Design, fabrication, and in vitro testing of novel three-dimensionally printed tympanic membrane grafts

Highlights

• Tympanic membrane grafts can be manufactured using 3D printing.

• Acoustic properties of 3D grafts are uniform, reproducible and similar to human TM.

• Mechanical properties of 3D grafts are resilient following stress.

• 3D graft scaffold architecture influences acoustic and mechanical properties.

• 3D printed TMs enable the focused study of discrete biomechanical properties.

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 (0.2–10 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.

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 of the hair cells, which convert mechanical energy into neuronal impulses. The dynamic functional properties of the TM are contained in a multi-layered structure that is less than 100 μm thick (Kuypers et al., 2006). A host of structural features, including collagen fiber arrangement and trilayer design, enables the conversion of environmental sound to mechanical motion (O'Connor et al., 2008). These unique structural features work in concert to enable the effective transmission of sound across a wide range of frequencies (humans: 20 Hz to 20,000 Hz) and result in complex movements that vary in magnitude from picometers to microns (Decraemer et al., 1991).

Damage to the TM, such as from chronic otitis media (COM) or traumatic perforation, results in hearing loss due to ineffective sound transmission (Strens et al., 2012). Suppurative COM affects over 30 million individuals worldwide each year, leading to a significant health care burden (Monasta et al., 2012). The most common long-term complication in patients with COM is persistent TM perforation and conductive hearing loss. Tympanoplasty is the surgical repair of the TM, a procedure performed tens of thousands of times each year in the United States (Rubin, 1982). Successful tympanoplasty re-establishes efficient sound transmission from the environment to the ossicular chain, while also recreating a robust barrier between the ear canal and middle ear.

Historically, cadaveric TMs (House et al., 1966, Marquet, 1971), bovine pericardium (Pfaltz and Griesemer, 1985), and simple synthetic matrices (Kohn et al., 1984, Levin et al., 2009) have been used as TM grafts with moderate success in tympanoplasty. However, due to infectious risk from cadaveric tissue and concerns over graft uptake in synthetic matrices, the most common materials used today are autologous temporalis fascia, perichondrium and cartilage, which can be harvested from the patient at the time of surgery (Cabra and Monux, 2010, Eviatar, 1978, Lyons et al., 2015). Further, studies have indicated the ability of fascia to remodel, thin and become translucent following placement (Szabo, 2006).

Despite its widespread use and proven efficacy to reconstruct the TM and restore hearing, fascia has inherent limitations as a graft material. Fascia may contain intrinsic defects rendering it susceptible to ongoing COM (Boedts et al., 1990, Hiraide et al., 1980). The small irregularities within temporalis fascia may not be perceptible at the time of surgery, leading to unpredictable outcomes. Like all graft materials, fascia is also susceptible to continued middle-ear pressure differences that can result in retraction or re-perforation of the TM. Recent studies demonstrate that perforations persist in greater than 15% of pediatric patients following primary tympanoplasty (Hardman et al., 2015) and revision surgery rates for patients with COM nearly double that rate to 28% (Kaylie et al., 2006). Furthermore, revision procedures often leave the surgeon without adequate graft materials for TM reconstruction, given limited quantities of autologous materials. Collectively, refinements in graft materials used in tympanoplasty may improve patient outcomes and decrease surgery-related morbidity in selected cases.

Recent advances in multi-material three-dimensional (3D) printing enable fabrication of complex microscale architectures with controlled composition and structure (Gratson et al., 2004, Mironov et al., 2003, Murphy and Atala, 2014, Sun et al., 2012). Of specific relevance is direct ink writing (DIW), an extrusion-based printing method that enables a broad range of viscoelastic materials to be patterned under ambient conditions (Barry et al., 2009, Gratson et al., 2004, Hanson Shepherd et al., 2011, Lewis, 2006). Here, we harness advanced 3D printing techniques in an attempt to design and fabricate a biomimetic TM graft with the goal of reproducing specific structural features of the human TM.