ORIGINAL ARTICLE
Long-term Viability and Mechanical Behavior
Following Laser Cartilage Reshaping
Amir M. Karam, MD; Dmitriy E. Protsenko, PhD; Chao Li; Ryan Wright, BS; Lih-Huei L. Liaw, MS;
Thomas E. Milner, PhD; Brian J. F. Wong, MD, PhD
Objective: To investigate the long-term in vivo effect
of laser dosimetry on rabbit septal cartilage integrity, viability, and mechanical behavior.
Methods: Nasal septal cartilage specimens (control and
irradiated pairs) were harvested from 18 rabbits. Specimens were mechanically deformed and irradiated with
an Nd:YAG laser across a broad dosimetry range (4-8 W
and 6-16 seconds). Treated specimens and controls were
autologously implanted into a subperichondrial auricular pocket. Specimens were harvested an average±SD of
208±35 days later. Tissue integrity, histology, chondrocyte viability, and mechanical property evaluations were
performed. Tissue damage results were compared with
Monte Carlo simulation models.
L
Author Affiliations: Beckman
Laser Institute (Drs Karam,
Protsenko, and Wong; Messrs Li
and Wright; and Ms Liaw),
Division of Facial Plastic and
Reconstructive Surgery,
Department of
Otolaryngology–Head and Neck
Surgery, Irvine Medical Center
(Drs Karam and Wong), and
Department of Biomedical
Engineering (Dr Wong),
University of California, Irvine;
and Department of Biomedical
Engineering, University of
Texas at Austin (Dr Milner).
Results: All laser-irradiated specimens demonstrated variable tissue resorption and calcification, which increased with
increased dosimetry. Elastic moduli of the specimens were
significantly either lower or higher than controls (all P⬍.05).
Viability assays illustrated a total loss of viable chondrocytes within the laser-irradiated zones in all treated specimens. Histologic examination confirmed these findings. Experimental results were consistent with damage profiles
determined using numerical simulations.
Conclusion: The loss of structural integrity and chondrocyte viability observed across a broad dosimetry range
underscores the importance of spatially selective heating
methods prior to initiating application in human subjects.
Arch Facial Plast Surg. 2006;8:105-116
ASER CARTILAGE RESHAPING
was introduced in 1993 by
Helidonis et al1 (as also reported in Sobol et al2). In laser cartilage reshaping, specimens are held in mechanical deformation
and then heated using laser radiation. Heat
generation accelerates the process of mechanical stress relaxation, which allows tissue to remain in stable new shapes and geometries. Although the mechanisms
underlying laser cartilage reshaping have
not been completely identified, numerous animal studies have investigated the
various biophysical mechanisms underlying shape change.3-10 In contrast to conventional surgical techniques, which rely
on sutures and scalpels to balance and relieve the forces that resist mechanical deformation,11-16 laser cartilage reshaping relies on heat generation to induce structural
changes in the tissue matrix. Several studies have demonstrated the feasibility of laser-assisted cartilage reshaping in porcine and rabbit auricles, as well as in canine
trachea.17-21
At the Russian Academy of Sciences in
Troitsk, clinical evaluation of laserassisted cartilage reshaping for correc-
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
105
tion of nasal septal deformities22 has now
involved more than 200 patients (Emil Sobol, PhD, oral communication, 2004).
While several studies23,24 report the viability of cartilage tissue following laser irradiation in vivo, the laser dosimetry that
results in effective laser reshaping remains
unknown. The problem is challenging in
that heat is needed to cause physical changes
in the tissue to produce shape change, yet
excess heat generation can result in significant cell death within the heated volume
of tissue. Sobol et al8 theorized that a dosimetry parameter space may exist where
both cell viability and shape change can be
achieved. In contrast to this theory, early
work by Karamzadeh et al,5,23 and later by
Mordon et al24 and Chiu et al,20,25 has shown
that shape change in cartilage may be a consequence of spatially selective heating, a
principle and technique that have been exploited successfully in dermatologic laser
applications where heat is delivered selectively to specific regions of the tissue to cause
intense thermal injury.5,23,24 In those applications, however, the surrounding tissues
are largely spared of injury, allowing for the
regeneration and regrowth of the tissue in
the damaged regions.
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
New Zealand White Rabbits
Submucous Resection
Reshaping Apparatus
Cartilage Cut Into
2 Uniform Slabs
Laser-Reshaped
Cartilage
Control
Specimens Reimplanted
in Auricular Pocket
Specimen Harvest
Mechanical Testing
Viability Assay
Histology
Figure 1. Schematic of the experimental protocol.
The present study aimed to investigate the effect of
laser dosimetry (ie, power density and irradiation time)
on cartilage mechanical properties and viability and to
compare results obtained for temperature elevation, thermal injury, and shape change with those predicted by numerical simulation.
METHODS
Eighteen Pasteurella-free New Zealand white rabbits (3.5-4.5
kg; 9-12 months old) were used in the study. All protocols and
experimental design settings were reviewed and approved by
the University of California, Irvine Institutional Animal Care
and Use Committee.
ANIMAL SURGERY
Anesthesia, preoperative preparation, and the surgical technique have been described elsewhere23,26 and only an overview will be provided here. Animals were induced by administering ketamine hydrochloride (35 mg of 100-mg/mL ketamine
hydrochloride per kilogram of rabbit body weight) and xylazine hydrochloride (5 mg/kg of 20-mg/mL xylazine hydrochloride) via an intramuscular injection, intubated, and then maintained on isoflurane gas (2%-4%), which was titrated to effect.
A midline 3.5-cm-long nasal dorsal incision, from the level
of the frontonasal suture to 1.0 cm above the nasal tip, was made
to provide exposure for a laterally based osteoplastic flap
(1.0⫻3.0 cm) centered over the septum. After the nasal cavity was exposed, bilateral submucoperichondrial flaps were elevated, and a 1.0⫻3.0-cm central segment of the septal cartilage was removed. Both sides of the septal mucosa were
approximated, the osteoplastic flap was closed, and the skin
incisions were sutured together.
LASER RESHAPING AND
TEMPERATURE MEASUREMENT
Figure 1 illustrates the experimental protocol. The excised cartilage graft was immediately divided into 2 rectangular slabs
(1⫻5⫻15 mm each) with a razor blade. One served as the control specimen and the other was irradiated with the laser. The
cartilage for laser irradiation was placed in a custom jig assembly designed to maintain the specimen in a curved semicircular
geometry (Figure 2).27,28 The jig was attached to a computercontrolled rotary translation stage, which positioned the specimen relative to the laser beam. Light from an Nd:YAG laser
(␭=1.32 µm, 5.4-mm-diameter spot size, 50-Hz pulse repetition rate; New Star Lasers Inc, Roseville, Calif) was directed perpendicularly at the specimen by using a multimode optical fiber
(600 µm) and a collimating lens. This chassis was coupled to an
infrared thermopile detector, which was used to estimate surface temperature.16 Thus, real-time measurements of surface temperature were recorded during the laser irradiation. Each specimen was irradiated using a different power–irradiation time pair
(4-8 W, 6-16 seconds) as indicated in Table 1. The laser was
directed at 3 predetermined positions in a nonoverlapping vertical arrangement along the surface of the specimen (Figure 2).
Signals from the thermopile were recorded with the use of an
analog-to-digital converter and a computer workstation.23 A 15second delay was programmed between each irradiation spot to
reduce overheating due to heat conduction. After irradiation, the
cartilage specimen was immersed in an ambient-temperature isotonic sodium chloride solution bath for 15 minutes while maintaining the semicircular geometry. The distance between the ends
of the slab was measured after rehydration and removal from the
jig. Control specimens were secured in the same jig for 15 minutes (while immersed) but not irradiated. After removal from the
jig and measurement, the control and laser-irradiated specimens were immersed 3 times (15 minutes each) in an antibiotic
solution containing phosphate-buffered saline with gentamicin
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
106
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
2
1
3
Figure 2. Laser irradiation pattern for rabbit nasal septal cartilage. Positions 1, 2, and 3 illustrate the sequence of laser irradiation along the surface of the cartilage
during reshaping.
Table 1. Laser Power, Irradiation Time, Presence of Calcification, Peak Surface Temperature, Bend Angle,
Presence of Viable Chondrocytes, and Fibroblast Infiltration Into the Matrix
Specimen
No.
Laser
Power, W
Time, s
Calcification
Peak Surface
Temperature, oC
Acute Bend Angle,
Degrees*
Viable
Chondrocytes
Fibroblast
Infiltration
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
8
8
8
16
12
10
8
6
10
16
12
10
8
6
6
8
10
12
6
8
10
Partial
Partial
Partial
Partial
Partial
Partial
Partial
Partial
Total
Total
Partial
Partial
Partial
Partial
Partial
Partial
Partial
Partial
75
71
69
70
68
60
93
76
74
68
64
77
81
85
74
94
116
120
55
38
35
25
16
45
65
54
45
33
26
62
59
61
77
62
67
71
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
*Measured according to the method of Wright et al.29
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
107
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
A
B
C
D
Perichondrium
Figure 3. Autologous reimplantation of the septal cartilage graft into the subperichondrial pocket in the base of the auricle. A, Incision site at the base of the
auricle. B, Positioning of the irradiated cartilage beneath the perichondrium. C, The cartilage (asterisk) in place. D, Final position of the control and
irradiated-cartilage specimens (black rectangles).
sulfate (200 mg/L) and amphotericin B (22.4 mg/L) under sterile conditions.
REIMPLANTATION
A small vertical incision was made along the base of the right
auricle. The reshaped specimen and paired control were then autologously reimplanted into a subperichondrial pocket overlying the auricular cartilage in a transverse orientation (Figure 3).
This location was selected because of the intrinsic favorable curvature of the auricle and an intact perichondrium—factors we
hypothesized would optimize graft survival and shape retention.23 The degree of curvature of the auricle is significantly less
than that of the acutely reshaped cartilage specimens. Placement of the curved specimens within the subperichondrial pocket
resulted in an overall reduction of the curvature. A single 6-0
blue polypropylene suture was placed through the center of the
irradiated specimen before implantation to facilitate identification at harvest. The perichondrium and overlying skin were closed
in separate layers. Following surgery, the animals were observed daily and examined for signs of pain, wound infection,
and other operative complications.
SPECIMEN RETRIEVAL
Specimens were removed following euthanasia an average±SD
of 208±35 days after implantation. Samples were dissected free
from the subperichondrial pockets with the use of a dissection
microscope. Specimens were evaluated for gross shape change,
presence of calcification, and overall mechanical integrity
(Table 1). Specimens were photographed and prepared for mechanical analysis, confocal imaging, viability assessment, and
histologic examination.
MECHANICAL PROPERTIES
Specimens were maintained in isotonic sodium chloride solution until just before mechanical testing (no longer than 15 minutes). The width, thickness, and length of each specimen were
measured with a digital caliper. The specimens were visually
examined and separated on the basis of presence and patterns
of calcification. Specimens that showed evidence of partial calcification (nonhomogeneous) were grouped together, and the
calcified segments were excised and analyzed separately from
the noncalcified regions. Likewise, specimens that showed homogeneous patterns of calcification or noncalcification were
grouped and evaluated together.
Figure 4 shows the experimental setup for measurement
of the elastic modulus of cartilage specimens.30,31 The cartilage
specimen was secured between plastic clamps and attached to
a motorized stage. The free end of the specimen was brought
in contact with a blunt ball-tipped pin, mounted on a calibrated load cell (Model LCL 227 g; Omega Engineering, Inc,
Stamford, Conn). The pin was blunted to prevent penetration
into the specimen and gliding across the surface. The cantilevered cartilage specimen was deflected by moving the encoded motorized stage (Model M-126 PD; Physiks Instruments, Karlsruhe/Palmbach, Germany), controlled by a personal
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
108
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
computer, back and forth a distance of 1 mm at a velocity of 5
mm/s. To reduce measurement errors and variation due to specimen nonuniformity, a micropositioner (Newport Corporation, Irvine) moved the pin-and-load cell assembly along the
specimen’s surface to several different positions separated by
0.5 to 1 mm. The experiment was performed at each location
with the application of 5 cycles of deflection.
The elastic modulus (E), of the cartilage specimen was estimated using the following equation for elastic flexure of a cantilevered bar:
Signal
Conditioner
A/D
Converter
PC
3
1
7
4
L
where ∆F is the change in force; ∆X, the change in displacement; L, the distance between the pin and the edge of the clamp;
and I, the moment of inertia32 For a cross section of a rectangular bar with a width w and thickness b, I=wb3/12. The calculated elastic modulus was averaged across all pin locations
and deflection cycles.
2
5
6
VIABILITY ANALYSIS
After mechanical analysis, an approximately 500-µm-thick crosssectional slice was cut lengthwise from the dissected tissue
sample. The tissue slice was incubated in a live/dead assay system for visualization with a confocal microscope. The 2-color
fluorescent viability dye solution was prepared using green fluorescent cyanine dye and red fluorescent ethidium homodimer-2 (SYTO 10 and DEAD Red, respectively; Molecular
Probes Inc, Eugene, Ore). Live/dead assay has previously been
used to identify live and dead cells in laser-irradiated cartilage
with flow cytometry10 and with confocal microscopy.33 The assay determines cell viability on the basis of cell membrane integrity, which is considered an accurate indicator of cell viability.34 The assay uses a system of 2 fluorescent nucleic acid stains
that weakly bind to DNA and RNA. SYTO 10 permeates all cells
in the sample and weakly binds to the cell’s nucleotides with
low affinity. SYTO 10 is excited at 488 nm and fluoresces green
at 520 nm. The second stain, DEAD Red, can only permeate
cells with compromised membranes, weakly binds to DNA and
RNA with high affinity, and thereby displaces any bound SYTO
10. DEAD Red is excited at 488 nm and fluoresces red at 615
nm. The sectioned specimens were placed in a live/dead assay
staining solution consisting of 4 µL of SYTO 10 and 2 µL of
DEAD Red in 1 mL of Hank balanced salt solution. This ratio
of dye concentration was found to be optimal for our experiments because STYO 10 tends to photobleach at a much higher
rate than DEAD Red does.
HISTOLOGIC EXAMINATION
The remaining portions of the specimens were fixed in formalin, serially dehydrated using graded ethanol solutions, and embedded in paraffin. The 6-µm sections were stained with hematoxylin-eosin and examined microscopically to correlate with
confocal microscopic findings.
Figure 4. Experimental setup for measurement of cartilage elastic modulus:
(1) cartilage specimen, (2) clamps for holding specimen, (3) motorized
stage, (4) blunt pin in contact with cartilage specimen, (5) load cell, (6)
micropositioner, (7) approximate positions of pin-cartilage contact sites. L is
the distance between the pin (4) and the edge of the clamp (2).
where ␶ is heating time, ⍀ is the thermal damage parameter
and ⍀ⱖ1 corresponds to 1; R, the universal gas constant; T,
the absolute temperature; A, a rate constant or frequency factor; and Ea, the activation energy per mole of molecules.36 The
values of A and Ea were previously estimated on the basis of
experiments that quantified the short-time decrease in viable
rabbit septal chondrocytes as a function of exposure time in
temperature-controlled isotonic sodium chloride baths.9 These
variables allow estimation of cell viability given knowledge of
the heating profile as a function of time.
The optical and thermal properties of cartilage, thermal damage settings, and geometry of the simulated cartilage specimen
used in the numerical model are listed in Table 2. The temperature profiles as a function of time and space were calculated for a single laser exposure using each of the dosimetry setting pairs listed in Table 1. These data were used to estimate the
peak temperature for each dosimetry pair as previously described.35 The model was used to estimate the spatial distribution of thermal injury by numerically integrating equation 2 and
permits identification of regions of intense thermal injury, ie,
where ⍀(␶) is greater than 1. Because a circular laser beam was
used in the experiments and simulations, the spatial extent of
severe thermal injury (ie, cell death) could be estimated. Results are presented as mean±SD unless specified otherwise.
NUMERICAL MODEL
OF CARTILAGE IRRADIATION
Laser irradiation (Nd:YAG, ␭=1.32 µm) of rectangular slabs
of cartilage was numerically modeled. A previously described
thermo-optical model35 was modified to include calculations
of thermal damage based on a rate process damage mechanism, as follows:
RESULTS
MACROSCOPIC TISSUE PROPERTIES
Table 1 gives measured bend angles immediately following laser irradiation for each dosimetry pair. A direct relationship between laser dosimetry and the degree of bend
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
109
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
angle was observed up to a power density of 20.4 W/cm2.
At greater doses, the degree of curvature flattened
(Table 1). Bend angles were derived from recent ex vivo
experiments designed to determine the relationship between laser dosimetry and the resultant bend angle. Details of these studies are provided elsewhere.29
All laser-irradiated specimens demonstrated some loss
of tissue integrity and calcification, increasing with increased dose (Figure 5). Nearly all irradiated specimens contained calcified zones associated with regions
where laser energy was directed. The extent of tissue calcification increased with laser dosimetry and generated
a radial pattern of calcification. Nonirradiated controls
showed relatively intact preservation of tissue bulk with
no evidence of calcification.
After tissue recovery at 208±35 days, irradiated specimens (both stiff [calcified] and soft [noncalcified]) did
not maintain curvature and conformed to the general
shape of the auricular base. Specimens that were very stiff
or very soft lacked any of the original curvature.
SURFACE TEMPERATURE MEASUREMENTS
Peak surface temperature measurements increased with
increasing dosimetry. The range of surface temperatures was between 60°C and 120°C (Table 1).
(Figure 6). The average elastic modulus for these samples
was 9±6 MPa (range, 1.6±0.1 MPa to 21±1.7 MPa for
the least and most stiff specimens, respectively).
Figure 7 shows the elastic moduli of the laserirradiated specimens compared with the average modulus of the control group. Data from the nonhomogeneous group (irradiated specimens 3, 5, 9, 10, and 11)
are arranged in pairs, showing the elastic modulus of noncalcified and calcified portions separately. The lowest elastic modulus of 0.17±0.03 MPa was measured in the soft
portion of the specimen from animal 3. Specimens from
animals 6, 7, 8, 14, and 16 demonstrated a high degree
of calcification. Mechanical flexing of these specimens
in an experimental apparatus resulted in random fracturing of calcified material, which prevented accurate estimation of elastic modulus. However, from data obtained before the fracturing occurred, it was possible to
estimate that the elastic modulus of these specimens exceeded 43 MPa.
Figure 8 shows elastic modulus as a function of the
laser energy delivered per a single irradiation spot for the
noncalcified and calcified portions of the nonhomogeneous and homogeneous specimens, respectively. The
elastic modulus of the calcified portions of the nonhomogeneous samples was enhanced with increasing energy level, from 1.4±0.1 MPa at 24 J to 15±5 MPa at 50
J. In the same energy range, the elastic modulus of non-
MECHANICAL MEASUREMENTS
We found no evidence of calcification in any of the 18
control specimens; however, specimen flexibility did vary
25
Length
15 ⫻ 10−3 m
Width
5 ⫻ 10−3 m
Thickness
Initial
temperature
Thermal
conductivity
Density
1.25 ⫻ 10−3 m
25°C
Heat capacity
Convection
coefficient
0.6 W/m per
Kelvin
1260 kg/m3
4000 J/(kg 䡠 K)
20 W/(m2 䡠 K)
100 m−1
Absorption
coefficient
Scattering
coefficient
Scattering angle
Tissue refraction
index
Laser beam
radius
Laser beam
profile
Frequency factor
Activation
energy
A
2.6 ⫻ 103 m−1
Elastic Modulus, MPa
20
Table 2. Cartilage Slab Geometry and Optical, Thermal,
and Thermal Damage Settings Used in the Numerical Model
for Cartilage Irradiation
0.9 radian
1.37
15
10
5
2 ⫻ 10−3 m
0
Flat-top
1.2 s−1
4.5 J 䡠 mol−1
C
1
2
3
4
5
7
8
9
10 11 13 14 15
6
16 17
Animal No.
Figure 6. Average (and standard deviation) elastic modulus of the control
specimens. The average modulus of the control specimens (C) is shown at
the far left for comparison.
B
1 cm
C
1 cm
1 cm
Figure 5. Range of tissue resorption and calcification following laser irradiation. A, A control specimen shows no evidence of calcification or resorption. B, A
sample irradiated at 4 W for 12 seconds shows loss of tissue integrity and heterogeneous patches of calcification. C, A specimen irradiated at 4 W for 16 seconds
shows extensive calcification extending beyond the borders of the native cartilage.
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
110
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
100
Elastic Modulus, MPa
calcified portions remained significantly lower (from
0.17±0.03 MPa to 0.61±0.07 MPa) than the control value
(9±6 MPa) (P = .048).
The nonhomogeneous specimens—specimens with
spatially separated calcified and noncalcified portions—
were present only at energy levels of 50 J or below.
Homogeneously calcified specimens with elastic moduli
greater than 10 MPa appeared at energy levels of 50 J
and above. However, soft homogeneous specimens with
elastic moduli varying from 0.25 ± 0.10 to 0.97 ± 0.06
MPa were present throughout the investigated energy
ranges.
E > 43 MPa
10
0
CHONDROCYTE VIABILITY
HISTOLOGIC FINDINGS
Review of the histologic images reinforced the confocal
microscopy observations. Images revealed empty lacunae with fibroblasts tunneling through a deteriorated matrix devoid of chondrocytes in all laser-irradiated specimens (Figure 10). The matrix was replaced by scar tissue
and calcification in the irradiated spots. The calcification density was the greatest at the center of the laser spot.
However, regions outside the laser-irradiated zone and
controls showed normal cellular and extracellular architecture. The lack of viable chondrocytes in the irradiated regions was consistent with numerical simulation
results.
NUMERICAL SIMULATION
OF CARTILAGE IRRADIATION
Figure 11 compares measured peak radiometric temperature on the irradiated cartilage surface during laser
irradiation (Texp) with values predicted by numerical simulation (Tsim). The experimental values are an average of
measurements recorded during the 3 consecutive irradiations on the specimen for a given set of laser settings.
The simulated and experimental values correlated well
at temperatures below 90°C (correlation coefficient, 0.95).
0.1
C 12 1
2
3
4
5
7
8
9 10 11 13 14 15 6 16 17
Animal No.
Figure 7. Average (and standard deviation) elastic modulus of
laser-irradiated specimens. The moduli of soft (noncalcified) (gray bars) and
stiff (calcified) (solid bars) portions are shown for nonhomogeneous
specimens 3, 5, 9, 10, and 11. The elastic modulus (E ) of specimens 6, 7, 8,
14, and 16 was greater than 43 MPa. The average modulus of the control
specimens (C) is shown at the far left for comparison. Rabbit 12 sustained
damage to the cartilage specimen (control) and therefore could not be
reliably evaluated. Rabbit 18 died before mechanical testing of the cartilage.
100.0
Elastic Modulus, MPa
Confocal microscopy using the live/dead assay revealed
no evidence of live, healthy chondrocytes within the laser-irradiated region of each specimen for any laser dosimetry pairs used in this experiment (Figure 9). Instead, the results consistently showed an extracellular
matrix devoid of chondrocytes that was in the process of
being invaded by fibroblasts (Figure 9). The distinction
between live and dead cells was ultimately of minimal
value because the assay tested the viability of the only
cell type present—invading fibroblasts. The pervading
red signal found in the confocal images represented dead
fibroblast tissue, which was vulnerable to desiccation
and readily perished during the staining procedure. The
importance of the live/dead assay is its utility in distinguishing fibroblasts from chondrocytes by way of their
respective cellular morphology. As the confocal images
of the null controls showed, live chondrocytes are round
and were evenly distributed throughout the matrix, in
contrast to elongated fibroblasts, which permeated the
dead matrix.
10.0
1.0
0.1
20
30
40
50
60
70
80
90
Delivery Energy, J
Figure 8. Average (and standard deviation) elastic modulus of
laser-irradiated specimens as a function of laser energy delivered to 1
irradiated spot of the soft (noncalcified) (open triangles) and stiff (calcified)
(open boxes) portions of the nonhomogeneous specimens and the
homogeneous specimens (solid circles). The ordinate is on the log scale.
When the radiometric temperature was above 90°C, the
simulated values were consistently lower.
Figure 12 correlates the radius of the region of thermal damage (ie, ⍀⬎1) estimated from the numerical simulation with the bend angle of cartilage specimens measured immediately after laser irradiation. The relationship
between bend angle and laser dosimetry using the apparatus described in this study has been described previously.29 A thermal damage radius exceeding 2.5 mm corresponds to the diameter of a damage zone that exceeds
the width of cartilage sample, although the simulation
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
111
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
A
B
C
Figure 9. Representative live/dead viability assay evaluated with the use of confocal microscopy. A, A control specimen demonstrates the homogeneous staining
pattern for green round cells, which represent intact, viable chondrocytes. B, A specimen irradiated at 4 W for 12 seconds exhibits the fingerlike infiltration pattern
of green-staining fibroblasts. C, A specimen irradiated at 8 W for 10 seconds shows a predominance of red-staining cells (chondrocytes) and a paucity of green
round cells (viable chondrocytes).
calculates damage longitudinally along the specimen and
places an upper limit of 7.5 mm on the radius. In experiments, damage zones from adjacent irradiation spots overlap because of heat diffusion; thus, numerical simulations underestimate damage. Figure 12 demonstrates that,
across the range of laser power and irradiation times, the
radius of the thermal damage varied linearly with bend
angle (correlation coefficient, 0.91).
COMMENT
In laser-assisted cartilage reshaping, heat creates physical changes within the tissue matrix, which leads to the
acceleration of stress relaxation and to the establishment of a new equilibrium shape. Heat also may injure
chondrocytes, the constitutive cells of cartilage tissue responsible for the generation and maintenance of a healthy
tissue matrix. An important question under debate is
whether a laser dosimetry parameter space exists where
competing objectives of shape change and cell viability
are achieved. Although the results of this study do not
provide any evidence of such a parameter space, the question may not be relevant to the clinical utility of laserassisted cartilage reshaping. Alternatively, the success of
laser-assisted cartilage reshaping in both animals and humans may rely on the spatially selective heating of cartilage tissue,5,23,24 where heat significantly alters the mechanical properties of discrete regions of tissue without
regard to cell viability. Adjacent tissue does not undergo significant thermal modification, and the net shape
change is produced as a consequence of focal tissue denaturation (and cell damage) combined with wound healing and tissue regeneration owing to the presence of normal surrounding tissue. To verify what occurs in clinical
laser-assisted cartilage reshaping, it is imperative that the
relationship between dosimetry and tissue viability be determined in a long-term in vivo model, because wound
healing, tissue regeneration, and tissue repair cannot be
adequately evaluated using ex vivo tissue specimens in
culture. Hence, this study focused on evaluating long-
term results with an emphasis on obtaining both physical and biological measurements of tissue behavior. In
previous investigations,3-5,7 the acute effect of laser dosimetry on bend angle in cartilage was studied exhaustively in rabbit and porcine nasal septal cartilage and has
provided some information on what might be effective
laser settings to reshape tissue when using Nd:YAG laser irradiation and volumetric heating. Recent work by
our group29 indicates that the maximum bend angle increases with laser dose immediately following laser irradiation, until the power density exceeds 20.4 W/cm2.
Above this threshold, the bend angle does not significantly increase with power density. The dosimetry settings used in this study ranged from levels too low to create shape change to those high enough to do so, based
on our ex vivo studies.
Cartilage is an avascular tissue and undergoes a wound
healing and tissue repair process, which is different from
most other soft tissues. Cartilage injury can result in depopulation of chondrocytes and subsequent changes in
the matrix, including depletion of proteoglycans, calcification, ossification, scar/fibrosis, and even repopulation by chondrocytes provided a stem cell source such
as perichondrium is close to the injured region.24
The laser wavelength used in this study (1.32 µm) produces relatively uniform longitudinal heating of the specimen. Similarly, large laser spots were used to reduce lateral temperature gradients.
Of note, systematic evaluation of each dosimetry pair
(Table 1) demonstrated that no viable populations of
chondrocytes were identified in the region of laser
energy deposition in any specimen studied. This
included dosimetry pairs that produced negligible shape
change (eg, 4 W and 6 seconds). Given that no dosimetry settings and corresponding temperature elevations
maintain cell viability, even under circumstances where
minimal shape change occurs, we believe that laser cartilage reshaping may not involve balancing shape
change and stress relaxation with the preservation of
cell viability within the same space. Effective shape and
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
112
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
A
B
C
D
E
F
Figure 10. Histologic cross sections of laser-irradiated specimens. A, The edge of the laser-irradiated zone of a specimen irradiated at 4 W for 10 seconds
(original magnification ⫻40). The asterisk represents a region on the periphery of the laser spot showing the presence of normal cartilage matrix and lacunae with
chondrocytes. The dashed line indicates the transition zone between nonirradiated (viable) and irradiated (nonviable) cartilage. The laser-irradiated zone
demonstrates the loss of chondrocytes and the empty lacunae. B, A cross section through a specimen irradiated at 4 W for 6 seconds illustrates an acellular
matrix and empty lacunae (original magnification ⫻20). C and D, Infiltration of fibrous tissue into the irradiated cartilage matrix (C, specimen irradiated at 4 W for
10 seconds; D, specimen irradiated at 4 W for 6 seconds; original magnifiiciation for both ⫻20). E, The presence of dense calcification within the cartilage matrix
of a specimen irradiated at 8 W for 8 seconds (original magnification, ⫻20). F, A control specimen illustrates normal cartilage microstructure and the presence of
chondrocytes within the lacunae (original magnification, ⫻40).
the preservation of cell viability are mutually exclusive
phenomena.
Our results underscore the importance of spatially selective heating in cartilage reshaping, which was the technique elegantly demonstrated by Mordon et al24 and Chiu
et al20,25 for reshaping rabbit ears. The concept of spatial
selectivity in laser therapy has become the cornerstone
of dermatologic laser surgery20,23-25 and was advocated for
use in laser cartilage reshaping as early as 1998.5
In a pioneering in vivo study, Mordon et al24 used nearinfrared laser irradiation in combination with contact cooling to reshape rabbit auricular cartilages. Biopsy specimens of the laser-treated region obtained at 1, 3, and 6
weeks demonstrated the presence of a layer of complete
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
113
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
130
120
R = 0.95
Temperature, Tsim, ° C
110
100
90
80
70
60
50
50
60
70
80
100
90
110
120
130
Temperature, Texp, ° C
Figure 11. Correlation of simulated maximal temperature at the cartilage
surface (Tsim) and experimentally measured values (Texp). Each data point
corresponds to a particular combination of laser power and irradiation time.
3.0
Damage Radius, mm
2.8
2.6
(2)
2.4
2.2
R = .091
(1)
2.0
1.8
10
20
30
40
50
60
70
80
Bend Angle, Degrees
Figure 12. Correlation of simulated values of thermal damage radius and
experimentally measured final bend angles of reshaped cartilage. Shown are
the levels of damage radius corresponding to the radius of the laser spot
(horizontal line 1) and also half the width of the cartilage sample
corresponding to half the distance between irradiation spots (horizontal line 2).
chondrocyte death adjacent to a layer of viable chondrocytes.24 Similar findings were reported by Chiu et al.20
The fact that this was accomplished without incisions or
graft removal means that an intact perichondrium was
preserved, either in appositional or adjacent regions. Perichondrium is rich in stem cells and highly vascularized.
In contrast, the present study used extracorporeal irradiation and reshaping of graft tissue with nearinfrared laser wavelengths that minimized axial temperature gradients (and hence any axial spatial selectivity) and
facilitated the study of the isolated effect of dosimetry (or
temperature) on tissue biophysical behavior alone. The
lack of any viable chondrocytes in the laser irradiation
zones suggests that the success of cartilage reshaping in
humans is the result of spatial selectivity of heating. Hence,
the task of reshaping cartilage focuses on delivering adequate laser energy to specific regions of the specimen
where heat can relieve internal stresses produced by mechanical deformation.
The results of our mechanical analysis further support this approach. A significant difference (P=.048) between the elastic moduli of control and irradiated samples
was observed. The presence of tissue calcification was observed in irradiated specimens only. The elastic moduli
of irradiated specimens were either significantly higher
or lower than those of control specimens. The presence
of focal zones of calcification tended to increase with
power density. Experimental specimens could be separated into the following 3 distinct subgroups: (1) very
soft without calcification, (2) very stiff with complete calcification, and (3) inhomogeneous with intermingled regions of soft (noncalcified) and stiff (calcified) tissue. Soft,
low-modulus noncalcified tissue was found in specimens irradiated at every power level; however, stiff, highmodulus calcified tissue was only present in samples exposed to a total energy of 50 J or greater. These findings
suggest that the calcification process may evolve in a stepwise pattern following laser irradiation. The initial laser
interaction may result in loss of viable chondrocytes followed by deterioration of matrix architecture, infiltration of fibroblasts, and subsequent collagen deposition.
The rate and degree of calcification follows a dosedependent relationship, although the mechanism of laserinduced mineralization is unclear. The effect likely results
from loss of tissue viability. This observed phenomenon
would have a deleterious impact on clinical outcome, especially in structures such as the external nose and ear.
The reasonable agreement between experimental and
simulated peak temperatures (Figures 11 and 12) further validates our numerical model as a tool to calculate
temperature distributions during laser heating of cartilage.35 Thermo-optical models have been used extensively in dermatologic laser applications and have been
invaluable in the optimization of dosimetry settings for
several clinical procedures, such as the treatment of vascular lesions. In the present study, the principal value of
the thermo-optical simulation is in its use in combination with equation 2, such that tissue damage can be estimated. Although the Arrhenius rate-process models assume a 1-step interaction, they have been surprisingly
accurate in estimating thermal damage. These results link
thermal injury with acute shape change and demonstrate that shape change and tissue injury are highly correlated. Given the strong correlation between thermal
damage and bend angle, the prospect of being able to
achieve significant shape change without thermal injury appears remote for the laser devices used in this study.
Hence, the clinical effectiveness of shape change is likely
a consequence of spatially selective heating, in which damaged tissue regions are small and surrounded by healthy
tissue or stem cell–rich perichondrium. Regardless, the
behavior of cartilage following any physical modification process is unpredictable, and even measures such
as morselization and carving can produce chondrocyte
death.37 Hence, even standard surgical approaches may
significantly traumatize the tissue and compromise cell
viability.
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
114
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
In clinical studies, Ovchinnikov et al22 reported the
successful use of holmium:YAG laser (␭=2.12 µm) wavelengths to alter the shape of nasal septal cartilage in humans. The thickness of human nasal septal cartilage has
been estimated to vary from 2 to 4 mm; the optical penetration depth of the laser wavelengths used by Ovchinnikov and colleagues (they later switched to erbiumdoped fiber lasers, which emit light at 1.54 µm) was
significantly less than these dimensions, and their irradiation was performed in a transmucosal fashion. In all
likelihood, the shallow absorption and short radiation
times resulted in the heating of only the superficial layers of the septum, producing physical changes only in
the most superficial regions of the cartilaginous septum. The deeper regions of the cartilaginous septum and
the contralateral septal mucosa were largely spared thermal injury, and the relatively small spot size ensured that
reasonable wound healing would occur owing to the presence of viable tissue surrounding the laser irradiation site.
CONCLUSIONS
We evaluated a broad range of laser dosimetry pairs (and
hence temperature profiles) that produced varying degrees of acute shape change in rabbit septal cartilage tissue. The bulk heating model used in this study resulted
in compromise of chondrocyte viability, loss of structural integrity, and drastic changes in elastic modulus after 7 months. These findings were observed even when
using dosimetry that did not produce significant acute
shape change. Because no appreciable axial temperature gradient was produced during Nd:YAG laser irradiation of the grafts, the results of our study provide information on the temperature-time criteria that lead to
cell death in septal cartilage and are in accord with estimates produced by our numerical models. Our study underscores the importance of spatially selective heating,
which is likely fundamental to the successful clinical application of this therapy in human subjects.
Accepted for Publication: November 15, 2005.
Correspondence: Brian J. F. Wong, MD, PhD, Beckman
Laser Institute and Medical Clinic, University of California, Irvine, 1002 Health Sciences Rd E, Irvine, CA
92612 ([email protected]).
Funding/Support: This study was supported in part by
an Undergraduate Research Opportunities Program Grant
from the University of California, Irvine; grants D00170,
DC005572, and RR-01192 from the National Institutes
of Health, Bethesda, Md; the American Society for Lasers in Surgery and Medicine, Inc, Wausau, Wis; and grant
FA9550-04-1-0101 from the Air Force Office of Scientific Research, Arlington, Va.
Previous Presentation: This study was presented in part
at the Combined Otolaryngological Spring Meetings,
American Academy of Facial Plastic and Reconstructive
Surgery; May 13, 2005; Boca Raton, Fla.
Acknowledgment: We thank George Peavey, DVM, and
Laurie Newman for their insights and assistance with the
surgical protocol and care of the animals. Tatiana Krasieva,
PhD, provided technical support for confocal imaging.
REFERENCES
1. Helidonis E, Sobol E, Kavvalos G, et al. Laser shaping of composite cartilage grafts.
Am J Otolaryngol. 1993;14:410-412.
2. Sobol EN, Bagratashvili VN, Omelchenko AI, et al. Laser shaping of cartilage. In:
Anderson RR, ed. Laser Surgery: Advanced Characterization, Therapeutics, and
Systems IV.. Vol 2128. Bellingham, Wash: International Society for Optical Engineering; 1994:43-49.
3. Wong JF, Milner TE, Kim HH, Nelson JS, Sobol EN. Stress relaxation of porcine
septal cartilage during Nd:YAG (␭= 1.32 mm) laser irradiation: mechanical, optical, and thermal responses. Basic J Biomed Opt. 1998;3:409-414.
4. Wong BJ, Milner TE, Harrington A, et al. Feedback-controlled laser-mediated cartilage reshaping. Arch Facial Plast Surg. 1999;1:282-287.
5. Karamzadeh AM, Rasouli A, Tanenbaum BS, Milner TE, Nelson JS, Wong BJ.
Laser-mediated cartilage reshaping with feedback-controlled cryogen spray cooling: biophysical properties and viability. Lasers Surg Med. 2001;28:1-10.
6. Wong BJ, Milner TE, Kim HH, et al. Critical temperature transitions in laser mediated cartilage reshaping. In: Anderson RR, Bartels KE, Bass LS, et al, eds. Lasers in Surgery: Advanced Characterization, Therapeutics, and Systems VIII. Vol
3245. Bellingham, Wash: International Society for Optical Engineering; 1998:
161-172.
7. Wong BJ, Milner TE, Anvari B, et al. Measurement of radiometric surface temperature and integrated back-scattered light intensity during feedback controlled laser-assisted cartilage reshaping. Lasers Med Sci. 1998;13:66-72.
8. Sobol EN, Sviridov AP, Omelchenko AI, et al. Mechanism of laser-induced stress
relaxation in cartilage. In: Jacques SL, ed. Laser-Tissue Interaction VIII. Vol 2975.
Bellingham, Wash: International Society for Optical Engineering; 1997:310315.
9. Diaz SH, Nelson JS, Wong BJ. Rate process analysis of thermal damage in cartilage.
Phys Med Biol. 2003;48:19-29.
10. Rasouli A, Sun CH, Basu R, Wong BJ. Quantitative assessment of chondrocyte
viability after laser mediated reshaping: a novel application of flow cytometry.
Lasers Surg Med. 2003;32:3-9.
11. Gillies HD. Plastic Surgery of the Face. London, England: Oxford University Press;
1920.
12. Gibson T, Davis W. The distortion of autogenous cartilage grafts: its cause and
prevention. Br J Plast Surg. 1958;10:257-274.
13. Fry H. Cartilage and cartilage grafts: the basic properties of the tissue and the
components responsible for them. Plast Reconstr Surg. 1967;40:526-539.
14. Fry H, Robertson W. Interlocked stresses in cartilage. Nature. 1967;215:53-54.
15. Fry HJ. Interlocked stresses in human nasal septal cartilage. Br J Plast Surg. 1966;
19:276-278.
16. Gunter JP, Clark CP, Friedman RM. Internal stabilization of autogenous rib cartilage grafts in rhinoplasty: a barrier to cartilage warping. Plast Reconstr Surg.
1997;100:161-169.
17. Wang Z, Pankratov MM, Perrault DF, Shapshay SM. Laser-assisted cartilage reshaping: in vitro and in vivo animal studies. In: Anderson RR, Watson GM, Steiner
RW, eds. Lasers in Surgery: Advanced Characterization, Therapeutics, and Systems V. Vol 2395. Bellingham, Wash: International Society for Optical Engineering; 1995:296-302.
18. Wang Z, Volk MS, Shapshay SM. Endoscopic laryngotracheoplasty and graft soldering with the carbon dioxide laser: an animal study. Ann Otol Rhinol Laryngol.
1997;106:989-994.
19. Jones N, Sviridov A, Sobol E, Omelchenko A, Lowe J. A prospective randomized
study of laser reshaping of cartilage in vivo. Lasers Med Sci. 2001;16:284290.
20. Chiu LL, Wong BJ, Chang JC. Minimizing superficial thermal injury using cryogen
spray cooling during laser reshaping of composite cartilage grafts. LaserExpress
Web site. May 2003. Available at: http://www.laserexpress.net/lasernews/lasernewsv3
.nsf/1abdb1fc1ac52224882568ab0066346e/efe50704720d36ff85256d33001ef9d9
?OpenDocument. Accessed January 15, 2004.
21. Wong BJ, Chao KK, Kim HK, et al. The porcine and lagomorph septal cartilages:
models for tissue engineering and morphologic cartilage research. Am J Rhinol.
2001;15:109-116.
22. Ovchinnikov Y, Sobol E, Svistushkin V, Shekhter A, Bagratashvili V, Sviridov A.
Laser septochondrocorrection. Arch Facial Plast Surg. 2002;4:180-185.
23. Karamzadeh AM, Chang JC, Diaz S, Milner TE, Wong BJF. Long-term in vivo stability of rabbit septal cartilage following laser cartilage reshaping: a pilot
investigation. Lasers Surg Med. 2005;36:147-154.
24. Mordon S, Wang T, Fleurisse L, Creusy C. Laser cartilage reshaping in an in vivo
rabbit model using a 1.54 microm Er:Glass laser. Lasers Surg Med. 2004;34:
315-322.
25. Chiu LL, Wong JF, Chang JC. Reduction of superficial thermal injury using cryogen cooling during laser-assisted cartilage reshaping of composite cartilage grafts:
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
115
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014
26.
27.
28.
29.
30.
preliminary investigation. In: Bass LS, Kollias N, Malek RS, et al, eds. Lasers in
Surgery: Advanced Characterization, Therapeutics, and Systems XIII. Vol 4949.
Bellingham, Wash: International Society for Optical Engineering; 2003:325334.
Chang JC, Diaz-Valdes S, Wong BJF. Preliminary investigations of lasermediated cartilage reshaping in the in-vivo rabbit model. In: Bartels KE, Bass LS,
de Riese WT, et al, eds. Lasers in Surgery: Advanced Characterization, Therapeutics, and Systems XII. Vol 4609. Bellingham, Wash: International Society for
Optical Engineering; 2001:326-330.
Johansen E, Burden BA, Wong JF. Determination of optimum laser parameters
for cartilage reshaping in porcine septum using Nd:YAG laser (␭=1.32 um). In:
Duncan DD, Jacques SL, Johnson PC, eds. Laser-Tissue Interaction XII: Photochemical, Photothermal, and Photomechanical. Vol 4257. Bellingham, Wash: International Society for Optical Engineering; 2001:205-212.
Burden BA, Johansen E, Wong JF. Design and construction of a precision cartilage reshaping device. In: Anderson RR, Bartels KE, Bass LS, et al, eds. Lasers
in Surgery: Advanced Characterization, Therapeutics, and Systems XI. Vol 4244.
Bellingham, Wash: International Society for Optical Engineering; 2001:366371.
Wright R, Protsenko DE, Diaz S, Ho K, Wong BJF. Shape retention in porcine
and rabbit nasal septal cartilage using saline bath immersion and Nd:YAG laser
irradiation. Lasers Surg Med. 2005;37:201-209.
Chao KK, Ho KH, Wong BJ. Measurement of the elastic modulus of rabbit nasal
31.
32.
33.
34.
35.
36.
37.
septal cartilage during Nd:YAG (␭=1.32 µm) laser irradiation. Lasers Surg Med.
2003;32:377-383.
Gaon MD, Ho KH, Wong BJ. Measurement of the elastic modulus of porcine septal cartilage specimens following Nd: YAG laser treatment. Lasers Med Sci. 2003;
18:148-153.
Eshbach OW, Lessells JM, Cherniak GS, Love JE. Mechanics of deformable bodies.
In: Eshbach OW, ed. Handbook of Engineering Fundamentals. New York, NY: John
Wiley & Sons; 1952:512-515.
Mainil-Varlet P, Monin D, Weiler C, et al. Quantification of laser-induced cartilage injury by confocal microscopy in an ex vivo model. J Bone Joint Surg Am.
2001;83:566-571.
Grogan SP, Aklin B, Frenz M, Brunner T, Schaffner T, Mainil-Varlet P. In vitro
model for the study of necrosis and apoptosis in native cartilage. J Pathol. 2002;
198:5-13.
Diaz SH, Aguilar G, Lavernia EJ, Wong BJ. Modeling the thermal response of
porcine cartilage to laser irradiation. IEEE J Quantum Electron. 2001;7:944951.
Pearce J, Thomsen S. Rate process analysis of thermal damage. In: Welch A,
van Gemert MJC, eds. Optical-Thermal Response of Laser-Irradiated Tissue. New
York, NY: Plenum Publishing Corp; 1995:561-606.
Wong BJ, Giammanco PF. The use of preserved autogenous septal cartilage in
“touch-up” rhinoplasty. Arch Facial Plast Surg. 2003;5:349-353.
(REPRINTED) ARCH FACIAL PLAST SURG/ VOL 8, MAR/APR 2006
116
WWW.ARCHFACIAL.COM
©2006 American Medical Association. All rights reserved.
Downloaded From: http://archpedi.jamanetwork.com/ on 10/28/2014