Field of Invention

The invention relates to the development of a concentrated hydrogel/hydrocolloid structure formed as a result of gelation of quince seeds with water to be used as a bioink in 3 dimensional (3D) bioprinting technique. The obtained bioink is used as tissue scaffold in tissue engineering and 3D cell culture studies.

Current Status of the Technique Related to the Invention

Mucilage, gum or hydrogel-like materials obtained from quince seeds have been recently used as tissue scaffolds in the field of tissue engineering. There are various kinds of extraction and production methods in different studies, and these methods cause serious inconsistencies in terms of obtaining the material, especially its use in tissue engineering. In addition, the use of toxic solvents for extraction can also be an obstacle for its use in tissue engineering.

When the quince seed hydrogel is obtained by water extraction at high temperatures, the number of process steps increases, and an additional solvent is included in the alcohol treatment. In other extractions, solvents known to be toxic to cells are used. In addition, obtaining powdered form of mucilage by lyophilization complicates the production stages.

Bioinks are fluid materials used in the 3D bioprinting method, usually containing biomaterials and/or cells. For a bioink to be suitable for printing, it is important to be fluid, easily printed on the surface, and be shaped, as well as biocompatible. Some polymers used as bioinks are not suitable for cell bioprinting because they are melted at high temperatures or require the usage of toxic solvents. However, in the 3D bioprinting method, it is extremely important that the cells can be encapsulated and printed in the developed bioink. For this purpose, hydrogels have started to be developed as bioinks. In order for a hydrogel to be used as a bioink in the 3D bioprinting technique, it should be suitable for printing in the form of a continuous filament without sticking to the printing head, and for this, it should have low adhesion and surface tension. Besides, it must be suitable for fast and non-toxic cross-linking methodology in order to retain its shape after printing and be suitable for cell printing. In addition to all these, it is also important that it is biocompatible, economical, and easily accessible, has quick gelation ability, and does not dissolve in the cell culture medium. However, it is observed that the materials used in tissue engineering do not provide the desired properties together in terms of mechanical, physical or biocompatibility properties to be developed as bioink. For this reason, there is a need for natural, biocompatible, easy to obtain and apply, innovative and cost-effective bioinks to be used in the bioprinting technique.

In the patent document numbered JPS59161402 (A), the invention relates to a method for producing quince seed gum. It was stated that the powder obtained by precipitation of the gum obtained from the quince seed using hydrophilic organic solvent such as methanol, ethanol or isopropyl alcohol was obtained with high whiteness and good quality. In this study, the gum obtained from the quince seed was not used in hydrogel form and as a bioink.

In the patent document numbered JPS59163312 (A), the invention is about a method designed to obtain high-quality powder extract from quince seeds. For this purpose, the extract obtained from the seeds at 60°C or higher temperature was concentrated, then the precipitate obtained by mixing with isopropyl alcohol was dried and characterized by pulverizing. In this study, powder extract was obtained from quince seed and was not used in hydrogel form.

In the patent document with number W02020086941 (Al), the invention is directly related to the development of mammalian, plant, microbial-derived or synthetic hydrogels and biocompatible polysaccharide gums with thickening properties in hybrid form for use as bioinks in 3D bioprinting technique. In the study, gelatin, silk, and chitosan were mixed with xanthan gum, alginate-xanthan gum, and glucomannan, respectively, and a bioink was developed, and characterized for use in tissue engineering and 3D cell culture studies. The hydrogels used in this invention have no similarity with the hydrogel obtained from quince seeds in terms of content.

The patent document numbered CN111032794 (A), includes the addition of any gelling agent such as agarose, agar gum, acacia, methylcellulose, quince seed, guar gum, xanthan gum to the mixture in order to eliminate the gelation problem encountered in the ink mixture which contains at least one of the urethane or urea group polymers. In addition, the ink will be used for image production, it has not been developed for 3D bioprinting and there are no biocompatibility studies. In the patent document number WO2018/071639, the invention is related to the development of undenatured collagen as a bioink, and then printing it with 3D printing technique for use in cell culture studies. In the study, undenatured collagen was linked with cross-linking agents. In the aforementioned invention, it is not a question to use hydrogels obtained from quince seeds as bioinks in the 3D bioprinting technique.

Patent document WO2019/173637 relates to the development of a bioink containing nanocellulose, water, alginate, and ionic crosslinking agents. The developed bioink was printed with a 3D extrusion based bioprinter and then evaluated in terms of rheology, morphology, and biocompatibility. In the aforementioned invention, it is not a question to use hydrogels obtained from quince seeds as bioinks in the 3D bioprinting technique.

In the study conducted by Maroufi and Ghorbani, curcumin was added to the hydrogel obtained by mixing chitosan and quince seed hydrogel, and an injectable hybrid material was developed for tissue engineering applications. In the study, quince seeds were treated with ethanol and dried, then mixed with distilled water at 35°C for 12 hours and finally centrifuged to obtain a hydrogel. The resulting hydrogel was not used as a bioink but was added to the chitosan solution. The study reported here is completely different from the method of the invention in terms of hydrogel production and use. (Maroufi, L. Y, & Ghorbani, M. (2021). Injectable chitosan-quince seed gum hydrogels encapsulated with curcumin loaded-halloysite nanotubes designed for tissue engineering application. International Journal of Biological Macromolecules .)

In the study by Aghmiuni et al, tissue scaffolds with a porous structure were produced and characterized by lyophilization method after the hydrogel obtained from quince seeds was mixed with PEG and PCL. In this study, the hydrogel was obtained as a result of incubation of quince seeds in deionized water for 24 hours, but it does not include a study on the use of hydrogel as a bioink. (Aghmiuni, A. I., Keshel, S. H., Sefat, F., & Khiyavi, A. A. (2020). Quince seed mucilage-based scaffold as a smart biological substrate to mimic mechanobiological behavior of skin and promote fibroblasts proliferation and h-ASCs differentiation into keratinocytes. International journal of biological macromolecules, 142, 668-679.)

In another study reported in 2020, the powder obtained by lyophilization method after 24 hours incubation of quince seeds with distilled water was dissolved in 1/3 volume of acetic acid/formic acid solution and 2% by weight was obtained. Then, it was mixed with the prepared PCL solution in different volumes and obtained by electrospinning technique and characterized. The hydrogel production method used in the study is quite different from the invention method and does not include any research on the use of the obtained hydrogel as a bioink. (Allafchian, A., Jalali, S. A. H., Mousavi, S. E., & Hosseini, S. S. (2020). Preparation of cell culture scaffolds using polycaprolactone/quince seed mucilage. International journal of biological macromolecules, 155, 1270-1276.)

In another study reported by §im§ek et al, mucilage obtained from quince seeds was dissolved in ultrapure water and then molded and obtained as tissue scaffold. Quince seed hydrogel was not used as a bioink in the study, it was processed by completely different technique than the one proposed invention and used in tissue engineering. (§im§ek, E., Karaca, B., & Arslan, Y. E. (2020). Bioengineered three-dimensional physical constructs from quince seed mucilage for human adipose-derived mesenchymal stem cells. Journal of Bioactive and Compatible Polymers, 35(3), 240-253.)

In another study reported in 2021, mucilage extraction from quince seed was performed with the help of a rotary evaporator and then lyophilized. Then, 3D porous silicon-quince seed mucilage (Si-QSM) cryogel was obtained by microwave-assisted sol-gel reaction and characterized for use in bone tissue engineering. The scaffold production method in this study is completely different from the invention, and the reported study is not a about the use of mucilage as a bioink. (Yilmaz, H. D., Cengiz, U., Arslan, Y. E., Kiran, F., & Ceylan, A. (2021). From a plant secretion to the promising bone grafts: Cryogels of silicon-integrated quince seed mucilage by microwave-assisted sol-gel reaction. Journal of Bioscience and Bioengineering .)

In the study by Ghafourian et al, quince seeds were mixed with distilled water at 50-60°C for 30 minutes, then cooled and filtered. It was then dried in an oven at 40°C and obtained in powder form, and then mixed with water and used in gel form. The gel obtained in the study was not developed as a bioink. In this respect, it is completely different from the proposed invention. (Ghafourian, M., Tamri, P, & Hemmati, A. (2015). Enhancement of human skin fibroblasts proliferation as a result of treating with quince seed mucilage. Jundishapur journal of natural pharmaceutical products, 10(\)j

In the study reported by Hemmati and Mohammadian, mucilage obtained by mixing quince seeds with distilled water and heating was examined in terms of its wound healing properties. In this study, the mucilage differs from the proposed invention in terms of obtaining the material, as it does not undergo any further processing after extraction. Also, reported material was not developed as a bioink. (Hemmati, A. A., & Mohammadian, F. (2000). An investigation into the effects of mucilage of quince seeds on wound healing in rabbit. Journal of herbs, spices & medicinal plants, 7(4), 41-46.)

In the study by Chinga-Carrasco et al, bioink was developed using cellulose nanofibrils obtained from sugarcane pulp and printed by 3D printing method. Although the invention seems partially similar to bioink with the presence of xylan in the bioink content, the content of the overall bioink formula obtained from cellulose nanofibril and the hydrogel bioink obtained from quince seed are completely different from each other. (Chinga-Carrasco, G., Ehman, N. V., Filgueira, D., Johansson, J., Vallejos, M. E., Felissia, F. E., ... & Area, M. C. (2019). Bagasse — A major agro-industrial residue as potential resource for nanocellulose inks for 3D printing of wound dressing devices. Additive Manufacturing, 28, 267-274.)

Although there are some examples where hydrogels obtained from quince seeds are used in tissue engineering studies in the known state of the technique mentioned in detail above, the use of glucuronoxylan-based hydrogel obtained from quince seed to be used as a bioink in 3D bioprinting technique is not included in the technique. In the state of the art, the necessity of using high temperature or toxic solutions to obtain hydrogel/hydrocolloid from quince seed prevents the obtained hydrogel from having high biocompatibility in tissue engineering applications. Processing the hydrogel obtained from quince seeds by methods such as lyophilization, electrospinning and molding limits the targeted structure, size or pore density of the produced scaffold.

Brief Description and Objectives of the Invention

The present invention relates to the production of glucuronoxylan-based hydrogel/hydrocolloid from quince seed for use as a bioink in the 3D bioprinting method and the development of bioink, which meets the above-mentioned requirements, eliminates all disadvantages, and brings some additional advantages.

The fact that the hydrogel obtained in the invention method is suitable for 3D bioprinting and that the bioprinting parameters are optimized for different concentrations makes it easier for the produced scaffold to have the desired shape, size, and pore density. In addition, a hydrogel bioink obtained from quince seed by the method of the invention has features such as being suitable for printing in the form of a continuous filament without sticking to the printing head, having low adhesion and surface tension, keeping its shape post printing, and being suitable for fast and non-toxic cross-linking methodology. In addition, being biocompatible and economical are other advantages it has.

The bioink, which was developed by utilizing the biocompatibility feature of the quince seed hydrogel with the invention, is used in 3D bioprinting by encapsulating the cells.

In the invention, hydrogel is obtained by incubating the quince seed shell in distilled water at room temperature for 24 hours. In the state of the art, hydrogel is obtained from quince seeds at high temperature or using toxic solvents in 12 hours or less. The hydrogel obtained at the end of this process is precipitated and dried, and the obtained extract is then dissolved in water and used in hydrogel form. However, since precipitation and drying processes are also included in the process, the process becomes longer and more complex, and toxic solvents are added to the process. In the method of the invention, the hydrogel is obtained in 24 hours at room temperature by using distilled water. The most important advantage of this method is that it does not require the use of high temperatures or toxic solvents.

After incubation, the resulting hydrogel was mixed with EDC-NHS crosslinking agents and developed for use as a bioink in a 3D bioprinter. One of the important advantages of the stated bioink is that the crosslinking methodology of the hydrogel is less toxic compared to other methods. It is another important advantage of the invention that the glucuronoxylan-based quince seed hydrogel is bioprinted 15 minutes after treatment with the crosslinking agent.

Owing to the developed bioink, tissue scaffolds can be designed in the desired shape and height, and printed with the 3D bioprinting technique. Another important advantage is the production of tissue scaffolds whose pore structure and size can be easily controlled.

Since the developed bioink is not a melt-printed polymer or a material prepared with toxic solvents, it is suitable for the cells to be encapsulated and printed in the bioink. In this context, another important advantage of the developed concentrated bioink is that it is suitable for printing in the range of 5.0-6.5 psi pressure values and does not affect cell viability.

In conclusion, the proposed Glucuronoxylan-based quince seed hydrogel is a natural, biocompatible, available, cost-effective, and innovative alternative to bioinks currently in commercial use. Definitions of Figures Explaining the Invention

The figures and related explanations necessary for a better understanding of the invention subject are as follows.

Figure 1: Bioprinted images of each concentration with optimized values (40 mg/mL, 2psi; 50mg/mL, 4psi; 60mg/mL, 6psi; lOOmg/mL, 6.8psi)

Detailed Description of the Invention

In the detailed explanation, the method of obtaining glucuronoxylan-based hydrogel/hydrocolloid from quince seed and developing bioink for 3D bioprinting method is explained only for better understanding of the subject and without any limitation.

A production method of bioink comprises following steps;

• Obtaining hydrogel by incubating the quince seed shell in distilled water at room temperature for 24-48 hours,

• Addition of 0.4M l-Ethyl-3 -(3 -Dimethylaminopropyl) Carbodiimide (EDC) and 0.1M N-hydroxysuccinimide (NHS) crosslinking agents in 1 : 1 volume into ImL hydrogel,

• Mixing until a homogeneous mixture is obtained.!. Obtaining Hydrogel from Quince Seed

First, the seeds are isolated from the quince fruit, the brown shell outside the core is broken off and gelled at room temperature (25°C) for 24 hours by mixing only with water. In this step, the concentration is calculated as mg/mL, and the hydrogel with a concentration of lOOmg/mL optimized for use in 3D bioprinting is prepared at room temperature using 500 mg of quince seed shell and 5mL of ultrapure water. There is no need to use a magnetic stirrer for the gelation process to take place. Owing to the gelling feature of the glucuronoxylan contained in the quince seed, a hydrogel form has been obtained and the required viscosity has been achieved to be used as a bioink.

2. Crosslinking with EDC-NHS and Obtaining Bioink

After 24 hours, the obtained hydrogel is separated from the shells with the help of a ImL syringe. Then, 0.4M lOOpl l-Ethyl-3 -(3 -Dimethylaminopropyl) Carbodiimide (EDC) and 0.1M lOOpl N-hydroxysuccinimide (NHS) crosslinking agents are added into ImL hydrogel to ensure that the bioprinted structure retains its shape after printing and to prevent it from dissolving in the cell culture medium. At this stage, crosslinking takes place with the help of EDC and NHS crosslinking agents between the carboxylic acid (-COOH) and amine (-NH2) groups in the chemical structure of the hydrogel obtained from the quince seed shell. EDC- NHS cross-linking methodology was preferred because it is less toxic than alternatives such as glutaraldehyde in the literature and does not affect cell viability in cell culture applications.

3. 3D Bioprinting

The obtained bioink and crosslinking agents are mixed as described in step 2, charged into the syringe of the 3D bioprinter, and printed with optimized printing parameters. Bioink with lOOmg/mL concentration is printed in the pressure range of 5.0-6.5psi with 25G (0.25mm) printing head, lOmm/s printing speed, 0.1cm layer height and 5% infill density. The pressure value required for 3D bioprinting of the bioink obtained from quince seed hydrogel is suitable for printing by encapsulating the cells into the bioink and it is below the pressure values that reduce cell viability (Nair, K., Gandhi, M., Khalil, S., Yan, K. C., Marcolongo, M., Barbee, K., & Sun, W. (2009). Characterization of cell viability during bioprinting processes. Biotechnology Journal: Healthcare Nutrition Technology, 4(8), 1168-1177.). Since the EDC- NHS cross-linking mechanism takes place within 15 minutes, the scaffolds are obtained in cross-linked form 15 minutes after the 3D bioprinting process.

Table 1. Optimized parameters for extrusion-based 3D bioprinting of quince seed hydrogel developed as a bioink