Ether-Oxygen Containing Electrospun Microfibrous and Sub-Microfibrous Scaffolds Based on Poly(butylene 1,4-cyclohexanedicarboxylate) for Skeletal Muscle Tissue Engineering

Abstract

We report the study of novel biodegradable electrospun scaffolds from poly(butylene 1,4-cyclohexandicarboxylate-co-triethylene cyclohexanedicarboxylate) (P(BCE-co-TECE)) as support for in vitro and in vivo muscle tissue regeneration. We demonstrate that chemical composition, i.e., the amount of TECE co-units (constituted of polyethylene glycol-like moieties), and fibre morphology, i.e., aligned microfibrous or sub-microfibrous scaffolds, are crucial in determining the material biocompatibility. Indeed, the presence of ether linkages influences surface wettability, mechanical properties, hydrolytic degradation rate, and density of cell anchoring points of the studied materials. On the other hand, electrospun scaffolds improve cell adhesion, proliferation, and differentiation by favouring cell alignment along fibre direction (fibre morphology), also allowing for better cell infiltration and oxygen and nutrient diffusion (fibre size). Overall, C2C12 myogenic cells highly differentiated into mature myotubes when cultured on microfibres realised with the copolymer richest in TECE co-units (micro-P73 mat). Lastly, when transplanted in the tibialis anterior muscles of healthy, injured, or dystrophic mice, micro-P73 mat appeared highly vascularised, colonised by murine cells and perfectly integrated with host muscles, thus confirming the suitability of P(BCE-co-TECE) scaffolds as substrates for skeletal muscle tissue engineering.

Advances in biomaterials for skeletal muscle engineering and obstacles still to overcome

“…Therefore, imaging is considered the most efficient preliminary tool to assess cell alignment and myotube formation. A number markers can be used, but the most common myogenic marker for IF is myosin heavy chain (MHC) [ 63 , 68 , 70 , 76 , 83 , 85 , 88 , 89 , 97 , 101 ]. Once myotubes are labeled with MHC, myotubes can be imaged, and fusion index (percent differentiation) can be calculated by dividing the number of nuclei (stained with a nuclear stain) colocated in MHC + myotubes by the total number of nuclei in the image field.…”

“…Other myogenic markers that are commonly used to visualize and assess myotube formation, include desmin [ 82 , 103 ] and α-sarcomeric actin [ 82 , 85 , 88 , 89 , 101 ]. In addition, a cytoskeletal actin stain can be utilized to assess overall biomaterial-guided cell alignment [ 50 , 70 , 72 , 76 , 84 , 87 , 98 ].…”

“…Common markers of myogenic gene expression that are quantified via PCR include MHC [ 50 , 70 , 82 , 88 , 89 , 94 , 97 , 101 ], myogenic factor 5 (Myf5) [ 89 , 97 , 101 ], myogenin [ 50 , 70 , 82 , 84 , 88 , 89 , 97 , 101 ], MyoD [ 70 , 82 , 84 , 88 , 89 , 94 , 97 , 101 ], and sarcomeric actin [ 84 ]. Although not as common, western blots and ELISAs have been used to assess production of early-stage myogenic proteins [ 68 , 70 ], which can provide a clearer assessment of myotube maturation. Finally, formation of NMJs is often assessed through contractile response to electrical stimulation [ 63 , 89 , 97 ] or staining for AChrR [ 97 ], which can further indicate myotube maturation.…”

“…From there, histology and IF staining can be performed. Hematoxylin and eosin (H&E) paired with Masson's trichrome histological stain can be used to identify the types of cells that have infiltrated the explant and assess the extent of muscle fiber formation versus fibrosis at the defect site [ 61 , 63 , 70 , 91 , 98 ]. IF labeling can be used to evaluate myogenic, angiogenic, and neurogenic differentiation within the defect site.…”

“…In a similar way, Bloise et al. used poly(butylene1,4-cyclohexandicarboxylate- co -triethylene cyclohexanedicarboxylate) [P(BCE-co-TECE)] as a tunable synthetic material [ 70 ]. Increasing the concentration of ether linkages present in the copolymer impacted hydrophilicity, mechanical properties, and degradation kinetics.…”