3D structural patterns in scalable, elastomeric scaffolds guide engineered tissue architecture

Abstract

Microfabricated elastomeric scaffolds with 3D structural patterns are created by semiautomated layer-by-layer assembly of planar polymer sheets with through-pores. The mesoscale interconnected pore architectures governed by the relative alignment of layers are shown to direct cell and muscle-like fiber orientation in both skeletal and cardiac muscle, enabling scale up of tissue constructs towards clinically relevant dimensions.

Keywords: alignment; heart; microfabrication; muscle; polyglycerol sebacate (PGS).

Figure 1

Fabrication of multi-layer scaffolds with unique 3D structural patterns. (a) Process flow for creating multi-layer scaffolds. To make polymer sheets with throughpores, Si wafers were etched with specific patterns to serve as reusable molds, on which poly(glyercol sebacate) (PGS) was cast, cured, and delaminated by dissolving a sacrificial layer of maltose. Multi-layer scaffolds were assembled using a semi-automatic process to align and bond the sheets into multi-layer structures. (b)-(i) Demonstration of two specific two-layered (2L) alignment patterns, short strut offset (SSoff) (b,d,f,h) and long strut offset (LSoff)(c,e,g,i), for PGS polymer sheets, each sheet with rectangular pores 250 µm×125 µm and thickness of 70 µm. (b-c) Schematic drawings with yellow line indicating pore interconnectivity pattern, (d-e) SEM showing internal pore structures, (f-g) 3D laser scanning micrographs indicating features in the third dimension via color bar, (h-i) bright-field micrographs indicating multi-layered alignment over a macro-scale on the order of several mm. Scale bars are (d,e) 100 µm (h,i) 1 mm.

Figure 2

Multi-scale alignment of C2C12 myoblasts cultured on 2L PGS scaffolds with different 3D structural patterns. (a,c,d,g,i) SSoff pattern (b,e,f,h,j) LSoff pattern. (a-b) Wide view fluorescent micrographs of F-actin stained tissue constructs, where macroalignment of muscle tissue bundles is indicated by arrows. (c-f) Cross sections stained with hematoxylin and eosin indicated tissue weaving throughout both scaffold layers (c) PD-section of SSoff (d) XD-section of SSoff (e) PD-section of LSoff (f) XD-section of LSoff. (g-j) Confocal micrographs of tissue constructs stained for F-actin (green) and counterstained for nuclei (blue) taken at the interface of the two scaffold layers demonstrating meso-scale pore interconnectivity patterns which guided tissue architecture and cell alignment. (k) Graphical representation of cellular alignment quantified via FFT analysis of n=6 circular masks cropped from confocal micrographs (see Supplementary Figure S1), with the axis lines representing the predominant alignment direction. (l) Orientation index (average of n=6) indicating that both distributions are equally ‘well-aligned,’ calculated by normalizing the percentage of cells aligned within ± 20° of the mean to the percentage of cells aligned within ±20° of the mean if cells were randomly oriented. Scale bars in all images are 100 µm.

Figure 3

Alignment and differentiation of neonatal rat heart cells cultured on 2L PGS scaffolds with different 3D structural patterns. (a,c) SSoff, (b,d)LSoff, shown at different magnifications and imaged by (a-b) fluorescence or (c-d) confocal microscopy (a,c-e) of F-actin stained (green) and nuclear DNA counter-stained or (b,f) sarcomeric α-actinin stained (red); arrows indicate striations in cardiac myocytes. (g,h) TEM images showing (g) abundant mitochondria (M) positioned between myofilaments (MF) with clearly defined sarcomeres and z-lines (z) and (h) a well-formed junctional complex. (i) Mechanical testing confirmed that mechanical anisotropy was independent of pore interconnectivity pattern for the 2L scaffolds (LSoff versus SSoff) and independent of the number of layers (2L versus 1L scaffolds). (j-k) As cell alignment and mechanical anisotropy can be effectively decoupled, tissue constructs can be implanted with muscle contraction from cell alignment (green arrows) and mechanical support (PD, black arrow) from PGS scaffolds in the (j) same or perpendicular (k) directions. Scale bars are (a-d) 100 µm (e,f) 10 µm (g,h) 500 nm.

Figure 4

Scale-up of multi-layer scaffolds for implantation of cardiac tissue constructs. (a) Bright-field image of a three-layered (3L) scaffolds with rectangular pores with “staircase” alignment pattern (b) Confocal cross-sections of heart cells culture on the 3Lscaffold shown in (a), stained for F-actin (green) and counterstained for nuclear DNA (purple), demonstrating seeding throughout all layers (c,d) SEM images of four-layered scaffolds in a short-strut offset alignment pattern, with number indicating individual layers (e,f) SEM images showing (e) longitudinal and (f) cross section views of a five-layered tissue construct including two layered scaffolds on either side of a central layer with channels instead of through-pores, which can be used for perfusion of nutrients and/or endothelialization. Channels are indicated by asterisks. Scale bars are (a) 350 µm (b-f) 100 µm.