Abstract
Synthetic polymers scaffolds often need to be coated with extracellular matrix (ECM) proteins to improve cell adhesion. For cultivated meat applications, coating should be avoided since it is necessary to eliminate expensive and animal-derived components. As cellulose acetate nanofibers is a low-cost cellulose-derived material, that induces cell adhesion and proliferation, we investigated its use associated with a bioactive annatto extract, a food-dye and potential meat preservative, as scaffolds for cultivated meat. Here, the bioactive electrospun nanofibers were evaluated through morphological, mechanical and biological characterizations. The results revealed that the scaffolds were porous with no specific alignment with an average fiber diameter of 420±212 nm. Molecular analyzes revealed that in contrast to cellulose acetate scaffold, annatto-loaded cellulose acetate scaffold favor a proliferative state of C2C12 mouse skeletal myoblasts. SEM microscopy images suggests that the nanofiber substrates can sustain long-term culture of the cells, up to 28 days. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as a scaffold for cultivated meat and muscle tissue engineering.
Keywords: cultivated meat, cellulose acetate, annatto, nanofiber, scaffold, muscle tissue engineering
1. Introduction
Cultivated meat is an emerging technology that aims to obtain meat products through animal muscle tissue growth in a controlled environment, applying tissue engineering techniques [1]. The interest in cultivated meat (also known as in vitro meat, clean meat, cultured meat, cell-based meat or lab-grown meat) is motivated by several factors: potential increase in future demand for animal protein, concerns about the environmental impact of current meat production methods and occupation of land to raise animals, greenhouse gas emissions resulting from meat production and animal welfare, which has become an important issue for many consumers [2,3].
Tissue engineering techniques are used to provide a suitable and complex environment for cells or tissue to be grown on scaffolds, biomaterials capable of mimicking the extracellular matrix of the animals, which can support the development and growth of complex skeletal muscle tissues [4,5]. One stage in meat cultivation is controlled growth and maturation of myoblasts in scaffolds [6,7]. Scaffolds are a key component for cell agriculture, serving as an integrated support network in which cells expand and differentiate in ways that depend on anchorage [10]. A fundamental approach for cultivated meat is to extend the replicative capacity of skeletal muscle precursor cells for expansion on an industrial scale [8,9]. In this sense, scaffolds for cultivated meat must support proliferation and long-term stable culture of muscle cells under conditions that mimic the animal’s body. Therefore, additional research is needed to improve the design of scaffolds appropriate for myoblast cultivation in order to obtain muscle fibers.
Electrospun nanofiber scaffolds present an interesting alternative for muscle cells cultivation because they can better simulate typical muscle fibrous architecture. The nanoscale structure mimics the extracellular matrix morphology and induce great cellular attachment due to nanofiber high aspect ratio, scaffold porosity and surface-to-volume ratio [11]. Previous studies have demonstrated the importance of the nanoscale structure and its anisotropy in synthetic polymers for the development of 3D matrices [18,19]. Besides this, the nanofibers contribute to rapid diffusion of oxygen and nutrients, as well as cell infiltration. In addition, nanofiber scaffolds have the ability to induce cell alignment along the fibers that may induce muscle fiber maturation [12]. Although various nanofiber scaffolds have been developed for biomedical applications, few investigations have been done for applications in cultivated meat.
Cellulose-based biomaterials offer some important advantages over conventional synthetic materials and show great scientific promise [13]. Several studies have demonstrated that the hydrophilic hydroxyl moieties of the cellulose and specialized cellulose binding domains provides sites that favor adhesion and proliferation [14,15]. Cellulose acetate (CA) is a modified natural polymer that has good solubility and can be easily controlled morphologically [16,17]. CA nanofibers are very interesting in cultivated meat applications because, in addition to being a biocompatible material, their fabrication by the electrospinning process is relatively easy [14]. Besides, contrary to scaffolds composed of plant-based materials, they do not need to be coated with ECM proteins or chemical modification to improve cell adhesion [13, 27]. Santos et al. (2021) demonstrated that it is possible to grow fibroblasts on cellulose acetate nanofibers without the need for coating [26]. Thus, the application of cellulose acetate nanofibers in a cultured meat production process may be a more economical option compared to other synthetic polymers. Nevertheless, studies with cellulose acetate nanofibers for applications in tissue engineering and cultured meat are still scarce.
Another important point in food production is preservation, which nowadays is focused on the use of natural products [20]. Recently, essential oils extracted from plants have received a lot of attention due to their meat protection properties. Antimicrobial properties of plant essential oils are derived from some main bioactive components such as phenolic acids, terpenes, aldehydes, and flavonoids that are present in essential oils [21]. Various mechanisms such as changing the fatty acid profile and structure of cell membranes and increasing the cell permeability as well as affecting membrane proteins and inhibition of functional properties of the cell wall are effective in antimicrobial activity of essential oils. [22].
Annatto is the fruit of the annatto (Bixa orellanna L.) native to South America. Annatto seeds are considered antibiotics of medicinal character, acting as an anti-inflammatory for bruises and wounds, also having use in the cure of bronchitis and external burns. In addition, annatto has a long history of use in the food industry as a natural dye [23,24,25]. Our research group, produced scaffolds from cellulose acetate nanofibers loaded with annatto extract and demonstrated that the scaffold maintained the viability of mouse fibroblasts after 48 h of culture in addition to the cells attached, spread and colonized the nanofiber scaffold [26].
Here, we investigated morphological, mechanical and biological features of a bioactive nanofiber scaffolds previously developed by our research group, to evaluate their potential for application in cultivated meat. We found that cellulose acetate nanofibers loaded with annatto extract favored cell adhesion and improved cell viability and long-term cell proliferation.
2. Materials and methods