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