Root-end Filling Materials Alter Fibroblast Differentiation

Abstract

Root-end filling materials are commonly used following endodontic surgical procedures; however, their effect on adjacent soft tissues is poorly understood. We predict that, due to the differences in their chemical composition, these materials will have profoundly different effects on the survival and differentiation of fibroblasts. Many of the root-end filling materials examined were initially cytotoxic to both PDL and gingival fibroblasts in co-culture experiments; however, this was reduced after the materials were washed in either mineral trioxide aggregate (MTA) or hybrid ionomere composite resin (HICR) for 2 wks. Additionally, PDL fibroblasts displayed enhanced proliferation on MTA and survival on amalgam when compared with gingival fibroblasts. MTA preferentially induced alkaline phosphatase expression and activity in both PDL and gingival fibroblasts. In contrast, HICR inhibited alkaline phosphatase expression and activity. In addition, MTA and HICR repressed pleiotrophin in PDL fibroblasts, while HICR repressed periostin in both fibroblasts. Thus, root-end filling materials differentially affect periodontal fibroblast differentiation. Abbreviations: mineral trioxide aggregate (MTA), zinc-oxide eugenol cement (ZOEC), hybrid ionomer composite resin (HICR), reverse-transcriptase polymerase chain-reaction (RT-PCR).

INTRODUCTION

Surgical endodontic therapy is performed to prevent egress of irritants from the root canal system into the periradicular tissues (Torabinejad et al., 1995c). This is accomplished through surgical exposure of the root, resection of the apical portion, and placement of a root-end filling material to seal the canal system. An ideal material to seal the root-end cavity should prevent leakage (Gartner and Dorn, 1992). It should have dimensional stability, adherence to the walls of the cavity, resistance to resorption, and moisture resistance (Torabinejad et al., 1995a); it should also be non-toxic and biocompatible to promote healing (Torabinejad et al., 1995c).

For many years, amalgam was accepted as the material of choice for endodontic surgery, but its use came into question when SEM studies showed gaps between the amalgam and the root canal wall, and when concerns about mercury toxicity developed (Dorn and Gartner, 1990). Zinc-oxide eugenol cements (ZOEC) have been suggested as alternate root-end filling materials (Torabinejad and Pitt Ford, 1996). ZOEC offers a better seal than amalgam and is not resorbable. It has high compressive and torsional strength, low solubility, is radiopaque, and has a neutral pH (Oynick and Oynick, 1978), and, in clinical examinations, the success rate was 75% for amalgam and 95% for ZOEC cement (Dorn and Gartner, 1990). A hybrid ionomer composite resin (HICR) (Geristore^®, DenMat, Santa Maria, CA, USA) has been shown in clinical trials to be effective in restoring subgingival areas (Shuman, 1999). It is well-tolerated when used for perforation repairs, and evidence of tissue attachment has been demonstrated histologically (Dragoo, 1997).

A more recently developed material, mineral trioxide aggregate (MTA), has also been advocated for use as a root-end filling material. The principle components of MTA are tricalcium silicate, tricalcium aluminate, tricalcium oxide, and silicate oxide (Torabinejad et al., 1995c). The sealing ability of MTA is superior to that of amalgam and ZOEC, and its seal was not adversely affected by blood contamination (Torabinejad et al., 1993, 1994, 1995b). It was also less cytotoxic than amalgam, ZOEC, and IRM (Torabinejad et al., 1995b). MTA has also been demonstrated to support cementum deposition (Torabinejad et al., 1995d) with reduced inflammation (Koh et al., 1998; Torabinejad et al., 1998).

Fibroblasts are the predominant resident cell type of the periodontal connective tissue (Bartold et al., 2000). Gingival fibroblasts maintain the integrity of gingival connective tissue, while periodontal ligament (PDL) fibroblasts with specialized functions are responsible for the formation and maintenance of periodontal ligament fiber attachments as well as repair, remodeling, and regeneration of the adjacent alveolar bone and cementum (Boyko et al., 1981). Cementogenesis is similar to osteogenesis, requiring the regulated action of specialized cells (cementoblasts and osteoblasts) and potentially fibroblasts (Saygin et al., 2000). While PDL-derived cells and osteoprogenitors are possible precursors for cementoblasts, their origins, formation, and orientation are not well-understood (Pitaru et al., 1994).

The purpose of the present study is to assess the effects of these root-end filling materials on the survival of and osteogenic gene expression in PDL and gingival fibroblasts. We also examined the effects of these materials on cellular alkaline phosphatase activity as a potential indicator of cementogenesis.

MATERIALS & METHODS

Tissue Culture

Periodontal and gingival fibroblasts were established from patients with healthy gingiva who underwent oral surgery at the Louisiana State University School of Dentistry for the purpose of removing impacted wisdom teeth. In all cases, tissues were obtained from subjects following informed consent as prescribed in an approved IRB protocol. PDL fibroblasts were obtained from the PDL remaining attached to extracted molars, while the gingival fibroblasts were obtained from loose gingival tissue that was free of epithelium and associated alveolar bone (Palaiologou et al., 2001). Fibroblasts between the 5th and 16th passages were used in this study.

Preparation of Root-end Filling Materials

Mineral Trioxide Aggregate (MTA) (ProRoot^™, Dentsply Tulsa Dental, Tulsa, OK, USA), Hybrid Ionomer Composite Resin (HICR) (Geristore^®, DenMat Corporation, Santa Maria, CA, USA), zinc oxide eugenol cement (ZOEC) (SuperEBA^™, Bosworth Corporation, Chicago, IL, USA), and amalgam (Tytin^® slow set, Kerr Corporation, Orange, CA, USA) were all prepared according to manufacturers’ instructions and allowed to set for 24 hrs at 37°C in 100% humidity. We then pulverized the materials to increase surface area for potential cell contact and used a sieve to separate them into particle sizes between 180 and 500 μm. Cortical bone (Musculoskeletal Transplant Foundation, Edison, NJ, USA), 150–450 μm, was also used as a particle control. In some cases, the particles were placed into 50-mL tubes containing 30 mL of HEPES buffer solution (N-[2-hydroxyethyl]piperazine-N′-[ethanesulfonic acid], pH 7.2, 100 mM NaCl). We changed the solution daily for 2 wks to allow for removal of toxic by-products before the materials were utilized in the experiments.

Cell Survival and Proliferation Assay

Approximately 2 × 10⁴ fibroblasts in a volume of 500 μL of MEM-α containing 10% fetal bovine serum were plated onto 24-well tissue culture plates. The plates then received either 50 mg of freshly mixed or washed materials. After incubation for 11 to 13 days, the relative number of cells in each sample was determined according to a fluorescent cell attachment protocol (Palaiologou et al., 2001). Briefly, a proprietary green fluorescent dye, CyQUANT GR (Molecular Probes, Eugene, OR, USA), exhibits strong fluorescence enhancement when bound to cellular nucleic acids. Under these conditions, this assay has a linear detection range extending from about 50 to 50,000 cells per microplate well, fluorescing at 530 nm when bound to DNA. The sample fluorescence is measured by means of a fluorescence microplate reader (FL500, BioTek Instruments, Winooski, VT, USA). All data were collected, each group of samples was averaged, and a mean and standard deviation were compared with the control value. ANOVA analysis was applied to all the values, and statistical significance was determined at p < 0.01.

Fibroblast Differentiation

PDL and gingival fibroblasts were plated in T-25 flasks at a density of 2 × 10⁵ cells in MEM-α medium containing 10% fetal bovine serum, penicillin, streptomycin, dexamethasone (100 nM), ascorbic acid (50 μg/mL), and β-glycerophosphate (10 mM) to promote differentiation along a mineralized (osteoblast-like) phenotype (Kuru et al., 1999; Lin et al., 1999). A 200-mg sample of each washed material was added to flasks of each cell type before incubation at 37°C in a humidified incubator gassed with 5% CO₂ with media changes every 3 to 4 days. By day 3, cells formed a loose monolayer covering the entire surface of the flask, with roughly 20% of the cells in direct contact with the materials. The cells were harvested by trypsinization at days 1, 3, 7, and 14. RNA was extracted with the use of guanidine thiocynate, used as a template for reverse-transcriptase polymerase chain-reaction (RT-PCR), and visualized on ethidium-bromide-stained agarose gels (Palaiologou et al., 2001). PCR primers used in this study were: Ribosomal Subunit S15 (5′-TTCCGC AAGTTCACCTACC-3′, 5′-CGGGCCGGCCATGCTTT ACG-3′, 361 Bp, Accession# NM_001018), β1 subunit of Integrin (5′-CAAAGGAACAGC AGAGAAGC-3′, 5′-GTGGAAAACACCA GCAGC-3′, 537 Bp, Accession# NM_002205), Vimentin (5′-ATG TTTCCAAG CCTGACC-3′, 5′-CT GTCCATCTCTAGTTT AAACC-3′, 584 Bp, Accession# X56134), Cellular Fibronectin (5′-ATTGATGCA CCATCCAACC-3′, 5′-TCTGAGAGAGAGCTTCTTGTCC-3′, 930 Bp, Accession# M10905), Collagen Type 1α1 (5′-GATTGA CCCCAACCAAGG-3′, 5′-AGTGA CGCTGTAGGTGAAGC-3′, 409 Bp, Accession# NM000088), Osteonidogen (5′-TCACACTACACCC TTAAGTCG-3′, 5′-TCTAAAGCAAA GAGCCAGC-3′, 432 Bp, Accession# D86425), Osteomodulin (5′-GTGATGTGTCCTTCTATTGACC-3′, 5′-AAAGTCCCCTT CTGCTCC-3′, 282 Bp, Accession# AB000114), Osteonectin (5′-TGTGTGACCCAGGACTACC-3′, 5′-CACCACTCATTGTTAG AAAGC-3′, 617 Bp, Accession# NM004598), TIED (5′-GAATT CCAGTGCGATATCACC-3′, 5′-ACTTCCCACAATGACAA GAA CC-3′, 652 Bp, Accession# AB008375), Pleiotrophin (5′-GGATGACCCCCAAATAGC-3′, 5′-GAAA GGCAGGATGAT GACC-3′, 637 Bp, Accession# AB004306), Periostin (5′-CACACTCTTTGCTCCCACC-3′, 5′-GAATCGCACCGTTT CTCC-3′, 650 Bp, Accession# D13665), Alkaline Phosphatase (5′-GCACCTGCCTTACTAACTCC-3′, 5′-CATGATCACGTC AATGTCC-3′, 626 Bp, Accession# AB011406), Osteopontin (5′-GCATCACCTGTGCCATACC-3′, 5′-CATTCAACTCCTCGC TTTCC-3′, 522 Bp, Accession# J04765), Bone Sialoprotein (5′-TTAGCTGCAATCCAGCTTCC-3′, 5′-CTCCCCCTCGTATT CAACG-3′, 408 Bp, Accession# NM004967), and Osteocalcin (5′-CATGAGAGCCCTCACAC TCC-3′, 5′-CAGCCAACTCG TCACAGTCC-3′, 254 Bp, Accession# X53698).

Alkaline Phosphatase Activity

PDL and gingival fibroblasts were plated into 24-well plates as described above and exposed to 50 mg of each root-end filling material for 1–17 days. Cells were rinsed three times in PBS and frozen to allow cells to undergo lysis. A 500-μL quantity of 1 mg/mL p-nitrophenol phosphate (PNPP) in 0.1 M diethanolamine (pH 8.3) was added to each well and incubated at 25°C for 30 min with gentle agitation. The enzymatic color reaction was stopped by the addition of 500 μL of 0.75 N NaOH, and the mixture was assayed for 405-nm absorbance in a microplate reader (FL600, BioTek, Winooski, VT, USA).

RESULTS

Cell Survival and Proliferation Assay

PDL and gingival fibroblasts grown on tissue culture plastic steadily proliferated through day 11 in culture, with PDL fibroblasts displaying greater proliferation than gingival fibroblasts (Fig. 1). PDL fibroblasts grown in the presence of MTA, HICR, or bone survived and proliferated similarly to those grown on plastic. In contrast, gingival fibroblasts grown on these same materials survived but did not proliferate. Cells were observed in direct contact with particles of all these materials, often overgrowing the particles. In general, cells exposed to washed materials showed greater proliferation than those cultured with fresh materials. Interestingly, washed MTA enhanced PDL fibroblast but not gingival fibroblast proliferation. In contrast, cells cultured with ZOEC died rapidly, regardless of cell type or washing of the materials. Cells also died rapidly when grown on amalgam, with the exception that PDL fibroblasts survived longer on washed amalgam. Cells were rarely observed in direct contact with either ZOEC or amalgam. Thus, PDL and gingival fibroblasts displayed different survival and proliferative responses to the root-end filling materials examined.

Root-end Filling Materials Differentially Affect Fibroblast Gene Expression

PDL fibroblasts

When PDL fibroblasts were grown in media containing dexamethasone to induce a more osteoblast-like phenotype (Kuru et al., 1999; Lin et al., 1999), changes in gene expression were noted (Fig. 2A), and the relative expressions of these transcripts were compared (Fig. 3 ). Cells grown on tissue culture plastic expressed β1 integrin, collagen type I (α1 chain), vimentin, fibronectin, TIED, periostin, osteonidogen, and BSP uniformly. Alkaline phosphatase and pleiotrophin expression increased after day 7 in culture. Cells cultured with amalgam and ZOEC did not survive past day 3 and rapidly lost expression of all of the transcripts examined (data not shown).

PDL fibroblasts cultured with MTA expressed alkaline phosphatase prematurely (by day 3), with declining expression at days 7 and 14. In contrast, periostin expression was reduced by day 14, while pleiotrophin and TIED expression was reduced at all times examined. In contrast, PDL fibroblasts cultured with HICR expressed pleiotrophin and periostin at reduced levels at all times examined, while osteonidogen and TIED expression was reduced only at day 3. In addition, the normal rise in alkaline phosphatase expression observed with plastic on days 7 and 14 was suppressed entirely.

Gingival fibroblasts

Gingival fibroblasts behaved similarly to periodontal ligament fibroblasts grown under these conditions (Figs. 2B , 3 ), with the exception that gingival fibroblasts grown on plastic or HICR did not express alkaline phosphatase or osteopontin, and the expression of both of these transcripts was induced only by the presence of MTA. In addition, osteonectin (whose expression increased with time on both plastic and MTA) was suppressed by HICR.

MTA Induces Alkaline Phosphatase Activity

In our assays, only MTA consistently induced significant levels of alkaline phosphatase activity, and did so for both gingival and PDL fibroblasts (Fig. 4 ). Maximal enzyme activity was detected after 9 days in culture for both cell populations in the presence of washed MTA, while this expression was delayed for 2–4 days with fresh MTA. In addition, PDL fibroblasts displayed roughly twice the alkaline phosphatase activity of gingival fibroblasts. In contrast, HICR could induce alkaline phosphatase activity in PDL fibroblasts only when washed, although this activity was significantly less than that induced by MTA. Interestingly, PDL fibroblasts grown on plastic expressed lower but detectable levels of alkaline phosphatase activity between 7 and 11 days in culture, while gingival fibroblasts did not express similar levels of enzyme activity until day 13 through day 17. Thus, root-end filling materials differentially induced alkaline phosphatase activity in periodontal fibroblasts.

DISCUSSION

The purpose of this study was to assess whether root-end filling materials would cause changes in cell survival/proliferation and in the expression of selected genes associated with hard-tissue formation in cultures of periodontal ligament and gingival fibroblasts. Our results with gingival and PDL cell cultures demonstrate that a different degree of toxicity exists between the materials. Cultures with HICR and bone showed survival rates similar to those of untreated fibroblasts, with the washed material exhibiting a higher level of proliferation than the fresh. ZOEC and amalgam were highly toxic, killing the majority of the cells the first day. In contrast, PDL fibroblasts cultured with MTA had enhanced proliferation compared with gingival cultures that had very little proliferation.

We hypothesized that MTA would induce fibroblasts to express genes associated with cementum formation. This is based on studies in both monkeys and dogs in which cementum deposition was histologically observed on this root-end filling material (Torabinejad et al., 1995d, 1997). Our results indicated that both periodontal ligament and gingival fibroblasts are capable of expressing genes indicative of osseous repair as well as cementum deposition, specifically alkaline phosphatase. Our results indicate that MTA induced premature and enhanced expression of alkaline phosphatase activity for both fibroblast populations. In contrast, HICR prompted alkaline phosphatase activity only in PDL fibroblasts, and to a lesser extent than MTA. Alkaline phosphatase is a phosphate-releasing protein, and its activity is considered an important indicator of bone formation and a phenotypic marker of osteoblasts (Kuru et al., 1999). The observation that alkaline phosphatase transcript levels peak prior to any detectable increase in alkaline phosphatase activity following exposure to MTA was not unexpected; however, the finding that enzyme activity levels increased after a decline in transcript levels was unexpected. Further studies will need to be performed for a more careful examination of the correlation of transcript and protein stability with respect to the regulation of functional levels of phosphatase activity.

Changes in expression patterns of several transcripts associated with hard-tissue formation were also detected. Periostin is an ECM protein that appears in cells with the ability to differentiate into osteoblasts. This protein is preferentially expressed in periosteum and periodontal ligament, indicating its potential role in bone and tooth formation and in the recruitment and attachment of osteoblast precursors in the periosteum (Horiuchi et al., 1999). This transcript was expressed in PDL and gingival fibroblasts cultured with MTA, but its expression was repressed when cultured with HICR. Osteonidogen is a member of a family of glycoproteins involved in the formation of a complex with type IV collagen and laminin and is thought to play a critical role in basement membrane assembly (Kang and Kramer, 2000). Osteonidogen was expressed when gingival fibroblasts were grown with MTA, but was reduced in the presence of HICR. In contrast, osteonidogen expression was delayed in PDL fibroblasts grown on HICR. Pleiotrophin (osteoblast-stimulating factor) promotes in vitro proliferation and differentiation of osteoblasts (Masuda et al., 1997). Its expression was repressed in PDL fibroblasts with both MTA and HICR. TIED expression was repressed in PDL fibroblasts in contact with HICR, but the function of this cell-surface protein related to the integrin β subunit is unknown (Berg et al., 1999).

Analysis of our data indicates that MTA generally induces an osteogenic phenotype (alkaline phosphatase, osteonidogen, osteonectin, and osteopontin), while HICR tended to inhibit the expression of osteogenic transcripts (alkaline phosphatase, periostin, pleiotrophin). Thus, different root-end filling materials influence the differentiation of PDL and gingival fibroblasts in different ways.

Figure 1.

Survival and proliferation of fibroblasts on root-end filling materials. The survival and proliferation of gingival and PDL fibroblasts grown (A) on tissue culture plastic were examined fluorometrically as a measure of total DNA content within these cells. The effects of several root-end filling materials (B, Bone; C, MTA; D, Amalgam; E, HICR; F, ZOEC) were also examined. Gingival fibroblasts (triangles) and PDL fibroblasts (squares) were compared for freshly prepared materials (solid lines; solid symbols) and those thoroughly washed for 2 wks (dashed lines; open symbols). The error bars represent the standard deviation of 4 samples for each material. Plastic represents the results of 2 independent experiments, neither involving washing. ANOVA analysis was applied to all the values, and statistical significance was determined at p < 0.01. Data points represent a typical experiment that was repeated in triplicate.

Figure 2.

Transcript alteration by root-end filling materials. Semi-quantitative analysis of transcript expression by periodontal ligament and gingival fibroblasts was performed with RT-PCR. The expression of transcripts associated with hard-tissue formation was examined when these fibroblasts were cultured with different root-end filling materials. β1 integrin is an ECM protein found in all cell types and was used as a standard control for template cDNA quality and quantity. Alk Phos = alkaline phosphatase; BSP = Bone sialoprotein; MTA = mineral trioxide aggregate.

Figure 3.

Changes in relative transcript expression. The relative expressions of specific transcripts were compared for cells (either gingival fibroblasts [GF] or periodontal ligament fibroblasts [PDLF]) treated with root-end filling materials (HICR and MTA) with respect to control cultures grown on plastic. All cells were treated with 10 mM dexamethasone to stimulate differentiation to a mineralized phenotype. The RNA levels in all samples were first normalized to depict uniform expression of the S15 ribosomal subunit. The Fig. depicts the relative expression of differentially expressed transcripts as the mean ± standard deviation of 3 independent experiments. The relative levels of expression for a specific transcript are expressed as a ratio of the expression by cells grown in the presence of the material and those grown on plastic, where a value of 1 indicates identical levels of expression. The dashed rectangle represents the area in which relative expression values are not statistically different from one another. In this study, we determined statistically that expression levels were significantly different if there was a five-fold or greater difference in expression, based upon the average variance between samples with a group (40%), the number of independent samples evaluated (4), and a confidence level of greater than 99% by ANOVA analysis. Each point represents the mean of 3 samples. Error bars represent standard deviation from the mean. Ob Spec = osteoblast specific factor-1, Ob Cys = osteoblast cysteine-rich protein, Ob Stim = osteoblast-stimulating factor, Alk Phos = alkaline phosphatase, BSP-2 = Bone sialoprotein-2, MTA = mineral trioxide aggregate.

Figure 4.

Effects of root-end filling material on alkaline phosphatase activity. Alkaline phosphatase activity was measured colorimetrically for PDL fibroblasts and gingival fibroblasts (GF) grown in the presence of plastic (solid diamonds), MTA (solid squares), HICR (solid triangles), and bone particles (circles) for from 1 to 17 days in culture. Enzyme activity was compared for PDL fibroblasts (A,C) and gingival fibroblasts (B,D) cultured in the presence of fresh (C and D) and washed (A and B) root-end filling materials. Note that the PDL fibroblasts expressed approximately twice the alkaline phosphatase activity of gingival fibroblasts. Each point represents the mean of 4 samples. Error bars represent standard deviation from the mean. ANOVA analysis was applied to all the values, and statistical significance was determined at p < 0.01. Data points represent a typical experiment that was repeated in triplicate.

Footnotes

Notes

A supplemental appendix to this article is published electronically only at .

Acknowledgements

We thank Amber Spencer for invaluable technical assistance. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF (2000–05)-04.

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