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Effect of chemical and physical properties on the in vitro degradation of 3D printed high resolution poly(propylene fumarate) scaffolds Jason M Walker, Emily Bodamer, Olivia Krebs, YuanYuan Luo, Alex P Kleinfehn, Matthew L. Becker, and David Dean Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00146 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017
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Effect of chemical and physical properties on the in vitro degradation of 3D printed high resolution poly(propylene fumarate) scaffolds Jason M. Walker1,2, Emily Bodamer1, Olivia Krebs1, Yuanyuan Luo3, Alex Kleinfehn3, Matthew L. Becker3, David Dean1,*
1
The Ohio State University, Department of Plastic Surgery, 460 West 12th Ave., Rm. 388. Columbus, OH 43210 2
Youngstown State University, Department of Mechanical and Industrial Engineering, 1 University Plaza, Youngstown, OH 44555 3
The University of Akron, Department of Polymer Science, 170 University Ave Akron, OH 44325-3909
*
Corresponding author, E-Mail:
[email protected] Phone: 614-688-9044 Fax: 614-688-2195
Keywords: Poly(propylene fumarate), Resorption, Degradation, Scaffold, Tissue Engineering
ABSTRACT Two distinct molecular masses of poly(propylene fumarate) (PPF) are combined with an additive manufacturing process to fabricate highly complex scaffolds possessing controlled chemical properties and porous architecture. Scaffolds were manufactured with two polymer molecular masses and two architecture styles. Degradation was assessed in an accelerated in vitro environment. The purpose of the degradation study is not to model or mimic in vivo degradation, but to efficiently compare the effect of modulating scaffold properties. This is the first study addressing degradation of chain-growth synthesized PPF, a process which allows for considerably more control over molecular mass distribution. It demonstrates that with greater process control, not only is scaffold fabrication reproducible, but the mechanical properties and degradation kinetics can be tailored by altering the physical properties of the scaffold. This is a clear step forward in using PPF to address unmet medical needs while meeting regulatory demands and ultimately obtaining clinical relevancy.
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INTRODUCTION Tissue engineering (TE) is a rapidly maturing field which aims to regenerate lost or damaged tissue. This approach typically relies on a degradable porous biomaterial that is combined with bioactive molecules and cells1. Often, synthetic polymers are used as the scaffolding material. Synthetic materials can be tailored to meet specific requirements such as biocompatibility, mechanical properties, microstructure, and degradation rate2. Selection of materials and manufacturing techniques are therefore paramount, as well as interdependent. Recently developed additive manufacturing (AM) techniques allow for greater control over the external and internal architectures of a TE scaffold though the use of computer-aided design (CAD). However, different materials are processed through different AM methods and, therefore, the selections of material and manufacturing technique must be considered together. Only a few polymeric biomaterials are available that are nontoxic, resorbable, and can be 3D printed. These materials include polylactides3-5, poly(ε-caprolactone)6-7, and poly(propylene fumarate)8. Among these, polylactides typically degrade within weeks, while poly(ε-caprolactone) degrades very slowly over the course of years9. Poly(propylene fumarate) (PPF) has been identified as a biomaterial for tissue engineering scaffolds that may degrade safely and controllably within a more useful timeframe; that is, with a rate that more closely matches the development of new tissue formation10,11. PPF, which was first reported in 1994, degrades via ester hydrolysis into propylene glycol and fumaric acid, two products which are cleared by normal metabolic processes12. Moreover, PPF is photocrosslinkable, meaning that complex, computer-designed structures can be manufactured directly via 3D printing. The manufacturing method is equally important to material selection in the development of TE scaffolds. Conventional methods of porous scaffold fabrication, including porogen leaching, gas foaming, and electrospinning13-15, offer a minimal amount of control over surface area, porosity, and pore size. These techniques produce stochastic porosities that lack repeatability in fabrication, potentially contain disjointed void spaces, and likely result in irregular degradation, mass transport, and stress-distribution. Furthermore, without the use of complex molds, the external shape of conventionally fabricated scaffolds 2 ACS Paragon Plus Environment
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are limited to simple geometries such as cylinders. Extrusion-based 3D printing enables more control over the internal and external architectures of tissue engineering scaffolds using direct-from-CAD manufacturing of thermoplastics, but offers comparably low resolution16. Stereolithography, a light-based 3D printing process which consists of curing a photosensitive liquid polymer into a solid polymer, generally offers the highest resolution17. Recent advances to light-based 3D printing include the use of digital micromirror devices (DMD) to project a complete cross-section at once, which has further increased efficiency and resolution18. Herein we demonstrate the use of AM for the fabrication of tissue engineering scaffolds out of PPF by DMD-based mask projection stereolithography. Furthermore, we seek to evaluate the effect of physical parameters such as molecular mass of the polymer and scaffold pore architecture on characteristics such as mechanical integrity and degradation rate. Three experimental groups are used to study the effects of the two properties. First, pore architecture is held constant while a variation in molecular mass is studied and, second, molecular mass is held constant while a variation in pore architecture is studied. In the past, such experiments were difficult, if not impossible, to undertake due to the lack of consistency in material synthesis and/or scaffold fabrication. Here, we utilize an innovative scaffold design method and a high resolution 3D printer to manufacture complex scaffolds with extremely fine features. Additionally, the poly(propylene fumarate) used in this study was synthesized by a new method resulting in significantly larger batches with a high level of control over the polymer molecular mass and mass distribution19. The aim of this study is to manufacture scaffolds with highly controlled physical and chemical properties and to explicate the relationships between scaffold properties and degradation kinetics.
MATERIALS AND METHODS Scaffold Design
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Each scaffold was cylindrical with a prescribed diameter of 6 mm and height of 10 mm. Schoen’s gyroid triply periodic minimal surface was used as the geometric foundation for the pore architecture. The gyroid is a mathematical surface that can be approximated to the first order by the nodal equation20: Φ = sinN cosN + sinN cosN + sinN cosN − C Using a custom algorithm implemented in MATLAB® (MathWorks, Natick, MA), the gyroid surface was used to separate a three dimensional object (i.e., the scaffold) into two complex and intertwined, but separate phases. Briefly, this is accomplished by implicitly describing both the gyroid surface and the solid object using scalar fields, then intersecting the scalar fields, and finally reconstructing an explicit surface from the result using a marching cubes algorithm21. Thereafter, one phase is established as a solid network of struts, while the second phase is void and constitutes the porous volume22. Kapfer et al. refer to this type of structure as a network solid23. This method is computationally efficient and enables full control over the pore and strut sizes within the scaffold. In this work, two porous architectures were chosen: a fine architecture and a coarse architecture. The gyroid parameters, N, and, C, are given in Table 1, in addition to the dimensions. Table 1. Pore design parameters of PPF scaffolds (SA = Surface Area) Architecture
Pore Size Porosity
SA (mm2)
N
C
Strut Size
Fine
1.9243π
-1.1476
200 µm
700 µm
88.2%
597.1
Coarse
0.9622π
-1.1476
400 µm
1400 µm
88.2%
312.7
The two pore structures are geometrically similar, possessing the same volume and percent porosity. Figure 1A shows a single unit cell of the coarse architecture with unit side length 2.08 mm, while Figure 1B shows eight unit cells of the fine architecture stacked in a 2x2x2 arrangement with total side length 2.08 mm.
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Figure 1. Comparison of (A) coarse pore architecture and (B) fine pore architecture. In the same volume, the fine architecture has exactly eight times the number of unit pore cells than the coarse architecture. Although cylindrical structures were used in this work, it is important to note that this same design methodology can be applied to arbitrarily shaped objects. Thus, anatomically shaped implants derived directly from patient CT-data can be produced.
Resin formulation Poly(propylene fumarate) (PPF) was synthesized by a ring-opening copolymerization method using a chain-growth mechanism19. The chain-growth mechanism produces a consistent molar mass with higher yields compared to traditional step growth methods, resulting in large batches with consistent mass and mass distribution properties. Two different batches of PPF were synthesized for comparison in this study: a low molecular mass (Mn = 1500 Da, Dm = 1.6) and a high molecular mass (Mn = 2450 Da, Dm = 1.6). The formulation of resin from both batches of PPF was carried out identically. All experiments were produced from these two batches of materials. To reduce the viscosity for 3D printing, PPF was diluted in diethyl fumarate (DEF, 1:1 wt%, Sigma-Aldrich, St. Louis, MO) at 65°C. DEF also possesses a carbon-carbon double bond that should allow it to participate in the crosslinking reaction24. Lastly, two photoinitiators, Irgacure 819 (3.0 wt%, BASF Corp., Florham, NJ) and Irgacure 784 (0.4%, BASF), and a UV absorber, oxybenzone (0.7%, Sigma-Aldrich), were added to the PPF/DEF mixture to enable and control photocrosslinking.
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Additive manufacturing Scaffolds were fabricated on an EnvisionTEC Perfactory P3 Mini Multi Lens DMD-based mask projection stereolithography unit (EnvisionTEC, Inc., Dearborn, MI). CAD models were digitally sliced into layers using the Perfactory software suite prior to manufacturing. The Perfactory P3 is an inverted system that projects upwards through a transparent glass plate into a reservoir containing the resin. After each projection, the build platform moves vertically upwards to accommodate resin inflow for the next layer. The effect of different build parameters on cure depth and quality was studied and the following combination was selected: layer thickness = 50 µm, irradiance = 350 mW/dm2, and projection time = 30 s. After printing, scaffolds were immediately rinsed with acetone, 70% ethanol (v/v), and distilled water for 15 s each using wash bottles. Following this, scaffolds were immersed in an ultrasonic bath for 5 min in acetone then 15 min in distilled water. Finally, scaffolds were post-cured in a ProCure 350 UV chamber (3D-Systems, Rock Hill, SC) for 480 min to ensure a fully crosslinked network. All scaffolds were manufactured with 4 mm tall supports which were manually removed after cleaning and post-curing.
Accelerated in vitro degradation Degradation was evaluated over 30 days in a sodium hydroxide solution (0.1 M NaOH). Scaffolds were submerged in 10 mL of solution in individual glass culture tubes, capped, and maintained at 37 °C. Throughout the study, the NaOH solution was replaced with fresh solution every second day. The previous solution was left to cool to room temperature and its pH was measured. At each time point (days 0, 2, 4, 6, 8, 10, 20, 30), five samples were removed for analysis and washed in deionized water for 15 s. The scaffolds were dried in an oven at 37 °C for 24 h. The external dimensions of all scaffolds were measured after drying with a digital caliper. For each dimension, three measurements were made and the average was recorded. The mass of each scaffold was taken prior to the experiment and again at their respective time points after drying using an electronic balance. The percent mass remaining was calculated for each sample by: 6 ACS Paragon Plus Environment
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% mass remaining = 1 −
M − M M
where Mi is the initial mass and Mf is the final mass. Mechanical properties were evaluated by uniaxial unconfined compression on a TestResources load frame with a 250 lbf load cell (TestResources, Shakopee, MN). Samples were compressed at a rate of 1.0% strain per second. The compressive stiffness was calculated as the slope of the linear portion of the stress-strain curve. The yield strength was evaluated with a 0.2% offset. Some samples exhibited brittle fracture at the top of the linear stress-strain region, in which case the fracture stress was used as the yield stress. Examples of brittle fracture and 0.2% offset are shown in Figure 2. Scaffolds were imaged using a light microscope with Motic Images Plus software (Motic North America, Richmond, BC, Canada).
Figure 2. Mechanical compression examples depicting yield strength on stress-strain curves by (A) brittle fracture and (B) plastic deformation with 0.2% offset
Statistical analysis Student’s two-tailed t-test and ANOVA methods were used to compare quantitative data across two or more groups (Minitab Inc., State College, PA, USA). p-Values of
