# Preparation of an Oxygen-Releasing Capsule for Large-Sized Tissue Regeneration
## 1 Introduction  
Necrosis often occurs under hypoxic conditions and is a major limiting factor in tissue engineering, particularly for large tissue sections [1]. One major obstacle to the development of clinically appropriate three-dimensional tissue structures is the lack of adequate nutrients and oxygen supply to these tissues prior to their in vivo vascularization; unlike living tissues in vivo, these grafts are unable to maintain cell viability and function at the core of dense engineered tissues. Diffusion only supplies nutrients and oxygen to cells at the surface of these artificial tissues, resulting in necrotic cell death from a lack of oxygen [2; 3]. These results indicate that the limits of oxygen diffusion become increasingly important as the biomass and cell number in these artificially generated tissues increases [4]. In normal human biological systems, tissues are located within close proximity to blood vessels, which ensures their survival via the transportation of nutrients and oxygen. New tissues often require neovascularization which can only extend between 100 and 200 \(\upmu\)m from the closest capillary [5; 6]. This means that new tissues require sustained angiogenesis to develop beyond 100-200 \(\upmu\)m in depth. However, in engineered tissue grafts, the distance between the cells and the blood vessels within the graft is often a few millimeters or even centimeters, as the grafts begin to grow after implantation. Therefore, larger-sized implanted tissues need to undergo rapid vascularization from the new capillary network within the tissue in order to survive. After transplantation, the host's blood vessels typically invade tissues in response to signals secreted by the transplanted cells in response to hypoxia, forming new capillary and blood vessel networks. However, since this spontaneous intravascular growth is often limited to tens of micrometers per day, the time required to fully vascularize a few millimeters of implant is weeks [7; 8; 9]. Inadequate vascularization during this period can result in increased malnutrition and hypoxia in these tissues. In addition, hypoxic conditions commonly occur within the deeper regions of these constructs as a result of inadequate oxygen supply in vitro [10]. This means that large scale tissue regeneration is often incomplete and very difficult to accomplish [11; 12]. Therefore, it is important to develop novel smart materials that can support the oxygenation of these larger tissues in vitro and in vivo. Previous studies have used hydrogen peroxide as a source of oxygen, but the release of cations into solution following its catalysis could adversely affect some cell types [13; 14; 15; 16]. For this reason, our group assessed whether hydrogen peroxide could be used as a source of oxygen and has successfully demonstrated that it is possible to encapsulate hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), a small water molecule, in a biocompatible single polymer shell [17; 18; 19; 20; 21]. In brief, we used a dual emulsion solvent evaporation method to encapsulate hydrophilic low molecular weight drugs (such as hydrogen peroxide) in PLGA microspheres and tested these microspheres in vitro in a 3D alginate matrix. In order for the hydrogen peroxide to decompose of only oxygen and water, catalase was immobilized to alginate by binding the -NH\({}_{2}\) group of catalase to the -COOH group of alginate by EDC(N-(3-Dimethylaminopropyl)-N\"-ethylcarbodiimide hydrochloride)/NHS (N-Hydrooxysuccinimide) chemistry [22; 23]. According to previous studies, cell survival was 60-65% in hypoxic conditions of 1% O\({}_{2}\), but the rate increased up to 100% when oxygen-producing microspheres were added in the same conditions [15; 16]. Thus, it was confirmed that oxygen was released in stable condition from polymeric matrix and supported cell survival in hypoxic condition and ultimately resulted in successful tissue regeneration (Figure 1).