Claims:
We Claim:

1. A formulation of biocompatible and degradable fabricating material for 3D bioprinting, the formulation comprising:
a) an alginate at a concentration range between 2%-4%;
b) an oxidized sodium alginate at a concentration of 10%; and
c) a gelatin at a concentration of 10%.

2. The composition as claimed in claim 1, wherein said AOAG hydrogel is biodegradable and biocompatible.

3. The composition as claimed in claim 1, wherein said AOAG hydrogel exhibits dual crosslinking mechanism.

4. The composition as claimed in claim 1, wherein said oxidized alginate of the AOAG hydrogel has faster degradation rate.

5. The composition as claimed in claim 1, wherein said AOAG hydrogel is partially crosslinked to modulate viscosity.
, Description:Preamble to the Description
[0001] The following specification describes the invention and the manner in which is to be performed:
DESCRIPTION OF THE INVENTION
Technical field of the invention
[0002] The present invention relates to a formulation of biocompatible and degradable fabricating material for 3-Dimensional (3D) bioprinting. More specifically, the invention relates to a formulation of AOAG hydrogel composition with desirable physical and chemical properties to be used in bio-fabrication for tissue and organ constructs.
Background of the invention
[0003] Tissue engineering and regenerative medicine aim to develop biomaterials, scaffolds, organs and implants. Biomaterials have been explored for their potential to support tissue repair and regeneration and to recreate the structure and/or function of tissue. The recent advancement in tissue engineering has led to regeneration of various organs and tissues including skin, heart kidney and liver.
[0004] The replacement of damaged organs and tissues with artificial grafts has been widely performed to cure end-stage organ failure. Since, a severe shortage of donated organs and complications that are associated after organ replacement, new strategies are needed for tissue repair and regeneration, including tissue engineering and cell therapy.
[0005] Bio-fabrication refers to the automated generation of biologically functional products with structural organization from living cells, bioactive molecules, biomaterials, cell aggregates, through bioprinting or bio-assembly and subsequent tissue maturation processes.
[0006] Bio-fabrication enables the fabrication of biological constructs with precise control over the positioning of cells and biomaterials. Bio-fabrication technology involves bioprinting and bio-assembly of constructs. These techniques deliver high level of biomimicry by recreating complexity of tissues and organs.
[0007] Development of novel biomaterial-based strategies that mimic native tissues and organs has led to advancement of tissue engineering. These biomaterials are capable of initiating cells to sense their local environment through cell-cell and cell-extracellular matrix (ECM) contacts.
[0008] 3D-bioprinting corresponds to layer-by-layer precise positioning of biomaterials, biochemicals and living cells with spatial control of functional components. The substrate is typically planar solid surfaces such as Petri dishes, glass slides, or wells of culture plates, also, nonplanar, nonsolid, and flexible substrates. Bioprinting and bio-assembly of biomaterials must allow modulation of cell-cell and cell-extracellular matrix (ECM) interactions.
[0009] Various printing and crosslinking strategies have been employed to print fidelity and resolution. 3D printing techniques frequently used for cell behaviour studies and tissue repair are inkjet-based bioprinting, pressure-assisted bioprinting, laser-assisted bioprinting and stereolithography.
[0010] Pressure-assisted bioprinting is based on extrusion principle to create desirable 3D constructs. It is one of the most commonly used techniques. The biomaterials such as solution, paste or dispersion are extruded by coordinating the motion of pneumatic pressure, plunger or screw-based extrusion in the form of continuous filament through a nozzle onto a stationary substrate. Biomaterials including polymers and ceramics, proteins and biomolecules, living cells, and growth factors as well as their hybrid structures can be printed.
[0011] This technique involves layer-by-layer application to complete 3D-patterns. The pressure-assisted bioprinting employs room temperature processing, direct incorporation of cells and homogenous distribution of cells. The mechanical integrity of the extruded structures is controlled through thermal or chemical cross-linking, or multi-material channel approaches postdeposition.
[0012] Biomaterials used for 3D-bioprinting are categorized into hydrogel, metallic, ceramic, polymeric and composite materials. The development of natural bio-inks such as collagen, gelatin, alginate etc. is desirable due to their inherent biocompatibility. Certain parameters such as viscosity, pore-size and interconnectivity influence the encapsulated cells. The rheological properties of the biomaterial, extrusion temperature, nozzle type used, and applied pressure are the critical parameters that affect the physical and biological characteristics of the printed construct.
[0013] Biocompatibility is a significant parameter of biomaterials. The cells involved in the regeneration process are influenced by the macro- and microarchitecture of the constructs. The biomaterial must be cyto-compatible and support cell-growth, proliferation, adhesion and migration. In addition, the biomaterial should be safe and do not cause any immunogenic reaction.
[0014] Multiple parameters such as pore size, shape, volume directly affect the behavior of cells in the scaffold. Porosity and interconnectivity play important roles in the growth of surrounding tissues. The pores of the biomaterial allow oxygen and nutrients to be transported into the interior and eliminate the waste generated by cellular metabolism.
[0015] Hydrogels are widely utilized in biomedical fields. Hydrogels possess a three-dimensional network structure that swells from the absorption of large amounts of water. Hydrogels represent one of the most common scaffolding materials in tissue engineering. Due to majority of water content, hydrogels provide similar in-vivo environment. Hydrogels are prepared using natural, synthetic, and composite biomaterials with high biocompatibility.
[0016] Hydrogels have many advantageous properties such as cytocompatibility, tissue mimetic water content, support of cell migration and tissue integration, sustained release of growth factors, and controllable physical properties. Hydrogels used in tissue engineering applications are predominantly based on natural derived polymers, including alginate, gelatin, collagen, chitosan, silk fibroin, fibrin, and hyaluronic acid.
[0017] Natural hydrogels have inherent excellent cytocompatibility, low toxicity, and susceptibility to enzymatic degradation. Natural hydrogels, derived from polysaccharide or proteins, offer inherent bioactivity except for agarose and alginate and display a chemical and structural resemblance to ECM. However, the synthetic hydrogels often lack response to biologic stimuli and requires modification to induce responses.
[0018] Hydrogels alone provides low mechanical strength, difficult to handle, needs high sterilized conditions and is expensive for treatment. The crosslinking of hydrogels enhances mechanical properties of these hydrogels.
[0019] During the printing process, maintaining suitable viscosity plays a major role and is challenging. While printing, chances of deformation and collapse are higher for materials with low viscosity and the materials with high viscosity blocks the nozzles while printing. The pressure and viscosity of the biomaterial play an important during the process of printing as they determine the extrusion output.
[0020] The crosslinking of hydrogels is essential for providing support and mechanical strength to the printable substance. Crosslinking is categorized into physical and chemical crosslinking. Chemical crosslinking has been widely used, which include thermal- or photochemical-induced radical polymerization (RP). Incomplete chemical crosslinking often leads to oxygen inhibition effect because of which mechanical performance of the hydrogel declines significantly. Heat induced deterioration is also commonly observed among chemical crosslinked hydrogels.
[0021] Physical crosslinking relies on noncovalent interactions such as hydrogen bonding, ionic bonding, metal coordination and so on. Most of printable physical crosslinked hydrogels have unsatisfactory mechanical properties such as bad stretchable structure and lack of efficient energy dissipation mechanism.
[0022] The Patent Application No. WO2011US29832 entitled “Biodegradable Scaffolds” discloses a composition of biodegradable polymer matrix. The biodegradable reinforcing particle consists of porous oxide particles and porous semi-conductor particles. The composition of polymer matrix comprises unsaturated biodegradable polymer. The hydrogen porogen comprises alginate, gelatin and fibrin. The composition contains combination of therapeutics, imaging agents, anti-inflammatory agents, antibiotics, proteins, platelet rich plasma, cells, degradation inducers of porous particles. The biodegradable polymers in the composition are poly (lactic-co- glycolic acid) (PLGA) and agarose which are dispersed in the matrix. The active agents present in the composition are associated with biodegradable polymer matrix. The biodegradable polymer matrix is used as scaffolds for treating bone defects.
[0023] The Patent Application No. EP19990923091 entitled “Biodegradable Sustained-Release Alginate Gels” discloses sustained-release formulations using biodegradable alginate delayed gels or and methods thereof. The composition comprises a sustained-release delayed gel with a biodegradable anionic polysaccharide; a biologically active agent; and at least one bound polyvalent metal ion, wherein the biologically active agent comprises a protein and wherein said biodegradable anionic polysaccharide is an alginate ester which is ionically crosslinked to provide an injectable, biodegradable, biocompatible alginate ester hydrogel. The composition contains ions of manganese, strontium, iron, magnesium, calcium, barium, copper, aluminium and zinc. The composition consists proteins such as leptin, G-CSF, SCF, BDNF, GDNF, NT3, GM-CSF, IL-Ira, IL2, TNF-bp, MGDF, OPG, interferons, erythropoietin, KFG, insulin and analogs.
[0024] The Patent Application No. CN2015138004 entitled “Biomedical composite hydrogel, and preparation method and applications thereof” discloses a a biomedical composite hydrogel. The composition comprises of hyaluronate sodium, sodium alginate, BDO glycidyl ether crosslinking agent and sodium hydroxide solvent. The hydrogel is applied in the field of cosmetic raw material or auxiliary material, tissue filling material, drug carrier or drug ingredient, wound or skin auxiliary material, tissue engineering scaffold material, anti-adhesion isolation material, and materials used for beautifying, wrinkle removing, and moisture retention. The preparation method of biomedical gel involves taking proportional quantity of hyaluronate sodium in the sodium hydroxide solution and dissolved completely. The hyaluronic mass concentration was made to be 1%-8%; In hyaluronic acid solution, sodium alginate solution was added and was agitated thoroughly; the BDO glycidyl ether crosslinking agent was added to make BDO glycidyl ether crosslinking agent volume concentration range be 0.1% ~ 2%; Solutions were placed in 40 ~ 60-degree Celsius and Homogeneous phase was made. 3 ~ 8h, then mixed solution drying at room temperature 2 ~ 5d, treat that moisture evaporation rate is 85 ~ 95%, obtain hyaluronic acid-sodium alginate transparent membrane; After residual alkali lye is removed in diaphragm cleaning, being placed on mass concentration is 0.2% ~ 12%CaCl 2 and soaked for 8 ~ 32 hours in solution, obtain hyaluronic acid-sodium alginate plural gel. Mechanical properties of the biomedical composite hydrogel are improved greatly without influencing gel biocompatibility and degradation properties; and biosecurity and degradability of the using amount of the crosslinking agent are ensured in single crosslinked membrane preparation.
[0025] The Patent Application No. CN202110231563 entitled “Medical hydrogel composition, medical hydrogel and preparation method of medical hydrogel” provides a medical hydrogel composition, medical hydrogel and a preparation method of the medical hydrogel. The hydrogel composition comprises hyaluronic acid or a derivative thereof modified with aldehyde groups; water-soluble chitosan or a derivative thereof and natural substances containing amino and alginate. Components of medical hydrogel composition are water-soluble natural macromolecules, the biocompatibility is good, the medical hydrogel composition can be well compounded with cells, degradation products are safe and non-toxic, and potential risks of anaphylactic reaction or toxic reaction do not exist.
[0026] The Patent Application No. CN20111371866 entitled “Oxidized sodium alginate/gelatin degradable hydrogel and preparation method thereof” relates to oxidized sodium alginate/gelatin degradable hydrogel and a preparation method thereof. The preparation method comprises the following steps: Oxidation treatment on the sodium alginate with an oxidizing agent so as to obtain oxidized sodium alginate and preparing an oxidized sodium alginate solution; Blending and defoaming the human body-absorbable fibres, a gelatin solution and the oxidized sodium alginate solution, standing an obtained mixture, cross-linking the mixture with a CaCl2 solution so as to obtain the hydrogel, and carrying out cutting processing, disinfection and packaging. The degradable hydrogel exhibits good compatibility with human bodies, a high degradation rate and the effects of stopping bleeding and accelerating healing of wounds. The preparation method has the advantages of short flow, simple operation, low cost, environmental friendliness and a high economic benefit.
[0027] The Patent Application No. WO2018US43643 entitled “HYDROGEL FOR TISSUE ENGINEERING AND BIOPRINTING” relates to a composition of a hydrogel that includes a plurality biodegradable natural polymer macromers crosslinked with a first agent and optionally a plurality of cells dispersed in the crosslinked macromers, the microgels are capable of being crosslinked with a second agent that is different than the first crosslinking agent. The hydrogel consists natural polymer macromers are ionically cross linkable.
[0028] The Patent Application No. TW20130142302 entitled “A hydrogel material and method of preparing the same” discloses a degradable cross-linked hydrogel material made of gelatin and gelatin[gamma]-poly glutamate salt, and the preparation method thereof. The preparation method includes pre-treatment of proanthocyanin and gelatin mixed polyglutamate, the treatment includes dispersion of proanthocyanin in an acidic solution, and preparation of a gelatin mixed polyglutamate solution with pH range of 1-7. The gelatin mixed polyglutamate solution are uniformly mixed and subjected to a crosslinking reaction at a non-specific temperature. The composition is biocompatible, biodegradable, and used for implantation into a human body and applied in tissue engineering.
[0029] Despite the significant effort in developing and improving process principles and demonstrating their feasibility for viable cell printing, several challenges need to be addressed to accelerate the successful clinical translation of bioprinting technology. There exists a lack of software design tools to engineer tissue and organ systems. The biomaterials used in 3D-printing are limited and expensive. Cell-free scaffolds have been printed in a wide variety of biomaterials, whereas development of newer printable biomaterials to accommodate cells are limited. After encapsulation of cells, the cells secrete extracellular matrix (ECM), until then the biomaterial must be able to provide similar environment. The development of hydrogels which maintain cell viability, activity and physical shape in the final printed construct plays a crucial role. The processability for bioprinting can be improved by improving viscosity and gelation of the hydrogel. The physical crosslinking is adopted over chemical crosslinking but the mechanical performance is often unreliable. The mechanism of dual crosslinking mechanism helps in improving mechanical properties of the hydrogel such as high strength, stretchability and toughness.
Summary of the invention
[0030] The invention overcomes the drawbacks of the existing prior arts by dual crosslinking mechanism and degradable properties. The formulation of the present invention modulates viscosity of hydrogel at various temperatures.
[0031] The invention discloses a formulation of Alginate, Oxidized Sodium Alginate and Gelatin (AOAG) hydrogel. The formulation comprises alginate at a concentration of 2%-4%, Oxidized sodium alginate at a concentration of 10% and gelatin at a concentration of 10%.
[0032] Alginate is a natural polymer with polymeric chains of mannuronic acid and glucuronic acid. Alginate polymers form a stiff gel by crosslinking with divalent cations such as calcium ions. Calcium crosslinked alginate has been known to be good wound dressers because of its low immunogenicity, biocompatibility, water retention and biodegradability property.
[0033] Gelatin is obtained from the hydrolysis of collagen. Gelatin exhibits good biocompatible and biodegradable property. It contains cell attachment ligands that help in the cell growth, proliferation and migration of cells around the matrix. Gelatin also forms a gel through thermal gelation at lower temperatures.
[0034] Composite hydrogels are made up of alginate and polymers. They contain cell attachment ligands that are used for tissue construct. The function of alginate in the composite hydrogel is to maintain the viscosity by partial crosslinking with low concentration calcium ions.
[0035] In the present invention, sodium alginate is oxidized using sodium periodate. The oxidized sodium alginate is mixed with gelatin to form mixed alginate hydrogel. Sodium alginate is added to the mixed alginate hydrogel to obtain AOAG hydrogel.
[0036] Bio-ink is prepared by mixing sterile hydrogel with DMEM (Dulbecco's Modified Eagle Medium) media and FBS (Fetal Bovine Serum) and suspended in calcium chloride to increase viscosity. The bio-ink is printed using 25G – 27G dispensing needle on a sterile platform containing sterile 200 – 400 millimolar calcium chloride bath for further crosslinking of the printed construct to maintain the structural integrity. The cell viability at various concentrations of calcium chloride was studied by MTT assay. The results indicated that 70% - 90% cell-viability was observed with 15 mins of incubation in medium containing calcium.
[0037] The present invention discloses a formulation of AOAG hydrogel. Since the composition consists natural polymers combined, the AOAG hydrogel exhibits inherent biocompatible and biodegradable properties. The AOAG hydrogel involves dual crosslinking mechanism which aids in modulation of viscosity. The AOAG hydrogel when inoculated with cell lines shows greater percentage of cell viability.
Brief description of the drawings
[0038] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings.
[0039] FIG 1 tabulates the composition of the AOAG hydrogel according to an embodiment of the invention.
[0040] FIG 2 illustrates flowchart for the preparation of AOAG hydrogel.
[0041] FIG 3 illustrates the periodate oxidation of sodium alginate.
[0042] FIG 4 illustrates the crosslinking of oxidized alginate and gelatin through Schiff’s base reaction.
[0043] FIG 5 illustrates the dual crosslinked hydrogel with crosslinking between oxidized alginate and gelatin via Schiff’s base reaction and divalent ionic crosslink between alginate-alginate and alginate-oxidized alginate.
[0044] FIG 6 illustrates flowchart for the process of preparation of the bio-ink.
[0045] FIG 7 tabulates the printing characteristics of the AOAG hydrogel composition.
[0046] FIG 8 illustrates 3D printed AOAG hydrogel with 2%-4% alginate, 10% oxidized alginate and 10% gelatin.
[0047] FIG 9 tabulates the composition of the AOAG hydrogel with 2% alginate, 5% oxidized alginate and 10% gelatin according to an embodiment of the invention.
[0048] FIG 10 tabulates the printing characteristics of the AOAG hydrogel with 2% alginate, 5% oxidized alginate and 10% gelatin .
[0049] FIG 11 illustrates 3D printed AOAG hydrogel with 2% alginate, 5% oxidized alginate and 10% gelatin.
[0050] FIG 12 tabulates the composition of the AOAG hydrogel with 4% alginate, 5% oxidized alginate and 10% gelatin according to an embodiment of the invention.
[0051] FIG 13 tabulates the printing characteristics of the AOAG hydrogel with 4% alginate, 5% oxidized alginate and 10% gelatin .
[0052] FIG 14 illustrates 3D printed AOAG hydrogel with 4% alginate, 5% oxidized alginate and 10% gelatin.
[0053] FIG 15 illustrates MTT assay results.
[0054] FIG 16 illustrates the graph of viscosity of AOAG hydrogel at different temperatures.
Detailed description of the invention
[0055] In order to make the matter of the invention clear and concise, the following definitions are provided for specific terms used in the following description.

[0056] The term “Biocompatibility” refers to the ability of a material to perform with an appropriate host response in a specific situation.

[0057] The term “3-D Bioprinting” refers to the three-dimensional printing of biological tissue and organs through the layering of living cells.

[0058] The term “Bio-ink” refers to any natural or synthetic polymer selected for its biocompatible components and favorable rheological properties.

[0059] The term “Extracellular matrix (ECM)” refers to a large network of proteins and other molecules that surround, support, and give structure to cells and tissues in the body.

[0060] The invention discloses a formulation of biocompatible and degradable fabricating material for 3D bioprinting applications. Specifically, the invention discloses a formulation of hydrogel with alginate, oxidized sodium and gelatin with improved biocompatibility and degradability.

[0061] A variety of materials have been developed to mimic specific cell and tissue niches. Hydrogels are the most commonly explored materials for fabricating the complex 3D cellular microenvironments. Hydrogels are biomaterials with good biocompatibility, nonimmunogenic and are widely used for bioprinting.
[0062] Hydrogels are divided into naturally derived hydrogels and synthetic hydrogels. Naturally derived hydrogels are advantageous because of their inherent biocompatibility, biodegradability and safety, including chitosan, alginate, hyaluronan, collagen and agarose, obtained from various renewable resources such as animal, plant, algae, and microorganisms.
[0063] Natural hydrogel polymers are often derived from native ECM components such as collagen, fibrin, hyaluronic acid (HA), also, they have been created from nonmammalian sources such as algae (alginate) and seaweed (agarose). Natural hydrogel polymer materials have the advantages of high biocompatibility and degradability through natural enzymatic or chemical processes. In addition, materials derived from mammalian ECM contain natural ligands that allow for cellular adhesions.
[0064] Mode of polymerization has critical action on resulting hydrogel structure and mechanics. Naturally derived polymers have their own inherent methods of gelation, such as thermal (agarose, collagen, and gelatin), pH (collagen and gelatin), ionic (alginate), or enzyme-based (fibrin) cross-linking. For these biomaterials, the mechanics of the resulting gel can be loosely controlled by adjusting the prepolymer density. Crosslinking of polymer chains aids in increasing mechanical strength and stability of the hydrogel. Natural polymer hydrogels have poor mechanical strength. Dual crosslinking increases overall strength of the hydrogel.
[0065] Alginate is a natural biopolymer consisting of ß-D-mannuronic acid and a-L-guluronic acid extracted from brown algae. Alginate crosslinks via ionic, covalent and thermal processes to obtain alginate hydrogels. Alginates undergo ionotropic gelation in water solution in the presence of divalent cations such as Ca2+, Mg2+, or Ba2+. These hydrogels are used in drug release and wound healing. Crosslinking alginate hydrogels improve in vivo synthesis of ECM. They facilitate adhesion, proliferation and growth of cells.
[0066] Gelatin is obtained by thermal denaturation of animal collagen. Gelatin hydrogels are biodegradable and release non-toxic degradation products. Gelatin hydrogels have applications in reconstructive surgery, drug delivery, wound healing and tissue regeneration. Gelatin helps in improving viscoelastic properties of the hydrogel. Due to poor thermal stability, gelatin hydrogels require need biomaterials to form composite hydrogels.
[0067] FIG 1 tabulates the composition of the AOAG hydrogel according to an embodiment of the invention. The formulation of the present invention comprises alginate at a concentration range of 2%-4%, oxidized sodium alginate at a concentration of 10% and gelatin at a concentration of 10%.
[0068] FIG 2 illustrates flowchart for the process of preparation of AOAG hydrogel according to an embodiment of the invention. The process (200) starts with a step (201) by preparing oxidized sodium alginate by periodate oxidation which starts with dissolving alginate in distilled water with 5% w/v mixture. At step (202), sodium periodate is added in the ratio of 0.8:1 to the glucuronate group present in sodium alginate dissolved in distilled water and continuing reaction for 6 hours. At step (203), ethylene glycol is added to stop the reaction. At step (204), the solution is dialysed with 12KDa dialysis membrane with distilled water with regular water change. The dialysed solution is freeze-dried to obtain oxidized alginate powder. At step (205), the oxidized alginate is dissolved in 5% - 10% w/v of 0.1M Phosphate Buffered Saline (PBS) () to which 2% - 4% w/v sodium alginate is added to make a mix alginate blend. At step (206), gelatin (bloom strength 225G) is prepared by dissolving gelatin in 6% - 10% w/v of distilled water at 50°C. At step (207), equal volume of gelatin is added to mixed alginate slowly with continuous stirring to facilitate the covalent crosslinking between oxidized alginate and gelatin. Finally, at step (208), the resultantant AOAG hydrogel is heated to 80°C followed with filter sterilization with 0.22 micron filter under aseptic conondition to remove micrbial contents.

[0069] FIG 3 illustrates the periodate oxidation of sodium alginate. where, the carbon-carbon carbon bond in the urinate residue of the alginate chain is cleaved with the addition of the di-aldehyde group. Sodium periodate is added to sodium alginate and dissolved in distilled water and the reaction is allowed to continue in dark for 6 hours. The reaction is stopped by adding ethylene. Obtained alginate solution is freezed dried to obtain oxidized alginate powder.
[0070] FIG 4 illustrates the crosslinking of oxidized alginate and gelatin through Schiff’s base reaction. The di-aldehyde group crosslinks with the amino group through Schiff’s base reaction to form an amide bond. Oxidized alginate crosslinks with gelatin to form a viscous hydrogel that is suitable for bioprinting. The oxidixed alginate was dissolved in 0.1M phosphate buffer saline to which sodium alginate was added to make alginate blend. Gelatin was prepared by dissolving gelatin in distilled water.
[0071] FIG 5 illustrates the dual crosslinked hydrogel with crosslinking between oxidized alginate and gelatin via Schiff’s base reaction and divalent ionic crosslink between alginate-alginate and alginate-oxidized alginate. Oxidized alginate and alginate ionically crosslinks with divalent cations such as calcium ions to form a gel. To the mixed alginate blend, equal volume of gelatin was added slowly with continuous stirring to facilitate covalent crosslinking.

[0072] After the preparation of AOAG hydrogel, bio-ink is prepared. The bio-ink is prepared by adding nutrient medium and supplements. The prepared bio-ink is added inoculated with cell culture and incubated for growth of cells.

[0073] FIG 6 illustrates flowchart for the process of preparation of bio-ink. The process (600) of preparation of bio-ink starts with a step (601) by mixing the sterile AOAG hydrogel with 10X nutrient medium containing growth supplements (Dulbecco’s Modified Eagle Medium (DMEM/ Ham F12)) and serum (Fetal Bovine Serum (FBS)) in the ratio of 8:1:1 (AOAG hydrogel : 10X media : FBS). At step (602), the resultant bio-ink is used to resuspend the cell pellet and then 20 – 50 milliMolar sterile calcium chloride is added to partially crosslink the AOAG hydrogel for increasing viscosity. At step (603), the bio-ink is loaded into the syringe cartridge and loaded into a bioprinter with dispenser temperature set to 20°C for 10 – 15 minutes to allow thermal gelling of gelatin to futher increase viscosity for optimal printing condition. At step (604), the cell laden bio-ink wasis printed 25G – 27G dispensing needle on a sterile platform with containing sterile 200 – 400 millimolar calcium chloride bath for further crosslinking of the printed construct to maintain the structural integrity. At step (605), the printed 3D tissue construct is allowed to crosslink for 5 – 10 minutes and then the the tissue construct is washed with sterile PBS thoroughly to remove excess calcium. At step (606), the tissue construct is incubated in growth medium for futher maturation and development of the tissue.

[0074] FIG 7 tabulates the printing characteristics of the formulation. The bio-ink is printed using nozzle of 0.26mm (25G nozzle) with fill density of 100%, print speed of 7mm/s and an input flow of 1500%.

[0075] FIG 8 illustrates 3D printed AOAG hydrogel with 2%-4% alginate, 10% oxidized alginate and 10% gelatin. The structure of the AOAG hydrogel can be varied by varying the concentration of the components
[0076] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.

Example 1: Composition of AOAG hydrogel with 2% alginate, 5% oxidized alginate and 10% gelatin
[0077] The composition of AOAG hydrogel comprises alginate at a concentration of 2%, oxidized sodium alginate at a concentration of 5% and gelatin at a concentration of 10%.
[0078] FIG 9 tabulates a composition of AOAG hydrogel with 2% alginate, 5% oxidized alginate and 10% gelatin according to an embodiment of the invention.
[0079] The prepared bio-ink is printed using printing characteristic such as nozzle diamneter of 0.26mm (25G nozzle) along with fill density of 20%, print speed of 7mm/s and an input flow of 2000%.
[0080] FIG 10 tabulates the printing characteristics of the AOAG hydrogel with 2% alginate and 5% oxidized alginate and 10% gelatin.
[0081] FIG 11 illustrates 3D printed AOAG hydrogel with 2% alginate, 5% oxidized alginate and 10% gelatin. The structure of the AOAG hydrogel can be varied by varying the concentration of the components.
Example 2: Composition of AOAG hydrogel with 4% alginate 5% oxidized alginate andd 10% gelatin
[0082] The composition of AOAG hydrogel comprises alginate at a concentration of 4%, oxidized sodium alginate at a concentration of 5% and gelatin at a concentration of 10%.
[0083] FIG 12 tabulates a composition of AOAG hydrogel with 4% alginate, 5% oxidized alginate and 10% gelatin according to an embodiment of the invention.
[0084] The prepared bio-ink is printed using printing characteristic such as nozzle diamneter of 0.26mm (25G nozzle) along with fill density of 20%, print speed of 7mm/s and an input flow of 2200%.
[0085] FIG 13 tabulates the printing characteristics of the AOAG hydrogel with 4% alginate, 5% oxidized alginate and 10% gelatin.
[0086] FIG 14 illustrates 3D printed AOAG hydrogel with 4% alginate, 5% oxidized alginate and 10% gelatin. The structure of the AOAG hydrogel can be varied by varying the concentration of the components.
Example 3: MTT Assaay of fibroblast cell lines to determine cell viability
[0087] MTT assay was performed on fibroblast cell lines which were incubated in the bio-ink according to an embodiment of the invention. Cell viability was tested for various calcium chloride concentrations against time. The AOAG bio-ink was printed on a higher concentration calcium bath (200-500 milliMolar) for further crosslinking of alginate for obtaining a 3D construct with good structural integrity. The crosslinking with higher concentration calcium was done for a time of 10 – 15 mins to avoid loss of cell viability.

[0088] FIG 15 illustrates MTT assay results. The viability of cells at a high concentration of calcium is not decreased (approximately 70 - 90% viability with 15 mins of incubation in medium containing calcium).

Example 4: Analysis of Viscosity of AOAG hydrogel at different temperatues

[0089] Viscosity of the AOAG hydrogel was measured at various temperatures such as 200 C, 280 C, and 370 C. Modualtion of viscosity is one of the main limiations of various AOAG hydrogels which be overcome by the current invention.
[0090] FIG 16 illustrates the graph of viscosity of AOAG hydrogel at various temperatures. The graph depicts constant viscosity throughout the change in shear rate.
[0091] The formulation of the present invention discloses biocompatible and degradable fabricating material for 3D bioprinting. The formulation comprises alginate, sodium alginate and oxidized sodium alginate hydrogel. The formulation uses natural polymers which possess inherent biocompatibility and biodegradability along with nonimmunogenic properties. Dual crosslinking mechanism is observed in the present invention. The dual crosslinking helps in modulating the viscosity of the AOAG hydrogel which is a crucial parameter for bioprinting. Oxidized alginate present in the AOAG hydrogel allows faster degradation of the 3D construct to facilitate the cells to secrete their extracellular matrix. The formulation also exhibits greater cell viability.