Biology

BIOMEDICINE

3D bioprinting of collagen to rebuild components of the human heart A. Lee1*, A. R. Hudson1*, D. J. Shiwarski1, J. W. Tashman1, T. J. Hinton1, S. Yerneni1, J. M. Bliley1, P. G. Campbell1,2, A. W. Feinberg1,3†

Collagen is the primary component of the extracellularmatrix in the humanbody. It has proved challenging to fabricate collagen scaffolds capable of replicating the structure and function of tissues and organs.We present a method to 3D-bioprint collagen using freeform reversible embedding of suspended hydrogels (FRESH) to engineer components of the human heart at various scales, from capillaries to the full organ. Control of pH-driven gelation provides 20-micrometer filament resolution, a porous microstructure that enables rapid cellular infiltration and microvascularization, and mechanical strength for fabrication and perfusion of multiscale vasculature and tri-leaflet valves.We found that FRESH 3D-bioprinted hearts accurately reproduce patient-specific anatomical structure as determined by micro–computed tomography.Cardiac ventricles printedwith human cardiomyocytes showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.

F or biofabrication, the goal is to engineer tissue scaffolds to treat diseases for which there are limited options, such as end-stage organ failure. Three-dimensional (3D) bio- printing has achieved important milestones

including microphysiological devices (1), pat- terned tissues (2), perfusable vascular-like net-

works (3–5), and implantable scaffolds (6). However, direct printing of living cells and soft biomaterials such as extracellular matrix (ECM) proteins has proved difficult (7). A key obstacle is the problem of supporting these soft and dy- namic biological materials during the printing process to achieve the resolution and fidelity

required to recreate complex 3D structure and function. Recently, Dvir and colleagues 3D- printed a decellularized ECM hydrogel into a heart-like model and showed that human car- diomyocytes and endothelial cells could be in- tegrated into the print and were present as spherical nonaligned cells after 1 day in culture (8). However, no further structural or functional analysis was performed. We report the ability to directly 3D-bioprint

collagen with precise control of composition and microstructure to engineer tissue components of the human heart at multiple length scales. Collagen is an ideal material for biofabrication because of its critical role in the ECM, where it provides mechanical strength, enables struc- tural organization of cell and tissue compart- ments, and serves as a depot for cell adhesion and signaling molecules (9). However, it is dif- ficult to 3D-bioprint complex scaffolds using collagen in its native unmodified form because gelation is typically achieved using thermally driven self-assembly, which is difficult to control. Researchers have used approaches including

RESEARCH

Lee et al., Science 365, 482–487 (2019) 2 August 2019 1 of 5

1Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA. 2Engineering Research Accelerator, Carnegie Mellon University, Pittsburgh, PA 15213, USA. 3Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA. *These authors contributed equally to this work. †Corresponding author. Email: feinberg@andrew.cmu.edu

Fig. 1. High-resolution 3D bioprinting of collagen using FRESH v2.0. (A) Time-lapse sequence of 3D bioprint- ing of the letters “CMU” using FRESH v2.0. (B) Schematic of acidified collagen solution extruded into the FRESH support bath buffered to pH 7.4, where rapid neutralization causes gelation and formation of a collagen filament. (C and D) Rep- resentative images of the gelatin microparticles in the support bath for (C) FRESH v1.0 and (D) v2.0, showing the decrease in size and polydispersity. (E) Histo- gram of Feret diameter distribution for gelatin microparticles in FRESH v1.0 (blue) and v2.0 (red). (F) Mean Feret diameter of gelatin micro- particles for FRESH v1.0 and v2.0 [N > 1200, data are means ± SD, ****P < 0.0001 (Student t test)]. (G) Storage (Gʹ) and loss (Gʺ) moduli for FRESH v1.0 and v2.0 support baths showing yield stress fluid behavior. (H) A “window-frame” print construct with single filaments across the middle, comparing G-code (left), FRESH v1.0 (center), and FRESH v2.0 (right). (I) Single filaments of collagen showing the variability of the smallest diameter (~250 mm) that can be printed using FRESH v1.0 (top) compared to relatively smooth filaments 20 to 200 mm in diameter using FRESH v2.0 (bottom). (J) Collagen filament Feret diameter as a function of extrusion needle internal diameter for FRESH v2.0, showing a linear relationship.

on A ugust 1, 2019

http://science.sciencem

ag.org/ D

ow nloaded from

chemically modifying collagen into an ultraviolet (UV)–cross-linkable form (10), adjusting pH, temperature, and collagen concentration to con- trol gelation and print fidelity (11, 12), and/or denaturing it into gelatin (13) to make it ther- moreversible. However, these hydrogels are typ- ically soft and tend to sag, and they are difficult to print with high fidelity beyond a few layers in height. Instead, we developed an approach that uses rapid pH change to drive collagen self- assembly within a buffered support material, enabling us to (i) use chemically unmodified collagen as a bio-ink, (ii) enhance mechanical properties by using high collagen concentra- tions of 12 to 24 mg/ml, and (iii) create complex structural and functional tissue architectures. To accomplish this, we developed a substantially improved second generation of the freeform reversible embedding of suspended hydrogels (FRESH v2.0) 3D-bioprinting technique used in combination with our custom-designed open- source hardware platforms (fig. S1) (14, 15). FRESH works by extruding bio-inks within a thermoreversible support bath composed of a gelatin microparticle slurry that provides support during printing and is subsequently

melted away at 37°C (Fig. 1, A and B, and movie S1) (16). The original version of the FRESH support

bath, termed FRESHv1.0, consisted of irregularly shaped microparticles with a mean diameter of ~65 mm created by mechanical blending of a large gelatin block (Fig. 1C) (16). In FRESH v2.0, we developed a coacervation approach to gen- erate gelatin microparticles with (i) uniform spherical morphology (Fig. 1D), (ii) reduced poly- dispersity (Fig. 1E), (iii) decreased particle diam- eter of ~25 mm (Fig. 1F), and (iv) tunable storage modulus and yield stress (Fig. 1G and fig. S2). FRESH v2.0 improves resolution with the ability to precisely generate collagen filaments and ac- curately reproduce complex G-code, as shown with a window-frame calibration print (Fig. 1H). Using FRESHv1.0, the smallest collagen filament reliably printed was ~250 mm in mean diameter, with highly variable morphology due to the rela- tively large and polydisperse gelatin micropar- ticles (Fig. 1I). In contrast, FRESH v2.0 improves the resolution by an order of magnitude, with collagen filaments reliably printed from 200 mm down to 20 mm in diameter (Fig. 1, I and J). Filament morphology from solid-like to highly

porous was controlled by tuning the collagen gelation rate using salt concentration and buffer- ing capacity of the gelatin support bath (fig. S3). A pH 7.4 support bath with 50 mM HEPES was the optimal balance between individual strand resolution and strand-to-strand adhesion and was versatile, enabling FRESH printing of mul- tiple bio-inks with orthogonal gelation mech- anisms including collagen-based inks, alginate, fibrinogen, andmethacrylated hyaluronic acid in the same print by adding CaCl2, thrombin, and UV light exposure (fig. S4) (15). We first focused on FRESH-printing a sim-

plified model of a small coronary artery–scale linear tube from collagen type I for perfusionwith a custom-designed pulsatile perfusion system (Fig. 2A and fig. S5). The linear tube had an inner diameter of 1.4 mm (fig. S6A) and a wall thick- ness of ~300 mm (fig. S6B), and was patent and manifold as determined by dextran perfusion (fig. S6, C to E, and movie S2) (15). C2C12 cells within a collagen gel were cast around the printed collagen tube to evaluate the ability to support a volumetric tissue. The static nonper- fused controls showedminimal compaction over 5 days (Fig. 2B), and a cross section revealeddead

Order now and get 10% discount on all orders above $50 now!!The professional are ready and willing handle your assignment.

ORDER NOW »»