The Laryngoscope
C 2014 The American Laryngological,
V
Rhinological and Otological Society, Inc.
Angiogenesis in Costal Cartilage Graft Laryngotracheoplasty:
A Corrosion Casting Study in Piglets
Lukas H. Kus, MD, MSc; Jaina Negandhi, MSc; Michael C. Sklar, MD; Antoine Eskander, MD;
Marvin Estrada, BSc; Robert V. Harrison, PhD, DSc; Paolo Campisi, MD, MSc, FRCSC;
Vito Forte, MD, FRCSC; Evan J. Propst, MD, MSc, FRCSC
Objectives/Hypothesis: To investigate the timing and degree of angiogenesis following anterior costal cartilage graft
laryngotracheoplasty in an animal model.
Study Design: Randomized controlled animal model.
Methods: Twelve pigs were included in this study. Three control pigs were perfused with intravascular methyl methacrylate, and overlying tissue was corroded with potassium hydroxide and hydrochloric acid, leaving only a cast of vessels. Nine
pigs underwent anterior costal cartilage graft laryngotracheoplasty and were survived for various lengths of time (3 for 48
hours, 3 for 10 days, 3 for 3 weeks) prior to corrosion casting. Transition zones between trachea and cartilage graft as well
as the graft itself were analyzed for signs of angiogenesis (budding, sprouting, intussusception) and hypoxic or degenerative
vessel features (extravasation, corrugation, circular constriction) using scanning electron microscopy.
Results: Angiogenesis peaked above control levels 48 hours after laryngotracheoplasty (P <.0001) and decreased 10
days and 3 weeks following surgery (P <.001, P <.0001, respectively) while remaining elevated above control levels
(P <.0001, P <.005, respectively). There was no difference in hypoxic or degenerative features across surgical and control
groups. Sprouting angiogenesis dominated over intussusception preoperatively (P <.0001) and 3 weeks following surgery
(P <.05). However, there was no difference in type of angiogenesis 48 hours and 10 days following surgery.
Conclusion: Angiogenesis peaked by 48 hours following costal cartilage graft laryngotracheoplasty and persisted for at
least 3 weeks (although decreased) after surgery in this animal model. Hypoxic or degenerative processes did not appear to
play a role in tracheal revascularization during the first 3 postoperative weeks.
Key Words: Angiogenesis, laryngotracheoplasty, animal model, corrosion casting, costal cartilage graft.
Level of Evidence: N/A.
Laryngoscope, 124:2411–2417, 2014
INTRODUCTION
Laryngotracheoplasty (LTP) is an airway reconstructive procedure for the treatment of subglottic stenosis. LTP can be categorized as expansion of the
subglottic framework using cartilage or resection of a
stenotic segment. Expansion techniques usually involve
placement of an autologous costal or thyroid ala cartilaginous graft. Although LTP success rates can be as high
Additional Supporting Information may be found in the online
version of this article.
From the Department of Otolaryngology–Head and Neck Surgery
(L.H.K., J.N., M.C.S., A.E., R.V.H., P.C., V.F., E.J.P.), Canada; and the Laboratory
Animal Services (M.E.), The Hospital for Sick Children, University of
Toronto, Toronto, Canada.
Editor’s Note: This Manuscript was accepted for publication
January 6, 2014.
Presented at the American Society of Pediatric Otolaryngology
Annual Meeting in Arlington, Virginia, U.S.A, April 25–28, 2013.
This work was funded by a Hospital for Sick Children Surgical
Services Innovation Grant. The authors have no other funding, financial
relationships, or conflicts of interest to disclose.
Send correspondence to Dr. Evan J. Propst, Department of Otolaryngology–Head and Neck Surgery, 6th Floor, Burton Wing, The Hospital
for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8,
Canada. E-mail: [email protected]
DOI: 10.1002/lary.24597
Laryngoscope 124: October 2014
as 90%,1 restenosis and failure to decannulate can be
devastating for patients when surgery is not successful.
Approximately one in 50 rib grafts necrose following
LTP.1 Revascularization is believed to be an important
factor in maintaining the viability of a cartilage graft
and therefore in preventing restenosis.2,3 The exact
mechanism and timing of angiogenesis in laryngotracheal wound healing is poorly understood.
Corrosion casting allows for the study of vascular
structures. A liquid plastic polymer is injected into an
organ’s blood supply and allowed to harden. Overlying
tissues are then corroded and the resulting vascular cast
can be analyzed using scanning electron microscopy
(SEM). The purpose of this study was to employ the
technique of corrosion casting to investigate angiogenesis following anterior costal cartilage graft laryngotracheoplasty. We hypothesized that there would be a
critical period of angiogenesis shortly after surgery that
would decline over the ensuing weeks.
MATERIALS AND METHODS
Animals
This study was approved by the Animal Care Committee
at the Hospital for Sick Children. Twelve Yorkshire piglets,
mean age 7.0 6 0.95 weeks (range 6–8 weeks) and mean weight
Angiogenesis in Costal Cartilage Graft Laryngotracheoplasty
2411
Fig. 1. Anterior costal cartilage graft laryngotracheoplasty in a porcine model. (A) Cartilage carved in the shape of a boat. (B) Cartilage graft inset into porcine airway. [Color figure can be viewed in
the online issue, which is available at www.laryngoscope.com.]
15.4 6 1.93 kg (range 13.2–19.8 kg) were included. Three control
animals were ventilated with a laryngeal mask airway and
underwent corrosion casting immediately. Nine experimental
animals underwent laryngotracheoplasty with anterior costal
cartilage graft and were randomized to one of three durations
of survival (48 hours, 10 days, 3 weeks) prior to corrosion
casting.
Laryngotracheal Reconstruction
Animals were sedated with an intramuscular injection of
Akmezine (0.2 mL/kg) and were placed under anaesthesia using
isoflurane (5%) by facemask. Animals were intubated with a
6.0-mm or 6.5-mm internal diameter cuffed Sheridan endotracheal tube (ETT; Teleflex Medical, Research Triangle Park, NC)
and anaesthesia was maintained using isoflurane (3%). ETT
cuff pressure was maintained at 20 cm H2O using a Magnehelic
manometer (Dwyer Instruments, Michigan City, IN). Heart
rate, respiratory rate, oxygen saturation, and carbon dioxide
levels were monitored throughout the procedure.
All surgeries were performed by the same surgeons (EJP,
LHK). A 10-cm vertical midline neck incision was made from 4
cm above the hyoid bone to the sternal notch, and subplatysmal
flaps were elevated using microbipolar cautery. The strap
muscles were divided in the midline. A vertical anterior cricoid
split was extended inferiorly through two tracheal rings (1.5 cm
length). Care was taken not to elevate mucosa or muscle off the
trachea in order to leave the vascular supply undisturbed.
The cartilaginous graft was harvested through a horizontal incision below the level of the axilla beginning 1 cm lateral
to the lateral border of the sternum and extending 4 cm laterally. Overlying muscle was divided without injuring costal cartilage. The perichondrium was incised sharply along its superior
and inferior borders. The posterior perichondrium was elevated
off the cartilage using a Freer elevator. The rib was divided
laterally at the osseocartilaginous junction and medially at the
lateral border of the sternum. The wound was filled with water
and there was no pleural breach. The wound was closed in
layers with 3-0 Vicryl sutures (Ethicon, Somerville, NJ) and a
running subcuticular 4-0 Monocryl suture (Ethicon, Somerville,
NJ). A Penrose drain was not used due to concerns that the animals would not tolerate it.
The graft was carved in standard fashion measuring 1.5
cm in length and 5 mm in width (distraction), leaving 2 mm
flanges on each side. The graft was sutured into the anterior
cricoid split (perichondrium facing into the airway) using 5-0
Laryngoscope 124: October 2014
2412
Polydioxanone mattress sutures (Ethicon, Somerville, NJ) with
knots tied on the tracheal surface to prevent interference with
vascular ingrowth from the overlying strap muscles (Fig. 1). An
air leak test was negative. The wound was closed in layers with
3-0 Vicryl sutures and a running subcuticular 4-0 Monocryl
suture. A Penrose drain was not used. The mean duration of
each procedure (anaesthetic and surgical time) was 175.0 6 23.5
minutes (range 135–205 minutes), with a mean surgical time of
100.0 6 19.5 minutes (range 95–125 minutes).
All animals were extubated immediately following surgery
and were assessed every 3 hours for 12 hours and then every
12 hours thereafter. Evidence of respiratory distress, pain, or
wound infection was monitored. Indicators for these included
poor feeding, low urinary or fecal output, panting, downward
head position, lethargy, coughing, or subcutaneous emphysema.
Pain management consisted of oral buprenorphine (0.05 mg/kg)
every 6 hours for 3 days, and then as needed every 6 hours as
well as oral Metacam (0.4 mg/kg) every 24 hours for 5 days.
Casting
Animals were anaesthetized for a second time at various
intervals of survival duration. They were ventilated through a
size 3 pediatric laryngeal mask airway (LMA North America,
San Diego, CA) to avoid further injury to the airway. We have
previously described this casting technique in detail.4 Essentially,
a 25-cm midline vertical thoracotomy was performed, the superior vena cava and aortic arch were dissected out, and the pig
was euthanized with an intracardiac injection of Euthanyl
(25 mg/kg). Contrary to humans, pigs have a solitary bicarotid
trunk that divides into both left and right common carotid
arteries.5 Satinsky clamps were placed on the superior vena cava
and on the aortic arch proximally and distally to the bicarotid
trunk to create a vascular circuit (Suppl. Fig. 1). The superior
vena cava and bicarotid trunk were cannulated with 20 French
catheters (Terumo Cardiovascular Systems, Ann Arbor, MI), the
aortic cannula was attached to a cardiac perfusion pump (Medtronic, Minneapolis, MN) and the venous cannula was left open
to air. The vascular loop was flushed with 3 L of heparinized
(15 units/mL) 0.9% saline at a rate of 150 mL/min until there
was no more blood in the circuit. The blue casting reagent
(methyl methacrylate) described elsewhere was perfused into the
aortic cannula until it extravasated from the venous cannula and
the animal’s skin color changed from pink to blue.4 The animal
was placed on ice for 3 hours while the casting resin solidified.
Corrosion
A section of upper airway tissue from the superior aspect
of the thyroid cartilage to the carina was harvested. The inferior borders of the vocal cords, the graft, and every three tracheal rings inferiorly were marked with 5-0 Nylon sutures
(Ethicon, Somerville, NJ), which are resistant to corrosion. The
specimen was sutured to a 6-mm diameter white polypropylene
drinking straw and was corroded in a 16% KOH bath at 45 C
for 2 days, washed three times in distilled water, decalcified in
a 2% HCl bath at 45 C for 1 day, then washed three times in
distilled water.
Scanning Electron Microscopy
Airway casts were freeze-dried overnight in a Micro Modulyo freeze dryer (Thermo Electron, Marietta, OH) and divided
vertically along their posterior aspect to visualize the grafted
area from within the tracheal lumen. Samples from the region
of the cartilage graft and the region where the graft approximated the trachea were excised and mounted on stubs. These
Angiogenesis in Costal Cartilage Graft Laryngotracheoplasty
three controls, five experimental (2 casted at 48 hours,
1 casted at 10 days, 2 casted at 3 weeks). Four casting
failures resulted from resin leakage at the aortic cannulation site due to the high viscosity of the polymer resin.
Normal Vascular Anatomy of the Trachea
We have previously described in detail the normal
vascular anatomy of the porcine trachea.4 Essentially,
two longitudinally oriented vessels near the tracheoesophageal groove gave rise to circumferential branches
that were interconnected by smaller vertical branches
(Suppl. Fig. 3).
Qualitative Description of Vasculature
Following Laryngotracheoplasty
Fig. 2. Scanning electron microscopy images of vascular features.
(A) Angiogenic and (B) hypoxic/degenerative features.
were sputter coated with gold and viewed using SEM (Hitachi
S570, Tokyo, Japan) under constant conditions (accelerating
voltage 5 kV, magnification 1903, working distance 18,400 lm).
Six images were obtained by one author (JN) for each animal
from a randomly selected area within the defined sample
region. Images were deidentified, coded, randomized and presented to two blinded observers (LHK, MS), each of whom counted
features in every image (or field of view). Angiogenic features6
(vessel budding, sprouting, and intussusception) and hypoxic or
degenerative features7 (resin extravasation, vessel corrugation,
and circular constriction of vessels) were recorded for each field
of view (Fig. 2; Suppl. Fig. 2).
Statistics
Counts for each feature for each set of six images were
averaged for each animal. Inter-rater reliability was assessed
using intraclass correlation (ICC) coefficient with an ICC
greater than 0.8 considered acceptable (high reliability). One
rater’s results were then used for the remainder of the analysis.
Based on analysis for normality, equality of variance, and
the relatively small sample size at each time point, nonparametric descriptive statistics were used (median and interquartile range). Comparisons of two imaging features (total
sprouting vs. intussusception; degenerative vs. vasospastic)
within a group at a given time point were performed using the
related-samples Wilcoxon signed rank test. Control and experimental animals as well as features at different time points
were compared using the independent samples Kruskal-Wallis
test with a Bonferroni correction for multiple comparisons.
The limit of significance was taken to be 5% (P <.05) for all
comparisons, and all analyses were performed using SPSS version 21 (IBM, Armonk, NY).
RESULTS
All 12 animals survived the surgery and their prescribed postoperative period until corrosion casting without respiratory distress, subcutaneous emphysema,
bleeding, or wound infection. Eleven animals had a
mean postoperative weight gain of 0.28 6 0.06 kg/day
(range 0.12–0.53 kg/day). Only one animal that survived
to 10 days did not gain weight following airway surgery.
Corrosion casting was successful in eight of 12 animals:
Laryngoscope 124: October 2014
Forty-eight hours post-LTP, there were no vessels
in the anterior subglottic region consistent with the
graft location. This was smaller after 10 days due to
ingrowth of wispy vessels. After 3 weeks, this region
was covered with vessels, although it appeared less
dense than controls. SEM imaging (Fig. 3) of the transition zone between normal trachea and costal cartilage
graft 48 hours postoperatively revealed a densely packed
vascular plexus with a predominance of round holes representing transcapillary tissue pillars (intussusceptions)
(Fig. 3A). Transition zone vessels became thinner, and
the vascular network became less dense 10 days and
3 weeks post-LTP whereby they more closely resembled
control trachea (Fig. 3B, 3C). In the region of the graft,
there was a mixture of thin capillaries and thick immature bulbous vessels suggesting that revascularization
in the graft region lagged behind the transition zone
(Fig. 3 D).
Quantitative Description of Vasculature
Following Laryngotracheoplasty
Vessel features in SEM images were counted by two
raters with an interclass correlation coefficient of 0.993,
indicating high reliability. Total angiogenesis (budding,
sprouting, and intussusception) peaked above control
levels in the area around the cartilage graft 48 hours following laryngotracheoplasty (P <.0001; Fig. 4). Total
angiogenesis decreased 10 days (P <.0001) and 3 weeks
(P <. 0001) following surgery but remained significantly
elevated compared to controls (P <.0001, P 5.005,
respectively). Although the density of casted vessels was
not high enough to evaluate the area of the graft
48 hours and 10 days following LTP, there was no difference in total angiogenesis between the area around the
graft and the area of the graft itself 3 weeks postoperatively (P 5.488). There was no difference in hypoxic or
degenerative features (resin extravasations, vessel corrugations, circular constrictions) across surgical and control groups at any time points, either in the area
surrounding the graft or in the area of the graft itself,
after correcting for multiple comparisons (Fig. 5).
Angiogenic features were further divided into sprouting features (vessel buds and sprouts) and intussusceptive
features (intussusceptions) and their relative counts were
Angiogenesis in Costal Cartilage Graft Laryngotracheoplasty
2413
Fig. 3. Scanning electron microscopy of corrosion casts after anterior costal cartilage graft laryngotracheoplasty. (A) At 48 hours following
surgery, buds, sprouts and intussusceptions are visible at the trachea–graft transition zone, with a general appearance of intussusceptive
predominance. (B) 10 days after surgery the capillary plexus begins to thin out; however regions of dense intussusceptions, buds and
sprouts remain in the trachea–graft transition zone. (C) Three weeks after surgery, the capillary plexus has thinned substantially in the trachea–graft transition zone and D) the area of the graft itself.
compared over time (Fig. 6). Sprouting angiogenesis predominated over intussusceptive angiogenesis preoperatively
(i.e., controls) (P <.0001) and 3 weeks following LTP both
around (P <.05) and within (P <.01) the area of the graft.
There was no difference in the amount of sprouting and
intussusceptive features 48 hours or 10 days following surgery (P 5.53, P 5.50, respectively), suggesting that surgery led to a relative increase in intussusceptive
angiogenesis. Hypoxic or degenerative features were also
further divided into degenerative features (resin extravasations) and vasospastic features (corrugations and circular constrictions) and compared over time; however, there
was no difference at any time point after correcting for
multiple comparisons (P >.05) (Fig. 7).
DISCUSSION
Laryngotracheoplasty (LTP) with costal cartilage
graft fails in approximately 10% of patients.1 Although
one hypothesis is that costal cartilage necroses due to
poor revascularization, leading to scar formation, the
exact mechanism and timing of angiogenesis following
costal cartilage graft LTP is poorly understood.2,3,8,9 The
present study investigated angiogenesis following anterior costal cartilage graft LTP by injecting the vasculature of pigs with a liquid plastic polymer after varying
durations of survival, corroding the surrounding tissue
and examining resultant vascular casts using SEM.
Laryngoscope 124: October 2014
2414
All animals survived LTP and their prescribed postoperative period until corrosion casting without adverse
events. This suggests that our porcine model for studying the effects of laryngotracheal wound healing is
feasible. All animals were extubated uneventfully immediately following surgery, a practice performed in
humans in some centers but not universally. None of the
animals developed subcutaneous emphysema, despite
not having a Penrose drain, suggesting that where there
is no air leak intraoperatively perhaps a drain is not
entirely necessary.
The normal vascular anatomy of control tracheas
revealed two large longitudinal vessels along the posterior aspect of the trachea, giving rise to circumferential
branches connected by vertical branches that penetrated
to form a plexus of fine capillaries on the lumenal side.
Although the present study is the first to describe porcine tracheal vasculature, it has been described previously in human fetuses,10 guinea pigs,11 sheep,12 and
dogs.13 Similarities between porcine and human tracheal
vasculature suggest that results from the present study
may be translatable to human patients.
Little is known about mechanisms of tracheal
revascularization. Angiogenesis in other organs occurs
by sprouting and intussusception. In sprouting angiogenesis, existing blood vessels develop a bud or outgrowth that narrows and extends into a new vessel
branch.14 This occurs by proteolytic degradation of
Angiogenesis in Costal Cartilage Graft Laryngotracheoplasty
Fig. 4. Box plots comparing total angiogenesis (buds, sprouts, intussusceptions) over time following anterior costal
cartilage graft laryngotracheoplasty. P
values represent a comparison between
controls and each of the different time
points.
extracellular matrix, followed by chemotactic migration
of endothelial cells, formation of a lumen, and endothelial maturation.15 During wound healing, capillary
sprouts invade fibrin-rich wound clot and reorganize
into a microvascular network.16 Intussusceptive angiogenesis involves the internal division of a preexisting
vascular plexus into mature capillaries by transcapillary
tissue pillars that form between the walls of the vascular plexus.17 Endothelial reorganization around these
pillars eventually leads to a perforation that elongates
until a septum is formed between two adjacent rows of
endothelial cells, creating the walls of two distinct capillary vessels.17 In comparison with sprouting, intussusceptive angiogenesis generates blood vessels more
rapidly and at a more favorable energetic and metabolic
cost.18 The dominant type of angiogenesis varies by
organ, although both sprouting and intussusceptive
angiogenesis may occur concurrently.15 Final steps of
revascularization involve pruning of the newly formed
vascular tree and remodeling of blood vessels until a
mature vasculature is formed.15
Results from the present study suggest that revascularization of the trachea following costal cartilage
graft LTP peaks as early as 48 hours postoperatively or
perhaps sooner. This suggests that special attention
should be paid to decreasing injury to the trachea during
this immediate postoperative period. Early extubation,
nasotracheal intubation rather than oral intubation (to
decrease the amount of endotracheal tube movement),
and withholding steroids are but a few ways to help preserve this tenuous blood supply. Total angiogenesis
decreased 10 days and 3 weeks following surgery but
remained significantly elevated compared to controls.
This suggests that the aforementioned precautions
should probably continue for at least for 3 weeks postoperatively; however, they may not be as necessary as they
are during the first 48 hours following surgery.
Sprouting angiogenesis predominated over intussusceptive angiogenesis preoperatively and 3 weeks following LTP but not 48 hours or 10 days following surgery,
suggesting that surgery led to a relative increase in
intussusceptive angiogenesis. Given that intussusceptive
angiogenesis generates blood vessels more rapidly and
with greater metabolic efficiency,18 one might hypothesize that this pattern of angiogenic switching could be
adaptive in early wound healing. Interestingly, this
Fig. 5. Box plots comparing total
hypoxic/degenerative features (extravasations, corrugations, circular constrictions) over time following anterior costal
cartilage graft laryngotracheoplasty. P
values represent a comparison between
controls and each of the different time
points.
Laryngoscope 124: October 2014
Angiogenesis in Costal Cartilage Graft Laryngotracheoplasty
2415
Fig. 6. Sprouting versus intussusceptive angiogenesis over time following
anterior costal cartilage graft laryngotracheoplasty. P values represent a
comparison between total sprouting
and intussusceptive vessel features
at each time point.
pattern has previously been described in tumor models
following radiation or chemotherapy with tyrosine
kinase inhibitors.18–20 To our knowledge, this is the first
description of such an angiogenic switch in a surgical
model.
We have designed an experimental animal model
that allows for the study of angiogenesis following costal
cartilage graft LTP. Future studies investigating the
effects of various pharmacological interventions on
angiogenesis following LTP are warranted. These could
include the administration of basic fibroblast growth factor as a promoter of angiogenesis7,9,21 or corticosteroid
as an inhibitor of wound healing.22
A limitation of this study is its small sample size
due to casting failures from resin leakage at the aortic
cannulation site. Obtaining an ideal resin viscosity is
difficult because resin must be thin enough to reach
small vessels yet thick enough not to crumble following
corrosion. Another limitation is the arbitrary selection of
survival durations prior to casting. We waited 48 hours
prior to casting to ensure that vessels were sealed to
prevent leakage of resin. Casting closer to the time of
surgery could provide more information about early
angiogenesis. Evaluating animals at more time points
within and beyond the 3-week window could provide
more specific information. Lastly, computer software
able to photograph, recreate, and analyze vascular casts
in 3D space may provide additional information regarding angiogenesis beyond what human beings can assess
by counting vascular features.
CONCLUSION
We have developed a porcine model for studying
angiogenesis following costal cartilage graft LTP. The
gross vascular anatomy of the porcine trachea closely
resembles that of the human. Angiogenesis appears to
peak by 48 hours following costal cartilage graft LTP
Fig. 7. Vasospastic versus degenerative
features over time following anterior
costal cartilage graft laryngotracheoplasty. P values represent a comparison
between degenerative and vasospastic
vessel features at each time point. After
controlling for multiple comparisons,
none of the P values were statistically
significant.
Laryngoscope 124: October 2014
2416
Angiogenesis in Costal Cartilage Graft Laryngotracheoplasty
and persists for at least 3 weeks (although decreased)
after surgery in this animal model. Results suggest that
special attention to decreasing injury to the trachea during this immediate postoperative period may lead to
decreased rates of graft failure and restenosis.
BIBLIOGRAPHY
1. Choi SS, Zalzal GH. Pitfalls in laryngotracheal reconstruction. Arch Otolaryngol Head Neck Surg 1999;125:650–653.
2. Walner DL, Heffelfinger SC, Stern Y, Abrams MJ, Miller MA, Cotton RT.
Potential role of growth factors and extracellular matrix in wound healing after laryngotracheal reconstruction. Otolaryngol Head Neck Surg
2000;122:363–366.
3. Schroeder JW, Jr., Rastatter JC, Walner DL. Effect of vascular endothelial
growth factor on laryngeal wound healing in rabbits. Otolaryngol Head
Neck Surg 2007;137:465–470.
4. Kus LH, Sklar MC, Negandhi J, et al. Corrosion casting of the subglottis
following endotracheal tube intubation injury: a pilot study in Yorkshire
piglets. J Otolaryngol Head Neck Surg 2013;42:52. doi: 10.1186/19160216-42–52.
5. Hammel JM, Deptula J, Hunt PW, Lang H, Duncan KF. Anoxic ventilation
improves systemic perfusion during extracorporeal circulation with
uncontrolled systemic-to-pulmonary shunt. Asaio J 2007;53:238–240.
6. Macchiarelli G, Jiang JY, Nottola SA, Sato E. Morphological patterns of
angiogenesis in ovarian follicle capillary networks. A scanning electron microscopy study of corrosion cast. Microsc Res Tech 2006;69:
459–468.
7. Ohtake M, Morino S, Kaidoh T, Inoue T. Three-dimensional structural
changes in cerebral microvessels after transient focal cerebral ischemia
in rats: scanning electron microscopic study of corrosion casts. Neuropathology 2004;24:219–227.
8. Nakanishi R, Hashimoto M, Yasumoto K. Improved airway healing using
basic fibroblast growth factor in a canine tracheal autotransplantation
model. Ann Surg 1998; 227:446–454.
Laryngoscope 124: October 2014
9. Albes JM, Klenzner T, Kotzerke J, Thiedemann KU, Schafers HJ, Borst
HG. Improvement of tracheal autograft revascularization by means of
fibroblast growth factor. Ann Thorac Surg 1994;57:444–449.
10. Strek P, Nowogrodzka-Zagorska M, Litwin JA, Miodonski AJ. The lung in
closeview: a corrosion casting study on the vascular system of human
foetal trachea. Eur Respir J 1994;7:1669–1672.
11. Miodonski A, Kus J, Tyrankiewicz R. Scanning electron microscopical
study of tracheal vascularization in guinea pig. Arch Otolaryngol 1980;
106:31–37.
12. Hill P, Goulding D, Webber SE, Widdicombe JG. Blood sinuses in the submucosa of the large airways of the sheep. J Anat 1989;162:235–247.
13. Laitinen A, Laitinen LA, Moss R, Widdicombe JG. Organisation and structure of the tracheal and bronchial blood vessels in the dog. J Anat 1989;
165:133–140.
14. Folkman J. Tumor angiogenesis. Adv Cancer Res 1985;43:175–203.
15. Risau W. Mechanisms of angiogenesis. Nature 1997;386:671–674.
16. Tonnesen MG, Feng X, Clark RA. Angiogenesis in wound healing. J Investig Dermatol Symp Proc 2000;5:40–46.
17. Burri PH, Tarek MR. A novel mechanism of capillary growth in the rat
pulmonary microcirculation. Anat Rec 1990;228:35–45.
18. Hlushchuk R, Riesterer O, Baum O, et al. Tumor recovery by angiogenic
switch from sprouting to intussusceptive angiogenesis after treatment
with PTK787/ZK222584 or ionizing radiation. Am J Pathol 2008;173:
1173–1185.
19. Drevs J, Muller-Driver R, Wittig C, et al. PTK787/ZK 222584, a specific
vascular endothelial growth factor-receptor tyrosine kinase inhibitor,
affects the anatomy of the tumor vascular bed and the functional vascular properties as detected by dynamic enhanced magnetic resonance
imaging. Cancer Res 2002;62:4015–4022.
20. Nakamura K, Taguchi E, Miura T, et al. KRN951, a highly potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, has
antitumor activities and affects functional vascular properties. Cancer
Res 2006;66:9134–9142.
21. Tani A, Tada Y, Takezawa T, et al. Regeneration of tracheal epithelium
using a collagen vitrigel-sponge scaffold containing basic fibroblast
growth factor. Ann Otol Rhinol Laryngol 2012;121:261–268.
22. Talas DU, Nayci A, Atis S et al. The effects of corticosteroids on the healing of tracheal anastomoses in a rat model. Pharmacol Res 2002;45:299–
304.
Angiogenesis in Costal Cartilage Graft Laryngotracheoplasty
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