STIMULATING CELLULAR ORTHODONTIC ADVANCEMENT

orthcell

“Cutting in Half Orthodontic Brace and Aligner Wear Time with Bioelectric Stimulation”

OrthodontiCell (patent pending) has the mission to reduce in half or more, the time that it takes to complete orthodontic treatment and then have the teeth become stable in their final position equally as rapidly.  This is all made possible by wearing a bioelectric stimulation mouthpiece only 3 times a week for 20 minutes.

Orthodontic braces and clear aligners work by applying force to teeth in order to gradually realign them.  This force naturally causes a demineralization (softening) of the bone, which allows the teeth to move. Although the time it takes for patients to wear braces or aligners varies considerably, it generally takes on average up to 2 years. To help shorten treatment time, Dr. John Marchetto of Weston, FL and Howard Leonhardt of Salt Lake City, UT has formed OrthodontiCell (Salt Lake City, Utah).  OrthodontiCell has developed the Tooth Movement Accelerator TM, which uses bioelectric energy to significantly increase the rate at which teeth move during orthodontic tooth movement. The Tooth Movement Accelerator TM is a removable and non-invasive appliance that a patient wears in the mouth for 20 minutes every 3 days.  OrthodontiCell is working with Dr. Jorge Genovese of Argentina (also a co-founder) and is about to commence animal studies utilizing the bioelectric energy, at the Forsyth Institute in Cambridge, MA.  This study will then be followed using the device and method in controlled clinical trials in the USA.

The technology is based on patent pending bioelectric stimulation technology originally developed to regenerate damaged hearts back in 1999 and is now adapted and reprogrammed for increasing the rate of tooth movement and thereby reducing orthodontic treatment time.  The OrthodontiCell Tooth Movement Accelerator TM emits small electric pulses that controls expressions of RANKL, OPG, TNF-alpha, SDF-1, HGF, IGF-1, VEGF, eNOS and M-CSF as well as stem cell differentiation.   Studies and patents (some with patents pending) have been completed for all these cells and proteins individually for various applications of regeneration.  Previously, a number of studies have demonstrated that regular needle injections of RANKL in the area of desired tooth movement, accelerates tooth movement by 300% and will decreases the time needed to wear braces or aligners.

This bioelectric stimulator, will achieve incredibly fast orthodontic treatment with less pain, in fact, the electrical stimulation actually has a pain reducing affect.   When compared to surgical corticotomies (the well documented surgical approach to acceleration tooth movement), it was faster, eliminates the morbidity, pain and suffering of surgery.  The key to the increased rate of tooth movement is drawing an abundance of the needed cells and proteins to the site of tooth movement.  These cells and proteins will accelerate the demineralization (softening) of bone, thereby allowing teeth to move faster.  Once the teeth are in their final proper position, signaling for an increase in specific cells and proteins will accelerate the remineralization (hardening) and shorten the time for retention.  One example is SDF-1, a key patented signal for homing stem cells from the surrounding tissue (bone marrow, gum tissue, fat cells and circulating blood) to come to the treated site to aide in tooth movement.  There are many other cells and proteins and cytokines that have an increased expression through specific patented signals, all working together to substantially increase the rate of tooth movement.

Howard J. Leonhardt the Founder and CEO of Leonhardt Ventures and Cal-X Stars Business Accelerator, Inc., Dr. John Marchetto an Orthodontist in Weston, Florida and Dr. Jorge Genovese a collaborative researcher from Buenos Aires, Argentina are co-inventors of this product.

“This invention addresses the desire to reduce in half or more, the time it takes to treat orthodontic patients.  The approach is to speed up the normal process of bone demineralization in order to accelerate tooth movement.  Millions of people that wear braces or aligners would love to cut their treatment time by more than half.  Other devices that use light or vibration fall very short in reducing treatment time because they do not provide a clear and reliable pathway to the underlying mechanism of action for tooth movement.  Additionally, they have no effect on helping to stabilize and improve retention after the orthodontic treatment is completed.  This invention is completely different than previous devices as it provides clear cut direct control for the release of essential cells and proteins needed for accelerating tooth movement, and with less pain”, states Dr. John Marchetto, co-Inventor.

Link to news release…

Device to Cut Orthodontic Treatment Time in Half …

https://www.prbuzz.com/…/372828-device-to-cut-orthodontic-treatment-…

20th, 2016 – PRBuzz – Leonhardt Ventures — a developer, manufacturer and … OrthodontiCell is incubating within Cal-X Stars Business Accelerator, Inc. The …

OrthodontiCell is a startup in the Leonhardt’s Launchpads Utah innovation accelerator a unit of Cal-X Stars Business Accelerator a Leonhardt Ventures Co.

OrthodontiCell

A Leonhardt Ventures Co.

@ Leonhardt’s Launchpads Utah

489E, 400 South, Unit 116

Salt Lake City, Utah 84111

954 401 0096 Howard Leonhardt

954 812 4238 Dr. John Marchetto

Email: howard.leonhardt@orthodonticell.com

john. marchetto@orthodonticell.com

Media Relations:  Brian Hardy – brian@fizzpopmedia.com

Bioelectric Stimulation Induced RANKL and OPG Release in Rats for Controlled Tooth Movement

Bioelectric Stimulation Induced RANKL and OPG Release in Rats for Controlled Tooth Movement

Principal Investigator: Alpdogan Kantarci

Co-investigators: Beyza Tagrikulu (Forsyth Institute), Sercan Akyalcin (TUFTS)

Institute: Forsyth Institute, 245 First Street, Cambridge, MA 02142

Dept:  Applied Oral Sciences

I- SPECIES INFORMATION AND NUMBERS

  1. Species to be used: Rat (Rattus norvegicus), Sprague-Dawley
  1. Describe your rationale for:
  2. The use of an animal model (rather than in vitro or human models):

Orthodontic tooth movement occurs in the presence of a mechanical stimuli followed by remodeling of the alveolar bone and periodontal ligament (PDL). Bone remodeling is a process of both bone resorption on the pressure site and bone formation on the tension site. RANKL and OPG are critical determinants in alveolar bone modeling and remodeling during and after orthodontic tooth movement [Davidovitch, 1991]. The paradigm is that RANKL expression accompanies alveolar bone resorption on the compressed side of the tooth and is affiliated with accelerated tooth movement when enhanced locally while OPG enhanced expression inhibits RANKL-mediated osteoclastogenesis and stimulates osteoblastic bone apposition [Alhashimi et al., 2001]. Thus, it is thought that the bone remodeling is controlled by a balance between RANK–RANKL binding and OPG production [Kanzaki et al., 2006].

Reducing orthodontic treatment duration is one of the main concerns in orthodontics. Long treatment time is one of the common drawbacks that faces orthodontist and the patient; increasing risks of caries, gingival recession, and root resorption. Various surgical and non-surgical methods have been suggested to reduce orthodontic treatment time such as increased use of brackets, increased force levels and relying on less friction bracket systems, pharmacological approaches, low-intensity laser irradiation, electrical stimulation, photobiomodulation, and surgical protocols such as corticotomy [Qamruddin et al., 2015].

Bioelectric stimulation is a new approach for specifically targeting molecules that play role in signaling and biological processes. This approach has been used in medicine for reversing the pathways that lead to pathological changes or activating wound healing. We hypothesized that the bioelectric stimulation can be an effective tool to specifically stimulate the bone turnover during orthodontic tooth movement and stabilize the outcomes of treatment. The approach will include delivering specific signals at specific time intervals to activate 1) the expression of receptor activator of nuclear factor kappa B ligand (RANKL) for accelerated tooth movement, and 2) osteoprotegerin (OPG) for stabilizing the position of the teeth. In order to test our hypothesis, histological evidence from an experimental animal model is required in a controlled time- and dose-dependent study design before applying the technique safely to humans.

2- The use of this species:

The rats are chosen because they present the smallest animal model with which we (and others) have successfully developed an orthodontic treatment model. Their resemblance to human teeth anatomy and alveolar bone is also suitable for this type of procedure. Likewise, rat models are widely used for acceleration of orthodontic tooth movement researches. Our previous work and studies by others on this animal model has shown that human tooth movement can be efficiently simulated. We have already established the protocol on experimental tooth movement in rat model.

  1. Provide a name for each procedure you will use

     Experimental procedure 1: Impact of bioelectric stimulation of RANKL on acceleration of orthodontic tooth movement and its stability

Number of animals per group : 10

x Number of groups:   2

= Total for procedure: 20

In this experiment, we will test if RANKL bioelectric stimulation (BES) will 1) accelerate the orthodontic tooth movement (TM) and 2) impact the stability of the TM. The results will be compared to those of experimental procedure #3 below. In total, 20 rats will be used and will be divided into two groups:

Group 1: Bioelectric Stimulation (BES) of RANKL for acceleration of TM (n=10). BES will be applied for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21.

Group 2: Retention of space after BES-stimulated RANKL for acceleration of TM (n=10). BES will be applied for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21 and will be followed by appliance removal and monitoring the animals for another 21 days.

Bioelectric signals will be delivered to the molars by using alligator clamps connected to a benchtop stimulator.

     Experimental procedure 2: Impact of bioelectric stimulation of OPG on acceleration of orthodontic tooth movement and its stability

Number of animals per group : 10

x Number of groups:   2

= Total for procedure: 20

In this experiment, we will test if OPG bioelectric stimulation (BES) will impact the stability of the TM. The results will be compared to those of experimental procedures #1 above and #3 below. In total, 20 rats will be used and will be divided into two groups:

Group 1: Bioelectric Stimulation (BES) of RANKL for acceleration of TM and OPG for stability of the treatment outcome (n=10). BES for RANKL will be applied for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21. BES for OPG induction will be applied for 20 minutes/day on days 13, 17 and 21.

Group 2: Bioelectric Stimulation (BES) of RANKL for acceleration of TM and OPG for stability of the treatment outcome (n=10). BES for RANKL will be applied for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21. BES for OPG induction will be applied for 20 minutes/day on days 13, 17 and 21. Appliances will be removed and animals will be monitored for another 21 days.

Bioelectric signals will be delivered to the molars by using alligator clamps connected to a benchtop stimulator.

     Experimental procedure 3: Conventional orthodontic tooth movement

Number of animals per group :   5

x Number of groups:   2

= Total for procedure: 10

In this phase of the study 10 young adult rats will be divided into two groups:

Group 1: The animals will receive conventional orthodontic treatment (TM) for 21 days. (n=5)

Group 2: The animals will receive TM for 21 days followed by appliance removal and monitoring for relapse for another 21 days. (n=5)

  1. BACKGROUND
  2. Purpose of study (background, questions, hypotheses; distinguish among experiments if relevant):

Orthodontic tooth movement has been described as a site-specific bone remodeling consisting of coupled bone resorption and bone formation (Baloul et al., 2011; Barfitt, 1984). The mechanical basis of orthodontic treatment is the application of force to the teeth using an appliance. This, in turn, leads to biologic reactions, including remodeling changes in the dental and periodontal tissues (Baloul et al., 2011; Krishnan et al., 2006).

Tooth movement requires the binding of receptor activator of nuclear factor kappa β ligand (RANKL) to RANK, a cell membrane protein found on osteoclast precursor cells [Yamaguchi et al., 2009]. RANKL is produced by the osteoblasts. During the orthodontic movement, RANKL is responsible for the generation and maintenance of osteoclasts by binding RANK [Alhadlaq et al., 2015]. Osteoprotegerin (OPG) acts as a decoy receptor that binds to RANKL and blocks osteoclastogenesis [Yamaguchi et al., 2009]. The regulation of osteoclast activity by the RANK/RANKL/OPG axis immediately following active tooth movement plays a critical role in alveolar bone maturation and post-orthodontic stability. Meanwhile, inhibition of osteoclastogenesis and osteoclast activity by local delivery of recombinant OPG protein could serve as a rational pharmacological approach for enhancing orthodontic retention and stability [Hudson et al., 2012].

There are two basic ways to accelerate the tooth movement and reduce the treatment duration. One approach is making the treatment mechanics more efficient. Another approach involves interventions to increase the velocity of orthodontic tooth movement by enhancing the bone remodeling. This intervention can be classified into three categories: (1) biochemical stimulation (2) mechanical or physical stimulation, and (3) surgical interventions. These methods range from the use of cyclic vibration [Kau et al., 2011], magnets [Kolahi et al., 2009], photobiomodulation [Shaughnessy et al., 2016], direct electrical current [Kolahi et al., 2009], to selective decortication  [Wilcko et al., 2009, Baloul et al. 2011].

Bioelectric stimulation (BES) is not a new concept but its application to enhance the rate of healing is novel. Since the 1960s, it has been increasingly recognized that the human body generates a host of low-level electric signals as a result of injury, stress and other factors; that these signals play a necessary part in healing and disease-recovery processes; and that such processes can be accelerated by providing artificially-generated signals which mimic the body’s own in frequency, waveform and strength. The application of electric fields and continuous currents similar to those generated physiologically by the organism can modify cell behavior [Agren et al., 2007] and induce changes in the skin [Neves et al., 2013], cartilage [Campos Ciccone et al., 2013, Zuzzi et al., 2013], tendons [Fujita et al., 1992, Lin et al., 2006], and bone [Martin et al., 1978, Mendonca et al., 2013]. Changes also occur in the transport of ions across cell membranes, as well as in the migration of leukocytes, macrophages, and keratinocytes and in the proliferation of vascular endothelial cells, osteoblasts, osteoclasts, chondrocytes, and fibroblasts [Chao et al., 2000, McCaig et al., 2005, Funk et al., 2006]. In vitro studies have suggested that BES affects cellular mechanisms such as ATP production and protein synthesis, exerts antioxidant effects, and promotes changes in blood flow and in transmembrane transport, as well as inducing the synthesis and release of epidermal and vascular growth factors and the expression of their respective receptors [Poltawski et al., 2009].

In orthodontics, the local application of exogenous electric currents were combined with tooth movement and shown to accelerate orthodontic treatment [Davidovitch et al., 1980, Kim et al., 2008]. In an experimental study, Hashimoto [1990] investigated the effects of microcurrent application (10 μA) on the tooth surface of cats and demonstrated that this treatment increased bone deposition. It was concluded that microcurrent application combined with a mechanical force may accelerate alveolar bone remodeling and orthodontic tooth movement. In a very recent study, Spadari et al (2017) suggested that  microcurrent application combined with induced tooth movement favored tissue responses, reduced the number of granulocytes and increased the number of fibroblasts, blood vessels, and osteoclasts. These studies however, did not target specific molecular pathways.

In this study, we hypothesized that a targeted BES will deliver specific signals at specific time intervals to induce an over-expression of RANKL for accelerated tooth movement, and after desired tooth movement is completed, OPG for stabilizing the position of the teeth.

The study groups will be as follows:

Group 1: Bioelectric Stimulation (BES) of RANKL (n=10). BES for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21.

Group 2: Retention of space after BES-stimulated RANKL (n=10). BES for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21 followed by appliance removal + monitoring for 21 days.

Group 3: Bioelectric Stimulation (BES) of RANKL and OPG (n=10). BES for RANKL for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21. BES for OPG for 20 minutes/day on days 13, 17 and 21.

Group 4: Bioelectric Stimulation (BES) of RANKL and OPG (n=10). BES for RANKL for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21. BES for OPG for 20 minutes/day on days 13, 17 and 21 followed by appliance removal + monitoring for 21 days.

Group 5: Conventional orthodontic treatment (TM) for 21 days. (n=5)

Group 6: Conventional orthodontic treatment (TM) for 21 days followed by appliance removal+ monitoring for 21 days. (n=5)

III. LITERATURE SEARCH

Keywords used: Experimental periodontitis, orthodontic tooth movement, photobiomodulation, animal model

Following papers evaluated the use of bioelectric stimulation for orthodontic tooth movement acceleration:

  • Spadari GS, Zaniboni E, Vedovello SA, Santamaria MP, do Amaral ME, Dos Santos GM, Esquisatto MA, Mendonca FA, Santamaria M Jr. Electrical stimulation enhances tissue reorganization during orthodontic tooth movement in rats. Clin Oral Invest (2017) 21:111–120.
  • Hashimoto H. Effect of micro-pulsed electricity on experimental tooth movement. Nihon Kyosei Shika Gakkai Zasshi. 1990 Aug; 49(4):352-61.
  • Stark TM, Sinclair PM. Effect of pulsed electromagnetic fields on orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 1987 Feb; 91(2):91-104.
  • Davidovitch Z, Finkelson MD, Steigman S, Shanfeld JL, Montgomery PC, Korostoff E. Electric currents, bone remodeling, and orthodontic tooth movement. I. The effect of electric currents on periodontal cyclic nucleotides. Am J Orthod. 1980 Jan; 77(1):14-32
  • Davidovitch Z, Finkelson MD, Steigman S, Shanfeld JL, Montgomery PC, Korostoff E. Electric currents, bone remodeling, and orthodontic tooth movement. I. The effect of electric currents on periodontal cyclic nucleotides. Am J Orthod. 1980 Jan; 77(1):14-32.

Following papers evaluated the effects of RANKL and OPG on orthodontic tooth movement and stability:

  • Hudson JB, Hatch N, Hayami T, Shin JM, Stolina M, Kostenuik PJ, Kapila S. Local delivery of recombinant osteoprotegerin enhances postorthodontic tooth stability. Calcif Tissue Int. 2012 Apr; 90(4):330-42.
  • Schneider DA, Smith SM, Campbell C, Hayami T, Kapila S, Hatch NE. Locally limited inhibition of bone resorption and orthodontic relapse by recombinant osteoprotegerin protein. Orthod Craniofac Res. 2015 Apr;18 Suppl 1:187-95.
  • Dunn MD, Park CH, Kostenuik PJ, Kapila S, Giannobile WV. Local delivery of osteoprotegerin inhibits mechanically mediated bone modeling in orthodontic tooth movement. Bone. 2007 Sep; 41(3):446-55.
  • Zhao N, Lin J, Kanzaki H, Ni J, Chen Z, Liang W, Liu Y. Local osteoprotegerin gene transfer inhibits relapse of orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 2012 Jan; 141(1):30-40.
  • Fujita S, Yamaguchi M, Utsunomiya T, Yamamoto H, Kasai K. Low-energy laser stimulates tooth movement velocity via expression of RANK and RANKL. Orthod Craniofac Res. 2008 Aug;11(3):143-55.
  • Nishijima Y, Yamaguchi M, Kojima T, Aihara N, Nakajima R, Kasai K. Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro. Orthod Craniofac Res. 2006 May;9(2):63-70.
  • Yamaguchi M. RANK/RANKL/OPG during orthodontic tooth movement. Orthod Craniofac Res. 2009 May;12(2):113-9.

Following papers evaluated different surgical and non-surgical methods to accelerate orthodontic tooth movement in animal models:

  • Baloul SS, Gerstenfeld LC, Morgan EF, Carvalho RS, Van Dyke TE, Kantarci A. Mechanism of action and morphologic changes in the alveolar bone in response to selective alveolar decortication–facilitated tooth movement. Am J Orthod Dentofacial Orthop.2011;139:83-101.
  • Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop. 2006;129:469.e1-32.
  • Yuan H, Zhu X, Lu J, Dai J, Fang B, Shen SG. Accelerated orthodontic tooth movement following le fort I osteotomy in a rodent model. J Oral Maxillofac Surg. 2014 Apr;72(4):764-72.
  • Cheung T, Park J, Lee D, Kim C, Olson J, Javadi S, Lawson G, McCabe J, Moon W, Ting K, Hong C. Ability of mini-implant-facilitated micro-osteoperforations to accelerate tooth movement in rats. Am J Orthod Dentofacial Orthop. 2016 Dec;150(6):958-967.
  • Dibart S, Yee C, Surmenian J, Sebaoun JD, Baloul S, Goguet-Surmenian E, Kantarci A. Tissue response during Piezocision-assisted tooth movement: a histological study in rats. Eur J Orthod. 2014 Aug;36(4):457-64.
  • Yadav S, Dobie T, Assefnia A, Gupta H, Kalajzic Z, Nanda R. Effect of low-frequency mechanical vibration on orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 2015 Sep;148(3):440-9.
  • Ekizer A, Uysal T, Güray E, Akkuş D. Effect of LED-mediated-photobiomodulation therapy on orthodontic tooth movement and root resorption in rats. Lasers Med Sci. 2015 Feb;30(2):779-85.
  • Soma S, Matsumoto S, Higuchi Y, Takano-Yamamoto T, Yamashita K, Kurisu K, Iwamoto M. Local and chronic application of PTH accelerates tooth movement in rats. J Dent Res. 2000 Sep;79(9):1717-24.
  • Akin E, Gurton AU, Olmez H. Effects of nitric oxide in orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop. 2004 Nov;126(5):608-14.
  • Hashimoto F, Kobayashi Y, Mataki S, Kobayashi K, Kato Y, Sakai H. Administration of osteocalcin accelerates orthodontic tooth movement induced by a closed coil spring in rats. Eur J Orthod. 2001 Oct;23(5):535-45.
  1. EXPERIMENTAL PROCEDURES

     Experimental procedure 1: Impact of bioelectric stimulation of RANKL on acceleration of orthodontic tooth movement and its stability

Animals will be purchased from Charles River Laboratories and will be acclimatized for 3 days prior to the study. These animals will be distributed as follows:

Group 1: Bioelectric Stimulation (BES) of RANKL for acceleration of tooth movement (n=10). BES will be applied for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21.

Group 2: Retention of space after BES-stimulated RANKL for acceleration of tooth movement (n=10). BES will be applied for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21 and will be followed by appliance removal and monitoring the animals for another 21 days.

All animals will receive orthodontic treatment and will undergo mesial tooth movement of the left maxillary 1st molar in a split mouth design. We will use our model that we have established in rats (Baloul et al. 2011). Briefly, the animals will be anesthetized, a mini-screw will be inserted into palatal bone lingual to the maxillary incisors, and a coil spring will be ligated to the left upper maxillary molar, activated for 10 mm and ligated around the mini-screw implant and incisors.  The mini-screw placement is a minimally invasive procedure; a flap reflection is not necessary. The screws that will be used are 1.5-diameter 2-mm long titanium mini-screws (Neuro MD screw, KLS Martin L.P., Jacksonville, FL) and will be inserted through the mesial eyelet of the coil spring palatal to the incisors into the maxillary bone avoiding the palatal bony suture.  This will be performed by manual manipulation. In humans, the process requires no special postoperative care. We have not observed any distress or any other symptoms in rats. Yet, in order to avoid any pain, all animals in all groups regardless of the mini-screw placement will receive buprenorphine. Shallow notches will be made around the left first molar crown and the incisor teeth using a small round bur in order to enhance the stability of the ligation. Flowable composite bonding material (Henry Schein Inc., Melville, NY, USA) will be placed over the ligature wire on the mesio-buccal-distal aspects of the incisor teeth.  This will be done to prevent appliance loss, and protect the animal’s lips form damage by the wire.

In addition to orthodontic treatment mechanics, animals in both groups will undergo bioelectric stimulation. Bioelectric signals will be delivered to the molars by using alligator clamps connected to a benchtop stimulator.

After 21 days of orthodontic tooth movement the orthodontic appliances will be removed in both groups. The animals in group 2 will be monitored for 21 more days.

At the day of euthanasia, ~ 4ml blood sample will be collected from each animal by cardiac venipuncture under anesthesia and will used to separate the serum for analyses of RANKL. OPG, calcium, PTH, and calcitonin. After the sacrifice, the maxillae and lower skull will be defleshed, dissected and placed in formalin solution for storage. Radiographic images will be taken on the dissected maxillae and any changes in the anatomic structures will be evaluated. Then, micro-CT images will be obtained for each animal to study the bone response at three-dimensional level. After imaging is completed, tissues will be processed for histological analyses; the maxillae will be decalcified, sectioned and stained using the TRAP staining. An osteoclast/pre-osteoclast count will be collected in the PDL area of the mesial root of 1st molar in three different vertical levels (most apical, middle and most coronal). H&E staining will allow histomorphometric appraisal of calcified osseous tissues surrounding the 5 roots of the upper first molars. mRNA isolation will prepare from each animal around the maxillary molar area harvested. Inter-group comparisons will be made using ANOVA with Bonferroni’s correction for multiple groups. Intra-group comparisons will be made using t-test for repeated measurements.

     Experimental procedure 2: Impact of bioelectric stimulation of OPG on acceleration of orthodontic tooth movement and its stability

In total, 20 rats will be used in this group and the rats will be divided into two groups:

Group 1: Bioelectric Stimulation (BES) of RANKL for acceleration of tooth movement and OPG for stability of the treatment outcome (n=10). BES for RANKL will be applied for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21. BES for OPG induction will be applied for 20 minutes/day on days 13, 17 and 21.

Group 2: Bioelectric Stimulation (BES) of RANKL for acceleration of tooth movement and OPG for stability of the treatment outcome (n=10). BES for RANKL will be applied for 20 minutes/day on days 1, 4, 7, 10, 13, 17, and 21. BES for OPG induction will be applied for 20 minutes/day on days 13, 17 and 21. Appliances will be removed and animals will be monitored for another 21 days.

All animals will receive orthodontic treatment and will undergo mesial tooth movement of the left maxillary 1st molar in a split mouth design. We will use our model that we have established in rats (Baloul et al. 2011). Briefly, the animals will be anesthetized, a mini-screw will be inserted into palatal bone lingual to the maxillary incisors, and a coil spring will be ligated to the left upper maxillary molar, activated for 10 mm and ligated around the mini-screw implant and incisors.  The mini-screw placement is a minimally invasive procedure; a flap reflection is not necessary. The screws that will be used are 1.5-diameter 2-mm long titanium mini-screws (Neuro MD screw, KLS Martin L.P., Jacksonville, FL) and will be inserted through the mesial eyelet of the coil spring palatal to the incisors into the maxillary bone avoiding the palatal bony suture.  This will be performed by manual manipulation. In humans, the process requires no special postoperative care. We have not observed any distress or any other symptoms in rats. Yet, in order to avoid any pain, all animals in all groups regardless of the mini-screw placement will receive buprenorphine. Shallow notches will be made around the left first molar crown and the incisor teeth using a small round bur in order to enhance the stability of the ligation. Flowable composite bonding material (Henry Schein Inc., Melville, NY, USA) will be placed over the ligature wire on the mesio-buccal-distal aspects of the incisor teeth.  This will be done to prevent appliance loss, and protect the animal’s lips form damage by the wire.

In addition to orthodontic treatment mechanics, animals in both groups will undergo bioelectric stimulation for tooth movement acceleration on days 1, 4, 7 and 10. This will be followed by OPG induction to increase stability of the tooth movement on days 13, 17 and 21. Bioelectric signals will be delivered to the molars by using alligator clamps connected to a benchtop stimulator.

At the end of 21 days the orthodontic appliances will be removed in both groups. In group 2, the animals will be monitored for another 21 days.

At the day of euthanasia, ~ 4ml blood sample will be collected from each animal by cardiac venipuncture under anesthesia and will used to separate the serum for analyses of RANKL. OPG, calcium, PTH, and calcitonin. After the sacrifice, the maxillae and lower skull will be defleshed, dissected and placed in formalin solution for storage. Radiographic images will be taken on the dissected maxillae and any changes in the anatomic structures will be evaluated. Then, micro-CT images will be obtained for each animal to study the bone response at three-dimensional level. After imaging is completed, tissues will be processed for histological analyses; the maxillae will be decalcified, sectioned and stained using the TRAP staining. An osteoclast/pre-osteoclast count will be collected in the PDL area of the mesial root of 1st molar in three different vertical levels (most apical, middle and most coronal). H&E staining will allow histomorphometric appraisal of calcified osseous tissues surrounding the 5 roots of the upper first molars. mRNA isolation will prepare from each animal around the maxillary molar area harvested. Inter-group comparisons will be made using ANOVA with Bonferroni’s correction for multiple groups. Intra-group comparisons will be made using t-test for repeated measurements.

     Experimental procedure 3: Conventional orthodontic tooth movement

In total,10 rats will be used. In this phase of the study 10 young adult rats will be divided into two groups:

Group 1: The animals will receive conventional orthodontic treatment for 21 days. (n=5)

Group 2:  The animals will receive conventional orthodontic treatment for 21 days followed by appliance removal and monitoring for relapse for another 21 days.

All animals will receive orthodontic treatment and will undergo mesial tooth movement of the left maxillary 1st molar in a split mouth design. We will use our model that we have established in rats (Baloul et al. 2011). Briefly, the animals will be anesthetized, a mini-screw will be inserted into palatal bone lingual to the maxillary incisors, and a coil spring will be ligated to the left upper maxillary molar, activated for 10 mm and ligated around the mini-screw implant and incisors.  The mini-screw placement is a minimally invasive procedure; a flap reflection is not necessary. The screws that will be used are 1.5-diameter 2-mm long titanium mini-screws (Neuro MD screw, KLS Martin L.P., Jacksonville, FL) and will be inserted through the mesial eyelet of the coil spring palatal to the incisors into the maxillary bone avoiding the palatal bony suture.  This will be performed by manual manipulation. In humans, the process requires no special postoperative care. We have not observed any distress or any other symptoms in rats. Yet, in order to avoid any pain, all animals in all groups regardless of the mini-screw placement will receive buprenorphine. Shallow notches will be made around the left first molar crown and the incisor teeth using a small round bur in order to enhance the stability of the ligation. Flowable composite bonding material (Henry Schein Inc., Melville, NY, USA) will be placed over the ligature wire on the mesio-buccal-distal aspects of the incisor teeth.  This will be done to prevent appliance loss, and protect the animal’s lips form damage by the wire.

At the end of 21 days the appliances will be removed in both groups. In group 2, the animals will be monitored for another 21 days.

At the day of euthanasia, ~ 4ml blood sample will be collected from each animal by cardiac venipuncture under anesthesia and will used to separate the serum for analyses of RANKL. OPG, calcium, PTH, and calcitonin. After the sacrifice, the maxillae and lower skull will be defleshed, dissected and placed in formalin solution for storage. Radiographic images will be taken on the dissected maxillae and any changes in the anatomic structures will be evaluated. Then, micro-CT images will be obtained for each animal to study the bone response at three-dimensional level. After imaging is completed, tissues will be processed for histological analyses; the maxillae will be decalcified, sectioned and stained using the TRAP staining. An osteoclast/pre-osteoclast count will be collected in the PDL area of the mesial root of 1st molar in three different vertical levels (most apical, middle and most coronal). H&E staining will allow histomorphometric appraisal of calcified osseous tissues surrounding the 5 roots of the upper first molars. mRNA isolation will prepare from each animal around the maxillary molar area harvested. Inter-group comparisons will be made using ANOVA with Bonferroni’s correction for multiple groups. Intra-group comparisons will be made using t-test for repeated measurements.

  1. EUTHANASIA

Following the cardiac puncture under anesthesia, animals will be euthanized by CO2 asphyxiation in the euthanasia chamber. Animals will be observed for complete cessation of breathing and beyond for one minute. Bilateral thoracotomy will be performed after confirmation of euthanasia and before tissue sampling. This method is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (AVMA 2013).

The effects of electrical current from a micro-electrical device on tooth movement

Korean J Orthod. 2008 Oct;38(5):337-346. Korean.
Published online October 30, 2008. https://doi.org/10.4041/kjod.2008.38.5.337
Copyright © 2008 Korean Association of Orthodontists
The effects of electrical current from a micro-electrical device on tooth movement
Dong-Hwan Kim, DDS, MSD,a Young-Guk Park, DDS, MSD, PhD,b and Seung-Gu Kang, DDS, MSD, PhDc
aPrivate practice.
bProfessor, Department of Orthodontics, School of Dental Medicine, Kyung Hee University, Korea.
cAssistant Professor, Department of Orthodontics, School of Dental Medicine, Kyung Hee University, Korea.

Corresponding author: Young-Guk Park. Department of Orthodontics, School of Dental Medicine, Kyung-Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul 130-702, Korea. +82 2 958 9310; Email: ygpark@khu.ac.kr
Received September 01, 2006; Revised May 30, 2008; Accepted June 03, 2008.

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Abstract

Objective
The purpose of this study was to determine whether an exogenous electric current to the alveolar bone surrounding a tooth being orthodontically treated can enhance tooth movement in human and to verify the effect of electric currents on tooth movement in a clinical aspect.

Methods
This study was performed on 7 female orthodontic patients. The electric appliance was set in the maxilla to provide a direct electric current of 20 µA. The maxillary canine on one side was assigned as the experimental side, and the other as control. The experimental canine was provided with orthodontic force and electric current. The control side was given orthodontic force only. Electrical current was applied to experimental canines for 5 hours a day. The amount of canine movement was measured with an electronic caliper every week.

Results
The amount of orthodontic tooth movement in the experimental side during 4 weeks was greater by 30% compared to that of the control side. The amount of increase in tooth movement in the experimental side was statistically significant. The amount of tooth movement in the experimental side during the first two weeks was greater than that in the following two weeks. The amount of weekly tooth movement in the control side was decreased gradually.

Conclusions
These results suggested that the exogenous electric current from the miniature electric device might accelerate orthodontic tooth movement by one third and have the potential to reduce orthodontic treatment duration.

Keywords: Electric appliance; Tooth movement; Canine retraction

Electrical stimulation enhances tissue reorganization during orthodontic tooth movement in rats

  • Gisele Sampaio Spadari
  • Ewerton Zaniboni
  • Silvia Amelia Scudeler Vedovello
  • Mauro Pedrine Santamaria
  • Maria Esméria Corezola do Amaral
  • Gláucia Maria Tech dos Santos
  • Marcelo Augusto Marretto Esquisatto
  • Fernanda Aparecida Sampaio Mendonca
  • Milton Santamaria-Jr
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  4. 4.
Original Article

Abstract

Objective

This study evaluated the effects of a low-intensity electric current on tissue reorganization during experimental orthodontic tooth movement.

Materials and methods

Thirty-two animals were divided into two groups evaluated on days 3 and 7: OTM—orthodontic tooth movement and OTM + MC—orthodontic tooth movement and microcurrent application (10 μA/5 min). The samples were processed for histological, morphometric, and Western blotting analysis.

Results

Analysis of the periodontal ligament (PL) showed a significantly smaller number of granulocytes in the OTM + MC group on day 7.The number of fibroblasts was significantly higher in the OTM + MC group on days 3 and 7. The area of birefringent collagen fibers was more organized in the OTM + MC group on days 3 and 7. The number of blood vessels was significantly higher in the OTM + MC group on day 7. Microcurrent application significantly increased the number of osteoclasts in the compression region of the PL. In the OTM + MC group on day 7 of tooth movement, the expression of TGF-β1 and VEGF was significantly reduced whereas the expression of bFGF was increased in PL.

Conclusions

Electrical stimulation enhances tissue responses, reducing the number of granulocytes and increasing the number of fibroblasts, blood vessels, and osteoclasts and modulates the expression of TGF-β1, VEFG, and bFGF.

Clinical relevance

This technique is used in many areas of medicine, but poorly explored in dentistry and orthodontics. This treatment is cheap and non-invasive and can be applied by own orthodontist, and it can improve the treatment with a faster and safe tooth movement, without pain.

Link to article

Effect RANKL Produced by Periodontal Ligament Cells on Orthodontic Tooth Movement

Effect RANKL Produced by Periodontal Ligament Cells on Orthodontic Tooth Movement 
Abstract The bone remodeling process involved in orthodontic tooth movement consists of bone resorption on the compression side and bone formation on the tension side of the teeth. Osteoclasts play an important role in bone remodeling and are necessary for orthodontic tooth movement. Receptor activator of nuclear factor-κB ligand (RANKL) is essential for osteoclast formation and differentiation. Several cell types have been reported to be capable of producing RANKL. We are interested in whether there is a dominant cell type which RANKL production is critical in generating orthodontic tooth movement. In this study, we used a Cre recombinase mouse model to study the effect of RANKL deletion in periodontal ligament cells on orthodontic tooth movement. We found RANKL deletion in periodontal ligament cells significantly decreased the amount of orthodontic tooth movement and reduced the number of osteoclasts formed on the compression side after subjecting the teeth to orthodontic force. It suggests RANKL production from periodontal ligament cells contributes greatly to orthodontic tooth movement and serves as an important source of RANKL in osteoclastogenesis during orthodontic tooth movement.

http://repository.upenn.edu/cgi/viewcontent.cgi?article=1015&context=dental_theses

Local delivery of recombinant osteoprotegerin enhances postorthodontic tooth stability.

Local delivery of recombinant osteoprotegerin enhances postorthodontic tooth stability.

https://www.ncbi.nlm.nih.gov/pubmed/22382900

Clinical Study Korea Results