Plant-based scaffolds and tunable hydrogels for bioprinting and methods thereof

Abstract

The invention relates to a bioink formulation that is prepared using a cellulose biomaterial that is derived from plant cells. The cellulose biomaterial is a scaffold such as an extracellular matrix derived from plant cells such as live plant cells, dead plant cells, decellularized plant cells or a plant cell-line that could be used to develop a biostable, biodegradable, bioprintable and tunable bioinkthat could be used for a variety of applications including bioprinting. A method of purifying the cellulose biomaterial by an acidic maceration and decelullarization technique is also provided along with a method of preparing the tunable bioink from the derived plant biomaterial.

Description

PLANT-BASED SCAFFOLDS AND TUNABLE HYDROGELS FOR BIOPRINTING AND METHODS THEREOFFIELD OF THE INVENTION

[0001] The invention relates to tunable bioink hydrogels comprising purified plant biomaterial. More specifically, the invention relates to bioink formulations that can be used for bioprinting applications and methods of preparing the formulations from plant-purified biomaterials.BACKGROUND OF THE INVENTION

[0002] In recent years, decellularized plants have gained popularity as bio-inspired and inexpensive materials in tissue engineering to support mammalian cell proliferation. These scaffolds possess 3D cellulose structures, are highly biocompatible, and can be utilized as guidance cues for cell proliferation and adhesion. More recently, green bioprinting and incorporating live plant cell suspension has initiated a new path to produce fully naturally derived renewable bio-scaffolds with promising potential. In this regard, various plant models have been used i.e.: basil, micro algae and for food industry the lettuce leaf cells for pectin based bioink, in addition to carrot. One of most recent studies used UV polymerizable hydrogel after extruding Oryza sativa (rice cells) and Zinnia elegans plant. In addition, decellularized Tobacco tissue, BY2 cells and rice created a three-dimensional environment for cell growth. Thus, cultured plant cells are an amazing candidate to custom matrices for biomedical applications.

[0003] 3D matrices, also known as scaffolds, are essential for supporting the growth of cells by providing physical support. What makes these matrices truly remarkable is their ability to enable 3D cell culture, which more closely mimics the in vivo microenvironment compared to 2D cell culture systems. This is particularly relevant in tissue engineering and drug discovery, where 3D cell culture system provides an ideal approach for developing scaffoldbased constructs. These constructs have immense potential for disease modeling, regenerative medicine, and studying the stem cell niche. By using 3D cell culture systems, researchers have made significant advancements in these fields, providing invaluable insights into cellular processes, and paving the way for future innovation.

[0004] Biomaterials are used in biomedical applications such as drug delivery, tissue engineering, and medical implants. It is critical that these materials elicit an appropriate immune response from the host to be considered biocompatible. To make this determination, the material’s immunogenicity, carcinogenicity, and mineralization must be taken into account. Biomaterials can be sourced from natural or synthetic origins, each with their own advantages and disadvantages. Natural biomaterials are derived from natural sources such as animal tissues,plant tissues, and microorganisms. Collagen is a widely used natural biomaterial due to its biocompatibility and biodegradability. It is commonly used in tissue engineering for applications such as promoting nerve regeneration. Silk fibroin is another natural biomaterial that has been used in medical implants such as an injectable hydrogel. Chitosan, a natural polymer derived from chitin, is currently being explored as a biomaterial for use in skin regeneration and cartilage repair.

[0005] Tissue engineering is a multidisciplinary field that combines principles from regenerative medicine and biology to create biological substitutes that assist in the repair and restoration of organ function. The process of creating functional tissue structures begins with the production of scaffolds that mimic the extracellular matrix (ECM). The extracellular matrix (ECM) plays a crucial role in providing structural characteristics and creating a microenvironment that regulates various cellular activities, including adhesion, proliferation, migration, differentiation, and apoptosis. These cellular processes are controlled through biochemical signaling mechanisms facilitated by the ECM. The primary objective of tissue engineering is to create substitutes for damaged or diseased tissues and organs that can effectively maintain, restore, or enhance their functions within the body. Mostly, cells are cultured on biodegradable materials that have been specifically designed as porous scaffolds. Over time, as the cells proliferate and secrete their own extracellular matrix, and as the scaffold material gradually degrades, a tissue-like structure is generated. This engineered tissue structure can be subsequently used for transplantation purposes, offering potential applications in regenerative medicine.

[0006] After Wichterle et al. suggested the use of a hydrated hydroxymethyl methacrylate (HEMA) network for contact lenses in the 1960s, there has been significant research interest in exploring the application of hydrogels in tissue engineering and biomedicine. Hydrogels are commonly utilized as scaffolds in tissue engineering due to their resemblance to the extracellular matrix, their favorable interaction with cells, tunable stiffness, and their unique property of being able to hold substantial amounts of water within their structure without getting dissolved in the water. This intriguing property of hydrogels makes it possible for them to mimic various characteristics of the extracellular matrix (ECM) found in tissues. They have been a popular choice because they can maintain a distinct 3D structure, provide mechanical support to cells in engineered tissues, and replicate the properties of the native extracellular matrix. Biocompatibility, mechanical properties, and cell-hydrogel interaction represent three fundamental design principles when developing hydrogels. Biocompatibility is essential for evaluating hydrogels in tissue engineering, indicating their ability to interact without adverse immune reactions. The mechanical properties of hydrogels are crucial for tissue engineering as they influence tissue formation, cell migration, proliferation, and differentiation. Scientists have extensively studied the relationship between hydrogel mechanical properties and cell behavior, employing methods such as adjusting molecule structures, cross-linking density, and polymer chain rigidity to control these properties. For example, Chu et al. studied the addition of alginate to the kidney decellularized extracellular matrix to enhance physical stiffness of the hydrogel. The findings highlightedthe importance of optimizing the composition to mimic the natural microstructure and create a favorable environment for cellular engineering and proliferation. Similarly, in another study by Ningrum et al, the mechanical properties of PVA hydrogels were enhanced through the addition of graphene oxide, demonstrating improved cytocompatibility with fibroblast cells.SUMMARY OF THE INVENTION

[0007] The invention describes a bioink formulation which can be employed for various applications, for example, bioprinting scaffolds, regenerative medicine etc. The bioink formulation may comprise a cellulose biomaterial derived, purified or isolated from plant cells and one or more pharmaceutically acceptable components. The cellulose biomaterial may be derived, purified or isolated from the cells by any known derivation, purification or isolation processes known in the art. For instance, the bioink formulation may be derived or purified from plant cells such as live plant cells, dead plant cells, decellularized plant cells or a plant cell-line. A person skilled in the art would understand that any plant cell, tissue or plant part could be employed to obtain the cellulose biomaterial. In some embodiments, well known plant cell lines such as Tobacco BY-2 cells or Arabidopsis thaliana T-87 cells could be utilized. In other embodiments, live or dead plant cells of any plant or plant part could be used.

[0008] The invention also teaches a method of deriving, purifying or isolating the biomaterial that is being used in the preparation of the bioink formulation. The biomaterial could be a cellulose biomaterial and could be derived from any plant parts, tissues or structural cells thereof. The biomaterial can be purified by macerating the plant cells with an acidic maceration mixture. The maceration step may be carried out at a high temperature. Various maceration techniques are known in the art and any of those could be utilized here. The inventors have proposed a novel maceration plus decellularizing protocol which worked well and is encompassed within the scope of this invention. The maceration step may be followed by a step of decellularizing the macerated plant cells to purify or isolate the biomaterial from the plant cells.

[0009] Lastly, the invention teaches a method of preparing a tunable bioink which could be used for the bioprinting applications. The method may comprise a step of purifying or isolating a biomaterial from plant cells according to the method steps described above and throughout the disclosure. This step is followed by a step of mixing or infusing the biomaterial with a synthetic biomaterial or one or more pharmaceutically acceptable components to prepare a mixture. The resultant mixture may comprise the components in various combinations and percentages. The mixing step may be followed by a step of sterilization or lyophilization. In certain embodiments, the next step is sterilizing the mixture to prepare the tunable bioink. The sterilization conditions and methods could vary greatly and any known sterilization treatment which works well with plant cells could be employed in this invention.BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 illustrates the steps of fabricating a 3D scaffold from BY2-derived hydrogel bioink at scale bar 1000 um.

[0011] Figure 2 shows BY2 Cells have a distinctive oblong shape. Figure 2(A) shows the native, live BY2 cells before decellularizing. Fig 2(B) shows BY2 cells after decellularizing. The decellularized cell suspension was stained with Congo red staining (0.2% in dH2O). Figure 2(C) shows BY2 cells after decellularizing and lyophilization. Lyophilization was done by labcono. Fig 2(E) shows the confocal image of the live BY2 cells, stained with Congo-red and Hoechst. Fig 2 (F) shows the confocal image of the decellularized BY2 cells stained with Congo-red and Hoechst.

[0012] Figure 3(A) provides a graph that shows the difference in the area of channels difference in each formulation. Images of 11 bioprinted scaffolds with formulations one that is the optimized formulation with BY2 (B), image of 1 of the optimized bioink printed scaffold with BY2 cells (C), Scaffolds without BY2 (D), (E) scaffolds with CNC slurry and without BY2 which is too soft and it is not printable with resolution.

[0013] Fig 4A. provides the mechanical properties of the 3 different formulations were measured after bioprinting and cross-linking with CaCh.

[0014] Figure 5 provides SEM image of control and 3 different formulations including the optimized formulation. Fig, 5A) provides cross section of the Alginic Acid formulation. Cross-section of formulation 2 that contains all content of formula 1 without BY2 cells is shown in Figure 5B. Formulation 3’s cross-section is shown in Figure 5C. The optimized formulation’s cross section is provided in Figure 5D.

[0015] Figure 6A, 6B, 6C, and 6D, show fibroblast growth, attached and spread out on scaffolds. Figure 6E shows quantification of cell density on scaffolds and revealed similar proliferation dynamics during the experimental time course.

[0016] Figure 7 shows SEM images of fibroblasts after 5 days of culture on the scaffolds. Cells proliferated on the surfaces of the scaffold as shown in Figure 7A and grew through channels of scaffold as shown in Figure 7B; cells proliferated in a sporadic manner and had patchy colonization. Figure 7C shows that cells created paths on the scaffold and grew throughout the whole scaffold surface. Figure 7D provides SEM data that visualizes NIH- 3T3-GFP Fibroblasts that created an extracellular matrix and created a niche on the biocompatible scaffold and started to merge and differentiated.

[0017] Figure 8A and 8B shows lyophilized decellularized T87. Figure 8A shows T87 plant cells Macerated. Figure 8B shows the lyophilized decellularized scaffolds where the same formulation was bioprinted with Live T87 cells.

[0018] Fig 9A shows the SEM image of surface area of a bioprinted scaffold with Live T87 cells with Gel-I and decellularized after bioprinting. Fig 9B shows the SEM image of a surface area of the bioprinted scaffold. Live T87 cells were bioprinted with Gel-I and decellularized after bioprinting. Figure 9C shows the SEM image, detail image of surface area of a bioprinted scaffold Live T87 cells bioprinted with Gel-I and decellularized after bioprinting.

[0019] Figure 10 shows the SEM data of T87 Live bioprinted scaffolds (homemade Alginate gel + Cell ink) and Decellularized. Specifically, Figure 10A shows the SEM image of a cross-section area of a bioprinted scaffold. Figure 10B and 10C shows the higher magnification of a cross-section area of a bioprinted scaffold. Figure 10D shows the higher magnification of a cross-section area, zoomed in on a T87 cell that is decellularized and clearly embraced within the cellulose nanofibrils.

[0020] Figure 11 shows the in vitro Biocompatibility test. Figure 11 A shows the scaffolds with formulation Bio ink- lll-no pectin, were seeded by Fibroblast GFP-3T3 cells. Figure 11 B shows the scaffolds with formulation Bioink- V-Pectin which were seeded by Fibroblast GFP-3T3 cells. Figure 11 C shows the scaffolds Bioink-I, seeded by Fibroblast GFP-3T3 cells. The cells grew and the formulation was biocompatible. Control test.

[0021] Figure 12 relates to the in vitro Biocompatibility tests. Figure 12A shows the scaffolds with formulation Bioink-Ill (no pectin). Figure 12B shows the same scaffold that is presented in Figure 12A, presented with 10x, lower magnification.

[0022] Figure 13 shows the cellulose signature is seen in NMR study. The NMR study analyzed the materials that the scaffolds are made from. A) The red graph represents data analysis from T87 scaffolds bioprinted with homemade gel III and shows the peaks related to cellulose, confirming the high concentration of cellulose. B) The blue graph represents analysis data of scaffolds made with BY2 decellularized and shows a high content of pectin, cellulose, and a weak presence of galacturonan / hemicelluloses.

[0023] Figure 14 shows printing of “LIVE” T87 cells into a scaffold. Figure 14A shows the scaffolds with formulation Bioink-Ill- No pectin, and live T8 bioprinted with BIOX. Figure 14B shows bioprinted scaffolds with formulation III no pectin with live T87. Figure 14C provides an illustration of live T87 cells in a bioprinted scaffold with Bioink III a D1. Scaffold stained with plant cells viability assay kit (Sigma PA0100). Figure 14D provides an illustration of live T87 cells in a bioprinted scaffold with Bioink III at D8.

[0024] Figure 15 shows in vitro biocompatibility test. Figure 15A shows the scaffolds with formulation Bioink-lll- No pectin. Figure 15B shows the scaffolds with formulation Bioink-IV+pectin formulation. Scaffolds were seeded by Fibroblast GFP-3T3 cells. Figure 15C shows the scaffolds with formulation Bioink-VI formulation. Scaffolds were seeded by Fibroblast GFP-3T3 cells. Figure 15D shows the bioprinted scaffold with Bioink I.

[0025] FIGURE 16 shows vitro Biocompatibility test of formulations with live T87 cells. Figure 16A & 16B show scaffolds after crosslinking with sterile CaCb, Ph: 5.8 CaCI2 100mM incubated in plant culture media. Figure 16B show Bioink VI that contains T87 cells. Plant cells survived and grew in the scaffold. The live cells are visualized in green colour as they are positive for fluorescein positive, and dead cells are Propidium iodide positive in red.

[0026] Figure 17 shows solid state31P-nmr. A) The NMR study for identifying the solid state 31 P-nmr is provided. The results confirm that T87 survived and thrived in the homemade gel for 8-14 days. 1) The graph shows data from scaffolds made by Live T87 and after incubation for 14 days in plant media, decellularized and investigated by NMR and showed high content of Phosphor. 2) The graph is from the sample of T87 live bioprinted scaffolds that decellularized on Day 1 , right after bioprinting. The graph in red is completely negative for Phosphor. 3) The graph in blue colour demonstrates the existence of phosphor in a sample from live bioprinted T87. This graph was from a bioprinted scaffold without decellularizing. (3)The similar peaks at 50 and 0 for graph 1 and 3 shows the trace of phosphor and it confirms the T87 cell survival.

[0027] Figure 18 illustrates a comparison between live BY-2 cells and decellularized once. Image A showcases live BY-2 cells stained with Hoechst 3342), while image B showcases BY-2 cells afterthatdecellularization protocol mentioned earlier, also stained with Hoechst 3342.

[0028] Figure 19 illustrates the Hydrogel scaffolds design. The shaped hydrogel scaffolds were designed with various pore sizes, using the Lumen-X bioprinter and viewed through stereoscope microscopy. Scaffold A showcases a haystack shaped 10x10 mm with 9 pores of 2x2 mm each. Scaffold B showcases a cylinder-shaped scaffold with a circumference of 3.17 cm with 9 porous of 3.7 mm of circumference. Scaffold C showcases a cylinder-shaped scaffold with a circumference of 3.17 cm with 21 porous of 1.2 mm of circumference. The scale bar in the image measures 1 mm.

[0029] Figure 20 shows growth of 3T3 mice embryonic fibroblasts expressing GFP on hydrogel scaffolds. Image A and B, captured through confocal microscopy using Nikon software and analyzed with Imaged, showcase the attachment of GFP positive cells on the cylinder-shaped scaffold. The scale bar in the image measures 100 pm.

[0030] Figure 21 show a) BY2 decellularization steps, and b) Steps for the fabrication of the FB scaffolds.

[0031] Figure 22 shows the compressive Young's modulus (YM) of the scaffolds was measured for four different concentrations of BY2 in FB scaffolds.

[0032] Figure 23, shows the frequency sweep (a,c) and amplitude sweep (b, and d) test for FB-0, FB-5, FB-10, and FB-20 samples.

[0033] Figure 24 provides an SEM Analysis of FB Scaffolds and BY2 Cell Distribution, a) Cross-sectional SEM images illustrating the interconnected porous structure of FB scaffolds and the uniform distribution of BY2 cells within FB-5, FB-10, and FB-20 scaffolds, b) SEM image of the surface area of FB-10 scaffold revealing BY2 cells embedded within the Fibrin matrix (BY2 cells highlighted in yellow, fibrin fibers in purple).

[0034] Figure 25 illustrates Cell Viability and Live Cell Count on Various Scaffolds, a) Live / Dead cell ratio for FB- 0, FB-5, FB-10, and FB-20 over a 5-day period, b) Number of live cells for FB-0, FB-5, FB-10, and FB-20 over a 5-day period. The n-value for each time point and each scaffold was n = 9. c) Representative confocal image of Hoechst-PI assay on FB-10 scaffold at day 5. Blue represents Hoechst-stained live cells, while red indicates Pl- stained dead cells.

[0035] Figure 26 provides SEM image of 3T3 cells on FB-10 scaffold after five days, demonstrating BY2 cells in yellow, 3T3 cells in sea-green, and fibrin fibers in purple.

[0036] Figure 27 provides frequency sweep (a) amplitude sweep (b), and creep compliance (c) versus time (oo = 15 Pa) for FB-0, FB-5, FB-10, and FB-20 samples. Storage modulus (G’) (- A-) consistently exceeds loss modulus (G”) (-•-), indicating solid-like behavior. Increased BY2 cell concentration correlates with higher moduli. Frequency sweep shows moduli increase with frequency, likely due to reduced deformation time and potential structural changes. Amplitude sweep reveals storage modulus decreases with strain rate, suggesting viscoelastic behavior. Each sample was tested with n = 6 replicates. The colors in the figure correspond to different samples: red indicates FB-0, blue denotes FB-5, green represents FB-10, and black signifies FB-20.

[0037] Figure 28 shows growth of 3T3 mice embryonic fibroblasts on hydrogel scaffolds.

[0038] Figure 29 shows results of a live I dead cell assay.

[0039] Figure 30 shows a comparison of Young’s Modulus in various scaffolds.

[0040] Figure 31 shows the biocompatibility and cell infiltration of the PEDGA+ BY-2 Scaffolds Implants.

[0041] Figure 32 shows a comparison between PEGDA + BY-2 and PEGDA only.

[0042] Figure 33 shows the gel production process.

[0043] Figure 34 shows examples of gel formulations based on the formulation with alginate and cellulosic material isolated from apples.

[0044] Figure 35 shows the experimental results of the extrusion experiment where the bioink formulations were extruded two times.

[0045] Figure 36 shows a 3D construct made with Alginate 1 % cellulosic isolate from apples at 3%, shaped as a grid with a syringe and 25G needle.

[0046] Figure 37 shows a 3D construct made with Alginate 1 %, and M3 stands for cellulosic isolate from apples at 3%, shaped as a hexagonal prism with a syringe and 25G needle.

[0047] Figure 38 shows 3D constructs made with Alginate at 4% and Alginate 1 % cellulosic isolate from apples at 3% shaped as a ring by molding.

[0048] Figure 39 shows test pieces for bioinks biocompatibility assessment, where A. is the dimensions, B. is the piece production in CaCh immersion for crosslinking, and C. test piece after crosslinking before mold detachment.

[0049] Figure 40(A) shows barrel opening of the test piece, Figure 40(B) shows cells were inoculated inside the test piece, and after 1 h Figure 40 (C) shows test pieces were covered with media.

[0050] Figure 41 shows results for A1 M3-HA where cells grow everywhere, covering the piece surface.

[0051] Figure 42 shows A1 M3 in day #9 with a clear change in the surface.

[0052] Figure 43 shows results for sample A1 M3 (A = Alginate 1 %, and cellulosic isolate from apples at 3%): Figure 43(A) shows test piece: longitudinal cut, 10x magnification. Figure 43(B) shows inside the barrel: longitudinal cut, magnification of 100x.

[0053] Figure 44 shows sample with Alginate 1 % + cellulosic isolate from apples at 3%: images of the Hoechst and Congo Red stained cells, with the red filter for cellulose, blue for the nuclear matter, and green for the fluorescent membrane of 3T3-gfp cells, and with the merged images at the bottom right, magnification 100x, scale bar 100 urn.

[0054] Figure 45 shows results for sample Alginate 1 %, and cellulosic isolate from apples at 3% + Collagen 30 mg / g: The composite bottom right image of the Hoechst and Congo Red stained cells, with the red filter forcellulose, blue for the nuclear matter, and green for the fluorescent membrane of 3T3-gfp cells, magnification 100x, scale bar 100 urn.

[0055] Figure 46 shows results of sample Alginate 1 %, and cellulosic isolate from apples at 3% + Hyaluronic Acid 15 mg / g: The composite image at right image, with stained cells with Hoechst and Congo Red, blue for the nuclear matter, and green for the fluorescent membrane of 3T3-gfp cells, magnification 100x, scale bar 100 urn.

[0056] Figure 47 shows (A-D) strain Vs stress chart for the crosslinked materials.

[0057] Figure 48 (A-D) shows the test piece for mechanical test.DETAILED DESCRIPTION

[0058] The following description is of preferred embodiments by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

[0059] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0060] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[0061] The following definitions supplement those in the art and are directed to the current application. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limitingDEFINITIONS

[0062] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0063] In this application, the use of "or" means "and / or" unless stated otherwise. The terms "and / or" and "any combination thereof and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any and all combinations are specifically contemplated. The term "or" can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0064] Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0065] Reference in the specification to "some embodiments," "an embodiment," "one embodiment" “alternate embodiment”, or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0066] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0067] The term "about" in relation to a reference numerical value and its grammatical equivalents as used herein can include the numerical value itself and a range of values plus or minus 10% from that numerical value. The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1 % of a given value. In another example, the amount "about 10" includes 10 and any amounts from 9 to 11 .

[0068] EMBODIMENTS

[0069] Without wishing to be bound by theory or experimental results, the following paragraphs describe the nature of the invention by way of examples only. The experiments, or specific examples of materials, promoters, enhancers, biomaterials, cell lines and hydrogels described should not be construed as limiting the scope of the invention. A person skilled in the art would readily understand and appreciate that other materials, promoters, enhancers, biomaterials, cell lines and hydrogels not specifically described also form part of the invention.

[0070] Provided herein are plant biomaterials that can be used to prepare tunable bioink formulations. The plant biomaterials could be hydrogels derived from and / or comprising decellularized plant or structural cells thereof. As described in detail herein below, hydrogels may be derived from and / or may comprise decellularized plant tissue or structural cells thereof, and which: may comprise plant structures and / or architectures of interest; may be produced by readily scalable production methods; may provide for a wide range of scaffold microstructures and / or macrostructures and / or biochemistry; may provide tunable mechanical properties; may provide tunable porosity; may be biocompatible in vitro and / or in vivo, may be stable to a variety of conditions or any combinations thereof. By using single structural cells, groups of structural cells, or both, derived from a plant tissue (the single structural cells or groups of structural cells having a decellularized 3-dimensional structure lacking cellular materials and nucleic acids of plant or fungal tissue), distributed within a carrier derived from one or more dehydrated, lyophilized, or freeze-dried hydrogels, a variety of aerogels, hydrogels, could be envisioned. In certain embodiments, the single structural cells, groups of structural cells, or both, may be derived from plant tissue (using mercerization treatment as described herein, which allows for reproducible and scalable production. Related methods and uses, as well as productions methods, are also described in detail herein.

[0071] In an embodiment of the invention, a bioink formulation is provided. The bioink formulation may comprise a cellulose biomaterial derived, purified or isolated from plant cells and one or more pharmaceutically acceptable components. The cellulose biomaterial may be derived, purified or isolated from the cells by any known derivation, purification or isolation processes known in the art. For instance, the bioink formulation may be derived or purified from plant cells such as live plant cells, dead plant cells, decellularized plant cells or a plant cell-line. A person skilled in the art would understand that any plant cell, tissue or plant part could be employed to obtain the cellulose biomaterial. In some embodiments, well known plant cell lines such as Tobacco BY-2 cells or Arabidopsis thaliana T-87 cells could be utilized. In other embodiments, live or dead plant cells of any plant or plant part could be used.

[0072] In certain embodiments, the biomaterial derived is a scaffold from the plant cells. The scaffold could be the extracellular matrix (ECM) or any other scaffold derived from the plant cells. The scaffold could be in the form of a hydrogel, or any other stable form, for instance powder, liquid or solid form.

[0073] In certain embodiments, the biomaterial could be mixed or infused with a synthetic biomaterial to prepare the bioink formulation. For instance, synthetic biomaterials such as poly(lactic-co-Glycolic acid) (PLGA), poly(glycolic acid) (PGA), polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), Fibrin or any other synthetic biomaterial or combination of one or more synthetic biomaterials could be used. A person skilled in the art would understand that any physiologically safe and compatible synthetic biomaterials could be used in this invention.

[0074] In some embodiments, the biomaterial may be a hydrogel and may comprise alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose , agar, pluronic acid, triblock PEO-PPO-PEO copolymers of polyethylene oxide) (PEO) and polypropylene oxide) (PPO), dissolved or regenerated plant cellulose, dissolved cellulose-based hydrogel, hyaluronic acid, extracellular matrix proteins (e.g. collagen, gelatin, or fibronectin, or any combinations thereof), monoacrylated polyethylene glycol), polyethylene glycol) diacrylate, polyethylene glycol) diacrylate (PEGDA)- co-PEGMA, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(lactic-co-glycolic acid), chitosan, chitin, xanthan gum, elastin, fibrin, fibrinogen, cellulose derivatives, carrageenan, or microcrystalline cellulose, or any combinations thereof; wherein the hydrogel is optionally cross-linked.

[0075] In some embodiments, the cellulose biomaterial and the synthetic biomaterial are formulated in a fixed ratio. In certain embodiments, the derived and synthetic biomaterials could be mixed in equal ratios. Depending on the need and requirements of the bioink formulation, i.e. depending on the required porosity, viscosity of stability, the concentrations of the biomaterials could be adjusted. In some embodiments, the bioink formulation may comprise the biomaterial, the synthetic biomaterial and the one or more pharmaceutically acceptable components.

[0076] The resultant bioink formulation or the components contained therein, in some embodiments, are biodegradable, biocompatible, bioprintable, biostable, customizable, reproducible or tunable. For instance, the cellulose biomaterial derived from plant cells is biodegradable, biocompatible, bioprintable, biostable, customizable, reproducible or tunable. In certain embodiments, the cellulose biomaterial may have a peculiar porosity, peculiar viscosity or a peculiar shape. For instance, the biomaterial may have a mesh-like porous structure with a hollow lattice.

[0077] In some embodiments, the biomaterial may be derived, purified or isolated by subjecting the plant cells to maceration treatment, oxidative treatment, decellularization treatment or a combination thereof. In certain embodiments, the biomaterial may be lyophilized by any known lyophilization technique in the art after it’s purified or derived from the plant cells. In some embodiments, the treated plant cells i.e. the macerated or oxidatively treated, or decellularized or lyophilized cells may have a shelf-life of at least one year. The biomaterial derived from the plant cells may have a shelf-life of about four to six months.

[0078] In some embodiments, the purified or derived biomaterial is substantially free of nicotine or any cellular content. The biomaterial may also be substantially free of signaling molecules that are capable of triggering an autoimmune response.

[0079] In certain embodiments, the bioink formulation may comprise one or more pharmaceutically acceptable components such as thickeners, stabilizers, viscosity agents, or pH stabilizers. A person skilled in the art would readily understand that any physiologically or pharmaceutically acceptable component used in the bioinks or for printing bioscaffolds could be utilized here. For instance, the one or more pharmaceutically acceptable components could be alginic acid or salt thereof, agarose, nanofibrillated cellulose (CNF), cellulose nanocrystals (CNC), agarose, pectin or any other cellulose nanomaterial. The one or more pharmaceutically acceptable components could be in any stable form, for instance, in a gel, slurry or a spray-dried form.

[0080] In some embodiments, the bioink formulation may additionally comprise live plant cells, dead plant cells, lyophilized plant cells, cellular adhesion promoters, growth factors, or a combination thereof. Any plant cells, promoters or growth factors known in the art that would work with the other bioink formulation components could be utilized here.

[0081] 2ln some embodiments, he bioink formulation may specifically comprising a) the cellulose biomaterial derived from the plant cells; b) alginic acid or salt thereof; c) nanofibrillated cellulose; and d) cellulose nanocrystals. For instance, the formulation may comprise, a) 1 wt% to 10 wt% of the cellulose biomaterial; b) 2 wt% to 5 wt% alginic acid or salt thereof; c) 2 wt% to 5 wt% nanofibrillated cellulose; and d) 2 wt% to 5 wt% cellulose nanocrystals. In some other embodiment, the formulation may comprise a) 1 wt% of the cellulose biomaterial; b) 2.8 wt% alginic acid or salt thereof; c) 3 wt% nanofibrillated cellulose; and d) 3 wt% cellulose nanocrystals. In certain embodiments, the formulation additionally comprises the one or more pharmaceutically acceptable components noted above.

[0082] In certain embodiments, the bioink formulation may have a Young’s modulus in the range of 10 Pa to 1 GPa. In some embodiments, it could be lower or within a specific range depending in the desired scaffold properties.

[0083] In some embodiments, the biomaterial, the one or more pharmaceutically acceptable components, or the bioink formulation itself could be crosslinked with a crosslinking agent. For instance, any know cross-linking agent known in the art could be used to crosslink the desired component. Crosslinking agent such as CaCI2, UV light, PEGDA or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), for instance, could be used.

[0084] In certain embodiments, the formulation is highly homogenous, highly resilient, and highly extrudable compared to a formulation that lacks the cellulose biomaterial derived from the plant cells. In certain embodiments, the bioink formulation could be employed for use in bioprinting, for use in production of bioprintable scaffolds, for culturing mammalian cells, for use in production of implantable scaffolds, for use in production of scaffolds with adefined shape, for use in in-vitro production of cell lines, for use in regenerative medicine, for use in reconstructive surgery, for use in tissue engineering, for use in biomedical application, for use in spinal cord, bone, ligament injury treatment, for use in wound healing, for use in cosmetic applications, for use in dentistry applications, for use in drug delivery, for use in skin or cartilage regeneration, or for use in manufacturing a dermal filler. Various other applications not specifically enlisted herein could be envisioned and are encompassed within the scope of this application.

[0085] In another embodiment, a method of purifying, deriving or isolating a biomaterial from plant cells is provided. The biomaterial could be a cellulose biomaterial and could be derived from any plant parts, tissues or structural cells thereof. The biomaterial can be purified by macerating the plant cells with an acidic maceration mixture. The maceration step may be carried out at a high temperature. Various maceration techniques are known in the art and any of those could be utilized here. The inventors have proposed a novel maceration plus decellularizing protocol which worked well and is encompassed within the scope of this invention. The maceration step may be followed by a step of decellularizing themacerated plant cells to purify or isolate the biomaterial from the plant cells.

[0086] In some embodiments, the maceration step may comprise various additional steps or any standard prepreparation steps which are encompassed within the scope of this invention. For instance, the maceration step may comprise an oxidative treatment of the plant cells by the acidic maceration mixture. In certain embodiment, the maceration step may comprise a step of treating the plant cells with a surfactant for a predetermined period of time. In certain embodiments, the maceration step may comprise a step of sterilizing the plant cells. In certain embodiments, the maceration step may be repeated one or more times to allow the acidic maceration mixture to weaken and remove the cell wall, protoplast, cytoplasm or other cellular content from the plant cells. In certain embodiments, the method may additionally comprise a step of lyophilization of the purified or isolated biomaterial.

[0087] In certain embodiments, the maceration and decellularization steps are performed with a specific time and temperature range. The conditions could be modified or adjusted depending in the need of the invention. In certain embodiments, the maceration step may be carried out at a temperature ranging from 40 °C to 60 °C. In certain embodiments, the maceration step may comprise a step of treating the plant cells with the acidic maceration mixture; and a step of incubating the plant cells and the acidic maceration mixture overnight at a temperature ranging from 40 °C to 60 °C.

[0088] In certain embodiments, the method may additionally comprise a step of centrifuging the plant cells prior to the step of macerating the plant cells. In certain embodiments, the method may additionally comprise a step of washing the plant cells of after the decellularizing step. The washing step may be followed by mixing thedecellu larized and washed plant cells overnight on a mixer or shaker. In certain embodiments, the incubation step may be carried out at a temperature ranging from 55 °C to 58 °C.

[0089] The acidic maceration mixture may comprise an oxidative component, an acidic componentand one of more industrially acceptable components. In some embodiments, the acidic maceration mixture may comprise glacial acetic acid or absolute glacial acetic acid, hydrogen peroxide, and one or more industrially acceptable excipients. For instance, the acidic maceration mixture may comprise 1 part of a 30% solution of hydrogen peroxide, 4 parts of distilled water, and 5 parts of absolute glacial acetic acid. This is merely an exemplary embodiment and many combination and percentages of components could be used to form the acid maceration mixture.

[0090] In certain embodiments, the decellularization step comprises treating the macerated plant cells with a surfactant for at least 24 hours at room temperature. This step may be followed by a step of washing the plant cells with one or more industrially acceptable solvents. Many surfactants are known in the art, some examples are sodium dodecyl sulphate (SDS), sodium lauryl sulphate (SLS), Triton X, Tween 20, Sodium Deoxycholate (SDC) or bleach.

[0091] In certain embodiments, the method may further comprise a step of storing the biomaterial overnight in a freezer at a temperature ranging from 0 °C to -195 °C. The method may additionally comprise a step of freeze drying or lyophilizing the biomaterial in a lyophilizer. The method may also comprise a step of sterilizing the biomaterial by one or more sterilization treatments. For instance, the biomaterial could be sterilized by autoclaving, ethanol treatment, gamma irradiation, UV treatment or Ethylene Oxide (ETO) treatment. These steps could be permed individually or in combination with each other.

[0092] In some embodiments, the above-recited method steps could be used or employed for purifying or isolating the cellulose biomaterial derived from live plant cells, dead plant cells or lyophilized plant cells.

[0093] In certain embodiments, a method of preparing a tunable bioink is provided. The method may comprise a step of purifying or isolating a biomaterial from plant cells according to the method steps described above and throughout the disclosure. This step is followed by a step of mixing or infusing the biomaterial with a synthetic biomaterial or one or more pharmaceutically acceptable components to prepare a mixture. The resultant mixture may comprise the components in various combinations and percentages. The mixing step may be followed by a step of sterilization or lyophilization. In certain embodiments, the next step is sterilizing the mixture to prepare the tunable bioink. The sterilization conditions and methods could vary greatly and any known sterilization treatment which works well with plant cells could be employed in this invention.

[0094] In certain embodiments, the mixing step may be performed or carried out using an industrial mixer, a rotary mixer, a shaker, mortar and pestle, rolling pin, vortex, pipette, blender, magnetic stirrer, centrifugation, homogenizer or sonicator. The mixing step may occur at room temperature or a temperature that is suited for the desired application. In the mixing step, the component may be mixed sequentially, simultaneously or in a specific sequence. The desired properties of the bioink would greatly influence that decision.

[0095] In certain embodiments, the sterilization step is carried out by autoclaving, ethanol treatment, gamma irradiation, UV treatment or Ethylene Oxide (ETO) treatment. A person skilled in the art would understand that many other sterilization options not explicitly recited herein are encompassed in this invention.

[0096] In certain embodiments, the method may comprise a step of crosslinking the biomaterial, the one or more pharmaceutically acceptable components, or the bioink formulation. The crosslinking step may be performed by any known method in the art. In some embodiments, the crosslinking step may be carried out using a crosslinking agent. The method may also comprise a step of lyophilizing the tunable bioink to prepare a powder of the bioink. In some embodiments, the method may also comprise s step of centrifuging the mixture to remove air pockets or air bubbles.

[0097] As recited hereinbefore, the one or more pharmaceutically acceptable components could be alginic acid or salt thereof, agarose, nanofibrillated cellulose (CNF), cellulose nanocrystals (CNC), agarose, pectin or any other cellulose nanomaterial. Any other components known to be physiologically and pharmaceutically acceptable could be used here. In certain embodiments, the isolated or purified biomaterial is the cellulose biomaterial derived from live plant cells, dead plant cells, decellularized plant cells or a plant cell line. Any plant cell, tissue or part thereof could be employed in this invention. In an exemplary embodiment, the isolated or purified biomaterial is the cellulose biomaterial derived from Tobacco BY-2 cells or Arabidopsis thaliana cells.

[0098] The derived biomaterial could be mixed or infused with any synthetic biomaterial known in the art. Some exemplary embodiments are poly(lactic-co-Glycolic acid) (PLGA), poly(glycolic acid) (PGA), polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), Fibrin or a combination thereof, some embodiments, the biomaterial may be a hydrogel and may comprise alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose , agar, pluronic acid, triblock PEO-PPO-PEO copolymers of polyethylene oxide) (PEG) and polypropylene oxide) (PPO), dissolved or regenerated plant cellulose, dissolved cellulose-based hydrogel, hyaluronic acid, extracellular matrix proteins (e.g. collagen, gelatin, or fibronectin, or any combinations thereof), monoacrylated polyethylene glycol), poly(ethylene glycol) diacrylate, polyethylene glycol) diacrylate (PEGDA)-co-PEGMA, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(lactic-co-glycolic acid), chitosan, chitin, xanthan gum, elastin, fibrin, fibrinogen,cellulose derivatives, carrageenan, or microcrystalline cellulose, or any combinations thereof; wherein the hydrogel is optionally cross-linked.

[0099] In certain embodiments, the method may additionally comprise a step of infusing the tunable bioink with live plant cells, dead plant cells, lyophilized plant cells, cellular adhesion promoters, growth factors, or a combination thereof. These components are well known in the art and any known live plant cells, dead plant cells, lyophilized plant cells, cellular adhesion promoters, growth factors that would work well could be employed here. In certain embodiments, the above recited method steps could be employed for preparing the bioink formulation defined above.

[0100] The crosslinking agents could be CaCI2, UV light, PEGDA or 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC). In still another embodiment of any of the above formulations biomaterials, hydrogels, at least some cellulose and / or cellulose derivative(s) of the hydrogel may be cross-linked by physical cross-linking (e.g. using glycine) and / or chemical cross-linking (e.g. using citric acid in the presence of heat); wherein at least some cellulose and / or cellulose derivative(s) of the aerogel or foam is functionalized with a linker (e.g. succinic acid) to which one or more functional moieties are optionally attached (e.g. amine-containing groups, wherein cross-linking may further optionally be achieved with one or more protein cross-linkers such as formalin, formaldehyde, glutaraldehyde, and / or transglutaminase); or any combinations thereof.

[0101] In some embodiments, the mixing step may additionally comprise a step of infusing the cellulose biomaterial in the synthetic biomaterial, wherein the step of infusing may be carried out by any known mixing or infusion protocols known in the art, for instance, UV crosslinking.

[0102] Biomaterials & Scaffolds

[0103] The potential of decellularized apple hypanthium tissue and other cellulose-based vegetal scaffolds as a lignocellulosic biocompatible material has been shown previously, which has similar architecture to the structure of mammalian tissue. The biocompatibility of plant-based cellulose in vivo and in vitro also has been studied as it exhibits limited foreign body response. In addition, investigations on the suitability of decellularized spinach and chive stem affirmed the concept of plant-derived decellularized scaffold models and theirsuitability for use in tissue engineering.

[0104] These studies proved that decellularized plants are not only easily renewable and biocompatible but also the rough cellulose-based Extracellular Matrix (ECM) has a structure that enhances cell attachment. This canadopt an abundant source of cellulose-based Bioink that is highly extrudable. Indeed, cellulose scaffolds have the potential to recapitulate the ECM well remarkably with appropriate modifications.

[0105] While decellularized plant-derived scaffolds have many benefits, the fixed nature of their 3D geometry is an important limitation. Recently, 3D bioprinting has become a populartool forthe creation of scaffolds with defined shapes. To create a 3D-printed scaffold, a bioink must be made formulated. One of the fundamental base ingredients of any bioprint blend is derivatives from algae including alginate, agarose or cellulose nanomaterials produced from bacterial generation or woody biomass. Nanocellulose can be obtained from wood, cotton, hemp, flax, wheat straw, sugar beet, potato tuber, mulberry bark, ramie, algae, and tunicin. There are three different nanocellulose categories: (1) Cellulose nanocrystals (CN ), (2) Nanofibrillated cellulose (NFC), and (3) Bacterial cellulose (BC) each of which have unique properties of viscosity and extrudability.

[0106] As with most biomaterials, CNF and CNC are used due to their mechanical and chemical features. However, the inventors were able to purify another plant-derived cellulose which also has the features of traditional cellulose-based biomaterial which are, biocompatibility, biodegradability and low toxicity. This plant-derived biomaterial recapitulates the ECM very well and is essential to develop a porous scaffold.

[0107] With the technique developed and proposed by the inventors, this plant-derived cellulose biomaterial can be derived from any plant cells. The biomaterial can then be used to develop tunable porous bioscaffolds that work great in any bioprinting application.

[0108] The properties of biomaterials can vary greatly depending on their source and processing method. For example, natural polymers can have different mechanical properties depending on the source tissue or animal species. Processing methods such as crosslinking can also influence the mechanical properties of natural and synthetic biomaterials. Porosity is another important factor in biomaterial design, as it can affect cell attachment and growth.

[0109] Previous studies have shown that natural plant-derived cellulose biomaterial scaffolds can be produced by employing common decellularization approaches which support 3D cell culture. Such scaffolds can be produced for repairing tissue damage and providing structural support. These structures are porous and biocompatible and excellent substrates for cell attachment.

[0110] Synthetic biomaterials that are synthesized in a laboratory may also be employed to obtain the tunable bioscaffolds using the proposed technique. They have the advantage of being tunable in terms of their properties and can be designed to meet specific mechanical requirements for targeted applications. Examples of synthetic biomaterials include poly(lactic-co-Glycolic acid) (PLGA), poly(glycolic acid) (PGA), polyethylene glycol (PEG),Polyethylene Glycol Diacrylate (PEGDA) and Fibrin. These work well for creating biocompatible and biodegradable polymeric devices that can be used for drug delivery purposes. Moreover, PEG and other synthetic biomaterials can also be utilized to bind with other molecules, thereby opening new avenues for various therapeutic applications. For example, the synthetic biomaterials can bind to the plant-derived cellulose biomaterial to create a stable, biocompatible and porous bioscaffold.

[0111] For ease of access, the inventors used plant-based cell lines for proof-of-concept studies. However, any plant material can be decellularized to obtain a suspension of cells for the proposed technique. Two cell lines were tested a) Tobacco BY-2 and b) T87 cell line.

[0112] The BY-2 cells are resilient and comparatively easy to work with. TBY-2 cell line has been established from the callus induced on a seedling of Nicotiana tabacum L. cv. Bright Yellow 2. The significance of BY2 is their higher growth rates and high homogeneity. Cell line T87 is derived from Arabidopsis thaliana.

[0113] The initial proof-of-concept study used a novel method to purify biopolymers from renewable plant sources, namely the Tobacco Bright Yellow-2 (BY-2) single cells suspension. This new method includes decellularizing plants and isolating the plants’ Extracellular Matrix (ECM). This biopolymer i.e. the plants’ Extracellular Matrix (ECM) can be used as the main base of a bio-ink. The printability of this biopolymer was investigated by assessing the resolution of prints additionally supported by SEM images demonstrating the porosity of this biopolymer. Furthermore, the biocompatibility of these scaffolds was assessed with an in vitro assay. The seeded scaffolds with mammalian NIH3T3- GFP fibroblast showed potential for cell adhesion. Moreover, the bio-ink formulation demonstrated stable behaviour as well as a very porous structure. This work reports the customizable and tunable behaviour of plant-derived biopolymers without swelling. Potential applications might include extrudable materials with the future potential of implantation or more commercial applications.

[0114] Decellularizing Technique

[0115] One method of creating a biomaterial scaffold is through the use of decellularized plant cells. Decellularization is the process of removing all the cells from a tissue, leaving behind a cellulosic scaffold. The scaffold is made up of mainly polysaccharides such as cellulose, hemicellulose, lignin and pectin. The following provides structural support, guidance, and porosity. After being shaped and processed, the scaffold can then be seeded with mammalian cells and cultured in vitro or implanted in vivo to promote tissue repair and regeneration. The use of plant-derived cellulosic scaffold has several potential advantages over animal-derived extracellular matrix, such as a lower risk of immune rejection and the ability to easily scale up production.

[0116] While decellularized plant-derived scaffolds have many benefits, the fixed geometry of such scaffolds is an important limitation. Recently, 3D printing with biomaterials (bioprinting) has become a very popular tool for the creation of scaffolds with controlled shapes. By using a specific type of 3D printer, a biomaterial scaffold can be created by printing layers of decellularized plant cells in a specific pattern to form the desired shape and size. Essentially, bioprinting is a process that involves layer-by-layer deposition of biologically formulated plant-derived cellulose ink. This method allows for precise control over the design and structure of the scaffold for different applications. To maintain the structure of the scaffold, it is necessary to both customize it and reinforce it.

[0117] The inventors have developed a novel method to purify a tunable biomaterial that can be customized and molded in any shape based on the organ of interest and application. This tunable biomaterial can be used as a dermal filler in reconstructive surgeries. In this method, any decellularized plant material can be used as a green and sustainable source of biomaterial.

[0118] TOBACCO BY-2

[0119] For initial proof-of-concept study, a new plant model was purified to produce a new green biopolymer derived from the Nicotiana tabacum BY-2 cell line ( rpcOOOOl ). This line was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan. Although it took significant experimentation, the bioink developed by the inventors created an extrudable ink with optimized extrusion, good viscosity and stability. After trying out various combinations and percentages of alginic acid, cellulose microfibrils were used. In bioprint formulation development, cellulose microfibrils contribute significantly to rigidity as well as the flexibility of the BY2 cell wall. Therefore, for the highest resolution printability of the proposed scaffold, various percentages and combinations of CNF and CNC were added.

[0120] After decellularizing the BY-2 cells, a fine and tunable paste is produced which can be used as a source of plant-based cellulose. The fine plant cell suspension is purified by a technique called maceration that have been used in food industries, wineries, medicinal plant and plant anatomists. This process is used to dissolve cementlike material that glued adjacent cells. It essentially involves multiple extraction steps to break down the plant tissue and softening plant leaves, stem or fruits.

[0121] The maceration technique was customized by adding to it a novel decellularizing protocol to achieve better and softer decellularized cell suspension. These attempts on decellularizing Tobacco callus and later BY2 cell line resulted into a novel maceration treatment based decellularizing technique. This method impacts the plant cell walls and results in a single cell suspension that is easier to decellularize and process into a biopolymer. This resultant Bioink was found to have optimum homogeneity and efficient extrudability.

[0122] Furthermore, BY2 cell pellet was treated with destructive oxidizing agents and concentrated acid at high temperatures. These procedures can cause changes in the volume of the cells and rigidity of the material. The process weakens the cell-walls and destroyed the cell membrane and protoplast. Therefore, the BY2 cell suspension was easily decellularized and produced a very fine plant ECM which is depleted from cell content, and is rich in cellulose. In some instances, the decellularized BY2 cells were dried freeze and then the powder like ECM was stored at room temperature for over a year. Furthermore, it was attempted to mix the extracted biomaterial I biopolymer with alginic acid which made the Bioink stronger and more tunable. In fact, it was observed that only the native structure of decellularized BY2 cells created the hollow lattice, and it could mimic the mammalian ECM, with heterogenous three-dimensional meshwork.

[0123] Materials and Methods

[0124] Plant cell culture and maintenance

[0125] An in vitro cell culture of BY2 (rpcOOOOl RIKEN BRC) was used for harvesting plant cells and purifying biopolymers to produce bioink for bioprinting. This cell line was imported from Japan and was used after acclimatizing it to the lab environment. The tobacco BY-2 cell line is derived from seedlings of N. tabacum L. cv. Bright Yellow 2 boasts a propagation rate of 80 to 100 fold within 1 week. BY2 cells were obtained from the S phase cell cycle. The adapted medium of Linsmaier and Skoog (mLS) was supplemented with 0.2 mg / L 2,4- dichlorophenoxyacetic acid (2,4-D), KH2PO4 and thiamine HCI and adjusted to pH 5.8 with NaOH. The BY-2 cell culture has been maintained in the dark at 27°C with rotary shaking at 130 rpm and sub cultured at 3-day intervals. The Tobacco BY-2 cells were harvested every 3 days.

[0126] Maceration and Decellularizing Nicotiana tabacum (BY-2)

[0127] The harvested Tobacco BY-2 cells sediment was centrifuged for 10 minutes at 4000 rpm and treated with a customized maceration technique that is originated from an established protocol (Yeung, 1998). In this method, the mixture of glacial acetic acid and hydrogen peroxide was used to permeabilize the cell wall and destroyed the protoplast and cell membrane. The maceration mixture includes 1 part of a 30% solution of hydrogen peroxide (Fisher 18603263), 4 parts of distilled water, and 5 parts of absolute glacial acetic acid (ACP chemicals A-0302). This maceration mixture was added to the BY2 cell pellet and incubated in a water bath overnight at 56 °C. It was then washed 3 times with dH2O and incubated overnight on the shaker in ddH2O at 13000 rpm, at room temperature. This step removes all cytoplasmic and protoplast content. If done properly the resulting pellet should be visibly white and have a minimal yellowish appearance. The BY2 cell pellets are added to a 0.1 % SDS solution (BP166-500) in Erlenemayer flask and agitated on a shaker for 1-2 days at room temperature. The SDS wasrefreshed each day, and on the second day, cells were added to the fresh CaCh 10OmM and shaker at 13000 rpm at room temperature for 2 days to clear and wash the SDS left over from the cells. After that, the sample was washed in PBS (MP Biomedicals) for 1 day and washed in ddH2O for 2 days. The decellularized sample was frozen at -80 C in an Ultra-Low-Temperature freezer overnight and then freeze-dried with the Labcono Lyophilizer. The material was then autoclaved for sterilization and was found to be stable in dry and at room temperature. The lyophilized purified decellularized BY2 was stable at room temperature and in dry conditions.

[0128] Fabrication of plant cell hydrogel based

[0129] The plant cell laden hydrogel was composed of decellularized BY2 and addition of alginic acid, a sodium salt-based gel, helped to reach a firmer texture following bioprinting. Alginic acid sodium salt-based gel was prepared under sterile conditions and the 2.8% w / v (Sigma 180947) was autoclaved and stored at 4 °C. At the time of printing, the alginate gel was acclimated to room temperature. In addition, the CNF-Slurry-SMC (Cellulose Nanofibrils 3 wt %) and 10% gel of CNC-SD (Cellulose Nanocrystals- spray dry) that were obtained from the supplier Cellulose Lab were used to add viscosity for a consistent homogenous plotting blend. Cellulose Nanocrystals were added to create better extrusion, viscosity and stability of constructs. All Bioink’s components were autoclaved (45 minute 122 KP) and used at room temperature by collecting in a 10ml syringe (BD leurlock 10ML 302995). The 3ml (0.264 mg) decellularized Lyophilized BY2 cells were added to a 2ml of 2.8wt % Alginic acid in this syringe. Then it was mixed by coupling with another syringe containing CNF-Slurry-SMC and CNC- SD. Following the mixing of the aforementioned components air pockets in the suspension were eliminated by centrifugation. The bioink developed from plant cell hydrogel was loaded in 3ml cartridges for a pneumatic bioprinter. In total, 4 ml of Cellulose Nanocrystals were used for 3 ml of cells and 2 ml of alginate. The plotting blend (Alginate / CNF-slurry-SMC + CNC-SD + Decell-Lyo-BY2) was found to form a homogeneous paste with precise deposition of uniform strands. Figure 1 illustrates the steps of fabricating a 3D scaffold from BY2-derived hydrogel bioink. The scale bar was as follows: Scale bar 1000 urn.

[0130] Construction of hydrogel scaffold

[0131] For extrusion-based 3D plotting, the BioX (Cellink) was used. The bioink / hydrogel pastes were dispensed through a beveled needle (0.41 mm) inner diameter operating a plotting speed of 3mms-1 and a head extrusion pressure of 12-25 kPa. The Haystack structure was used as the model given its reproducible structure. This structure is designed in 1x1x3mm and printed with a pneumatic printing probe. The scaffolds were plotted on a plastic petri dish and crosslinked with 100 mM CaCh following printing. Scaffold was then stored in 70% Ethanol to sterilize the material for subsequent assays.

[0132] Mechanical properties

[0133] The compressive moduli of the 3 different formulations were measured after bioprinting and cross-linking samples (10 x 10 * 3 mm) with CaCh using the UniVert testing machine (CellScale) at a crosshead speed of 1 mm / min-1 . For all samples, Young’s modulus was calculated from the linear region of the strain curve, and the compression strengths were reported as the amount of stress at 70% total strain. The values reported were the average of 11 specimens for each formulation for a total 33 samples for this test.

[0134] Cell culture

[0135] In this study, the NIH3T3-GFP mouse fibroblasts were used (obtained from the American Type Culture Collection (ATCC)). The GFP- 3T3 cells were cultured in the standard cell culture media (high glucose DMEM ( Cytvia Hyclone), with 10% fetal bovine serum (10% FBS Cytiva HyClone SH30396), 1 % penicillin / streptomycin (HyClone SH30022FS) and at 37°C and 5% CO2 in T75 flasks (Thermo Scientific) Culture media was exchanged every second day and the cells subcultured at 80% confluence.

[0136] In vitro cell culture on cellulose scaffolds

[0137] The scaffold seeding procedure took place in 12 well tissue culture plates. All scaffolds after bioprinting and cross-linking with CaCh 100mM were sterilized in 70% Ethyl alcohol for 1 hour, then washed with sterile PBS in sterile condition and incubated overnight with standard cell culture media. Scaffolds were then seeded with 100x103 GFP-3T3 cells and after seeding were incubated for 3 hours then 2.5 mL of standard media was added to each well. For continued cell proliferation, the culture media was changed every 2 days. These experiments were conducted over 14 days and scaffolds were collected on days 1 , 7, 14 and day 21 for immunofluorescence staining and imaging and cell quantification.

[0138] Immunofluorescence staining

[0139] The GFP-3T3 cells were stained for actin cytoskeleton and nucleus of mammalian cells based on established protocols. For this purpose, scaffolds were fixed with 4% paraformaldehyde, washed with PBS 3 times and permeabilized with Triton X-100 at 37°C. As the scaffolds are very sensitive after 30 days of culture and are mainly cellulose based, the cross-linking of alginate was weakened over time, therefore CaCh 10mM was added in the last wash to make the scaffold harder. The phalloidin conjugated to Alexa Fluor 546 (Invitrogen) was used for actin staining and nuclei was stained by labelling the DNA with DAPI (Invitrogen). Samples were then mounted in Vectashield mounting media (BioLynX VECTH1000) prior to imaging.

[0140] Optical Microscopy

[0141] Confocal imaging was performed on an A1 R high-speed laser scanning confocal system on a TiE inverted optical microscope platform (Nikon, Canada) with appropriate laser lines and filter sets. Transmitted light images were acquired on an inverted TiE microscope (Nikon, Canada) with phase contrast optics. Images were analyzed using Imaged open-access software (http: / / rsbweb.nih.gov / ij / ). Brightness and contrast adjustments were the only manipulations performed on images.

[0142] Scanning Electron Microscopy

[0143] Two different methods were used for the preparation of samples. Scaffolds without NIH-3T3 cells were first prepared with the freeze fraction technique being immersed in a -120 °C on liquid nitrogen and broken into 4 pieces then were fixed overnight in 2% Glutaraldehyde (G7651-10MLSigma Aldrich, UK) and dehydrated with a series of graded concentrations (35%, 50%, 70%, 95% and 99, 100%) of ethanol. The dehydrated sample was then incubated overnight in a fume hood in 100% of Hexamethyldisilazane (Sigma 440191-100 ML) and left overnight to be dried. The Bioprinted scaffolds crosslinked and seeded with NIH-3T3 GFP cells for 2 days and 4 weeks in culture. They were gently washed with PBS (PH:7), then the same as non-seeded scaffolds were prepared. Following processing, samples were gold sputter-coated at a current of 15 mA for 3 minutes with a Hitachi E-1010 ion sputter device. SEM imaging was conducted at voltages ranging from 2.00-10.0 kV on a JEOL JSM-7500F FESEM.

[0144] Results

[0145] Decellularized B Y2 cells as plant cell-laden hydrogel

[0146] The biopolymer developed by the inventors was a result of the maceration and decellularization technique used by the inventors where the BY2 cells exclusively contained cell wall structure. The decellularized cells were stained with Congo red staining that dye the plants cell wall. A comparison of BY2 live images with decellularized cells demonstrates that BY2 cells as well as the sieve body structure of BY2 remained untouched. As the cell wall is composed of cellulose, it would be positive for Congo red. The Hoechst 3342 (Thermo Fisher H3570) staining was used for the investigation of any remained protoplast and cell nuclei. As it is shown in Figure2 (E and F), the results showed no significant presence of any cell content and nuclei, which confirmed the efficiency of the maceration and decellularizing procedure.

[0147] Figure 2 shows BY2 Cells have a distinctive oblong shape. Figure 2(A) shows the native, live BY2 cells before decellularizing. The BY2 cells were stained with Plants cells viability assay kit (Sigma PA0100). The livecells are green and fluorescein-positive express within the cytoplasm. The dead cells are Propidium iodide positive and express red colour fluorescence. Fig 2(B) shows BY2 cells after decellularizing. The decellularized cell suspension was stained with Congo red staining (0.2% in dH2O). Figure 2(C) shows BY2 cells after decellularizing and lyophilization. Lyophilzation was done by labcono. Fig 2(E) shows the confocal image of the live BY2 cells, stained with Congo-red and Hoechst. Fig 2 (F) shows the confocal image of the decellularized BY2 cells stained with Congo-red and Hoechst. Images were acquired with Nikon T1 eclipse Confocal microscope and analyzed with NIS element and Fiji.

[0148] Bioink formulation and optimization

[0149] The 2.8% W / V Alginate solution was quickly mixed with lyophilized BY2. This concentration was selected after trying various Alginate concentrations. Alginate was chosen after testing 1 , 1 .5, 2, 2.5, 5, and 10% of Alginate. The lower concentrations were insufficiently robust to cross-link with CaCh and demonstrated low stability. In contrast, higher concentration showed very high viscosity and at room temperature it was impossible to mix and dissolve it with BY2-derived hydrogel.

[0150] The higher alginate was very viscous and couldn’t fuse with cellulose nanocrystals. In addition, among available Cellulose Nano Crystals in the market, two types were picked for ease of access. The CNF Slurry SMC comes in 3 wt% and is added to the blend of Alginate and BY2 hydrogel, to develop a homogenous printing paste. As CNC-slurry-SMC comes with 3wt %, the different bioink blend prepared based on volume of CNC with the total mixture of BY2 and Alginate. The BY2 hydrogel was lyophilized to keep the control of moist and maximize blend stability. The above materials merely provide exemplary embodiments and other weight percentages of ingredients are well encompassed within the scope of this inventionsTable 1 . Exemplary bioink formulationsConcentration Alginic CNF- CNC- CNC- Decellularized Printability Resol Stability of materials Acid Slurry- slurry SD cells (BY2) ution 2.8% SMC10%Formulation 1 + 0 + + Yes +++ YesFormulation 2 0 + 0 Yes ++ Not strongFormulation 3 Yes N

[0151] Printability testing

[0152] For print resolution using the three different exemplary formulations of CNF-Slurry-SMC + CNC-SD + AG + BY2 and CNF-Slurry-SMC + CNC-SD + AG and CNF-Slurry-SMC + CNC-slurry + CNC-SD + Alginate was tested by printing a haystack 10x10x3 mm, 5 layers tall (300 mm) with 3% rectilinear infill and crosslinked with 100mM CaCh. By using the Fiji software the size of channels per scaffold was measured for each formulation, a total of 6 scaffolds used for each formulation and per scaffold 16 channels area was measured as well as the whole surface area of each scaffold. An analysis was done by GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA). The statistical analyses showed that formulation 1 with decellularized BY2 cells demonstrated a better resolution and well-shaped channels, and homogenous results compared to the other formulations. In addition, statistics showed a significant difference between Formula 1 and Formula 2 and 3. In fact, the addition of decellularized BY2 made this printability significantly different from the 2 other formulations. The number of biological replicates is indicated in each figure legend. The value of p<0.05 was considered statistically significant.

[0153] Figure 3(A) provides a graph that shows the difference in the area of channels difference in each formulation. By using the Fiji software the size of channels per scaffold was measured for each formulation, a total of 6 scaffolds were used for each formulation and per scaffold 16 channels are measured as well as the whole surface area of each scaffold. An analysis was done by GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA). The statistical analyses showed that formulation 1 with decellularized BY2 cells has the best resolution and well-shaped channels, and homogenous data. In addition, statistics showed a significant difference between Formula 1 and Formula 2 and 3. In fact, the addition of decellularized BY2 made this significantly different from the 2 other formulations. The number of biological replicates is indicated in each figure legend. The value of p<0.05 was considered statistically significant. The P< 0.001 = *** and P< 0.039 = *. Images of 1 1 bioprinted scaffolds with formulations one that is the optimized formulation with BY2 (B), image of 1 of the optimized bioink printed scaffold with BY2 cells (C), Scaffolds without BY2 (D), (E) scaffolds with CNC slurry and without BY2 which is too soft and it is not printable with resolution.

[0154] Mechanical test

[0155] The mechanical properties of the 3 different formulations were measured after bioprinting and crosslinking with CaCL. As is shown in Figure 4 (A) the n-values were 11 for each formulation. The results demonstrate that significant difference in Young’s modulus between formulation 1 and formulation 2 and 3 variabilities (** p=0.003 and *** p= 0.0003 ). As is shown a clear difference in mechanical properties of Formulation 1 with BY2and other formulations without BY2 although there is a slight difference between bioink with CNF-Slurry-SMC + CNC-SD + AG and CNF-Slurry-SMC + CNC-slurry + CNC-SD + AG. Also, Figure4 (B) shows the compressive strength for three different formulations. As it is shown in the Figure 4 (B), there is a significant difference in the compressive strength for the formula 1 (28.77 kPa) in comparison to the formula 2 (34.67 kPa). This difference could be due to the porous nature of the BY2 cells, which consequently has resulted in a porous nature of the formula 1 . Therefore, this porous structure could have resulted in a lower compressive strength in comparison to the formula 2, which has a more compact structure.

[0156] Fig 4A. provides the mechanical properties of the 3 different formulations were measured after bioprinting and cross-linking with CaCh. As is shown in Figure 4 the n-values are 1 1 for each formulation. Figure 4B shows the compressive strength for three different formulations. The results demonstrate that significant difference in Young’s modulus between formulation 1 and formulation 2 and 3 variabilities (p = 0.003 and p= 0.0003). As is shown there’s a clear difference in mechanical properties of Formulation 1 with BY2 and other formulations without BY2, although there is a slight difference between bioink with CNF-Slurry - SMC + CNC-SD + Alg and CNF-Slurry- SMC + CNC-slurry + CNC-SD + Alg.

[0157] Porous scaffold

[0158] The SEM images evidently showed the porous structure of the developed hydrogel. Significantly, the Bioink containing BY2 exhibited a very consistent porous structure. Specifically, Figure 5D in comparison with only Alginate (Figure 5A) and two other formulations without BY2 and with CNC-Slurry, (Figure 5B, 5C).

[0159] Formulation 2, (Figure 5B) showed that the Cellulose and alginate blend cannot produce a very precise and homogenous porous structure. This blend was very fragile and had few porous-like structures. Moreover, formulation 3, (Figure 5C), shows just the fibrillar structure of Cellulose Nanofibrils and no consistent porous structure. These findings aligned with the behaviour of this bioink as a very stable, and tunable bioink that created scaffolds with resolution without any swelling or collapsing trend.

[0160] Figure 5 provides SEM image of control and 3 different formulations including the optimized formulation. Fig, 5A) provides cross section of the Alginic Acid formulation. Cross-section of formulation 2 that contains all content of formula 1 without BY2 cells is shown in Figure 5B. Formulation 3’s cross-section is shown in Figure 5C. The optimized formulation’s cross section is provided in Figure 5D.

[0161] Biocompatibility

[0162] As explained above, GFP-3T3 cells were cultured and used for Biocompatibility test. For each scaffold 100000 cells used for seeding per 1 cm2, for each scaffold. After day 1 , 7 and 21 , scaffolds were collected, fixed and stained for DAPI and phalloidin. In this regard, cell compatibility and survival were observed after 1 day and this trend continued for21 days. For each scaffold formulation (n=3 pertime point, per formulation), three randomly chosen and 1 xi cm2 areas were imaged and the total number of cell nuclei was counted in each region and averaged as shown in Figure 6. To study the GFP-3T3 cell's growth and survival; the phalloidin conjugated to Alexa Fluor 546 (Invitrogen) used for actin and nuclei was stained by labelling the DNA with DAPI (Invitrogen). Samples were then mounted in Vectashield mounting media (BioLynX VECTH1000). Images were taken for 10X and 40X magnification. The cell number was quantified with the Fiji cell counting command and a graph was prepared accordingly.

[0163] As is shown in Figure 6A, 6B, 6C, and 6D, fibroblast grew, attached and spread out on scaffolds. Figure 6E shows quantification of cell density on scaffolds and revealed similar proliferation dynamics during the experimental time course. There was no significant difference in cell density between the scaffolds on Day 1 and day 7. However, by Day 14, the cell density on scaffolds was significantly different (p = 0.0194). Cells were calculated for 3 different areas per scaffold, and for each time point, 3 scaffolds (n=9) were used. The cell’s growth continued and kept a growing curve. Initially, on Day 1 , cell density on the scaffolds was (p =0.0157) at 131 cells / cm2.

[0164] SEM imaging on the seeded scaffold revealed cells covering the surface as well as proliferating inside of the pores (as shown in Figure 7). In addition, it showed that the cells grew after 14 days, and started to create cell patches after 2 weeks. As cell density on the scaffold increased over time, the bigger cells colony tend to organize the separated colonies.

[0165] Figure 7 shows SEM images of fibroblasts after 5 days of culture on the scaffolds. Cells are attached on the surface of cellulose-based bioprinted scaffolds with the formulation that was coated with FBS prior to seeding. For each scaffold, 100000 cells were used. Cells proliferated on the surfaces of the scaffold as shown in Figure 7A and grew through channels of scaffold as shown in Figure 7B; cells proliferated in a sporadic manner and had patchy colonization. Figure 7C shows that cells created paths on the scaffold and grew throughout the whole scaffold surface. Figure 7D provides SEM data that visualizes NIH-3T3-GFP Fibroblasts that created an extracellular matrix and created a niche on the biocompatible scaffold and started to merge and differentiated.

[0166] T87 CELL LINE

[0167] This study evaluated a proof-of-concept bio-ink, by examining print fidelity and the structural integrity of resulting scaffolds. The overall shape fidelity of bioprinted constructs was assessed by measuring deposited material in the x- and y-axes, and the dimension as the resolution in the z-axis. Additionally, the microstructure and porosity of the bioprinted scaffolds was characterized with electron microscopy. Moreover, the compressive young modulus was measured, and the biocompatibility of these scaffolds assessed in vitro through seeding scaffolds with mouse fibroblast cells and examining cell proliferation. In addition, biocompatibility was also examined in a preliminary in vivo study in which scaffolds were implanted subcutaneously. Assessment of cell infiltration and foreign body response was then carried out after several weeks inside of the animal. The results indicate that this plant-based bioink has bioprinting potential due to its biocompatibility, biodegradability, and versatility in creating complex structures.

[0168] In an exemplary embodiment, the Bioink is composed of 3 main parts, A) Alginic acid sodium salt based gel and B) Cellulose biomaterial and C) Live cells of plant of interest. It is pertinent that the Bioink’s material is sterile and autoclaved (45 minute 122 KP) and working condition is in the Laminar Flow Hood.

[0169] In the above example, the A) component is readied by mixing Alginic acid sodium salt. 2.8% (Sigma 180947) and Ultra Pure™ Agarose (Fisher 16500100) 1.5 % and both are dissolved in 100ml of the Arabidopsis plants media (Master mix-T87) and then Autoclaved and stored at 4 °C.

[0170] The B) component, which is mostly a mix of two different cellulose variations is readied. In this example, 20 ml of CNF-Slurry-SMC (Cellulose Nanofibril 3%), CNC-Slurry (Cellulose Nanocrytal 6%) and 10% gel of CNC- SD (Cellulose Nanocrystals- spray dry) in dH2O was autoclaved.

[0171] Several variations of B) are possible. The B) component can be customized or changed by adding various concentrations of Cellulose Nanocrystals or Cellulose Nanofibrils. In some embodiments, Pectin may also be added. For instance, in Bioink-IV, 15% of Pectin from apple (Sigma #93854-100G) was added. The Bioink IV had higher viscosity, and therefore, pectin made the Bioink thicker and less printable with low extrusion. In other words, it requires higher pressure and higher temperature for smooth extrusion and which could be lethal for Live T87 Bioprinting.

[0172] On the other hand, component C was the Bioink developed by the inventors from T87 cells using laminar flow hood and sterile cell culture techniques. 50ml of T87 cell suspension was collected from each Erlene mayer flask and kept on constant shaking movement to have proper aeration (130rpm), at the temperature of 27 °C under fluorescent light with 40-80 PPFD and capped with sterile cotton cork or silicone sponge (Chem glass# CLS- 1490-028). In total 200-300 ml T87 cell suspension was collected from an assigned flask as harvest flask, whichpassed through an autoclaved and sterile Sieve Stainless Steel Wire Cloth (0.5mm, Adamas-Beta 0203725) that fitted a 600ml tall Becker to collect and separate larger cell clumps. This provided a fine and homogenous cell suspension without any dead cells colony or clump which was capable of passing through 20G needle. The cells were centrifuged for 5 minute at 4000RPM at 27 °C to separate cell pellet and discard the leftover of liquid T87 media. The T87 cell line was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.

[0173] In the next step, one 10 ml sterile leur lock syringe was filled with 5 ml of T87 cell pellet and mixed with 4 ml of CNF-Slurry-SMC (Cellulose Nanofibril 3%). After that, warm 2.5 ml of Alginic acid 2.8% + Agarose 1.5 was collected in another 10 ml syringe and mixed with cellulose gels, 2.5 ml of CNC-SD (Cellulose Nanocrytals- spray dry 10% gel) or in case of Bioink II Cellulose Slurry (CNC-Slurry 6%) was used.

[0174] To warm the alginate acid and agarose gel, microwave was used for 1 minute and after cooling down, it became safe for cells and did not solidify (37 °C). It was then collected with wide bore pipettes. The syringe that contains 5 ml of cells pellet is connected with the 4 ml of CNF-Slurry-SMC (Cellulose Nanofibril 3%) with a coupler to another 10 ml syringe with 2.5 ml of CNC-SD (Cellulose Nanocrytals-spray dry) and warm homemade gel. The syringes were connected with a coupler and the loaded gels were then mixed with cells by pushing and pulling the plunger of syringes. Attempt was made to mix the contents very well. The mixed Bioink with cells looked light green when very homogenous. In addition to shaking the mixture, when all cells and Bioink (homemade gels and Cellulose gel) were mixed and looked homogenous, a cap was used to close or seal the tip of the syringe. The syringe was then placed on vortex after ensuring that the bioink inside is very well mixed and completely homogenous. These steps were performed quickly to keep the cells alive and to avoid the Agarose based gel solidification. If solidified, the agarose would create clumps and hardened particles. 5 ml cell ink empty cartridge was loaded slowly to avoid any bubbles and after sealing and placing end closing rubbers the cartridge was vortexed to de-gass them and to take out all possible small air bubbles. In orderto ensure that minimum air bubbles are there, each cartridge was placed in 50 ml tubes and centrifuged at 27C, 5-10 minute of 4000 tpm prior to loading into Biox probe head, Pneumatic 5ml and printing.

[0175] Haystack structure was used as the Bioprinting model. After Bioprinting with BioX, if the structure of a Haystack, or mesh with size of Maximum: 10x10x3 cm, I minimum. 1x1x3mm, then treat with decellularizing protocol would follow.

[0176] Decellularizing scaffolds

[0177] The bioprinted scaffolds are left in 0.1 % SDS (in autoclaved dH2O) and incubated in room temperature for 2-4 days in 0.1 % SDS (BP166-500). Then the scaffolds are washed for 2-3 days in CaCI2 100mM at room temperature followed by washing 2 days in dH20. For insertion of scaffolds subcutaneously, the scaffolds are sterilized in 70% Ethanol for minimum 1 day and before insertion the scaffolds are washed in sterilized 0.9% Saline solution.

[0178] The following Table provides a summary of various gel formulations with ingredient weight percentages that were developed by the inventors.

[0179] Table 2: The summary of various formulations

[0180] Some of the formulations were found to be optimal in terms of biocompatibility and printability. A few formulations even showed T87 plant cell growth for 14 days where the plant cells survived and propagated. In some embodiments, the scaffolds were prepared using “DEAD” decellularized, lyophilized T87 cells in hydrogels

[0181] Figure 8A and 8B shows Lyophilized decellularized T87. Figure 8A shows T87 plant cells Macerated (30% solution of hydrogen peroxide (Fisher 18603263), DH2O and absolute glacial acetic acid (ACP chemicals A-0302) in 57C for 24 hours, after multiple washes with DH20 and decellularizing with 0.1 % SDS solution (BP166-500) for 2 days and washed with fresh CaCI2 100mM 2-3 days, and after washing with DIH20, Lyophilized and the powder like structure produced. Figure 8B shows the lyophilized decellularized scaffolds where the same formulation was bioprinted with Live T87 cells. The decellularizing technique discussed above was used. After washing with DH2O, the scaffolds were lyophilized.

[0182] Fig 9A shows the SEM image of surface area of a bioprinted scaffold with Live T87 cells with Gel-I and decellularized after bioprinting. Fig 9B shows the SEM image of a surface area of the bioprinted scaffold. Live T87 cells were bioprinted with Gel-I and decellularized after bioprinting. Figure 9C shows the SEM image, detail image of surface area of a bioprinted scaffold Live T87 cells bioprinted with Gel-I and decellularized after bioprinting.

[0183] Figure 10 shows the SEM data of T87 Live bioprinted scaffolds (homemade Alginate gel + Cellink) and Decellularized. Specifically, Figure 10A shows the SEM image of a cross-section area of a bioprinted scaffold. The scaffold was bioprinted with Live T87 cells and then decellularized. The T87 cells were clearly observed in the cross-section structure and embraced with cellulose nanofibrils. This resulted in a very homogenous and porous structure. Figure 10B and 10C shows the higher magnification of a cross-section area of a bioprinted scaffold. The scaffold was bioprinted with Live T87 cells and then decellularized. The T87 cells are clearly observed in the cross-section structure and embraced closely within the cellulose nanofibrils and has created a mesh like and porous structure throughout the inside of scaffolds. Figure 10D shows the higher magnification of a cross-section area, zoomed in on a T87 cell that is decellularized and clearly embraced within the cellulose nanofibrils.

[0184] Figure 11 shows the in vitro Biocompatibility test. Figure 11A shows the scaffolds with formulation Bio ink- lll-no pectin, were seeded by Fibroblast GFP-3T3 cells. The cells grew and the formulation was biocompatible. The images show the Fibroblast GFP-3T3 cells attached to the scaffold surface and growing and creating the layer of cells. The cells expressing GFP is visualized in green with confocal microscopy. To study the GFP-3T3 cell’s growth and survival; the phalloidin conjugated to Alexa Fluor 546 (Invitrogen) used for actin to visualize the cytoplasm in red and nuclei was stained by labelling the DNA with DAPI (Invitrogen). Samples were then mounted in Vectashield mounting media (BioLynX VECTH1000). Images were taken for 40X on Day 18. The scaffolds with formulation Gel-Ill were contained with T87-Live cells and after bioprinting, the scaffold was decellularized. The seeding number of cells was 100K cells.

[0185] Figure 1 1 B shows the scaffolds with formulation Bioink- V-Pectin which were seeded by Fibroblast GFP- 3T3 cells. The cells grew and the formulation was biocompatible. The images show the Fibroblast GFP-3T3 cells attached to the scaffold surface and growing and creating the layer of the cell. The cells expressing GFP are visualized in green with confocal microscopy. The GFP-3T3 cell’s growth the phalloidin conjugated to Alexa Fluor 546 (Invitrogen) was used for actin to visualize the cytoplasm in red and nuclei were stained by labelling the DNA with DAPI (Invitrogen). Samples were then mounted in Vectashield mounting media (BioLynX VECTH1000). Images were taken for 140X on Day 18 and the scaffolds with formulation V+Pectin were contained with T87-Live cells and after bioprinting the scaffold was decellularized. The seeding number of cells was 100K cells.

[0186] Figure 11 C shows the scaffolds Bioink-I, seeded by Fibroblast GFP-3T3 cells. The cells grew and the formulation was biocompatible. Control test. The images show the Fibroblast GFP-3T3 cells had lower attachment were not equally adhesive as the other formulations, but they was biocompatible. The Fibroblast GFP-3T3 cells are visualized in green as they express GFP. The cells expressing GFP were visualized in green, phalloidin conjugated to Alexa Fluor 546 (Invitrogen) was used for actin to visualize the cytoplasm in red and nuclei were stained by labelling the DNA with DAPI (Invitrogen). Samples were then mounted in Vectashield mounting media (BioLynX VECTH1000). Images were taken for 40X on Day 18.

[0187] Figure 12 relates to the in vitro Biocompatibility tests. Figure 12A shows the scaffolds with formulation Bioink-Ill (no pectin). This formulation was seeded by Fibroblast GFP-3T3 cells. The cells grew and the formulation was biocompatible. The images show the Fibroblast GFP-3T3 cells attached to the scaffold surface and growing and creating the layer of cells. The cells expressing GFP and visualized in green with confocal microscopy. To study the GFP-3T3 cell’s growth and survival; the phalloidin conjugated to Alexa Fluor 546 (Invitrogen) used for actin to visualize the cytoplasm in red and nuclei was stained by labelling the DNA with DAPI (Invitrogen). Samples were then mounted in Vectashield mounting media (BioLynX VECTH1000). Images were taken for 40X on Day 18. The scaffolds with formulation Gel-Ill were contained with T87-Live cells and after bioprinting the scaffold was decellularized. The seeding number of cells was 100K cells.

[0188] Figure 12B shows the same scaffold that is presented in Figure 12A, presented with 10x, lower magnification. The cells expressing GFP and visualized in green with confocal microscopy. To study the GFP-3T3 cell’s growth and survival; the phalloidin conjugated to Alexa Fluor 546 (Invitrogen) used for actin to visualized the cytoplasm in red and nuclei was stained by labelling the DNA with DAPI (Invitrogen).

[0189] NMR analysis using NIR-MIR spectroscopy (Nuclear Magnetic Resonance spectroscopy, commonly known as NMR spectroscopy or Magnetic Resonance Spectroscopy, MRS) is a powerful tool for material analysis. It provides valuable information about chemical composition, structure, and other material properties. Figure 13 shows the cellulose signature is seen in NMR study. The NMR study analyzed the materials that the scaffolds are made from. A) The red graph represents data analysis from T87 scaffolds bioprinted with homemade gel III and shows the peaks related to cellulose, confirming the high concentration of cellulose, b) The blue graph represents analysis data of scaffolds made with BY2 decellularized and shows a high content of pectin, cellulose, and a weak presence of galacturonan / hemicelluloses.

[0190] Figure 14 shows printing of “LIVE” T87 cells into a scaffold. Figure 14A shows the scaffolds with formulation Bioink-ll I- No pectin, and live T8 bioprinted with BIOX.

[0191] Figure 14B shows bioprinted scaffolds with formulation III no pectin with live T87. They maintained in sterile condition in plant media for 2 weeks. Haystack structure was printed in 10x10x3. The Bioink contains: Live T87 cells + Cellulose Nanofibrils(CNF) 3% + Cellulose Nanocrystals (CNC) 6% , Sodium Alginic Acid (2.8%), Agarose(1 .5%). Scaffolds after bioprinting were cross linked with autoclaved and Ph: 5.8 CaCh 100mM then incubate in a sterile T87 media (with double amount of Sucrose and Organic solutions in the master mix), and kept at RT(21 C) and under 60 PPFD fluorescent light. The scaffolds stayed sterile and cells were alive for about 2 weeks. BIOX bioprinter setting information used was needle size 20 G( light pink needle) , Speed 3mm / s, Pressure: 12 kPa, Infill 4%. The above settings and specifics are merely exemplary and other possibilities are encompassed within the invention.

[0192] Figure 14C provides an illustration of live T87 cells in a bioprinted scaffold with Bioink III a D1. Scaffold stained with plant cells viability assay kit (Sigma PA0100). The 3D scaffolds imaged with Confocal microscopy, 10X. The live cells are in green and flouescein positive, and dead cells are Propidium iodide positive.

[0193] Figure 14D provides an illustration of live T87 cells in a bioprinted scaffold with Bioink III at D8. Scaffold were kept in plant culture media and at D8 stained with plant cells viability assay kit (Sigma PA0100). The 3D scaffolds imaged with Confocal microscopy, 10X. The live cells are in green and flouescein positive, and dead cells are Propidium iodide positive. T87 cells remained alive and propagate.

[0194] Figure 15 shows in vitro biocompatibility test. Figure 15A shows the scaffolds with formulation Bioink-Ill- No pectin. The seeded Fibroblast GFP-3T3 cells grew and proliferated at the surface of scaffold. The testing was done for 18 days, 100k cells, seeding. The Fibroblast GFP-3T3 cells visualized in green as they express GFP. The phalloidin conjugated to Alexa Fluor 546 (Invitrogen) used for actin to visualized the cytoplasm in red and nuclei was stained by labelling the DNA with DAPI (Invitrogen). Figure 15B shows the scaffolds with formulation Bioink-IV+pectin formulation. Scaffolds were seeded by Fibroblast GFP-3T3 cells. The cells grew and the formulation were biocompatible. The images shows the Fibroblast GFP-3T3 cells attached on the scaffold surface and grew and created the layer of cell. The phalloidin conjugated to Alexa Fluor 546 (Invitrogen) used for actin to visualized the cytoplasm in red and nuclei was stained by labelling the DNA with DAPI (Invitrogen). Figure 15C shows the scaffolds with formulation Bioink-VI formulation. Scaffolds were seeded by Fibroblast GFP-3T3 cells. The cells grew and the formulation were biocompatible. The images shows the Fibroblast GFP-3T3 cells has less attachment on the scaffold surface and grew and created the layer of cells. The Fibroblast GFP-3T3 cells visualized in green as they express GFP. The phalloidin conjugated to Alexa Fluor 546 (Invitrogen) used for actin to visualized the cytoplasm in red and nuclei was stained by labelling the DNA with DAPI (Invitrogen). Figure 15D shows the bioprinted scaffold with Bioink I. The phalloidin conjugated to Alexa Fluor 546 (Invitrogen) used for actin to visualized the cytoplasm in red and nuclei was stained by labelling the DNA with DAPI (Invitrogen). TheFibroblast GFP-3T3 cells visualized in green as they express GFP. Samples were then mounted in Vectashield mounting media (BioLynX VECTH 1000). Images were taken for 10X at Day 18. The scaffolds with formulation III, and VI and IV were contained with T87-Live cells and after bioprinting the scaffolds were decellularized after bioprinting and after sterilizing used for seeding of Fibroblast GFP-3T3. The seeding number of cells for all scaffolds was 100K cells.

[0195] FIGURE 16 shows vitro Biocompatibility test of formulations with live T87 cells. Homemade Bioink+cellink. The live cells are visualized in green colour as they are positive for fluorescein positive, and dead cells are Propidium iodide positive in red. Figure 16A & 16B show scaffolds after crosslinking with sterile CaCh, Ph: 5.8 CaCI2 100mM incubated in plant culture media. The incubation was within a sterile T87 media (with double the amount of Sucrose and Organic solutions in the master mix), and kept at Room temperature (21 C) and under 60 PPFD fluorescent light. The scaffolds stayed sterile and cells were alive for about 2 weeks. The staining of 3d Bioprinted scaffolds was done with plant cells viability assay kit (Sigma PA0100). The 3D scaffolds were imaged with Confocal microscopy, 10X. Figure 16B show Bioink VI that contains T87 cells. Plant cells survived and grew in the scaffold. The live cells are visualized in green colour as they are positive for fluorescein positive, and dead cells are Propidium iodide positive in red.

[0196] Figure 17 shows solid state31P-nmr. A) The NMR study for identifying the solid state 31 P-nmr is provided. The results confirm that T87 survived and thrived in the homemade gel for 8-14 days. 1) The graph shows data from scaffolds made by Live T87 and after incubation for 14 days in plant media, decellularized and investigated by NMR and showed high content of Phosphor. That is interpreted as the sign of T87 cell survival and propagation in the bioprinted scaffold. 2) The graph is from the sample of T87 live bioprinted scaffolds that decellularized on Day 1 , right after bioprinting. The graph in red is completely negative for Phosphor. 3) The graph in blue colour demonstrates the existence of phosphor in a sample from live bioprinted T87 that named Pristine here. This graph was from a bioprinted scaffold without decellularizing. The observing traces of phosphor in 1 is aligned with data of fresh bioprinted scaffold with live T87 without decellularizing (3). The similar peaks at 50 and 0 for graph 1 and 3 shows the trace of phosphor and it confirms the T87 cell survival.

[0197] PEGDA AND BY2 CELLS

[0198] Regenerative medicine for tissue repair is an important and intense area of research. The ultimate goal of regenerative medicine is to repair or replace tissues in patients that have suffered from traumatic accidents or degenerative diseases, often by utilizing biomaterial scaffolds or matrices to direct or stimulate cell growth. There are numerous approaches for producing 3D matrices that support the growth of mammalian cells. One method of creating a biomaterial scaffold is through the use of decellularized plant cells. Decellularization is the process ofremoving all the cells from a plant tissue, leaving behind a cellulosic scaffold. It has been previously demonstrated that natural plant-derived cellulose scaffolds can be produced by employing common decellularization approaches, which will support 3D cell culture.

[0199] In the present proof-of-concept study the inventors focused on bringing together plant decellularization techniques with 3D bioprinting to create decellularized plant scaffolds with controllable architectures. More specifically, this study focused on the decellularization of the BY-2 cell line, a Japanese line that originates from the Tobacco plant. The resulting cellulose was then mixed with polyethylene glycol diacrylate (PEGDA), to create biocompatible ink, which was then incorporated in the Lumen-X 3D bio-printer. Scaffolds were then custom printed, giving a versatility of possible scaffold shapes, pore sizes, making them suitable for different applications in tissue engineering. Additionally, the study showed that mammalian cells could successfully attach on and colonize the scaffolds, and therefore, the structures were found to be a great support for newly formed tissues in receiving organism.

[0200] It was envisioned by the inventors that plant decellularization techniques can be combined with 3D bioprinting to create decellularized plant scaffolds with controllable architectures. This would involve using a biocompatible ink made by blending decellularized plant cells with polyethylene glycol diacrylate (PEGDA), which can be printed using UV curable technology. UV-crosslinking is a process in which a polymer is exposed to UV light to form covalent bonds between the polymer chains, resulting in a more stable and mechanically stronger material. This process can be used to crosslink PEGDA, a water-soluble, biocompatible polymer, to create biocompatible scaffolds. The Bio-printer Lumen-X is a 3D printing technology was used for proof-of-concept study to print and bond PEGDA using UV-crosslinking. By controlling the printing parameters and UV light intensity, the mechanical properties and pore size of the scaffolds can be optimized for specific tissue engineering applications. UV-crosslinking with PEGDA is known to be an effective method for creating mechanically stable scaffolds with high water retention, making it an attractive hydrogel.

[0201] It was envisioned that incorporating decellularized BY-2 cells into the 3D ink will help improve the biocompatibility. The addition of cellulose to a scaffold offers structural support for cells to grow and proliferate, acting as a template for the formation of new tissue. Cellulose also provides the necessary mechanical properties to maintain the integrity of the scaffold, resulting in a more stable and durable structure. In addition, the decellularized BY-2 cells have a microporous structure that enhances the attachment of mammalian cells. It would therefore be possible that this porous structure can absorb a large amount of biological fluids40, making it an ideal environment for simulating the properties of living tissues41 .

[0202] The BY-2 tobacco cell line is a well-established tool in plant tissue culture and biotechnology research, making it a potential candidate for this application. However, as noted earlier, any live, dead or decellularized plant cells can be used instead of BY-2 and this cell line was merely used for ease of access.

[0203] BY-2 cells have a short generation time, making them perfect for large scale production. With their porous structure and soft consistency, decellularized BY-2 cells can help create a scaffold that mimics the properties of living tissues. Therefore, in this proof-of-concept study the inventors brought together plant decellularization techniques with 3D bioprinting to create decellularized plant scaffolds with controllable architectures. They optimized the techniques of decellularization of BY-2 Tobacco, to specifically extract their cellulose structure. The resulting cellulose was then mixed with PEGDA to create biocompatible ink, which was incorporated in the Lumen- X 3D bio-printer. Scaffolds were then printed and their biological resistance and compatibility was assessed. The said scaffolds were found to form great structures for newly formed tissues in receiving organism.

[0204] For the formulation of hydrogel, a combination of decellularized BY-2 and PEGDA was used. The exposure time to UV light using a Lumen-X device was optimized. After several iteration, it was found that various percentage combinations of decellularized BY-2 and PEGDA mixture, resulted in the favorable hydrogel. For instance, a 30% decellularized BY-2 and 70 % PEGDA mixture provided a stable structure. The printer Lumen-X was used to control the UV exposure time, which meant that it can adjust the duration of the UV light exposure according to specific request. After trying different times, it was found that some time ranges worked better than the others. For instance, with 19 seconds of UV per lay of light exposure, it was found that the timed exposure approach worked nicely.

[0205] The inventors aimed to incorporate as much of the decellularized BY-2 in PEGDA as possible, while still maintaining a solid structure to find the appropriate balance between PEGDA and BY-2 amounts. Since the decellularized BY-2 cells are known to increase the porosity of the hydrogel at the microscale level and PEGDA is known for the mechanical properties and robustness of the final composite hydrogel, it was envisioned that the combination would provide stable porous scaffolds.

[0206] Figure 18 illustrates a comparison between live BY-2 cells and decellularized once. Image A showcases live BY-2 cells stained with Hoechst 3342), while image B showcases BY-2 cells afterthat decellularization protocol mentioned earlier, also stained with Hoechst 3342. This stain provides the investigation of any remained protoplast and cell nuclei. Notably, Image B exhibits an absence of nuclei. The images were obtained using Nikon confocal microscopy and analyzed with Imaged software. The scale bar in the image measures 50 pm.

[0207] Figure 19 illustrates the Hydrogel scaffolds design. The shaped hydrogel scaffolds were designed with various pore sizes, using the Lumen-X bioprinter and viewed through stereoscope microscopy. Scaffold A showcases a haystack shaped 10x10 mm with 9 pores of 2x2 mm each. Scaffold B showcases a cylinder-shaped scaffold with a circumference of 3.17 cm with 9 porous of 3.7 mm of circumference. Scaffold C showcases a cylinder-shaped scaffold with a circumference of 3.17 cm with 21 porous of 1.2 mm of circumference. The scale bar in the image measures 1 mm.

[0208] Figure 20 shows growth of 3T3 mice embryonic fibroblasts expressing GFP on hydrogel scaffolds. The cells were cultured for 10 days on the selected hydrogels scaffolds before being fixed with 10% formalin. Image A and B, captured through confocal microscopy using Nikon software and analyzed with Imaged, showcase the attachment of GFP positive cells on the cylinder-shaped scaffold. The scale bar in the image measures 100 pm.

[0209] The above results shows the versatility of hydrogel scaffolds, which were capable of being designed with various shapes as well as different porous sizes for different applications in tissue engineering. Additionally, it was evident the mammalian cells were able to successfully attach on the scaffold.

[0210] Cell adhesion experiment: This experiment on seeding cells aimed to investigate whether mammalian cells could adhere to the scaffold and proliferate. This evaluated the biocompatibility of the scaffolds with the NIH 3t3 cell line, specifically mouse embryonic fibroblast, chosen for their significant role in tissue healing and extracellular matrix placement. Following a 7-day incubation period, the NIH 3t3 mammalian cells exhibited attachment and elongation, particularly evident on the porous regions created during printing. Figure 28 demonstrates the attachment of the cell onto the scaffold, aided by triple staining of the actin, nuclei, and the cellulose scaffold. Figure 28A provides a comprehensive view of the scaffold’s porous structure, highlighting its cellulose composition. Meanwhile, figure 28B provides a closer look at the actin stain, facilitating the visualization of cell morphology and cytoskeleton organization.

[0211] Growth of 3T3 mice embryonic fibroblasts on hydrogel scaffolds: Figure 28: This figure shows the growth of 3T3 mice embryonic fibroblasts on hydrogel scaffolds. The cells were seeded at 1x105 cell / ml, cultured for 7 days on the hydrogel’s scaffolds, fixed with 10% formalin, and stained with DAPI (blue for nuclei), Phalloidin 546 (green for actin), and Congo Red (red for cellulose). Confocal microscopy was used to capture the images, which were then analyzed using Nikon software and Imaged. The images evidently demonstrate cell attachment. The scale bars were as follows: Scale bar A: 200 pm. Scale bar B: 100 pm.

[0212] Live / dead assay experiment: To evaluate cell proliferation, of PEGDA and PEGDA +BY-2 the scaffold samples were assessed at day 1 , 5 and 10, staining them with Hoechst 3332 and Propidium Iodide (PI). Hoechst,capable of permeating cell membranes to label DNA in both living and dead cells, was employed to mark the total number of cells. Conversely, PI selectively infiltrated cells with compromised plasma membranes, highlighting only dead cells in red30. In Figure 29A, 29B and 29C, the two colors filters were separated in the software Fiji, facilitating cell counting. To determine the number of live cells the inventors counted 3 field of view (FOV), n=9 per condition, and subtracted the dead cell count from the total.

[0213] Live / Dead Assay: Figure 29 provides a live / dead cell assay. Graph A illustrates the comparative analysis between PEGDA and PEGDA +BY-2 scaffolds over multiple time points (day 1 , 5, and 10), depicting cell density per 1 .6 pm2 FOV using Hoechst 33342 staining for all cells and Propidium iodide for dead cells. Graph B and C showcases the live and dead cells between 3 different time points (day 1 , 5, and 10). Figure D, E, and F present sequential confocal microscopy images of PEGDA + BY-2 scaffolds at each time point, with 3 scaffolds per condition and 3 distinct FOV analyzed per time point. This experiment was replicated 3 times for robustness. Confocal microscopy captured images, followed by analysis using Nikon software and Imaged. The scale bar for Figure D, E and F: 200 pm.

[0214] Young’s Modulus Understanding: Young’s Modulus in tissue engineering is important for comprehending tissue behavior, particularly given the diverse compositions of various scaffolds, which can significantly affect their level of resistance and elasticity. In Figure 30A, the comparison between PEGDA alone and PEGDA combined with BY-2 decellularized plant cells revealed an important insight. Notably, PEGDA alone exhibited more variation, with compressive modulus (Young’s modulus) values ranging from ±12.75 kPa to ±96.50 kPa, resulting in a larger standard deviation and less consistency. In comparison, PEGDA combined with BY-2 cells demonstrated a narrower range, with compressive modulus values spanning from ±47.00 kPa to ±71 .75 kPa. This highlights greater resilience of PEGDA combined with BY-2 cells, suggesting that PEGDA alone may be more prone to brittleness. This aligned with the objective of creating a hydrogel that maximizes elasticity which ensures that the scaffold can withstand pressure within the body without fracturing.

[0215] The bioprinting method offers a high degree of customizability, as highlighted in the introduction through the mention of Young’s Modulus. Figure 30B demonstrates the impact of varying UV light exposure time on the formulation of PEDGA + BY-2 scaffolds, as photo-crosslinking can be adjusted to modulate the compression modulus. The graph depicts exposure times of 4s, 10s, and 12s. Notably, the compressive modulus distinctly changed; at 4s, it registered a lower average of ±34.45 kPa; at 10s, an average of ±57.67 kPa was observed; and the 12s, the average rose to ±65.88 kPa. These findings underscore the tunability of the bioprinting process, enabling precise control over scaffold properties to meet specific application requirements.

[0216] Comparison of Young’s Modulus in various scaffolds: Figure 30 shows the comparison of Young’s modulus in various scaffolds. Graph A showcase the comparison between scaffolds composed of PEGDA only and PEGDA combined with BY-2 decellularized cells under a 10-second UV light exposure. Graph B illustrates the impact of different UV light exposure durations (4, 10 and 12 seconds) form the Lumen-X bio printer on the Young’s Modulus of the scaffolds. These assessments were conducted using the UniVert testing machine (CellScale) with the stress measured at 10 % total strain and a speed of 3 mm.min-1 . Each tested with 10 scaffolds.

[0217] Following the in vitro assays, the inventors then carried out in vivo experiments using female Sprague Dawley rats. In these experiments, 3D-printed scaffolds were placed subcutaneously in the rats. The study included two types of scaffolds: PEGDA and PEGDA+BY-2. Each type was tested at 3 different time point-4 weeks, 8 weeks, and 12 weeks- with for rats per condition per time point, totaling 24 rats. At each time point, the rats were euthanized, and the scaffolds were collected and fixed with 10% formalin. The samples were then embedded in paraffin, sectioned into 4 pm slices, and stained with Hematoxylin and Eosin (H&E), Masson’s Trichrome (MT), anti-CD31 , and anti-CD45.

[0218] Biocompatibility and Cell Infiltration of PEDGA+ BY-2 Scaffolds Implants: Figure 31 shows biocompatibility and cell infiltration of the scaffold implants. This figure presents axial sections of representative 3D-printed cellulose-based scaffolds stained with H&E and anti-CD45. These images illustrate acute moderate to severe foreign body reaction at 4 weeks (A) and at 8 weeks (B), followed by assimilation of the cellulose scaffold into the native rat tissue at 12 weeks (C). Higher magnification regions of interest as shown in D-F, as indicated in inset (A-C), provided detailed observation of cell population dynamics during biomaterial assimilation processes. The immune response observed with H&E staining ws further confirmed using anti-CD45 antibody, a known marker of leukocytes (G-l). The population of cells within the scaffold were mainly macrophages, multinucleated cells, and active fibroblasts. The scale bars were for D-l = 500 pm.

[0219] Comparison between PEGDA + BY-2 and PEGDA only: Figure 32 shows a comparison between PEGDA + BY-2 and PEGDA only. This figure compares axial sections of representative 3D-printed scaffolds at week 12. Image (A) shows the PEGDA scaffold stained with MT, with close-up images (E-l) that illustrates H&E and MT staining. Image (C) showcases the PEGDA + BY-2 scaffold stained with MT, with close -up images (G) for H&E stain and (K) for MT stain. Histochemical staining was used to facilitate a better comparison between the 2 scaffold types. Image (B) showcases anti-CD45 staining to show blood vessels infiltration, compared to Image (D), where the PEGDA+BY-2 scaffold exhibits slightly more infiltration. Image F and Image H are zoomed-in images of anti-CD45 and anti-31 are provided for both PEGDA and PEGDA+BY-2 scaffolds and shown in Image J and Image L. These results indicate that the PEGDA + BY-2 scaffold maintained better structural integrity and show enhanced cell infiltration. The scale bars for E-L = 500 pm.

[0220] FIBRIN AND BY-2 CELLS

[0221] In this proof-of-concept study, the inventors investigated the effect of fabricating composite scaffolds composed of decellularized Nicotiana Tabacum BY-2 cells bound in a Fibrin matrix. Such scaffolds were easily producible and moldable into many shapes / sizes and possessed tuneable mechanical properties. Results demonstrated that an increase in BY-2 volume fraction lead to a significant increase in the compressive Young modulus, while SEM imaging revealed an increased level of interconnected pores due to the distribution of BY-2 cellular structures in the Fibrin matrix. Rheological measurements featured G' > G" condition, confirming their gellike behavior, with a storage modulus that increased with increasing BY-2 volume fraction. To assess biocompatibility and cell proliferation, NIH3T3 / GFP fibroblast cells were cultured on the scaffolds. This proof-of- concept study demonstrates the potential use of these composite biomaterials produced from plant and protein polymers in tissue engineering applications.

[0222] The development of tissue engineering scaffolds with enhanced mechanical properties and biocompatibility is crucial for successful tissue regeneration. In this study, the incorporation of decellularized Bright Yellow-2 tobacco (BY2) cells into fibrin-based scaffolds was investigated and evaluated their impact on mechanical properties, morphology, and cell compatibility. Four different concentrations of BY2 cells (0 g / L, 5 g / L, 10 g / L, and 20 g / L) were incorporated into fibrin scaffolds. The compressive Young's modulus (YM) of the scaffolds increased with increasing BY2 concentration, with the highest YM observed for the 20 g / L BY2 concentration. The addition of BY2 cells acted as a reinforcing agent, strengthening the hydrogel structure, and increasing the fiber content. Rheological measurements demonstrated the solid-like behavior of the scaffolds, with both storage modulus (G') and loss modulus (G") increasing with higher BY2 concentrations. Scanning electron microscopy (SEM) images revealed a porous structure with uniform distribution of BY2 cells within the scaffolds, and the surfaces of the BY2 cells were coated with fibrin fibers. Live-dead cell analysis showed higher cell viability and attachment on scaffolds with higher BY2 concentrations. Overall, the incorporation of BY2 cells enhanced the mechanical properties and biocompatibility of the fibrin scaffolds, making them promising candidates for tissue engineering applications. These findings provide valuable insights for the design and optimization of plant-based scaffolds for tissue regeneration.

[0223] There has been significant research that focused on developing fibrin-based hydrogels for tissue engineering applications. Fibrin hydrogels have found broad application in tissue engineering due to their characteristics of high water content resembling tissues, strong biocompatibility, mechanical properties similar to soft tissues, effective transport of waste and nutrients, ability to uniformly enclose cells, and ability to gel after injection as a liquid. Fibrinogen and fibrin have multiple functions that overlap and affect blood clotting, tissue repair, inflammation, abnormal tissue growth, and interactions with cells and surfaces. These functions arecontrolled by different sites on fibrin, which may change during fibrin formation or interactions with other molecules. The structure of fibrin is essential for various biological functions, including thrombin binding, regulation of factor XIII, growth factor binding, and interactions with different cells such as platelets, fibroblasts, leukocytes, and endothelial cells. The process of how fibrin is formed from its constituent parts can be broken down into several stages. The first step is the removal of one or both of each type of fibrinopeptide. Depending on the speed at which the A and B fibrinopeptides are released, different paths of fibrin formation may occur. The path that fibrin takes is also affected by external factors such as pH, temperature, ionic strength, and the presence of other molecules and proteins. Fibrin is the final outcome of the blood clotting process, while fibrinolysis is a process that prevents blood clots from growing and causing problems.

[0224] Researchers have explored the unique characteristics of fibrin to develop novel strategies in tissue engineering, regenerative medicine, and drug delivery systems. Itosaka et.al. investigated the potential of using a fibrin matrix as a scaffold for bone marrow stromal cell (BMSC) transplantation in spinal cord injury treatment. Results showed that BMSCs could survive in fibrin matrix for 2 weeks, and animals treated with BMSC-fibrin matrix construct showed significant improvement in neurologic function recovery compared to those treated with vehicle or BMSC alone. Another study by Zhang et.al. revealed that aligned fibrin hydrogel (AFG) was helpful in the restoration of fibers in SCI lesions, leading to promoted functional recovery and limited glial scarring. Tractography and histological examination demonstrated the effectiveness of AFG in the rehabilitation of injured axons. In another study Nihouannen et.al. demonstrated that the combination of fibrin glue and microporous biphasic calcium phosphate granules could lead to new bone fillers with enhanced properties making them potentially useful as scaffolds for bone tissue engineering. Also, platelet-rich fibrin has shown the potential to eliminate microorganisms from wounds, and stimulate the formation of new blood vessels, which can be utilized for wound healing applications. These findings suggest that fibrin matrix could be a promising, minimally invasive scaffold for use in biomedical engineering applications.

[0225] Moreover, the integration of plant-based biomaterials into hydrogel systems offers a promising avenue for the development of sustainable and biocompatible tissue engineering scaffolds. Plant-based biomaterials have emerged as a promising alternative to traditional biomaterials for tissue engineering applications due to their unique advantages, such as biocompatibility, biodegradability, and cost-effectiveness. Modulevsky et.al. showed that 3D cellulose scaffolds made from apple hypanthium tissue can support the in vitro 3D culture of fibroblasts, muscle myoblasts, and epithelial cells. They showed that these cellulose scaffolds provide a favorable environment for the cells to adhere, invade and proliferate. In another study Hickey et.al showed that apple-derived cellulose scaffolds are also highly biocompatible and retain vascularization properties in vivo. Fontana et.al. showed that Human dermal fibroblasts (hDFs) cells follow the topographical cues of cellulose scaffolds derived from Solenostemon and parsley plants when cultured in-vitro. Cheng et.al showed that the Orientation OrderParameter of C2C12 cells grown on the surfaces of cellulose scaffolds derived from green onion was observed to be higher than that for the control (tissue culture plate). Finally, in a study that was conducted by, Modulevsky et.al. showed that Asparagus derived cellulose scaffold, due to their biocompatibility and vascularized structure, can significantly increase motor function in fully transacted spinal cord injured rats compared to control animals. These studies show that cellulose-based biomaterials have the potential for numerous applications in tissue engineering.

[0226] Biomaterials can be categorized as either biodegradable or non-biodegradable, indicating their ability to break down overtime or remain intact without dissolving. In this proof-of-concept study, the objective was to create a scaffold that struck a balance between these two properties. By achieving this balance, the inventors aimed to create a versatile scaffold that combined the advantages of both biodegradable and non-biodegradable materials. To explore this approach, Applicants incorporated decellularized Tobacco Bright Yellow 2 (BY2) cells into a fibrin matrix. It was speculated that the fibrin matrix would naturally degrade while the BY2 cells would provide longterm support for cell growth within the implant. After successful decellularization of the BY2 cells as discussed above, four samples with varying concentrations of decellularized BY2 cells were fabricated and embedded in fibrin. Through testing these samples, it was illustrated that the addition of decellularized BY2 cells can enhance the mechanical properties of these highly moldable and easily shapeable fibrin scaffolds. Additionally, by culturing 3T3 fibroblast cells on the scaffolds, their cell-supportive nature was demonstrated. Overall, the results presented in this study shed light on the promising potential of these fibrin-BY2 (FB) scaffolds for tissue engineering applications.

[0227] Materials and Methods

[0228] BY-2 cell culture

[0229] The culturing media with a composition of 4.3 g M&S salts media, 30 g sucrose, 100 mg inositol, 1 mg thiamine (1 ml, 1 mg / ml stock), 0.2 mg 2,4-D (20 pl, 10 mg / ml stock), 200 mg KH2PO4, with a total volume of 1 liter were prepared and the pH was adjusted to 5.8. Then, the prepared media were autoclaved and kept in the fridge (3 °C) for future use. Bright yellow-2 tobacco (BY-2) (Nicotiana tabacum) cell line was cultured as described previously (Nagata et al. 1992). The culturing was done every three days, where 2-5 ml of the cells were transferred to a new flask containing 95 ml of the culturing media and cultured in a rotary shaker at 130 rpm and 30 °C. The rest of the cells were harvested and centrifuged at 4000 rpm for 5 min. Subsequently, the supernatant was removed, and the cells were kept in PBS for further use.

[0230] Decellularizing BY-2 cells

[0231] In order to permeabilize the cell wall of the BY-2 cells, the cell harvest was kept in a -80 °c freezer overnight as a means to mechanically permeabilize the plant cell wall through the formation of ice crystals. The decellularization procedure was done according to the well-established protocol to remove DNA and cellular material from BY-2 cells while keeping the cellulose in the cell wall. After thawing the cell harvest at room temperature for 3 hours, the cells were centrifuged, the supernatant was removed, was added to 0.25 % (w / w) sodium dodecyl sulphate (SDS) solution, and kept on a stirrer (700 rpm) for four days with the SDS being refreshed each day. On the fourth day, to facilitate the removal of SDS by breaking the cells were centrifuged, moved to 400 ml of 100 mM CaCh solution being stirred overnight. Afterwards, the cells were washed one time with DI water to remove extra CaCh, then was stirred in 400 ml of fresh DI water overnight (700 rpm). Finally, the decellularized BY2 cells were centrifuged, the supernatant was removed, resuspended in PBS, sterilized in an autoclave and kept in a sealed container for further use.

[0232] Fabrication of Fibrin BY2 scaffolds

[0233] To investigate the impact of BY2 addition to fibrin on scaffold's mechanical properties, morphology, and biocompatibility, four different samples with varying concentrations of BY2 (20 g / L, 10 g / L, 5 g / L, and 0 g / L) were prepared. To maintain constant concentrations of other components, four different dilutions of decellularized BY2 in PBS was prepared. These dilutions were used to achieve the desired BY2 concentration in each final scaffold. For each sample, 40 pL of decellularized BY2 suspension was mixed with 11 pL of commercially available fibrin sealant Tisseel's solution 2, which contains 500 units / mL of thrombin and 40 pmol / mL of calcium chloride. The resulting solution was then combined with 11 pL of Tisseel's solution 1 , which contains 86.5 mg / mL of human fibrinogen and 3000 KlU / mL of fibrinolysis inhibitor. The final solution was immediately transferred to a sterilized custom-built Teflon mold with a 4 mm diameter (a), which was attached to a sterile culture plate. After 15 minutes, the mold was detached from the culture plate (b), and the scaffolds adhered to the underneath plate and were ready for use. Figure 21 show a) BY2 decellularization steps, and b) Steps for the fabrication of the FB scaffolds.

[0234] NIH3T3 Cell Culture

[0235] In this study, NIH3T3 mouse fibroblasts that had been genetically modified to express GFP were used. The cells were incubated at 37 °c and 5% CO2 in high-glucose Dulbecco's Modified Eagle Medium (DMEM) (Hyclone Laboratories Inc., Logan, UT) supplemented with 10% of fetal bovine serum (Hyclone Laboratories Inc., Logan, UT) and 1 % of penicillin-streptomycin (Hyclone Laboratories Inc., Logan, UT). The culture media was changed every 48 hours, and the cells were passaged when they reached 70% confluency. Fibrin-BY2 scaffolds were soaked in complete media for 1 hour before seeding to promote adhesion, and a 40 pL droplet containing 1 .0 x 10A4 cells was placed on top of each scaffold in a 24-well plate. In order to allow the cells to adhere to thescaffold, the samples were incubated for 4-5 hours. Subsequently, 2 mL of culture media was added to each well, and the samples were incubated at 37 °c and 5% CO2. The culture media was replaced every 48 hours, and the cells were cultured on the scaffolds for five days.

[0236] Live-Dead Cell Analysis

[0237] After 1 , 3, and 5 days of culturing, the culturing media was removed and each scaffold were stained with a 0.5 ml of solution of 10 pg / mL of Hoechst 33342 (Invitrogen) and 1 pg / mL of Propidium Podide (Invitrogen) in PBS and were incubated for 5 minutes to stain the cell nuclei of live + dead and dead cells, respectively, which was followed by another 5 minutes of washing with PBS. Confocal microscopy was then utilized to capture images of the biomaterials and the images were thresholded using Imaged (Fiji) adaptive threshold plug-in, then the number of the live and dead cells were counted using the analyze particles plug-in in Imaged.

[0238] Scanning electron microscopy

[0239] To prepare the samples for SEM imaging, the first step involved fixing them in 2.5% Glutaraldehyde. Next, they were dehydrated using a series of ethanol washes with increasing concentrations (35%, 50%, 70%, 95%, and 99%). The samples were dried by utilizing a PVT 3D critical point dryer (Tousimis) and the dried samples were then coated with gold using an ion sputter device and imaged using a dEOL dSM-7500 F FESEM at 2.00 kV. For scaffolds that were cultured with 3T3 cells, a different protocol was used to avoid disrupting their structure during critical point drying. In this case, the ethanol-dehydrated samples were treated with HMDS for 4 hours and air-dried overnight in a fume hood.

[0240] Young’s modulus measurements

[0241] The mechanical properties of the scaffolds were evaluated by subjecting them to compression using UniVert (CellScale) mechanical testing device. The compression was carried out at a rate of 3 mm / min and up to a maximum of 90% strain. The obtained force-displacement curves were converted to stress-strain curves, and the Young's modulus was determined by fitting the slope in a linear regime between 10 and 20% compression of the stress-strain curve.

[0242] Rheological measurements

[0243] The FB scaffolds' dynamic shear moduli were evaluated by subjecting them to strain-controlled oscillatory shear deformation using a Paar Physica rheometer (MCR 301) with a parallel plate geometry. Initial strain amplitude sweeps were conducted af fixed frequencies of co = 0.1 , 1 , and 10 rad / s to determine the linear responserange. Subsequently, frequency sweeps were performed over the range of 0.1 < co < 10 s-1 at a low strain amplitude (y0 = 0.1 %), both before and after undergoing large shear amplitude deformation (y0 = 100% at co = 1 s-1).

[0244] Results

[0245] Young modulus

[0246] The compressive Young's modulus (YM) of the scaffolds was measured for four different concentrations of BY2 in FB scaffolds (Figure 22). The results demonstrate a gradual increase in YM as the BY2 concentration is increased. Initially, starting with a zero gr / L BY2 concentration, the addition of 5 gr / L of BY2 causes a slight increase in YM from 1 1.74 kPa to 13.23 kPa, which is not statistically significant. However, when the BY2 concentration is further increased from 5 gr / L to 10 gr / L, YM shows a noticeable increase from 13.23 kPa to 15.98 kPa. This trend continues, with a significant increase in YM from 15.98 kPa to 24.09 kPa as the BY2 concentration is further increased to 20 gr / L in the FB scaffold. Figure 2 clearly shows that the YM for the 20 gr / L concentration is significantly higher than that for the other samples.

[0247] There are several possible reasons for this observation. Firstly, the additional BY2 acts as a reinforcing agent, thereby strengthening the hydrogel structure. Secondly, the addition of BY2 may increase the fiber content in the scaffold, as evident from the SEM images in the next section, where BY2's cell walls are composed of fibrous cellulose. This increase in fiber content enhances the mechanical properties of the hydrogel by facilitating more effective intermolecular interactions between the cellulose fibers and the fibrin network. Another possibility is that the increased cellulose content affects the porosity of the final hydrogel. Typically, hydrogels have a high-water content, which makes them relatively soft and deformable. However, the presence of BY2 can reduce the overall porosity of the hydrogel, resulting in a denser and stiffer structure.

[0248] Figure 22 shows the effect of BY2 Concentration on Compressive Young's Modulus (YM) of FB Scaffolds. Young’s Modulus gradually increased with higher BY2 concentrations. Young’s Modulus showed a noticeable increase from 13.23 kPa to 15.98 kPa as BY2 concentration increases from 5 gr / L to 10 gr / L. Young’s Modulus significantly increased from 15.98 kPa to 24.09 kPa at a BY2 concentration of 20 gr / L. (* indicates p < 0.05, *** indicates p < 0.005, **** indicates p < 0.001)

[0249] Rheological measurements

[0250] Figure 23, shows the frequency sweep (a,c) and amplitude sweep (b, and d) test for FB-0, FB-5, FB-10, and FB-20 samples. As shown in this figure, the storage modulus (G') consistently exceeded the loss modulus(G"), indicating the solid-like nature of the FB scaffolds, and the solid-like behavior was maintained along all the range of frequencies. As it is obvious from the figure, for both frequency and amplitude sweep with increasing the in the concentration of the BY2’s in the samples, both storage modulus (G') and loss modulus (G") increases. Each sample was tested with n = 6 replicates.

[0251] Frequency sweep results show that with increasing the frequency, storage modulus and loss modulus increases. This increase in the storage modulus at higher frequencies can be attributed to the fact that, as the frequency increases, the available time for the material to deform decreases. As a result, the material exhibits a stiffer response, leading to an increase in the storage modulus. Another reason could be the structural changes. At higher frequencies, the sample may undergo structural changes due to the rapid deformation cycles. This can result in an increase in the number of physical or chemical cross-links, entanglements, or other network structures and interactions between the BY2 cells and fibrin fibers in the FB scaffolds, leading to an increase in the storage modulus.

[0252] Also, from amplitude sweep curves it can be deduced that the storage modulus decreases dramatically with increasing the strain rate. A drop in storage modulus in the amplitude sweep test at higher strain rates can occur due to several reasons. One possible explanation could be related to changes in the structure. When subjected to higher strain rates, the material may experience changes in its structure caused by the intense shear forces. This can result in the weakening of the interactions between BY2 cells and fibrin fibers, leading to a decrease in the storage modulus and increase in the loss modulus. It can be inferred from this decline that FB scaffolds exhibit viscoelastic behavior where the viscous and elastic properties change with the rate of deformation. At higher strain rates, the viscous behavior may dominate, leading to a decrease in the storage modulus.

[0253] Frequency sweep shows moduli increase with frequency, likely due to reduced deformation time and potential structural changes. Amplitude sweep reveals storage modulus decreases with strain rate, suggesting viscoelastic behavior.

[0254] Figure 27 shows the frequency sweep (a) and amplitude sweep (b) tests for FB-0, FB-5, FB-10, and FB- 20 samples. As shown in the figure, the storage modulus (G1) consistently exceeded the loss modulus (G"), indicating the solid-like nature of the FB scaffolds. This is further supported by a low loss tangent (tan 6 < 0.14) across the entire frequency range, indicating the sustained solid-like behavior throughout the frequency spectrum. As shown in the figure, both storage modulus (G1) and loss modulus (G") increase with concentration of the BY2 cells in all rheological experiments. Also, the frequency sweep measurements show a modest increase of the storage and loss moduli with frequency for all samples. Moreover, the amplitude sweep measurements show thatthe storage modulus decreases substantially for all samples with increasing the strain amplitude, an indication of shear softening behaviour. Interestingly, this shear softening is accompanied by an increase in the loss moduli with increasing strain amplitude in those samples containing BY2 cells (while the pure fibrin sample G" is relatively insensitive to strain amplitude over the range studied here). The inventors also characterized sample deformation in response to a steady applied shear stress, Go .

[0255] Figure 27 (c) shows plots of the time-dependent creep compliance, J(t)=y(t) / oo, for the FB-0, FB-5, FB- 10, and FB-20 samples. As illustrated in the figure, it's evident that J(t) is a decreasing function of BY2 cell concentration at all times t, in accordance with the resulting storage moduli shown in Fig 3(a). Moreover, the time dependence of J(t) is sublinear in time, indicating that the system is not fluid-like at the applied stress (oo=15Pa), but rather undergoes a gradual internal restructuring process in response to applied stress.

[0256] These rheological measurements provide insights into the gel-like behavior of the FB scaffolds and the influence of BY2 concentration on their mechanical properties. The results from the frequency sweep indicate that as the frequency increases, both the storage modulus and loss modulus show an upward trend. The increase in the storage modulus at higher frequencies can be attributed to the fact that, as the frequency increases, the available time for the material to deform decreases. As a result, the material exhibits a stiffer response, leading to an increase in the storage modulus. Another reason could be the structural changes. At higher frequencies, the sample may undergo structural changes due to the rapid deformation cycles. This can result in an increase in the number of physical or chemical cross-links, entanglements, or other network structures and interactions between the BY2 cells and fibrin fibers in the FB scaffolds, leading to an increase in the storage modulus.

[0257] Additionally, according to the amplitude sweep curves, the storage modulus undergoes a notable decrease as the strain rate increases. A drop in storage modulus in the amplitude sweep test at higher strain rates can occur due to several reasons. One possible explanation could be related to changes in the structure. When subjected to higher strain, the material may experience changes in its structure caused by the intense shear forces. This can result in the weakening of the interactions between BY2 cells and fibrin fibers, leading to a decrease in the storage modulus and increase in the loss modulus. It can be inferred from this decline that FB scaffolds exhibit viscoelastic behaviorwhere the viscous and elastic properties change with the rate of deformation. At higher strain rates, the viscous behavior may dominate, leading to a decrease in the storage modulus.

[0258] Furthermore, creep compliance tests were conducted to investigate the long-term deformation response of the scaffold under a constant load. The results present significant insights into how BY2 concentration influences the mechanical properties of the hydrogel. FB-20 demonstrate the lowest creep compliance among all examinedconcentrations. This behavior indicates that a higher particle concentration enhances the hydrogel's resistance to long-term deformation under a constant load. As the amount of BY-2 in the scaffold was decreased the creep compliance increased in line with expectations for a hydrogel with no particle reinforcement. Surprisingly, FB-10 displayed higher creep compliance than FB-5. The fact that creep compliance tests involve long-term deformation under a constant load, which inherently takes into account prolonged stress conditions, could explain these unexpected results. The hydrogel with 10% BY-2 might exhibit higher creep compliance due to polymer-particle interactions and network disruption. This doesn't necessarily contradict the higher storage and compression moduli, as these moduli reflect the short-term response to stress.

[0259] Scanning Electron Microscopy (SEM)

[0260] To investigate the surface and cross-sectional characteristics of the scaffolds, SEM imaging was conducted for each sample. The SEM images in Figure 24 (a) depicts the cross-sectional area, revealing an interconnected porous structure present in the FB scaffolds. It is evident from the images that BY2 cells are uniformly distributed within the FB-5, FB-10, and FB-20 scaffolds. The SEM analysis also highlights the microscale size of the BY2 cells, ranging approximately between 40 to 130 micrometers, while the fibrin fibers exhibit a nano-scale size, approximately between 90 to 300 nanometers. The cross-sectional images further demonstrate that the FB scaffolds containing BY2 cells exhibit a distinct phenomenon: the surfaces of the BY2 cells are coated with fibrin. This occurrence can be attributed to the sequential process in which BY2 cells are introduced to the scaffold prior to fibrinogen coagulation, facilitating the formation of fibrin fibers on the BY2 cells. Furthermore, the surfaces of the BY2 cells are predominantly covered with smaller-sized fibrinogen fibers. Figure 24 (b) showcases the surface morphology observed in the FB-10 scaffolds revealing that the BY2 cells are embedded within the Fibrin matrix. As the concentration of BY2 increases, the surface roughness becomes more pronounced. Notably, FB-20 exhibits the roughest surface morphology, attributed to a higher concentration of BY2 cells compared to the other three samples. Conversely, the FB-0 scaffold demonstrates the smoothest surface among the samples.

[0261] Figure 24 provides an SEM Analysis of FB Scaffolds and BY2 Cell Distribution, a) Cross-sectional SEM images illustrating the interconnected porous structure of FB scaffolds and the uniform distribution of BY2 cells within FB-5, FB-10, and FB-20 scaffolds. BY2 cells are depicted in micro-scale size (40-130 pm), while fibrin fibers exhibit a nano-scale size (90-300 nm). The images also reveal the coating of BY2 cell surfaces with fibrin, attributed to the sequential introduction of BY2 cells prior to fibrinogen coagulation. Smaller-sized fibrinogen fibers predominantly cover the surfaces of BY2 cells, b) SEM image of the surface area of FB-10 scaffold revealing BY2 cells embedded within the Fibrin matrix (BY2 cells highlighted in yellow, fibrin fibers in purple)

[0262] Live dead cell analysis

[0263] To assess the biocompatibility of the FB scaffolds, 3T3-GFP cells were seeded onto the scaffold surface for 1 , 3, and 5 days. Live and dead cells were labeled and counted using Hoechst and PI stains at each time point. Hoechst is capable of permeating both live and dead cell walls, binding to the DNA in the nucleus, while PI only penetrates the cell walls of dead cells. Figure 25(a) illustrates the live / dead cell ratios for different concentrations of BY2 in FB scaffolds at 1 , 3, and 5 days while Figure 25(b) represents the total cell number of the 3T3 cells over time. An increase in BY2 concentration from 0 to 10 g / L is associated with an increase in the live / dead cell ratio, with FB-10 scaffold displaying the highest ratio. However, a decline in the live / dead cell ratio becomes evident when the BY2 concentration exceeds 20 g / L. Furthermore, Figure 25(c) exhibits a representative confocal image of a 3T3-GFP-seeded FB scaffold, highlighting live cells in blue and dead cells in red. Additionally, Figure 26 displays an SEM image of a 5-day-seeded FB-10 scaffold with 3T3-GFP, showing a strong attachment of 3T3 cells (colored in sea green) on BY2 cells (colored in yellow) after the degradation of surface fibrin fibers.

[0264] Figure 25: Cell Viability and Live Cell Count on Various Scaffolds, a) Live / Dead cell ratio for FB-0, FB-5, FB-10, and FB-20 over a 5-day period, b) Number of live cells for FB-0, FB-5, FB-10, and FB-20 over a 5-day period. The n-value for each time point and each scaffold was n = 9. c) Representative confocal image of Hoechst- Pl assay on FB-10 scaffold at day 5. Blue represents Hoechst-stained live cells, while red indicates Pl-stained dead cells.

[0265] Figure 26: SEM image of 3T3 cells on FB-10 scaffold after five days, demonstrating BY2 cells in yellow, 3T3 cells in sea-green, and fibrin fibers in purple.

[0266] These findings can be explained as follows: Fibroblast cells are known to express integrin, a protein that facilitates binding to fibrinogen. This indicates that a favorable surface coated with fibrin promotes cell adhesion and proliferation. As revealed by SEM analysis, FB scaffolds containing BY2 cells exhibit a coating of fibrin on the surfaces of the BY2 cells within the scaffolds. Additionally, as the concentration of BY2 in the scaffold increases, the scaffold's surface becomes rougher, providing more surface area for 3T3 cells to attach and grow on. Consequently, an increase in the live / dead cell ratio can be observed with increasing BY2 concentration, up to 10 g / L. However, once the BY2 concentration exceeds 10 g / L and reaches 20 g / L, the scaffolds become excessively dense, as evidenced in the young modulus section, impeding cell spreading and proliferation. As a result, the live / dead cell ratio decreases compared to lower concentrations of BY2.

[0267] It is important to note that the goal of this study was to achieve a balance between biodegradable and non-biodegradable materials by introducing non-biodegradable BY2 cells into the biodegradable fibrin matrix. Figure 6 clearly demonstrates that by day 5, fibrin has degraded from the surface of the FB-10 scaffold, and the 3T3 cells have attached to the BY2 cells. This balance holds potential benefits, particularly in scenarios like spinalcord injury, where a scaffold providing temporary support that eventually degrades could aid in the regeneration of damaged tissue.

[0268] In conclusion, this study investigated the mechanical properties, rheological behavior, surface morphology, and biocompatibility of fibrin-BY2 (FB) scaffolds with varying concentrations of BY2. The results demonstrated that the addition of BY2 to the fibrin scaffold significantly influenced its characteristics. The compressive Young's modulus (YM) of the scaffolds increased gradually with an increase in BY2 concentration up to 10 gr / L, followed by a bigger jump from 10 gr / L to 20 gr / L. This increase in YM can be attributed to the reinforcing effect of BY2 and the enhanced fiber content in the scaffold, resulting in more effective intermolecular interactions. The increased cellulose content may also contribute to the denser and stiffer structure of the hydrogel. Rheological measurements revealed that the FB scaffolds exhibited solid-like behavior, with the storage modulus (G') consistently exceeding the loss modulus (G"). Both storage and loss moduli increased with an increase in BY2 concentration. Higher frequencies led to an increase in storage modulus, indicating a stiffer response of the material. The amplitude sweep test showed a drop in storage modulus at higher strain rates, suggesting viscoelastic behavior of the FB scaffolds. SEM images displayed an interconnected porous structure in the FB scaffolds, with uniform distribution of BY2 cells. The surfaces of BY2 cells were coated with fibrin, indicating successful incorporation of BY2 into the scaffold. As the BY2 concentration increased, the surface roughness became more pronounced, with FB-20 exhibiting the roughest surface morphology. Live-dead cell analysis demonstrated the biocompatibility of the FB scaffolds. An increase in BY2 concentration up to 10 g / L correlated with a higher live / dead cell ratio compared to that for neat fibrin, indicating better cell viability of Fibrin-BY2 scaffolds. Overall, the findings of this study highlight the potential of BY2 in fibrin-based scaffolds. The addition of BY2 improved the mechanical properties, influenced the rheological behavior, and affected the surface morphology of the scaffolds. Further investigations can focus on optimizing the BY2 concentration to achieve the desired properties for specific tissue engineering applications.

[0269] In this study, the new method to purify a sustainable cellulose-based Bioink was tested. In the research using BY2 cells, the challenging issue of reproduction of plant-based material was resolved as this organism is an immortalized plant line. In addition, the production and purification of this model was cheaper and less labor intensive than other wood, pulp and bacterial-derived cellulose. The presented method showed the developed plant-derived biomaterial is as efficient as other available acellular materials along with lower prices and challenges in its production. Like most acellular materials, the developed biomaterial was a porous structure. This porosity is a key factor in designing biostable scaffolds, it allows cell migration, vascularization, tissue growth, and cell viability inside constructs. Based on previous works, acellular materials that have been used in bioink formulations combine collagen, fibrin, nanocellulose, chitosan, and polycaprolactone (PCL). Most of these common acellular materials mostly are not consciously sourced requiring extensive processing. The naturally derived materials purified fromvertebrates. In addition, most of animal / human-derived ECM have risk factors of allergenicity and contamination. Therefore, the plant-based scaffolds have an advantage in terms of lacking the signalling molecules to induce an autoimmune response. The other very popular ECM is nanocellulose and has a plant-based source; however, it has low stability. Therefore, in this study the scaffolds were fabricated with the novel blend of decellularized BY2 cells. For better gelation, alginate was used and nanocellulose was added, and cross-linked it with Ca, as a result, a very stable, biocompatible bioink developed.

[0270] The efficient technique to decellularize plant tissue as noted above was utilized which has added an acidic maceration step in the decellularizing protocol and lyophilized the decellularized plants to have better stability. This technique allowed harvesting a high number of cells and possibility of conserving them at room temperature for over a year. It was relatively easy to handle and carry as well in comparison to another naturally cellulose biomaterial.

[0271] Through biocompatibility tests and studying microscopic analyses of mammalian cells NIH 3T3-GFP on scaffold and mechanical test, it was found that the formulation with BY2 decellularized cells were more manageable and could shape structures with higher resolution. In addition, it was biocompatible and non-toxic. Although, this biopolymer is biologically inactive, it could mimic the ECM and is degradable. The novel CNF-Slurry- SMC + CNC-SD + AG + BY2 blend showed appropriate printing and enabled the fabrication of construct with open micropores. This technique could be optimized to produce bioink in less than a month in a biology lab without substantial resource investments or refinement with toxic chemicals. It also doesn’t require complex cross linking. The scaffolds can store after dry freeze in room temperature. As a result, application of BY2 cells or any live, dead, or decellularized cells, or plant cell lines, show a great potential for tissue engineering purpose and are a great alternative for other biomaterial sources open new horizon toward greener and Eco-friendly biopolymers. This method and fabricated biomaterial can replace other carbonaceous materials. Indeed, the reproduction of BY2 or other plant cells and purification of Biopolymers from that is cheaper and faster than other Carbonaceous Materials that are derived from Biomasses such as Chitosan. Furthermore, purification of Nanocellulose from pulp and wood and other agricultural waste requires capital costs and utilizing land with the cost of transportation and workforce. Indeed, the lab-grown BY2 with a significant growth rate; 100 fold per week. Moreover, if media has more phosphate by threefold compared to the common MS medium from Nagata’s formulation 17, BY2 cells grow more biomass and as a result faster and cheaper this biomass can be used for developing cellulose based biopolymers. Indeed, growing these cells in the lab is more feasible and manageable in regards to their ambient temperature, feeding regiment, logistics, and monitoring in comparison with the purification of biopolymers from cellulose sources in plants, corps, woods, algae, sea squirts ( Ascidiacea ), and Bacteria . Indeed, as ancient biomaterials humankind has been using this most abundant renewable organic matter in our biosphere. The annual biosynthetic production of cellulose is estimated to be over 1011 tons. Consequently, this model of using plant cells anddecellularizing method have a promising prospect in the field of green bioprinting and green biofabrication. Hence, this novel biomass purification method and designed biopolymer have vast potential for use in biomedical and tissue engineering applications. In conclusion, the presented method can use in any research lab that can maintain BY2 cells and therefore, prepare an in-house biopolymer at less cost and complications.

[0272] Crosslinkable biocompatible formulation with Collagen and Hyaluronic Acid

[0273] The bioink needs to be extrudable through the bioprinter injection needles, flowable and stable as a gel to keep the structure shape for posterior hardening by crosslinking. Formulations with these characteristics can be obtained by mixing biocompatible croslinkable gels with stabilizers, that creates a structured matrix for cell scaffolding.

[0274] The inventors tested gel formulations that can be packed in syringes to be injected or extruded. These compound mixtures, as a semi-solid but substantially dilute cross-linked system to be used in physiological environments, in vitro or in vivo.

[0275] The bioink extrusion procedure generally must compromise plasticity and provide self-supporting, flow, and structure. The material must be such that possesses an inherent organization that can keep the printed shape for a given time stabilized by the structural lattice created by the mixture components. The inventors attempted to prepare a formulation that’s resistant but not rigid and which is obtained by the biomaterial’s flexibility and dynamic interactions.

[0276] A bioprinted structure that is resistant to compression can be obtained by crosslinking formulation components. As described earlier, alginate has several positive aspects such as simplicity, reversibility, and biocompatibility. Another aspect to keep in mind with bioprinting application is calcium release that softens the 3D construct leading to desegregation.

[0277] Cellulose is a natural polymer isolated from plants. Consequently to the arrangement of the enzyme machinery, plants produce it with a structure consistently standardized to the genera to which it belongs. Cellulose is the main component of cell walls and is the structure that creates protection, compartmentalizing metabolic structures and compounds.

[0278] The function that cellulose serves in nature can be used fortissue engineering. Cellulose is a non-charged molecule, insoluble but polar, interacting highly with water and other biological compounds. These characteristics lead to easy interactions with the animal tissue environment within cells promoting adherence (Entcheva et al.2004; Modulevsky et al. 2014, 2016; Ferreira et al., 2020) and differentiation (Li et al., 2014; Courtenay et al., 2019).

[0279] Cellulose forms stable suspensions relying on its hydroxyl groups, absorbing water at different degrees depending on the particle aspect ratio (Etale et al., 2023). The swelling of cellulose depends on crystal arrangements and particle size after the extraction method; in our experiments cellulosic isolate from apples absorbed 98% of water creating viscous and stable gels. Thus, the cellulosic isolate of apples or similar plants I fruits is a high adherent surface for animal cells, improving the biocompatibility and tissue integration.

[0280] The bleached cellulosic isolate from decellularized apple tissue is a biocompatible and highly water absorbent. It creates dynamic interactions forming stable suspensions with flow and viscosity. Its physicochemical behavior is similar to nanocelluloses, being a versatile chemically adaptable material which makes it perfect for bioink applications.

[0281] The inventors present proof of concept formulations based on decellularized and bleached cellulosic isolate from apples, merely as an example, to construct printable and moldable pieces, parts or arquiteted structures for implanting, repairing, or improving animal tissues. Its extensive surface area as a consequence of the unique laminar morphology, promotes the water interaction through weak bonds to create stable self-structured construct in formulations with gelling agents,

[0282] The inventors also present a stepwise process to create ordered and homogeneous stable suspensions for this purpose. It also presents proof of concept constructs for biocompatibility assessment showing improved cell adherence.

[0283] Experimental Data

[0284] Several high and low concentrations (w / w) of formulations were tested where calcium-free PBS was used as a solvent. In the following section the abbreviations “Co” relates to Collagen, the abbreviation “HA” relates to Hyaluronic acid and “q.s” means quantum satis, meaning “as much as is enough”.

[0285] Hyaluronic acid is a very effective gelling agent to mix with crosslinkable polymers such as Alginate (or Alginic Acid) and its water retention is similar to cellulosic isolate from apples in the range of 0.5 - 0.7 g.g-1 .

[0286] The following table provides various trial formulations and their concentrations.

[0287] Production methodology

[0288] Materials were weighted and mixed vigorously using a vortex inside a falcon tube. The mixing process was completed by repeated laminar flux between two syringes. The stopper was removed from the barrel and the mixtures were kept for degassing inside a vacuum chamber for 5 minutes with the pump on. Repeatedly (until no bubbles formed) gels were seated again by impact / inertia (impacting against the bench) in the syringe bed horizontally to have a surface area for degassing as big as possible. Figure 33 shows the gel production process.

[0289] Figure 34 shows examples of gel formulations based on the formulation with Alginate (or Alginic Acid) and cellulosic material isolated from apples where A1.5: Alginate 1.5%, A0.5M1.5: Alginate 0.5% Cellulosic isolate 1.5%, A1 M1.5: Alginate 1% Cellulosic isolate 1.5%, A1 M3: Alginate 1% Cellulosic isolate 3%. The concentrations provided are merely representative and not binding in any way.

[0290] These bioink formulations were tested by being extruded using a syringe, two consecutive times, stacking each injection with approximately 100 pg. Formulations that kept injected material stacked were considered selfstructured as perthe eligibility criteria. Figure 35 shows the experimental results of the extrusion experiment where the bioink formulations were extruded two times. Where AO.5% = Alginate at 0.5% and M 1 .5% = cellulosic isolate from apples at 1 .5%; A1 % = Alginate at 1 % and M 3% = cellulosic isolate from apples at 3%.

[0291] It was noted that the following bioink prototypes I formulations i.e. Alginate 1 % + cellulosic isolate from apples at 3%, Alginate 1 %, and cellulosic isolate from apples at 3% + Collagen 30 mg / g, Alginate 1 % + cellulosic isolate from apples at 3% + Hyaluronic acid 15 mg / g, performed well as self-structured drops. These concentrations are merely representative and not binding in any way.

[0292] Table 3 provides trials and formulation with representative concentrations which were tested as gel and to see if they were extrudable, self-structured and printable.

[0293] Figure 36 shows a 3D construct made with Alginate 1 % cellulosic isolate from apples at 3%, shaped as a grid with a syringe and 25G needle. Figure 37 shows a 3D construct made with Alginate 1 %, and M3 which stands for cellulosic isolate from apples at 3%, shaped as a hexagonal prism with a syringe and 25G needle.

[0294] Furthermore, several pieces were constructed by molding and crosslinking on CaCh submersion. Rings were produced using a syringe with an 18.81 mm diameter, with a solution of 4% alginate, and other with Alginate 1 % and cellulosic isolate from apples at 3%). After 24h of crosslink in CaCh solution the pieces shrinked. A reduction in diameter of 5% was observed for the piece produced with Alginate at 4%, and a contraction of 15% was observed for the one produced with Alginate 1 % cellulosic isolate from apples at 3%, as shown in Figure 38. Figure 38 shows 3D constructs made with Alginate at 4% and Alginate 1 % cellulosic isolate from apples at 3% shaped as a ring by molding. Table 4 shows the experiments that were carried out for validation of the various bioink formulations.

[0295] Bioink Biocompatibility: Several bioink prototypes were tested as shown in the table below. The table provides trial formulations with representative concentrations.

[0296] Test-piece production and validation: The biocompatibility assessment was conducted using a modified syringe as a mold to produce pieces with reproducible dimensions, a barrel volume of 20 uL and a surface area of ~296 mm2as observed in Figure 39. Figure 39 shows test pieces for bioinks biocompatibility assessment, where A. is the dimensions, B. is the piece production in CaCh immersion for crosslinking, and C. test piece after crosslinking before mold detachment. Note that pieces were placed in DMEM solution for a week for degradation in this media.

[0297] Results: The test pieces were placed in sterile 24 well plates, 3T3-gfp cells (50k for each 20 uL barrel) after 1 h covered with media (DMEM, glucose). The experiments were performed during 9 days for cell growth. The experiment was realized as described below and as shown in Figure 40. Figure 40. (A) shows barrel opening of the test piece, (B) shows cells were inoculated inside the test piece, and after 1 h (C) shows test pieces were covered with media. The cell development was monitored every day.

[0298] Figure 41 shows results for A1 M3-HA where cells grow everywhere, covering the piece surface. Figure 42 shows A1 M3 in day #9 with a clear change in the surface. After 9 days the pieces were fixed and stained with Hoechst than Congo red using the protocol as described on methods session. The picture in Figure 9 presents a test piece based on the formulation Alginate 1 % + cellulosic isolate from apples at 3% after Hoechst staining with the visible change on the surface.

[0299] Results for the sample A1 M3: Cells are seen to be covering the surface of the piece. The cellulosic scaffold supports cellular growth at the top. Figure 43 shows results for sample A1 M3 (A = Alginate 1 %, and cellulosic isolate from apples at 3%): (A) Test piece: longitudinal cut, 10x magnification. (B) Inside the barrel: longitudinal cut, magnification of 100x.

[0300] Figure 44 shows sample with Alginate 1 % + cellulosic isolate from apples at 3%: images of the Hoechst and Congo Red stained cells, with the red filter for cellulose, blue for the nuclear matter, and green for thefluorescent membrane of 3T3-gfp cells, and with the merged images at the bottom right, magnification 100x, scale bar 100 urn.

[0301] Results for the sample with Alginate 1 %, and cellulosic isolate from apples at 3% + Collagen 30 mg / g: Cells are seen to cover the surface in great extension. Cells are always close to the cellulose: cell adherence.

[0302] Figure 45 shows results for sample Alginate 1 %, and cellulosic isolate from apples at 3% + Collagen 30 mg / g: The composite bottom right image of the Hoechst and Congo Red stained cells, with the red filter for cellulose, blue for the nuclear matter, and green for the fluorescent membrane of 3T3-gfp cells, magnification 100x, scale bar 100 urn.

[0303] Results for the sample with Alginate 1 %, and cellulosic isolate from apples at 3% + Hyaluronic Acid 15 mg / g: Cells are seen to cover the surface in great extension. Visually, many more cells are apparent. Presence of glicocalix can also be seen.

[0304] Figure 46 shows results of sample Alginate 1 %, and cellulosic isolate from apples at 3% + Hyaluronic Acid 15 mg / g: The composite image at right image, with stained cells with Hoechst and Congo Red, blue for the nuclear matter, and green for the fluorescent membrane of 3T3-gfp cells, magnification 100x, scale bar 100 urn.

[0305] Physico-chemical parameters: Viscosity results

[0306] For this analysis the equipment used was Anton Paar ViscoQC 300, and spindle type was SC4-21 , with shear rate: 0.5 / s, run time: 1 min, and 6 readings for each sample for material homogenization.

[0307] The values of the viscosity for various bioink formulations can be found below.

[0308] Conclusion: Alginate and cellulose are non-newtonian materials as more shear thinning results in lower viscosity when more force is applied. The pressure applied to the plunger and flow speed also influence the viscosity parameter. The viscosity impacts the maximum flow rate and clogging potential.

[0309] Young modulus - mechanical test: For this analysis, several formulations were tested. 50N with Alginate 4%, and 10N a) Alginate 1 %, and cellulosic isolate from apples at 3%, b) Alginate 1 %, and cellulosic isolate from apples at 3% + Collagen 30 mg / g, and c) Alginate 1 %, and cellulosic isolate from apples at 3% + Hyaluronic acid 15 mg / g.

[0310] The test pieces of crosslinked bioinks, triplicate measurements, 10% compression, 3.5 to 4.5 mm high, were analyzed with Origin. The Young’s modulus can be defined as the slope (gradient) of the trendline expressing the ratio, E = Tensile Stress / Tensile Strain. The values obtained for the Young’s modulus are in table 7, and the charts where the values were obtained are in Figure 47.Table 7. Value of Young’s modulus for the samples of bioink prototypes.

[0311] Figure 47 shows strain Vs stress chart for the crosslinked materials, (A) Alginate 4%, (B) Alginate 1 %, and cellulosic isolate from apples at 3%, (C) Alginate 1 %, and cellulosic isolate from apples at 3% + Collagen 30 mg / g, (D) Alginate 1 %, and cellulosic isolate from apples at 3% + Hyaluronic acid 15 mg / g. The gradient from thetrendline equation is Young’s modulus, E = Tensile Stress / Tensile Strain, the material stiffness in the elastic region of the chart.

[0312] Conclusion: By translating the elastic strain, it is evident that higher values mean harder pieces. The values obtained show that the formulations can be modulated to generate constructs with a range of mechanical strengths. Cellulosic isolate from apples is a good platform to accept different compounds creating pieces with different degrees of hardness.

[0313] Cell Culture Methods: The principal steps used for this animal cell culture were; Part A: Culture media preparation; Part B: Cell culturing; Part C: Cell passage - test pieces inocula; and Part D: Microscopy

[0314] All work was developed in a sterile environment and with sterile materials, in biological hood (BSC), and manipulated with the best practices to minimize the risk of contamination.

[0315] Cell Culture Materials: The following key chemicals and solutions were used.

[0316] Part A: Culture media preparation

[0317] Culture media• MEM Alpha (1X) (w / L-Glutamine, ribonucleosides and deoxyribonucleosides), Gibco, REF A10490-1 (500 mL bottle) (for MC3T3-E1 / Subclone 4 cells)• DMEM / HIGH GLUCOSE (w / 4.00 mM L-Glutamine, 4500 mg / L Glucose and Sodium pyruvate), HyClone, Cat No SH30243.01 (500 mL bottle) (for 3T3 cells)• Foetal Bovine Serum (FBS)• Penicillin and Streptomycin (P / S)

[0318] Part B: Cell culturing:• Completed culture media (w / FBS and P / S)• Frozen cell line from the -80°C freezer

[0319] Part C: Cell passageCulture medium (w / FBS et P / S)• 0,05% trypsin (in sterile PBS)• Sterile Phosphate Buffered Saline (PBS)

[0320] Procedure: Part A: Culture media preparation• Preheat a water bath to 37°C.• Place a bottle of sterile medium, FBS and P / S in the water bath for about 20 minutes.• Sterilize the BSC and turn on the ventilation.• Sterilize all the material and place it in the BSC.• Remove 55 mL of medium from the bottle (there will be 445 mL remaining).• Add 50 mL of FBS (final concentration of 10%) and 5 mL of P / S (final concentration of 1%) to the content of the bottle.• Make sure the medium is well mixed.• The medium can be used now. Store at 4°C for further usage.

[0321] P roce d u re : Part B: Cell culturing• Preheat a water bath to 37°C.• Place a bottle of prepared medium in the water bath for about 20 minutes.• Sterilize the BSC and turn on the ventilation.• Sterilize the equipment before putting it in the BSC.• Activate the vacuum pump and sterilize the hose line for liquid collection from pasteur pipettes.• Take a tube of the desired cells from the freezer (-80°C) and let them thawTransfer the cells in a Falcon tube.• Add 2 ml of preheated medium in the tube.• Centrifuge for 3 minutes at 1000 RPM.• Return the tube in the BSC (disinfect the tube before inserting).• Remove the supernatant with a glass disposable Pasteur pipette and a vacuum pump.• Resuspend the cells (pellet) in 1 mL of medium.• Transfer the cellules in a sterile petri dish.• Add 9 mL of medium to the petri dish.• Gently mix the content of the petri dish (in 8 shapes) to make sure that the cells are dispersed on the surface of the dish.• Write the date, type of cells, passage number and initial on the lid of the petri dish with a permanent marker.• Place the cells in the incubator at 37°C with 5% CO2.

[0322] Procedure: Part C: Cell passage• Preheat a water bath to 37°C.• Place a bottle of sterile medium, trypsin and PBS in the water bath for about 20 minutes.• Take a look at the cells under the microscope.• Sterilize the BSC and turn on the ventilation.• Sterilize all the material and place it in the BSC.• Activate the vacuum pump and sterilize the hose line for liquid collection from pasteur pipettes.• Aspirate the culture medium with a glass pasteur pipette and a pump.Add 2 mL of PBS to the petri dish to wash the cells.• Aspirate the PBS with a glass pasteur pipette and a pump.• Add 2 mL of 0,05% trypsin to the cells.• Place the petri dish containing the cells and trypsin in an incubator at 37°C and 5% CO2 for about 3-4 minutes.• Look at the cells under the microscope to ensure that the cells are well detached from the bottom of the petri dish before proceeding to the next steps.• Add 5 mL of medium to the petri dish and shake gently to suspend the cells.• Transfer the cells to a Falcon tube.• Collect an aliquot of the cell suspension from the Falcon tube to do a cell count.• Centrifuge for 4 minutes at 1000 RPM and during the waiting time, carry out the cell count (see Part D: Cell count).• Aspirate the supernatant with a glass pasteur pipette and a pump.• Resuspend the cells (pellet) in a volume of culture medium (see the calculation example above in section D).• Add the cells and a volume of culture medium to a sterile petri dish (the volume depends on the desired cell concentration).• Gently mix the content of the petri dish (8 shape) to make sure that the cells are dispersed on the surface of the dish.• Write the date, cell type, passage number and initials on the lid of the petri dish with a permanent marker.• Place the cells in an incubator at 37°C and 5% CO2

[0323] Procedure: Part D: Cell count• Clean an hemocytometer and the coverslip that comes with the hemocytometer with a small amount of EtOH 70%. Wipe with Kimwipes to not damage the glass, and to use the coverslip with the hemacytometer because a normal slip cannot be used.• Place the coverslip in the centre of the hemocytometer (above the grid).• Pipette around 20 pL of the cell suspension (do not forget to resuspend).• Place the tip in the slot on the side of the hemocytometer and eject a volume of the cell suspension quickly to fill all the space under the coverslip without creating air bubbles.• Repeat the previous step on the other side of the hemocytometer if needed.• Place the hemocytometer under the microscope.• Identify the squares that will be used for the cell count (see Figure 2).• Count cells one square at a time. Always count the cells in the squares from top to bottom, not counting the cells touching the bottom and right lines (see Figure 3 for a schematic representation).

[0324] A person skilled in the art would understand that the above procedures and material concentrations are merely representative and one can modify the steps and / or concentrations to achieve similar output / results. In order to calculate the total number of cells the following formula was used: Total # of cells = (average of cells counted) x 10 000 x (final volume in the Falcon tube before centrifugation)

[0325] For example: In the 4 squares, the number of cells were 110, 95, 102 and 99. The average therefore would be 101 .5 cellules ((110 + 95 + 102 + 99) Z 4 =101 .5). Before centrifugation, a final volume of 7 mL was there. Therefore, the number of cells in the Falcon tube is 7.105x 10A6 (101 .5 x 10 000 x 7 = 7.105x 10A6 cells).

[0326] With the total number of cells, one can resuspend the cells in a certain volume of medium to obtain the desired cell concentration. For example: if one wants to resuspend cells to obtain a concentration of 2x10A6 cells / mL, with 7.105x 10A6 cells in the Falcon tube, one will have to add 3.55 mL of culture medium (7,105x 10A6 cells / 2x10A6 cells / mL = 3.55 mL).

[0327] Finally, clean the hemocytometer and coverslip with EtOH 70% and Kim wipes, then place them in their appropriate bag and case. Dispose of the cell aliquot and pipette tips in a Biohazard waste bag.

[0328] Cell Staining: The key chemicals and solutions were involved.• Paraformaldehyde (4%) (PFA)• Phosphate Buffered Saline (PBS)• Congo Red (CR)• Hoechst

[0329] Sample preparation1 . Before imaging, it is important to prepare the sample properly.2. If you are doing live cell imaging, make sure the imaging is complete within 30 minutes. Keep the sample hydrated, and do not remove it from the sterile culture dish if it is to be returned to the incubator.3. If the samples are to be fixed, remove the cell culture media under aseptic conditions and gently wash with PBS 3X.4. Add 4% PFA to the sample and ensure that it is completely covered. Note: the fixation time depends on the size of the sample. Cells on Petri dishes require ~ 10 minutes. Thick samples may require 30 min, 1 h, or overnight if they are very large. Caution: check the requirements of the stain before performing the fixation. It is rare, but some stains require very short fixation times <3 min.5. After the fixation, remove the PFA, and dispose of it in the waste beaker in the fume hood. 6. Wash the samples 3X with PBS.

[0330] Congo Red staining (scaffold)1. Prepare a 0.1% CR (m / v) solution in dH2O, media, or PBS depending on the intended application.2. Some of the CR will not fully dissolve. Remove particulate matter with a 0.22 pm filter and a syringe.3. Add the CR to the sample. Ensure the sample is fully submerged in the dye. Incubate at room temperature for 30 min.4. Note: when finished, parafilm the CR solution and store in the fridge for further use.5. Remove the stain and wash gently with PBS 3X.

[0331] Hoechst staining (cell nuclei)1 . Note: the Hoechst stain can be used for live and fixed cells. Therefore, it must be kept sterile at all times; if you are using it for fixed cells, it still must be opened in the BSC. Hoechst is light-sensitive and should be returned to the fridge as soon as possible.2. Make a dilution of 1 :1000 of the Hoechst stain in media or PBS. Mix well with a pipette. 3. Add the solution to the sample and ensure full coverage. Incubate for 30 minutes in the dark. 4. Remove the stain and wash gently with PBS 3X.

[0332] Viscosity: Viscosimetric analysis was performed on an Anton Paar model ViscoQC 300 equipment, using an SC4-21 spindle, running time experiments of 1 minute.

[0333] Mechanical test: The Young’s modulus for the 3D constructs was obtained using CellScalle equipment, processing with UniVert software.

[0334] Test pieces were produced using an adapted cuvette with a rubber ending for expelling the construct (Figure 48. A). Formulations were injected to fill all the space; it was placed inside a vacuum chamber to air removal for the sake of piece homogeneity then dropped in a CaCI2 2% aqueous solution (figure 48, B). After 24h the molded pieces were unmolded and cut to precise contact area measurement, Figure 48, C and D. Figure 48 shows the test piece for mechanical test.

[0335] A 10% deployment was measured for each material in triplicate. Force by deployment data were used to calculate the Strain and Stress based on area and construct the graphics. Young’s modulus was the slope for the graph produced using the Strain versus stress curve.

[0336] Thus, the cellulosic isolate creates a highly adherent matrix for bioink formulations that is self-structured after the extrusion process in layered bioprinting, as well as for molding of engineered structures. The cellulosic isolate based gels creates self-structured constructs by extrusion without need of immediate crosslinking.

[0337] Cellulosic isolate particle size may range from 150 pm wide to 250 pm wide, with an average of 220 pm wide, in a thin rounded shape morphology, and presents less than 1 % of particles under 20 pm, reducing the chance of requests for specific toxicological assessment by regulatory agencies.

[0338] The particle size of the cellulosic isolate from apples or similar plants or fruits is small and flows through some of the most commonly used needles employed for bioprinting. The use of the cellulosic isolate from apples or similar plants or fruits for cell growth is advantageous as a highly biocompatible gelling matrix for bioink formulations, Cell adherence was found to be more pronounced for the formulation with cellulosic isolate from apples or similar plants or fruits than with alginate solely, showing that it can be a modulating factor as well as collagen and hyaluronic acid for cell adhesion.

[0339] This invention as described refers to the use of cellulosic isolate from fruits and vegetables, and all the results exposed were based on the material isolated from apples for the sake of clarity. The formulations produced presents improved physicochemical and cell interaction properties. It was found that the formulations based on cellulosic isolate are compatible with the bioink criteria of extrudability, flowability, and crosslinkable properties.

[0340] The cellulosic isolate from apples generates stable suspensions with viscosity and gel behavior that creates self structured constructs after extrusion. The polar nature of cellulosic isolate of apples interacts with gelling agents and proteins to create homogeneous gels, such as Hyaluronic acid and Alginate and Collagen. The cellulosic isolate from apples creates an adherent surface for cells, alone or mixed with Hyaluronic acid and Collagen. Thus it is an inexpensive and effective way to incorporate biocompatible architected structures as healing or cosmetic parts.

[0341] In conclusion, our based on cellulosic isolate from apples creates bioinks that amplify the bioperformance of engineered constructs for healing, reparatory or cosmetic applications considering the inherent cell adherence of this material. The bioprinting technology could prove useful for partial allografts substitution, to create reparatory and cosmetic implants of engineered constructs, or for food bioprinting.

[0342] The invention has been described with regard to one or more embodiments, however, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined by the claims. All citations and / or references recited herein are hereby incorporated by reference in their entirety.

[0343] REFERENCES1. Mao, A. S. & Mooney, D. J. Regenerative medicine: Current therapies and future directions. Proc Natl Acad Sci U S A 112, 14452-14459 (2015).2. Padmalayam, I. & Suto, M. J. 3D Cell Cultures. Mimicking In Vivo Tissues for Improved Predictability in Drug Discovery, in Annual Reports in Medicinal Chemistry vol. 47 367-378 (Academic Press Inc., 2012).3. Modulevsky, D. J., Lefebvre, C., Haase, K., Al-Rekabi, Z. & Felling, A. E. Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture. PLoS One 9, e97835 (2014).4. Modulevsky, D. J., Cuerrier, C. M. & Felling, A. E. Biocompatibility of Subcutaneously Implanted Plant- Derived Cellulose Biomaterials. PLoS One 11 , e0157894 (2016).5. Hickey, R. J., Modulevsky, D. J., Cuerrier, C. M. & Felling, A. E. Customizing the Shape and Microenvironment Biochemistry of Biocompatible Macroscopic Plant-Derived Cellulose Scaffolds. ACS Biomater Sci Eng 4, 3726-3736 (2018).6. Modulevsky, D. J., Lefebvre, C., Haase, K., Al-Rekabi, Z. & Felling, A. E. Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture. PLoS One 9, e97835 (2014).7. Gershlak, J. R. et al. Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds. Biomaterials 125, 13-22 (2017).8. Jansen, K. et al. Spinach and Chive for Kidney Tubule Engineering: the Limitations of Decellularized Plant Scaffolds and Vasculature. AAPS Journal 23, (2021).9. Carter, N., Grant, I., Dewey, M., Bourque, M. & Neivandt, D. J. Production and Characterization of Cellulose Nanofiber Slurries and Sheets for Biomedical Applications. Frontiers in Nanotechnology 3, (2021).10. Das, S., Ghosh, B. & Sarkar, K. Nanocellulose as sustainable biomaterials for drug delivery. Sensors International 3, (2022).11. Lin, N. & Dufresne, A. Nanocellulose in biomedicine: Current status and future prospect. European Polymer Journal vol. 59 302-325 Preprint at https: / / doi.Org / 10.1016 / j.eurpolymj.2014.07.025 (2014).12. Seidel, J. et al. Green bioprinting: Extrusion-based fabrication of plant cell-laden biopolymer hydrogel scaffolds. Biofabrication 9, (2017).13. Landerneau, S., Lemarie, L., Marquette, C. & Petiot, E. Green 3D bioprinting of plant cells: A new scope for 3D bioprinting. Bioprinting vol. 27 Preprint at https: / / doi.Org / 10.1016 / j.bprint.2022.e00216 (2022).14. Park, S. M., Kim, H. W. & Park, H. J. Callus-based 3D printing for food exemplified with carrot tissues and its potential for innovative food production. J Food Eng 271 , (2020).15. Beckwith, A. L., Borenstein, J. T. & Velasquez-Garcia, L. F. Tunable plant-based materials via in vitro cell culture using a Zinnia elegans model. J Clean Prod 288, (2021).16. Phan, N. v., Wright, T., Rahman, M. M., Xu, J. & Coburn, J. M. In Vitro Biocompatibility of Decellularized Cultured Plant Cell-Derived Matrices. ACS Biomater Sci Eng 6, 822-832 (2020).17. Nagata, T., Nemoto, Y. & Hasezawa, S. Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants. Int Rev Cytol 132, 1-30 (1992).18. Numata, K. et al. Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Sci Rep 8, (2018).19. Guolla, L., Bertrand, M., Haase, K. & Pelling, A. E. Force transduction and strain dynamics in actin stress fibres in response to nanonewton forces. J Cell Sci 125, 603-613 (2012).20. Bose, S., Roy, M. & Bandyopadhyay, A. Recent advances in bone tissue engineering scaffolds. Trends in Biotechnology vol. 30 546-554 Preprint at https: / / doi.Org / 10.1016 / j.tibtech.2012.07.005 (2012).21. Seror, J., Stern, M., Zarka, R. & Orr, N. The potential use of novel plant-derived recombinant human collagen in aesthetic medicine. Plast Reconstr Surg 148, 32S-38S (2021).22. Hospodiuk, M., Dey, M., Sosnoski, D. & Ozbolat, I. T. The bioink: A comprehensive review on bioprintable materials. Biotechnology Advances vol. 35 217-239 Preprint at https: / / doi.Org / 10.1016 / j.biotechadv.2016.12.006 (2017).23. Biswal, T., BadJena, S. K. & Pradhan, D. Sustainable biomaterials and their applications: A short review, in Materials Today: Proceedings vol. 30274-282 (Elsevier Ltd, 2020).24. Varma, R. S. Biomass-Derived Renewable Carbonaceous Materials for Sustainable Chemical and Environmental Applications. ACS Sustainable Chemistry and Engineering vol. 7 6458-6470 Preprint at https: / / doi.Org / 10.1021 / acssuschemeng.8b06550 (2019).25. Nagata T, K. F. Plant cell biology through the window of the highly synchronized tobacco BY-2 cell line. (1999).26. Kargarzadeh, H., loelovich, M., Ahmad, I., Thomas, S. & Dufresne, A. Methods for Extraction of Nanocellulose from Various Sources. (2017).27. loelovich Michael. Plant_Biomass_as_a_Renewable_Source_of_B. Lambert Academic Publishing (2013).28. Doran, P. M. Send Orders of Reprints at reprints@benthamscience.net Current Medicinal Chemistry, vol. 20 (2013).29. Hakkinen, S. T. et al. Tobacco BY-2 media component optimization for a cost-efficient recombinant protein production. Front Plant Sci 9, (2018).30. Mao, A. S. & Mooney, D. J. Regenerative medicine: Current therapies and future directions. Proceedings of the National Academy of Sciences 112, 14452-14459 (2015).31. Padmalayam, I. & Suto, M. J. 3D Cell Cultures, in 367-378 (2012). doi:10.1016 / B978-0-12-396492- 2.00024-2.32. Fontoura, J. C. et al. Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance. Materials Science and Engineering C 107, (2020).33. Jeon, J., Lee, M. S. & Yang, H. S. Differentiated osteoblasts derived decellularized extracellular matrix to promote osteogenic differentiation. Biomater Res 22, (2018).34. Assungao, M. et al. Cell-Derived Extracellular Matrix for Tissue Engineering and Regenerative Medicine. Frontiers in Bioengineering and Biotechnology vol. 8 Preprint at https: / / doi.org / 10.3389 / fbioe.2020.602009 (2020).35. Khan, H. M. et al. Smart biomaterials and their potential applications in tissue engineering. Journal of Materials Chemistry B vol. 10 6859-6895 Preprint at https: / / doi.org / 10.1039 / d2tb01106a (2022).36. Janani, G., Pillai, M. M., Selvakumar, R., Bhattacharyya, A. & Sabarinath, C. An in vitro 3D model using collagen coated gelatin nanofibers for studying breast cancer metastasis. Biofabrication 9, 015016 (2017).37. Ghasemi-Mobarakeh, L., Kolahreez, D., Ramakrishna, S. & Williams, D. Key terminology in biomaterials and biocompatibility. Curr Opin Biomed Eng 10, 45-50 (2019).38. Chen, X. et al. Functional Multichannel Polypropylene Fumarate)-Collagen Scaffold with Collagen- Binding Neurotrophic Factor 3 Promotes Neural Regeneration After Transected Spinal Cord Injury. Adv Healthc Mater 7, 1800315 (2018).39. Zheng, H. & Zuo, B. Functional silk fibroin hydrogels: preparation, properties and applications. Journal of Materials Chemistry B vol. 9 1238-1258 Preprint at https: / / doi.org / 10.1039 / d0tb02099k (2021).40. Islam, M. M., Shahruzzaman, M., Biswas, S., Nurus Sakib, M. & Rashid, T. U. Chitosan based bioactive materials in tissue engineering applications-A review. Bioactive Materials vol. 5 164-183 Preprint at https: / / doi.Org / 10.1016 / j.bioactmat.2020.01 .012 (2020).41 . Reddy, M. S. B., Ponnamma, D., Choudhary, R. & Sadasivuni, K. K. A comparative review of natural and synthetic biopolymer composite scaffolds. Polymers vol. 13 Preprint at https: / / doi.org / 10.3390 / polym13071105 (2021).42. Sonam, S., Chaudhary, H., Arora, V., Kholi, K. & Kumar, V. Effect of physicochemical properties of biodegradable polymers on nano drug delivery. Polymer Reviews 53, 546-567 (2013).43. Elmowafy, E. M., Tiboni, M. & Soliman, M. E. Biocompatibility, biodegradation and biomedical applications of poly(lactic acid) / poly(lactic-co-glycolic acid) micro and nanoparticles. Journal of Pharmaceutical Investigation vol. 49 347-380 Preprint at https: / / doi.org / 10.1007 / s40005-019-00439-x (2019).44. Kiss, E., Kutnyanszky, E. & Bertoti, I. Modification of Poly(lactic / glycolic acid) Surface by Chemical Attachment of Polyethylene glycol). Langmuir 26, 1440-1444 (2010).45. Makadia, H. K. & Siegel, S. J. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel) 3, 1377-1397 (2011).46. Zarrintaj, P., Saeb, M. R., Jafari, S. H. & Mozafari, M. Application of compatibilized polymer blends in biomedical fields, in Compatibilization of Polymer Blends 511-537 (Elsevier, 2020). doi:10.1016 / B978-0-12- 816006-0.00018-9.47. Han, F. et al. Tissue Engineering and Regenerative Medicine: Achievements, Future, and Sustainability in Asia. Frontiers in Bioengineering and Biotechnology vol. 8 Preprint at https: / / doi.org / 10.3389 / fbioe.2020.00083 (2020).48. Modulevsky, D. J., Lefebvre, C., Haase, K., Al-Rekabi, Z. & Pelling, A. E. Apple derived cellulose scaffolds for 3D mammalian cell culture. PLoS One 9, (2014).49. Adamski, M. et al. Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications. Journal of Visualized Experiments (2018) doi:10.3791 / 57586.50. Abdul Khalil, H. P. S. et al. A Review on Plant Cellulose Nanofibre-Based Aerogels for Biomedical Applications. Polymers (Basel) 12, 1759 (2020).51 . Salimi, S., Sotudeh-Gharebagh, R., Zarghami, R., Chan, S. Y. & Yuen, K. H. Production of Nanocellulose and Its Applications in Drug Delivery: A Critical Review. ACS Sustainable Chemistry and Engineering vol. 7 15800- 15827 Preprint at https: / / doi.org / 10.1021 / acssuschemeng.9b02744 (2019).52. Huerta, R. R., Silva, E. K., Ekaette, I., El-Bialy, T. & Saldana, M. D. A. High-intensity ultrasound-assisted formation of cellulose nanofiber scaffold with low and high lignin content and their cytocompatibility with gingival fibroblast cells. Ultrason Sonochem 64, (2020).53. Modulevsky, D. J., Cuerrier, C. M. & Pelling, A. E. Biocompatibility of Subcutaneously Implanted Plant- Derived Cellulose Biomaterials. PLoS One 11 , (2016).54. Phan, N. v., Wright, T., Rahman, M. M., Xu, J. & Coburn, J. M. In Vitro Biocompatibility of Decellularized Cultured Plant Cell-Derived Matrices. ACS Biomater Sci Eng 6, 822-832 (2020).55. Modulevsky, D. J., Lefebvre, C., Haase, K., Al-Rekabi, Z. & Pelling, A. E. Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture. PLoS One 9, e97835 (2014).56. Hickey, R. J., Modulevsky, D. J., Cuerrier, C. M. & Pelling, A. E. Customizing the Shape and Microenvironment Biochemistry of Biocompatible Macroscopic Plant-Derived Cellulose Scaffolds. ACS Biomater Sci Eng 4, 3726-3736 (2018).57. Hickey, R. J., Leblanc Latour, M., Harden, J. L. & Pelling, A. E. Designer Scaffolds for Interfacial Bioengineering. Adv Eng Mater (2022) doi:10.1002 / adem.202201415.58. Holmes, J. T. et al. Homemade bread: Repurposing an ancient technology for in vitro tissue engineering. Biomaterials 280, (2022).59. Mariani, E., Lisignoli, G., Borzi, R. M. & Pulsatelli, L. Biomaterials: Foreign bodies ortuners forthe immune response? International Journal of Molecular Sciences vol. 20 Preprint at https: / / doi.org / 10.3390 / ijms20030636 (2019).60. Szymanski, T. et al. Hyaluronic acid and multiwalled carbon nanotubes as bioink additives for cartilage tissue engineering. Sci Rep 13, 646 (2023).61. Jin, S. et al. Recent advances in PLGA-based biomaterials for bone tissue regeneration. Acta Biomater 127, 56-79 (2021).62. Chia, H. N. & Wu, B. M. Recent advances in 3D printing of biomaterials. J Biol Eng 9, (2015).63. Murphy, S. v. & Atala, A. 3D bioprinting of tissues and organs. Nature Biotechnology vol. 32 773-785 Preprint at https: / / doi.org / 10.1038 / nbt.2958 (2014).64. Parhi, R. Cross-linked hydrogel for pharmaceutical applications: A review. Advanced Pharmaceutical Bulletin vol. 7 515-530 Preprint at https: / / doi.org / 10.15171 / apb.2017.064 (2017).65. Eshel-Green, T., Eliyahu, S., Avidan-Shlomovich, S. & Bianco-Peled, H. PEGDA hydrogels as a replacement for animal tissues in mucoadhesion testing. Int J Pharm 506, 25-34 (2016).66. Magalhaes, L. S. S. M. et al. Nanocomposite Hydrogel Produced from PEGDA and Laponite for Bone Regeneration. J Funct Biomater 13, (2022).67. Cristovao, A. F. et al. Customized tracheal design using 3D printing of a polymer hydrogel: influence of UV laser cross-linking on mechanical properties. 3D Print Med 5, (2019).68. Patil, S. S., Shinde, V. S. & Misra, R. D. K. pH and reduction dual-stimuli-responsive PEGDA / PAMAM injectable network hydrogels via aza-michael addition for anticancer drug delivery. J Polym Sci A Polym Chem 56, 2080-2095 (2018).69. Calo, E. & Khutoryanskiy, V. v. Biomedical applications of hydrogels: A review of patents and commercial products. European Polymer Journal vol. 65 252-267 Preprint at https: / / doi.Org / 10.1016 / j.eurpolymj.2014.11.024 (2015).70. Ahmed, E. M. Hydrogel: Preparation, characterization, and applications: A review. Journal of Advanced Research vol. 6 105-121 Preprint at https: / / doi.Org / 10.1016 / j.jare.2013.07.006 (2015).71 . Phan, N. v., Wright, T., Rahman, M. M., Xu, J. & Coburn, J. M. In Vitro Biocompatibility of Decellularized Cultured Plant Cell-Derived Matrices. ACS Biomater Sci Eng 6, 822-832 (2020).72. Nagata, T., Nemoto, Y. & Hasezawa, S. Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants. Int Rev Cytol 132, 1-30 (1992).73. Nagata, T., Nemoto, Y. & Hasezawa, S. Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants, in 1-30 (1992). doi:10.1016 / S0074-7696(08)62452-3.74. Abasalizadeh, F. et al. Alginate-based hydrogels as drug delivery vehicles in cancer treatment and their applications in wound dressing and 3D bioprinting. Journal of Biological Engineering vol. 14 Preprint at https: / / doi.Org / 10.1 186 / sl 3036-020-0227-7 (2020).75. Gangolphe, L. et al. Electrospun microstructured PLA-based scaffolds featuring relevant anisotropic, mechanical and degradation characteristics for soft tissue engineering. Materials Science and Engineering C 129, (2021).76. Waqanivavalagi, S. W. F. R., Ground, M. B., Alarcon, C., Milsom, P. & Cornish, J. Subcutaneous surgical rat models for the evaluation of decellularised heart valve immunogenicity: A systematic review. Materialia (Oxf) 21 , (2022).77. Khorramirouz, R. et al. A novel surgical technique for a rat subcutaneous implantation of a tissue engineered scaffold. Acta Histochem 120, 282-291 (2018).78. Doench, Ingo, et al. "Cellulose nanofiber-reinforced chitosan hydrogel composites for intervertebral disc tissue repair." Biomimetics 4.1 (2019): 19.79. Howard, D., et al., Tissue engineering: strategies, stem cells and scaffolds. Journal of anatomy, 2008. 213(1): p. 66-72.80. Vacanti, J.P. and R. Langer, Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. The lancet, 1999. 354: p. S32-S34.81. Fernandes, H., et al., Extracellular matrix and tissue engineering applications. Journal of materials chemistry, 2009. 19(31): p. 5474-5484.82. Khademhosseini, A. and R. Langer, A decade of progress in tissue engineering. Nature protocols, 2016. 11 (10): p. 1775-1781.83. Khademhosseini, A. and R. Langer, Microengineered hydrogels for tissue engineering. Biomaterials, 2007. 28(34): p. 5087-5092.84. Wichterle, O. and D. Lim, Hydrophilic gels for biological use. Nature, 1960. 185(4706): p. 117-118.85. Hoffman, A.S., Hydrogels for biomedical applications. Advanced drug delivery reviews, 2012. 64: p. 18-23.86. Zhao, Y., et al., Supramolecular adhesive hydrogels for tissue engineering applications. Chemical Reviews, 2022. 122(6): p. 5604-5640.87. Mantha, S., et al., Smart hydrogels in tissue engineering and regenerative medicine. Materials, 2019. 12(20): p. 3323.88. Naahidi, S., et al., Biocompatibility of hydrogel-based scaffolds for tissue engineering applications. Biotechnology advances, 2017. 35(5): p. 530-544.89. Alakpa, E.V., et al., Tunable supramolecular hydrogels for selection of lineage-guiding metabolites in stem cell cultures. Chem, 2016. 1 (2): p. 298-319.90. Chu, T.L., et al., In-vitro and in-vivo biocompatibility of dECM-alginate as a promising candidate in cell delivery for kidney regeneration. International Journal of Biological Macromolecules, 2022. 211 : p. 616-625.91. Ningrum, D.R., et al., In Vitro Biocompatibility of Hydrogel Polyvinyl Alcohol / Moringa oleifera Leaf Extract / Graphene Oxide for Wound Dressing. Polymers, 2023. 15(2): p. 468.92. Huang, S. and X. Fu, Naturally derived materials-based cell and drug delivery systems in skin regeneration. Journal of Controlled Release, 2010. 142(2): p. 149-159.93. Janmey, P.A., J.P. Winer, and J.W. Weisel, Fibrin gels and their clinical and bioengineering applications. Journal of the Royal Society Interface, 2009. 6(30): p. 1-10.94. Bayer, I.S., Advances in fibrin-based materials in wound repair: a review. Molecules, 2022. 27(14): p. 4504.95. Litvinov, R.I., et al., Polymerization of fibrin: specificity, strength, and stability of knob-hole interactions studied at the single-molecule level. Blood, 2005. 106(9): p. 2944-2951.96. Itosaka, H., et al., Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering. Neuropathology, 2009. 29(3): p. 248-257.97. Zhang, Z., et al., Effect of hierarchically aligned fibrin hydrogel in regeneration of spinal cord injury demonstrated by fractography: A pilot study. Scientific reports, 2017. 7(1): p. 40017.98. Le Nihouannen, D., et al., Micro-architecture of calcium phosphate granules and fibrin glue composites for bone tissue engineering. Biomaterials, 2006. 27(13): p. 2716-2722.99. Cieslik-Bielecka, A., et al., The application of L-PRP in AIDS patients with crural chronic ulcers: A pilot study. Advances in medical sciences, 2018. 63(1): p. 140-146.100. Mohammadinejad, R., et al., Status and future scope of plant-based green hydrogels in biomedical engineering. Applied Materials Today, 2019. 16: p. 213-246.101. Indurkar, A., et al., Plant-based biomaterials in tissue engineering. Bioprinting, 2021. 21 : p. e00127.102. Modulevsky, D.J., et al., Apple derived cellulose scaffolds for 3D mammalian cell culture. PloS one, 2014. 9(5): p. e97835.103. Hickey, R.J., et al., Customizing the shape and microenvironment biochemistry of biocompatible macroscopic plant-derived cellulose scaffolds. ACS Biomaterials Science & Engineering, 2018. 4(11): p. 3726- 3736.104. Fontana, G., et al., Biofunctionalized plants as diverse biomaterials for human cell culture. Advanced healthcare materials, 2017. 6(8): p. 1601225.105. Cheng, Y.-W., et al., Engineering aligned skeletal muscle tissue using decellularized plant-derived scaffolds. ACS biomaterials science & engineering, 2020. 6(5): p. 3046-3054.106. Modulevsky, D.J., et al., Plant Scaffolds Support Motor Recovery and Regeneration in Rats after Traumatic Spinal Cord Injury. bioRxiv, 2020: p. 2020.10. 21 .34780710729. Geevarghese, R., et al., Biodegradable and Non-Biodegradable Biomaterials and Their Effect on Cell Differentiation. International Journal of Molecular Sciences, 2022. 23(24): p. 16185.107. Modulevsky, D.J., C.M. Cuerrier, and A.E. Pelling, Biocompatibility of subcutaneously implanted plant- derived cellulose biomaterials. PloS one, 2016. 11 (6): p. e0157894.108. Aarstad, O., et al., Mechanical properties of composite hydrogels of alginate and cellulose nanofibrils. Polymers, 2017. 9(8): p. 378.109. Yang, J. and C. Han, Mechanically viscoelastic properties of cellulose nanocrystals skeleton reinforced hierarchical composite hydrogels. ACS applied materials & interfaces, 2016. 8(38): p. 25621-25630.110. Doench, I., et al., Cellulose nanofiber-reinforced chitosan hydrogel composites for intervertebral disc tissue repair. Biomimetics, 2019. 4(1): p. 19.111. Gailit, J., et al., Human fibroblasts bind directly to fibrinogen at RGD sites through integrin avp3. Experimental cell research, 1997. 232(1): p. 118-126.112. Bensaid, W., et al., A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials, 2003. 24(14): p. 2497-2502.

Claims

CLAIMS1. A bioink formulation comprising a cellulose biomaterial derived from plant cells and one or more pharmaceutically acceptable components.

2. The bioink formulation of claim 1 , wherein the plant cells are live plant cells, dead plant cells, decellularized plant cells or a plant cell-line.3 The bioink formulation of claim 1 or 2, wherein the plant cells are Apple cells, Tobacco cells or Arabidopsis thaliana cells.4 The bioink formulation of any one of claims 1-3, wherein the biomaterial is a scaffold derived from the plant cells.5 The bioink formulation of any one of claims 1-4, wherein the biomaterial is a hydrogel that comprises the scaffold of the plant cells.6 The bioink formulation of any one of claims 1-5, wherein the scaffold is an extracellular matrix (ECM) derived from the plant cells.7 The bioink formulation of any one of claims 1-6, wherein the biomaterial comprises at least one synthetic or natural polymer.8 The bioink formulation of claim 7, wherein the at least one synthetic or natural polymer is poly(lactic-co- Glycolic acid) (PLGA), poly(glycolic acid) (PGA), polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), Fibrin, collagen, hyaluronic acid or a combination thereof.9 The bioink formulation of claim 7 or 8, wherein the formulation comprises the biomaterial and the synthetic and / or natural polymer in a fixed ratio.10 The bioink formulation of any one of claims 7-9 wherein the formulation comprises the biomaterial, the synthetic and / or natural polymer and the one or more pharmaceutically acceptable components.11 The bioink formulation of any one of claims 1-10, wherein the biomaterial is biodegradable, biocompatible, bioprintable, biostable, customizable, reproducible or tunable.

12. The bioink formulation of any one of claims 1-11 , wherein the biomaterial has a mesh-like porous structure with a hollow lattice.

13. The bioink formulation of any one of claims 1-12, wherein the biomaterial is derived by subjecting the plant cells to maceration treatment, oxidative treatment, decellularization treatment or a combination thereof.

14. The bioink formulation of claim 13, wherein the derived biomaterial is lyophilized.

15. The bioink formulation of any one of claims 1-14, wherein the biomaterial is a hydrogel.

16. The bioink formulation of claim 13, wherein the treated plant cells has a shelf-life of at least one year.

17. The bioink formulation of any one of claims 1-16, wherein the biomaterial has a shelf-life of about four to six months.

18. The bioink formulation of any one of claims 1-17, wherein the biomaterial is substantially free of nicotine.

19. The bioink formulation of any one of claims 1-18, wherein the biomaterial is substantially free of cellular content.

20. The bioink formulation of any one of claims 1-19, wherein the biomaterial is substantially free of signaling molecules that trigger an autoimmune response.21 . The bioink formulation of any one of claims 1-20, wherein the one or more pharmaceutically acceptable components are thickeners, stabilizers, viscosity agents, or pH stabilizers.

22. The bioink formulation of any one of claims 1-20, wherein the one or more pharmaceutically acceptable components are alginic acid or salt thereof, agarose, nanofibrillated cellulose (CNF), cellulose nanocrystals (CNC), agarose, pectin or any other cellulose nanomaterial.

23. The bioink formulation of any one of claims 1-22, wherein the one or more pharmaceutically acceptable components are in a gel, slurry or a spray-dried form.

24. The bioink formulation of any one of claims 1-23, further comprising live plant cells, dead plant cells, lyophilized plant cells, cellular adhesion promoters, growth factors, or a combination thereof.

25. The bioink formulation of claim 1 , comprising:a) the cellulose biomaterial derived from the plant cells; b) alginic acid or salt thereof; c) nanofibrillated cellulose; and d) cellulose nanocrystals.

26. The bioink formulation of claim 25, wherein the formulation comprises: a) 1 wt% to 10 wt% of the cellulose biomaterial; b) 2 wt% to 5 wt% alginic acid or salt thereof; c) 2 wt% to 5 wt% nanofibrillated cellulose; and d) 2 wt% to 5 wt% cellulose nanocrystals.

27. The bioink formulation of claim 25, wherein the formulation comprises: a) 1 wt% of the cellulose biomaterial; b) 2.8 wt% alginic acid or salt thereof; c) 3 wt% nanofibrillated cellulose; and d) 3 wt% cellulose nanocrystals.

28. The bioink formulation of any one of claims 25-27, wherein the formulation additionally comprises the one or more pharmaceutically acceptable components.

29. The bioink formulation of any one of claims 1-28, wherein the formulation has a Youngs modulus in the range of 10 Pa to 1 GPa.

30. The bioink formulation of any one of claims 1 -29, wherein the formulation is biodegradable, biocompatible, bioprintable, biostable, customizable, reproducible or tunable.

31. The bioink formulation of any one of claims 1-30, wherein the biomaterial, the one or more pharmaceutically acceptable components, or the bioink formulation is crosslinked with a crosslinking agent.

32. The bioink formulation of claim 31 , wherein the crosslinking agent is CaCh, UV light, PEGDA or 1 -ethyl- 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

33. The bioink formulation of any one of claims 1-24, wherein the formulation is highly homogenous, highly resilient, and highly extrudable compared to a formulation that lacks the cellulose biomaterial derived from the plant cells.

34. The bioink formulation of any one of claims 1-25 for use in bioprinting, for use in production of bioprintable scaffolds, for culturing mammalian cells, for use in production of implantable scaffolds, for use in production of scaffolds with a defined shape, for use in in-vitro production of cell lines, for use in regenerative medicine, for use in reconstructive surgery, for use in tissue engineering, for use in biomedical application, for use in spinal cord, bone, ligament injury treatment, for use in wound healing, for use in cosmetic applications, for use in dentistry applications, for use in drug delivery, for use in skin or cartilage regeneration, or for use in manufacturing a dermal filler.

35. The bioink formulation of any one of claims 1-34, wherein storage modulus (G') and loss modulus (G") of the formulation increases with increase in concentration of the cellulose biomaterial.

36. The bioink formulation of any one of claims 1-34, wherein the formulation’s resistance to deformation under a constant load increases with increase in concentration of the cellulose biomaterial.

37. The bioink formulation of any one of claims 1-36, wherein the formulation comprises the cellulose biomaterial and a pharmaceutically acceptable component and optionally at least one natural polymer.

38. The bioink formulation of claim 37, wherein the formulation comprises the cellulose biomaterial and alginic acid.

39. The bioink formulation of claim 37, wherein the formulation comprises the cellulose biomaterial and alginic acid and optionally hyaluronic acid or collagen.

40. The bioink formulation of claim 39, wherein the concentration of the cellulose biomaterial ranges from 0 25% - 5%, the concentration of alginic acid ranges from 0.25% - 2%, and the concentration of hyaluronic acid or collagen ranges from 10mg / g to 50 mg / g.41 The bioink formulation of any one of claims 37-40, wherein the formulation has a tensile stress I tensile strain ranging from 20 Pa to 400 Pa.

42. A method of purifying or isolating a biomaterial from plant cells comprising: a) macerating the plant cells with an acidic maceration mixture at a high temperature; b) decellularizing the plant cells of step a) to purify or isolate the biomaterial from the plant cells.

43. The method of claim 42, wherein the maceration step comprises an oxidative treatment of the plant cells by the acidic maceration mixture.

44. The method of claim 42, wherein the maceration step comprises a step of treating the plant cells with a surfactant for a predetermined period of time.

45. The method of claim 42, wherein the maceration step comprises a step of sterilizing the plant cells.

46. The method of any one of claims 42-45, wherein the maceration step is repeated one or more times to allow the acidic maceration mixture to weaken and remove the protoplast, cytoplasm or other cellular content from the plant cells.

47. The method of any one of claims 42-46, wherein the method further comprises a step of lyophilization wherein the purified or isolated biomaterial is lyophilized.

48. The method of any one of claims 42-47, wherein the maceration step is carried out at a temperature ranging from 40 °C to 60 °C.

49. The method of any one of claims 42-48, wherein the maceration step comprises: c) treating the plant cells with the acidic maceration mixture; and d) incubating the plant cells and the acidic maceration mixture overnight at a temperature ranging from 40 °C to 60 °C.

50. The method of any one of claims 42-49, wherein the method comprises: e) centrifuging the plant cells prior to the step a).51 . The method of claim 50, further comprising: f) washing the plant cells of step b) and mixing overnight on a mixer or shaker.

52. The method of claim 42, wherein the incubation step is carried out at a temperature ranging from 55 °C to58 °C.

53. The method of any one of claims 42-52, wherein the acidic maceration mixture comprises glacial acetic acid or absolute glacial acetic acid, hydrogen peroxide, and one or more industrially acceptable excipients.

54. The method of any one of claims 42-53, wherein the acidic maceration mixture comprises 1 part of a 30% solution of hydrogen peroxide, 4 parts of distilled water, and 5 parts of absolute glacial acetic acid.

55. The method of any one of claims 42-54, wherein the decellularization step comprises: g) treating the macerated plant cells with a surfactant for at least 24 hours at room temperature; and, h) washing the plant cells of step g) with one or more industrially acceptable solvents.

56. The method of claim 55, wherein the surfactant is sodium dodecyl sulphate (SDS), sodium lauryl sulphate (SLS), Triton X, Tween 20, Sodium Deoxycholate (SDC) or bleach.

57. The method of any one of claims 42-56, wherein the method further comprises storing the biomaterial overnight in a freezer at a temperature ranging from 0 °C to -195 °C.

58. The method of any one of claims 42-57, wherein the method further comprises freeze drying or lyophilizing the biomaterial in a lyophilizer.

59. The method of any one of claims 42-58, wherein the method further comprises sterilizing the biomaterial by one or more sterilization treatments.

60. The method of claim 59, wherein the biomaterial is sterilized by autoclaving, ethanol treatment, gamma irradiation, UV treatment or Ethylene Oxide (ETO) treatment.61 . The method of any one of claims 42-60, wherein the method is for purifying or isolating a cellulose biomaterial from live plant cells, dead plant cells or lyophilized plant cells.

62. A method of preparing a tunable bioink comprising: a) purifying or isolating a biomaterial from plant cells according to the method of any one of claims 42 tob) mixing the biomaterial with at least one synthetic or natural polymer or one or more pharmaceutically acceptable components to prepare a mixture; and c) sterilizing the mixture to prepare the tunable bioink.

63. The method of claim 62, wherein the mixing step is carried out using an industrial mixer, a rotary mixer, a shaker, mortar and pestle, rolling pin, vortex, pipette, blender, magnetic stirrer, centrifugation, homogenizer or sonicator.

64. The method of claim 62 or 63, wherein the mixing step occurs at room temperature.

65. The method of any one of claims 62-64, wherein the components are mixed sequentially in the mixing step.

66. The method of claim any one of claims 62-65, wherein the sterilization step is carried out by autoclaving, ethanol treatment, gamma irradiation, UV treatment or Ethylene Oxide (ETO) treatment67. The method of any one of claims 62 to 66, wherein the method further comprises: crosslinking the biomaterial, the one or more pharmaceutically acceptable components, or the bioink formulation with a crosslinking agent, or lyophilizing the tunable bioink to prepare a powder of the bioink.

68. The method of any one of claims 62 to 67, wherein the method further comprises: centrifuging the mixture to remove air pockets or air bubbles.

69. The method of any one of claims 62-68, wherein the one or more pharmaceutically acceptable components are alginic acid or salt thereof, agarose, nanofibrillated cellulose (CNF), cellulose nanocrystals (CNC), agarose, pectin or any other cellulose nanomaterial.

70. The method of any one of claims 62-69, wherein the isolated or purified biomaterial is a cellulose biomaterial derived from live plant cells, dead plant cells, decellularized plant cells or a plant cell line.

71. The method of any one of claims 62-70, wherein the isolated or purified biomaterial is a cellulose biomaterial derived from Tobacco cells, Apple cells, or Arabidopsis thaliana cells.

72. The method of any one of claims 62-71 , wherein the at least one synthetic or natural polymer is poly(lactic- co-Glycolic acid) (PLGA), poly(glycolic acid) (PGA), polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), Fibrin, collagen, hyaluronic acid or a combination thereof.

73. The method of any one of claims 62-72, wherein the method further comprises a step of infusing the tunable bioink with live plant cells, dead plant cells, lyophilized plant cells, cellular adhesion promoters, growth factors, or a combination thereof.

74. The method of claim any one of claims 57-73, wherein the method is for preparing the bioink formulation defined in claims 1-41 .

75. The method of claim 67, wherein the crosslinking agent is CaCh, UV light, PEGDA or 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDO).

76. The method of claim 62, wherein the mixing step involves a step of infusing the cellulose biomaterial in the synthetic biomaterial.

77. The method of claim 76, wherein the step of infusing is carried out by UV crosslinking.

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