# Improvement of Mechanical Properties of 3D Bioprinted Structures through Cellular Overgrowth
## 1 Introduction  
3D bioprinting is a modern field of additive manufacturing classified at the intersection of biomedical science, biology, mechanical engineering, and materials science [1]. The technology is based on the manufacturing of three-dimensional structures through precise layer-by-layer deposition of a bioink that is a combination of living cells, biomaterials, and bioactive molecules [2, 3, 4]. Three-dimensional bioprinting techniques, thanks to the fabrication of structures that mimic native tissues, can find a variety of applications in the treatment of injuries, congenital defects, whole organ disorders, and cancer therapy. Today, 3D bioprinting technologies are the basis for in vitro drug testing and the creation of physiological tissue models of disease [5; 6; 7]. The dynamic development of 3D bioprinting technology started at the beginning of the 21 st century, and, since then, intensive research work has been carried out on the possibility of printing various types of tissue in an effort to reduce the number of autologous transplants [3]. We can distinguish several 3D bioprinting technologies, which include extrusion-based bioprinting, droplet bioprinting, stereolithography-based bioprinting, and laser-assisted bioprinting [1; 2; 3; 4; 5]. Due to its high versatility, low cost, low material consumption, ability to use high densities of biological material, and high cell viability directly after the manufacturing process using bioinks that have the property of thinning by shearing, the most widely used 3D bioprinting technology is direct extrusion-based bioprinting [1; 3; 8]. The extrusion bioprinting process is carried out by extruding bioink through a nozzle using a mechanical or pneumatic drive. Continuous microfilaments are created during printing, which are deposited on the substrate and shaped into printouts with the desired geometry. The final printout structure is influenced by extrusion temperature, nozzle diameter, extrusion pressure, nozzle and extrusion speed, and path spacing between paths [1]. The numerous advantages of 3D extrusion bioprinting enable the creation of structures with compositional and structural complexity [3; 8]. In the processes of 3D extrusion bioprinting, bioink compositions play a key role. Generally, hydrogels are one of the most commonly used materials for their production [5]. Their common use in 3D extrusion bioprinting techniques is dictated by their unique properties, among which the most important from the point of view of promoting the development of cellular functions in 3D in vitro cultures are their ability to retain large amounts of water and the possibility of modifying their rigidity and mechanical properties to mimic the natural extracellular matrix [9; 10; 11]. In addition, among the advantages of using hydrogel bioinks based on hydrogel materials in 3D bioprinting techniques, one should also emphasize their high permeability to oxygen, nutrients, and other water-soluble compounds, as well as their ability to allow free migration of the cells in any direction, unlike polymer scaffoldings [12]. The compatibility of hydrogel materials with various cell types (including fibroblasts, chondrocytes, hepatocytes, smooth muscle cells, adipocytes, and stem cells) and tissues creates the potential to create biomimetic structures that mimic the natural physiological environment, thereby increasing this way the range of their applications in the field of regenerative medicine [11; 13]. Among the key parameters during the 3D bioprinting process of hydrogel bioinks, we can mention printability, mechanical properties, biodegradability, the presence of modifiable functional groups on the surface, and the way that the structures cross-link [14].