaku Corp., Japan) in 0.05 M tris-HCl buffer, pH 7.8, containing 5 mM CaCl, for 12 h at 55’C. Then 50 ~1 of water containing 5pg of chondroitin sulphate (Wake Chemical Co., Japan) and 500 111of 2 mM MgSO, were added. Glycosaminoglycans were precipitated for 1 h with 500 ~1 of 1% cetylpyridinium chloride in 0.02 M NaCl. The precipitate was collected onto a filtration membrane (HA-Millipore, 0.45 itrn pore size, Millipore Corp., Bedford. MA, U.S.A.) held in a membrane holder (Millipore Corp., Bedford, MA, U.S.A.). The non-precipitable glycoproteins were washed through with 1% cetylpyridinium chloride in 0.02 M NaCl. The radioactivity was counted in 10 ml of scintillation cocktail (ACS Il. Amersham, Arlington Heights, IL, U.S.A.). The data were expressed as counts/min/pg protein. Alkaline phosphatase
assay
On day 7 during the proliferating stage, day 14 during the stationary stage and day 28 during multilayered stage, the cultures were stimulated by growth factors at a final concentration of 5 ngiml in DMEM in the presence and absence of 10% calf serum for 48 h. Cell layers were extracted in IO mM tris-HCl buffer, pH 7.4, containing 0.1% Triton X-100 and then sonicated. Enzyme activity was assayed (Lowry et al., 1954) using PNP phosphate (Sigma Chemical Co., St Louis, MO, U.S.A.) as the substrate incubating at 37°C for 30 min. The reaction was stopped by the addition of 1 ml of 0.05 N NaOH and the absorbances were measured at 410 nm. The data were expressed as pmol PNP/h/mg protein. Protein concentrations were determined (Lowry et al., 1951). RESULTS
DNA synthesis
PDGF and IGF-I stimulated [“‘I]-deoxyuridine incorporation at a concentration above 1 ng/ml, and IGF-II above 5ng/ml. Acidic FGF and bFGF showed a similar dose dependent curve, and [‘2Sl]deoxyuridine incorporation gradually increased at a concentration of 0.1-50 ng/ml. EGF had a little stimulatory effect on DNA synthesis at a concentration above 0.5 ng/ml. TGF-/l inhibited DNA synthesis above 5 ng/ml (Fig. 1). At a concentration of 50 ng/ml, PDGF, IGF-I, IGF-II, aFGF, bFGF and EGF stimulated [“‘I]-deoxyuridine incorporation about 13., 6.2-, 4.6-, 3.8-, 3.1- and 1.2.fold, respectively, above control value with a remarkable statistical significance (p < 0.001, except EGF, p < 0.01). Only TGF-fi decreased (‘2sl]-deoxyuridine incorporation about 30% (p < 0.001). Ten per cent calf serum stimulated about a 2300% increase in [‘*‘I]-deoxyuridine incorporation as compared with 0.2% BSA because it provided an enriched supplement of growth factors and nutrients. It was chosen, therefore, to examine the inhibitory effect of growth factors. In 10% calf
Growth factors on pulp cell
233
Table 1. Effects of the seven types of growth factor and 10% calf serum on [‘?+sulphate incorporation in the proliferating and stationary stage
Control aFGF bFGF PDGF TGF-fl IGFI IGFII EGF lO%CS
Proliferating stage
Stationary stage
2830 k 312 9356 k 797’ 4006 + 340* 3015 + 532 3925 L-431’ 4831 f 322. 4631 + 698. 2593 & 453 1824 * 107’
1687 + 197 3035 f 225* 1567 + 22 1221 * 790 1166+ 163’ 1127 + 154* 1624+78 1081 & 1IO’ 1229 f 90*
Results expressed in terms of counts/min/pg protein, mean *SD of six determinations from one of three experiments. *Significantly different from control value (p < 0.001).
0,
0
0.01 0.1
0.5
I
5
nglml Fig. I. Dose-response of primary pulp cells stimulated by aFGF (O-O), bFGF (0.. O), PDGF (A.-A), TGF-j (A---A), IGF-I (I--m), IGF-II (Cl--0) and EGF (+ --- +) in 0.2% BSA-supplemented culture. Symbols represent counts per min per well of [12JI]-deoxyuridine incorporation in DNA, mean f SD of four wells from one of three experiments. *p c 0.01, l p c 0.001 compared with control. serum-supplemented culture, TGF-j3 showed an inhibition of DNA synthesis at a concentration above 5 n&ml in two of three separate assays. Other factors had no effect (Fig. 2). Proteoglycan
synthesis
stage of culture, aFGF, IGF-I, IGF-II, bFGF, and TGF-/I increased [“S]sulphate incorporation 231, 71, 64, 42 and 39%, respectively, as compared with control (p < 0.001). Ten per cent calf serum significantly decreased the During
the proliferating
incorporation 37% (p < 0.001). PDGF and EGF had no significant effect (Table 1). In stationary-stage cultures, EGF, IGF-I, TGF-/?, 10% calf serum and PDGF decreased [“S]-sulphate incorporation 36, 33, 31, 28 and 27%. respectively (p c 0.001). Only aFGF significantly increased [“S]sulphate incorporation 80% (p < 0.001) (Table 1). Alkaline phosphatase
PDGF, aFGF and bFGF caused 6540% inhibition of alkaline phosphatase activity as compared with controls in both serum-free and 10% calf serumsupplemented culture of proliferating, stationery and multilayered stages except in serum-free culture of stationary stage (p < 0.001 and 0.01). TGF-fl significantly decreased alkaline phosphatase activity by 65 and 40%, respectively, in serum-free cultures of proliferating and stationary stages (p < 0.001 and 0.01). The other growth factors had no significant effect (Table 2). Table 2. Effects of the seven types of growth factor on alkaline phosphatase activity in the absence and the presence of 10% calf serum in proliferating, stationary and multilayered stages Proliferating stage Control aFGF bFGF PDGF TGF-8 IGF-I IGF-II EGF
nglml Fig. 2. Doscresponse of primary pulp cells stimulated aFGF (O-O), bFGF (0. * .O), PDGF (A.-A), TGF-B (A---A), IGF-I (W-B), IGF-II (O--o), and EGF (+ --- +) in 10% calf serum-supplemented culture. Symbols represent counts per min per well of [‘“Ij-deoxyuridine incorporation in DNA, mean f SD of four wells from one of three experiments. l p < 0.001 compared with control.
actioity
Control aFGF bFGF PDGF TGF-fl IGF-I IGF-II EGF
11.31 2 5.71 5 4.16 2 6.79 i 4.09 + 11.622 10.61 2 9.19 i
Stationary stage
Absence 2.31 3.70 & 0.52 0.97* 3.32 + 0.44 0.39t 3.28 + 0.48 0.62* 2.76 + 0.29 0.23t 2.57 rf:0.24’ 1.62 3.10 f 0.39 1.74 3.09 f 0.24 0.75 3.47 + 0.53
Multi-layered stage 5.97 k 1.32 3.01 + 0.59* 3.01 & 0.16* 2.37 + 0.21. 5.65 k 1.30 5.45 f 0.60 4.51 & 0.68 3.99 & 0.35
Presence 3.02 k 0.31 1.23& 0.23 1.86 5 0.22t 0.68 5 0.09’ 1.96 & 0.27* 0.54 + 0.13* 1.52 i 0.31t 0.51 * 0.10’ 3.05 k 0.39 0.78 k 0.22 2.42 & 0.14 1.34 kO.18 2.11 kO.40 1.18 kO.15 2.66 k 0.52 0.93 * 0.14
Results expressed in terms of pmol/h. mg protein, mean k SD of four determinations from one of two experiments. *Significantly different from control value (p < 0.01). tSignificantly different from control value (p < O.OOI).
bl.
234
NAKASHIMA
DISCLSSION
I had earlier characterized the primary pulp cells used here (Nakashima, 1991). The trypsin pretreatment/collagenase separation was chosen because a large enough number of primary cells can be obtained from one isolation, so enabling multiple analyses, Primary cell culture is considered to retain more of the phenotypic characteristics of the original pulp cells than with subcultured and cloned cells. The pulp cell likeness of these ceils has been shown by the presence of high alkaline phosphatase activity and parathyroid hormone-stimulated, CAMP production (Nakashima, 1991). In long-term culture, nodules are seen in which initial calcification occurs around preodontoblast-like cells, suggesting that the isolated pulp cell may be able to differentiate into an odontoblast in uifro. Therefore, one may speculate about the reaction of pulp cells to growth factors in wound healing by extrapolation from the effects of these factors on DNA synthesis, proteoglycan synthesis and alkaline phosphatase activity in the primary pulp cell culture. EGF stimulates in citro proliferation of dental mesenchymal cells isolated from tooth germs (Steidler and Reade, 1981; Thesleff, Ekblom and Keski-Oja, 1983; Partanen, Ekblom and Thesleff, 1985; Partanen and Thesleff, 1989), while FGF and PDGF have no effect (Partanen et al., 1985). In cloned pulp cells, EGF stimulated DNA synthesis; whereas TGF-8 did not (Liang et al., 1990). PDGF (Ross, Raines and Bowen-Pore, 1986), aFGF (Uhlrich et al., 1986), bFGF (Delli-Bovi et al., 1987, 1988), IGFs (Rechler ef al., 1974; Haselbacher and Humbel, 1976; Phillips and Vassilopoulou-Sellin, 1980a, b) and EGF (Canalis and Raisz, 1979) are known to stimulate proliferation of fibroblasts. My pulp cells were isolated from permanent bovine incisors whose root development was complete. After 6 days of culture the cells were predominantly stellate-like fibroblasts with a few short processes. PDGF, IGF-I, IGF-II, aFGF, bFGF and EGF were all potent mitogens for these pulp cells, showing the similarity between cultured pulp cells and fibroblasts. The difference in the effect of FGF and PDGF may be due to the developmental stage of the pulp, the culture system, or both. TGF-fl can act either as a growth inhibitory or a mitogenic factor, depending on cell source, cell population, cell density, presence or absence of serum, culture conditions and whether other growth factors are present (Bonewald and Mundy, 1990). TGF-j? inhibited DNA synthesis in cultured pulp cells in the absence of serum. Discrepancies were observed in cultures supplemented with 10% calf serum where TGF-fl inhibited DNA synthesis in two of three separate assays. TGF-/? and IGFs increase the expression of sulphate proteoglycan present in the extracellular matrix and culture medium of various cell types including fibroblasts (Phillips and Vassilopoulou-Sellin, 1980a, b; Chen, Hoshi and Mckeehan, 1987; Bassols and Massagtie, 1988). The effect of FGF on sulphate proteoglycan synthesis by chondrocytes varies depending upon the stage of differentiation of cells and culture conditions (Sachs et al., 1982; Kato and Gospodarowicz, 1985; Hamerman, Sasse and
Klagsbrun, 1986). Water-soluble dentine extracts increased proteoglycan synthesis in embryonic chick limb mesenchymal cells and stimulated chondrogenesis as detected by metachromatic staining with toluidine blue (Rabinowitz, Syftestad and Caplan, 1990). In my work, aFGF, IGF-I, IGF-II, bFGF and TGF-P stimulated proteoglycan synthesis during the proliferating stage, while EGF, IGF-I, TGF-/? and PDGF inhibited proteoglycan synthesis and only aFGF stimulated the synthesis during the stationary stage. Thus, the effects were dependent on the degree of differentiation of the cells. The acquisition of alkaline phosphatase activity may be a prerequisite for the differentiation and possible specialization of pulp ceils in cico (Miller, Everett and Cramer, 1976) because alkaline phosphatase activity in the subodontoblastic layer is higher than in the odontoblastic layer and highest in the pulp (Nuki and Bonting. 1961; Goggins and Fullmer, 1967; Yoshiki and Kurahashi, 1971). Alkaline phosphatase as a pulp-capping agent stimulates the pulp tissue to form dentine matrix (Seltzer, 1959; Seltzer, Bender and Kaufman, 1961). Therefore, I used alkaline phosphatase activity as a marker to indicate the effect of growth factors on the differentiation of pulp cells; changes in alkaline phosphatase activity may reflect their state of maturation. Inhibition of alkaline phosphatase activity by aFGF has been reported in calvarial cell cultures and organ cultures (Rodan et al., 1987; Shen et al., 1989; Nicolas et al., 1990) and by EGF (Nicolas et al., 1990). In an osteosarcoma cell line, bFGF similarly inhibits alkaline phosphatase activity (Miller and Puzas, 1988). There are discrepancies in the reported effects of TGF-fi on osteoblastic cell lines (Noda and Rodan, 1986; Pfeilschifter, D’Souza and Mundy, 1987). It has been suggested that changes in alkaline phosphatase activity may reflect the maturation state of the osteoblast (Bonewald and Mundy, 1990). TGF-P and EGF also show suppressive effects in cloned pulp cells (Liang et al., 1990). In my study, TGF-fi, bFGF, aFGF and PDGF inhibited alkaline phosphatase activity. These growth factors may control the expression of alkaline phosphatase in vitro by preventing differentiation during periods of active cell proliferation and regulating the expression of mature cell phenotype. These results suggest that in pulp wound healing, proliferation of mesenchymal pulp cells may be stimulated mainly by PDGF and IGF-I, and the production of extracellular matrix proteoglycan may be enhanced by aFGF, IGF-I and IGF-II. Furthermore, TGF-8, PDGF, aFGF and bFGF may be possible regulators of the differentiation of pulp cells into odontoblasts. Acknowledgements-1
wish to thank Professor T. Nakamura, Faculty of Science, Kyushu University, in his instruction and advice, and the gift of human PDGF. I also thank Professor H. Nagasawa for a critical reading of this manuscript. REFERENCES Antoniades H. N., Scher C. D. and Stiles C. D. (1979) Purification of human platelet-derived growth factor. Proc. natn. Acad. Sci., U.S.A. 76, 1809-1813.
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