Research paper Design, fabrication, and in vitro testing of novel three-dimensionally printed tympanic membrane grafts * Elliott D. Kozin a, b, c , Nicole L. Black d, e , Jeffrey T. Cheng a, b, c , Max J. Cotler d, e , Michael J. McKenna a, b, c , Daniel J. Lee a, b, c , Jennifer A. Lewis d, e , John J. Rosowski a, b, c , Aaron K. Remenschneider a, b, c, * a Department Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, MA, USA b Eaton Peabody Laboratories, Massachusetts Eye and Ear Infirmary, Boston, MA, USA c Department of Otology and Laryngology, Harvard Medical School, Boston, MA, USA d Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA e Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA article info Article history: Received 21 December 2015 Received in revised form 7 March 2016 Accepted 9 March 2016 Available online xxx Keywords: Tympanic membrane 3D printing Holography Tympanoplasty Biomimetic Tissue engineering abstract The tympanic membrane (TM) is an exquisite structure that captures and transmits sound from the environment to the ossicular chain of the middle ear. The creation of TM grafts by multi-material three- dimensional (3D) printing may overcome limitations of current graft materials, e.g. temporalis muscle fascia, used for surgical reconstruction of the TM. TM graft scaffolds with either 8 or 16 circumferential and radial filament arrangements were fabricated by 3D printing of polydimethylsiloxane (PDMS), flex- polyactic acid (PLA) and polycaprolactone (PCL) materials followed by uniform infilling with a fibrin- collagen composite hydrogel. Digital opto-electronic holography (DOEH) and laser Doppler vibrometry (LDV) were used to measure acoustic properties including surface motions and velocity of TM grafts in response to sound. Mechanical properties were determined using dynamic mechanical analysis (DMA). Results were compared to fresh cadaveric human TMs and cadaveric temporalis fascia. Similar to the human TM, TM grafts exhibit simple surface motion patterns at lower frequencies (400 Hz), with a limited number of displacement maxima. At higher frequencies (>1000 Hz), their displacement patterns are highly organized with multiple areas of maximal displacement separated by regions of minimal displacement. By contrast, temporalis fascia exhibited asymmetric and less regular holographic patterns. Velocity across frequency sweeps (0.2e10 kHz) measured by LDV demonstrated consistent results for 3D printed grafts, while velocity for human fascia varied greatly between specimens. TM composite grafts of different scaffold print materials and varied filament count (8 or 16) displayed minimal, but measurable differences in DOEH and LDV at tested frequencies. TM graft mechanical load increased with higher filament count and is resilient over time, which differs from temporalis fascia, which loses over 70% of its load bearing properties during mechanical testing. This study demonstrates the design, fabrication and preliminary in vitro acoustic and mechanical evaluation of 3D printed TM grafts. Data illustrate the feasibility of creating TM grafts with acoustic properties that reflect sound induced motion patterns of the human TM; furthermore, 3D printed grafts have mechanical properties that demonstrate increased resistance to deformation compared to temporalis fascia. © 2016 Elsevier B.V. All rights reserved. 1. Introduction The tympanic membrane (TM) captures and directs sound from the environment to the ossicular chain of the middle ear, enabling transformation of sound pressure waves to mechanical motion. Mechanical motion is subsequently transmitted by the ossicles to the inner ear, where movement of perilymph results in stimulation Commonly used abbreviations: COM, Chronic otitis media; DOEH, Digital opto- electronic holography; DIW, Direct ink writing; DMA, Dynamic mechanical anal- ysis; PLA, Flex-polyactic acid; LDV, Laser Doppler vibrometry; PCL, Poly- caprolactone; PDMS, Polydimethylsiloxane; 3D, Three-dimensional; TM, Tympanic membrane * This work was presented at MEMRO 2015 in Aalborg Denmark. * Corresponding author. Department Otolaryngology, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA, 02118, USA. E-mail address: Aaron_Remenschneider@meei.harvard.edu (A.K. Remenschneider). Contents lists available at ScienceDirect Hearing Research journal homepage: www.elsevier.com/locate/heares http://dx.doi.org/10.1016/j.heares.2016.03.005 0378-5955/© 2016 Elsevier B.V. All rights reserved. Hearing Research xxx (2016) 1e13 Please cite this article in press as: Kozin, E.D., et al., Design, fabrication, and in vitro testing of novel three-dimensionally printed tympanic membrane grafts, Hearing Research (2016), http://dx.doi.org/10.1016/j.heares.2016.03.005