Diagram of chick chorioallantoic
membrane preparation procedure to receive tissue implant. A, Aspiration of
albumin; B, removal of eggshell apex; and C, placement of specimen in Teflon
ring and Petri dish cover.
Cartilage specimens enveloped
by chick chorioallantoic membrane vasculature. A, Native cartilage (×40);
B, laser-irradiated cartilage (×40); and C, boiled cartilage (×40).
Asterisks indicate the Teflon retaining ring.
Histological sections (hematoxylin-eosin)
of a native cartilage specimen enveloped by the chick chorioallantoic membrane
at ×10 (A), ×20 (B), and ×40 (C) magnifications.
Wong BJF, Karamzadeh AM, Hammer-Wilson MJ, Liaw LL, Nelson JS, Milner TE. Angioresistance of Thermally Modified Cartilage Grafts in the Chick Chorioallantoic Membrane Model. Arch Facial Plast Surg. 2001;3(1):24-27. doi:
From the Beckman Laser Institute and Medical Clinic, University of
California, Irvine (Drs Wong and Nelson, Mr Karamzadeh, and Mss Hammer-Wilson
and Liaw); Division of Facial Plastic Surgery, Department of Otolaryngology–Head
and Neck Surgery, University of California, Irvine, Orange, Calif (Dr Wong);
and Biomedical Engineering Program, Department of Electrical and Computer
Engineering, University of Texas at Austin (Dr Milner).
Background The chick chorioallantoic membrane (CAM) model allows direct observation
of vascularization acutely in explanted or cultured tissues in an immunologically
isolated environment. In vivo, angioinvasion of the tissue matrix does not
occur in viable cartilage tissue, whereas denatured or nonviable grafts are
readily vascularized and/or resorbed.
Objective To determine, using the CAM model, whether angioinvasion of thermally
altered cartilage explants occurs acutely.
Materials and Methods Porcine septal cartilage specimens were removed from freshly killed
animals and divided into 3 groups (n = 10): an untreated control group, a
group in which cartilage was boiled in isotonic sodium chloride solution (normal
saline) for 1 hour, and a laser-irradiated group (Nd:YAG, λ = 1.32µ
m, 30.8 W/cm2, irradiation time = 10 seconds). Tissue specimens
were then washed in antibiotic solutions, cut into small cubes (approximately
1.5 mm3), placed on the surface of 30 CAMs (7 days after fertilization),
and allowed to incubate for an additional 7 days. After incubation, the membranes
and specimens were fixed in situ with formaldehyde and then photographed using
a dissection microscope.
Results Examination with a dissecting microscope showed no obvious vascular
invasion of the cartilage or loss of gross tissue integrity in any of the
3 experimental groups, although all specimens were completely enveloped by
the CAM vascular network. No vascular invasion of the tissue matrix was observed
Conclusion These experiments demonstrate that cartilage specimens remain acutely
resistant to angioinvasion or metabolism by the immunologically immature CAM
whether native unmodified tissue, completely denatured (boiled), or thermally
modified following laser irradiation.
SURGICAL reconstruction in the head and neck for the correction of congenital,
traumatic, or oncologic defects often requires the use of autogenous nonvascularized
cartilage grafts harvested from heterotopic sites. Recent advances in biomedical
laser technology have led to the development of nonablative surgical procedures
that can be used to reshape cartilage without the need for morselization,
suturing, or carving.1 Photothermal heating
during laser irradiation with nonablative power densities results in a temperature-dependent
acceleration of mechanical stress relaxation within the tissue matrix that
allows the cartilage to be reshaped into new stable configurations.2-5 Although
this technique has prompted a great deal of interest and research focused
on determining the mechanism of reshaping,6-8
tissue viability following laser irradiation has received only limited study.9-10
Although animal models provide the best method to examine the integrated
vascular and immunologic responses to implanted materials, the preliminary
nature of laser-assisted cartilage reshaping makes such studies at this point
impractical. As an alternative to live animals, we used the chick chorioallantoic
membrane (CAM) model to examine the effect of laser irradiation and intense
thermal heating (boiling) on acute graft viability. The CAM model is a low-cost
hybrid ex vivo and in vivo system that allows direct observation of vascularization
in explanted or cultured tissues placed on the surface. Furthermore, because
the chick immune system is not competent during the first 17 days after fertilization,
angiogenesis can be studied in the absence of both cellular and humoral immune
responses. In the 1970s, the CAM model was used to study angiogenesis in various
Cartilage was observed to resist angiogenesis from the CAM.14-15
Although the explanted tissue would be enveloped by the CAM, no vascular invasion
of the cartilage matrix was observed histologically.16-18
These and other experiments led to the discovery of angioresistant proteins
native to cartilage tissues.19-21
Vascularization of cartilage tissue in the CAM occurs when the tissue is depleted
of its soluble proteoglycan pool and antiangiogenic factors that inhibit neovascularization.14-15 Extraction is accomplished using
guanidine hydrochloride (1, 2, and 3 mol/L) followed by washing in saline.
The process does not denature or solubilize matrix collagen fibers. While
neovascularization is inhibited in frozen cartilage tissue specimens,22 to our knowledge, the effect of moderate or intense
heating has not been investigated. In this study, the CAM model was used to
evaluate the effects of thermal modification on the angioresistant properties
of porcine septal cartilage, because angioinvasion is the first step toward
graft resorption, which would result in clinically devastating outcomes.
Fresh porcine septal cartilage was obtained from a local abattoir (Clougherty
Packing Company, Vernon, Calif) and harvested as previously described.4 The specimens were cut into disks 6 mm in diameter
and 1 mm thick and divided into 3 groups (n = 10). Negative control specimens
did not undergo any thermal modification. Positive control specimens were
boiled in saline solution for 60 minutes at 100°C. Laser-irradiated specimens
were exposed to Nd:YAG laser (λ = 1.32 mm, 30.8 W/cm2) radiation
for 10 seconds. Following laser irradiation, the specimens were rinsed for
45 minutes in antibiotic solutions (amphotericin, 20 mg/L, and gentamycin,
200 mg/L, in phosphate-buffered saline solution) 3 consecutive times.
The specimens from each group were then cut into small cubes (1.5 mm3) under sterile conditions. The CAMs were prepared as previously described,
as illustrated in Figure 1A through
C.23 On the
fourth day of incubation in a 38°C, 66% humidified incubator (Profi I;
Lyon Electric, Chula Vista, Calif), an 18-gauge needle and syringe were used
to aspirate approximately 4 mL of albumin and create an air pocket (Figure 1A). On the seventh day of embryo
development, a 20-mm-diameter hole was made by cutting and removing the apex
of the eggshell (Figure 1B). A Teflon
ring was placed on the CAM surface to stabilize and limit the movement of
transplanted samples (Figure 1C).
While viewing with a dissection microscope (×15), the specimens were
gently placed in the center of the retaining ring. A total of 30 cartilage
specimens were placed on 30 CAMs. A sterile Petri dish was positioned to cover
the hole in the eggshell. The eggs were replaced in a static incubator and
allowed to continue development until after fertilization day 14. At the end
of this period, the CAMs were removed from the incubator. The specimens were
fixed in situ by adding drops of formaldehyde to the central portion of each
ring over the cartilage specimen. Using microdissection techniques, the retaining
ring was gently dissected free from the membrane and the specimen immersed
in a 10% formaldehyde solution. Preserved CAMs were photographed under bright-field
microscopy (×30 magnification) with a dissection microscope. Following
fixation, specimens were serially dehydrated using graded ethanol solutions
and subsequently embedded in paraffin. The microsections were sectioned (6µ
m thickness) and stained with hematoxylin-eosin and examined microscopically
Figure 2A through C is a photographic montage of native, laser-irradiated,
and boiled specimens as visualized with a dissecting microscope at ×40
magnification, respectively. Specimens, although completely enveloped by the
CAM vascular network, show no gross loss of structural integrity. Figure 3 A through C is a photographic montage
of stained histological sections of a native cartilage specimen at ×10,×
20, and ×40 magnifications, respectively. No evidence of angioinvasion
is observed within the cartilage matrix, although blood vessels are clearly
visible in the enveloping CAM. Similar findings were noted in the laser-irradiated
specimens and boiled cartilage specimens, where histological integrity of
the tissue is maintained.
Xenographic studies can be performed in the CAM because its immune system
does not develop until approximately day 17 after fertilization; graft-vs-host
reactions do not occur. As a consequence, the CAM can be used to assess the
angiogenic properties of tissues following biochemical and physical modifications.
Since the membrane and developing blood vessels are directly visible, the
CAM permits observation of the developing vascular network. As illustrated
in Figure 2 and Figure 3, all specimens retained histological integrity, despite
having been enveloped by the CAM vasculature. Gross and microscopic tissue
integrity was maintained during the 7-day incubation period even in specimens
boiled for 1 hour.
These findings suggest that even nonviable cartilage can acutely resist
vascular invasion. Albeit, in the true animal model, thermally modified cartilage
grafts would be observed for substantially longer periods in an immunocompetent
host. Resorption of denatured nonviable grafts would likely occur along with
an intense inflammatory response. Inasmuch as few clinical procedures that
involve the heating of cartilage tissue exist, few studies that focus on the
viability of thermally modified cartilage tissue have been reported. Although
laser-mediated cartilage reshaping uses nonablative power densities, significant
tissue temperature elevations of up to 70°C occur. Although clinical trials
using laser radiation to reshape cartilage are under way, the safety of this
procedure has not been fully established. The focus of this pilot study was
to determine whether heated (with laser or via boiling) cartilage grafts would
survive in vivo. Although the results of this pilot investigation show that
gross and histological structural integrity were maintained without angioinvasion
of the tissue matrix, further animal studies will be needed to determine whether
tissue viability is maintained. Even though preliminary biochemical studies
demonstrate that laser reshaping can be performed with preservation of a significant
fraction of chondrocytes within the cartilage matrix, viability in vivo depends
on how the host immune system responds to thermally altered tissue regardless
of whether it is autologous or heterogeneous. Heat denatures proteins, and
these macromolecules may serve as potent antigens, provoking profound host
inflammatory response. Vascularization of such tissues would result in resorption.
Although the study of thermal effects in mesenchymal tissues is still
in its infancy, tissue modification and engineering of cartilage tissue and
cartilaginous frameworks have been extensively studied for more than 2 decades.
With growing interest in tissue engineering cartilage autografts, the need
for animal model studies is pressing.
Accepted for publication April 26, 2000.
This work was supported in part by grants from the National Institutes
of Health (1 K08 DC 00170-01, AR-43419, RR-01192, and HL-59472), Office of
Naval Research (N00014-94-0874), Whitaker Foundation, Rosylyn, Va (WF-21025),
and Department of Energy (95-3800459).
Presented in part at the annual meeting of the Society of Photo-optical
and Instrumentation Engineers, San Jose, Calif, January 23, 1999.
Corresponding author: Brian J. F. Wong, MD, Beckman Laser Institute
and Medical Clinic, University of California, Irvine, 1002 Health Sciences
Rd E, Irvine, CA 92612 (e-mail: email@example.com).