Combined technologies for microfabricating elastomeric cardiac tissue engineering scaffolds

Abstract

Polymer scaffolds that direct elongation and orientation of cultured cells can enable tissue engineered muscle to act as a mechanically functional unit. We combined micromolding and microablation technologies to create muscle tissue engineering scaffolds from the biodegradable elastomer poly(glycerol sebacate). These scaffolds exhibited well defined surface patterns and pores and robust elastomeric tensile mechanical properties. Cultured C2C12 muscle cells penetrated the pores to form spatially controlled engineered tissues. Scanning electron and confocal microscopy revealed muscle cell orientation in a preferential direction, parallel to micromolded gratings and long axes of microablated anisotropic pores, with significant individual and interactive effects of gratings and pore design.

Figure 1. Micropatterning of poly(glycerol sebacate)(PGS)

(a) schematic depicting UV light projection through a mask onto a silicon wafer, (b) interferometry of resulting master showing double grooves, (c,d) SEMs at low-and high-magnification of membranes with micropatterning. Scale bars: (c) 500 μm, (d) 50 μm.

Figure 2. Laser microablation of PGS

(a) schematic of method (laser directed through back side of patterned membrane), (b,c,d,e) SEMs at low- and high magnification of membranes with micropatterning and (b,c) square pores or (d,e) anisotropic rectangular pores. Scale bars: (b-d) 500 μm, (c-e) 200 μm.

Figure 3. Mechanical properties of PGS membranes and porous scaffolds, as determined by uniaxial tensile testing

(a) Elastic modulus (E) and (b) Ultimate tensile strength (UTS) measured for three types of specimens with gratings: PGS membranes (BULK LINE) and PGS scaffolds from the SQ LINE and RECT LINE groups. Tensile stretch was applied in two directions: parallel or orthogonal to the gratings (preferred direction, PD, and cross-preferred direction, XD). Data represent average ± standard deviation. * = p < 0.05, ** = p < 0.01.

Figure 4. Scanning electron micrographs of C2C12 muscle cells cultured on PGS scaffolds

(a, b) Square and rectangular pores without micropatterning, respectively; (c, d) square and rectangular pores with gratings, respectively. Scale bars: (a-d) 100 μm. Arrows (c,d) indicate grating direction.

Figure 5. Confocal laser micrographs of C2C12 muscle cells cultured on PGS scaffolds after actin-phalloidin actin staining

(a,b) Square pores without micropatterning; (c,d) square pores with gratings; (e,f) rectangular pores without micropatterning; (g,h) rectangular pores with gratings. Scale bars: (a,c,e,g) 100 μm; (b,d,f,h) 50 μm. Arrows (c,d,g,h) indicate grating direction.

Figure 6. Orientation of C2C12 muscle cells cultured on PGS scaffolds

(a) Schematic of cell long axis angle deviation relative to grating direction. Cell alignment (within a 10 degree angle deviation threshold) based SEM images (b) for all four groups of scaffolds, (c) square pores without micropatterning, (d) square pores with gratings, (e) rectangular pores without micropatterning; (f) rectangular pores with gratings. Data represent average ± standard deviation. * = p < 0.05, ** = p < 0.01.