Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
R- and Z‑Axis Patterned Scaffolds Mimic Tracheal Circumferential Compliance and Longitudinal Extensibility Elizabeth M. Boazak, Jamie M. Benson, and Debra T. Auguste*,† Department of Biomedical Engineering, The City College of New York, Steinman Hall, 160 Convent Avenue, New York, New York 10031, United States ABSTRACT: There remains no routine treatment for congenital tracheal abnormalities affecting more than 1/3 of the length. Natural and artificial prostheses are plagued by mechanical failure and inconsistent outcomes. Mimicking native tissue mechanics in an engineered replacement may improve functional and patient outcomes. We synthesized tubular constructs comprising photocross-linked methyl acrylate-co-methyl methacrylate, p(MA-coMMA), with patterned r- and z-axes in order to achieve mechanical properties similar to lamb tracheae. Hard and soft alternating bands, and a soft vertical section, mimic tracheal architecture. Patterned constructs were capable of 46% elastic longitudinal extension. The construct longitudinal composite modulus, 0.34 ± 0.09 MPa, was not significantly different from ovine tracheae. The superior of two geometries evaluated supports up to a 46% reduction of internal volume within the physiological range of transmural pressures. Thus, these patterned hydrogels yielded longitudinal elasticity and radial rigidity while allowing for radial deformation required for effective coughing. KEYWORDS: trachea replacement, compliance, trachea mechanics, biomimetic
1. INTRODUCTION
in contrast to frequent complications observed in response to full circumferential internal stents.11 We have previously investigated the use of bioinspired ring patterns to create radially rigid and longitudinally extensible tubes.12 Through this work, we found that a 1:2 thickness ratio of hard to soft rings was optimal. The Young’s modulus of hard and soft rings were similar to tracheal cartilage (1−20 MPa13,14) and ligament (13−255 kPa15), respectively, which resulted in a construct with longitudinal and radial mechanical properties similar to neonatal trachea. In this paper, we build on our previous patterning studies in order to make a longitudinally extensible construct with physiologic compliance. We hypothesized that incorporation of a soft vertical region mimicking the architecture of the trachealis muscle may produce constructs with matched compliance. In a departure from our previous work with HEMA hydrogels, these constructs were synthesized from poly(methyl acrylate-comethyl methacrylate), abbreviated p(MA-co-MMA). p(MA-coMMA) is reported to have high long-term toughness under physiological conditions, is noncytotoxic, and can be photopolymerized to achieve a wide range of Young’s moduli.16,17 As described herein, p(MA-co-MMA) also offers superior yield properties as compared to pHEMA.12 To compare our constructs to native organ mechanical properties, we have also performed tensile, cyclic compression, and compliance
The trachea exists for the sole purpose of transporting air. Yet, the physiology that enables this seemingly simple function is surprisingly complex. Mechanical anisotropy plays an important role in supporting respiration. Three tissue types principally contribute to trachea mechanics: cartilage, smooth muscle (trachealis), and annular ligament. Cartilage rings maintain a patent airway,1 whereas the annular ligament engenders longitudinal extensibility, which is required for normal respiration.2 The native trachea extends up to 20% in adults and 46% in neonates during normal respiration.3 Trachealis muscle actively controls airway cross-sectional area, and contributes most significantly to tracheal compliance.4,5 Compliance is defined as the change in volume for a given change in transmural pressure. The ability to deform at relevant transmural pressures is essential for productive coughing.6,7 Reduction in cross-sectional area allows for an increase in air velocity sufficient to clear mucus. Chronically retained secretions result in increased susceptibility to infection, as well as airway obstruction and respiratory discomfort.8,9 A rigid implant with isotropic mechanical properties may result in complications because of mechanical mismatch resulting in pulling at the attachment sites and failure to clear mucus effectively. To avoid such complications and ensure implant success, matching native compliance and extensibility may be required. Supporting this approach, an external C-shaped tracheal splint, which would allow for trachealis deformation, resulted in no subsequent hospitalizations or complications,10 © XXXX American Chemical Society
Received: August 31, 2017 Accepted: November 12, 2017 Published: November 13, 2017 A
DOI: 10.1021/acsbiomaterials.7b00641 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 1. Schematic of scaffold synthesis method, showing the components of the prepolymer solution which are mixed along with the photoinitiator AIBN, and inserted into a tubular mold sealed at either end with PDMS caps. A photomask is wrapped around the mold, which is then placed into a photopolymerization box. mold was exposed for an additional 40 min, changing the water in the inner cylinder every 10 min. Constructs were washed and stored in water for a minimum of 3 days before testing in order to remove unreacted components and achieve stable mechanical properties. 2.3. Compliance Testing. The tapered ends of two 25 mL serological pipettes were filled with Sylegard elastomer to create plugs to seal the ends of the patterned constructs. One plug contained an embedded segment of plastic tubing. The plugs were secured using wire, and vacuum grease helped achieve a tight seal. The sealed construct was then placed in a rigid chamber and creating a setup similar to that used by Croteau & Cook.18 Airway compliance is defined as the change in airway volume over the change in transmural pressure, (ΔVA/ΔPtm, the slope of a volume pressure curve).19 Volume pressure curves report a ΔVA normalized by the original volume to better enable comparisons across samples of different sizes, and to literature values which are regularly reported in a normalized form. Change in volume was recorded in 10 cm H2O increments for tubes with uniform wall thickness and ovine tracheae, and in 5 cm H2O increments up to a 30 cm H2O collapsing pressure for tubes with nonuniform wall thickness. At each increment, the construct was allowed to equilibrate until the reading was stable. According to Costantino et al., a 1 min equilibration time is sufficient for stable readings.20 Rubber stoppers were used to seal the ends of the tracheae. 2.4. Tensile and Cyclic Radial Compression Tests. An Instron 5543 mechanical testing system outfitted with a BioBath and 10 and 500 N load cells was used to perform tensile testing at a strain rate of 1 mm/min. All samples were submerged in 1× phosphate buffered saline (PBS) for the duration of testing. The composite modulus was calculated to 0.5% strain for polymer constructs. For ovine trachea, the toe-region modulus was typically calculated between 2 and 4% strain and the linear region between 25%−35%. Toughness was calculated as the area under the normalized load vs composite strain curve until failure. The terms “stress” and “strain” are avoided, as the values do not accurately represent the internal stresses or strains in specific regions of a composite/multitissue sample. The cross sectional area of samples cut from tubular constructs and sheep tracheae was measured with calipers and used for normalized load calculations. All longitudinal samples subjected to tensile testing contained ≥5 bands/alternating ring and ligament segments. Tubes with uniform wall thickness were used for mechanical testing. Samples were cut from the center of the hard and soft rings to determine the modulus of hard and soft regions. To evaluate yielding, we elongated banded longitudinal strips cut from tubular constructs to 46% composite
tests on sheep trachea. We chose to match the mechanical properties of sheep trachea because the gestation period is similar to humans, making sheep a suitable model for fetal studies.
2. MATERIALS AND METHODS 2.1. Materials. Methyl acrylate (MA), methyl methacrylate (MMA), polyethylene glycol dimethacrylate (PEGDMA) Mn = 550, and 2,2′-Azobis(2-methylpropionitrile) (AIBN) were purchased from Sigma-Aldrich (St. Louis, MO). Glass tubing for the construction of the molds used to synthesize tubular constructs was purchased at custom lengths from Greatglas, Inc. (Wilmington, DE). Sylgard 184 Elastomer kit was purchased from VWR (Bridgeport, NJ). Tracheae from sheep aged 8−36 months were purchased commercially from Senat Poultry, Inc. (Patterson, NJ). 2.2. Scaffold Synthesis. Custom molds were prepared using two glass cylinders with a 20.2 mm inner diameter (outer cylinder) and a 14 mm outer diameter (inner cylinder). A detachable base was cast using the Sylgard elastomer kit to hold cylinders concentrically, in an either centered or off-center configuration, in order to produce tubular constructs with uniform and nonuniform wall thickness while keeping the construct wall volume constant. Before each use of the mold, the exterior of the inner cylinder and the interior of the outer cylinder were treated with Rainex for 10 min, at which time the cylinders were wiped dry. Vacuum grease was used to create a tight seal between the glass and elastomer base. A prepolymer solution was prepared by mixing 36% (v/v) MA and 54% (v/v) MMA (monomers), 10% (v/v) PEGDMA (cross-linker), and 0.6% (w/v) AIBN (photoinitiator), and subsequently degassed for 10 min. All materials were used as received. The solution was then used to fill the space between the glass cylinders, and the top was sealed with a second PDMS spacer to create an airtight environment (Figure 1). The center cylinder was filled with room temperature water in order to prevent overheating. The mold was then placed in the center of an RMR-600 Photochemical Reactor, containing eight 15.24 cm (6 in.) 368 nm UV lights, and the built-in fan turned on. After 3 min of UV exposure, the water in the center cylinder was changed, and matching photomasks created with an opaque tape were wrapped around the mold exterior and inserted into the center tube. The photomask design allowed for additional exposure to 4 mm bands and masked 8 mm bands and a soft vertical segment comprising 1/5 of the cylinder’s circumference. After application of the photomasks, the B
DOI: 10.1021/acsbiomaterials.7b00641 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 2. (A) p(MA-co-MMA) constructs were produced with uniform and nonuniform wall thicknesses. Both geometries were spatially patterned with alternating hard and soft bands and a soft vertical region, mimicking the cartilage, ligament, and smooth muscle composition of native trachea. (B) There is a 4-fold difference in tensile young’s modulus between the hard and soft regions. (C) Constructs are visibly patterned, where “soft” regions are white and “hard” regions are translucent.
defined an elastic modulus of 195 ± 41 kPa for neonatal lamb trachealis muscle, which is very comparable to the 230 ± 70 kPa modulus of construct the soft regions. We note that the elastic modulus of the hard segment was at the lower limit of tracheal cartilage. While we can achieve a wider range of properties in unpatterned p(MA-co-MMA) polymers (data not shown), the relatively long UV exposure time required for cross-linking hard bands (e.g., 40 min) affects patterning resolution. Despite being masked, the soft regions continue to cross-link with increased exposure time for the hard bands, as evidenced by increasing Young’s modulus. This may be due to photoinitiator diffusion and/or construct heating. With the current band size ratios, the highest modulus we were able to achieve for hard regions while keeping the soft regions within the range of annular ligament was 0.78 MPa, an approximately 4-fold increase. Submersion of patterned constructs in ddH2O resulted in polymer compaction, which was more significant in softer regions. As such, soft regions took on a white opaque appearance, while hard segments remained translucent (Figure 2C). The disproportionate compaction of hard and soft regions resulted in a construct with a hard:soft band size ratio of approximately 1:1 (4 mm:4 mm), despite the initial 1:2 ratio of the photomask. In our previous, work we found that a 1:1 band size ratio did not result in a significant change in longitudinal Young’s modulus as compared to a 1:2 ratio, although it did reduce the patterned construct yield strain. Because of the more elastomeric nature of p(MA-co-MMA) as compared to HEMA, we anticipated that a 1:1 hard:soft ratio would provide sufficient longitudinal mechanics. Furthermore, the lower modulus of the hard regions, as compared to our earlier work, may necessitate a greater percentage of hard regions for sufficient resistance to radial deformation. Our earlier patterning studies were used as a basis for the construct design; the use of a new material required evaluation of the longitudinal and radial mechanics of the tubular constructs in addition to the role of patterning and geometry in defining tubular compliance. 3.2. Longitudinal Properties. Longitudinal sections of patterned constructs were evaluated under tensile loading and compared to ovine trachea specimens. All mechanical analysis was performed under physiological conditions (submerged in PBS at 37 °C). As shown in Figure 3A, the composite modulus of the constructs was 0.34 ± 0.09 MPa, which was not
strain at 1 mm/min, after which loading was removed. Samples were left unloaded for 5 min before beginning the subsequent reloading. Segments of tubular constructs and ovine tracheae were subjected to 250 cycles of compression to a deformation comprising 15% of the specimen outer diameter. Testing was performed at 0.833 Hz, mimicking the 1.2 s/breath average neonatal respiratory rate. It was confirmed that patterned p(MA-co-MMA) tubular constructs reached a plateau in peak load at 15% radial deformation by cycle 29 ± 15, where the plateau point is defined as a difference in peak load
