In Vivo Electromechanical Reshaping of Ear Cartilage in A Rabbit Model
In Vivo Electromechanical Reshaping of Ear Cartilage in A Rabbit Model
In Vivo Electromechanical Reshaping of Ear Cartilage in A Rabbit Model
Author Manuscript
JAMA Facial Plast Surg. Author manuscript; available in PMC 2014 August 05.
Published in final edited form as:
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Sepehr Oliaei, MD, Cyrus Manuel, BS, Badran Karam, BS, Syed F. Hussain, BS, Ashley
Hamamoto, BS, Dmitriy E. Protsenko, PhD, and Brian J. F. Wong, MD, PhD
Department of Otolaryngology–Head and Neck Surgery (Dr Oliaei) and Beckman Laser Institute
and Medical Clinic (Messrs Manuel, Karam, and Hussain, Ms Hamamoto, and Drs Protsenko and
Wong), University of California, Irvine.
Abstract
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Objective—To report the first successful study to date of in vivo electromechanical reshaping of
ear cartilage in a rabbit model.
Methods—Ears of New Zealand white rabbits were re-shaped using percutaneous needle
electrode electromechanical reshaping (5 V for 4 minutes) and were then bolstered for 4 weeks.
Ten ears were treated, with 2 undergoing sham procedures and serving as controls. The treatment
was performed using a platinum array of electrodes consisting of 4 parallel rows of needles
inserted across the region of flexures in the ear. After 4 weeks, the animals were killed, and the
ears were photographed and sectioned for conventional light microscopy and confocal microscopy
(live-dead fluorescent assays).
Results—Significant shape change was noted in all the treated ears (mean, 102.4°; range,
87°-122°). Control ears showed minimal shape retention (mean, 14.5°; range, 4°-25°). Epidermis
and adnexal structures were preserved in reshaped ears, and neochondrogenesis was noted in all
the specimens. Confocal microscopy demonstrated a localized zone of nonviable chondrocytes
(<2.0 mm in diameter) surrounding needle sites in all the treated ears.
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Conclusions—Electromechanical reshaping can alter the shape of the rabbit auricle, providing
good creation and retention of shape, with limited skin and cartilage injury. Needle electrode
electromechanical reshaping is a viable technique for minimally invasive tissue reshaping, with
potential applications in otoplasty, septoplasty, and rhinoplasty. Further studies to refine dosimetry
parameters will be required before clinical trials.
Congenital external ear malformations, including the protruding ear, occur in 5% of the
population.1 Such deformities can lead to long-term psychological trauma from childhood
onward.1 Because much of the ear growth is complete by age 3 years2 and because the
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geometry and dimensions of the adult auricular cartilage are reached by age 6 to 7 years,3
surgical correction of the external ear is recommended before the child is of school age, a
time at which teasing and ridicule among children begins.
The underdeveloped antihelix and a large conchal bowl are 2 underlying malformations that
require surgery. Classic otoplasty seeks to address these 2 conditions. Otoplasty is a
technically demanding procedure, with a steep learning curve in which poor aesthetic
outcomes or failure is common. The lack of consensus on a single effective otoplasty
technique has led to the development of more than 40 different operations.4 Of these, the
corrective method found to be most appropriate has been approached subjectively, with
minimal quantifiable data.5 To overcome the limitations of conventional surgery, minimally
invasive reshaping techniques (including laser,6 radiofrequency,7 and enzymatic digestion8)
have been developed. Electromechanical reshaping (EMR) of cartilage is a minimally
invasive technique that was introduced previously9 but is still experimental. It is a novel
technique that has significant potential for use in facial reconstructive surgery.
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Previous ex vivo experiments have shown that direct current electrical fields around the area
of active stress concentration can affect permanent change in cartilage.9 Nonthermogenic
redox reactions at the tissue-electrode interface change the composition of cartilage,
resulting in relaxation of internal stresses and sustained shape change, without the need for
surgical incisions.9-11 Accordingly, surgical embodiments of EMR would use percutaneous
or transmucosal needles inserted into the regions of stress distribution in mechanically
deformed or reshaped cartilage specimens, and the feasibility of this approach in ex vivo
tissues has been previously discussed.12,13
Given that a set of voltage and time parameters has been identified that produce effective
shape change in intact ex vivo auricular specimens,12 further optimization of electrical
dosimetry variables and electrode configurations in vivo is needed to move this technology
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toward clinical use.10,12 This study aimed to evaluate the in vivo effects of EMR of ear
cartilage in a rabbit model to determine the dependence of shape change, mechanical
stability, and tissue injury on dosimetry (voltage and application time) and electrode
configuration.
METHODS
The protocol was approved and performed under the guidance of the Institutional Animal
Care and Use Committee at the University of California, Irvine. All the regulations were
followed throughout the duration of the study.
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EMR PROCEDURE
Ten New Zealand white rabbits (Western Oregon Rabbit Company), weighing 3.8 to 4.0 kg,
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agitation; photographic documentation of the ears was obtained at regular intervals. After 4
weeks, the animals were killed with a lethal dose of intraperitoneal pentobarbital, and the
splints were removed. The auricles were then harvested in preparation for the subsequent
procedures listed in this subsection.
Photography—With the tip pointing superiorly and inferiorly while being supported from
the base, each ear was photographed and videotaped. Analysis of the degree of shape change
in each ear was measured using a commercially available software package (Photoshop
version 9.0; Adobe Systems, Inc).
cure. This base was then mounted onto the upper platen of the mechanical testing platform.
Ears were bent 90°, and the bottom platen of the platform was gradually raised until its edge
was in contact with the deflected portion of the ear 10.0 mm away from the bend axis of the
ear. A static reaction force was recorded at this moment. The upper platen moved up and
down at a constant velocity of 2.5 mm/s to a maximum amplitude of 2.5 mm, flexing the ear
at its bend (resulting from reshaping). A stationary load cell rated to 250 N attached to the
bottom platen measured the integrated reaction force produced by the ear in response to
flexing.
Displacement and integrated reaction force were recorded during flexing. The interval where
this force changes linearly with displacement was identified, and the integrated elastic
modulus was calculated. The mechanical response of control ears (no EMR) was evaluated
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in a manner similar to that of the treatment ears. In both treatment and control ears, the
initial reaction force registering at the moment of contact with the bottom platen and
corresponding to the reaction to 90° bending was recorded and was subtracted from the
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Specimens were cut in serial cross-section with a razor blade and were prepared under
minimal lighting conditions because the aforementioned dyes are photoreactive. Both dyes
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For histologic analysis, the specimens were formalin fixed and paraffin embedded. A
microtome was used to serially section the paraffin blocks, which were then stained with
hematoxylin-eosin. Once on the slides, sections were digitized and montaged to reconstruct
the specimen. Three individuals (S.O., C.M., and S.F.H.) blinded to the study were trained
to analyze each histologic specimen. Cartilage thickness was measured at the apex of the
bend using available software (ImageJ; National Institutes of Health). Evidence of scar
tissue and any signs of chondrocyte regeneration were noted.
To assess the viability of chondrocyte cells, specimens were stained with calcein
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acetomethoxy and ethidium homodimer 1 and were viewed using confocal microscopy.
Within 48 hours of euthanasia, the harvested ears were stained under low light to prevent
premature fluorescence of the dyes used. A 480-nm argon laser confocal microscope was
used to image stained specimens at ×10 magnification. One individual (C.M.) blinded to the
study was trained to analyze each confocal result. Data were presented blinded, and this
individual measured cartilage thickness, width of tissue injury at each electrode, and the
width of live cells between electrodes.
RESULTS
All the rabbits tolerated EMR and survived for the duration of the study. Initial exudative
scabs at perforation sites resolved by postoperative day 11 (Figure 2); fully intact
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epithelium was noted in all the ears at the time of euthanasia. No infection, scarring, or
bleeding complications were noted. All the treated ears showed significant shape change
(mean, 102.4°; range, 87°-122°), exceeding that of control ears (mean, 14.5°; range, 4°-25°)
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(Table).
HISTOLOGIC EXAMINATION
Histologic examination of the electroformed sites showed an intact epidermis and adnexal
structures (Figure 3). Platinum needle electrode (30-gauge) perforations in the ear created a
discontinuous cartilaginous layer spanning 0.3 to 0.8 mm. A chondroblastic proliferation
was observed near the area of the cartilaginous defect. This
correspondstoasubperichondrialformationofcartilagearound the periphery of the site where
EMR was applied.
CONFOCAL MICROSCOPY
Confocal microscopy demonstrated a localized zone of nonviable chondrocytes less than 2.0
mm in diameter surrounding the point of insertion of needle electrodes in all the treated ears
(Figure 3). Within this zone was evidence of fibrocartilage scar tissue. The anodic and
cathodic rows were similar in appearance for live-dead fluorescent assays.
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COMMENT
In this pilot study, we demonstrated that EMR can be used to alter the shape of the ear in an
in vivo rabbit model. The low cost, technically simple, and nonthermogenic features of
EMR make this modality an appealing alternative to other minimally invasive systems, such
as laser and RF-mediated reshaping. To advance toward clinical trials, the safety and
efficacy of EMR on living tissue must be assessed.14 This study demonstrated that EMR
technology can be applied along the entire width of the auricle to create shape change,
without complications of major skin injury, necrosis, or infection. Skin injury merits
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discussion because the electrodes were in contact with the skin and cartilage during the
procedure. A major concern was that EMR performed without the use of an insulation layer
covering portions of the needle electrode not in contact with cartilage might lead to
cutaneous injury and adverse outcomes. On inspection and with histologic analysis, we
found no sustained injury, despite the observation of crusting and serous exudates
immediately after EMR was performed. Based on this information, we conclude that
epithelial injury with the present protocol will not be a major hurdle for using this
technology.
The dosimetry parameters were derived from prior ex vivo investigations12 as a starting
point for producing effective shape change in live rabbit ears. Future experiments will
further examine the dependence of shape change on dosimetry in vivo, with the broad goal
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Histologic analysis of the ear showed patterns promising to remodeling that lead to
permanent shape change with EMR. Normal tissue regions that were 2.0 mm away from the
electrodes showed no signs of necrosis, fibrotic changes, or other damage. Regions near the
vicinity of each electrode (<2.0 mm away) showed chondrocyte proliferation. The cells were
most dense by the needle sites and where curvature was greatest. Needle insertion sites there
were replaced with a fibrotic process or remained unchanged, indicating that a longer study
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period is warranted to observe complete closure. The toxic pH gradient created by the EMR
procedure seems spatially limited and perhaps may be mitigated in part by blood flow
through vascularized tissues. Blood does not flow through cartilage tissue, and this fact is
exploited when performing EMR.
Assessing chondrocyte viability using laser confocal microscopy and live-dead fluorescent
assays is a well-established technique,15,16 aiding the optimization of EMR dosimetry and
geometry of electrode placement. Confocal microscopy identified some regions of tissue
injury, which were spatially limited to the immediate vicinity around each needle electrode
up to 1.2-mm away. The tissue injury surrounding each electrode insertion was consistent
among all the specimens, but these regions were small and represented a fraction of the total
tissue volume in the specimen. In addition, the visualization of chondrocyte neogenesis
coincided with the histologic results of the same ear. Previous in vitro investigations12
examining cell injury in EMR-altered cartilage specimens showed results similar to our in
vivo investigations. This degree of tissue injury is limited in its spatial extent and is on par
with results using conventional techniques, such as cartilage morselization. Like
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radiofrequency and laser cartilage reshaping, EMR achieves shape change at the expense of
cell injury.
The objective herein was to assess the safety and efficacy of EMR in vivo. To our
knowledge, this is the first study to demonstrate that shape change can be achieved via mild
injury to the cartilage, with no persistent damage to the epithelial lining. Future studies will
examine longer survival times, which will provide more information on the long-term in
vivo response of cartilage tissue (and overlying skin) to EMR.
In conclusion, EMR altered the shape of the rabbit auricle, providing good creation and
retention of shape, with limited skin and cartilage injury. Needle electrode EMR is a viable
technique for minimally invasive tissue reshaping, with potential applications in otoplasty,
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septoplasty, and rhinoplasty. Further studies to refine dosimetry parameters and to assess
reshaping over time with the auricular splints removed will be required before clinical trials.
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Acknowledgments
Funding/Support: This study was supported by grant ERT 44380-29302 from the National Institutes of Health (Dr
Wong).
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Figure 1.
In vivo electromechanical reshaping of ear cartilage in a rabbit model. A, Schematic
representation. B, Acrylic jig used to mechanically deform ears during the procedure. DC
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Figure 2.
Electromechanical reshaping results. A, Typical appearance of a bolstered-treated ear on
postoperative day 5, with dried exudative scabs. By day 11 (×1.5 magnified inset), the scabs
are gone, and the underlying skin appears healthy and intact. B and C, Appearance of ears
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after removal of the bolster on day 28 (after euthanasia) in a control ear (B) and in a treated
ear (C).
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Figure 3.
Histologic findings. A, Control ear shows normal-appearing cartilage and epithelial lining.
B, Live-dead fluorescent assay confocal microscopy shows viable chondrocytes (green)
throughout the control specimen. C, Treated ear shows curved geometry with intact
epithelial lining and adnexal structures (short blue arrow). Neochondrogenesis is noted
along the undersurface of the cartilage in the periphery of perforated sites (long blue arrow
and ×2 magnified inset). D, Live-dead fluorescent assay confocal microscopy shows limited
zones of nonviable chondrocytes (red) around needle electrode sites, with live cells along
the undersurface of the cartilage, presumably representing ingrowth of new chondrocytes
(×10 magnified inset). The gross section is shown on the left.
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Table
Bend Angles After In Vivo Electromechanical Reshaping of Ear Cartilage in a Rabbit Model
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Ear No. a
Bend Angle
Control
1 25
2 4
Treated
1 108
2 87
3 97
4 119
5 99
6 97
7 90
8 122
a
Calculated as degrees of bend from a vertical plane.
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