Functional 3‐D cardiac co‐culture model using bioactive chitosan nanofiber scaffolds

Abstract

The in vitro generation of a three-dimensional (3-D) myocardial tissue-like construct employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration, drug testing, and tissue engineering applications. Despite significant progress in this field, current cardiac tissue models are not yet able to stably maintain functional characteristics of cardiomyocytes for long-term culture and therapeutic purposes. The objective of this study was to fabricate bioactive 3-D chitosan nanofiber scaffolds using an electrospinning technique and exploring its potential for long-term cardiac function in the 3-D co-culture model. Chitosan is a natural polysaccharide biomaterial that is biocompatible, biodegradable, non-toxic, and cost effective. Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the extracellular matrix (ECM) in vivo. The chitosan fibers were coated with fibronectin via adsorption in order to enhance cellular adhesion to the fibers and migration into the interfibrous milieu. Ventricular cardiomyocytes were harvested from neonatal rats and studied in various culture conditions (i.e., mono- and co-cultures) for their viability and function. Cellular morphology and functionality were examined using immunofluorescent staining for alpha-sarcomeric actin (SM-actin) and gap junction protein, Connexin-43 (Cx43). Scanning electron microscopy (SEM) and light microscopy were used to investigate cellular morphology, spatial organization, and contractions. Calcium indicator was used to monitor calcium ion flux of beating cardiomyocytes. The results demonstrate that the chitosan nanofibers retained their cylindrical morphology in long-term cell cultures and exhibited good cellular attachment and spreading in the presence of adhesion molecule, fibronectin. Cardiomyocyte mono-cultures resulted in loss of cardiomyocyte polarity and islands of non-coherent contractions. However, the cardiomyocyte-fibroblast co-cultures resulted in polarized cardiomyocyte morphology and retained their morphology and function for long-term culture. The Cx43 expression in the fibroblast co-culture was higher than the cardiomyocytes mono-culture and endothelial cells co-culture. In addition, fibroblast co-cultures demonstrated synchronized contractions involving large tissue-like cellular networks. To our knowledge, this is the first attempt to test chitosan nanofiber scaffolds as a 3-D cardiac co-culture model. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiac structure and function. These studies will provide useful information to develop a strategy that allows us to generate engineered 3-D cardiac tissue constructs using biocompatible and biodegradable chitosan nanofiber scaffolds for many tissue engineering applications.

Strategies for improving the physiological relevance of human engineered tissues

“…Co-cultures lead to increased extracellular matrix deposition over mono-cultures, including fibronectin deposits in glomerular tissue [19] and collagen deposition and mineralization in bone tissue constructs [20]. Improved function has been demonstrated by co-cultures including beating cardiomyocytes, which increased fluctuations in intracellular calcium ion concentrations not achieved in mono-cultures [21]. Additionally, proper morphology has been observed in co-cultures for cardiomyocytes [21], endothelial cells [18], and epithelial cells [19] not observed when the cells were cultured individually.…”

“…Improved function has been demonstrated by co-cultures including beating cardiomyocytes, which increased fluctuations in intracellular calcium ion concentrations not achieved in mono-cultures [21]. Additionally, proper morphology has been observed in co-cultures for cardiomyocytes [21], endothelial cells [18], and epithelial cells [19] not observed when the cells were cultured individually. Improvements in vascular structures can also be achieved with co-cultures over mono-cultures [17, 18, 22].…”

Fibers for hearts: A critical review on electrospinning for cardiac tissue engineering

“…Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the ECM in vivo. Hussain et al studied ventricular CMs from neonatal rats in various culture conditions (i.e., mono-and co-cultures) for their viability and function by using electrospun chitosan-based nanofiber scaffolds [68]. The results demonstrated that the chitosan nanofibers retained their cylindrical morphology in long-term cell cultures and exhibited good cellular attachment and spreading in the presence of the adhesion molecule fibronectin.…”

“…The scaffolds are incubated in solutions containing the biological substance, which coats the surface of the fibers. Fibronectin is the most widely used biological substance for cardiac cells and has been coated on chitosan [68], PCL [153] [84], PLA [154] and PU [99]. Collagen type-I was also used on P(L-D,L)LA [111] and laminin on PLGA [121].…”

Nanofibrous Scaffolds as Promising Cell Carriers for Tissue Engineering

“…However, pure polymeric nanofibers are suitable mainly for soft tissue engineering, such as reconstruction and regeneration of blood vessels [13,22,23,31,32], myocardium [33,34], heart valves [35,36], skeletal muscle [37,38], skin [15, [39][40][41], tendon and ligament [42,43], intestine [44,45], tissues of the respiratory system, such as trachea and bronchi [46,47], components of urinary tract, such as bladder [48] and urethra [49], visceral organs, such as liver [50,51] or pancreas (pancreatic islets [52,53]), central nervous system, such as brain [6,54,55], spinal cord [56,57], optic system, such as optical nerve [58] and retina [59], and peripheral nervous system [17,60]. Nanofibrous scaffolds can be associated with another advanced technique in recent tissue engineering-controlled delivery of various types of stem cells, such as bone marrow mesenchymal stem cells [51,[61][62][63], adipose tissue-derived stem cells [64,65], neural tissue-derived stem cells [57], and induced pluripotent stem cells …”