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Biomaterials & Bioengineering |
Effects of Echistatin and an RGD Peptide on Orthodontic Tooth Movement
C. Dolce*,
A. Vakani,
L. Archer,
J.A. Morris-Wiman and
L.S. Holliday
Department of Orthodontics, College of Dentistry, University of Florida, Box 100444, JHMHC, Gainesville, FL 32610-0444;
Correspondence: * corresponding author, cdolce{at}dental.ufl.edu
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ABSTRACT
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We tested whether orthodontic tooth movement (OTM) could be blocked by local administration of echistatin or an arginine-glycine-aspartic acid (RGD) peptide, agents known to perturb bone remodeling, adjacent to maxillary molars in rats. These molecules were incorporated into ethylene-vinyl acetate (ELVAX), a non-biodegradable, sustained-release polymer. In vitro experiments showed that the echistatin and RGD peptide were released from ELVAX in active forms at levels sufficient to disrupt osteoclasts. Biotinylated RGD peptide was released from ELVAX into the PDL after surgical implantation. ELVAX loaded with either RGD peptide or echistatin and surgically implanted next to the maxillary molars inhibited orthodontic tooth movement (p < 0.01). The RGD peptide also reduced molar drift (p < 0.05). This study shows the feasibility of using ELVAX to deliver integrin inhibitors adjacent to teeth to limit local tooth movement in response to orthodontic forces.
Key Words: integrins • orthodontic tooth movement • echistatin • RGD
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INTRODUCTION
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Histological examination of the alveolar bone has demonstrated that, during orthodontic tooth movement (OTM), alveolar bone modeling (apposition of bone on its surface to shape and size it) predominates on the tension side, and alveolar bone remodeling (activation of osteoclast precursors, then osteoclastic bone resorption followed by bone formation to repair the defect) predominates on the pressure side (King et al., 1991b). The kinetics of OTM occurs in three phases: the initial tipping phase (activation of cells), a lag phase (recruitment of osteoclasts to the site, which starts to resorb bone), and finally the post-lag phase, where tooth movement occurs (Reitan, 1967).
Integrins have been directly linked to the cellular response to mechanical strain (Schwartz and Ingber, 1994). Both osteoclasts (the bone-resorbing cells) and osteoblasts (the bone-forming cells) express multiple integrins and bind many RGD-containing proteins, including osteopontin and cleaved type I collagen, which are abundant in bone (Teitelbaum, 2000). Agents such as echistatin, an RGD-containing peptide derived from snake venom, and short RGD-peptides interfere with aspects of integrin-mediated signal transduction, which are important for bone remodeling. Echistatin can induce the disruption of cell-matrix interactions and appears to cause an early reduction of pp125FAK phosphorylation. This results in the disassembly of actin cytoskeleton and of focal adhesions (Staiano et al., 1997). In vivo studies have demonstrated that echistatin is a potent inhibitor of bone resorption in thyroparathyroidectomized rats (Fisher et al., 1993; Masarachia et al., 1998). Similarly, in oophorectomized rats, which experience bone loss and have been used to mimic the post-menopausal patient, both echistatin (Fisher et al., 1993; Yamamoto et al., 1998) and SC56631 (Engleman et al., 1997) (an RGD peptidomimetic) inhibited bone resorption. The effects of echistatin were attributed to a decrease in osteoclast function, whereas the effects of SC56631 were attributed to a decrease in osteoclast number. Integrins have been localized in the periodontium and have been shown to play a role in both periodontal homeostasis and disease pathogenesis (Steffensen et al., 1992).
The purpose of this study was to use a well-characterized in vivo model for OTM (King et al., 1991a) and follow tooth movement kinetics in the presence of the echistatin or a short RGD peptide placed subcutaneously, adjacent to the maxillary teeth. We achieved localized delivery of these agents by incorporating them into ELVAX 40, a non-biodegradable, non-inflammatory sustained-release polymer (Niemi et al., 1985). We conducted in vitro studies to show that functional echistatin and RGD peptides were released from ELVAX 40 at levels sufficient to disrupt the formation of actin rings. The effects of localized delivery of echistatin and RGD peptides on OTM were then characterized.
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MATERIALS & METHODS
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Reagents
The RGD peptide GRGDSP was obtained from Life Technologies (Gaithersburg, MD, USA). ELVAX 40 was obtained from DuPont (Wilmington, DE, USA). Sulfo-NHS-LC-biotin was obtained from Pierce (Rockford, IL, USA). Echistatin and other reagents were obtained from Sigma (St. Louis, MO, USA) unless otherwise noted.
Preparation of Polymers Containing RGD Peptides and Echistatin
Implants of the polymer ELVAX were used to deliver echistatin or the GRGDSP peptide subgingivally next to the maxillary molars. This polymer has been used to deliver biologically active substances in significant quantities over time in several other experimental paradigms (Ling et al., 1995; Jones et al., 1996; BenEzra et al., 1997). Polymers containing synthetic peptides were prepared according to the protocol of Langer and Folkman (1976). Briefly, 0.05 mg of echistatin or 2 mg GRGDSP were mixed with 100 µL of 10% ELVAX (DuPont, Wilmington, DE, USA) in dichloromethane. The resulting dispersion was quick-frozen on dry ice and then dried under vacuum to remove any solvent. The resultant material was cut into strips (1 mm height x 1 mm width x 3 mm in length).
Actin Ring Formation
Actin rings are characteristic of mature bone-resorbing osteoclasts. For these studies, mouse marrow osteoclasts were generated as described previously (Holliday et al., 1995), and actin rings were detected with fluorescein-tagged phalloidin stain (Lee et al., 1999).
Surgical Insertion of ELVAX Strips into Rats
Either ELVAX was loaded with GRGDSP or echistatin, or unloaded controls were placed at the time of spring activation (day 0) next to the apices of the maxillary first molars via a 2- to 3-mm incision in the vestibule. Once the incision was made, a sharp instrument was used to elevate the periosteum so that the polymer could be placed adjacent to the bone. The incision was then closed with one or two sutures (braided silk 6-0).
Biotinylation of RGD Peptide
In this study, the peptide was biotinylated (EZ-LINK Sulfo-NHS-LC-Biotinylation Kit, Pierce, Rockford, IL, USA) for ease of visualization. ELVAX was loaded with synthetic biotinylated peptides and implanted next to the maxillary first molars in 3 rats. These rats were killed at day 6, and the diffusion of this biotinylated peptide was histologically assessed. Tissue sections were incubated with avidin conjugated to horseradish peroxidase (HRP) and then visualized by treatment with 3,3'-diaminobenzidine (DAB). As control, the ELVAX without the peptide was inserted into the buccal vestibule.
Animals for OTM Experiments
Sprague-Dawley rats (5–7 animals/group) were purchased from Charles River Breeding Laboratories (Wilmington, MA, USA). The rats were shipped by air freight and allowed to acclimate for 2 wks under experimental conditions, including being housed in plastic cages, eating a diet of ground laboratory chow, drinking distilled water ad libitum, and being maintained on a standard 12-hour light/dark cycle. Young male (40- to 50-day-old) rats were used because they rapidly recover from the surgeries required to place the appliances. Moreover, male rats were chosen to eliminate the hormonal changes associated with estrus. The University of Florida Institutional Animal Care and Use Committee approved the animal protocol.
Orthodontic Tooth Movement
Orthodontic tooth movement was accomplished as previously described (King et al., 1991a). Preparatory sessions consisted of the following sequence of events: (a) Animal weights were recorded; (b) anesthesia was attained via intra-peritoneal injections of ketamine (87 mg/kg) and xylazine (13 mg/kg); (c) modified orthodontic cleats were bonded bilaterally to the occlusal surfaces of the maxillary first molars; (d) all 4 incisors were pinned to prevent further eruption and minimize movement of the anchorage; (e) the lower incisors were reduced slightly; and (f) the mandibular first molars were extracted to prevent appliance damage. The animals were then allowed to recover for 3 wks, during which wound healing and weight gain were monitored. (Experience from previous studies had indicated that the rats would fully recover in 3 wks.)
We activated the appliances by positioning the rats in a head restrainer and placing orthodontic springs. One end of a Nickel Titanium closed-coil spring (light; #10-000-06; GAC, Central Islip, NY, USA) was ligated to the molar cleat, while the other was attached to a 40-gram suspended weight. This force was chosen since it has been shown to demonstrate the typical OTM kinetics and acceptable balance between bone formation and resorption. The anterior end of the coil was then bonded with autocuring methacrylate to the acid-etched lateral surfaces of the maxillary incisors, followed by removal of the weight and excess coil spring. This method ensured both a precise and reproducible initial orthodontic force (designed to tip the maxillary first molars mesially) and appliances with equivalent decay rates. The strain environments created are primarily compression on the mesial surface and tension on the distal. The control group received all the procedures except spring placement.
Measurement of Tooth Movement
Four cephalometric x-rays of the head were taken at day 0 and at days 1, 3, 7, and 10. Radiographic exposures were made with the use of a head-holding device. This consists of ear rods for positioning the rat face down on the base. The cephalograms were then digitized at 600 dpi by means of the NIH imaging program ImageJ. OTM was measured from the molar cleat to the distal of the pin on the incisor.
Statistical Analysis
The data for tooth movement and drift are expressed as means ± SEM. A one-way analysis of variance (ANOVA) was used for testing the significance between the groups.
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RESULTS
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Previous studies have shown that treatment of osteoclasts with either RGD peptide or the snake-venom-derived integrin inhibitor echistatin disrupts actin rings that are characteristic of bone-resorptive osteoclasts (Sato et al., 1990; Horton et al., 1991). To test whether these reagents were incorporated into ELVAX so that they were released in active form, we incubated mouse marrow osteoclasts with ELVAX alone or loaded with GRGDSP or echistatin. Osteoclasts on slices of sperm whale dentin (US Department of Fisheries, San Diego, CA, USA) were incubated under the following conditions: untreated (Fig. 1A ), treated with ELVAX (Fig. 1B), ELVAX loaded with GRGDSP (Fig. 1C), or ELVAX loaded with echistatin (Fig. 1D). After 90 min, the cells were fixed and examined for the presence of actin rings, a crucial marker for functional osteoclasts, with the use of fluorescently tagged phalloidin. Untreated osteoclasts showed the typical actin ring (Fig. 1A). The overall morphology of the osteoclasts on dentine slices was not disrupted when treated with either echistatin or GRGDSP (Figs. 1C, 1D). However, quantitative analysis showed that treatment with either echistatin or GRGDSP dramatically decreased the number of actin rings without changing the number of osteoclasts (p < 0.001) (Fig. 1E). These experiments confirmed that the GRGDSP peptide and echistatin were incorporated into ELVAX so that they would be released in an active form at levels sufficient to disrupt osteoclast activity in their vicinity.
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Figure 1. RGD peptide and echistatin released from ELVAX disrupt osteoclasts on coverslips or bone slices. Mouse marrow osteoclasts were grown to maturity, loaded on slices of sperm whale dentin, and cultured for 2 days to allow the osteoclasts to adhere and become active. The osteoclasts were then left untreated (A), treated with ELVAX alone (B), or with ELVAX loaded with echistatin (C) or RGD peptide (D). After 90 min, the cells were fixed with 2% formaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS, and stained with fluorescein-tagged phalloidin. Representative cells from the various treatment conditions are shown. Arrows point to the edges of the respective cells. The cell in each pane is approximately the same size, and all were photographed at the same magnification. The dramatic difference in appearance is the result of disruption of actin rings resulting from RGD-peptide and echistatin treatment. The bar is 5 µm. "I" shows quantitative analysis of the number of osteoclasts with actin rings on dentin slices after 90 min. The data represent mean ± SD of 4 experiments.
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Next we tested whether the GRGDSP peptide was released in vivo and determined the dispersion area of the released peptide. To do this, we surgically inserted into the rats ELVAX loaded with GRGDSP peptide that had been covalently tagged with biotin. After 6 days, the rats were killed, and the tissues around the teeth were fixed, embedded, and stained with streptavidin-horseradish peroxidase (HRP). The biotinylated peptide GRGDSP was localized within the PDL (Fig. 2).
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Figure 2. Localization of the biotinylated RGD peptide in alveolar tissues. ELVAX was loaded with synthetic biotinylated RGD peptide (A) or alone (B) and implanted next to the maxillary first molars in rats. The rats were killed at day 6, and the diffusion of this biotinylated peptide was assessed. Tissue sections were incubated with avidin conjugated to horseradish peroxidase (HRP) and then visualized by treatment with 3,3'-diaminobenzidine (DAB). PDL, periodontal ligament. Note the increased staining in (A), indicating the presence of the biotinylated RGD peptide.
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To test whether ELVAX-RGD and ELVAX-echistatin affected OTM, we inserted, next to the maxillary molars, either ELVAX alone or ELVAX loaded with either GRGDSP peptide or echistatin. The rats gained weight during the experimental procedure, suggesting that they tolerated the procedure well. In the control group, 3 phases of OTM were observed: initial tipping, which lasted up to day 1; then the lag phase, from days 1 to 7; and finally tooth movement began again (Fig. 3). The tooth movement at day 10 in the control group was significantly different (p < 0.01) from that in the other two groups. Both GRGDSP and echistatin inhibited OTM, since no movement occurred after day 1 (Fig. 3). The only other notable difference among the three groups was that more initial tipping took place at appliance activation in the echistatin group (p < 0.01).
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Figure 3. Tooth movement kinetics in the control, echistatin-, or RGD-peptide-treated rats. Each point represents the mean ± SEM of 5–7 animals. *p < 0.01.
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The control rats exhibited distal drift of the molar. In the presence of GRGDSP, this process was inhibited up to day 1 (p < 0.05) (Fig. 4).
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Figure 4. Tooth drift kinetics in the control or RGD-peptide-treated rats. Each point represents the mean ± SEM of 5–7 animals. *p < 0.05.
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DISCUSSION
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The present study demonstrated that OTM could be inhibited by the local administration of integrin inhibitors. Disruption of integrin signaling also temporarily inhibited molar drift, a naturally occurring process in rats (Tran-Van et al., 1982).
Perturbation of the interaction of osteoblasts with its integrin receptors by using antibodies or RGD-like fragments suppresses the formation of mineralized nodules in vitro and delays the expression of tissue-specific genes, including osteocalcin (Clover et al., 1992). Also, apoptosis of differentiated osteoblastic cells is triggered by interfering with their interaction with fibronectin (Frisch and Francis, 1994). Peptides containing the RGD motif have been shown to inhibit osteoclast-mediated bone resorption in vitro and prevent osteoporosis in vivo (Engleman et al., 1997). These can potentially aid in the treatment of osteoporosis and possibly in modulating OTM. In fact, in a recent study, a decrease in the number of osteoclasts as well as a decrease in the number of TRAP-positive cells was demonstrated when an RGD-containing peptide was injected locally during OTM (Terai et al., 1999).
We tested two types of integrin inhibitors. The RGD peptide has a low affinity for the principal integrin of osteoclasts,  vβ3. We loaded a high concentration of this peptide into ELVAX. In contrast, echistatin has an affinity for vβ3 three orders of magnitude higher, and relatively low concentrations were loaded into ELVAX. Both variants disrupted actin rings in osteoclasts in cell culture, and inhibited OTM. Echistatin and the RGD peptide both bind multiple integrin receptors, and the various receptors they bind may not completely overlap. Moreover, because the integrin receptors are present in many cell types, the above-observed effects may not be direct and not solely on osteoclasts.
An unexpected observation in those animals that were treated with echistatin was the greater initial tipping at appliance activation. The loaded ELVAX was placed about 30 min before appliance activation. We speculate that either echistatin disruption of the PDL probably occurred earlier than that of the RGD peptide, due to its high affinity to vβ3, or echistatin may affect a different subset of integrin receptors than the RGD peptide, which would lead to a greater disruption of the PDL. The tooth movement which we observed was slightly greater that reported by other investigators using the same experimental model (King et al. 1991a; Gibson et al., 1992; Konoo et al., 2001). This may be due to mild inflammation due to the placement of the ELVAX polymer or the polymer itself.
Another interesting observation was the inhibition of molar drift for the first day. This suggests that the RGD peptide may be inhibiting osteoclasts from resorbing bone and consequently temporarily preventing distal drift. Future studies will now be required to determine the optimal combination of inhibitor affinity and concentration to limit OTM while reducing potential deleterious effects.
In summary, in this report we show that ELVAX 40 can be used to administer integrin inhibitors locally to the pressure side of rat incisors. We found that OTM was markedly inhibited by ELVAX-delivered echistatin or RGD peptides in a rat model system. This study provides a "proof in principle" for limiting the OTM of specific teeth by using a non-biodegradable, non-inflammatory sustained-release polymer to deliver integrin inhibitors.
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ACKNOWLEDGMENTS
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This work was supported by NIH grants DE13857 (CD) and AR47959 (LSH).
Received for publication July 9, 2002.
Revision received May 23, 2003.
Accepted for publication June 6, 2003.
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Journal of Dental Research, Vol. 82, No. 9,
682-686 (2003)
DOI: 10.1177/154405910308200905
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ABSTRACT
INTRODUCTION