TY - JOUR
AU - Yadav,, Sumit
AB - Summary Background Accelerating orthodontic tooth movement (OTM) through biologically effective methods, such as increasing osteoclast-mediated alveolar resorption, could effectively shorten treatment time. Objective To evaluate an injectable formulation containing receptor activator of nuclear factor kappa-B ligand (RANKL) on the OTM. Materials and methods We fabricated a RANKL formulation from 100 µl of 100 µg/ml RANKL adsorbed on 10 mg of poly(lactic acid-co-glycolic acid) microspheres embedded in a 10 wt% aqueous hydroxyethyl cellulose carrier gel. We characterized these formulations for the rate of RANKL release, and then tested for bioactivity using in vitro cell culture. In vivo OTM studies were conducted using 15 week old male Wistar rats for 14 days. We injected the RANKL formulations palatal to the left maxillary first molar and accomplished OTM with a nickel–titanium (NiTi) coil spring applying 5–8 g force. Control groups involved the application of NiTi coil spring with and without placebo formulation. The outcome measure included the distance of tooth movement, bone volume fraction, tissue density, and root volume determined with micro-computed tomography. We determined the amount of osteoclast activity using tartrate-resistant acid phosphatase (TRAP) staining. Results These formulations were able to sustain the release of RANKL for more than 30 days, and the released RANKL showed a positive effect on mice osteoclast precursor cells (RAW 264.7). Reported injectable RANKL formulations were effective in accelerating OTM compared with other control groups, with 129.2 per cent more tooth movement than no formulation and 71.8 per cent more than placebo formulation, corresponding with a significant increase in the amount of TRAP activity. We did not observe any significant differences in root resorption between the groups. Conclusion Our study shows a significant increase in OTM with injectable formulations containing RANKL. Introduction Reduction of orthodontic treatment time is one of the current initiatives of orthodontic research. Orthodontic treatment averages around 20 months (1) and is influenced by factors including the severity of malocclusion, treatment mechanics, patient compliance, and patient biology (2). Increased treatment duration presents a burden to the clinician and the patient, causing white spot lesions, caries, root resorption, and poor periodontal health (3–5). The benefits of shortening orthodontic treatment duration have resulted in the advent of various modalities to accelerate orthodontic tooth movement (OTM). Minimally invasive methods, such as vibration, low-level light energy, or low-intensity pulsed ultrasound, have not been shown to accelerate OTM effectively (5, 6). Invasive surgical interventions, such as alveolar decortication, piezocision, corticision, and corticotomy, have limited evidence to support effectiveness in accelerating OTM (5,7–9). However, the effects of invasive surgical interventions are transient, so the surgical insult must be repeated throughout treatment (7, 8, 10, 11). OTM is an aseptic inflammatory response dependent on bone modelling and remodelling (12, 13). The rate-determining factor of tooth movement is bone resorption carried out by osteoclasts on the compression side of tooth movement at the bone and periodontal ligament interface (14). The activation of osteoclasts depends on stromal and osteoblast-derived factors. One such factor, receptor activator of nuclear factor kappa-B ligand (RANKL), is secreted by osteoblasts to enable binding to RANK on the surface of developing osteoclast cells, allowing for activation, differentiation, and survival of osteoclasts (10). The activity of RANKL is countered by osteoprotegerin (OPG), secreted by osteoblasts to act as a decoy receptor of RANKL (15). The RANKL/OPG ratio and RANK expression by osteoclasts control differentiation of osteoclasts essential for the initial phase of bone remodelling (16). Systemically, the RANKL/RANK/OPG pathway has a central role in determining bone mass as it releases osteoclast progenitors into circulation. RANKL-induced osteoclast activation is essential to homeostasis through progenitor recruitment linking bone remodelling with haematopoiesis regulation (17). During tooth movement, RANKL expression increases in the gingival crevicular fluid in humans with compression force in adolescent patients (18, 19). Local delivery of RANKL increases osteoclast production on the compression side of tooth movement in comparison with control, corresponding to an increase in tooth movement (20, 21). RANKL is, therefore, a key acceleratory factor in OTM, acting by inducing continuous bone resorption on the compression side of tooth movement. Poly(lactic acid-co-glycolic acid; PLGA) is a routinely used biodegradable polymer in tissue engineering and drug delivery (22, 23). This polymer offers a wide range of physicochemical properties and allows for encapsulation of a variety of bioactive molecules (24). Preparation of PLGA with other hydrophilic polymers such as polyvinyl alcohol (25) and polyethylene glycol (26) allows desirable drug release profiles (27). Sydorak et al. reported on the use of PLGA microspheres for the controlled release of OPG to enhance the orthodontic tooth anchorage (28). Similarly, Lanao et al. used PLGA-calcium phosphate formulations for the controlled release of RANKL to stimulate osteoclast formation (29). In this article, we explore delivery of RANKL through PLGA microspheres embedded in a carrier gel for accelerating OTM using in vitro and in vivo studies. We hypothesize that localized and sustained delivery of RANKL would cause a continuous acceleration of OTM without systemic side-effects. Materials and methods RANKL formulation with PLGA-aqueous hydroxyethyl cellulose microspheres We fabricated hollow, porous, microspheres with PLGA (50:50 ratio of PLA:PGA; Lakeshore Biomaterials™, Birmingham, Alabama, USA) using standard double emulsion technique (29–31). We sieved the microspheres to control particulate size between 250 and 425 μm, and then sterilized the microspheres with ethylene oxide for 12 hours. We adsorbed rat soluble RANKL (sRANKL, Prospec, East Brunswick, New Jersey, USA) on sterilized microspheres at a 1 μg RANKL:1 mg microsphere ratio using a concentration of 100 μg/ml RANKL in phosphate-buffered saline (PBS) at room temperature for 30 minutes, then lyophilized them overnight to produce RANKL-PLGA microspheres. Subsequently, we embedded RANKL-PLGA microspheres in 10 per cent aqueous hydroxyethyl cellulose (HEC) gel (Sigma-Aldrich, St. Louis, Missouri, USA) at a 1:3 ratio (RANKL-PLGA:HEC, w/v) in PBS at room temperature under sterile conditions to produce the RANKL formulation. RANKL release study from RANKL-PLGA microspheres and RANKL formulation RANKL concentrations of 30 ng/ml, ranging from 10 to 100 ng/ml, induce the formation of osteoclasts from murine hematopoietic cells in a dose-dependent manner (32). We fabricated a controlled-release RANKL formulation consisting of PLGA microspheres dispersed in HEC gel that would achieve a release profile of 10–30 ng/ml over 30 days through degradation and diffusion mechanisms (31). We chose a period of 30 days to exceed the length and ensure a steady release of RANKL for the duration of the subsequently proposed in vivo study. Material characterization We characterized the RANKL-loaded microspheres for surface morphology and porosity with scanning electron microscopy (SEM; JEOL JSM-6335F; JEOL Inc., Massachusetts, USA). We detected the in vitro release behaviour in PBS (pH 7.4) at physiologic conditions (37°C) using standard methods (29). We placed 6 mg of RANKL-loaded PLGA-microspheres in 12 μl of HEC gel in the insert of a Transwell plate (Corning Inc., Corning, New York, USA) to isolate the formulation, loaded 200 μl of PBS in each well, then obtained a 100 μl sample at designated time points with complete replenishment. We measured RANKL concentration through the use of the CBQCA protein quantitation kit (CBQCA Protein Kit C6667; Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s specification. In vitro cell culture studies We tested the retention of RANKL bioactivity of the formulation in vitro on murine osteoclast precursor cells (29). We initially grew RAW 264.7 (Sigma-Aldrich) to confluence in Dulbecco’s modified Eagle’s medium (Sigma Aldrich) with 10 per cent foetal bovine serum (FBS) and 1 per cent penicillin–streptomycin (P/S) at 37°C and 5 per cent CO2. We trypsinized and seeded the cells at 4000 cells/cm2 (7600 cells/well) in a 24-well Transwell (Corning) to isolate the formulation from the cell culture. We induced osteoclast differentiation for 5–7 days, as shown previously in the literature, in Alpha Minimal Essential Medium medium with 10 per cent FBS and 1 per cent P/S. We used a positive control consisting of RANKL added directly to the cells at 1300 ng/ml in a total volume of 500 μl. We used two negative controls, 1. no RANKL added and 2. a placebo formulation consisting of 1 mg of empty PLGA microspheres in 3 μl of HEC loaded on a Transwell plate insert. The experimental group consisted of RANKL formulation (1 mg of RANKL-PLGA microspheres in 3 μl of HEC) loaded in the insert of the Transwell plate. We fixed the cells using 2.5 per cent glutaraldehyde and performed tartrate-resistant acid phosphatase (TRAP) staining according to manufacturer protocol (TRAP-staining kit 387; Sigma Aldrich). We subsequently evaluated each group for the number of osteoclasts that differentiated from the osteoclast precursor cells. In vivo effect of RANKL on the rate of tooth movement Ethical statement and animals The institutional care committee of the University of Connecticut Health Center approved all the experimental procedures involving the Wistar rats (IACUC Approval #101842-0819). All rats had 1 week of acclimatization and stayed in the animal facility in ventilated cages with a photoperiod of 12:12. Study design We randomly divided twenty-four 15-week-old male Wistar rats (Charles River Laboratories, Wilmington, Massachusetts, USA) weighing 500–600 g into three groups (Figure 1): Figure 1. Open in new tabDownload slide Experimental Design of in vivo study, depicting randomization of the 24 male Wistar rats into three groups. Figure 1. Open in new tabDownload slide Experimental Design of in vivo study, depicting randomization of the 24 male Wistar rats into three groups. Group 1: orthodontic spring with no microparticulate formulation (no formulation, n = 8) Group 2: orthodontic spring with placebo microspheres (placebo formulation, n = 8) Group 3: orthodontic spring with microparticulate formulation with RANKL (RANKL formulation, n = 8) The experimental procedures followed a previous study (33). We placed the animals under general anaesthesia with xylazine (13 mg/kg) and ketamine (87 mg/kg). We used a closed-coil nickel–titanium spring to deliver 5–8 g, as measured with a force gauge, of mesial force to the left maxillary molar using the maxillary incisors as anchorage. We attached the spring to the left maxillary first molar and grooved maxillary incisors using 0.008-in stainless steel wire, then secured with self-etching primer (Transbond Plus; 3M Unitek, Monrovia, California, USA) and light-cure adhesive resin cement (Filtek Supreme; 3M Unitek). In the experimental group, we made a flapless osteoperforation with a ¼ round bur and high-speed handpiece on the mesiopalatal of the left first maxillary molar. We injected the RANKL formulation (1 μg RANKL:1 mg microsphere in 3 μl 10 per cent HEC gel) or the placebo formulation (1 mg microsphere in 3 μl 10 per cent HEC gel) in the osteoperforation, then sealed the gingiva with cyanoacrylate tissue adhesive (Vetbond; 3M Unitek, St. Paul, Minnesota, USA) without the need for sutures. We allowed the animals to recover in incandescent light for warmth after the injection of formulation and spring placement and closely monitored them for pain and discomfort. We checked the appliance twice a week. After the 14 days experimental period, we euthanized the rats by inhalation of carbon dioxide followed by cervical dislocation. We chose 14 days for the in vivo study to sufficiently reach the linear phase of tooth movement in a rodent model (34). Micro-computed tomography analysis After the euthanasia, we dissected and fixed the maxilla from each animal in 10 per cent formalin for 5 days, then dehydrated them with a series of alcohol changes. We performed micro-computed tomography (micro-CT) at the UConn Health MicroCT imaging facility with a μCT40 instrument (Scanco Medical AG, Bruttisellen, Switzerland; 55 kV, 145 mA, 1000 projections/rotation, 300 ms) to analyse bone volume fraction (BVF), tissue density (mineralized tissue in the region of interest), trabecular spacing, root volume, and intermolar distance. We constructed three-dimensional images using standard convolution and back projection algorithms with Shepp and Logan filtering and rendered within a 12.3 mm field of view at a discrete density of 578 704 voxel/mm3 (isometric 12 mm voxels). We used the serial images for quantitative analysis of alveolar bone changes in the region of interest (ROI). We defined the ROI vertically as the most occlusal point of the furcation to the apex of the maxillary roots. The transverse boundaries of the ROI formed a rectangular conformation bounded distally by the distal portion of the distobuccal and distopalatal root, and mesially by the distal part of the mesial root. We determined regional effects of the formulation using the same ROI in the contralateral maxilla for the RANKL formulation group and the no formulation group. Histological staining For the histological studies, we rehydrated and decalcified the samples with 14 per cent ethylenediaminetetraacetic acid for 4 weeks at 4°C. Subsequently, we processed the samples for paraffin embedding, using serial sagittal sections 5–7 μm in thickness to show the mesial and distobuccal roots. We performed TRAP staining using a leukocyte acid phosphatase (TRAP) kit (386-1 KT; Sigma-Aldrich) according to manufacturer’s instructions. We determined the TRAP-positive, multinucleated cells on the alveolar bone surfaces on the mesial sides of the distobuccal roots. We identified the ROI for osteoclast quantification on the alveolar bone as a rectangle parallel to the sagittal axis of the distobuccal root of the first molar with 200 μm width and with the length extending from the bifurcation to the apex. We counted the osteoclast numbers in three sections from four rats in each group and averaged the values for each animal. Statistical analysis We used descriptive analysis to summarize the data. We examined the outcome variables of OTM, BVF, tissue density, osteoclast number (fluorescent intensity), root resorption (root volume), and RANKL activity and kinetics. The distance of OTM was the primary outcome. We used the D’Agostino and Pearson omnibus normality test to examine the normality of the data distribution. The outcome measured was normally distributed. All statistical comparisons were made using one, two-way analysis of variance and post hoc Tukey’s honestly significant difference with a significance level of P value of less than 0.05. We based the selected sample size for each experiment on a power analysis. For an α value of 0.05, a β value of 0.1, and a standard deviation of 10 per cent, we determined the sample size for in vivo studies to be 8 for a power of 0.8 (33). Results Characterization of PLGA microspheres Supplementary Figure 1 shows the overall chemical structure of the RANKL formulation. We fabricated a gel of PLGA microspheres that was uniformly dispersed in a HEC aqueous solution at 37ºC. The RANKL formulation was stable up to 40 days in vitro under physiological conditions. SEM imaging showed that microspheres were spherical with a rough surface morphology, with a heterogeneous size distribution of 250–425 μm (Supplementary Figure 2). Release of RANKL from PLGA and PLGA-HEC-RANKL microspheres We depict the RANKL release profile from PLGA microspheres in the absence of HEC gel in Figure 2a. The release of RANKL from PLGA microspheres followed a typical pattern of diffusion, with 8 per cent of RANKL released during the initial 24 hours, then decreasing from day 2 to 7. The release rate increased from day 7 to the end of the study, with 79 per cent release at 28 days. We show the RANKL release from RANKL formulation over 28 days in Figure 2b. The release of RANKL from RANKL formulation was found to be 14 per cent on the first day and steadily increased, reaching 82 per cent at day 28. The release data have been normalized to the total amount of RANKL adsorbed on the microspheres and formulation. Figure 2. Open in new tabDownload slide (A) RANKL release profile from RANKL-PLGA microspheres. (B) RANKL release profile from RANKL formulation. Release characterization obtained over 28 days using 6 mg of RANKL formulation. Figure 2. Open in new tabDownload slide (A) RANKL release profile from RANKL-PLGA microspheres. (B) RANKL release profile from RANKL formulation. Release characterization obtained over 28 days using 6 mg of RANKL formulation. Kinetic release studies We calculated the drug release mechanism of RANKL microspheres and RANKL formulation using values of the correlation coefficient (r2) and rate constants (k) shown in Table 1. The results were fit into two kinetics models using Higuchi and Korsmeyer–Peppas methods. Higuchi model describes the release of drugs as a square root of time based on Fickian diffusion: Table 1. Cumulative RANKL release data from RANKL-PLGA microspheres and RANKL formulation fit to the Higuchi, and Korsmeyer–Peppas drug release models Formulations Kinetic models Parameters PLGA-RANKL PLGA-RANKL-HEC Higuchi-equation r2 0.9726 0.9896 KH 2.49 11.68 Korsmeyer–Peppas r2 0.8347 0.9704 n 0.3874 0.2604 KHP 0.04938 0.1321 Formulations Kinetic models Parameters PLGA-RANKL PLGA-RANKL-HEC Higuchi-equation r2 0.9726 0.9896 KH 2.49 11.68 Korsmeyer–Peppas r2 0.8347 0.9704 n 0.3874 0.2604 KHP 0.04938 0.1321 HEC: hydroxyethyl cellulose; PLGA: poly(lactic acid-co-glycolic acid; RANKL: receptor activator of nuclear factor kappa-B ligand. Open in new tab Table 1. Cumulative RANKL release data from RANKL-PLGA microspheres and RANKL formulation fit to the Higuchi, and Korsmeyer–Peppas drug release models Formulations Kinetic models Parameters PLGA-RANKL PLGA-RANKL-HEC Higuchi-equation r2 0.9726 0.9896 KH 2.49 11.68 Korsmeyer–Peppas r2 0.8347 0.9704 n 0.3874 0.2604 KHP 0.04938 0.1321 Formulations Kinetic models Parameters PLGA-RANKL PLGA-RANKL-HEC Higuchi-equation r2 0.9726 0.9896 KH 2.49 11.68 Korsmeyer–Peppas r2 0.8347 0.9704 n 0.3874 0.2604 KHP 0.04938 0.1321 HEC: hydroxyethyl cellulose; PLGA: poly(lactic acid-co-glycolic acid; RANKL: receptor activator of nuclear factor kappa-B ligand. Open in new tab Q = kH t1/2 (1) where kH is a constant reflecting the design variables of the system. The release data of RANKL was fit to the Korsmeyer–Peppas model to determine the mechanism of drug release as follows: MtM∞=Ktn (2) where Mt/M∞ is the fraction of drug released at time t, K is rate constant and n is the diffusion exponent characteristic of the release mechanism. The value of n indicates the release mechanism of drug, where 0.5 > n > 1.0 indicates anomalous transport kinetics, whereas n ≈ 0.5 indicates pure diffusion-controlled (Fickian) mechanism. A value of n < 0.5 indicates drug diffusion partially through a swollen matrix and water-filled pores (35). RANKL activity after release from PLGA microspheres in HEC gel We measured the activity of the RANKL formulation by its ability to transform RAW 264.7 murine osteoclast precursors into osteoclast-like cells in vitro. We used TRAP staining to determine the presence of active RANKL, indicated by the formation of multinucleated osteoclast-like cells. The negative controls included addition of no RANKL, and addition of placebo formulation during the differentiation of RAW 264.7 cells. The positive control included the addition of 130 ng/ml of RANKL. We performed the differentiation in a Transwell plate, which isolated the microparticles and formulation while allowing RANKL to pass freely to the cell culture. Addition of the RANKL formulation successfully allowed differentiation of RAW 264.7 cells into osteoclast-like cells, whereas the addition of the placebo formulation did not contribute to any differentiation of the cells (Figure 3). Quantification of the results shows that direct addition of RANKL, or the positive control, had 5.5 times (P < 0.05) the number of differentiated osteoclasts in comparison with the RANKL formulation (Figure 4). Figure 3. Open in new tabDownload slide In vitro analysis of RANKL formulation bioactivity. RANKL-PLGA-HEC formulation maintained bioactivity and ability to induce differentiation of RAW 264.7 murine pre-osteoclast cells into osteoclast-like cells. (A) No RANKL added control. (B) Blank PLGA-HEC formulation control. (C) RANKL PLGA-HEC formulation (1.5 mg/4.5 µl). (D) RANKL addition positive control (140 ng/ml). (E) Quantification of in vitro activity. Quantification shows formation of approximately 5.5 times more osteoclasts with direct addition of RANKL (positive control) in comparison with the RANKL formulation. Figure 3. Open in new tabDownload slide In vitro analysis of RANKL formulation bioactivity. RANKL-PLGA-HEC formulation maintained bioactivity and ability to induce differentiation of RAW 264.7 murine pre-osteoclast cells into osteoclast-like cells. (A) No RANKL added control. (B) Blank PLGA-HEC formulation control. (C) RANKL PLGA-HEC formulation (1.5 mg/4.5 µl). (D) RANKL addition positive control (140 ng/ml). (E) Quantification of in vitro activity. Quantification shows formation of approximately 5.5 times more osteoclasts with direct addition of RANKL (positive control) in comparison with the RANKL formulation. Figure 4. Open in new tabDownload slide Coronally reconstructed micro-computer tomography images with depiction of the region of interest (ROI), in which bone volume fraction and tissue density were analysed. (A) orthodontic tooth movement (OTM) only, (B) OTM + blank formulation, (C) OTM + RANKL formulation. RANKL: receptor activator of nuclear factor kappa-B ligand. Figure 4. Open in new tabDownload slide Coronally reconstructed micro-computer tomography images with depiction of the region of interest (ROI), in which bone volume fraction and tissue density were analysed. (A) orthodontic tooth movement (OTM) only, (B) OTM + blank formulation, (C) OTM + RANKL formulation. RANKL: receptor activator of nuclear factor kappa-B ligand. Effect of RANKL formulation on OTM Overall weight and health of the rats All rats used in the study were 15 weeks old at the start of the experiments and 17 weeks old when euthanized. The rats in all the groups for the entire duration remained healthy and had a slight increase in body weight. Inter-molar distance (orthodontic tooth movement) MicroCT analysis of OTM shows the amount of OTM in all of the groups over the 14 day period (Figure 5). No formulation, placebo formulation, and RANKL formulation had 0.24 ± 0.05 mm, 0.32 ± 0.1 mm, and 0.55 ± 0.25 mm tooth movement, respectively, in 14 days (Figure 6). RANKL formulation has significantly more tooth movement 129.17 per cent (P < 0.05) than no formulation and 71.8 per cent more than placebo formulation. Statistical analysis shows significant difference between the RANKL formulation group (P < 0.05) with the no formulation and the placebo formulation group but did not reveal a statistical difference between the placebo formulation and the no formulation groups. Figure 5. Open in new tabDownload slide Bar graph showing significant decrease in bone volume fraction (BVF) and tissue density, along with significant increase in amount of orthodontic tooth movement (OTM) with receptor activator of nuclear factor kappa-B ligand (RANKL) formulation (*P < 0.05). Figure 5. Open in new tabDownload slide Bar graph showing significant decrease in bone volume fraction (BVF) and tissue density, along with significant increase in amount of orthodontic tooth movement (OTM) with receptor activator of nuclear factor kappa-B ligand (RANKL) formulation (*P < 0.05). Figure 6. Open in new tabDownload slide (A) Histological analysis and quantification of osteoclast formation in orthodontic tooth movement (OTM) only, OTM + Blank, and OTM + RANKL groups. F: direction of force, D: distal root, C: cementum, PDL: periodontal ligament, AB: alveolar bone. (B) Bar Graph showing significant increase (*P < 0.001) in tartrate-resistant acid phosphatas (TRAP) activity with RANKL formulation. RANKL: receptor activator of nuclear factor kappa-B ligand. Figure 6. Open in new tabDownload slide (A) Histological analysis and quantification of osteoclast formation in orthodontic tooth movement (OTM) only, OTM + Blank, and OTM + RANKL groups. F: direction of force, D: distal root, C: cementum, PDL: periodontal ligament, AB: alveolar bone. (B) Bar Graph showing significant increase (*P < 0.001) in tartrate-resistant acid phosphatas (TRAP) activity with RANKL formulation. RANKL: receptor activator of nuclear factor kappa-B ligand. Effect on bone parameters The BVF and tissue density were significantly less in the RANKL formulation (P < 0.05) in comparison with the no formulation and placebo formulation controls. The BVF was approximately 58.06 ± 6.34 in the no formulation group, 50.50 ± 6.18 in the placebo formulation group, and 25.68 ± 11.45 in the RANKL formulation group (Figure 6). The BVF in the RANKL formulation group decreased by 55.77 per cent (P < 0.05) when compared to no formulation group and by 49.15 per cent when compared to the placebo formulation. However, the difference between no formulation and the placebo formulation control was not significant (P > 0.05). Similarly, for tissue density, the no formulation group had 1115 ± 48.95 mg hydroxyapatite/cm3 (mg HA/cm3), the placebo formulation had 1032 ± 80.87 mg HA/cm3, and the RANKL formulation had 952.3 ± 63.50 mg HA/cm3, with the only significant difference between the RANKL formulation (P < 0.05) with the other two groups. The tissue density in the RANKL formulation group was decreased by 14.59 per cent when compared to no formulation group and by 7.72 per cent when compared to the placebo formulation. Effect on osteoclast formation We obtained histological analysis and quantification of osteoclast formation using TRAP staining for all three groups (Figure 7), with the direction of force, distal root, cementum, periodontal ligament, and alveolar bone as labelled. The RANKL formulation had approximately twice the amount of TRAP activity in comparison with the other groups. We also depict the quantification of TRAP activity in Figure 7, where there is a significant increase (P < 0.001) in TRAP activity with RANKL formulation. Figure 7. Open in new tabDownload slide (A) Determination of distant effects and systemic effects of the RANKL formulation was assessed through analysis of the contralateral (right) maxilla for the animals (B) which underwent OTM + RANKL formulation administration. No difference in bone volume fraction (BVF) or osteoclast formation in comparison with control. OTM: orthodontic tooth movement; RANKL: receptor activator of nuclear factor kappa-B ligand. Figure 7. Open in new tabDownload slide (A) Determination of distant effects and systemic effects of the RANKL formulation was assessed through analysis of the contralateral (right) maxilla for the animals (B) which underwent OTM + RANKL formulation administration. No difference in bone volume fraction (BVF) or osteoclast formation in comparison with control. OTM: orthodontic tooth movement; RANKL: receptor activator of nuclear factor kappa-B ligand. Distant effects of RANKL formulation We determined the distant effects of the RANKL formulation through analysis of the contralateral (right) maxilla for the animals which underwent RANKL formulation administration (Figure 7A and B). There was no significant difference (P > 0.05) between the BVF of contralateral maxilla of the RANKL formulation group and the no formulation group. No difference in TRAP activity was noted on the contralateral maxilla between both groups as well. Effects on root resorption We calculated the root volume (RV) of buccal root separately as it was the leading root during orthodontic force application with the RANKL formulation injected in its proximity. Our microCT quantification of buccal RV showed 5.54 per cent increase in root resorption (P > 0.05; decrease in RV) in RANKL formulation group (RV: 75.6 ± 1.79) in comparison with no formulation group (RV: 80.03 ± 0.03), and a 4.12 per cent (P > 0.05) increase in root resorption in comparison to the placebo formulation (RV: 78.85 ± 2.27; Figure 8). Figure 8. Open in new tabDownload slide microCT reconstructed image of the root resorption in (A) OTM; (B) OTM + Blank; (C) OTM + RANKL and; (D) Bar graph showing no significant difference in the root volume in between different groups. OTM: orthodontic tooth movement; RANKL: receptor activator of nuclear factor kappa-B ligand. Figure 8. Open in new tabDownload slide microCT reconstructed image of the root resorption in (A) OTM; (B) OTM + Blank; (C) OTM + RANKL and; (D) Bar graph showing no significant difference in the root volume in between different groups. OTM: orthodontic tooth movement; RANKL: receptor activator of nuclear factor kappa-B ligand. Discussion Targeted and controlled release revolutionized medical and dental care by improving accuracy while reducing side-effects to the site of interest (36, 37). Its use in orthodontics relatively novel and has potential to maximize efficiency and quality of orthodontic treatment (29, 38). There is currently a need for an effective method of accelerating OTM and shortening treatment duration (3, 20, 39). Previous studies of gene therapy in rats have shown that a constant, raised level of RANKL would allow for the continuous increase in OTM (20, 21). As gene therapy is currently unfeasible for orthodontic applications, our formulation providing a localized and sustained dose of RANKL could potentially be a novel therapeutic to biologically and effectively accelerate OTM to shorten treatment duration. The RANKL release rate from our microsphere formulations depends on the amount of RANKL present on the surface and deep inside the microspheres. Our RANKL formulation releases 14 per cent of total RANKL on the first day, reaching 82 per cent on day 28. The initial burst release may be due to the drug that is present on the surface or diffusion of the drugs from smaller particles following quicker water uptake (40, 41). Our use of porous PLGA microspheres minimized initial burst release of RANKL for a steadier release rate. We found that embedding the microspheres in HEC gel allowed for better retention of RANKL administration, despite a slightly higher initial RANKL release. The Higuchi and Korsmeyer–Peppas release kinetic models indicate quasi-Fickian diffusion of RANKL from our RANKL formulation, where partial diffusion enables RANKL release from PLGA microspheres as diffusion of RANKL occurs when water penetration hydrates the PLGA matrix (42, 43). Degradation and diffusion mechanisms are central to the RANKL release characteristics of the formulation. We expect the degradation rate of our formulation to concur with previous studies, which have shown degradation of PLGA 50:50 microspheres to be 6–8 weeks in physiological conditions (44–46). Our RANKL formulation maintained its ability to transform RAW 264.7 cells to osteoclast-like cells comparable to a positive control of direct addition of RANKL. Direct addition of RANKL had 5.5 times the number of differentiated osteoclasts in comparison with the RANKL formulation. This is likely due to the controlled release of RANKL with the formulation, resulting in a lower initially released dose, despite comparable total amount of RANKL contained in the formulation with that added directly. RANKL expression is increased on the compression side of tooth movement and is associated with osteoclastogenesis required for bone resorption (15). In our study, we noted a significant increase in OTM with our RANKL formulation in comparison with control. RANKL formulation has 129.2 of tooth movement achieved in the no formulation group and 71.8 per cent more than placebo formulation in 14 days. Kanzaki has previously shown that overexpression of RANKL through gene therapy increases OTM 135.8 per cent of control, which is in agreement with our findings despite slight differences in methodology (20). Iglesias-Linares also shows similar results, with overexpression of RANKL through gene therapy producing 41.27 per cent more tooth movement than control (21). Despite that we did not compare the effects of our formulation with direct addition of RANKL in vivo, we conducted our study over 14 days, so we expected our formulation to produce similar results in tooth movement as direct addition of RANKL. Though 14 days is sufficient to reach the linear phase of tooth movement in rats, it may not adequately reflect a significant difference between direct addition of RANKL and sustained release of our formulation (34). Iglesias-Linares has shown that RANKL levels dropped between day 10 and day 30 with corticotomy in rats, despite conducting the procedure at the initiation of the study (21). We noted that the increase in OTM with our RANKL formulation was inversely proportional with the change in bone volume and tissue density and corresponded to a significant increase in TRAP activity and osteoclast formation, consistent with previous studies (20, 21). Although most of the TRAP activity appears to be within the alveolar bone, some of the activity is localized near the cementum. An increase in RANKL is associated with increase in external apical root resorption (15). However, we found no significant difference in the amount of RV between different groups in our study. We assessed the distant effects of the RANKL formulation by analysis of the BVF and TRAP activity on the contralateral maxilla of the no formulation group with the RANKL formulation group. No significant difference in BVF and TRAP activity was noted, suggesting there are negligible distant effects of the RANKL formulation administration. Although we did not examine the effects of our formulation on an extraoral site, we surmised that there would be limited systemic effects if the formulation had a minimal impact on the contralateral side of the maxilla. This study demonstrated a significant increase in OTM with the RANKL formulation, but care must be taken to extrapolate of our findings to a clinical setting due to limitations in a rodent model. Rats do not experience osteonal (secondary) remodelling and have a natural distal drift of molars. Nevertheless, rats still serve as a good model for OTM as long as we address these shortcomings in the experimental design (34). Further studies looking at the long-term effects and possibility of adverse, long-term systemic effects are needed to bring this therapeutic from bench to chairside. Conclusions In vitro: In this study, we developed a novel RANKL-loaded formulation, which could control the release of RANKL to biologically and effectively accelerate OTM. To use RANKL as a potential clinical therapeutic, we developed a RANKL-loaded injectable therapeutic that could accelerate OTM in a sustained and localized manner. We determined that our formulation consisting of RANKL adsorbed on PLGA microspheres embedded in HEC gel as a suitable vehicle for RANKL delivery. We achieved a linear release over 28 days and minimized burst release. The kinetic release mechanism showed quasi-Fickian release mechanism through diffusion. Our formulation also maintained the bioactivity of RANKL by enabling osteoclast differentiation of RAW 264.7 murine preosteoclast cells, similar to control. In vivo: Our study showed that our formulation effectively accelerates and achieves more OTM than no formulation and placebo formulation in adult male Wistar rats over 14 days. 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TI - Injectable RANKL sustained release formulations to accelerate orthodontic tooth movement
JF - The European Journal of Orthodontics
DO - 10.1093/ejo/cjz027
DA - 2020-06-23
UR - https://www.deepdyve.com/lp/oxford-university-press/injectable-rankl-sustained-release-formulations-to-accelerate-DQGDjr4sdD
DP - DeepDyve
ER -