A Bioprinting Process Supplemented with In Situ Electrical Stimulation Directly Induces Significant Myotube Formation and Myogenesis

Abstract

Electric field stimulation has supported biophysical and biological cues for tissue regeneration approaches to affect cell morphology, alignment, and even cellular phenotypes types. Here, an innovative bioprinting approach supported by in situ electrical stiumlation (E-printing) is used to fabricate a bioengineered skeletal muscle construct composed of human adipose stem cells and methacrylated decellularized extracellular matrix (dECM-Ma) derived from porcine muscle. To obtain highly ordered myofiber-like structures, various parameters of the printing process are optimized. The E-printed structure exhibits higher cell viability and fully aligned cytoskeleton than the conventionally printed cell-bearing structures, due to activation of voltage-gated ion channels that affect various signaling pathways. When using the E-printed structure, expression of myogenesis-related genes is upregulated by 1.9-2.5-fold higher than when using a dECM-Ma structure produced without electrical stimulation. Furthermore, when implanted into a rat model of volumetric muscle loss, the structure yields outstanding myogenesis relative to the conventionally bioprinted structure.

A bioprinted complex tissue model for myotendinous junction with biochemical and biophysical cues

“… 40 , 41 Those studies revealed that the hASCs containing muscle‐derived dECM were fully differentiated into the myogenic lineage due to the biochemical components, transforming growth factor β1, insulin‐like growth factor 1, and vascular endothelial growth factor, among others. 24 , 25 , 42 , 43 In addition, the tenogenic differentiation of hASCs could be clearly observed due to the increase in several integrin subunits and TGF‐β/SMAD pathways in the hASCs, compared to the collagen control. 42 …”

“…The attained genes were used to show whether the biochemical cue released from the bioinks and the alignment cue provided by the bioprinting process can encourage myogenic or tenogenic differentiation of hASCs cultured in growth medium alone, DMEM/low‐G, FBS, and penicillin–streptomycin. The results showed that the hASCs were well differentiated along the myogenic and tenogenic lineage due to the biochemical and biophysical cues, thus mimicking the microenvironmental conditions of the native muscle and tendon tissues, whereas the stem cells processed with the pure collagen bioink were differentiated into myogenic lineages within a low range due to biophysical cues, 24 but did not fully differentiate into tenogenic lineages.…”

“…Before preparing the muscle‐specific and tendon‐specific bioinks, skeletal muscle, and tendon tissues, isolated from the lower limbs of Yorkshire pigs (female; 10–15 months old), were decellularized using previously described protocols, described in Supplementary Information. 23 , 24 , 25 …”

“…Before preparing the muscle-specific and tendon-specific bioinks, skeletal muscle, and tendon tissues, isolated from the lower limbs of Yorkshire pigs (female; 10-15 months old), were decellularized using previously described protocols, described in Supplementary Information. [23][24][25] For the muscle bioink (M-bioink), mdECM was dissolved in 0.1 M acetic acid solution and mixed with 10Â enriched DMEM (Sigma-Aldrich) at a ratio of 1:1. Fibronectin (2.5 mg/ml) and hASCs (2 Â 10 7 cells/ml) were mixed with the neutralized mdECM hydrogel (3 wt%) to obtain the M-bioink.…”

In vivo bioprinting: Broadening the therapeutic horizon for tissue injuries

“…Dattatry et al revealed the relationship between different loading parameters and the rate of bone regeneration through deep learning [ 192 ], and guided the rehabilitation of patients undergoing bone defects repair. Similar strategies have been applied to other tissue's post-operative recovery, including cardiac muscles [ 193 , 194 ] and skeletal muscles [ [195] , [196] , [197] ] etc., by exerting appropriate stimulation parameters, such as the amplitude and frequency of myoelectric excitation. These investigations assist in the physiological functional demonstration of printed tissue scaffolds and confer the in vivo bioprinting veritable feasibility in clinical application.…”