High-Frequency Acceleration: Therapeutic Tool to Preserve Bone following Tooth Extractions

Abstract

A common problem in clinical dentistry is the significant and rapid bone loss that occurs after tooth extraction. Currently there is no solution for the long-term preservation of alveolar bone. Previously, we showed that high-frequency acceleration (HFA) has an osteogenic effect on healthy alveolar bone. However, it is not known if HFA can preserve alveolar bone after extraction without negatively affecting wound healing. The purpose of this study was to evaluate the effect of HFA on alveolar bone loss and the rate of bone formation after tooth extraction. Eighty-five adult Sprague-Dawley rats were divided into 3 groups: control, static (static load), and HFA. In all groups, the maxillary right third molar was extracted. The HFA group received HFA for 5 min/d, applied through the second molar. The static group received the same magnitude of static load. The control group did not receive any stimulation. Some animals received fluorescent dyes at 26 and 54 d. Samples were collected on days 0, 7, 14, 28, and 56 for fluorescence microscopy, micro–computed tomography, histology, RNA, and protein analyses. We found that HFA increased bone volume in the extraction site and surrounding alveolar bone by 44% when compared with static, while fully preserving alveolar bone height and width long-term. These effects were accompanied by increased expression of osteogenic markers and intramembranous bone formation and by decreased expression of osteoclastic markers and bone resorption activity, as well as decreased expression of many inflammatory markers. HFA is a noninvasive safe treatment that can be used to prevent alveolar bone loss and/or accelerate bone healing after tooth extraction.

Introduction

Alveolar bone supports teeth; protects nerves, vessels, and glands; and supports muscles of mastication and facial expression. One common cause of alveolar bone loss is tooth extraction. Bone loss is greatest in the first 3 to 12 mo postextraction, with a loss of nearly 40% in height and 60% in width. This continues until only a narrow ridge remains or the alveolar bone is lost entirely. Approximately 50% of people aged 25 to 44 y and 70% of people aged 45 to 64 y have lost ≥1 teeth (excluding third molars), while 22% of people aged ≥75 y are completely edentulous (Dye et al. 2012; Slade et al. 2014). Therefore, alveolar bone loss negatively affects the oral health and quality of life for a significant portion of the US population (Trulsson et al. 2002; Emami et al. 2013).

The current options to preserve or improve postextraction alveolar bone dimensions are surgical bone grafts and guided bone regeneration (Bartee 2001; Wang and Lang 2012). Unfortunately, these procedures preserve bone height but not width (Ten Heggeler et al. 2011; Leblebicioglu et al. 2013). Additionally, bone grafting carries potential postoperative complications due to excessive inflammation or delayed healing (Morjaria et al. 2014), and cost may prevent some people from receiving these treatments. Thus, partially or fully edentulous patients are at risk for the devastating sequelae of alveolar bone loss, thus creating an urgent need for an affordable, conservative, and efficacious therapy that preserves alveolar bone long-term (Mardas et al. 2015; Willenbacher et al. 2015).

Mechanically stimulating teeth using high-frequency acceleration (HFA) induces alveolar bone formation in dentate rats (Alikhani et al. 2012). HFA triggers skeletal adaptation to the altered levels and patterns of mechanical loading. It is unknown if HFA can simultaneously improve bone formation and decrease bone resorption in pathologic conditions, for example, during bone healing following tooth extraction. The objective of this investigation is to assess HFA as a noninvasive cost-effective treatment to maintain or improve alveolar bone after extractions.

Materials and Methods

Animal Study
The maxillary right third molar was extracted in 85 male Sprague-Dawley rats (400 g, 4 mo old) under intraperitoneal xylazine and ketamine-HCL anesthesia. Buprenorphine (anal- gesic) and Baytril (antibiotic) were given for 3 d postopera- tively (protocol conforms to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and was approved by New York University Institutional Animal Care and Use Committee). Rats were randomly divided into 3 groups. The HFA group received HFA (120 Hz, 0.3 g) applied to the occlusal surface of the maxillary right second molar for 5 min/d under 3% isoflurane anesthesia. This HFA regimen produced 4με (microstrain) in the surrounding bone. The static group received 4με of static load. The control group received no strain.

HFA and Static Force Application
Devices for HFA stimulation with low-magnitude force were prepared and calibrated at the mechanical engineering depart- ment of the Polytechnic Institute of Viseu–Portugal as described previously (Alikhani et al. 2012). Static load of 4με was delivered with the same device without activation (Appendix Fig. 1).

Micro–computed Tomography and Histomorphometry Analysis
Maxillae were scanned (μCT40; Scanco Medical, Bassersdorf, Switzerland), and histomorphometry data were generated with μCT V6.0 software on the HP open platform (OpenVMS Alpha Version 1.3-1 Session Manager). Bone fill was measured as bone volume/total volume (BV/TV) in defined boundaries on axial and sagittal slices through the entire volume of the right third molar extraction site (Fig. 1A). The boundaries were out- lined on each axial slice as 2 lines parallel to the buccal and palatal bony plates (following the outline of the socket) and 2 parallel lines at the most mesial and distal points on the mesio- buccal and distobuccal roots, respectively. On each sagittal slice, the mesial and distal boundaries contacted the most mesial and distal points on the socket and were perpendicular to 2 reference lines: the incisal reference line ran through the furcation of the first and second molars; the apical reference line ran parallel to the incisal reference line along the most api- cal portion of the alveolus.

Micro–computed tomography quantification (Table) and alveolar bony changes were assessed in the region apical to the distal root. On each sagittal slice, a line was drawn at the most apical point of the distal root, perpendicular to the long axis of the root. A second line was drawn parallel to the first line, per- pendicular to the long axis of the root, and tangent to the most apical point on the alveolus and the distance between these lines averaged (Fig. 2A). Alveolar height changes were evalu- ated on coronal slices through the extraction site. We averaged the greatest distance between the incisal and apical bone on the buccal and palatal plates to generate a single value for alveolar bone height on each slice (Fig. 2C).

Random and systematic errors were calculated according to Dahlberg and Houston (Da Souza Galvão et al. 2012). The ran- dom and systematic errors were found to be not significantly different for intraobserver (0.016 and 0.019 mm) and interob- server (0.020 and 0.024 mm) measurements.

Histology and Fluorescence Microscopy
Hemimaxillae were fixed in 4% paraformaldehyde, demineral- ized (14% EDTA for 2 wk), dehydrated in alcohol series, embedded in paraffin, and 5-μm slices cut. Five sections were immunostained (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA, USA) with antibodies for tartarate-resistant acid phosphatase (TRAP; Zymed antibodies; Invitrogen, Carlsbad, CA, USA) to identify osteoclasts. As a negative con- trol, sections were exposed to preimmune serum. Sections were scanned on a Scan Scope GL optical microscope (Aperio, Bristol, UK) at 20× magnification. Osteoclasts were defined as TRAP+ multinuclear cells on periosteal or endosteal surfaces from the distobuccal root of the maxillary second molar to the socket of third molar distobuccal root (apical and incisal refer- ence lines described earlier). The number of TRAP+ cells per 1 mm2 of alveolar bone was determined in 5 sections and aver- aged. Endochondral bone formation was assayed with tolu- idine blue staining of intermediate sections.

Rats received calcein (15 mg/kg, intraperitoneal) and xyle- nol orange (90 mg/kg, intraperitoneal) alternating on days 0, 28, and 54 and were euthanized on day 56. Hemimaxillae were fixed in formalin, washed overnight, dehydrated in alcohol, cleared in xylene, and embedded in methyl methacrylate (Erben 1997). Sections (7 to 10 μm thick; Leica RM2265 Microtome; Leica Biosystems, Buffalo Grove, IL, USA) were viewed by fluorescence microscopy (Nikon Microscopy; NIS- Elements Software, Tokyo, Japan) and photographed.

Quantitative Reverse Transcription Polymerase Chain Reaction Analysis
RNA was extracted from hemimaxillae (n = 5) on days 0, 3, and 14 as described previously (Oliveira et al. 2009). Inflammatory, osteogenic, and osteoclastic genes were ana- lyzed with rat-specific primers via a QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, CA, USA) on a DNA Engine Optican 2 System (MJ Research, Waltham, MA, USA). Relative mRNA levels were calculated and normalised to GAPDH and acidic ribosomal protein mRNA levels.

Protein Analysis
Total protein levels from hemimaxillae (n = 5) were quantitated with a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Enzyme-linked immunosorbent assay was used to measure activity of inflammatory markers (MyBiosource, San Diego, CA, USA) and a colorimet- ric assay used for alkaline phosphatase (Teixeira et al. 1995).

Statistics
Experiments were repeated at least 3 times. Data are reported as mean ± SEM. Significant differences between test groups and controls were assessed by analysis of variance. Pairwise multi- ple comparison analysis was performed by Tukey’s post hoc test. Two-tailed P values were calculated, with statistical significance set at P < 0.05.

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Results

HFA Accelerates Bone Healing in the Tooth Extraction Socket
Qualitative assessment of the third molar extraction site demonstrated
marked increase in bone fill in the HFA group as compared with the baseline (day 0) and static group at day 28 and preservation of alveolar bone height at day 56 (Fig. 1B). Bone fill in the HFA and static groups was significantly different at day 14. To quantify the bone fill, BV/TV was compared in the extraction site among all groups (Table). The BV/TV in the area of the socket (Fig.1A) increased from 27% (day 0) to 33% and 46% in static and HFA groups, respectively, 28 d after extraction. The differences between HFA and the other groups were statistically significant. No difference between static and control groups was detected (Appendix); therefore, for simplic- ity we present the data for static and HFA groups only.
Evaluation of the socket changes suggested that bone filling proceeded apically to cervically (Fig. 1B; compare 28-d sagit- tal and coronal slices for HFA and static groups). Indeed, at day 28, the HFA group versus the static group had significantly greater trabecular thickness, less trabecular spacing, and greater tissue mineral density in the bone of the apical third of the distal root (Table). We further examined the apical bone height (Fig. 2A) at days 0, 14, 28, and 56. At days 28 and 56 in comparison with day 0, the static group had 1.4- and 2.6-fold increases, respectively, while the HFA group had 4.6- and 5.3- fold increases in height, respectively. No differences between static and control groups were observed (data not shown), and the difference between static and HFA groups at days 28 and 56 was statistically significant.

Application of HFA in the Long Term Preserves Alveolar Bone
At day 56, BV/TV in the extraction socket and surrounding area (Fig. 1A) was 18% and 62% for the static and HFA groups, respectively. In the bone of the apical third of the distal root, the trabecular thickness was 63% greater, trabecular spacing 37% less, and bone mineral density 28% greater in the HFA group versus the static group (Table). The differences between HFA and the other groups were statistically significant for all parameters studied except trabecular number. Confirming the enhanced apical bone response and showing long-term preservation of alveolar bone, we found that coronal sections at day 56 (Fig. 2B) showed 58%, 49%, and 38% BV/TV in apical (zone 1), middle (zone 2), and cervical (zone 3) regions, respectively, of the HFA group, which were statistically dif- ferent from the corresponding regions in the static group (38%, 33%, and 28%, respectively). Moreover, the apical region showed significantly greater BV/TV than the cervical region for both HFA and static groups.
HFA also significantly increased and preserved alveolar bone height over the long term when compared with the static group at days 28 and 56 (Fig. 1C). When quantitated, alveolar bone height was significantly greater in the HFA group versus the static group at both times (Fig. 2C). Comparing days 28 and 56 with day 0, the static group lost 4% and 52.9% of their alveolar bone height, respectively, while alveolar bone in the HFA group remained unchanged. No difference between static and control group was observed (data not shown). Importantly, the differences between static and HFA groups at days 28 and 56 were statistically significant and were due entirely to alveolar bone loss in the static group.

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Discussion

Tooth loss leads invariably to progres- sive irreversible alveolar bone atrophy due to varying combinations of jaw anatomy, age, sex, hormonal changes, local inflammation, and masticatory habits (Kingsmill 1999; Clementini et al. 2014). Another key contributor to postextraction alveolar bone resorption is loss of masticatory forces transmitted through tooth roots to the surrounding alveolar bone (Ulm et al. 1997). Logically, restoring mechanical stimula- tion around the extraction site should preserve postextraction alveolar bone. We demonstrated here one approach to providing this treatment.
The major obstacle in developing a mechanical treatment that preserves postextraction alveolar bone is deliver- ing enough osteogenic strain (0.1%) to produce sufficient matrix deformation (Frost 1987; Turner et al. 1994) without fracturing the fragile alveolar bone. Interestingly, applying low strain (<0.001%) at high frequency produces osteogenic matrix deformation in weight-bearing bones (Rubin et al. 2001; Oxlund et al. 2003). Fortunately, osteogenic strain can also be manipulated by vary- ing the strain magnitude (Rubin and Lanyon 1984), strain rate (O’Connor et al. 1982), electrokinetic streaming currents (Pollack et al. 1984), piezoelectric currents (Bassett 1968), fluid shear flow (Weinbaum et al. 1994), or strain energy den- sity (Aguado et al. 2015).
The mechanism by which low strain is osteogenic is unknown. The probability that matrix deformation (or its by-products—e.g., streaming potentials, fluid drag, enhanced nutrient transport; Qin et al. 2003; Malone et al. 2007) is the sole contributor to strain-induced osteogenesis is unlikely because matrix deformation at low strain is too small to be detected by cells. This suggests that mechanical stimulation may produce other important osteogenic signals. Physical acceleration of a cell may be such a signal (Garman et al. 2007).
Bone, cartilage, and ligament cells respond to markedly dif- ferent ranges of strain, yet the resulting cellular accelerations fall within a narrow range. It should be emphasized that higher accelerations usually require higher strains, which sets limits on the osteogenic response if acceleration is the only treatment being considered. However, manipulating acceleration fre- quency is a safe compensation for this shortcoming. Indeed, HFA application, independent of matrix deformation, enhances bone formation in weight-bearing bones (Garman et al. 2007) and increases bone-healing rates (Omar et al. 2008; Goodship et al. 2009). Likewise, we significantly increase osteogenesis in alveolar bone using HFA with almost no matrix deformation (Alikhani et al. 2012).
Interestingly, HFA’s osteogenic effect on alveolar bone extends beyond the point of HFA application (Alikhani et al. 2012). Our finding that HFA applied to the second molar sig- nificantly improved bone fill in the third molar socket confirms this phenomenon. This is clinically significant because it veri- fies that HFA can safely increase bone formation in fragile areas such as extraction sites. While the gradient boundaries of HFA’s osteogenic effect remain to be determined, we predict that alveolar bone density will produce a relatively tight gradi- ent. Delineating the HFA’s “reach” is important for clinicians who must carefully consider unwanted side effects on adjacent teeth or delicate mucosa when designing devices and applying HFA treatments.
Some studies have shown the expression of cartilage- specific genes during bone healing of extraction sockets (Ting et al. 1993; Shyng et al. 1999). Because bone and cartilage cells can respond to mechanical stimulation, we decided to evaluate both intramembranous and endochondral pathways in the healing process in response to HFA. Mechanistically, our results suggest that HFA primarily induced intramembranous bone formation. This is in spite of increased Col2a1 expression in both control and HFA groups. Col2a1 is the primary extra- cellular matrix protein in cartilage, is highly expressed during endochondral bone healing, and is expressed at early stages of mesenchymal stem cell differentiation before commitment to the intramembranous pathway (Miljkovic et al. 2008; Palomares et al. 2009). Increased Foxo1 activation in early stages of socket healing also demonstrates commitment of these cells toward the osteogenic pathway (Teixeira et al. 2010).
While it is clear that HFA promotes skeletal wound healing, the molecular and physical mechanisms regulating this are not clear. It has been suggested that oscillating nuclei activate the cytoskeleton, which then increases osteoblast activity. Indeed, osteoblast markers (Col1a1, osteopontin, and osteocalcin) all increased significantly in our HFA-treated animals. This is in agreement with previous work demonstrating HFA’s osteo- genic effect in weight-bearing bones (Rubin et al. 2001). These data support the conclusion that HFA directly promotes molec- ular and physical responses in osteoblast during wound healing.
Interestingly, HFA also decreased RANKL expression and the number of osteoclasts. The antiresorptive effect of high- frequency, low-magnitude forces has been ascribed to osteo- cyte activation and release of TGF-β, nitric oxide, and prostaglandins that decrease osteoclast formation (Lau et al. 2010). We also found decreased inflammatory marker expres- sion suggesting that HFA affects all local cells (inflammatory and noninflammatory cells, including osteocytes), which can decrease RANKL expression and osteoclast activation. Since HFA did not affect OPG expression significantly, our results suggest that the decrease in RANKL, rather than OPG compe- tition, is the primary reason why osteoclast numbers declined following HFA treatment.
Our study supports HFA as a simple treatment to promote alveolar bone formation. Applying HFA for just 5 min/d as part of a home care regimen may significantly improve outcomes of procedures such as tooth extraction, implant and bone graft integration, and postorthodontic retention, as well as diseases such as osteoporosis and periodontal disease. Further research in this field will help uncover the mechanism of skeletal adap- tation to mechanical stimulation and how to manipulate HFA to treat bone loss and injuries in non-weight-bearing bones.

Acknowledgments

Funding for this project was provided by the Consortium for Translational Orthodontic Research. The original work on the osteogenic effect of high-frequency acceleration in alveolar bone resulted in a patent filled by New York University in which 2 of the authors are named as inventors: Mani Alikhani and Cristina Teixeira. The authors declare no further potential conflicts of interest with respect to the authorship and/or publication of this article.

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