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Osteogenic Gene Expression by Human Periodontal Ligament Cells under Cyclic Tension

¹ Department of Oral Sciences, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin, New Zealand;
² Department of Zoology, Division of Sciences, University of Otago, Dunedin, New Zealand; and
³ Faculty of Dentistry, National University of Singapore, Level 3 National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074

Correspondence: ^* corresponding author, pndmcm{at}nus.edu.sg

	ABSTRACT

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The forces that orthodontic appliances apply to the teeth are transmitted through the periodontal ligament (PDL) to the supporting alveolar bone, leading to the deposition or resorption of bone, depending upon whether the tissues are exposed to a tensile or compressive mechanical strain. To evaluate the osteogenic potential of PDL cells, we applied a 12% uni-axial cyclic tensile strain to cultured human PDL cells and analyzed the differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism by real-time RT-PCR array technology. Sixteen genes showed statistically significant changes in expression in response to alterations in their mechanical environment, including cell adhesion molecules and collagen fiber types. Genes linked to the osteoblast phenotype that were up-regulated included BMP2, BMP6, ALP, SOX9, MSX1, and VEGFA; those down-regulated included BMP4 and EGF. This study has expanded our knowledge of the transcriptional profile of PDL cells and identified several new mechanoresponsive genes.

Key Words: RT-PCR array • mechanical strain • osteogenic genes • periodontal ligament cells

	INTRODUCTION

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Orthodontic tooth movement is dependent upon the ease with which the periodontal ligament (PDL) can be remodeled by mechanical means, the forces applied to the teeth being transmitted through the PDL to the supporting alveolar bone. Changes in the metabolic and proliferative activity of the cellular constituents of the PDL are initiated, leading eventually to the deposition or resorption of bone, depending upon whether the tissues are exposed to a tensile or compressive mechanical strain (for a recent review, see Meikle, 2006).

New bone formation on the tension side of the tooth has traditionally been attributed to osteogenic cells originating from the alveolus. However, PDL cells are functionally heterogeneous and have been shown to: (1) exhibit osteogenic potential when cultured in the presence of ascorbic acid, sodium β-glycerophosphate, and dexamethasone (Cho et al., 1992; Basdra and Komposch, 1997); and (2) contain a subpopulation of cells able to produce the osteoblast-related matrix proteins osteopontin, alkaline phosphatase (ALP), and bone sialoprotein (BSP; Lekic et al., 2001; Murakami et al., 2003). It seems likely, therefore, that, under strained conditions, PDL cells have the capacity to differentiate into osteoblasts, as originally hypothesized by Roberts and Chase (1981), following experimental tooth movement in the rat.

For the present study, we proposed that PDL cells exposed to tensile mechanical strain during orthodontic tooth movement would express multiple genes involved in osteogenesis. We therefore applied intermittent tensile strain to human PDL cells in monolayer culture and quantified the differential expression of 78 genes implicated in osteogenesis and bone metabolism, using real-time polymerase chain-reaction (PCR) array analysis.

	MATERIALS & METHODS

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Preparation of Human PDL Cells
Human PDL fibroblasts were prepared from premolar teeth (extracted for orthodontic reasons) as described previously (Somerman et al., 1988), a procedure which has been widely used for studies of PDL cells. Human PDL cells constitute a heterogeneous cell population (Lekic et al., 2001) and have the potential to differentiate into various phenotypes, including osteoblasts, chondrocytes, and adipocytes (Gay et al., 2007). Teeth were washed with phosphate-buffered saline (PBS), and the PDL attached to the middle third of the root was removed with a scalpel. Tissue explants were plated onto 1.9-cm², 24-well Nunclon multidishes in Dulbecco’s modification of Eagle’s medium (DMEM; Invitrogen, Auckland, NZ) supplemented with 10% fetal calf serum (GIBCO) and antibiotic-antimycotic reagent (10,000 units penicillin, 10,000 µg streptomycin, and 25 µg/mL amphotericin B; Invitrogen), 100 mMol L-glutamine (Invitrogen), and Gentamycin (10 mg/mL, GIBCO) and cultured at 37°C in a humidified atmosphere of 5% CO₂/95% air. On reaching confluence, the cells were trypsinized and serially passaged through 25-cm², 75-cm², and 175-cm² tissue culture flasks (Cellstar; Greiner Bio-One AG, Frickenhausen, Germany). Stocks of cells were frozen in Cell Culture Freezing Medium (GIBCO) at –80°C and then transferred to liquid nitrogen for long-term storage. Several human PDL cell lines have been established, and fourth-passage cells from a single individual were used in this study. Approval to harvest human tissue from extracted teeth with the consent of the donor and/or parent was granted by the University of Otago Ethics Committee (Reference: 05/069).

Application of Tensile Strain to PDL Cells
Human PDL cells (3 x 10⁵/well) were subcultured into six-well, 35-mm flexible-bottomed Uniflex culture plates with a centrally located rectangular portion (15.25 mm x 24.18 mm) coated with type I collagen designed to provide a uniform uni-axial strain, and subjected to an intermittent deformation of 12% for 6 sec every 90 sec with a Flexercell FX-4000 Strain Unit (Flexcell Corporation, Hillsborough, NC, USA), as described previously (Garcia-López et al., 2005). Uni-axial strain was chosen to represent more closely the deformation to which PDL cells are exposed in vivo during occlusal loading and orthodontic tooth movement. The strain value of 12% was based on numerical data derived from a finite element model (Natali et al., 2004). This suggested that maximal PDL strains for horizontal displacements of a human maxillary central incisor under physiological loading conditions lie in the vicinity of 8–25%, depending upon the apico-crestal position; a value of 12% correlates well with strain conditions predicted at the mid-root. Four plates (2 experimental and 2 control) were allocated to each of the 3 time intervals (6, 12, and 24 hrs).

Isolation of RNA
At the end of each time-course, media were removed, and total RNA was extracted by a modification of the method described by Chomczynski and Sacchi (1987). Briefly, 0.5 mL of Trizol reagent (Invitrogen, Auckland, NZ) was added to each well of the Uniflex plate. After a five-minute incubation period, the cell lysate was added to a tube containing 0.1 mL of chloroform and shaken vigorously by hand for 15 sec. A further incubation of 2–3 min at room temp was required prior to centrifugation of the samples at 12,000 x g for 15 min at 4°C. Following centrifugation, a 300-µL quantity of the clear aqueous phase was added to an equal volume of 70% ethanol and vortexed to disperse the precipitate. Samples were then purified by means of the Purelink Micro-to-Midi Total RNA Purification System (Invitrogen). Samples were processed in accordance with the manufacturer’s instructions. RNA samples were eluted from the columns in 50 µL of RNase-free water and stored at –80°C. The concentration and purity (A₂₆₀/A₂₈₀) of the samples were determined by a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies, Rockland, DE, USA).

Real-time PCR Microarray Analysis
Total RNA samples were assessed for degradation status by denaturing agarose gel electrophoresis, prior to analysis by Superarray Bioscience Corporation (Frederick, MD, USA). Contaminating genomic DNA was removed from total RNA samples by DNaseI digestion prior to first-strand synthesis. First-strand synthesis was performed with the RT² PCR array First Strand Kit (Superarray Bioscience Corporation, Frederick, MD, USA). Samples were then screened for the expression of 78 genes of osteogenic significance by means of the RT² Profiler PCR Array System (Superarray Bioscience Corporation, Frederick, MD, USA). To obtain statistically robust data, we analyzed control and experimental samples at each of the 3 time-points in triplicate.

Statistical Methods
Changes in the expression of target genes were measured relative to the mean critical threshold (CT) values of 5 different calibrator genes (GAPDH, β-2-microglobulin, β-actin, HPRT1, and RPLI3A), by the CT method described previously (Livak and Schmittgen, 2001). T tests were used for statistical comparison of the control and experimental groups, with mean CT values derived from the triplicate samples (Yuan et al., 2006).

	RESULTS

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Evaluation of the plates by light microscopy showed that by 6 hrs the PDL cells in the experimental group had become reoriented away from the direction of stretch, as previously reported (Neidlinger-Wilke et al., 2002), demonstrating the responsiveness of the cells to the magnitude and frequency of the applied force. The 78 genes that were screened and the effects of intermittent strain on their differential expression are shown in the APPENDIX Table, a feature being the constitutive expression of numerous genes linked to osteoblast differentiation and bone metabolism. A gene was regarded as being constitutively expressed if it was detected at a CT of less than 35, and the dataset reflects a pattern of constitutive expression for the majority of genes screened (Fig. 1). CT values greater than 35 lie outside the detection threshold of the system; the gene was therefore considered not to have been expressed.

View larger version (10K): [in this window] [in a new window]   Figure 1. Histogram showing the mean cycle threshold (CT) distribution (± 1 SD) for experimental and control groups at 6 hrs. The mean values were determined from 3 replicate microarray plates. Low CT values (< 25) represent genes present at high transcript copy number. Genes with CT values of greater than 35 (absent calls) were considered to lie outside the detection threshold of the system. The cycle threshold distribution for the six-hour time-point was representative of the pattern observed at 12 and 24 hrs.

View larger version (49K): [in this window] [in a new window]   Figure 2. 3-D profile of genes showing a statistically significant (P < 0.05) up- or down-regulation of mRNA expression in response to a time-dependent exposure to a uni-axial, intermittent tensile mechanical strain of 12%. Two cell adhesion molecules, 3 BMPs, MSX1, and SOX9 were among the genes whose regulation appears to be sensitive to changes in their mechanical environment. Fold differences are represented on a logarithmic scale. Values on the graph floor in parentheses are displayed for negatively regulated genes and correspond to those represented in the Table.

	DISCUSSION

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Cultured human PDL cells have been widely used to study the effects of tensile mechanical strain on the expression of individual genes of interest at both the message and protein levels (Saito et al., 1991; Shimizu et al., 1994; Howard et al., 1998; Long et al., 2001; Chiba and Mitani, 2004; Yang et al., 2006). The results can sometimes be ambiguous, however, depending upon the culture conditions, stress regime, and the design of the mechanical deformation apparatus. For example, cyclic tension has been reported to both inhibit (Chiba and Mitani, 2004) and stimulate (Yang et al., 2006) the synthesis of ALP.

One advantage of real-time PCR microarray technology is that the quantitative expression of a large number of genes can be screened at the same time under identical experimental conditions, and by measuring control and experimental samples in triplicate, one can obtain statistically robust and therefore more meaningful data. The custom to date has been to express data in terms of fold-change, with a T/C ratio of ± 2 being regarded as representing a significant change in expression. Our findings suggest that this will both under- and over-report the number of genes influenced by the applied force, resulting in false-negatives and false-positives. For the majority of genes recording a statistically significant change, the T/C ratio was less than 2, and for several others for which the T/C ratio was greater than 2, the difference did not reach statistical significance.

Cells respond to mechanical forces via two specialized structures: focal adhesions linking cells to the surrounding extracellular matrix, and adherens junctions linking adjacent cells. In addition to functioning as cell adhesion molecules, these cell-matrix and cell-cell contacts initiate intracellular signaling cascades (outside-in signaling), linked to the cytoskeleton, that regulate many aspects of cell physiology (Wang et al., 1993). As one might expect, subjecting PDL cells to mechanical deformation significantly altered the expression of 2 adhesion molecules, ICAM1 (up) and the integrin ITGB1 (down). I-CAM1 is a cell-cell adhesion molecule, suggesting that the effect of tensile strain was to increase intercellular contact. Integrinβ1 is a cell-matrix adhesion molecule that binds to collagen and laminin, the reduction in ITGB1 expression being consistent with the re-orientation of the cells, observed by light microscopy.

Several genes involved in osteoblast differentiation and function—including BMP2, BMP6, and ALP—were up-regulated by mechanical deformation. BMP2 is a member of the TGF-β superfamily and plays a key regulatory role as a cell-cell signaling molecule during bone formation and repair (Wozney et al., 1988; Wozney and Rosen, 1998). BMP2-responsive genes include ALP, and the transcription factors SOX9, required for the differentiation of chondrocytes from mesenchymal stem cells (Bell et al., 1997), and RUNX2 (also known as core-binding factor alpha-1; CBFA1), which is essential for osteoblast differentiation and bone formation (Komori et al., 1997; Otto et al., 1997). Interestingly, it was SOX9 that was up-regulated along with the COL2A1 gene, and not RUNX2, which suggests that, during the time-course of the experiment, some of the cells were committed to the chondrogenic, and not the osteoblastic, pathway. However, this may be a tissue culture artifact related to the level of oxygenation of the cells. When BMPs in a suitable carrier are implanted into extraskeletal sites, they first induce chondrogenesis, thereby mimicking the normal developmental events of endochondral ossification (Urist, 1965). Only after the implants have become vascularized does bone form. In other words, BMPs behave initially as cartilage morphogenetic proteins. The PDL is a highly vascular tissue, and the angiogenic factor VEGF, a mitogen that acts primarily on vascular endothelial cells, appears to be a major regulator of blood vessel formation in the PDL (Kohno et al., 2003). VEGF expression was significantly elevated by mechanical strain by 24 hrs.

A further complication of relevance to the question of the particular differentiation pathway the cells may be following is their functional heterogeneity, the precise function of which is poorly understood (Lekic et al., 2001). Given its location, however, one might expect the PDL to provide progenitor cells for osteogenesis, cementogenesis, and fibrogenesis. Matsuda et al.(1998), in a study of mechanical stress-induced osteoblast differentiation by human PDL cells in vitro, suggested that the EGF/EGFR system acts as a negative regulator of osteoblast differentiation. Our finding that EGF expression was down-regulated, as was the EGFR gene (although not quite to statistical significance, P = 0.0664), is consistent with this observation, and suggests that some PDL clones were differentiating along the osteoblastic pathway.

Given the multiple effects and overlapping biological activities (redundancy) shown by the BMPs, whether the down-regulation of BMP4 expression has functional significance for the PDL is unclear. One of the distinctive features of the PDL is that it does not normally undergo ossification; ankylosis in the permanent dentition is uncommon, raising the question, Do ligament cells produce an osteo-inhibitory message, preventing ossification of the PDL? While occlusal loading during mastication no doubt plays a role, the molecular basis of the craniosynostoses, a family of disorders characterized by premature fusion of the craniofacial sutures, may offer some clues. Analysis of our data shows that human PDL cells constitutively express mRNAs for FGFR1, a gene that is over-expressed in Pfeiffer syndrome, as well as TWIST, a gene in which loss-of-function mutations cause Saethre-Chotzen syndrome (Baraitser and Winter, 2001). Kim et al.(1998) found that when BMP4 protein beads were applied to sagittal sutures in mice, induction of Msx1 and Msx2 expression and an increase in tissue volume resulted. This is a significant finding, because Boston-type craniosynostosis is caused by a gain-of-function mutation in the MSX2 gene. We did not measure MSX2, but found that down-regulation of BMP4 by mechanical loading was followed by a decrease in MSX1 expression.

Two fibrillar collagen genes, COL3A1 and COL11A1, were down-regulated by small but statistically significant amounts. Type XI collagen is essential for the formation of cartilage collagen fibrils, as well as for the differentiation and spatial organization of growth plate chondrocytes (Li et al., 1995). Its expression, however, is not limited to cartilage (Bernard et al., 1988). The down-regulation of COL3A1 was unexpected. Previously, we had shown, in a rabbit calvarial suture model, that sutural fibroblasts under tensile strain responded by synthesizing significant quantities of type III collagen, in addition to type I (Meikle et al., 1982). Type III collagen is commonly found in tissues undergoing rapid turnover, and in vitro studies have shown that cells respond to mechanical strain by increased proliferation (Brunette, 1984; Buckley et al., 1988). The difference may reflect the behavior of cells in monolayer culture as opposed to those in situ with an intact extracellular matrix.

In summary, the results of this quantitative real-time array analysis suggest that human PDL cells exposed to cyclic mechanical strain in vitro can express an osteogenic and chondrogenic transcriptional profile. Although message is not always translated into protein, analysis of these data should facilitate future studies of the involvement of these genes in remodeling the periodontium in vivo. This will require assaying culture supernatants for the expressed proteins of interest by ELISAs, and testing their biological activity with the appropriate bioassays, followed by immunolocalization of the proteins in situ with specific antibodies in animal models of tooth movement.

	ACKNOWLEDGMENTS

 
We thank the New Zealand Lottery Grants Board, the NZ Dental Association Research Foundation, the NZ Association of Orthodontists, and the University of Otago Research Committee for their generous financial support.

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received for publication April 30, 2007. Revision received September 14, 2007. Accepted for publication September 17, 2007.

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Journal of Dental Research, Vol. 86, No. 12, 1212-1216 (2007)
DOI: 10.1177/154405910708601214


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