DESCRIPTION

Technical Field

The present invention relates to a live microalgae-loaded scaffold that aims to supply the oxygen photosynthetically required by injured or damaged living tissues during the regeneration process and to accelerate the treatment process, and to a method for producing said live microalgae-loaded tissue scaffold via 3-dimensional bioprinting.

The present invention possesses the potential of finding use in the field of healthcare as a biomaterial. The present invention may be used as a wound dressing or a similar biomaterial particularly for skin wounds in the discipline of tissue engineering. A wound dressing comprising live, and photosynthesizing algae, will be produced, wherein said wound dressing has the capability of supplying oxygen to wounds, thereby enabling wounds to heal quickly in cases involving non-healing wounds and hypoxia (oxygen deficiency).

Known State of the Art (Prior Art)

Although applications of tissue engineering offer a plethora of opportunities in the field of regenerative medicine, these applications suffer from certain fundamental issues, the most important of which is the incapability of supplying enough oxygen to 3-dimensional tissue cultures. Moreover, cellular apoptosis or microtissue necrosis may be observed following the implantation of bioengineered microtissues/cell-loaded biomaterials in in vivo applications due to oxygen deficiency (hypoxia). Various oxygen-releasing materials such as sodium percarbonate, magnesium peroxide, calcium peroxide, and hydrogen peroxide have been used as sources of oxygen in tissue scaffolds and microspheres with the aim of reducing hypoxia. However, these materials are cytotoxic for cells because of the decomposition of salt byproducts and inappropriate release profiles, which induce adverse effects on cell viability. Thus, microalgae, which are capable of producing oxygen photosynthetically, and therefore, can serve as an uninterrupted source of oxygen for cells, have recently risen to prominence as an innovative approach. This approach incorporates photosynthetically supplying oxygen to human cells via algae even if an external source of oxygen is unavailable. However, microalgae are required to be immobilized to scaffold by means of a suitable material and method. In recent years, the 3D bioprinting method, which is a layered production technique, has been used many times for immobilizing microalgae in specific structures with the aim of producing photosynthetic materials.

Loading microalgae to 3-dimensional structures by means of 3D bioprinting method and conducting investigations on this subject have become an increasingly studied subject in the scientific literature only in the recent years, and the information regarding the diversity of used materials and the efficacy thereof is quite limited. Alginate is one of the most common biopolymers used in three-dimensional bioprinting because it can be cross-linked readily in order to render 3 -dimensional structures more stable and durable following the bioprinting process. It is known to have been used previously with the intention of producing structures loaded with microalgae. Furthermore, alginate is not harmful to living cells since the gelation process occurs rapidly under significantly mild conditions. Methylcellulose has been used alongside alginate in order to increase viscosity and ensure 3-dimensional printability. However, in addition to being an agent that increases viscosity, methylcellulose has no positive effects on cells. Using collagen biopolymer instead of methylcellulose increases cell proliferation and improves cell attachment to tissue scaffold.

In the known state of the art, the matrix (tissue scaffold) has a double-layered structure, consists of collagen and silicon materials, collagen is located on the surface of the scaffold that comes into contact with the tissue, while silicon is located on the outer portion, however, this matrix is not individual- specific as it is a ready-made matrix. Since the dimensions of the matrix are standard, the production offers no flexibility. Microalgae cells are cultivated afterwards in the ready-made matrix, therefore, the microalgae cell concentration in the structure is either not constant or cannot be measured precisely. Brief Description and Purposes of the Invention

The present invention relates to a method for producing microalgae-loaded tissue scaffold for tissue engineering via three-dimensional bioprinting that meets all the requirements mentioned above, overcomes all disadvantages, and brings further advantages.

The present invention enables producing algae-loaded tissue scaffold of different shapes and sizes. For instance, depending on the sensitivity of the device, and the material that are being used, production of a structure may be performed in a range between 5x5 mm and 200x200 mm . Since the use of 3D printer was not present in the prior art, the produced matrix is dimensioned by being cut by people who employ the technique. However, the precision of the device used with the present invention enables performing dimensioning in a precise and accurate manner.

In the present invention, since the bio-ink, which is the main material of the tissue scaffold comprises algae cells, cell immobilization is ensured to occur homogeneously in the structure. Thus, cell concentration is rendered more controllable. In the prior art, microalgae cells are impregnated into the tissue scaffold after the tissue scaffold is produced. This method, however, does not enable homogeneously distributing microalgae cells to every region of the tissue scaffold. However, microalgae cells that are attempted to be impregnated into the tissue scaffold may slip out of the tissue scaffold during the process, thereby inducing losses and/or changes in cell concentration. In the present invention, microalgae cells are introduced into the biopolymer mixture in a specific concentration on the onset of the process, thereby obtaining a homogeneous mixture, and the microalgae cells are homogeneously distributed to every region of the tissue scaffold that is being printed by means of a 3D printer.

Description of the Illustrative Figures

Respective figures and their corresponding descriptions are given below in order to provide a better understanding of the subject of the present invention. Figure 1 illustrates the vectorial view of the structure having a grid-fill shape to be produced by means of 3D printing. (1 - Crosswise View. 2 - Top View. 3 - Side View.)

Figure 2 illustrates the top and crosswise vectorial views of the structures with different fill shapes and heights producible via 3D printing. (1 - Grid Fill. 2 - Hexagonal Fill. 3 - Triangular Fill. 4 - 3-Layered Structure. 5 - 6-Layered Structure. 6 - 12-Layered Structure.)

Figure 3 illustrates the crosswise vectorial view of the microalgae-loaded 6-layered structure with grid fill shape produced by means of 3D printing. (1 - After 1-day Incubation. 2 - After 4-day Incubation.)

Figure 4 illustrates the macroscopic and microscopic images taken after a 7-day incubation period of the Microalgae-loaded 10-layered structure with grid fill shape produced by means of 3D printing. (1 - Macroscopic Image from the top. 2 - 4X light microscope image. 3 - 10X light microscope image. 4 - 10X light microscope image.)

Figure 5 illustrates the vectorial view of the final product produced by means of 3D printing. (1 - Microalgae-loaded tissue scaffold produced by means of 3D Printing. 2 - Thermoplastic polyurethane tape produced by means of 3D Printing.)

Detailed Description of the Invention

The present invention relates to a method for producing microalgae-loaded tissue scaffold in a microextrusion 3D bioprinter by means of a homogeneous bio-ink mixture comprising alginate and collagen biopolymers and microalgae cells.

The method for producing microalgae-loaded tissue scaffold comprises the process steps of;

• Dissolving alginate and collagen biopolymers separately in distilled water at room temperature at pH 7 such that the concentration ratios are 1-5% (mass/volume) of alginate and 5-12% (mass/volume) of collagen; • Adding the collagen solution to the alginate solution under 50-60°C and at 200-300 rpm;

• Adding microalgae cells, which are prepared as l-10xl0 ⁶ cell/gram material, into the polymeric mixture;

• Attaching a mixing syringe that is filled with the obtained bio-ink into the three- dimensional printer, and performing the printing process of tissue scaffold having the dimensions of 5x5 mm ² - 200x200 mm ² ;

The present invention comprises producing live microalgae-loaded structures, which are intended to photosynthetically supply the oxygen required by injured or damaged living tissues during the regeneration process and to accelerate the treatment process, by means of the 3-dimensional bioprinting method. A microextrusion 3D bioprinter is used in the production of the materials. Microextrusion printers are preferred as these printers are costefficient and suitable for live cell printing. The bio-ink to be used in the 3D printer is composed of alginate and collagen polymers. Both polymers may be used easily by being dissolved in distilled water or in solutions with distilled water content. The content of the bio-ink used in the production of materials is composed of 1-5% (mass/volume) of alginate, 5-12% (mass/volume) collagen polymers, and live microalgae cells (l-10xl0 ⁶ cell/gram material). Alginate is preferred as it may readily be gelated ionically following the printing process.

Since collagen ensures cell adhesion and cell differentiation, and particularly, affects cellular activity of fibroblasts in tissue regeneration, it is a natural polymer that is essential for cells of mammals. Collagen features natural biocompatibility, reduced cytotoxicity and high water-retaining capabilities as it is one of the main constituents of the extracellular matrix (ECM). Therefore, it is a promising material used in 3D bioprinting. Moreover, collagen was determined to be more resistant to deformation than alginate in a study in which an alginate/collagen mixture was used. Considering collagen's viscosity at room temperature and changes in viscosity thereof depending on temperature, it is highly convenient for 3D bioprinting performed at room temperature and even enables printing solutions with high viscosity owing to reduced viscosity at increased temperatures. Because of such characteristics, collagen is used both as a viscosity-increasing agent instead of methylcellulose and to accelerate the process of tissue regeneration. The microalgae-loaded tissue scaffold to be obtained as a final product is to be located on a transparent thermoplastic polyurethane (TPU) tape that is also printed by using a 3D printer, and that may optionally have a porous or a non-porous structure. The flexible structure of thermoplastic polyurethane (TPU) tape enables applying the microalgae- loaded tissue scaffold onto the skin in an effective way.

In addition, collagen is the main element that ensures the printability thereof on the material at room temperature. Optimal printability is ensured by changing the concentration of collagen. Additionally, collagen can produce positive effects on the treatment process by improving adhesion and proliferation of living cells during the healing period of wounds. 3D printing is carried out under atmospheric conditions and at room temperature since the material comprises living cells, and due to collagen's viscosity, which changes depending on temperature. After the preparation of the material is complete, the material is filled into 10 ml syringes and prepared for use in printing by using blunt syringe tip caps of different sizes. For instance, in this study, 24G syringe tips were generally employed. Structures to be printed may have different shapes and sizes. In general, however, structures with square- and grid- shaped fills were produced as examples. Prints were printed out so as to feature a porous structure with the aim of increasing the oxygen uptake. Prints may be completely non-porous, may have pores with different geometrical shapes, may have different width x length data, and different height. A tape with adhesive capabilities is required to be used so that the material may be rendered usable on the surface of the skin. This tape may be produced from transparent thermoplastic polyurethane. The tape is quite suitable for being used on the skin in terms of both biocompatibility and flexibility. Moreover, the tape may be produced so as to have a non-porous structure or to have structure that features different porosity ratios. The transparent tape allows the algae to photosynthesize by enabling them to be exposed to both sunlight and to external artificial lighting available in the environment. In addition, the tissue scaffold may be adhered firmly to the surface of the skin by means of said transparent tape.

The material features high printability owing to its collagen content. Thus, substantially thick or thin structures may be obtained. Furthermore, exceedingly precise prints can be achieved on the x-y plane in the printed structures. Thus, various shapes may be produced so as to have different sizes in a broad range. Collagen is capable of providing a compatible medium both for microalgae and human cells. Thus, microalgae-loaded wound dressings or other similar products produced from this material will be able to provide effects that accelerate and facilitate the treatment process. The tissue scaffold may be readily adhered to the surface of the skin by means of the transparent tape made of thermoplastic polyurethane.