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.
  1. Material and Methods:
  2. 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:
  1. Histological analysis
  2. DNA quantification
  3. Mechanical testing
  4. Scanning Electron Microscope
  5. 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.