Massachusetts
Eye and Ear
Chronic Otitis Media (COM)
• Most common long-term complications in patients with COM are
persistent tympanic membrane (TM) perforation and conductive
hearing loss (Fig 1), which can be corrected via tympanoplasty
Tympanoplasty
• Successful tympanoplasty recreates a robust barrier between
the canal and middle ear, as well as re-establishes sound
transmission to the ossicular chain
• Revision surgery rates for patients with COM are 10-30%, and
major limitations of tympanoplasty using autologous tissues
include re-retraction and re-perforation of grafts
3D Printing
• Advances in 3-dimensional (3D) biologic printing allow the
creation of structures with impressive complexity on a micron
scale
Background
1. Biomimetic tympanic membrane composite grafts were
successfully fabricated with direct extrusion technique
• Tympanic membrane graft scaffolds composed of PDMS, PLA
and PCL-based materials were fabricated with two filamentary
configurations: 8 radial (R) and 8 circumferential (C) filaments
or 16R and 16C filaments (Fig 3), each with a total diameter of
25mm (Fig 4)
• Scaffold thicknesses (orthogonal to the planar ring) prior to
infill was 56 ± 12µm for PDMS, 32 ± 6 µm for PLA and 48 ± 10
µm for PCL
• For comparison, moist temporalis fascia is approximately
750µm (n=3) in thickness, and the human TM varies between
50 and 150 µm
• 3D printing of graft scaffolds was performed via direct ink
extrusion technique through fine deposition nozzles under
ambient conditions in filamentary form (Fig 3)
• Biodegradable fibrin/collagen hydrogel was used to infill the
filamentary skeleton of the TM graft scaffolds
Acoustic Testing
• TM composite grafts, fascia and cadaveric human TM were
mounted and subjected to pure tones across the human
range of sound perception: 400Hz, 1kHz, 3kHz and 6kHz
• Digital Opto-Electronic Holography (DOEH) was used to
provide qualitative and quantitative information on sound
induced surface motion patterns
• Laser Doppler vibrometry (LDV) was used to obtain
measurements of the sound-induced velocity at the center of
TM composite grafts, fascia and cadaveric human TMs
using frequency sweeps from 200 Hz-10 kHz
Methodology Con’t
• 3D printers can fabricate “biomimetic” TM grafts with DOEH and LDV
measured acoustic properties that approximate the human TM, and
are more consistent than temporalis fascia
• Data have implications for the clinical application of 3D printed TMs that have the potential to improve tympanoplasty outcomes
Conclusions
Results
3-Dimensionally Printed Biomimetic Tympanic Membrane Approximates Sound Induced Motion of Human Eardrum
Elliott Kozin, MD1-3; Nicole Black4; Jeffrey Cheng, PhD2,3; Michael McKenna, MD1-3; Daniel Lee, MD1-3; Jennifer Lewis4, ScD; John Rosowski, PhD2,3, Aaron Remenschneider, MD MPH1-3
1Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, 2Department of Otology and Laryngology, Harvard Medical School, 3Eaton Peabody Laboratory, Harvard Medical School, 4Wyss Institute for Biologically Inspired Engineering,
Harvard School of Engineering and Applied Sciences
Contact: Elliott Kozin ([email protected]) Aaron Remenschneider ([email protected]) Harvard Department of Otolaryngology Boston, MA
References: 1. Kaylie, D., et al. Otolaryngol Head Neck Surg 134, 443-50, 2006. 2. Shimada, T., Lim, D. Ann Otol Rhinol Laryngol. 80, 210-7, 1971.
Rationale
• Temporalis fascia, perichondrium and cartilage are commonly
used as grafting materials, but these materials do not posses
similar structural arrangements as the native TM and may have
intrinsic defects rendering them susceptible to COM
• 3D printing may allow for creation of a biomimetic TM with
improved acoustic and mechanical properties
Hypothesis
• 3D printing techniques can be utilized to design and fabricate
biomimetic TM grafts that may reproduce the human TM’s
acoustic characteristics
Rational and Hypothesis
Fig 1. Human Tympanic Membrane. (A) Normal left sided TM. (B) Perforation results in a propensity for infections, pain and conductive hearing loss. (C) Left TM showing a dimeric membrane with complex retraction and perforation.
Fig 3. 3D printing of tympanic membrane composite grafts. (A) Multi-material 3D printing apparatus. (B) Layered circumferential and radial filaments are printed first using a 100µm nozzle. (C) The same material (with blue colorant) is then printed to create the outer border region. (D) The printed and cured scaffold is then infilled with 100µL of fibrin/collagen matrix.
8C/8R
16C/16R
PCL
8C/8R
16C/16R
PLA
8C/8R
16C/16R
PDMS
Borderregion
Radialfiber(R)
Circumferentialfiber(C)
A B
CD 15mm
25mm
Fig 4. Printed tympanic membrane scaffolds with different materials and designs. TMs in the first column of each box have a total diameter of 25 mm. The next two columns show higher magnification images, 50x with a scale bar of 1 mm and 100x with a scale bar of 500 µm, respectively. (D) Scaffold highlighting design features.
Results Con’t 2. Composite grafts demonstrate simple holographic motion at lower
frequencies with more complex patterns at frequencies >1000Hz
• DOEH illustrate simple modal motion patterns at 400Hz, with one to
three displacement maxima distributed over the membrane surface for
all three TM composite grafts, the temporalis fascia and the intact TM
• At higher frequencies (≥1000Hz) the motions of all the materials
compared become more complex, with multiple areas of maximal
displacement separated by regions of reduced displacement (Fig 5)
Fig 5. Digital opto-electronic holography (DOEH) fringe patterns of PDMS-based biomimetic tympanic membrane composite grafts, human fascia and tympanic membrane controls. Top row shows the displacement patterns of the visible 9 mm diameter section of an 8C/8R PDMS graft, the second row a 16C/16R PDMS graft, the third row a sheet of fresh human temporalis fascia, the fourth row a human TM with intact middle ear. The four columns show measurements at different frequencies. Color bars are standardized at each frequency. Displacement is normalized by sound pressure, and units are dB re 1µm/Pa.
8C/8R
16C/16R
400Hz 1000Hz 3000Hz 6000Hz
Decibels(dB)re1µm/Pa
Frequency
TemporalisFascia
HumanTM
Controls
TMCompositeGrafts(PDMS)
Methodology Design
• Classical scanning and transmission electron micrographic (EM)
studies suggest a radial, circumferential, and parabolic fibrous
scaffold of the TM (Fig 2)
Fabrication
• Based EM studies, radial and circumferential fibrous
arrangements of two separate filament counts (8 circumferential
[C] x 8 radial [R] or 16C x 16R) were chosen as a 3D printed TM
scaffold
3. TM composite grafts have consistent, uniform velocities when compared
to temporalis fascia
• PDMS, PLA, and PCL-based TM composite grafts have uniform surface
velocity compared to temporalis fascia (Fig 6)
• The mean of measurements on three different samples of each printed
material have velocities that are within an order of magnitude of each
other with near coincident peaks in the tested frequency range
• Comparison with intact human TM specimens with an associated middle
ear load shows, as expected, an umbo velocity lower than graft velocities
at frequencies <1 kHz, and with a velocity proportional to frequency
below 800 Hz
PLA Fascia
A B
Fig 6. Differences in normalized surface velocity among PLA-based tympanic membrane composite grafts and fascia. (A) 8C/8R PLA TM composite grafts have uniform surface velocity. PDMS and PCL also have consistent velocities among graft samples. (Data not shown.) (B) Human temporalis fascia samples yield variable velocity patterns despite identical harvest.
Future Directions Wound Healing: Chronic Perforation Model
“Tunable” Cellular Ingrowth
5
0 µL bFGF 2 µL bFGF
Fig 7. Ongoing in vitro studies preliminarily demonstrate fibroblast growth along 3D printed TM scaffolds. Confluency increases with addition of targeted growth factors, e.g. bFGF included in membrane hydrogel (right panel).
Fig 8. Pilot in vivo experiments demonstrate successful placement of a 3D printed “TM patch” in a chronic perforation chinchilla model. Evaluation of graft effects on the inner ear and potential for wound healing will be important next steps in advancing these constructs towards clinical applications.
Funding: American Otological Society Research Grant, 2015 National Institutes of Health (T32)
A B C
5mm
Fig 2. 3D printed biomimetic TM prototype based on EM studies: Schematic of fiber arrangement. A. Arrangement of fibrous layer of the human TM. (Shimada & Lim, 1971). B. TM prototype without collagen filler printed with SE 1700 PDMS ink through a 200μm nozzle. A B
3. Lewis, J. Advanced Functional Materials 16, 2193-2204, 2006. 4. Cheng, J, et al. Hear Res. 263, 66-77, 2010.
