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, 14 (1), e0211004
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Therapeutic Effect of Localized Vibration on Alveolar Bone of Osteoporotic Rats

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Therapeutic Effect of Localized Vibration on Alveolar Bone of Osteoporotic Rats

Mani Alikhani et al. PLoS One.

Abstract

Objectives: Vibration, in the form of high frequency acceleration (HFA), stimulates alveolar bone formation under physiologic conditions and during healing after dental extractions. It is not known if HFA has an anabolic effect on osteoporotic alveolar bone. Our objective is to determine if HFA has a regenerative effect on osteoporotic alveolar bone.

Methods and materials: Adult female Sprague-Dawley rats were divided into five groups: 1) Ovariectomized Group (OVX), 2) Sham-OVX Group that received surgery without ovariectomy, 3) OVX-HFA Group that was ovariectomized and treated daily with HFA, 4) OVX+Static Force Group that was ovariectomized and received the same force as HFA, but without vibration, and 5) Control Group that did not receive any treatment. All animals were fed a low mineral diet for 3 months. Osteoporosis was confirmed by micro-CT of the fifth lumbar vertebra and femoral head. HFA was applied to the maxillary first molar for 5 minutes/day for 28 and 56 days. Maxillae were collected for micro-CT, histology, fluorescent microscopy, protein and RNA analysis, and three-point bending mechanical testing.

Results: Micro-CT analysis revealed significant alveolar bone osteoporosis in the OVX group. Vibration restored the quality and quantity of alveolar bone to levels similar to the Sham-OVX group. Animals exposed to HFA demonstrated higher osteoblast activity and lower osteoclast activity. Osteogenic transcription factors (RUNX2, Foxo1, Osterix and Wnt signaling factors) were upregulated following vibration, while RANKL/RANK and Sclerostin were downregulated. HFA did not affect serum TRAcP-5b or CTx-1 levels. The osteogenic effect was highest at the point of HFA application and extended along the hemimaxillae this effect did not cross to the contra-lateral side.

Conclusions: Local application of vibration generated gradients of increased anabolic metabolism and decreased catabolic metabolism in alveolar bone of osteoporotic rats. Our findings suggest that HFA could be a predictable treatment for diminished alveolar bone levels in osteoporosis patients.

Conflict of interest statement

The original work on the Osteogenic effect of High Frequency Acceleration on alveolar bone resulted in a patent filed by New York University in which two of the authors are named as inventors: Mani Alikhani and Cristina Teixeira (Apparatus and method to increase craniofacial bone density, US Patent Application 12/555,964). The authors declare no further potential conflicts of interest with respect to employment, consultancy, patents, products in development or modified products. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Study time line.
Schematic of study time line. At 17 weeks, animals were ovariectomized and fed a low mineral diet for 12 weeks. Study of the HFA effect was then initiated and samples were collected 4 and 8 weeks later, at 33 and 37 weeks, respectively.
Fig 2
Fig 2. Region of interest (ROI).
(A) Inter-radicular bone area used for bone quantity and quality measurements (MB, mesial-buccal root; IB, intermediate buccal root; DB, disto-buccal root; MP, mesial-palatine root; DP, disto-palatine root). (B) ROI in the area of the maxillary first molar is limited from below with a horizontal plane parallel to the occlusal plane (Plane B) passing through the alveolar ridge crest; from the top by a horizontal plane passing through the apex of the mesio-buccal root and disto-buccal root (Plane T). The area between Planes B and T was divided equally into occlusal, middle and apical thirds to measure bone quantity and quality. The rectangular boxes have different dimensions, increasing in size apically as the roots diverge.
Fig 3
Fig 3. Mechanical testing of hemimaxilla.
Schematic view of three-point bending test of the rat hemimaxilla. Hemi-maxillae were placed buccal-side upward. The central loading point was aligned at the first molar midpoint. Maximum load, yield load and stiffness were computed at a displacement rate of 1mm/min.
Fig 4
Fig 4. Ovariectomized rats on low mineral diet developed osteoporotic alveolar bone.
(A) 3D μCT images of the fifth lumbar vertebral bodies and femoral head from Control and OVX rats at 29 weeks (Day 0). Note the marked loss of trabeculae in the cancellous bone of OVX animals. (B) Axial sections of 3D μCT scans through the maxillary alveolar bone from Control, Sham-ovariectomized (Sham), and ovariectomized rats (OVX) at 29 weeks (Day 0). Arrows point to loss of cancellous bone in the maxillary alveolar bone of the OVX animals. (C) Parametric values for intra-radicular bone of the maxillary first molar at Day 28. Each value represents the mean ± SEM of 6 samples.
Fig 5
Fig 5. HFA reestablished the density of osteoporotic alveolar bone in rats.
(A) Axial sections of 3D μCT images through the maxillary alveolar bone at Day 56 (20 weeks after ovariectomy) from Sham, OVX, OVX + HFA, OVX + Static groups. Significant osteoporotic changes can be appreciated in the OVX and OVX + Static groups compared to the Sham and OVX + HFA groups. (B) Parametric values for inter-radicular bone of the maxillary first molar at Day 56. Each value represents the mean ± SEM of 6 samples. (C) Comparison of BV/TV of different groups at Day 28 and Day 56. Each value represents the mean ± SEM of 6 samples (* significantly different from Sham, # significantly different from OVX group).
Fig 6
Fig 6. HFA increased expression of osteogenic markers in alveolar bone.
Gene expression of osteogenic markers (Foxo1, Runx2, Osterix, alkaline phosphatase (ALP), osteocalcin (OCN), collagen Iα1 (COL)) were measured by real time RT-PCR. Data shows mean fold change in mRNA levels compared to OVX, 56 days after application of HFA to the maxillary right first molar. Each value represents the mean ± SEM of 6 animals (* Significantly different from Sham group; # significantly different from OVX group).
Fig 7
Fig 7. Expression of members of Wnt and BMP2 signaling pathways decreased, while SOST increased, in alveolar bone in response to HFA.
Gene expression of Wnt signaling genes (β-catenin, Lrp6, Wnt3a), BMP2 and SOST was measured by real time RT-PCR. Data show mean fold change in mRNA levels compared to OVX, 56 days after application of HFA to the maxillary right first molar. Each value represents the mean ± SEM of 6 animals (* Significantly different from Sham group; # significantly different from OVX group).
Fig 8
Fig 8. HFA decreased expression of osteoclastic markers in alveolar bone.
Gene expression of RANKL, OPG, RANK and CTSK was measured by real time RT-PCR. Data shows mean fold change in mRNA levels compared to OVX, 56 days after application of HFA to the maxillary right first molar. Each value represents the mean ± SEM of 6 animals (* Significantly different from Sham group; # significantly different from OVX group).
Fig 9
Fig 9. HFA reduced osteoclast numbers in alveolar bone.
(A) Light microphotographs of H&E and immunohistochemically stained sections for TRAcP-5b. Images were collected close to the mid-root area of the first maxillary molar after 56 days of HFA application. Osteoclasts are identified as TRAcP-5b red cells in sections from different experimental groups. (B) Mean number of osteoclasts present in 5 consecutive sections of the ROI of the maxillary first molar. Each value represents the mean ± SEM of 6 animals (* Significantly different from Sham group; # significantly different from OVX group).
Fig 10
Fig 10. HFA had a gradient effect and restored the mechanical properties of osteoporotic alveolar bone.
(A) BV/TV was calculated form μCT analysis for inter-radicular bone of maxillary first, second and third molars 56 days after HFA application for Sham, OVX and OVX+HFA groups. Data are expressed as the mean 6 samples ± SEM (* significantly different from the Sham group). (B) Rats received demeclocycline (50mg/kg) at Day 0 and Day 54 and calcein green (15mg/kg) at Day 28. Animals were euthanized on Day 56. Fluorescence microscopy of sagittal sections at Day 56 shows increased intensity of the fluorescent labels in OVX+HFA maxillae compared to OVX maxillae, showing extensive bone formation especially at the point of HFA application (red arrow). (C) Fluorescence microscopy and μCT images of axial sections of maxillae in animals that received HFA on one side (HFA positive) and not the other side (HFA negative). Note the increase of the osteogenic signal only on the side that received HFA.
Fig 11
Fig 11. Local application of HFA did not affect systemic markers of bone resorption in OVX rats.
Serum levels of Tartrate resistant acid phosphatase-5b (TRAcP-5b) and C terminal telopeptide of type 1 collagen (CTX-1) were measured at Day 56 by ELISA. Data are expressed as the mean of 6 samples ± SEM (* significantly different from Sham group).

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Grant support

This project was supported by Small Business Technology Transfer Grant(STTR)#R41 DE025518 from NIDCR (nidcr.nih.gov) that was granted to Mani Alikhani and C.T. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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