Abstract
Surgery of the entire ear pinna even today presents a challenge to
reconstructive surgeons, in the absence of a universally acceptable,
quality construct for clinical use. In this article, the authors present
a technique to generate a flexible, human size ear with the aim to meet
this limitation for ear reconstructive surgeries. The construct was
engineered by using a decellularized goat ear cartilage. This was
characterized by hematoxylin-eosin (H/E), diamidino-2-phenylindole
(DAPI), Masson’s trichrome (MT), Alcian Blue (AB) staining and Scanning
Electron Microscopy (SEM) for extracellular matrix (ECM) analysis. The
decellularization protocol followed yielded complete removal of all
cellular components without changing the properties of the ECM. In vivo
biocompatibility of the ear pinna showed demonstrable recellularization.
Recellularization was tracked using HE, DAPI, MT, AB staining, toluidine
staining, SEM, vascular-associated protein (VAP) and
CD90+ expressing cells. VAP expression revealed
specific vasculogenic pattern (angiogenesis). CD90+expression reflected the presence of the stromal cell. The graft
maintained the properties of ECM and displayed chondrocyte recruitment.
In summary, the decellularized goat ear pinna (cartilage) exhibited
xenograft biocompatibility, stable mechanical properties and in vivo
chondrocyte recruitment. Subsequently developed tissue-engineered ear
pinna offer potential for cartilage flexibility and individualization of
ear shape and size for clinical application.
Keywords: Ear pinna, Cartilage, Reconstructive surgery,
decellularized scaffold, Biocompatibility, recellularization
Introduction:
Congenital deformities of the external ear (pinna or auricle and
external auditory canal [EAC]) are collectively termed microtia.
Congenital auricular malformations range from smaller than normal pinna
(Grade I), partial ear with closed-off external ear canal (Grade II),
small peanut shaped cartilage with relatively well-formed ear lobe
(Grade III) and total absence of ear (Grade IV). Higher grades are
accompanied by different levels of hearing loss and EAC abnormalities
too (Arora, Sahoo, Gopi, & Saini, 2016)(Barron & Pandit, 2003).
Occurrence reported varies from 0.83 to 17.4 per 10,000 births with
higher incidence reported in Hispanics, Asians, and native Americans and
associated consequences such as lack of confidence, poor interpersonal
skills and antagonism increasing with age have been reported (Duisit et
al., 2018).
Current therapies use the autologous intercostal cartilage hand-carved
to serve as grafts for ear reconstruction. Remodeling of cartilage to
the appropriate shape falls to the operating surgeon. However, this
autologous graft is associated with several unwanted ramifications such
as inflammation, lack of neo-cartilage maturation, donor site morbidity,
risk of pneumothorax and costochondritis (Hasan et al., 2014)(Kim &
Evans, 2005)(Jeevitaa Kshersagar, Kshirsagar, Desai, & Bohara,
2018)(Reighard, Hollister, & Zopf, 2017). A xenogeneic graft would
demonstrate enhanced compliance but is accompanied with the higher
chance of immune rejection with reduced patency (Assaw, 2018).
Allografts, though promising, also have their own limitations such as
host versus graft reaction, chances of opportunistic infectious diseases
or premature degradation of the implants. Non-biological source implants
or alloplastic/prosthetics made of silicone, hydroxyapatite, nonporous
materials, polyethylene implants do not appear natural but can be
biometric. The synthetic implants have a higher chance of secondary
tissue infection at the site of implantation, framework fracture and
extrusion (Tardalkar, Desai, Adnaik, Bohara, & Joshi, 2017)(Ten Koppel,
Van Osch, Verwoerd, & Verwoerd-Verhoef, 2001) Recent trends in
reconstructive surgery involve use of preformed implants to construct an
external ear or custom source implants at the time of surgical
implantation. These are plagued with the challenge of ensuring
mechanical stability of the implant (Arora et al., 2016). The present
status of cell-based therapies, with the dangers of dedifferentiation,
preclude their application in creation of an intact and complete
functioning model (Youngstrom, Barrett, Jose, & Kaplan, 2013).
Tissue engineering [TE] provides a viable alternative for
reconstructive surgeries of the external ear (Borrelli, Hu, Longaker, &
Lorenz, 2020). TE involves combining living cells with a
natural/synthetic support or scaffold to build a three-dimensional
living construct that is functionally, structurally and mechanically
equal to or better than the tissue that is to be replaced (Hasan et al.,
2014; Kim & Evans, 2005). Tissue Engineered technologies, where the
scaffold is generated through a decellularization process followed by
cell seeding, could meet scientific and clinical needs faster than other
methods. (Duisit et al., 2018).
Cartilage tissue engineering is an area that warrants greater attention
in this respect. A nascent area of investigation, it faces the dilemmas
of scaffold maintenance, unstable biomechanical properties, preservation
of ECM and biocompatibility(Barron & Pandit, 2003). The decellularized
ECM scaffolds serve as a natural template for organ regeneration without
immunogenic reaction (Díaz-Moreno et al., 2018; J. Kshersagar,
Kshirsagar, Desai, Bohara, & Joshi, 2018; Youngstrom et al., 2013).
They have the ability to support cell adherence and proliferation by
allowing nutrition and gas exchange resulting in new tissue generation
in the requisite shape. The efficiency of the TE ear pinna scaffold has
been evaluated in in-vivo experiments mainly at subcutaneous
implantation sites (Ten Koppel et al., 2001).
In this study, a chemical and enzymatic method of decellularization of
goat ear cartilage to generate a scaffold, was used. The processes did
not alter the cartilage-specific ECM. The derived construct was
evaluated in xeno transplant model in vivo with reference to
biocompatibility, functional recellularization and tensile strength
using histology, SEM and mechanical testing.
- Material and Methods:
- Sample collection:
Goat ear pinna (n=30) samples were harvested at the slaughterhouse. The
entire dermis and epidermis were removed carefully. All cartilages were
rinsed in 70% alcohol followed by normal saline containing antibiotics
solution of 100 ug/ml Streptomycin (Abbott Healthcare, India),100 U/ml
Penicillin (Alembic Pharmaceuticals Ltd, India), and 2.5 ug/ml
Gentamycin (Cadilla Pharmaceuticals Ltd, India). The rinsed and dried
cartilages were stored at -40°C until further use.
Decellularization of goat ear pinna:
Goat ear cartilage samples were decellularized using protocol developed
in-house to remove all
cellular components from ear pinna to make it nonimmunogenic.
Decellularization comprised of cycles carried out by five methods
(Figure 1). For the five methods, the goat ear cartilages were treated
using a sterile box in different solutions on shaker (REMI RX-12R-DX,
India) at 180 rpm. (Figure 1) Method 1 (M1) used a solution of 2%
sodium dodecyl sulphate (SDS) for 12 hours followed by distilled water
for 12 hours. Method 2 (M2) used 2% sodium deoxycholate for 6 hours
followed by 2% SDS treatment for 6 hours. Method 3 (M3) used 5%
Dimethyl sulfoxide (DMSO) (v/v) for 6 hours followed by 2% SDS
treatment for 6 hours. Method 4 (M4) used distilled water treatment for
2 hours followed by freeze and thaw and overnight treatment with 5%
DMSO thereafter. Method 5 (M5) used 1% trypsin (v/v) treatment for 12
hours followed by distilled water for 12 hours. At completion of the
treatments as specified, the ear cartilages were stored at
-40oC in the deep freezer overnight. All samples were
thawed the next morning at room temperature and the cycle repeated till
complete decellularization was achieved. After decellularization,
prepared scaffolds (Figure 2A) were stored in distilled water containing
antibiotics at - 40°C.
Testing of decellularized ear pinna scaffolds:
The decellularized ear pinna scaffolds were tested as follows:
- Histological analysis
- DNA quantification
- Mechanical testing
- Scanning Electron Microscope
- Histological analysis:
Native ear pinna of goat serve as a Control and decellularized ear pinna
were investigated at every 10th cycle by histology.
Specimens were fixed with 10% neutral-buffered formalin, dehydrated
through alcohol grades and embedded in paraffin wax. The sections were
stained for chondrocyte nuclear structures using haematoxyline and Eosin
(HE). Sections were stained with DAPI (Invitrogen, CA, USA) to stain
adenine–thymine rich regions in DNA. Demonstration of collagen was made
by Masson’s trichrome (MT) stain (Suvik & Effendy, 2012).
Glycosaminoglcans (GAG) content of samples was determined using Alcian
blue (AB pH-2.5) (Sigma, A5268) staining.
DNA quantification:
DNA was quantified spectrophotometrically with optical densities at
260nm and 280 nm to yield purity of nucleic acid. Residual DNA was
quantified in control and decellularized ear pinna using UV
Spectrophotometer (UV-1800 UV-VIS Spectrophotometer) at 260 nm as
elaborated here. Samples were collected at concentration of 1 mg/ml and
allowed to freeze at - 20°C. After 15 minutes all samples were crushed
with mortar pestle. 40 µl of DNA solution was transferred to 3.96 ml of
DW in 4 ml cuvette. Utmost care was taken to ensure that solution was
air bubble free. Solution was kept for 10 minutes to ensure the complete
diffusion of DNA throughout the solution. This represented a 1: 100
dilution of the standards and DNA samples. The spectrometer was set at
260 nm and reading was noted down (Tardalkar et al., 2017).
Mechanical testing:
Mechanical testing was performed by using displacement controlled setup.
Control and decellularized scaffold were cut into required
cylindrical-shaped 4 cm strips to obtain specimens for mechanical
analysis. Burst pressure of scaffold was derived using a modified
syringe pump with gradual increase in applied pressure. The values were
digitally recorded at each pressure. This was used to find out
mechanical strain, stress, strength, deformation and elasticity of
scaffold (Konig et al., 2009).
Scanning electron microscopy (SEM):
SEM was performed on cross sections of goat ear pinna cartilage
scaffolds to investigate cellular architecture and its porosity. After
dehydration in oven at 60oC, the decellularized
scaffold samples were fixed to the stage using double sided tape. Images
of scaffolds in varying magnification from 80X to 1700 X were taken
using JEOL JSM 6360 SEM model at Department of Physics, Shivaji
University, Kolhapur.
Fabrication of human ear pinna mould:
The auricle mold for the creation of human ear-like cartilage construct
was prepared by a Poly Vinyl Chloride (PVC) which is a non-toxic and
chemically inert material. PVC sheets were converted into proper
anatomical 3D human ear pinna mold in different sizes such as 55mm,
65mm, and 75mm using a patented method (Rahman, Griffin, Naik, Szarko,
& Butler, 2018).
Biocompatibility evaluation of the scaffold
The animal study and all experiments were approved by Institutional
Animal Ethical Committee (IAEC) (Ref. - 6/IAEC/2017) of D Y Patil
Education Society Deemed University, Kolhapur.
Twenty-four six-month old male mice (Mus Musculus ) weighing
approximately 150 to 160 g were used for study. The animals were divided
into control and experimental groups. The experimental mice were kept
according to principles of laboratory animal care at control room
temperature condition and humidity (~52%). After 4
weeks, animals were sacrificed by cervical dislocation.