Introduction

During orthodontic treatment, compressive forces were applied to the tooth and transmitted to the periodontal ligament. The main cell type, human periodontal ligament fibroblasts (HPdLFs), responds with the release of inflammatory cytokines, promoting bone resorption and periodontal remodeling to enable orthodontic tooth movement [1].

Interestingly, the early phase of orthodontic tooth movement consists of an acute inflammatory response [2]. Mechanical force causes capillary vasodilatation, followed by migration of leukocytes and the release of cytokines. One cytokine playing a crucial role in orthodontics is IL-1ß. Blocking IL-1ß by a soluble receptor inhibits orthodontic tooth movement [3]. IL-1ß amplifies the inflammatory response by the release of various pro-inflammatory factors in osteoblasts and HPdLFs. Orthodontic relevant pro-inflammatory factors are prostaglandin E-2 (PGE-2), cytokines like IL-6 and TNF-α, and proteases like the matrix-metalloproteinase 8 (MMP-8). All factors are upregulated in the gingival cervical fluid under compressive force during orthodontic treatment and associated with bone and periodontal turnover [4,5,6].

Bisphosphonates were traditionally used in the therapy of metabolic bone diseases like osteoporosis. With the increasing quantity of adults seeking for orthodontic therapy, patients taking bisphosphonates during orthodontic therapy will increase [7].

The principal therapeutic effect of bisphosphonates results from a suppressed bone resorption by a direct inhibition of osteoclast activity. Due to their mechanism of action, they can be classified in two groups. The non-nitrogen-containing bisphosphonate inhibits bone resorption by synthesizing cytotoxic ATP derivatives, which induce apoptosis of osteoclasts [8]. Clodronate is a first-generation non-nitrogen-containing bisphosphonate, often prescribed and subsequently subject of studies for over 30 years [9]. In addition to the principal inhibitory effect on osteoclasts, various studies demonstrate an interaction of clodronate with other cell types including HPdLFs, endothelial cells, and osteoblasts [10, 11]. Clodronate is often discussed for its anti-inflammatory properties. In connection with this, recent studies examine the use of clodronate in the treatment of destructive inflammatory diseases like periodontitis, osteoarthritis, and acute pancreatitis [12,13,14]. In vitro, clodronate is known to change the expression of various pro-inflammatory molecules like NO [15], MMPs [14], IL-1ß [16], and TNF-α [17].

In vivo studies observed a decelerated orthodontic tooth movement under clodronate intake [18, 19]. Lotwala et al. described a prolonged treatment time and increased numbers of incomplete extraction space closure in patients taking bisphosphonates [20]. Further studies demonstrated reduced osteoclast numbers during orthodontic treatments in rats by clodronate [19]. Searching the causal molecular mechanism, Liu et al. consider the inhibitory effect of clodronate on COX-2-dependent PGE-2 production in periodontal ligament cells [21]. The interaction between compressive force and clodronate regarding their effects on cell metabolism is not clear yet.

The aim of the study was to investigate the anti-inflammatory potential of clodronate on HPdLFs under compressive force. For this, HPdLFs were stimulated by IL-1ß in vitro and exposed to clodronate as well as compressive force.

Materials and methods

Cell culture

Commercially available HPdLFs (Lonza, Basel, Switzerland) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, USA) containing 10% fetal calf serum (FCS; Life Technologies GmbH, Darmstadt, Germany), 500 ng/mL beta fibroblast growth factor (bFGF; Life Technologies GmbH, Darmstadt, Germany), and 1% penicillin/streptomycin/neomycin. Cell characterization was provided by the manufacturers and verified via light microscopy and PCR detecting specific markers [22]. HPdLFs were incubated in a cell culture incubator with 5% CO2 and 95% air at 37 °C. When the cells reached confluence, they were detached by using 0.05% trypsin-EDTA solution (PAA, Pasching, Austria).

Bisphosphonates and interleukin-1ß

HPdLFs from passage three to six were used for this study. They were cultured in 6-well plates in the concentration of 100,000 cells/well. After 24 h of cell adhesion, medium was replaced by starvation medium containing 1% FCS. Depending on the group, recombinant human IL-1ß (R&D Systems, Minneapolis, USA) in a concentration of 10 ng/mL was added to the medium. This concentration has also been used in several studies to mimic inflammation in vitro [23,24,25]. The non-nitrogen-containing bisphosphonate clodronate (Bonefos, Bayer AG, Leverkusen, Germany) was added in a concentration of 5 μM. Previous studies showed that a concentration higher than 5 μM starts to affect cell viability significantly and lower concentrations showed absent effect [10, 26,27,28]. The incubation period was 48 h before mechanical compression.

Simulation of compressive force

Compressive force was applied on the 6-well plates by a centrifuge with swing-out rotor (Heraeus Labofuge 400, Heraeus Holding GmbH, Hanau, Germany) for 3 h at 124 g. The centrifugation force of 34.9 g/cm2 was calculated as described in the literature [29, 30].

Isolating RNA and cDNA

The peqGOLD Total RNA KIT (peqLab Biotechnology GmbH, Erlangen, Germany) was used to isolate the messenger RNA (mRNA) of the HPdLFs. The manufacturer’s protocol includes a DNase digestion step. To guarantee the quality, concentration (260) and purity (ratio, 260/280) of the mRNA were measured with the NanoDrop-Spectrophotometer ND-100 (peqLab Biotechnology GmbH, Erlangen, Germany). Reverse transcription of RNA (100 ng) was performed by standard protocol with the iScript cDNA Synthesis Kit (Biorad Laboratories, Hercules, USA) in a total volume of 20 μL and incubation in a thermal cycler (peqSTAR 96 Universal Gradient, peqLab Biotechnology GmbH, Erlangen, Germany).

Real-time PCR

The primers COX-2, IL-6, TNF-α, MMP-8, and TIMP-1 (Eurofins MWG Operon, Ebersberg, Germany) were designed with the NCBI-nucleotide library and Primer3-design. All primers had been matched to the mRNA sequences of the target genes (NCBI Blast software). As housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected, because of the most stable expression detected by the freeware called GeNorm (Table 1).

Table 1 Oligonucleotide primer sequences used for real-time PCR
Full size table

Quantitative real-time PCR was performed with the IQ5-I-Cycler and IQ5 Optical System software version 2.0 (Bio-Rad Laboratories, Hercules, USA) according to the manufacturer’s instructions. The initial denaturation was induced at 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s (denaturation), 56 °C for 30 s (annealing), and 71 °C for 20 s (elongation). Q-PCR amplification was conducted with a reaction mixture containing SYBR Green Supermix (Biorad Laboratories, Hercules, USA), an appropriate amount of paired primers, and 2 μL template cDNA. The background was to determine the threshold at the SYBR green fluorescence curve at the exponential part. This method was applied to calculate the cycle number and CT value for quantitation.

Furthermore, the CT values of GAPDH housekeeping gene and the individual primer efficacy were taken into account. Single product formation was confirmed by melting point analysis. Data were obtained from three individual experiments and normalized to the CT of GAPDH. CDNA from individual cell experiments was analyzed in triplicate PCR. The relative expression levels of each mRNA were evaluated by using a modification of the ΔΔCT method [31].

Enzyme-linked immunosorbent assay

To verify changings on the gene expression levels of COX-2, IL-6, MMP-8, and TIMP-1, an ELISA was added for IL-6, MMP-8, TIMP-1, and the synthesis product of COX-2 and PGE-2. With regard to the time lag between gene and protein synthesis, cell culture supernatants were collected 6 h after the application of compressive force. Protein synthesis level of IL-6, MMP-8, and TIMP-1 was quantified by the DuoSet ELISA Development Kit (R&D Systems, Minneapolis, USA) and protein synthesis of PGE-2 with the Prostaglandin E2 Parameter Assay Kit (R&D Systems, Minneapolis, USA). Optical density was determined using a microplate reader (VersaMax Microplate Reader; Molecular Devices, Sunnyvale, USA) set to 450 nm, and wavelength correction was set to 540 nm. Supernatants of untreated cells were provided as controls. The experiments were performed in triplicate.

Statistical analysis

SPSS 22.0 (IBM-SPSS, Ehningen, Germany) was used for statistical analyses. Normal distributions of the results were verified by Shapiro-Wilk’s test (p > 0.05). To detect the difference between the groups, one-way ANOVA was used with the post hoc Tukey test. A p value ≤ 0.05 was considered statistically significant.

Results

Gene expression of COX-2, IL-6, and TNF-α

Stimulation of HPdLFs with IL-1ß and clodronate led to an increase of COX-2 gene expression about 71-fold compared with the control group. Compressive force induced a 4.4-fold overexpression of COX-2 compared with the control, further enhanced by the stimulation of IL-1ß (61.8-fold compared with the control cells). Combination of compressive force, IL-1ß, and clodronate reduced the overexpression to a 41.1-fold increase (Fig. 1a).

Fig. 1
figure 1

Gene expression of COX-2 (a), IL-6 (b), and TNF-α (c) in HPdLFs exposed to 5 μM clodronate in combination with 10 ng/mL IL-1ß for 48 h and to 3 h of compressive force (compression) as fold of untreated cells (control). Data were obtained from three individual experiments. Asterisks indicate significant difference compared with untreated control cells (*p < 0.05)

Full size image

IL-1ß increased the IL-6 gene expression (73.6-fold compared with the control cells) in HPdLFs. Compressive force did not affect IL-6 gene expression significantly. When stimulated by IL-1ß, compressive force had an additive effect on IL-6 gene expression in HPdLFs up to a 161-fold increase compared with the control. When combined with clodronate, the IL-1ß-induced IL-6 gene expression increase was abolished (85.6-fold compared with the control cells) (Fig. 1b).

Gene expression of TNF-α was nearly doubled by IL-1ß when compared with the control, whereas the combination of IL-1ß and clodronate led to a smaller increase (1.6-fold compared with the control cells), both results without statistical significance. Compressive force had no statistical significant influence on TNF-α gene expression. Stimulation of HPdLFs with IL-1ß and compressive force slightly enhanced TNF-α gene expression with (1.3-fold compared with the control cells) and without (1.4-fold compared with the control cells) clodronate (Fig. 1c).

Protein synthesis of PGE-2 and IL-6

The PGE-2 protein levels in the supernatants substantiate the results at the gene expression level of its synthesizing enzyme COX-2. Neither control HPdLFs nor HPdLFs exposed to compressive force showed any verifiable PGE-2 level (≤ 9.7 pg/mL). IL-1ß-stimulated HPdLFs slightly enhanced PGE-2 protein synthesis to a detectable level with and without clodronate (134.5 pg/mL IL-1ß; 185.1 pg/mL IL-1ß and clodronate). In the presence of compressive force, IL-1ß led to a significant increase of the PGE-2 protein synthesis (1673.5 pg/mL) and combining with clodronate showed a slightly reduced effect (1436.2 pg/mL) compared with the control (Fig. 2a).

Fig. 2
figure 2

Protein synthesis of PGE-2 (a) and IL-6 (b) in HPdLFs exposed to 5 μM clodronate in combination with 10 ng/mL IL-1ß for 48 h and to 3 h of compressive force (compression) as fold of untreated cells (control). Data were obtained from three individual experiments. Asterisks indicate significant difference compared with untreated control cells (*p < 0.05)

Full size image

The protein synthesis of IL-6 increased significantly in HPdLFs stimulated with IL-1ß (25,705.6 pg/mL) compared with the control (15.5 pg/mL), whereas combining with clodronate had no additional effect (25,971.7 pg/mL). Compressive force had no influence on the protein level of IL-6 (21.7 pg/mL) compared with the control. IL-1ß-induced increase of IL-6 protein synthesis with (27,149.9 pg/mL) and without (27,643.6 pg/mL) clodronate was not affected by compressive force (Fig. 2b).

Gene expression and protein synthesis of MMP-8 and TIMP-1

IL-1ß increased the gene expression of MMP-8 about 8.5-fold compared with the control group; clodronate reduced the IL-1ß-induced gene expression to a 5.4-fold increase. Compressive force led to a 2.6-fold increase and had an additive effect on IL-1ß-induced increase (9.9-fold), whereas clodronate in turn reduced gene expression level of MMP-8 to a 7.6 increase (Fig. 3a). Gene expression of TIMP-1 was reduced in all examined groups. Compressive force led to the lowest significant TIMP-1 expression (0.3-fold) followed by the combinations of IL-1ß with clodronate (0.4-fold) and compressive force with IL-1ß (0.5-fold) (Fig 3b).

Fig. 3
figure 3

Gene expression of MMP-8 (a), TIMP-1 (b), ratio of MMP-8/TIMP-1 (c), and protein synthesis of TIMP-1 (d) in HPdLFs exposed to 5 μM clodronate in combination with 10 ng/mL IL-1ß for 48 h and to 3 h of compressive force (compression) as fold of untreated cells (control). Data were obtained from three individual experiments. Asterisks indicate significant difference compared with untreated control cells (*p < 0.05)

Full size image

The protein level of MMP-8 in the supernatants of all examined groups was below the measurable quantity (< 62.5 pg/mL; data not shown). Protein synthesis of TIMP-1 confirmed the results at the gene level. TIMP-1 protein level was significantly reduced in all groups compared with the control group (2197 pg/mL). Compressive force decreased protein level (1052.7 pg/mL) as well as stimulation with IL-1ß (1417.7 pg/mL). Clodronate had no further significant influence on TIMP-1 protein level in IL-1ß-stimulated HPdLFs (Fig. 3d).

The highest MMP-8/TIMP-1 gene expression ratios were measured in IL-1ß-stimulated HPdLFs (14.3-fold), particularly in combination with compressive force (21.2-fold). Clodronate diminished IL-1ß-induced upregulation with (11.5-fold) and without (12.5-fold) compressive force (Fig. 3c).

Discussion

The present study investigates the influence of clodronate on compressed HPdLFs with regard to inflammatory cytokines. Clodronate causes an anti-inflammatory effect of stimulated HPdLFs under compressive force particularly with regard to the COX-2 expression and the periodontal remodeling, represented by the MMP-8/TIMP-1 ratio.

COX-2 and PGE-2 are known to play a central role in osteoclastic bone resorption, essential for orthodontic tooth movement [32]. In accordance with this, our study detected an enhanced COX-2 expression in compressed HPdLFs. PGE-2 was under detectable level, but IL-1ß-stimulated HPdLFs with compressive force elevated PGE-2 synthesis up to the highest detected level. Studies with a similar experimental setup confirmed a COX-2-induced PGE-2 expression [22, 33].

Clodronate reduced the compression-induced COX-2 and PGE-2 expressions in IL-1ß-stimulated HPdLFs. Liu et al. also showed that clodronate could reduce the compression-induced production of PGE-2 and RANKL in the periodontal ligament [21]. Inhibition of the COX-2 expression and its derivatives could be the reason for decelerate orthodontic tooth movement. An analogical mechanism is known by NSAIDs, a potent inhibitor of the COX family. This inhibition causes decelerate tooth movement in vivo [34]. Our findings supported by the results of Liu et al. indicate that the inhibitory effect of clodronate on orthodontic tooth movement in vivo may result in part by the inhibition of COX-2-derived PGE-2 by clodronate.

In a previous study, we observed an enhancing effect of the nitrogen-containing bisphosphonate zoledronate on the IL-1ß-induced pro-inflammatory response of HPdLFs. Mechanical strain increased this pro-inflammatory effect additionally [24]. In the presented study, clodronate reduced the compression-induced pro-inflammatory response of HPdLFs to compressive force. Therefore, our results indicate that the pro-inflammatory effect of the nitrogen-containing bisphosphonates cannot be transferred to non-N-containing bisphosphonates such as clodronate. Interestingly, a recent study evidenced a protective role of clodronate toward the inflammatory N-BP side effects via inhibition of selective phosphate transporters in the cellular membrane [35]. However, the extra- and intracellular mechanisms by which clodronate exhibits its anti-inflammatory properties are still unknown. Tsubaki et al. showed an interaction of BP with the Ras/MEK/ERK and the p38MAPK pathways. The same signaling pathways are involved in the mechanotransduction of periodontal tissue remodeling markers during orthodontic tooth movement [36]. For analyzing the extra- and intracellular pathways, further studies have to be conducted to understand the way how the different factors interfere with each other.

Various cell types respond to stress factors like mechanical force by releasing the pro-inflammatory cytokine IL-1ß. IL-1ß, in turn, leads to the expression of further pro-inflammatory cytokines like IL-6 and TNF-α. IL-6 is known as an early biomarker of tooth movement [37]. It was upregulated in our study on gene expression level by compressive force combined with IL-1ß. Clodronate diminished the reaction, suggesting a slightly anti-inflammatory response of compressed HPdLFs with regard to IL-6. Clodronate without compressive force did not alter the IL-6 synthesis. Confirming this, Dehghani et al. detected a significantly reduced secretion of several pro-inflammatory cytokines in microglial cells by clodronate [17]. One of the inhibited pro-inflammatory cytokines of Dehghani et al. was TNF-α. TNF-α is a potent cytokine for osteoclast recruitment, often enhanced by mechanical stress. In accordance with other studies, compressive force did not affect TNF-α in our study [38,39,40]. Different results may take place because of different study designs and a sensitive regulation of TNF-α. With regard to the potential anti-inflammatory role of clodronate, we detected a slightly reductive effect of clodronate on IL-1ß-induced TNF-α expression.

MMP-8 levels increase in periodontal inflammation as well as in orthodontic tooth movement [41]. Llavaneras et al. investigated in vivo the effect of clodronate on bacterial-induced periodontal inflammation in a rat model [14]. They detected a reduction of the pathological elevated levels of MMPs through clodronate, accompanied by reduced tooth mobility and alveolar bone loss. We detected an inhibitory effect of clodronate on the IL-1ß-induced MMP-8 expression in the HPdLFs. Consequently in vivo, MMP-8-mediated collagen degradation may slow down and alveolar bone loss and tooth mobility might be reduced.

Mechanical force enhances MMP-8 expression in vitro and in vivo [41, 42]. Our study complements a synergistically effect of compressive force combined with IL-1ß on the MMP-8 expression, consequently inducing the highest MMP-8/TIMP-1 ratio. The balance of tissue remodeling switched to collagen degradation. Clodronate, particularly combined with compressive force, reduced the synergistically enhanced MMP-8/TIMP-1 ratio. Knowing the limits of an in vitro study and the gap to the complex in vivo situation, the downregulation of tissue remodeling genes by clodronate might be a reason for the decelerated orthodontic tooth movement.

Conclusion

This study provides a contribution to determine the role of clodronate in inflammatory processes. Clodronate demonstrated a slightly anti-inflammatory effect under compressive force with regard to COX-2, PGE-2, and IL-6. In particular, the periodontal remodeling presented by the MMP-8/TIMP-1 ratio was diminished by clodronate. This suggests apart from the direct osteoclastic inhibition an indirect influence of clodronate on the periodontal remodeling by affecting intercellular signaling molecules.