After skeletal muscle damage, satellite cells move towards the injured area to assist in regeneration. However, these cells are rare as their numbers depend on the age and composition of the injured muscle. This regeneration method often results in scar tissue formation along with loss of function. Although several treatment methods have been investigated, no muscle replacement treatment currently exists. Tissue engineering attempts to create, repair, and/or replace damaged tissue by combining cells, biomaterials, and tissue-inducing substances such as growth factors. Electrospinning produces a non-woven scaffold out of biomaterials with fiber diameters ranging from nanometers to microns to create an extracellular-like matrix on which cells attach and proliferate. Our focus is on synthetic polymers, specifically poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), and poly(ε-caprolactone) (PCL). Skeletal muscle cells grown on electrospun scaffolds tend to elongate and fuse together thus, mimicking natural tissue. Electrical stimulation has been shown to increase the number of cells fused in culture and decreased the time needed in culture for cells to contract. Therefore, a conductive element was added to each scaffold, specifically polyaniline (PANi), gold nanoparticles (Au Nps), and multi-walled carbon nanotubes (MWCNT). Our project goal is to create a polymeric, conductive, and biocompatible scaffold for skeletal muscle regeneration.

PANi and PDLA were mixed to form the following solutions 24% (83% PDLA/17% PANi), 24% (80% PDLA/20% PANi), 22% (75%PDLA/25% PANi), 29% (83% PDLA/17% PANi), and 29% (80% PDLA/20% PANi). Only the 75/25 electrospun scaffold was conductive and had a calculated conductivity of 0.0437 S/cm. Scaffolds with larger amounts of PANi were unable to be electrospun. PDLA/PANi scaffolds were biocompatible as primary rat skeletal muscle cells cultured in vitro did attach. However, the scaffolds shrunk, degraded easily, and became brittle. Although PDLA/PANi scaffolds were easily manufactured, our results indicate that this polymer mixture is not appropriate for skeletal muscle scaffolds.

PLLA and Au Nps were electrospun together to form three composite scaffolds: 7% Au-PLLA, 13% Au-PLLA, and 21% Au-PLLA. These were compared to PLLA electrospun scaffolds. Measured scaffold conductivities were 0.008 ± 0.015 S/cm for PLLA, 0.053 ± 0.015 S/cm for 7% Au-PLLA, 0.076 ± 0.004 S/cm for 13% Au-PLLA, and 0.094 ± 0.037 S/cm for 21% Au-PLLA. It was determined via SEM with a Bruker energy dispersive x-ray spectrometer (EDS) that the Au Nps were not evenly distributed within the scaffolds as they had agglomerated. Rat primary muscle cells cultured on the three Au-PLLA scaffolds displayed low cellular activity. A second cell study was conducted to determine Au NPs toxicity. The results show that the Au Nps were not toxic to the cells and the low cellular activity may be a marker for myotube fusion. Elastic modulus and yield stress values for the three Au-PLLA scaffolds measured on days 0, 7, 14, 21, and 28 were much larger than skeletal muscle tissue. Due to the larger mechanical properties and Au Nps agglomeration, a third polymer and conductive element scaffold was investigated.

PCL was chosen as the new synthetic polymer as it had a lower elastic modulus and high elongation. MWCNT were chosen as the conductive element as they disperse well within PCL when acid functionalized. A third component was added to the scaffold to help it move similar to skeletal muscle. Ionic polymer gels (IPG) are hydrogels that respond to an external stimulus such as temperature, pH, light, and electric field. A poly(acrylic acid)/poly(vinyl alcohol) (PAA/PVA) mixture is one type of IGP that responds to an electric field. The scaffolds were coaxially electrospun so that each fiber had a PCL-MWCNT interior with a PAA/PVA sheath. These scaffolds were compared to electrospun PCL and PCL-MWCNT ones. The addition of MWCNT to the PCL did increase scaffold conductivity. Actuation of the PCL-MWCNT-PAA/PVA scaffold occurred when 15V and 20V were applied. All three scaffolds had rat primary skeletal muscle cells attached but, more multinucleated cells with actin interaction were seen on PCL-MWCNT-PAA/PVA scaffolds. Once again the mechanical properties were greater than muscle, but because of its ability to actuate we believe the PCL-MWCNT-PAA/PVA scaffold has potential as a bioartificial muscle.

Further characterization of the PCL-MWCNT-PAA/PVA included varying the ratios of PAA/PVA, smaller crosslinking times, and lower amounts of MWCNT. Four ratios, 83/17, 60/40, 50/50, and 40/60, were successfully coaxially electrospun with PCL and MWCNT. Overall, very few differences were seen between the four ratios in conductivity, cellular biocompatibility, actuation angular speed, and mechanical properties. The 83/17 and 40/60 ratios were chosen for additional investigation into mechanical properties and actuation. As the mechanical properties of the two types of scaffolds did not change significantly through degradation, lower PVA crosslinking times were tested. No significant effects were found and it was hypothesized that the evaporation of the solution played a role in the crosslinking process. The smaller MWCNT amount scaffolds also did not significantly affect the mechanical properties or the actuation angular speeds. More work into lowering the scaffold mechanical properties while increasing the actuation angular speed is necessary.

Though the mechanical properties for the 83/17 and 40/60 scaffolds remained high compared to skeletal muscle, we also looked for differences in in vivo biocompatibility. Both scaffolds were implanted into the right vastus lateralis muscle of Sprague-Dawley rats. The left vastus lateralis muscle served as either the PBS injected sham surgery or an unoperated control. Biocompatibility was evaluated using enzymes, creatine kinase (CK) and lactate dehydrogenase (LDH), levels, fibrosis formation, inflammation, scaffold cellular infiltration, and neovascularization on days 7, 14, 21, and 28 post-implantation. Fibrotic tissue formation, inflammation, and elevated CK and LDH levels were observed initially but responses decreased during the four week study. Cells infiltrated the scaffolds and histological staining showed more fibroblasts than myogenic cells initially but over time, the fibroblasts decreased and myogenic cells increased. Neovascularization of both scaffolds was also recorded. PCL-MWCNT-PAA/PVA scaffolds were determined to be biocompatible, but some differences between the two types were noted. The 83/17 scaffolds caused less of a response from the body compared to the 40/60 scaffolds and had more myogenic cells attached. However, the 40/60 scaffolds had a larger number of blood vessels running through the scaffold. In conclusion, we have successfully fabricated a polymeric, conductive, and biocompatible scaffold that can actuate for skeletal muscle tissue engineering. Although our results are promising, more work is necessary to continue developing and refining the scaffold.