3D printing a biocompatible elastomer for modeling muscle regeneration after volumetric muscle loss

Abstract

Volumetric muscle loss (VML) injuries due to trauma, tumor ablation, or other degenerative muscle diseases are debilitating and currently have limited options for self-repair. Advancements in 3D printing allow for the rapid fabrication of biocompatible scaffolds with designer patterns. However, the materials chosen are often stiff or brittle, which is not optimal for muscle tissue engineering. This study utilized a photopolymerizable biocompatible elastomer - poly (glycerol sebacate) acrylate (PGSA) - to develop an in vitro model of muscle regeneration and proliferation into an acellular scaffold after VML injury. Mechanical properties of the scaffold were tuned by controlling light intensity during the 3D printing process to match the specific tension of skeletal muscle. The effect of both geometric (channel sizes between 300 and 600 μm) and biologic (decellularized muscle extracellular matrix (dECM)) cues on muscle progenitor cell infiltration, proliferation, organization, and maturation was evaluated in vitro using a near-infrared fluorescent protein (iRFP) transfected cell line to assess cells in the 3D scaffold. Larger channel sizes and dECM coating were found to enhance cell proliferation and maturation, while no discernable effect on cell alignment was observed. In addition, a pilot experiment was carried out to evaluate the regenerative capacity of this scaffold in vivo after a VML injury. Overall, this platform demonstrates a simple model to study muscle progenitor recruitment and differentiation into acellular scaffolds after VML repair.

Keywords: 3D printing; DLP; Poly glycerol sebacate acrylate; Skeletal muscle; Volumetric muscle loss.

Figure 1.

A.) Schematic of the DLP-based 3D printing system. (1) a light source (385nm) is reflected off of a DMD chip (2) from which masks with arbitrarily complex geometry (3) are continuously uploaded. The reflected light from the DMD chip goes through a series of projection lenses (4) into a prepolymer reservoir (5) containing PGSA. The pattern of light reflected into the prepolymer reservoir crosslinks PGSA and sticks to a moving probe (6) allowing for continuous smooth structures to be fabricated. B) Schematic outlining the cell seeding strategy to replicate muscle progenitor cell infiltration into a PGSA scaffold after VML injury. C) SEM of a PGSA scaffold with 450μm diameter channels (Scale bar = 200μm). D) SEM of a sectioned PGSA scaffold coated with dECM (small specular objects), demonstrating the longitudinal microgrooves that are formed during the continuous 3D printing process (Scale bar = 10μm). E) Effective Young’s modulus of PGSA as a function of the light exposure intensity. A light exposure of 5.6 mW cm⁻² was used in this study, which simultaneously allowed for stiffness similar to that normal skeletal muscle (107kPa-225kPa)[–52] as well as the ability to print fine structures without overpolymerization. Sidak post hoc tests identified significant differences between each light exposure intensity (bar, p<0.0175).

Figure 2.

Creating a transfected cell line to assess nuclear staining of cells in a PGSA scaffold. A) PGSA’s autofluorescence in the DAPI channel along with the material’s physical adsorption of far-red nuclear dye DRAQ5 makes it challenging for visualization of cells within the microchannel construct due to low signal-to-noise ratio. B) A schematic of the lentivirus donor plasmid showing the DNA elements destined to be integrated into the genome of C2C12 cells. The expression of H2B-iRFP is under cytomegalovirus (CMV) promoter while the expression of puromycin resistant gene is under SV40 promoter. C) Phase (left) and immunofluorescent (middle) images of the iRFP nuclear transfected C2C12 cell line used in these experiments. D) iRFP nuclear transfected C2C12 cells in the scaffold (left) and coverslip control (right) demonstrate clear imaging of the nucleus with no fluorescent noise from the scaffold itself. Scale bar = 300μm (A, D). Scale bar = 100μm (C).

Figure 3.

3D reconstructions at various viewing angles (A-C) of iRFP transfected C2C12 myoprogenitor cells in a 450μm diameter, dECM coated PGSA scaffold 3 days after seeding. Actin-red, KI67-yellow, nuclei-blue.

Figure 4.

C2C12 migration, proliferation, and alignment within the PGSA scaffolds. A-F) confocal images of cells migrating and proliferating (KI67⁺ - green) in 450μm diameter channels without dECM coating (A-C) and with dECM coating (D-F) at days 1 (left column), 3 (middle column) and 7 (right column) after seeding. White dashed lines indicate the border of each scaffold. Cell proliferation was measured as %KI67+ cells at day 1 (G), day 3 (H), and day 7 (I) after seeding. Scaffolds with 300μm (red), 450μm (green), and 600μm (blue) diameter channels were assessed with and without dECM coating at all timepoints. Cell alignment (0° = parallel to channel, 90° = perpendicular to channel) was assessed at day 1 (J), day 3 (K), and day 7 (L) after seeding. Scale bar = 450μm. n = 3.

Figure 5.

C2C12 MHC expression at 12 days after seeding in scaffolds without dECM coating (A) and with dECM coating (B) of PGSA scaffolds with 600μm diameter channel. C) The area of MHC+ expression normalized by actin+ expression was used to assess myofiber maturation in 3D. ** = p< 0.01. Scale bar = 600μm. n = 6-12.

Figure 6.

Preliminary histological assessment of a 3D printed PGSA scaffold to treat a VML injury in a rat. A.) Top down SEM of the implanted scaffold demonstrating the long hollow channels present in the scaffold. B.) Representative image of a volumetric muscle loss injury in a rat. C.) and D.) Representative images of a Masson’s Trichrome stained histology of a PGSA scaffold that was not coated with dECM in the middle of the scaffold (2.5mm from the border). E.) and F.) Representative images of a Masson’s Trichrome stained histology of a PGSA scaffold that was coated with dECM in the middle of the scaffold (2.5mm from the border). Green arrows indicate the presence of muscle fascicle formation deep within the scaffold. Scale bar = 1mm.