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 (Elliott_Kozin@meei.harvard.edu) Aaron Remenschneider (Aaron_Remenschneider@meei.harvard.edu) 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.