Aligned ovine diaphragmatic myoblasts overexpressing human connexin-43 seeded on poly (l-lactic acid) scaffolds for potential use in cardiac regeneration

Abstract

Diaphragmatic myoblasts (DMs) are precursors of type-1 muscle cells displaying high exhaustion threshold on account that they contract and relax 20 times/min over a lifespan, making them potentially useful in cardiac regeneration strategies. Besides, it has been shown that biomaterials for stem cell delivery improve cell retention and viability in the target organ. In the present study, we aimed at developing a novel approach based on the use of poly (L-lactic acid) (PLLA) scaffolds seeded with DMs overexpressing connexin-43 (cx43), a gap junction protein that promotes inter-cell connectivity. DMs isolated from ovine diaphragm biopsies were characterized by immunohistochemistry and ability to differentiate into myotubes (MTs) and transduced with a lentiviral vector encoding cx43. After confirming cx43 expression (RT-qPCR and Western blot) and its effect on inter-cell connectivity (fluorescence recovery after photobleaching), DMs were grown on fiber-aligned or random PLLA scaffolds. DMs were successfully isolated and characterized. Cx43 mRNA and protein were overexpressed and favored inter-cell connectivity. Alignment of the scaffold fibers not only aligned but also elongated the cells, increasing the contact surface between them. This novel approach is feasible and combines the advantages of bioresorbable scaffolds as delivery method and a cell type that on account of its features may be suitable for cardiac regeneration. Future studies on animal models of myocardial infarction are needed to establish its usefulness on scar reduction and cardiac function.

Aligned Carbon Nanotubes Reduce Hypertrophic Scar via Regulating Cell Behavior

“…Aligned collagen hydrogels, collagen fibers, nanotopography scaffolds, and natural–synthetic polyblend nanofibers are reported to direct multiple cell growth and improve nerve regeneration. − Aligned polymers and collagen fibers could affect extracellular matrix texture and promote the quiescent keratocyte phenotype, − which hold the promise for scar prevention. Fiber aligned poly- l -lactic acid scaffolds have also been proven potent in cardiac regeneration and scar prevention . These studies suggest that aligned structure materials with proper fiber diameter and interval are capable of inducing oriented cell growth.…”

“…Fiber aligned poly-Llactic acid scaffolds have also been proven potent in cardiac regeneration and scar prevention. 19 These studies suggest that aligned structure materials with proper fiber diameter and interval are capable of inducing oriented cell growth. However, the synthesis procedures of mentioned materials might be relatively complicated and costly, which may hinder their efforts toward mass production.…”

Effect of poly (l-lactic acid) scaffolds seeded with aligned diaphragmatic myoblasts overexpressing connexin-43 on infarct size and ventricular function in sheep with acute coronary occlusion

“…Obtention, processing and transduction of ovine DM have been reported previously [11]. Briefly, a sterile right minithoracotomy was performed in the 6th intercostal space under general anesthesia to obtain a diaphragm biopsy ($2 cm 2 ).…”

“…As cardiac synchronous contraction depends on the propagation of the action potentials through the syncytium, it is necessary to stimulate inter-cellular connections of the cells to be implanted. We have recently shown that DM modified to overexpress cx43 and seeded on aligned nanofibers of poly (l-lactic acid) (PLLA) that mimic the topography of cells in skeletal muscle increased inter-cellular connectivity in vitro [11]. However, whether or not this approach affords cardioprotection in vivo remains to be demonstrated.…”

Applications of poly(lactic acid) in bone tissue engineering: A review article

“…Some synthetic polymers are hydrophobic, whereas others are hydrophilic properties 19 . Owing to their ease of synthesis and modification, synthetic polymers have attracted much attention, 20 among which PLA, an FDA‐approved polymer, 21 is a promising candidate for biomedical applications. In this short review, we summarize the synthesis and application of PLA in bone tissue engineering, based on PubMed, ScienceDirect, and Scopus databases, using standard keywords including “bone tissue engineering,” “osteogenesis,” “PLA,” “bone regeneration,” and “poly(lactic acid).” Due to the PLA synthesis process and properties, its can be considered the most promising linear aliphatic biopolymer in medical applications (Figure 2).…”