# Improvement of Mechanical Properties of 3D Bioprinted Structures through Cellular Overgrowth
## 1 Introduction
Hydrogel materials used in 3D bioprinting processes can be classified according to their origin (synthetic, natural, or hybrid/modified), ionic charge (non-ionic, cationic, anionic, or ampholytic), biodegradability (biodegradable or non-biodegradable materials), physical properties (smart or conventional) and type of cross-linking (physical, chemical or radiation cross-linking) [15; 16]. In recent years, often used on their own or in combination, hydrogel bioink compositions have frequently used materials of natural origin, among which we can distinguish gelatin, sodium alginate, collagen, silk, hyaluronic acid, chitosan, fibrinogen, and agarose [17; 18; 19; 20; 21]. Control of cellular interactions in natural hydrogel materials can occur by changing the used polymers' physical, chemical, biochemical, or physiological properties [21]. One of the most important requirements for materials intended for the 3D bioprinting process is the correct choice of bioink composition, which is characterized by suitable structural and mechanical properties; the composition must also protects cells from damage during the printout manufacturing process and provide them with an environment favorable for controlling their phenotype and maintaining their vital functions [22]. Due to their high biocompatibility, non-immunogenicity, ability to optimize rheological properties, and bioactivity, sodium alginate-based polymer solutions are one of the most widely used hydrogel compositions [9; 23; 24]. Sodium alginate is a naturally occurring anionic, hydrophilic polysaccharide that is obtained from brown algae or seaweed. It consists of \(\alpha\)-L-galuronic acid (G-block) and \(\beta\)-D-manuronic acid (M-block), linked by 1-4 glycosidic bonds. The cross-linking process of alginate hydrogels most often occurs using divalent cations, among which calcium ions are generally used to build ionic bridges between G-units to form a thermostable three-dimensional gel network with an \"egg-box\" structure [17; 25; 26; 27; 28; 29]. Thanks to the control of the degree of cross-linking, alginate concentration, and its molecular weight, it is possible to modify the rigidity of the hydrogel because the higher the content of G units, the higher the rigidity and porosity of the polymer matrix, which are key parameters for the viability and proliferation of cells contained inside the material [17; 25; 30; 31]. Therefore, alginate's physical properties can potentially be tailored directly to three-dimensional cell growth and differentiation in both in vitro and in vivo models [22]. The non-toxicity and high biocompatibility of alginate make it a hydrogel that is now widely used in various industrial fields, including tissue engineering and in the pharmaceutical and food industries. Some of the disadvantages of alginate are its slow and uncontrolled degradation in mammalian organisms and low cell bioadhesion, which generate the need for modification with adhesive ligands with the purpose of obtaining suitable biological properties [29; 32; 33; 34]. Using alginate hydrogels in combination with other biomaterials of natural origin, such as gelatin or collagen, improves its bioactivity and modifies its physical and mechanical properties dedicated to the application requirements of 3D bioprinted structures [35; 36]. Gelatin is a natural, soluble protein obtained by partial hydrolysis of collagen derived from the body's connective tissues. We can distinguish more than a dozen types of gelatin, depending on the source of collagen and hydrolytic treatment. Due to the specific properties of gelatin, such as biodegradability, biocompatibility, non-toxicity, and low antigenicity, it has been approved by the Food and Drug Administration as a biomaterial suitable for clinical applications. Gelatin of porcine, bovine, or fish origin find their use in a wide range of regenerative medicine applications.