Archs oral Bid. Vol. 35, No. 3, pp. 241-247, 1990 Printed in Great Britain. All rights reserved
0003-9969/90$3.00+ 0.00 Copyright 0 1990Pergamon Press plc
CHARACTERISTICS OF HUMAN PERIODONTAL LIGAMENT CELLS IN T/ITRO M . J . SOMERMAN 9'*M F. YOUNG,~ R. A. FOSTER,'J. M. MOEHRING,~G. IMM’ and J. J. SAUK’ ‘Departments of Periodontics/Pharmacology, 2Department of Oral Pathology, 3Department of Oral Health Care De’livery, University of Maryland Dental School, Baltimore, MD 21201 and 4NIDR, NIH Bethesda, MD 20852, U.S.A. (Accepted 30 August 1989) Summary-Periodontal ligament cells may have a role in the regulation of hard and soft periodontal tissues, but their specific function has yet to be determined. To evaluate further their role in periodontal homeostasis, they were examined for osteoblast-like behaviour; in uifro no characteristic osteoblastic responsiveness was found. Periodontal ligament cells gave a PGE,- and isoproterenol-mediated CAMP response, but did not respond in a similar fashion to calcitonin or PTH. When exposed to PGE,, isoproterenol, or 1,25(OH), vitamin D,, they did not exhibit an increase in protein production, as measured by [‘%I-methionine incorporation. Immunofluorescent localization indicated that periodontal ligament cells produce a bone-associated protein, osteonectin. In addition, mRNA levels for osteonectin and bone proteoglycan I (biglycan) were detected in these cells, in vitro. This information should help to clarify the role such cells play in the regulation of periodontal tissues. Key words: periodontal ligament, immunohistochemistry,
INlrRODUCTION In order for periodontal regeneration to occur, the appropriate cells must migrate and subsequently attach at the healing site. They must then proliferate, differentiate and synthesize specific components, i.e. mineralized and soft connective tissues. Cells in the periodontal tissues, including the periodontal ligament, may have the capacity to regenerate tissues lost because of disease (Bowers, Schallhorn and Mellonig,
1982; Polson and Caton, 1982; Femyhough and Page, 1983; Melcher, 1976; Knox and Aukhil, 1988; Melcher and Cheong, 1988; Isidor et al., 1986). This possibility has stimulated an interest in the development of regenerative clinical procedures which focus on the regulation of cells of periodontal origin. Essential information needed for accomplishing such procedures includes the characterization of such cells so that the specific role each type plays in regeneration can be understood. Several researchers have cultured cells of periodontal origin and determined their biological properties in oitro (Rao, Moe and Heersche, 1978; Blomlof and Otteskog, 1981; Narayanan and Page, 1983; Ragnarsson, Carr and Daniel, 1985; Kawase et al., 1986; Rose et al., 1987; Sornerman et al., 1987a,b; Maeder, Carnes and Graves, 11988;Otsuka et al., 1988; Piche, Carnes and Graves, 1989). Such studies have indi-
cell culture, osteonectin, bone proteoglycan.
cated that periodontal ligament cells have osteoblastlike characteristics, which include high levels of alkaline phosphatase, production of predominantly type I collagen and production of osteonectin (Wasi et al., 1984), a bone-associated protein (Fisher and Termine, 1985). Information on the osteoblast-like properties of periodontal ligament cells is important to clinical investigations directed at stimulating periodontal regeneration. We have now attempted further characterization of the osteoblast-like properties of human periodontal ligament cells in vitro. Hormones known to stimulate CAMP and protein production in osteoblast-like cells were evaluated for their ability to increase CAMP levels and total protein production in periodontal ligament cells. In addition, indirect immunofluorescence studies and mRNA assays were performed to determine whether such cells produce three bone-associated extracellular matrix constituents, osteonectin, bone proteoglycan I (biglycan) (Fisher et al., 1987) and bone sialoprotein I (Prince and Butler, 1987; Somerman et al., 1987~). These are proteins for which antisera are available and which reflect osteoblastic activity; they are not specific to osteoblast-like cells, but are present in high concentrations in such cells. Moreover, both osteonectin and bone sialoprotein I are hormonally regulated in OSteoblast-like cells (Prince and Butler, 1987; Robey and Termine, 1985).
*Address correspondence to: Dr M. J. Somerman, Department of Pharmacology, University of Maryland Dental School, 666 W. Baltimore St, Baltimore, MD 21201, U.S.A. Abbreuiafions: BSA, bovine serum albumin; DMEM, Dulbecco’s Modified Eagle medium; FBS, fetal bovine serum; IBMX, 3-isobutyl-l-methyl xanthine; PBS, phosphate-buffered saline; PGE,, prostaglandin E,; PTH, parathyroid hormone
MATERIALS AND METHODS Derivation
of periodontal
cells
Connective tissue cells were obtained from the healthy periodontal ligaments of premolar teeth extracted for orthodontic reasons, as detailed by Somerman et al. (1987a). In brief, periodontal liga241
242
M. J. SOMERMAN et al.
ments were minced, dispersed on glass slides, inverted and placed in Leighton tubes and incubated with biopsy medium (DMEM with 250 pg/ml gentamicin sulphate, 1.16 g/1 glutamine, 5 pgg/ml amphotericin B, 100 U/ml penicillin, 100 p g/ml streptomycin and 10% FBS) at 37°C in a humidified atmosphere of 5% carbon dioxide-95% air. The following day, biopsy medium was replaced with culture medium (DMEM with 100 U/ml penicillin, 100 pg/ml streptomycin, 1.16 g/l glutamine and 10% FBS). When ceils surrounding the explant reached confluence, they were transferred to 75cm’ flasks; this transfer was designated as T, (first transfer). Cells were used between the third and eleventh passage in culture. CAMP assay
Periodontal ligament cells were seeded at a concentration of 50,000 cells per 35 mm Petri dish in culture medium and allowed to incubate overnight. The cell layers were then rinsed with 1 ml of PBS and allowed to incubate for 20 min in DMEM supplemented with 0.5% BSA and 0.5 mM IBMX. After this, the media were discarded and cells incubated with DMEM containing BSA, IBMX and the appropriate hormone for a designated time; the hormones evaluated were PGE,, PTH (hPTH l-34), salmon thyrocalcitonin and isoproterenol (Sigma Chem. Co., St Louis, MO, U.S.A.). The selected doses were based on previous experiments in vitro (Butler, 1985; Aubin et al., 1982). Appropriate control wells were also run. Media were then discarded again, cell layers rinsed twice with PBS and I ml of 90% propanol added to each well. After placing wells at 4°C for 1 h, propanol was removed from the cell layer and evaporated to dryness. CAMP analysis was based on competitive binding between tritiated and unlabelled CAMP to a protein having a high affinity for CAMP (Amersham kit TRK.432). Concurrent cultures were prepared for determination of total cell numbers per well. Thus, data were expressed as a ratio of treated to control CAMP production per lo6 cells. Determination of total protein
The methods for labelling cells have been reported (Somerman et al., 1987b; Hassell and Stanek, 1983). In brief, 2 x lo4 cells per test well (Corning No. 25820) were seeded in culture medium and incubated overnight. On the following day, the medium was removed and cells incubated for 24 h in DMEM containing 1.0% FBS, 0.5 mM IBMX, the appropriate hormone (isoproterenol or PGE2), and 50 pCi/ml [35S]-methionine (New England Nuclear, Boston, Mass., U.S.A.). For studies with vitamin D, periodontal ligament cells were incubated for 24 h in DMEM, containing 1.0% FBS, lo-‘M 1,25-dihydroxyvitamin D, and 50 pCi/ml [35S]-methionine. (The 1,25_dihydroxyvitamin D, was provided by Dr Uskokovic, Hoffman, LeRoche.) Then, cells and media were collected and dialysed extensively against water. After dialysis, aliquots of the samples were counted in a liquid scintillation spectrophotometer (Packard Tri Carb 460C) in Aquasol- (New England Nuclear). The resulting radioactive counts represented total non-dialysable radioactive material and were taken as a measure of total protein production. Non-radioactive wells were run in parallel so that
the total number of cells/well at the time of harvest could be obtained. These cells were removed with 0.08% trypsin/0.04% EDTA and total cells per well were determined electronically by Coulter counter. Thus, radioactive protein data were expressed as counts/min per lo6 cells. All assays and experiments were performed in triplicate. The statistical significance of the effects of agents on CAMP production and total protein production was determined by analysis of variance and Duncan’s multiple range test. Immunofluorescence microscopy
Immunofluorescence staining was performed on periodontal ligament cells grown on tissue culture chamber slides (Lab-Tek No. 4802). Staining for the bone-associated extracellular matrix constituent, osteonectin, and a bone-associated proteogiycan, biglycan (proteoglycan I), was done on 2- and 4-day non-confluent cultures. Antibodies to osteonectin and biglycan were generously provided by Dr Larry W. Fisher, NIDR/NIH, Bethesda, Md, U.S.A. (Fisher et al., 1987). Cells were rinsed twice in 50 mM PBS and fixed for 1 min in absolute methanol and air-dried. They were then rehydrated in PBS and pre-immune rabbit serum at 1: 100 dilution added at room temperature for 1 h. The cells were then layered with rabbit anti-human osteonectin, or rabbit antihuman biglycan at a 1: 10 or 1: 50 dilution, and with PBS for a pre-immune control, all for I h at room temperature. These concentrations were based on earlier studies with these antibodies on bone cells (Whitson et al., 1984). After incubation, cells were rinsed in PBS and then the staining and rinsing procedure was repeated with a 1:20 dilution of fluorescein-conjugated IgG, Fc fragment [goat antirabbit (Kirkegaard and Perry Labs, Inc., Gaithersburg, Md, U.S.A.)]. Antibodies were visualized and photographed with a Leitz epifluorescence microscope. mRNA levels
Total RNA was extracted from periodontal ligament cells with guanidine thiocyanate by the method of Ausubel et al. (1987). Northern blot analysis was performed using 1.2% agarose formaldehyde gels followed by transfer of RNA onto nitrocellulose. Nick-translated inserts of cDNA probes were then radioactively labelled and the blots hybridized. The nitrocellulose blots were washed under standard and stringent conditions to remove unbound probe, and exposed to X-ray film. RESULTS
Both PGE, and isoproterenol enhanced significantly CAMP production, whereas PTH and salmon thyrocalcitonin did not (Table 1). The stimulatory effect elicited by PGE, and isoproterenol increased in a dose-response manner. PGE, stimulated the CAMP response at a dose of 0.5 pgg/ml, with maximum response at 2.5 pgg/ml (Text Fig. 1). A CAMP response by cells exposed to isoproterenol was seen at lo-‘M, with a maximum response at 10m5M (Text Fig. 2). To determine the rate of hormone-mediated CAMP response, a time course was run (Text Fig. 3).
243
Characteristics of human periodontal ligament cells Table 1. Effect of hormones on CAMP production periodomal ligament cells CAMP production (treated to control ratio/lo6 cells)
DOSe
Hormone Control Calcitonin Isoproterenol PTH PGE,
by
1.0*0.1 1.1 f 0.1 5.0 k 0.6 1.0+0.1 3.1 + 0.0
(4OtlmU) (IavM)
(100 ng/ml) (5 krg/ml)
To measure CAMP produ’ztion, agents were added to triplicate dishes of periodontal ligament cells and incubated for 10 min, then the c:ells were processed to determine CAMP production.
The increase in CAMP production by cells exposed to PGE, or isoproterenol was rapid, with a maximal response at 10min. Ros 17/28 cells (well-characterized osteoblast-like cells derived from rat tumour) were run routinely as positive controls; these had a PTH- and isoproterenol-mediated CAMP response, as reported by Rodan and Rodan (1981). When the ability of PGE,, isoproterenol or 1,25(OH), vitamin D3 to alter total protein production by periodontal ligament cells was evaluated (Table 2) at the chosen dose [5 pg/ml PGE,; 10m5M isoproterenol; 10e8 M 1,25(OH),D,] and time (24 h exposure), these agents had no effect beyond that observed for untreated periodontal ligament cells. Radioactive proteins produced under these conditions were subjected to gel electrophoresis and analysed by autofluorographs. There were no significant changes in proteins produced by periodontal ligament cells under any of these treatments (data not shown). Periodontal ligament cells were immunoreactive to antibodies against osteonectin [Plate Fig. 4(a)]; elongated cells had pole-pole cytoplasmic staining for osteonectin without compartmentalization, and a similar diffuse cytoplasmic staining was also observed in spread cells. Cells did not stain for biglycan [Plate Fig. 4(b)]. Northern-blot analysis indicated substantial amounts of messenger RNA for osteonectin and biglycan (Plate Fig. 5’1.
_V z5 E
t
Control
0.01
0.05
0.5
2.5
5.0 I
1 Prostoglondin Ep
Fig. 1. CAMP dose-response by periodontal ligament cells exposed to PGE,. For CAMP assays, PGE, at the appropriate dose, was added to triplicate dishes of periodontal ligament cells and incubated for 10min and then the cells processed to determine CAMP production. Control, no hormone added. Results are expressed as a ratio of treated to control CAMP production per lo6 cells, &SD (error bars). *p =z0.05 significantly different from control cells.
Control
lo*
1ov
1o-r
10-e
10-s
Isoproteranol
Fig. 2. CAMP dose-response by periodontal ligament cells exposed to isoproterenol. For CAMP assays, isoproterenol, at the appropriate dose, was added to triplicate dishes of periodontal ligament cells and incubated for 10min and then the cells processed to determine CAMP production. Control, no hormone added. Results are expressed as a ratio of treated to control cAMP production per IO6cells + SD (error bars). *p iO.05 significantly different from control CellS. DISCUSSION
We have shown that human periodontal ligament cells have a PGE,- and isoproterenol-mediated CAMP response, but do not respond in a similar fashion to calcitonin or PTH. This is in agreement with the studies of Rao et al. (1978), who reported that porcine periodontal ligament cells exhibit increased levels of CAMP in response to PGE, but not in response to PTH or salmon thyrocalcitonin. In contrast, Piche et al. (1989) reported a PTH-mediated CAMP response for cells isolated from human periodontal ligament cells. However, as they stated, their method for obtaining cells, i.e. removal of periodontal ligament as well as cementum from unerupted third molars, may have resulted in the isolation of cementoblast-like cells. At present no markers are available for determining cementoblast-like cells in vitro. In addition, periodontal ligament cells isolated from unerupted third molars may behave differently than periodontal ligament cells isolated from functional regions. It is possible that by second passage, the earliest time at which periodontal ligament cells have been evaluated, they may have lost their PTHresponsiveness or have been selected for a population of cells that do not exhibit such a response. Alternatively, periodontal ligament cells may not have receptors for PTH. The ability of PGE, to alter periodontal ligament cell function indicates that this hormone, which is enhanced during inflammation, plays a role in regulating that function. No correlation was observed between the hormone-mediated increases in CAMP and total protein production (Table 2). Also, 1,25(0H)2 vitamin D,, a bone-regulatory hormone which can increase production of bone-associated proteins by osteoblasts (Prince and Butler, 1987; Butler, 1985), did not increase total protein production by periodontal ligament cells. The enhanced CAMP levels in cells exposed to PGE, and isoproterenol, which were without similar increases in [%I-methionine incorporation, can be attributed to many factors. Several findings suggest a correlation between an agent’s ability to increase CAMP levels and its ability to increase cellular synthesis of glycoproteins, as measured by
M. J. SOMERMAN
244
et al.
5
5 ”
4
G d
3
z L Y
2
!$
’
u
0 0
Control
1
5
10
0
30
I
I lsoproterenol
1
5
10
30
t
k Prortaqlandin
E,
Fig. 3. Time-course CAMP production by periodontal ligament cells exposed to IO-’ M isoproterenol, 2.5 pg/ml PGE, or control (no additions). To measure CAMP production, agents were added to wells of periodontal ligament cells and incubated for 0, 1, 5, 10 or 30 min and then cells processed to determine CAMP production. Each point on the bar graph is the mean k SD (error bars) of triplicate determinations. Results expressed as treated to control ratio per IO6cells. *p i 0.05 significantly different from control Cells.
Table 2. Effect of hormones on protein production by periodontal ligament cells Dose
Hormone Control PGE,
(5 pgfml) Control isoproterenol (IO-‘M) Control 1,25(OH), vitamin D,
(lo-’ M)
[3H]-mannose incorporation, without a similar increase in protein production (Firestone and Heath, 1980; Imada, Imada and Weiss, 1980; Okamoto et al., 1983; Kousvelari et al., 1984; Somerman et al., 1987d). We used immunofluorescent localization procedures and mRNA levels to determine whether periodontal ligament cells produce three bone-associated matrix constituents, bone sialoprotein 1, osteonectin and biglycan. Recent studies have suggested that bone-associated proteins can serve, at least in part, as markers for osteoblast-like cells (Whitson et al., 1984; Yoon, Buenaga and Rodan, 1988). Our findings (Plate Figs 4 and 5) indicate that periodontal ligament cells produce osteonectin and have mRNA levels for both osteonectin and biglycan. The precise role of any bone-associated proteins in the regulation of mineralization has not been established but they are thought to play a critical role in the control of bone homeostasis (reviewed by Butler, 1985). Osteonectin, a phosphorylated glycoprotein which has a high affinity for collagen, calcium and hydroxyapatite, may participate in the initiation of mineralization (Fisher and Termine, 1985). Osteonectin has been identified in tissues other than bone (Wasi et al., 1984; Nomura et al., 1988) and structural analysis has revealed similarities between this protein and SPARC
Total protein production (counts/min x lo-‘/lo6 cells) 10,366 f 11,447 f 151,093 f 155,457 * 185,921 f 166,601 f
8 1,662 10,004 9,340 29,637 9,016
(secreted protein acidic and rich in cysteine), bovine brain calmodulin and the calcium-binding proteins of muscle (Bolander et al., 1988). The wide distribution of osteonectin-like proteins has led researchers to suggest a calcium-dependent role for this protein in the regulation of matrix assembly (Bolander et al., 1988). Clearly, such a protein would be important for periodontal ligament cells in health, as well as during regeneration. Biglycan, a small bone proteoglycan, appears to be specific for bone and dentine (Fisher et al., 1987), although detailed immunocytochemical and mRNA studies have not been done. The significance of mRNA levels for biglycan in periodontal ligament cells is not known. Bone sialoprotein I or its mRNA are localized in selected cells of bone, kidney, decidua and brain (Mark et al., 1988; Nomura et al., 1988; Yoon ef al., 1988). Bone sialoprotein I [also called osteopontin (Oldberg, Franzen and Heinegard, 1986), 44 kDa bone phosphoprotein (Prince et al., 1987) and 2ar (Smith and Denhardt, 1987)], promotes attachment and spreading of connective tissue cells (Somerman et al., 1987a-d, 1989; Oldberg et al., 1986) via an R-G-D cell-binding sequence (Oldberg et al., 1986; Somerman et al., 1988). Furthermore, bone sialoprotein I appears more selective for connective tissue
Plate 1 Fig. 4. Indirect immunofluorescent micrographs of 4-day cultures of periodontal ligament cells exposed to antibodies to human osteonectin (a), biglycan (b), and a representative pre-immune serum control (c). x400. Fig. 5. Northern blots to identify mRNA for (a) osteonectin and (b) biglycan.
245
Characteristics of human periodontal ligament cells
-2.2kb
-2.4kb
5( b 1 Biglycan
5 ( a 11 Osteonectin
Plate 1
M. J. SOMERMAN et al.
246
cells than fibronectin, which enhances attachment of connective tissue cells and epithelial-like cells (Somerman et al., 1989). Bone sialoprotein I promotes attachment of periodontal ligament cells and gingival fibroblasts in oitro (Somerman et al., 1989), but mRNA for bone sialoprotein I was not detectable in periodontal ligament cells or gingival fibroblasts. Thus, human periodontal ligament cells do exhibit some osteoblast-like characteristics, but our studies suggest that they do not behave like classical osteoblast-like cells in vitro. Periodontal ligament cells have high alkaline phosphatase activity when compared with gingival tibroblasts (Kawase et al., 1986; Somerman et al., 1987a; Maeder et al., 1988). Furthermore, when human periodontal ligament cells and human gingival fibroblasts from the same patient are passaged over time, periodontal ligament cells become senescent faster as measured by proliferation, e.g. by passage seven periodontal ligament cells have a decreased proliferation rate, whereas gingival fibroblasts do not exhibit decreased proliferation with passaging (Somerman et af., unpublished observa-
tion). However, to date we have found no other differences between human periodontal ligament cells and human gingival fibroblasts. All our findings for periodontal ligament cells were similar to those for gingival fibroblasts (data not shown). The information obtained should help in understanding the factors that regulate neriodontal ligament cells both in health
and
during’tissue
regeneration.
Acknowledgements-We thank MS Jo Ann Walker for her skilful secretarial support. This study was supported by grants from the U.S. Public Health Service NIDR, NIH DE-07512 and DE-08648. REFERENCES Aubin J., Heersche J., Merriless M. and Sodek J. (1982) Isolation of bone cell clones with differences in growth, hormone response and extracellular matrix production. J. Cell B&l. 92, 452461. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A. and Struhl K. (Eds) (1987) Current Protocols in Molecular Biology, Chap. 4. Wiley, New York. Blomlof L. and Otteskog P. (1981) Composition of human periodontal ligament cells in tissue culture. &and. J. dent. Res. 89, 4346. Bolander M. E., Young M. F., Fisher L. W., Yamada Y. and Termine J. D. (1988) Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid). Proc. natn. Acad. Sci. U.S.A. 85, 2919-2923. Bowers G. M., Schallhorn R. G. and Mellonig J. T. (1982) Histologic evaluation of new attachment in human intrabony defects. A literature review. J. Periodont. 53, 509-5 16. Butler W. T. (Ed.) (1985) The Chemistry and Biology of Mineralized Tissues. Ebsco Media, Birmingham, Ala. Fernyhough W. and Page R. C. (1983) Attachment, growth anh synthesis by human gingival fibroblasts on demineralized or fibronectin-treated normal and diseased tooth roots. J. Periodont. 54, 133-140. Firestone G. L. and Heath E. C. (1980) The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes. Archs Biochem. Biophys. 201, 453461. Fisher L. W. and Termine J. D. (1985) Noncollagenous
proteins influencing the local mechanisms of calcification. Clin. Orthop. Related Res. 200, 362-385. Fisher L. W.,Hawkins G. R., Tuross N. and Termine J. D. (1987) Purification and uartial characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin from the mineral compartment of developing human bone. J. Cell Biol. 262, 2599265. Hassell T. M. and Stanek E. (1983) Evidence that healthy human gingiva contains functionally heterogeneous fibroblast subpopulations. Archs oral Biol. 28, 617625. Imada M., Imaha S. and Weiss D. (1980) Induction of surface glycoprotein expression by cyclic AMP in Chinese hamster ovary cells. Biochim. biophys. Acta 632, 47-53. Isidor F., Karring T., Nyman S. and Lindhe J. (1986) The significance of coronal growth of periodontal ligament tissue for new attachment formation. J. c/in. Periodonr. 13, 145-150. Kawase T., Sato S., Yamada M., Hirayama A., Miako K. and Saito S. (1986) Human periodontal ligament cells in vitro. Characterization of alkaline phosphatase. J. Bone Mineral. Res. Suppl. 1 1, 63A. Knox B. and Aukhil I. (1988) Ultrastructural study of experimental cementum regeneration in rats. J. periodont. Res. 23, 6&67. Kousvelari E. E., Grant S. R., Banerjee D. K., Newby M. J. and Baum B. J. (1984) Cyclic AMP mediates /3-adrenergic-induced increases in N-linked protein glycosylation in rat parotid acinar cells. Biochem. J. 222, 17-24. Maeder C. L., Carnes D. L. and Graves D. T. (1988) Alkaline phosphatase and osteocalcin levels in cells from periodontal explants. J. dent. Res. 67, 232A. Mark M. P., Butler W. T., Prince C. W., Finkelman R. D. and Ruth J.-V. (1988) Developmental expression of 44kDa bone ohosnhoorotein (osteonontin) and bone v-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat. Difirentiation 37, 123-126. Melcher A. H. (1976) On the repair potential of periodontal tissues. J. Periodont. 47, 256260. Melcher A. H. and Cheone T. 119881 Fibroblast-like cells depress formation of bone-like tissues in vitro. J. dent. Res. 67, 1419A. Narayanan A. S. and Page R. C. (1983) Connective tissues of the periodontium: a summary of current work. Collagen Rel. Res. 3, 33-64. Nomura S., Wills A. J., Edwards D. R., Heath J. K. and Hogan B. L. M. (1988) Developmental expression of 2ar (osteopontin) and spare (osteonectin) RNA as revealed by in situ hybridization. J. Cell Biol. 106, 44450. Okamoto Y., Sakai H., Sato J. and Akamatsu N. (1983) Effects of dibutyryl cyclic AMP on the synthesis of dolichol-linked saccharides and glycoproteins in cultured hepatoma cells. Biochem. J. 212, 859-867. Oldberg A., Franzen A. and Heinegard D. (1986) Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc. natn. Acad. Sci. U.S.A. 83, 8819-8823. Otsuka K., Pitara S., Overall C. M., Aubin J. E. and Sodek J. (1988) Biochemical comparison of fibroblast populations from different periodontal tissues: Characterization matrix protein and collagenolytic enzyme synthesis. B&hem. Cell Biof. 66, 167-I 76. Piche J. E., Carnes D. L. and Graves D. T. (1989) Initial characterization of cells derived from human periodontia. J. dent. Res. 68, 761-767. Polson A. M. and Caton J. (1982) Factors controlling periodontal repair and regeneration. J. Periodont. 53, 617-625. Prince C. W. and Butler W. T. (1987) 1,25-Dihvdroxvvitamin D, regulates the biosynthesis of osteoponiin, a bonederived cell attachment protein in clonal osteoblast-like osteosarcoma cells. Collagen Rel. Res. 7, 305-313. Prince C. W., Oosawa T., Butler W. T., Tomama M., Bhown A. S., Bhown M. and Schrohenloher R. E. (1987)
Characteristics of human periodontal ligament cells Isolation, characterization and biosynthesis of a phosphorylated glycoprotein from rat bone. J. biol. Chem. 262, 259-265. Ragnarsson B., Carr G. and Daniel J. C. (1985) Isolation and growth of human periodontal ligament cells in uifro. J. dent. Res. 64, 10261030. Rao L. G., Moe H. K. and Heersche J. N. M. (1978) In-vitro culture of porcine periodontal ligament cells: response of fibroblast-like and epithelial-like cells to prostaglandin E,, parathyroid hormone and calcitonin and separation of a pure population of fibroblast-like cells. Archs oral Biol. 23, 957-964. Robey P. G. and Termine J. D. (1985) Human bone cells in vitro. Calc. Tiss. Inr. 37, 453-460.
Rodan S. B. and Rodan Gr. A. (1981) Parathyroid hormone and isoproterenol stimulation of adenylate cyclase in rat osteosarcoma clonal cells. Biochim. biophys. Acta 673, 4652.
Rose G. G., Yamasaki A., Pinero G. J. and Mahan C. J. (1987) Human periodontal ligament cells, in vitro. J. periodonr.
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Smith J. H. and Dendhardt D. T. (1987) Molecular cloning of a tumor promoter-:mducible mRNA found in JB6 mouse epidermal cells: Induction is stable at high, but not at low. cell densities. J. Cell Biochem. 34. 13-22. Somerman M. J., Archer S. Y., Imm G. R. and Foster R. A. (1987a) A comparative study of human periodontal ligament cells and gingival ftbroblasts in uitro. J. dent. Res. 67, 6670.
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R. A. (1987b) Enhancement by extracts of mineralized tissues of protein productivity by human gingival fibroblasts. Archs oral Biol. 32, 879-883. Somerman M. J., Prince C. W., Sauk J. J., Foster R. A. and Butler W. T. (1987~) Mechanism of fibroblast attachment to bone extracellular matrix: Role of a 44 kilodalton bone nhosnhovrotein. J. Bone Mineral Res. 2. 259-265. Somerman-M. J., Shteyer A., Bowers M. .R. and Santora A. C. (1987d) Stimulation of mannose incorporation into rat osteoblastic osteosarcoma cells by parathyroid hormone. Archs oral Biol. 32, 535-538. Somerman M. J., Fisher L. W., Foster R. A. and Sauk J. J. (1988) Human bone sialoprotein I and II enhance fibroblast attachment in oitro. Calc. Tiss. Inf. 43, 5Ck53. Somerman M. J., Prince C. W., Butler W. T., Foster R. A., Moehring J. M. and Sauk J. J. (1989) Cell attachment activity of the 44 kilodalton bone phosphoprotein is not restricted to bone cells. Matrix 9, 4954. Wasi S., Otsuka K., Yao K.-L., Tung P. S., Aubin J. E., Sodek J. and Termine J. D. (1984) An osteonectin-like protein in porcine periodontal ligament and its synthesis by periodontal ligament fibroblasts. Can. J. Biochem. Cell Biol. 62, 47&478.
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0003-9969/90$3.00+ 0.00 Copyright 0 1990Pergamon Press plc
CHARACTERISTICS OF HUMAN PERIODONTAL LIGAMENT CELLS IN T/ITRO M . J . SOMERMAN 9'*M F. YOUNG,~ R. A. FOSTER,'J. M. MOEHRING,~G. IMM’ and J. J. SAUK’ ‘Departments of Periodontics/Pharmacology, 2Department of Oral Pathology, 3Department of Oral Health Care De’livery, University of Maryland Dental School, Baltimore, MD 21201 and 4NIDR, NIH Bethesda, MD 20852, U.S.A. (Accepted 30 August 1989) Summary-Periodontal ligament cells may have a role in the regulation of hard and soft periodontal tissues, but their specific function has yet to be determined. To evaluate further their role in periodontal homeostasis, they were examined for osteoblast-like behaviour; in uifro no characteristic osteoblastic responsiveness was found. Periodontal ligament cells gave a PGE,- and isoproterenol-mediated CAMP response, but did not respond in a similar fashion to calcitonin or PTH. When exposed to PGE,, isoproterenol, or 1,25(OH), vitamin D,, they did not exhibit an increase in protein production, as measured by [‘%I-methionine incorporation. Immunofluorescent localization indicated that periodontal ligament cells produce a bone-associated protein, osteonectin. In addition, mRNA levels for osteonectin and bone proteoglycan I (biglycan) were detected in these cells, in vitro. This information should help to clarify the role such cells play in the regulation of periodontal tissues. Key words: periodontal ligament, immunohistochemistry,
INlrRODUCTION In order for periodontal regeneration to occur, the appropriate cells must migrate and subsequently attach at the healing site. They must then proliferate, differentiate and synthesize specific components, i.e. mineralized and soft connective tissues. Cells in the periodontal tissues, including the periodontal ligament, may have the capacity to regenerate tissues lost because of disease (Bowers, Schallhorn and Mellonig,
1982; Polson and Caton, 1982; Femyhough and Page, 1983; Melcher, 1976; Knox and Aukhil, 1988; Melcher and Cheong, 1988; Isidor et al., 1986). This possibility has stimulated an interest in the development of regenerative clinical procedures which focus on the regulation of cells of periodontal origin. Essential information needed for accomplishing such procedures includes the characterization of such cells so that the specific role each type plays in regeneration can be understood. Several researchers have cultured cells of periodontal origin and determined their biological properties in oitro (Rao, Moe and Heersche, 1978; Blomlof and Otteskog, 1981; Narayanan and Page, 1983; Ragnarsson, Carr and Daniel, 1985; Kawase et al., 1986; Rose et al., 1987; Sornerman et al., 1987a,b; Maeder, Carnes and Graves, 11988;Otsuka et al., 1988; Piche, Carnes and Graves, 1989). Such studies have indi-
cell culture, osteonectin, bone proteoglycan.
cated that periodontal ligament cells have osteoblastlike characteristics, which include high levels of alkaline phosphatase, production of predominantly type I collagen and production of osteonectin (Wasi et al., 1984), a bone-associated protein (Fisher and Termine, 1985). Information on the osteoblast-like properties of periodontal ligament cells is important to clinical investigations directed at stimulating periodontal regeneration. We have now attempted further characterization of the osteoblast-like properties of human periodontal ligament cells in vitro. Hormones known to stimulate CAMP and protein production in osteoblast-like cells were evaluated for their ability to increase CAMP levels and total protein production in periodontal ligament cells. In addition, indirect immunofluorescence studies and mRNA assays were performed to determine whether such cells produce three bone-associated extracellular matrix constituents, osteonectin, bone proteoglycan I (biglycan) (Fisher et al., 1987) and bone sialoprotein I (Prince and Butler, 1987; Somerman et al., 1987~). These are proteins for which antisera are available and which reflect osteoblastic activity; they are not specific to osteoblast-like cells, but are present in high concentrations in such cells. Moreover, both osteonectin and bone sialoprotein I are hormonally regulated in OSteoblast-like cells (Prince and Butler, 1987; Robey and Termine, 1985).
*Address correspondence to: Dr M. J. Somerman, Department of Pharmacology, University of Maryland Dental School, 666 W. Baltimore St, Baltimore, MD 21201, U.S.A. Abbreuiafions: BSA, bovine serum albumin; DMEM, Dulbecco’s Modified Eagle medium; FBS, fetal bovine serum; IBMX, 3-isobutyl-l-methyl xanthine; PBS, phosphate-buffered saline; PGE,, prostaglandin E,; PTH, parathyroid hormone
MATERIALS AND METHODS Derivation
of periodontal
cells
Connective tissue cells were obtained from the healthy periodontal ligaments of premolar teeth extracted for orthodontic reasons, as detailed by Somerman et al. (1987a). In brief, periodontal liga241
242
M. J. SOMERMAN et al.
ments were minced, dispersed on glass slides, inverted and placed in Leighton tubes and incubated with biopsy medium (DMEM with 250 pg/ml gentamicin sulphate, 1.16 g/1 glutamine, 5 pgg/ml amphotericin B, 100 U/ml penicillin, 100 p g/ml streptomycin and 10% FBS) at 37°C in a humidified atmosphere of 5% carbon dioxide-95% air. The following day, biopsy medium was replaced with culture medium (DMEM with 100 U/ml penicillin, 100 pg/ml streptomycin, 1.16 g/l glutamine and 10% FBS). When ceils surrounding the explant reached confluence, they were transferred to 75cm’ flasks; this transfer was designated as T, (first transfer). Cells were used between the third and eleventh passage in culture. CAMP assay
Periodontal ligament cells were seeded at a concentration of 50,000 cells per 35 mm Petri dish in culture medium and allowed to incubate overnight. The cell layers were then rinsed with 1 ml of PBS and allowed to incubate for 20 min in DMEM supplemented with 0.5% BSA and 0.5 mM IBMX. After this, the media were discarded and cells incubated with DMEM containing BSA, IBMX and the appropriate hormone for a designated time; the hormones evaluated were PGE,, PTH (hPTH l-34), salmon thyrocalcitonin and isoproterenol (Sigma Chem. Co., St Louis, MO, U.S.A.). The selected doses were based on previous experiments in vitro (Butler, 1985; Aubin et al., 1982). Appropriate control wells were also run. Media were then discarded again, cell layers rinsed twice with PBS and I ml of 90% propanol added to each well. After placing wells at 4°C for 1 h, propanol was removed from the cell layer and evaporated to dryness. CAMP analysis was based on competitive binding between tritiated and unlabelled CAMP to a protein having a high affinity for CAMP (Amersham kit TRK.432). Concurrent cultures were prepared for determination of total cell numbers per well. Thus, data were expressed as a ratio of treated to control CAMP production per lo6 cells. Determination of total protein
The methods for labelling cells have been reported (Somerman et al., 1987b; Hassell and Stanek, 1983). In brief, 2 x lo4 cells per test well (Corning No. 25820) were seeded in culture medium and incubated overnight. On the following day, the medium was removed and cells incubated for 24 h in DMEM containing 1.0% FBS, 0.5 mM IBMX, the appropriate hormone (isoproterenol or PGE2), and 50 pCi/ml [35S]-methionine (New England Nuclear, Boston, Mass., U.S.A.). For studies with vitamin D, periodontal ligament cells were incubated for 24 h in DMEM, containing 1.0% FBS, lo-‘M 1,25-dihydroxyvitamin D, and 50 pCi/ml [35S]-methionine. (The 1,25_dihydroxyvitamin D, was provided by Dr Uskokovic, Hoffman, LeRoche.) Then, cells and media were collected and dialysed extensively against water. After dialysis, aliquots of the samples were counted in a liquid scintillation spectrophotometer (Packard Tri Carb 460C) in Aquasol- (New England Nuclear). The resulting radioactive counts represented total non-dialysable radioactive material and were taken as a measure of total protein production. Non-radioactive wells were run in parallel so that
the total number of cells/well at the time of harvest could be obtained. These cells were removed with 0.08% trypsin/0.04% EDTA and total cells per well were determined electronically by Coulter counter. Thus, radioactive protein data were expressed as counts/min per lo6 cells. All assays and experiments were performed in triplicate. The statistical significance of the effects of agents on CAMP production and total protein production was determined by analysis of variance and Duncan’s multiple range test. Immunofluorescence microscopy
Immunofluorescence staining was performed on periodontal ligament cells grown on tissue culture chamber slides (Lab-Tek No. 4802). Staining for the bone-associated extracellular matrix constituent, osteonectin, and a bone-associated proteogiycan, biglycan (proteoglycan I), was done on 2- and 4-day non-confluent cultures. Antibodies to osteonectin and biglycan were generously provided by Dr Larry W. Fisher, NIDR/NIH, Bethesda, Md, U.S.A. (Fisher et al., 1987). Cells were rinsed twice in 50 mM PBS and fixed for 1 min in absolute methanol and air-dried. They were then rehydrated in PBS and pre-immune rabbit serum at 1: 100 dilution added at room temperature for 1 h. The cells were then layered with rabbit anti-human osteonectin, or rabbit antihuman biglycan at a 1: 10 or 1: 50 dilution, and with PBS for a pre-immune control, all for I h at room temperature. These concentrations were based on earlier studies with these antibodies on bone cells (Whitson et al., 1984). After incubation, cells were rinsed in PBS and then the staining and rinsing procedure was repeated with a 1:20 dilution of fluorescein-conjugated IgG, Fc fragment [goat antirabbit (Kirkegaard and Perry Labs, Inc., Gaithersburg, Md, U.S.A.)]. Antibodies were visualized and photographed with a Leitz epifluorescence microscope. mRNA levels
Total RNA was extracted from periodontal ligament cells with guanidine thiocyanate by the method of Ausubel et al. (1987). Northern blot analysis was performed using 1.2% agarose formaldehyde gels followed by transfer of RNA onto nitrocellulose. Nick-translated inserts of cDNA probes were then radioactively labelled and the blots hybridized. The nitrocellulose blots were washed under standard and stringent conditions to remove unbound probe, and exposed to X-ray film. RESULTS
Both PGE, and isoproterenol enhanced significantly CAMP production, whereas PTH and salmon thyrocalcitonin did not (Table 1). The stimulatory effect elicited by PGE, and isoproterenol increased in a dose-response manner. PGE, stimulated the CAMP response at a dose of 0.5 pgg/ml, with maximum response at 2.5 pgg/ml (Text Fig. 1). A CAMP response by cells exposed to isoproterenol was seen at lo-‘M, with a maximum response at 10m5M (Text Fig. 2). To determine the rate of hormone-mediated CAMP response, a time course was run (Text Fig. 3).
243
Characteristics of human periodontal ligament cells Table 1. Effect of hormones on CAMP production periodomal ligament cells CAMP production (treated to control ratio/lo6 cells)
DOSe
Hormone Control Calcitonin Isoproterenol PTH PGE,
by
1.0*0.1 1.1 f 0.1 5.0 k 0.6 1.0+0.1 3.1 + 0.0
(4OtlmU) (IavM)
(100 ng/ml) (5 krg/ml)
To measure CAMP produ’ztion, agents were added to triplicate dishes of periodontal ligament cells and incubated for 10 min, then the c:ells were processed to determine CAMP production.
The increase in CAMP production by cells exposed to PGE, or isoproterenol was rapid, with a maximal response at 10min. Ros 17/28 cells (well-characterized osteoblast-like cells derived from rat tumour) were run routinely as positive controls; these had a PTH- and isoproterenol-mediated CAMP response, as reported by Rodan and Rodan (1981). When the ability of PGE,, isoproterenol or 1,25(OH), vitamin D3 to alter total protein production by periodontal ligament cells was evaluated (Table 2) at the chosen dose [5 pg/ml PGE,; 10m5M isoproterenol; 10e8 M 1,25(OH),D,] and time (24 h exposure), these agents had no effect beyond that observed for untreated periodontal ligament cells. Radioactive proteins produced under these conditions were subjected to gel electrophoresis and analysed by autofluorographs. There were no significant changes in proteins produced by periodontal ligament cells under any of these treatments (data not shown). Periodontal ligament cells were immunoreactive to antibodies against osteonectin [Plate Fig. 4(a)]; elongated cells had pole-pole cytoplasmic staining for osteonectin without compartmentalization, and a similar diffuse cytoplasmic staining was also observed in spread cells. Cells did not stain for biglycan [Plate Fig. 4(b)]. Northern-blot analysis indicated substantial amounts of messenger RNA for osteonectin and biglycan (Plate Fig. 5’1.
_V z5 E
t
Control
0.01
0.05
0.5
2.5
5.0 I
1 Prostoglondin Ep
Fig. 1. CAMP dose-response by periodontal ligament cells exposed to PGE,. For CAMP assays, PGE, at the appropriate dose, was added to triplicate dishes of periodontal ligament cells and incubated for 10min and then the cells processed to determine CAMP production. Control, no hormone added. Results are expressed as a ratio of treated to control CAMP production per lo6 cells, &SD (error bars). *p =z0.05 significantly different from control cells.
Control
lo*
1ov
1o-r
10-e
10-s
Isoproteranol
Fig. 2. CAMP dose-response by periodontal ligament cells exposed to isoproterenol. For CAMP assays, isoproterenol, at the appropriate dose, was added to triplicate dishes of periodontal ligament cells and incubated for 10min and then the cells processed to determine CAMP production. Control, no hormone added. Results are expressed as a ratio of treated to control cAMP production per IO6cells + SD (error bars). *p iO.05 significantly different from control CellS. DISCUSSION
We have shown that human periodontal ligament cells have a PGE,- and isoproterenol-mediated CAMP response, but do not respond in a similar fashion to calcitonin or PTH. This is in agreement with the studies of Rao et al. (1978), who reported that porcine periodontal ligament cells exhibit increased levels of CAMP in response to PGE, but not in response to PTH or salmon thyrocalcitonin. In contrast, Piche et al. (1989) reported a PTH-mediated CAMP response for cells isolated from human periodontal ligament cells. However, as they stated, their method for obtaining cells, i.e. removal of periodontal ligament as well as cementum from unerupted third molars, may have resulted in the isolation of cementoblast-like cells. At present no markers are available for determining cementoblast-like cells in vitro. In addition, periodontal ligament cells isolated from unerupted third molars may behave differently than periodontal ligament cells isolated from functional regions. It is possible that by second passage, the earliest time at which periodontal ligament cells have been evaluated, they may have lost their PTHresponsiveness or have been selected for a population of cells that do not exhibit such a response. Alternatively, periodontal ligament cells may not have receptors for PTH. The ability of PGE, to alter periodontal ligament cell function indicates that this hormone, which is enhanced during inflammation, plays a role in regulating that function. No correlation was observed between the hormone-mediated increases in CAMP and total protein production (Table 2). Also, 1,25(0H)2 vitamin D,, a bone-regulatory hormone which can increase production of bone-associated proteins by osteoblasts (Prince and Butler, 1987; Butler, 1985), did not increase total protein production by periodontal ligament cells. The enhanced CAMP levels in cells exposed to PGE, and isoproterenol, which were without similar increases in [%I-methionine incorporation, can be attributed to many factors. Several findings suggest a correlation between an agent’s ability to increase CAMP levels and its ability to increase cellular synthesis of glycoproteins, as measured by
M. J. SOMERMAN
244
et al.
5
5 ”
4
G d
3
z L Y
2
!$
’
u
0 0
Control
1
5
10
0
30
I
I lsoproterenol
1
5
10
30
t
k Prortaqlandin
E,
Fig. 3. Time-course CAMP production by periodontal ligament cells exposed to IO-’ M isoproterenol, 2.5 pg/ml PGE, or control (no additions). To measure CAMP production, agents were added to wells of periodontal ligament cells and incubated for 0, 1, 5, 10 or 30 min and then cells processed to determine CAMP production. Each point on the bar graph is the mean k SD (error bars) of triplicate determinations. Results expressed as treated to control ratio per IO6cells. *p i 0.05 significantly different from control Cells.
Table 2. Effect of hormones on protein production by periodontal ligament cells Dose
Hormone Control PGE,
(5 pgfml) Control isoproterenol (IO-‘M) Control 1,25(OH), vitamin D,
(lo-’ M)
[3H]-mannose incorporation, without a similar increase in protein production (Firestone and Heath, 1980; Imada, Imada and Weiss, 1980; Okamoto et al., 1983; Kousvelari et al., 1984; Somerman et al., 1987d). We used immunofluorescent localization procedures and mRNA levels to determine whether periodontal ligament cells produce three bone-associated matrix constituents, bone sialoprotein 1, osteonectin and biglycan. Recent studies have suggested that bone-associated proteins can serve, at least in part, as markers for osteoblast-like cells (Whitson et al., 1984; Yoon, Buenaga and Rodan, 1988). Our findings (Plate Figs 4 and 5) indicate that periodontal ligament cells produce osteonectin and have mRNA levels for both osteonectin and biglycan. The precise role of any bone-associated proteins in the regulation of mineralization has not been established but they are thought to play a critical role in the control of bone homeostasis (reviewed by Butler, 1985). Osteonectin, a phosphorylated glycoprotein which has a high affinity for collagen, calcium and hydroxyapatite, may participate in the initiation of mineralization (Fisher and Termine, 1985). Osteonectin has been identified in tissues other than bone (Wasi et al., 1984; Nomura et al., 1988) and structural analysis has revealed similarities between this protein and SPARC
Total protein production (counts/min x lo-‘/lo6 cells) 10,366 f 11,447 f 151,093 f 155,457 * 185,921 f 166,601 f
8 1,662 10,004 9,340 29,637 9,016
(secreted protein acidic and rich in cysteine), bovine brain calmodulin and the calcium-binding proteins of muscle (Bolander et al., 1988). The wide distribution of osteonectin-like proteins has led researchers to suggest a calcium-dependent role for this protein in the regulation of matrix assembly (Bolander et al., 1988). Clearly, such a protein would be important for periodontal ligament cells in health, as well as during regeneration. Biglycan, a small bone proteoglycan, appears to be specific for bone and dentine (Fisher et al., 1987), although detailed immunocytochemical and mRNA studies have not been done. The significance of mRNA levels for biglycan in periodontal ligament cells is not known. Bone sialoprotein I or its mRNA are localized in selected cells of bone, kidney, decidua and brain (Mark et al., 1988; Nomura et al., 1988; Yoon ef al., 1988). Bone sialoprotein I [also called osteopontin (Oldberg, Franzen and Heinegard, 1986), 44 kDa bone phosphoprotein (Prince et al., 1987) and 2ar (Smith and Denhardt, 1987)], promotes attachment and spreading of connective tissue cells (Somerman et al., 1987a-d, 1989; Oldberg et al., 1986) via an R-G-D cell-binding sequence (Oldberg et al., 1986; Somerman et al., 1988). Furthermore, bone sialoprotein I appears more selective for connective tissue
Plate 1 Fig. 4. Indirect immunofluorescent micrographs of 4-day cultures of periodontal ligament cells exposed to antibodies to human osteonectin (a), biglycan (b), and a representative pre-immune serum control (c). x400. Fig. 5. Northern blots to identify mRNA for (a) osteonectin and (b) biglycan.
245
Characteristics of human periodontal ligament cells
-2.2kb
-2.4kb
5( b 1 Biglycan
5 ( a 11 Osteonectin
Plate 1
M. J. SOMERMAN et al.
246
cells than fibronectin, which enhances attachment of connective tissue cells and epithelial-like cells (Somerman et al., 1989). Bone sialoprotein I promotes attachment of periodontal ligament cells and gingival fibroblasts in oitro (Somerman et al., 1989), but mRNA for bone sialoprotein I was not detectable in periodontal ligament cells or gingival fibroblasts. Thus, human periodontal ligament cells do exhibit some osteoblast-like characteristics, but our studies suggest that they do not behave like classical osteoblast-like cells in vitro. Periodontal ligament cells have high alkaline phosphatase activity when compared with gingival tibroblasts (Kawase et al., 1986; Somerman et al., 1987a; Maeder et al., 1988). Furthermore, when human periodontal ligament cells and human gingival fibroblasts from the same patient are passaged over time, periodontal ligament cells become senescent faster as measured by proliferation, e.g. by passage seven periodontal ligament cells have a decreased proliferation rate, whereas gingival fibroblasts do not exhibit decreased proliferation with passaging (Somerman et af., unpublished observa-
tion). However, to date we have found no other differences between human periodontal ligament cells and human gingival fibroblasts. All our findings for periodontal ligament cells were similar to those for gingival fibroblasts (data not shown). The information obtained should help in understanding the factors that regulate neriodontal ligament cells both in health
and
during’tissue
regeneration.
Acknowledgements-We thank MS Jo Ann Walker for her skilful secretarial support. This study was supported by grants from the U.S. Public Health Service NIDR, NIH DE-07512 and DE-08648. REFERENCES Aubin J., Heersche J., Merriless M. and Sodek J. (1982) Isolation of bone cell clones with differences in growth, hormone response and extracellular matrix production. J. Cell B&l. 92, 452461. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A. and Struhl K. (Eds) (1987) Current Protocols in Molecular Biology, Chap. 4. Wiley, New York. Blomlof L. and Otteskog P. (1981) Composition of human periodontal ligament cells in tissue culture. &and. J. dent. Res. 89, 4346. Bolander M. E., Young M. F., Fisher L. W., Yamada Y. and Termine J. D. (1988) Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid). Proc. natn. Acad. Sci. U.S.A. 85, 2919-2923. Bowers G. M., Schallhorn R. G. and Mellonig J. T. (1982) Histologic evaluation of new attachment in human intrabony defects. A literature review. J. Periodont. 53, 509-5 16. Butler W. T. (Ed.) (1985) The Chemistry and Biology of Mineralized Tissues. Ebsco Media, Birmingham, Ala. Fernyhough W. and Page R. C. (1983) Attachment, growth anh synthesis by human gingival fibroblasts on demineralized or fibronectin-treated normal and diseased tooth roots. J. Periodont. 54, 133-140. Firestone G. L. and Heath E. C. (1980) The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes. Archs Biochem. Biophys. 201, 453461. Fisher L. W. and Termine J. D. (1985) Noncollagenous
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Smith J. H. and Dendhardt D. T. (1987) Molecular cloning of a tumor promoter-:mducible mRNA found in JB6 mouse epidermal cells: Induction is stable at high, but not at low. cell densities. J. Cell Biochem. 34. 13-22. Somerman M. J., Archer S. Y., Imm G. R. and Foster R. A. (1987a) A comparative study of human periodontal ligament cells and gingival ftbroblasts in uitro. J. dent. Res. 67, 6670.
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R. A. (1987b) Enhancement by extracts of mineralized tissues of protein productivity by human gingival fibroblasts. Archs oral Biol. 32, 879-883. Somerman M. J., Prince C. W., Sauk J. J., Foster R. A. and Butler W. T. (1987~) Mechanism of fibroblast attachment to bone extracellular matrix: Role of a 44 kilodalton bone nhosnhovrotein. J. Bone Mineral Res. 2. 259-265. Somerman-M. J., Shteyer A., Bowers M. .R. and Santora A. C. (1987d) Stimulation of mannose incorporation into rat osteoblastic osteosarcoma cells by parathyroid hormone. Archs oral Biol. 32, 535-538. Somerman M. J., Fisher L. W., Foster R. A. and Sauk J. J. (1988) Human bone sialoprotein I and II enhance fibroblast attachment in oitro. Calc. Tiss. Inf. 43, 5Ck53. Somerman M. J., Prince C. W., Butler W. T., Foster R. A., Moehring J. M. and Sauk J. J. (1989) Cell attachment activity of the 44 kilodalton bone phosphoprotein is not restricted to bone cells. Matrix 9, 4954. Wasi S., Otsuka K., Yao K.-L., Tung P. S., Aubin J. E., Sodek J. and Termine J. D. (1984) An osteonectin-like protein in porcine periodontal ligament and its synthesis by periodontal ligament fibroblasts. Can. J. Biochem. Cell Biol. 62, 47&478.
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