Decellularized cell wall structures of plants and fungi and their use as scaffold materials

Abstract

Provided herein are scaffold biomaterials comprising a tissue-removed cellular material and a decellularized plant or fungal tissue of nucleic acid, the decellularized plant or fungal tissue comprising a cellulose or chitin-based three-dimensional porous structure. The invention also provides methods of making such scaffold biomaterials, and uses thereof as implantable scaffolds for supporting animal cell growth, promoting tissue regeneration, promoting angiogenesis, tissue replacement procedures, and / or as structural implants for cosmetic procedures. Also described are therapeutic treatment and / or cosmetic methods using such stents.

Description

Field of the Invention

[0001] The present invention generally relates to scaffold biomaterials and their uses. More specifically, the present invention relates to decellularized plant or fungal tissues and their use as scaffold biomaterials. Background Art

[0002] The biomaterials industry is estimated to have a market value of $9 billion and is driven by novel materials derived from natural sources, synthetic polymers, metals, and ceramics. These materials can form three-dimensional, highly porous scaffolds with nano- and micro-scale structures that are biocompatible and promote the growth of living cells. For example, there is significant interest in novel biomaterials that support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine.

[0003] Biomaterial scaffolds are used in a variety of fields, including dental and cosmetic surgery, clinical and medical treatments (e.g., regenerative medicine, wound healing, tissue engineering and repair, etc.), and research and development (including industrial and academic research in biomedical sciences).

[0004] Commercial biomaterials often require complex and time-consuming production methods, resulting in high costs for end users, even if they are not approved for human use. Furthermore, most commercial biomaterials are derived from human or animal sources, leading to the potential for rejection by the body, adverse immune responses, and / or disease transmission. Sourced materials may also have negative environmental impacts and raise concerns about unethical sourcing. Furthermore, some commercial biomaterials lose their shape after implantation, potentially reducing the success rate of tissue repair / replacement.

[0005] The development of novel biomaterials for tissue engineering strategies is currently under intensive investigation [1-3]. Biomaterials are being developed for the local delivery of therapeutic cells to target tissues [4,5], regeneration of damaged or diseased tissues [6-9], or replacement of entire organs [10-15]. In their most prevalent form, biomaterials provide three-dimensional (3D) scaffolds that attempt to mimic the in vivo cellular environment [14,16]. Methods have been developed to modify the mechanical [17-24], structural

[25] , and biochemical properties of these scaffolds of varying complexity [26-29]. Similarly, significant efforts are underway to ensure that such implanted biomaterials are biocompatible and stimulate only a minimal immune response. Efforts in biomaterial research are driven by the significant demand for replacement organs and tissues. As the population ages, the gap between the number of patients awaiting organ transplantation and the number of available donor organs is rapidly increasing

[30] . Although the clinical application of biomaterials is somewhat limited, physicians have successfully utilized synthetic biomaterials to treat a variety of damaged tissues and structures, such as skin, gums, cartilage, and bone [31-36].

[0006] Biomaterial scaffolds can take a variety of forms, such as powders, gels, films, and pastes [1,2]. Such polymer or hydrogel formulations can be molded or 3D printed to produce therapeutically valuable forms [37-39]. An alternative to these synthetic strategies is whole organ decellularization [10,12-16]. Indeed, it has been shown that it is possible to separate cells from donated organs, leaving behind a scaffold matrix, often referred to as a ghost organ

[14] . Ghost organs lack any cells from the donor and can subsequently be cultured with cells derived from the patient or other sources. This approach has been used to repair and replace defective tissues [40-42]. In the past few years, many body parts have been created using synthetic and decellularized approaches, including urethra, vagina, ear, nose, heart, kidney, bladder, and neural tissue [14,38,39,43-47].

[0007] However, these approaches are not without drawbacks

[48] . Synthetic techniques can involve animal products, and decellularization strategies still involve donor tissue and organs. The development of resorbable biomaterials has also been intensively investigated

[49] . In these cases, the goal is to provide the body with a temporary 3D scaffold on which healthy tissue can form. After weeks or months, the implanted scaffold will be absorbed, leaving behind completely natural healthy tissue [26,29,50,51]. While this is an attractive approach, many non-resorbable biomaterials (ceramics, titanium) have been successfully used in clinical settings and play an important role in numerous therapies [2,49,52-57]. Importantly, resorbable biomaterials are hampered by the fact that regenerated tissue often collapses and deforms due to loss of structure [58-62]. For example, decades of research on engineered cartilage ear reconstruction has shown that biomaterial implants eventually collapse and deform as the implanted scaffold breaks down and resorbs

[63] . However, more recent successful approaches rely on the use of resorbable collagen scaffolds embedded in permanent titanium wire scaffolds [53,64,65]. Therefore, the need for non-resorbable yet biocompatible scaffolds continues to exist in the field of tissue and organ engineering.

[0008] Recent complementary approaches have utilized scaffold materials that are not derived from human organ donors or animal products, including various forms of cellulose.[66–77] Nanocrystals, nanofibers, and bacterial cellulose constructs and hydrogels have also been investigated.[78–83]

[0009] Orthogonal but complementary approaches to organ decellularization and cellulosic biosynthesis strategies have also been investigated. These initial in vitro studies investigated cellulosic biomaterials derived from decellularized apple inflorescence tissue

[27] .

[0010] The problem of in vivo biocompatibility, alternative biomaterials and other methods of biomaterial production still exists.In summary, the industry still needs alternative, additional and / or improved biomaterials, methods for their preparation and / or their use. Summary of the Invention

[0011] It is therefore an object of the present invention to provide a biomaterial that can be used as a scaffold or implant in a variety of applications, including but not limited to surgical, clinical, therapeutic, cosmetic, developmental and / or other suitable applications.

[0012] Thus, in certain embodiments, provided herein are biomaterials produced from plant or fungal species. The biomaterials can be modified, for example, by (i) adding structures (i.e., other parts of plants or fungi, or living cells), drugs, or artificial structures (resorbable or non-resorbable materials); (ii) modifying their structure using mechanical or chemical methods to change the shape or composition of the original product to suit different applications; (iii) adding a matrix to or in the original scaffold product (e.g., collagen, fibronectin, or any other substrate) to modify cell adhesion or any other beneficial components of cell science, such as growth factors.

[0013] The biomaterials, preparation methods, and potential uses are described in more detail below. In certain embodiments, the biomaterials can be relatively low-cost and / or can use relatively efficient and / or time-compressed production procedures. In addition, by using complex structures as functional scaffolds, there are many possibilities for producing complex structures. The biomaterials can have the ability to maintain shape, can have a relatively minimal footprint (i.e., the scaffold is almost invisible before and / or after angiogenesis), can be highly biocompatible, can induce rapid angiogenesis, and / or can produce a minimized or almost non-existent immunogenic response.

[0014] In certain embodiments, the biomaterial can be derived from plants or fungi and thus can exhibit a relatively low environmental impact, and / or can be considered organic and / or biodegradable. In certain instances, the biomaterial can be produced from food waste, thereby providing an alternative route to waste products.

[0015] In one embodiment, provided herein is a scaffold biomaterial comprising a decellularized plant or fungal tissue from which cellular material and nucleic acids of the tissue have been removed, the decellularized plant or fungal tissue comprising a cellulose or chitin-based porous structure.

[0016] In another embodiment, provided herein is a scaffold biomaterial comprising a decellularized plant or fungal tissue from which cellular material and nucleic acids of the tissue have been removed, the decellularized plant or fungal tissue comprising a cellulose or chitin-based three-dimensional porous structure.

[0017] In one embodiment of the above-mentioned scaffold biomaterial, the decellularized plant or fungal tissue may comprise a plant or fungal tissue that has been decellularized by heat shock, treatment with detergent, osmotic shock, freeze drying, physical lysis, electrical disruption or enzymatic digestion, or any combination thereof.

[0018] In another embodiment of the above-mentioned scaffold material, the decellularized plant or fungal tissue may comprise a plant or fungal tissue that has been decellularized by treatment with a detergent or surfactant. In certain embodiments, examples of detergents may include, but are not limited to, sodium dodecyl sulfate (SDS), Triton X, EDA, alkyl treatment, acid, ionic detergent, nonionic detergent, or zwitterionic detergent, or a combination thereof.

[0019] In another embodiment of the above scaffold material, the decellularized plant or fungal tissue may comprise a plant or fungal tissue that has been decellularized by treatment with SDS.

[0020] In another embodiment of the above scaffold material, residual SDS can be removed from the decellularized plant or fungal tissue by washing with a divalent saline solution.

[0021] In another embodiment of the above scaffold material, residual SDS can be removed by precipitating / separating salt residues containing SDS micelles from the solution / scaffold using a divalent salt solution, and salt residues and / or SDS micelles can be removed using dH2O, acetic acid, dimethyl sulfoxide (DMSO) or sonication.

[0022] In another embodiment of the above scaffold material, the divalent salt of the divalent salt solution may include MgCl2 or CaCl2.

[0023] In another embodiment of the above scaffold material, the plant or fungal tissue can be treated with an SDS solution of 0.01 to 10%, such as about 0.1% to about 1%, or about 0.1% SDS or about 1% SDS in a solvent such as water, ethanol or other suitable organic solvents. Residual SDS can be removed by using an aqueous solution of CaCl2 at a concentration of about 100 mM followed by incubation in dH2O.

[0024] In certain embodiments, the SDS solution may have a concentration higher than 0.1%, which may facilitate decellularization and may be accompanied by enhanced washing to remove residual SDS.

[0025] In another embodiment of the above scaffold material, the decellularized plant or fungal tissue can be functionalized on at least some of the free hydroxyl functional groups by acylation, alkylation or other covalent modification to provide a functionalized scaffold biomaterial.

[0026] In another embodiment of the above scaffold material or materials, the decellularized plant or fungal tissue may be treated to introduce further structure and / or microstructure and / or may be functionalized on at least some of the free hydroxyl functional groups by acylation, alkylation or other covalent modification to provide a functionalized scaffold biomaterial.

[0027] In another embodiment of the above scaffold material or materials, the decellularized plant or fungal tissue can be processed to introduce microchannels and / or can be functionalized with, for example, collagen, factors that promote cell specificity, cell growth factors, or pharmaceutical agents.

[0028] In another embodiment of the above scaffold material or materials, the decellularized plant or fungal tissue may be functionalized with collagen.

[0029] In another embodiment of the above-mentioned scaffold material or material, the plant or fungal tissue can include apple inflorescence (Malus pumila) tissue, fern (Monilophytes) tissue, radish (Brassica rapa) root tissue, ginkgo branch tissue, horsetail (equisetum) tissue, daylily hermocallis hybrid leaf tissue, kale (Brassicaoleracea) stem tissue, conifer Douglas fir (Pseudotsuga menziesii) tissue, cactus fruit (pitaya) flesh tissue, spotted periwinkle (Maculata Vinca) tissue, water lotus (Nelumbo nucifera) tissue, tulip (Tulipa gesneriana) petal tissue, plantain (Musa paradisiaca) tissue, broccoli (Brassicaoleracea) stem tissue, maple leaf (Acer psuedoplatanus) stem tissue, beet (Beta vulgaris) primary root tissue, onion (Allium cepa) tissue, Orchidaceae tissue, Radish (Brassica rapa) stem tissue, Leek (Allium ampeloprasum) tissue, Maple (Acer) branch tissue, Celery (Apium graveolens) tissue, Onion (Allium cepa) stem tissue, Pine tissue, Aloe vera tissue, Watermelon (Citrullus lanatus var.lanatus) tissue, Meadowgrass (Lysimachia nummularia) tissue, Cactae tissue, Arctic Campion (Lychnis Alpina) tissue, Rhubarb (Rheum rhabarbarum) tissue, Pumpkin (Cucurbita pepo) tissue, Dracena (Asparagaceae) stem tissue, Tradescantia virginiana stem tissue, Asparagus (Asparagus officinalis) stem tissue, Mushroom (Fungi) tissue, Fennel (Foeniculum vulgare) tissue, Rose (Rosa) tissue, Carrot (Daucus carota) tissue or pear (Pomaceous) tissue.

[0030] In certain embodiments, the plant or fungal tissue can include genetically altered tissues prepared by direct genomic modification and / or by selective breeding to produce additional plant or fungal structures that are configured to physiologically mimic tissue and / or functionally promote the effects of the target tissue. Those skilled in the art, considering the teachings herein, will be able to select appropriate scaffold biomaterials to suit a particular application.

[0031] In another embodiment of the above scaffold material or materials, the scaffold biomaterial may further comprise living animal cells adhered to the cellulose or chitin-based three-dimensional porous structure. In another embodiment, the living animal cells may be mammalian cells. In another embodiment, the living animal cells may be human cells.

[0032] In another embodiment, provided herein is a method for preparing a decellularized plant or fungal tissue, wherein cellular material and nucleic acids of the tissue are removed, the decellularized plant or fungal tissue comprising a cellulose or chitin-based three-dimensional porous structure, the method comprising:

[0033] providing a plant or fungal tissue having a predetermined size and shape; and

[0034] decellularizing the plant or fungal tissue by heat shock, treatment with detergents, osmotic shock, freeze drying, physical lysis, electrical disruption, or enzymatic digestion, or any combination thereof,

[0035] Cellular material and nucleic acids are thereby removed from the plant or fungal tissue to form a decellularized plant or fungal tissue comprising a three-dimensional porous structure based on cellulose or chitin.

[0036] In another embodiment of the above method, the step of decellularization can include treating the plant or fungal tissue with a detergent or surfactant. In certain embodiments, examples of detergents can include, but are not limited to, sodium lauryl sulfate (SDS), Triton X, EDA, alkyl treatment, acid, ionic detergents, non-ionic detergents, or zwitterionic detergents, or combinations thereof. In certain embodiments, the step of decellularization can include treating the plant or fungal tissue with sodium lauryl sulfate (SDS).

[0037] In another embodiment of the above method, the decellularized plant or fungal tissue can include plant or fungal tissue that has been decellularized by treatment with a detergent. Examples of detergents can include, but are not limited to, sodium dodecyl sulfate (SDS), Triton X, EDA, alkyl treatments, acids, ionic detergents, nonionic detergents, zwitterionic detergents, or combinations thereof.

[0038] In another embodiment of the above methods, the decellularized plant or fungal tissue may comprise plant or fungal tissue that has been decellularized by treatment with SDS.

[0039] In another embodiment of the above method, residual SDS may be removed from the decellularized plant or fungal tissue by washing with a divalent saline solution.

[0040] In another embodiment of the above method, residual SDS can be removed by precipitating / disintegrating salt residues containing SDS micelles from the solution / scaffold using a divalent salt solution, and dH2O, acetic acid, dimethyl sulfoxide (DMSO) or sonication can be used to remove salt residues and / or SDS micelles. In another embodiment, the divalent salt of the divalent salt solution can include MgCl2 or CaCl2.

[0041] In another embodiment of the above method, the plant or fungal tissue may be treated with an SDS solution containing 0.01 to 10%, such as about 0.1% to about 1%, or about 0.1% SDS or about 1% SDS in a solvent (such as water, ethanol or other suitable organic solvent) and decellularized. Residual SDS may be removed by using an aqueous solution of CaCl2 at a concentration of about 100 mM followed by incubation in dH2O.

[0042] In certain embodiments, the SDS solution may have a concentration higher than 0.1%, which may facilitate decellularization and may be accompanied by enhanced washing to remove residual SDS.

[0043] In another embodiment of the above method, the decellularization step may include treatment with an SDS solution containing about 0.1% SDS in water, and residual SDS may be removed after decellularization using an aqueous CaCl2 solution having a concentration of about 100 mM followed by incubation in dH2O.

[0044] In another embodiment of the above method, the method may further include the step of functionalizing at least some of the free hydroxyl groups of the decellularized plant or fungal tissue by acylation, alkylation or other covalent modification. In certain embodiments, the hydroxyl groups of the decellularized plant or fungal tissue may be functionalized with collagen.

[0045] In another embodiment of the above method, the method may further include the step of treating the decellularized plant or fungal tissue to introduce further structure and / or microstructure, and / or functionalizing at least some of the free hydroxyl groups of the decellularized plant or fungal tissue by acylation, alkylation, or other covalent modification. In certain embodiments, the decellularized plant or fungal tissue may be treated to introduce microchannels, and / or the hydroxyl groups of the decellularized plant or fungal tissue may be functionalized with, for example, collagen, factors that promote cell specificity, cell growth factors, or pharmaceutical agents.

[0046] In another embodiment of the above method, the method may further include the step of introducing living animal cells into the cellulose- or chitin-based three-dimensional porous structure and allowing the living animal cells to adhere to the cellulose- or chitin-based three-dimensional porous structure. In certain embodiments, the living animal cells may be mammalian cells. In certain embodiments, the living animal cells may be human cells.

[0047] In another embodiment, provided herein is a scaffold biomaterial comprising decellularized plant or fungal tissue prepared by any of the above methods.

[0048] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials as an implantable scaffold for supporting animal cell growth, promoting tissue regeneration, promoting angiogenesis, tissue replacement procedures, or as a structural implant for cosmetic surgery.

[0049] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials as a structural implant for repair or regeneration following spinal cord injury.

[0050] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials as a structural implant for tissue replacement surgery and / or post-operative tissue regeneration.

[0051] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials as a structural implant for skin grafting and / or skin regeneration procedures.

[0052] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials as a structural implant for regeneration of vasculature in a target tissue or region.

[0053] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials as a bone replacement, bone filler, or bone graft material and / or for promoting bone regeneration.

[0054] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials as a tissue replacement for skin, bone, spinal cord, heart, muscle, nerve, blood vessel, or other damaged or deformed tissue.

[0055] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials in hydrogel form as a vitreous humor replacement.

[0056] In another embodiment, provided herein is a use of any of the above-described scaffold biomaterials as an artificial bursa, wherein the scaffold biomaterial forms a sac-like structure containing the scaffold biomaterial in hydrogel form.

[0057] In another embodiment, provided herein is the use of any of the above-described scaffold biomaterials as a structural implant for cosmetic surgery.

[0058] In yet another embodiment of any of the above uses, the scaffold biomaterial may be a scaffold biomaterial wherein the decellularized plant or fungal tissue of the scaffold biomaterial is configured to physiologically mimic a subject's tissue and / or functionally promote the effects of a target tissue of a subject.

[0059] In another embodiment, provided herein is a method for supporting animal cell growth, promoting tissue regeneration, promoting angiogenesis, tissue replacement, promoting angiogenesis, or providing a structural scaffold for cosmetic surgery in a subject in need thereof, the method comprising:

[0060] Providing a scaffold biomaterial according to any of the above scaffold biomaterials; and

[0061] The scaffold biomaterial is implanted into a subject.

[0062] In another embodiment of the above method, the scaffold biomaterial can be implanted into the spinal cord to promote repair or regeneration after spinal cord injury.

[0063] In another embodiment of the above method, the scaffold biomaterial can provide a structural implant for tissue replacement and / or tissue regeneration in a subject.

[0064] In another embodiment of the above method, the scaffold biomaterial can provide a structural implant for skin grafting and / or skin regeneration in a subject.

[0065] In another embodiment of the above method, the scaffold biomaterial can provide a structural implant for regeneration of vasculature in a target tissue or region in a subject.

[0066] In yet another embodiment of the above method, the scaffold biomaterial can provide a bone replacement, bone filler, or bone graft material for a subject, and / or can promote bone regeneration.

[0067] In another embodiment of the above method, the scaffold biomaterial can provide a tissue replacement for skin, bone, spinal cord, heart, muscle, nerve, blood vessel, or other damaged or deformed tissue in a subject.

[0068] In yet another embodiment of the above methods, a scaffold biomaterial in the form of a hydrogel can provide vitreous humor replacement to a subject.

[0069] In another embodiment of the above method, the scaffold biomaterial can provide an artificial bursa to a subject, wherein the scaffold biomaterial forms a sac-like structure comprising the scaffold biomaterial in hydrogel form.

[0070] In another embodiment of the above method, the scaffold biomaterial can provide a structural implant for use in cosmetic surgery.

[0071] In another embodiment of the above method, the step of providing the scaffold biomaterial may further comprise:

[0072] The scaffold biomaterial as described above is selected, wherein the decellularized plant or fungal tissue of the scaffold biomaterial is configured to physiologically mimic a tissue of a subject and / or functionally promote an effect of a target tissue of a subject.

[0073] In another embodiment, provided herein is a kit comprising a scaffold biomaterial as described above and at least one container or instructions for performing a surgical or cosmetic method as described above. In certain embodiments, the kit can be a surgical kit.

[0074] In another embodiment, provided herein is a kit comprising one or more of an SDS solution, a CaCl2 solution, or a PBS solution, and optionally further comprising instructions for practicing the method for preparing decellularized plant or fungal tissue as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] These and other features of the present invention will become more apparent from the following description, with reference to the following drawings:

[0076] Figure 1 Decellularized cellulose scaffolds. A) Phase contrast image (optical microscopy) of the cellulose cell wall structure in a decellularized apple tissue sample. Dark lines correspond to distinct cellulose structures forming a three-dimensional matrix. Overlapping dark structures highlight the 3D porous structure of the decellularized scaffold. B) SEM image of a similar cellulose scaffold, demonstrating its three-dimensional nature and large cavities, highlighting the various depths of the internal pockets that comprise the scaffold. Scale bar = 200 μm.

[0077] Figure 2 Cellulose scaffolds of various structures and sources. These novel scaffolds are obtained from plants (e.g., apple, asparagus, fennel) and fungi (e.g., white button mushrooms) by using a decellularization method.

[0078] Figure 3: Apple scaffold implantation mouse model (in vivo). Two cellulose scaffolds (5×5×1 mm) were subcutaneously implanted in the back of C57BL / 10 mice. The dorsal skin was then carefully excised and fixed in 10% formalin solution one (A) and four (B) weeks after surgery. Histological analysis of the implants was performed using hematoxylin and eosin (H&A) staining, and each implant was analyzed. After one week, cellular infiltration was visible, and complete infiltration was achieved with the presence of functional blood vessels (angiogenesis) after four weeks;

[0079] Figure 4 Scaffold footprint and complete cellular infiltration and angiogenesis (in vivo). A) The high porosity and thin-walled structure (<100 nm) of the apple-derived scaffold can be readily observed in a photograph taken in the middle of the implant one week after surgery. B) Complete cellular infiltration and angiogenesis accompanied by functional vessel formation four weeks after implantation. Cellulose scaffolds are not visible and require specific cellulose staining for visualization.

[0080] Figure 5 : Fixed and stained images of the actin cytoskeleton of cells cultured in 3D cellulose scaffolds, and SEM artistic images. A) NIH3T3, B) C2C12, and C) HeLa cells were cultured on cellulose scaffolds for 2 weeks, after which actin (green) and nuclei (blue) were stained. The actin cytoskeleton and nuclei of mammalian cells cultured on glass or in scaffolds were stained according to previous protocols (Guolla, Bertrand, Haase, & Pelling, 2012; Modulevsky, Tremblay, Gullekson, Bukoresthliev, & Pelling, 2012). Briefly, samples were fixed with 3.5% paraformaldehyde and permeabilized with Triton X-100 at 37°C. Actin was stained with phalloidin conjugated to Alexa Fluor 488 (Invitrogen), and nuclei were stained by labeling DNA with DAPI (Invitrogen). The samples were then mounted in Vecta-shield (Vector Labs). NIH3T3 and C2C12 cells display characteristic actin stress fibers found in cultured cells. HeLa cells also exhibit characteristic actin structures, including fewer prominent stress fibers and extensive cortical actin localization. The presence of stress fibers indicates mammalian cell adhesion to the surface of the cell wall scaffold and is present in vivo. Scale bar = 25 μm, applicable to all images. D) and E) are SEM images processed with artistic cell staining to highlight cell attachment to the cellulose scaffold.

[0081] Figure 6Figure 3: Cell wall structures found in the plant and fungal kingdoms. These examples of cellulose scaffolds were excised from animals 4 weeks after their implantation and stained with hematoxylin / eosin. This figure shows cell wall structure and its relationship to tissue function, which can guide biomaterial selection. Cell wall structures found in the plant and fungal kingdoms exhibit a variety of structures that may resemble tissues such as bone, skin, and nerves. Depending on the target tissue, the plant source of the biomaterial can be determined based on the plant's physical and chemical properties.

[0082] Figure 7 : Examples of histological results showing cell infiltration 1, 4, and 8 weeks after implantation (hematoxylin / eosin staining);

[0083] Figure 8 A) Collagen deposition (blue) and observed blood vessels (red cells are erythrocytes) within the cellulose biomaterial (white). B) Graph showing the quantitative representation of the pro-angiogenic properties of the scaffold (functional blood vessels were observed within 4 weeks after implantation);

[0084] Figure 9 : Non-resorbable properties of cellulose scaffolds after implantation as a function of time after transplantation;

[0085] Figure 10 : Improved cell attachment and proliferation by washing with calcium chloride;

[0086] Figure 11 : Cellulose scaffold preparation. Macroscopic appearance of freshly cut apple inflorescence tissue (A) and macroscopic appearance of translucent cellulose scaffold biomaterial after decellularization and lack of all native apple cells or cell fragments (B). H&E staining of a cross-section of decellularized cellulose scaffold (C). Cell wall thickness and loss of native apple cells after decellularization are shown. Scanning electron microscopy clearly revealed the 3D acellular and highly porous cellulose scaffold structure (D). Scale bars: AB = 2 mm, CD = 100 μm;

[0087] Figure 12 : Cellulose scaffold implantation and resection. Subcutaneous implantation of cellulose scaffold biomaterials was performed on the back area of a C57BL / 10ScSnJ mouse model through a small skin incision (8 mm) (A). Each implant was measured for scaffold area comparison before implantation (B). Cellulose scaffolds were excised 1 week (D), 4 weeks (E) and 8 weeks (F) after surgery, and macroscopic photographs were taken (control skin in C). Changes in cellulose scaffold surface area over time are presented (G). The area of the scaffold before implantation was 26.30 ± 1.98 mm 2 After implantation, the stent area decreased to 20.74±1.80mm after 1 week. 2 , and decreased to 16.41±2.44mm after 4 weeks2 , and decreased to 13.82±3.88mm after 8 weeks 2 After 8 weeks of implantation, the surface area of the cellulose scaffold was significantly reduced by approximately 12 mm 2 (48%) (*=P<0.001; n=12-14);

[0088] Figure 13 : Biocompatibility and cellular infiltration. Cross-sections of representative cellulose scaffolds stained with H&E and anti-CD45. These global views show an acute, moderate-severe, expected foreign body reaction at 1 week (A), a mild, chronic immune response and subsequent clearance at 4 weeks (B), and finally, the assimilation of the cellulose scaffold into native mouse tissue at 8 weeks (C). Higher magnification regions of interest (DF) allow observation of all cell type populations during the biomaterial assimilation process. At 1 week, granulocyte populations were observed, specifically polymorphonuclear leukocytes (PMNs) and eosinophils, characteristic of an acute, moderate to severe immune response, which is a normal response to the implantation procedure (D). At 4 weeks, a reduced immune response (mild to low-grade immune response) was observed, and the cell population within the epidermis surrounding the scaffold now contained higher levels of monocytes and lymphocytes, characteristic of a chronic response (E). Finally, at 8 weeks, the immune response was completely resorbed, and the epidermal tissue now appeared normal. The immune response observed with H&E staining was confirmed using an anti-CD45 antibody, a well-known leukocyte marker (GI). The cell population within the scaffold at this time is mainly macrophages, multinuclear cells and activated fibroblasts. Scale bars: AC = 1 mm, DF = 100 μm, GI = 500 μm;

[0089] Figure 14 : Extracellular matrix deposition. Cross-sections of representative cellulose scaffolds stained with Masson's trichrome (AC). One week after implantation, magnification of the region of interest in (A) shows the loss of collagen structure within the collagen scaffold (D, G). As fibroblasts begin to invade the scaffold, sparse collagen deposits can be observed within the cellulose scaffold after 4 weeks (E, H). Activated fibroblasts (spindle-shaped cells) are observed within the cellulose scaffold, and a collagen network is clearly visible in the lumen after 8 weeks (F, I). Scale bars: AC = 1 mm, DF = 100 μm, GI = 20 μm. * = collagen fibers; black arrows = cellulose cell walls; white arrows = fibroblasts;

[0090] Figure 15: Vascularization and angiogenesis. Blood vessels were directly visualized in the surrounding tissue surrounding the cellulose scaffold (A). Angiogenesis within the cellulose scaffold was confirmed by observing multiple cross-sections of blood vessels in micrographs of H&E staining (B) and Masson's trichrome staining (C). The angiogenic process was also confirmed by identifying endothelial cells within the cellulose scaffold by anti-CD31 staining (D). Scale bars: A = 1 mm, B = 50 μm, CD = 20 μm. White arrow = blood vessel;

[0091] Figure 16: Fixed and stained NIH3T3, C2C12 and HeLa cells cultured on natural 3D cellulose scaffolds. Specific fluorescent staining of (A) NIH3T3, (B) C2C12 and (C) HeLa mammalian cells within natural unmodified cellulose scaffolds. Mammalian cells and natural cellulose cell walls were stained with target-specific fluorescent stains, showing cellulose structure (red), mammalian cell membranes (green) and nuclei (blue). Prior to staining and imaging, cells were cultured for four weeks within decellularized cellulose scaffolds. To stain cellulose scaffolds and mammalian cells simultaneously, we first fixed the samples as described above and then washed the 4-week cultured samples three times with PBS. To label the cell wall, an established protocol was used (Truernit & Haseloff, 2008). The samples were rinsed with water and incubated in 1% periodic acid (Sigma-Aldrich) at room temperature for 40 minutes. The tissue was rinsed again with water and incubated for 2 hours with 100 mg / mL propidium iodide (Invitrogen) in Schiff reagent (100 mM sodium metabisulfite and 0.15 N HCl). The samples were then washed with PBS. To visualize mammalian cells within plant tissue, the samples were incubated in a solution of HBSS (20 mM HEPES, pH 7.4; 120 mM NaCl; 5.3 mM KCl; 0.8 mM MgSO4; 1.8 mM CaCl2; and 11.1 mM dextrose) containing 5 mg / mL wheat germ agglutinin (WGA) 488 (Invitrogen) and 1 mg / mL Hoechst 33342 (Invitrogen). WGA and Hoechst 33342 are live cell dyes that label mammalian cell membranes and nuclei, respectively. The cell wall scaffold was then transferred to a microscope slide and mounted in a chloral hydrate solution (4 g chloral hydrate, 1 mL glycerol, and 2 mL water). The slide was kept overnight in a closed environment at room temperature to prevent dehydration. The sample was then placed in PBS until ready for imaging. Clearly, mammalian cells were distributed over the entire biomaterial surface. Specifically, mammalian cells were observed to grow in aggregate within the cell wall cavity. The orthogonal view (ZY plane) shows the penetration depth of mammalian cells within the biomaterial. Green (cell membrane) and blue (nucleus) can be seen deep within the biomaterial, and the imaging penetration depth of the microscope was observed. Confocal volumes were acquired and projected in the XY and ZY planes. The ZY orthogonal view demonstrated the depth of cell proliferation within the cellulose scaffold. The top and bottom surfaces of the scaffold are shown. Scale bars: XY = 300 mm, ZY = 100 mm. In D), the biomaterial was sliced to reveal that the internal structure of the biomaterial exceeded the penetration imaging depth limit of the confocal microscope. SEM images of a cross-section of a cellulose scaffold that was allowed to proliferate for four weeks after inoculation with C2C12 cells.The cells were digitally stained to increase the contrast between the cells and the cellulose structure (scale bar: 50 mm). SEM imaging was performed on internal sections and revealed that the mammalian cells were present throughout the biomaterial, not just on the surface. The scaffolds containing the mammalian cells were first fixed with 3.5% paraformaldehyde as described above and then gently washed repeatedly with PBS. The samples were then dehydrated through a continuous ethanol gradient (50%, 70%, 95% and 100%) and dried in a freeze dryer. The samples were then gold coated using a Hitachi E-1010 ion sputtering unit at a current of 15 mA for 3 minutes. SEM imaging was performed on a JEOL JSM-7500F FESEM at a voltage of 2.00-10.0 kV;.

[0092] Figure 17 : Changes in cell proliferation and viability over time. A) NIH3T3, C2C12, and HeLa cells were cultured individually in cellulose n=3 scaffolds for 1, 8, and 12 weeks and then imaged using a confocal microscope after staining with Hoechst 33342. Cells were quantified at each time point using ImageJ open access software (http: / / rsbweb.nih.gov / ij / ). An increase in the cell population of all three cell types was observed. It should be noted that the increase in cell count can only be the result of proliferation, as the scaffolds were only seeded with the corresponding cell types at the beginning of the experiment. B) After 12 weeks of culture, C2C12 cells were fixed and stained with Hoechst 33342 (blue: living cells) and propidium iodide (PI) (red: apoptotic / necrotic cells). The copolymer volume was acquired and projected in the XY and ZY planes, revealing that cells proliferated throughout the structure during the 12-week culture period. Apoptotic / necrotic cells were found in the deeper areas of the scaffold. The top and bottom surfaces of the scaffold are shown. The number of live (Hoechst(+)) and dead (Hoechst / PI(+)) cells was counted, and 98% of the cells within the scaffold were viable. Data are shown for C2C12 cells, but are similar for NIH3T3 and HeLa cells (data not shown). Scale bars: B = 200 mm for XY, B = 100 mm for ZY;

[0093] Figure 18CaCl2 optimization. Phase contrast images: A, C, E, G, I, K, M, O. Hoechst (nuclear stain) fluorescence images: B, D, F, H, J, L, N, P. No CaCl2: A-D, 10 mM CaCl2: E-H, 100 mM CaCl2: IL, 1000 mM CaCl2: MP. No cells: A, B, E, F, I, J, M, N. Cells (C2C12 myoblasts): C, D, G, H, K, L, O, P. Improved cell growth occurs at 100 mM CaCl2 and above. Dark spots on the cellulose in the 100 mM and 1000 mM CaCl2 samples are precipitated by salt, as evidenced by the different positioning of nuclei in the fluorescence images and their presence in the absence of cells. Cells were grown on the scaffolds prior to imaging. Scale bar: 200 μm. The figure shows phase contrast images (A, C, E, G, I, K, M, O) and Hoechst fluorescence staining images (B, D, F, H, J, L, N, P) of decellularized scaffolds without any cultured cells and without CaCl2; C2C12 myoblasts cultured in scaffolds without CaCl2 (C, D); scaffolds treated with 10 mM CaCl2 (E, F); C2C12 myoblasts cultured in scaffolds treated with 10 mM CaCl2 (G, H); scaffolds treated with 100 mM CaCl2 (I, J); C2C12 myoblasts cultured in scaffolds treated with 100 mM CaCl2 (K, L); scaffolds treated with 1000 mM CaCl2 (M, N); and C2C12 myoblasts cultured in scaffolds treated with 1000 mM CaCl2 (O, P);

[0094] Figure 19 : Removal of salt residues. Residual SDS was removed from a cellulose scaffold using 100 mM CaCl2. (A) CaCl2 salt / SDS micelles were precipitated onto the biomaterial surface; phase contrast image. (B) Incubation with dH2O effectively removed salt residues. It should be noted that sonication, acetic acid incubation, and DMSO incubation produced equivalent results (see Figure 20 ). Scale bar = 200 μm;

[0095] Figure 20: Cell growth after desalting. Cells grew well for each desalting treatment. dH2O incubation: A, B; dH2O and sonication: C, D; acetic acid incubation: E, F; and DMSO incubation: G, H. Phase contrast images (A, C, E, G) show that there are no salt residues in the scaffold. Hoechst (nuclear staining) fluorescence images (B, D, F, H) show significant cell growth after 2 days of culture. Scale: 200 μm. The figure shows phase contrast (A, C, E, G) and Hoechst fluorescence staining (B, D, F, H) of decellularized apple scaffolds with C2C12 cell growth for 2 days in cultures washed with different salts. In A and B, the scaffold was incubated with dH2O. In C and D, the scaffold was incubated with dH2O and sonicated. In E and F, the scaffold was incubated with acetic acid. In G and H, the scaffold was incubated with DMSO;

[0096] Figure 21 : Various salts can be used to remove residual SDS. Different salt compounds can be used to accomplish the same task of removing residual SDS from biological materials. PBS, KCl, CaCl2, and MgCl2 (all 100mM) were used as salt washes to clean biological materials. C2C12 cell nuclei were stained with Hoechst on decellularized apples washed with different salts. Each salt treatment allowed cell growth; however, salts with divalent cations (CaCl2 and MgCl2) promoted greater cell growth. The figure shows histological images of C2C12 nuclei (2 days of growth) stained with Hoechst on decellularized apple scaffolds washed with 100mM PBS, KCl, CaCl2, MgCl2, CuSO4, KH2PO4, MgSO4, Na2CO3, and sodium ibuprofen. Different salt compounds can be used to accomplish the task of removing residual SDS from biological materials. PBS, KCl, CaCl2, MgCl2, CuSO4, KH2PO4, MgSO4, Na2CO3, and sodium ibuprofen (all 100 mM) were used as salt washes to clean the biomaterial and remove residual SDS. Each salt treatment shown in the figure allowed cell growth; however, salts with divalent cations (CaCl2 and MgCl2) and carbonate anion groups promoted greater cell growth.

[0097] Figure 22 Secondary wall staining of apple and asparagus scaffolds is shown. Different components of the cell wall can be used in biomaterials. The cinnamaldehyde groups of lignin were stained with Wiesner stain (light purple). Pectin and lignin were stained with Toluidine Blue O. Cellulose and β-(1-4)-glucan were stained with Congo Red.

[0098] Figure 23:Here we show that native cellulose can support mammalian cells, including C2C12 myoblasts, 3T3 fibroblasts, and human epithelial HeLa cells. However, functional biomaterials can also be chemically and mechanically tuned to suit specific intended uses. In these experiments, two different techniques were used to modify the stiffness of decellularized cellulose scaffolds. Additionally, phase contrast images demonstrate that the biomaterials continue to support mammalian cell culture after chemical and physical modification. A) Local mechanical elasticity of native tissue, decellularized (SDS), collagen-functionalized (SDS+Coll), and glutaraldehyde (SDS+GA) cross-linked cellulose scaffolds. Native tissue and unmodified scaffolds did not show any significant differences in mechanical properties. Both collagen-functionalized and chemically cross-linked scaffolds showed a significant increase in elasticity compared to DMEM scaffolds (***=p<0.001). (B) Decellularized (SDS), (C) collagen-functionalized (SDS+Coll), and (D) glutaraldehyde-cross-linked (SDS+GA) scaffolds all supported C2C12 cell growth. Scale bar = 200 mm;

[0099] Figure 24 : Reverse molding technique. Cellulose ring constructs from decellularized apple scaffolds were excised using a biopsy punch. C2C12 myoblasts were cultured on the scaffolds for 2 weeks. The biomaterial was completely invaded by cells. These rings were also used in combination with temporary reverse molding using gelatin (B) and permanent reverse molding using collagen (C). Both gave comparable cell growth to the bare cellulose scaffold (A). C2C12 cell nuclei were stained with Hoechst (blue), C2C12 cell membranes were stained with WGA (green), and cellulose was stained with Schiff's reagent and propidium iodide (red). Scale bar = 1000 μm. The first column shows C2C12 cell nuclei stained with Hoechst. The second column shows C2C12 cell membranes stained with WGA, used in combination with temporary reverse molding using gelatin. The third column shows cellulose from cultured C2C12 cells, stained with Schiff's reagent and propidium iodide, used in combination with permanent reverse molding using collagen. The fourth column shows the merge of the images in each of rows A, B, and C;

[0100] Figure 25 : Cell growth and reverse molding. Confocal imaging of C2C12 cells on native biomaterial (A), temporary reverse molding biomaterial using gelatin (B), and permanent reverse molding biomaterial using collagen (C). Xy and zymax projections are shown. The three different conditions gave the same results: complete invasion and high proliferation. Cellulose (red) was stained with propidium iodide using Schiff's reagent, and cell nuclei (blue) were stained with Hoechst. Scale: 200 μm;

[0101] Figure 26Figure 3: Cell invasion and proliferation with reverse molding techniques. Cell proliferation was estimated by calculating the total nuclear area for each molding technique (the control was native cellulose) (A). There were no significant differences between native cellulose, gelatin-molded, and collagen-molded samples. Cell invasion was estimated using the top:bottom nuclear area ratio (B). There were no significant differences between the three conditions. Consequently, under these experimental conditions, reverse molding did not alter cell invasion and proliferation.

[0102] Figure 27 : Establishing artificial microstructures in cellulose scaffolds derived from apples. Two different microstructures were generated within decellularized cellulose scaffolds to demonstrate the feasibility of using biomaterials for specific purposes to produce different microstructures, such as increasing host cell migration into cellulose scaffolds. In A), a 1 mm biopsy punch was used to create five negative cylindrical spaces within a cellulose scaffold derived from apples as the first example of an artificial microstructure. In contrast, in B), a 3 mm biopsy punch was used to create a single centered negative space. After only 4 weeks of implantation, increased vascularization was observed in the 1 mm and 3 mm examples directly derived from the artificially derived negative spaces (C and D). In C), blood vessels were located in each of the four corners of the biomaterial, suggesting increased vascularization within the artificially derived negative spaces. Similarly, in D), blood vessels could be observed at the top of the cellulose scaffold, suggesting that blood vessels pass through the cellulose scaffold. Cross-sections of representative cellulose scaffolds stained with hematoxylin and eosin (H&E) (EF);

[0103] Figure 28:Show pictures depicting cellulose scaffolds from various sources, and their resections and histology after 4 and 8 weeks are shown in the figure. Cellulose scaffolds derived from various plants were subcutaneously implanted in mice to evaluate the biocompatibility of 4 weeks and / or 8 weeks. Selective tissues of various plants were implanted for 4 or 8 weeks to demonstrate the biocompatibility of plant-derived cellulose and plant structures for host cell migration in vivo. In all instances, cell migration and proliferation were observed in the cellulose scaffold, highlighting the biocompatibility of the plant-derived cellulose scaffolds in these experiments. Subcutaneous implantation of cellulose scaffold biomaterials was performed on the back area of a C57BL / 10ScSnJ mouse model through a small skin incision (8 mm). Each implant was measured before implantation for scaffold area comparison (first column: cellulose scaffold). As shown, cellulose grafts were excised at 4 or 8 weeks (second column: resection). Continuous 5 μm thick sections were cut 1 mm inside the cellulose scaffold and stained with hematoxylin-eosin (H&E) (third column: histology). To assess cell infiltration, micrographs were captured using a Zeiss MIRAXMIDI Slide Scanner (Zeiss, Toronto, Canada) equipped with a 40× objective and analyzed using Pannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) and ImageJ software;

[0104] Figure 29 : Cellulose scaffold implantation and resection. Subcutaneous implantation of cellulose scaffold biomaterial was performed on the dorsal region of a C57BL / 10ScSnJ mouse model through a small skin incision (8 mm) (A). Each implant was measured before implantation for scaffold area comparison (B). The cellulose scaffolds were excised 1 week (D), 4 weeks (E) and 8 weeks (F) after surgery and macroscopic photographs were taken (control skin in C). At each time point, the blood vessels were clearly integrated with the cellulose implant, demonstrating biocompatibility. Similarly, there was no acute or chronic inflammation in the tissue surrounding the implant. The surface area of the cellulose scaffold was shown to change over time (G). The area of the scaffold before implantation was 26.30±1.98mm 2 After implantation, the stent area decreased to 20.74±1.80mm after 1 week. 2 , and decreased to 16.41±2.44mm after 4 weeks 2 , and decreased to 13.82±3.88mm after 8 weeks 2 After 8 weeks of implantation, the surface area of the cellulose scaffold was significantly reduced by approximately 12 mm 2 (48%) (*=P<0.001; n=12-14);

[0105] Figure 30: Biocompatibility and cellular infiltration. Cross-sections of representative cellulose scaffolds stained with H&E and anti-CD45. These global views show an acute, moderate-to-severe, expected foreign body reaction at 1 week (A), a mild, chronic immune response and subsequent clearance at 4 weeks (B), and finally, the assimilation of the cellulose scaffold into native mouse tissue at 8 weeks (C). Higher magnification regions of interest (DF), see insets (AC), allow for visualization of cell type populations during the biomaterial assimilation process. At 1 week, we can observe granulocyte populations, specifically polymorphonuclear leukocytes (PMNs) and eosinophils, characteristic of an acute, moderate to severe immune response, which is a normal response to the implantation procedure (D). At 4 weeks, a reduced immune response (mild to low-grade immune response) is observed, and the cell population within the epidermis surrounding the scaffold now contains higher levels of monocytes and lymphocytes, characteristic of a chronic response (E). Finally, at 8 weeks, the immune response is completely resorbed, and the epidermal tissue now appears normal (F). The immune response observed with H&E staining (GI) was confirmed using anti-CD45 antibodies, a well-known leukocyte marker. The cell population within the scaffold at this point was primarily composed of macrophages, multinucleated cells, and activated fibroblasts. Scale bars: AC = 1 mm, DF = 100 μm, GI = 500 μm.

[0106] Figure 31 : Extracellular matrix deposition. Cross-sections of representative cellulose scaffolds stained with Masson's trichrome (AC). One week after implantation, magnification of the region of interest in (A), see inset, shows the loss of collagen structure within the collagen scaffold (D, G). As fibroblasts begin to invade the scaffold, collagen deposits can be sparsely observed within the cellulose scaffold after 4 weeks (E, H). Activated fibroblasts (spindle-shaped cells) are observed within the cellulose scaffold, and a collagen network is clearly visible in the lumen after 8 weeks (F, I). Scale bars: AC = 1 mm, DF = 100 μm, GI = 20 μm. * = collagen fibers; black arrows = cellulose cell walls; white arrows = fibroblasts;

[0107] Figure 32 : Vascularization and angiogenesis. Blood vessels were directly visualized in the surrounding tissue surrounding the cellulose scaffold (A). Angiogenesis within the cellulose scaffold was confirmed by observing multiple cross-sections of blood vessels in micrographs of H&E staining (B) and Masson's trichrome staining (C). The angiogenic process was also confirmed by identifying endothelial cells within the cellulose scaffold by anti-CD31 staining (D). Scale bars: A = 1 mm, B = 50 μm, CD = 20 μm. White arrow = blood vessel;

[0108] Figure 33A) Two-photon confocal image of xylem structures (*) in decellularized asparagus (bar = 0.1 mm), using a cellulose-specific stain (red) to visualize fine structures within the plant. B) Phase contrast image of a single continuous xylem microchannel (*) in plant xylem (bar = 0.1 mm). C) SEM image of a freeze-fractured xylem microchannel (bar = 20 μm). D) General view of a decellularized plant plug prepared for implantation.

[0109] Figure 34 : A) Primary neurons grown in vitro along the walls of xylem microchannels of a decellularized plant scaffold (stained green with a cell membrane dye). Cross-sectional images (2 μm thick) at 1 mm depth were obtained within a 3 mm long plug (bar = 0.1 mm). B) H&E staining of a decellularized plant scaffold implanted subcutaneously 4 weeks later (bar = 1 mm). Inset: Cross-section of a xylem microchannel (bar = 0.2 mm). C) General view of a 3 mm decellularized plant graft (arrow) implanted into the spinal cord;

[0110] Figure 35 : A) MRI axial view, (i) upper and (iii) lower part of (ii) graft (arrow). (ii) Laminectomy at T8 / 9 resulted in loss of nerve roots visible in (i) and (iii). B) After 8 weeks, the spinal cord and brain were removed. The graft (arrow) appeared well integrated with no signs of infection, calcification or fibrosis. C) Motor recovery 8 weeks after implantation was demonstrated in (i-iii) coordinated stepping and treadmill weight bearing (illustrated) and on a flat BBB platform. D) BBB scores of control (red, n=4) and graft- (blue, n=7) rats on a flat BBB platform. E) Staining shows myelinated nerves from the spinal cord (sc) growing into the microchannels of the biomaterial (bm) (bar = 200 μm). The interface is indicated by a dotted line;

[0111] Figure 36 A global view of a whole spinal cord graft implanted in the T8-T9 region of the spine. The tissue was stained with H&E-LFB, which shows nuclei as dark purple and myelinated tissue as light blue. Importantly, microchannels spanning the entire length of the graft are infiltrated with both host cell types.

[0112] Figure 37: Ventral sections around the transection site (top cranial; bottom caudal) of control and spinal graft implanted rats were stained for neurofilament markers (NF200 green) and nuclei (Hoechst blue). In A), the dark area represents the location of the biomaterial. Interestingly, around the spinal graft, green filaments can be observed extending in the ventral direction (red arrows). These filaments represent mature neurons within the transection site of rats after 12 weeks in vivo. In contrast, in the control B), no organized neurofilaments can be observed, indicating a lack of mature filaments within the control transection site. In addition, Hoechst staining showed a significant increase in the number of nuclei, and as such cells, around the spinal graft within the transection site compared to the control;

[0113] Figure 38 Decellularization of apple syringa tissue and processing for skin grafting. The dorsal skin of C57BL / 10ScSnJ mice was shaved and prepared for surgery. (A) A 10 mm outer diameter rubber pad was sutured to the dorsal skin to prevent wound closure. (B) A 5 mm diameter piece of decellularized apple syringa tissue was placed in the center of the rubber pad and covered with a semipermeable adhesive. (C) Photographs were taken 4 days later to measure the extent of host cell infiltration during wound healing.

[0114] Figure 39 :Plant-derived cellulose scaffolds for bone transplantation. Each cylindrical (5 mm diameter, 1 mm thick) implant was measured for scaffold area comparison before implantation (A). Cellulose scaffold implants were implanted into rat skull defects and positioned to remain within the skull defect. The skin was then positioned on the graft and sutured to keep the scaffold in place. (B) The scaffold and surrounding bone tissue were separated and macroscopic photographs were taken 4 weeks after implantation (C). The separated tissue was then decalcified and processed / embedded in paraffin. Continuous 5 μm thick sections were cut 1 mm from the inside of the cellulose scaffold and stained with hematoxylin-eosin (H&E) (D). To evaluate bone regeneration, micrographs were captured using a Zeiss MIRAX MIDI Slide Scanner (Zeiss, Toronto, Canada) equipped with a 40x objective lens and analyzed using Pannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) and ImageJ software.

[0115] Figure 40: Different cellulose formulations, physical properties, and functionalizations. Cellulose can be used as blocks with different shapes (A) or dehydrated and ground into a powder form, which can then be rehydrated to the desired consistency to produce a gel (C, D) or paste (E, F). If the cellulose contains carboxymethyl cellulose, it can be easily cross-linked with citric acid and heat (B). Cellulose from different plants can also be combined, blended, cross-linked, etc.; and

[0116] Figure 41 : The figures show (A) a graph showing the survival of mice (n=190) and rats (n=12) 1 week, 4 weeks and 8 weeks after implantation of biomaterials (from various sources). (B) a graph showing the rejection rate of biomaterials at the same time points as in (A). DETAILED DESCRIPTION

[0117] This article describes a scaffold biomaterial comprising a decellularized plant or fungal tissue from which the cellular material and nucleic acids of the tissue have been removed, wherein the decellularized plant or fungal tissue comprises a porous structure based on cellulose or chitin. Also provided are methods for preparing such a scaffold biomaterial, and uses thereof, as an implantable scaffold for supporting animal cell growth, promoting tissue regeneration, promoting angiogenesis, tissue replacement procedures, promoting angiogenesis, and / or as a structural implant for cosmetic surgery. In addition, therapeutic treatments and / or cosmetic methods using such a scaffold are described, as well as other applications that may include, for example, veterinary applications. It should be understood that the embodiments and examples are provided for the purpose of illustrating to those skilled in the art and are not intended to be limiting in any way.

[0118] In certain embodiments, biomaterials are described herein that can have applications, for example, in biomedical laboratory research and / or clinical regenerative medicine. Such biomaterials can be effective as scaffolds, which can be used as research tools for biomedical implants, sensing devices, and drug delivery vehicles by industrial / academic biomedical researchers, and / or other suitable applications in which scaffolds can be used.

[0119] In certain embodiments, the biomaterials described herein can be derived from cell wall structures found in the plant and fungal kingdoms to produce complex 3D scaffolds that can promote cell infiltration, cell growth, angiogenesis, tissue repair and / or tissue reconstruction, etc. (e.g., see Figure 1 As will be appreciated, biomaterials as described herein can be produced from any suitable part of a plant or fungal organism, including, for example, seeds, roots, bark, leaves, stems, fruit, pulp, cores, and in certain embodiments, can be produced in various shapes (e.g., sheets, vessels, blocks, tubes, pores, etc.) or formulations (including, for example, pastes, granules, blocks, etc.) (see, for example, Figure 2). Biomaterials may include, for example, substances such as cellulose, chitin and / or any other suitable biochemicals / biopolymers found naturally occurring in these organisms.

[0120] In certain embodiments, the resulting scaffolds can also be: chemically modified to introduce custom surface chemistry; cut into solid blocks, injectable / extrudable pastes and / or slurries; and / or can offer a range of construction possibilities in the micron to centimeter range that can replace / mimic several living tissue environments.

[0121] As described herein, the use of such plant / fungus derived biomaterials can produce highly porous scaffolds that can have extremely thin walls (<100 nm) (see, e.g., Figure 1 In certain embodiments, this can provide a minimal footprint for the scaffold material (ie, when living cells fully invade, the cell to scaffold volume ratio can be significantly high).

[0122] In certain embodiments, the scaffold biomaterials as described herein can be biocompatible. As described in further detail below, following subcutaneous implantation of exemplary scaffold biomaterials in a mouse model, complete cellular infiltration and angiogenesis with functional vascularization were observed within 4 weeks after implantation (see, e.g., Figure 3 and 4 As demonstrated in experiments detailed below, when the scaffolds were implanted in vivo, the minimal footprint promoted cellular infiltration, angiogenesis, and tissue repair, and under the conditions tested, only a minimal inflammatory response (primarily resulting from the procedure itself, not the scaffold) was observed.

[0123] The experiments described below show that the plant / fungus derived biomaterials as described herein are fully biocompatible in vivo under the conditions tested. They are also fully compatible with in vitro studies, e.g. Figure 5 As shown, and Modulevsky, DJ, Lefebvre, C., Haase, K., Al-Rekabi, Z., and Pelling, AE “Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture.” Plos One, 9, e97835 (2014) (incorporated herein by reference).

[0124] In certain embodiments, unlike many commercial biomaterials, biomaterials derived from plants / fungi as described herein can be non-resorbable or difficult to resorb (i.e., they do not substantially decompose and are absorbed by the body). The non-resorbable properties of these scaffolds can provide certain benefits. For example, in certain embodiments, biomaterials as described herein can resist shape changes, and / or can maintain their expected geometry for a long time. In certain embodiments, because they can have minimal footprints compared to some other products, it can be considered that they are actually invisible to the body and hardly cause an immune response. When resorbable biomaterials decompose, their by-products typically cause adverse immune responses, and induce oxidative stress and cause the pH in the restored tissue to increase, which can be avoided by using non-resorbable biomaterials.

[0125] As will be understood, the meanings / definitions of the kingdom Plantae and Fungi used herein are based on the Cavalier-Smith classification (1998), unless otherwise indicated.

[0126] Scaffold biomaterials

[0127] In one embodiment, provided herein are scaffold biomaterials comprising decellularized plant or fungal tissue from which the cellular material and nucleic acids of the tissue have been removed, the decellularized plant or fungal tissue comprising a cellulose or chitin-based three-dimensional porous structure. As will be appreciated, in certain embodiments, the scaffold biomaterial may comprise substances foreign to the host that may provide infrastructure, support, and / or foundational structure for host cell infiltration, invasion, and / or proliferation.

[0128] In certain embodiments, the scaffold biomaterial can comprise a substantially solid state, block or other rigid shape, can be dehydrated and ground into a powder or granular form, can be in a cross-linked form (particularly when the scaffold biomaterial comprises a cellulose-based tissue, the tissue contains carboxymethyl cellulose, which can be easily cross-linked with citric acid and heat), or can be in a gel or paste form. Such a gel or paste can be produced by, for example, rehydrating the tissue in powder form to the desired consistency to produce a gel or paste. Additionally, in certain embodiments, compression molding can be used to generate a cellulose-based biomaterial sheet, optionally with various additives to enhance cross-linking. These additives can include, but are not limited to, oxalic acid, malonic acid, succinic acid, malic acid or citric acid, which can be added to the pulp or sprayed as a catalyst with sodium dihydrogen phosphate.

[0129] As will be appreciated, the decellularized plant or fungal tissue can comprise any suitable biological material derived or produced from a suitable plant or fungal derivative or direct tissue sample. In certain embodiments, such material can comprise a base structure and / or a reticular support structure, which can be produced by a suitable combination or single method to remove, lyse, or enzymatically treat native cells from plant or fungal tissue.

[0130] In certain embodiments of the above-mentioned scaffold material, the plant or fungal tissue can include apple inflorescence (Malus pumila) tissue, fern (Monilophytes) tissue, radish (Brassica rapa) root tissue, ginkgo branch tissue, horsetail (equisetum) tissue, hermocallis hybrid leaf tissue, kale (Brassica oleracea) stem tissue, conifer Douglas fir (Pseudotsuga menziesii) tissue, cactus fruit (pitaya) flesh tissue, spotted periwinkle (Maculata Vinca) tissue, water lotus (Nelumbo nucifera) tissue, tulip (Tulipagesneriana) petal tissue, plantain (Musa paradisiaca) tissue, broccoli (Brassica oleracea) stem tissue, maple leaf (Acer psuedoplatanus) stem tissue, beet (Beta vulgaris) primary root tissue, onion (Alliumcepa) tissue, orchidaceae (Orchidaceae) tissue, radish (Brassica rapa) stem tissue, leek (Allium ampeloprasum) tissue, maple (Acer) branch tissue, celery (Apium graveolens) tissue, onion (Allium cepa) stem tissue, pine tissue, aloe vera tissue, watermelon (Citrullus lanatus var. lanatus) tissue, meadowgrass (Lysimachia nummularia) tissue, cactae tissue, Arctic campion (Lychnis Alpina) tissue, rhubarb (Rheum rhabarbarum) tissue, pumpkin flesh (Cucurbita pepo) tissue, Dracena (Asparagaceae) stem tissue, tradescantia virginiana stem tissue, asparagus (Asparagus officinalis) stem tissue, mushroom (Fungi) tissue, fennel (Foeniculum vulgare) tissue, rose (Rosa) tissue, carrot (Daucus carota) tissue, or pear (Pomaceous) tissue.

[0131] In certain embodiments, the plant or fungal tissue can be genetically altered, either by direct genomic modification or by selective breeding, to produce additional plant or fungal structures that can be configured to physiologically mimic the tissue and / or functionally promote the effects of the target tissue. A skilled artisan, considering the teachings herein, will be able to select an appropriate scaffold biomaterial to suit a particular application.

[0132] In certain embodiments, suitable tissues for a particular application can be selected based on, for example, physical characteristics (e.g., size, structure (porous / tubular), stiffness, strength, hardness, and / or ductility), which can be determined and consistent with the particular application. In addition, chemical properties such as reactivity, coordination number, enthalpy of formation, heat of combustion, stability, toxicity, and / or type of bond can also be considered in selecting tissues suitable for a particular application. These characteristics (physical and chemical) can also be modified directly before or after decellularization and / or functionalization for specific applications. In addition, in certain embodiments, cellulose can be derived from different plants and can be combined and mixed, cross-linked, etc. using the chemical methods outlined below.

[0133] In certain embodiments, the scaffold biomaterial can be such a scaffold biomaterial, wherein the decellularized plant or fungal tissue of the scaffold biomaterial is configured to physiologically simulate the tissue of the subject and / or functionally promote the effect of the target tissue of the subject. In certain embodiments, the method of using such a scaffold biomaterial as described herein can include the step of selecting a scaffold biomaterial as described herein, wherein the decellularized plant or fungal tissue of the scaffold biomaterial is configured to physiologically simulate the tissue of the subject and / or functionally promote the effect of the target tissue of the subject. A technician taking into account the teachings of this article will be able to select an appropriate scaffold biomaterial to suit a particular application.

[0134] As a non-limiting example, Figure 6 Several examples of scaffold biomaterials are provided, demonstrating histological cell wall structures and their correspondence to certain tissues / tissue functions, which, in certain embodiments, can be used to guide the selection of scaffold biomaterials suitable for specific applications. As will be appreciated, cell wall structures found in the plant and fungal kingdoms exhibit a variety of structures that can resemble tissues such as bone, skin, and nerves. Depending on the target tissue, the plant or fungal origin of the biomaterial can be determined based on the physical and / or chemical characteristics of the plant and / or the physical and / or chemical characteristics of the resulting scaffold biomaterial.

[0135] As will be understood, cellular material and nucleic acids may include intracellular contents, such as organelles (e.g., chloroplasts, mitochondria), nuclei, cellular nucleic acids, and cellular proteins. These may be substantially removed, partially removed, or completely removed from the scaffold biomaterial. It will be appreciated that trace amounts of such components may still be present in the decellularized plant or fungal tissue described herein.

[0136] As will be appreciated, in certain embodiments, a three-dimensional (3D) porous structure may comprise a suitable structure that provides a base structure, support and / or infrastructure for foreign cell infiltration, invasion and / or proliferation while providing a continuous supply of culture medium / nutrients by passive diffusion.

[0137] Various methods can be used to produce the scaffold biomaterials described herein. For example, in certain embodiments of the scaffold biomaterials described above, the decellularized plant or fungal tissue may comprise a plant or fungal tissue that has been decellularized by the following methods: heat shock, treatment with a detergent (e.g., SDS, Triton X, EDA, alkaline treatment, acid, ionic detergent, non-ionic detergent, and zwitterionic detergent), osmotic shock, freeze drying, physical lysis (e.g., hydrostatic pressure), electrical destruction (e.g., non-thermal irreversible electroporation), or enzymatic digestion, or any combination thereof. In certain embodiments, the biomaterials described herein can be obtained from plants and / or fungi by a decellularization method, which can include any one of several methods (alone or in combination), including but not limited to heat shock (e.g., rapid freeze-thaw), chemical treatment (e.g., detergent), osmotic shock (e.g., distilled water), freeze drying, physical lysis (e.g., pressure treatment), electrical destruction, and / or enzymatic digestion.

[0138] In certain embodiments, the decellularized plant or fungal tissue may comprise a plant or fungal tissue that has been decellularized by treatment with a detergent or surfactant. Examples of detergents may include, but are not limited to, sodium dodecyl sulfate (SDS), Triton X, EDA, alkyl treatments, acids, ionic detergents, nonionic detergents, and zwitterionic detergents.

[0139] In still further embodiments, the decellularized plant or fungal tissue may comprise plant or fungal tissue that has been decellularized by SDS treatment.

[0140] In yet another embodiment, residual SDS can be removed from decellularized plant or fungal tissue using a divalent saline solution, which can be used to precipitate / disintegrate salt residues containing SDS micelles from the solution / scaffold, and dHO, acetic acid, dimethyl sulfoxide (DMSO), or sonication can be used to remove salt residues and / or SDS micelles.

[0141] In certain embodiments, the divalent salt of the divalent salt solution may include, for example, MgCl 2 or CaCl 2 .

[0142] In another embodiment, the plant or fungal tissue may be treated with an SDS solution containing 0.01 to 10%, such as about 0.1% to about 1%, or for example about 0.1% SDS or about 1% SDS in a solvent (such as water, ethanol or other suitable organic solvent) and decellularized. Residual SDS may be removed using an aqueous solution of CaCl2 at a concentration of about 100 mM, followed by incubation in dH2O.

[0143] In certain embodiments, the SDS solution can have a concentration higher than 0.1%, which can facilitate decellularization and can be accompanied by enhanced washing to remove residual SDS.

[0144] In certain embodiments, the plant or fungal tissue may have been decellularized by treatment with an SDS solution containing about 0.1% SDS in water, and residual SDS may be removed using an aqueous CaCl2 solution at a concentration of about 100 mM prior to incubation in dH2O.

[0145] Examples of experimental protocols for preparing biomaterials as described herein are provided in further detail in the "Scaffold Biomaterial Preparation Methods" section below and in Example 1.

[0146] In another embodiment of the above scaffold material, the decellularized plant or fungal tissue can be functionalized at least some of the free hydroxyl groups by acylation, alkylation or other covalent modification to provide a functionalized scaffold biomaterial. In certain embodiments, for example, the decellularized plant or fungal tissue can be functionalized with collagen.

[0147] In another embodiment of the above scaffold material or materials, the scaffold biomaterial may further comprise living animal cells adhered to the cellulose or chitin-based three-dimensional porous structure. In another embodiment, the living animal cells may be mammalian cells. In another embodiment, the living animal cells may be human cells.

[0148] Preparation method of scaffold biomaterial

[0149] In one embodiment, provided herein is a method for preparing a decellularized plant or fungal tissue, wherein cellular material and nucleic acids of the tissue are removed, the decellularized plant or fungal tissue comprising a cellulose or chitin-based three-dimensional porous structure, the method comprising:

[0150] providing a plant or fungal tissue having a predetermined size and shape; and

[0151] decellularizing the plant or fungal tissue by heat shock, treatment with detergents, osmotic shock, freeze drying, physical lysis, electrical disruption, or enzymatic digestion, or any combination thereof,

[0152] Cellular material and nucleic acids are thereby removed from the plant or fungal tissue to form a decellularized plant or fungal tissue comprising a three-dimensional porous structure based on cellulose or chitin.

[0153] In certain embodiments, the step of decellularizing the plant or fungal tissue may comprise decellularizing by treatment with a detergent. Examples of detergents may include, but are not limited to, sodium dodecyl sulfate (SDS), Triton X, EDA, alkyl treatments, acids, ionic detergents, nonionic detergents, or zwitterionic detergents.

[0154] In further embodiments, the step of decellularizing the plant or fungal tissue may comprise decellularizing the plant or fungal tissue by treatment with SDS.

[0155] In yet another embodiment, the step of decellularizing the plant or fungal tissue can remove residual SDS from the decellularized plant or fungal tissue by washing with a divalent salt solution. The divalent salt solution is used to precipitate / disintegrate salt residues containing SDS micelles from the scaffold, and dHO, acetic acid, dimethyl sulfoxide (DMSO), or sonication can be used to remove the salt residues or SDS micelles. The divalent salt of the divalent salt solution can include, for example, MgCl or CaCl.

[0156] In a specific embodiment, the decellularization step may include treatment with an SDS solution containing about 0.1% SDS in water, and after decellularization, residual SDS may be removed using an aqueous CaCl2 solution at a concentration of about 100 mM, followed by incubation in dH2O.

[0157] In another embodiment of the above method, the method can further include the step of functionalizing at least some of the free hydroxyl groups of the decellularized plant or fungal tissue by acylation, alkylation or other covalent modification. In certain embodiments, the hydroxyl groups of the decellularized plant or fungal tissue can be functionalized with collagen.

[0158] In another embodiment of the above method, the method may further include the step of introducing living animal cells into the cellulose- or chitin-based three-dimensional porous structure and allowing the living animal cells to adhere to the cellulose- or chitin-based three-dimensional porous structure. In certain embodiments, the living animal cells may be mammalian cells. In certain embodiments, the living animal cells may be human cells.

[0159] Application of scaffold biomaterials

[0160] In certain embodiments, for example, the biomaterials described herein can have applications in biomedical laboratory research and / or clinical regenerative medicine in human and / or veterinary applications. Such biomaterials can be effective as scaffolds, which can be used as research tools for biomedical implants, sensing devices, and drug delivery vehicles for industrial / academic biomedical researchers, and / or other suitable applications in which scaffolds can be used.

[0161] In certain embodiments, the scaffold biomaterials as described herein can be used as implantable scaffolds for supporting animal cell growth, promoting tissue regeneration, promoting angiogenesis, tissue replacement procedures, or as structural implants for cosmetic surgery.

[0162] In certain embodiments, the scaffold biomaterials as described herein can be used as a structural implant for repair or regeneration after spinal cord injury; as a structural implant for tissue replacement surgery and / or postoperative tissue regeneration; as a structural implant for skin grafting and / or skin regeneration surgery; as a structural implant for regeneration of the vascular system in a target tissue or region; as a bone replacement, bone filler or bone graft material, and / or for promoting bone regeneration; as a tissue replacement for skin, bone, spinal cord, heart, muscle, nerve, blood vessel or other damaged or deformed tissue; as a vitreous humor replacement (in hydrogel form); as an artificial bursa in which the scaffold biomaterial forms a sac-like structure containing the scaffold biomaterial in hydrogel form; and / or as a structural implant for cosmetic surgery.

[0163] In certain embodiments, the scaffold biomaterials described herein can be used as breast implants. Thus, the scaffold can be formulated to match the mammary glands / tissue found in a human breast and then used as a filling material, for example, for a breast implant.

[0164] In certain other embodiments, the scaffold biomaterial as described herein can be used as a cartilage replacement: thus, the scaffold can be formulated and designed to mimic cartilage tissue and used to replace certain body parts, such as the ears and nose.

[0165] In certain embodiments, the scaffold biomaterial as described herein can be used as a skin graft. The cellulose scaffold can be used as a skin graft to protect, repair and / or regenerate skin (epithelial / endothelium) after skin surgery (e.g., gums, etc.) or injury events (e.g., burns, etc.). In certain embodiments, it can be used to protect damaged tissue from external infection and / or directly regenerate tissue.

[0166] In certain embodiments, the scaffold biomaterials described herein can be used for regeneration of vascular systems. The wide range of cellulose structures available can allow for the artificial generation of vessel-like structures and / or can provide conditions suitable for angiogenesis (natural blood vessel formation).

[0167] In another embodiment, the scaffold biomaterials as described herein can be used for bone replacement or bone filling. Thus, the cellulose scaffolds can be formulated and designed to mimic bone tissue and then used to replace bones and bone parts, such as in dentistry, skull, fractures, hip replacement (bone or prosthetic fillers, etc.) and / or other such applications.

[0168] In certain embodiments, the scaffold biomaterials as described herein can be used as simple or complex tissues. For example, the scaffold can be used to replace simple (skin, bone) or complex (spinal cord, heart, muscle, nerve, blood vessel, etc.) tissues after an accident, deformity, aesthetics, injury, or other tissue damage.

[0169] In other embodiments, the scaffold biomaterials described herein can be used as vitreous humor materials. For example, a cellulose scaffold in the form of a hydrogel is a translucent gel. The consistency and clarity can be adjusted to match that of natural vitreous humor.

[0170] In certain embodiments, the scaffold biomaterials described herein can be used as bursae. Artificial bursae and their corresponding fluids can be made from the biomaterials described herein. The bursae can be made from solid cellulose, while the fluid can be formed from, for example, a cellulose hydrogel.

[0171] In certain embodiments, provided herein are methods for supporting animal cell growth, promoting tissue regeneration, promoting angiogenesis, replacing tissue, or providing a structural scaffold for cosmetic surgery in a subject in need thereof, the methods comprising:

[0172] Providing a scaffold biomaterial according to any of the above scaffold biomaterials; and

[0173] The scaffold biomaterial is implanted into a subject.

[0174] In certain embodiments, the scaffold biomaterial can be implanted into the spinal cord and promote repair or regeneration after spinal cord injury; can provide a subject with a structural implant for tissue replacement and / or tissue regeneration; can provide a subject with a structural implant for skin grafting and / or skin regeneration; can provide a subject with a structural implant for regeneration of the target tissue or region or vascular system in the subject; can provide a subject with a bone replacement, bone filler or bone graft material, and / or can promote bone regeneration; can provide a subject with a tissue replacement for skin, bone, heart, muscle, nerve, blood vessel or other damaged or deformed tissue; can provide a subject with a vitreous humor replacement (when in hydrogel form); can provide a subject with an artificial bursa, wherein the scaffold biomaterial forms a sac-like structure containing the scaffold biomaterial in hydrogel form; and / or can provide a structural implant for cosmetic surgery.

[0175] In certain embodiments, the scaffold biomaterial can be implanted into the spinal cord and can promote repair and / or regeneration following acute and / or chronic spinal cord injury in the central and / or peripheral nervous system.

[0176] Example 1 - Example of an experimental protocol for the production of scaffold biomaterials

[0177] In this example, two experimental protocols are described for preparing scaffold biomaterials as described herein from apple syringa tissue (Malus pumila). It should be understood that these protocols are provided as illustrative, non-limiting examples for those skilled in the art. A skilled person, considering the teachings herein, will appreciate various modifications, additions, substitutions, and / or other variations that may be made to these exemplary protocols.

[0178] The initial experimental protocol described below was successfully used to prepare scaffold biomaterials. However, this protocol required several weeks to provide complete cell infiltration under the conditions tested. Therefore, a modified protocol was subsequently developed that included the use of a calcium chloride wash (CaCl2) that gave similar results to the scaffold biomaterials prepared with the first protocol, but within a week (see Figure 9 and 10 ).

[0179] Initial protocol for in vivo (animal model) studies:

[0180] 1. Cut the apple slices into the desired shape and size

[0181] a. Cut the apple in half

[0182] b. Soak half of the apple in PBS, cut side down

[0183] c. Adjust the mandolin slicer to obtain the appropriate thickness (1.2 mm in this example)

[0184] d. Take an even slice of the apple without the core visible and place it on a metric cutting board

[0185] e. Cut one side of the apple to further process into cubes and keep the other piece in PBS

[0186] f. Cut the apple tissue into cubes using the guide wire (5mm X 5mm).

[0187] g. Place the cut slices into a 1.5 mL microcentrifuge tube

[0188] h. Measure the cut surface of unused sections at least 10X and record in the laboratory manual.

[0189] 2. Add 1 mL of 0.1% SDS (in autoclaved dH2O) and incubate on a shaker at 180 RPM RT for 2 days (room temperature)

[0190] a. Check that the apple block is not floating.

[0191] b. If the apple still floats, continue with SDS processing

[0192] 3. Place the processed apples in the microcentrifuge tube into the biosafety cabinet.

[0193] 4. Remove 0.1% SDS solution (room temperature) with a Pasteur pipette

[0194] 5. Wash the apple slices 4 times with autoclaved PBS (room temperature)

[0195] a. During washing, try to place the Pasteur pipette as close to the apple as possible without touching it. This is to allow the water to flow through the apple tissue.

[0196] b. When there is no liquid in the tube, continue to use the Pasteur pipette to draw liquid solution from the apple

[0197] c. As you wash more, you will see less "soap suds" remaining when you suck it through the straw

[0198] d. Do not stop washing until you see no more "soap bubbles" coming out of the apples

[0199] e. Apples should not float

[0200] 6. Place the required samples in a sterile microcentrifuge tube in relative positions.

[0201] 7. Remove the last PBS wash from the microcentrifuge tube and replace it with 70% ethanol.

[0202] 8. Place in 70% ethanol for 30 minutes to 1 hour

[0203] 9. Remove 70% ethanol

[0204] 10. Continue washing the apple slices with sterile PBS using the same technique as above.

[0205] a. Make sure to replace the Pasteur pipette

[0206] 11. Continue washing with PBS until the apple slices stop floating (at least 4 times)

[0207] 12. Remove PBS and replace with 1% penicillin / streptomycin PBS

[0208] 13. Implantation into animal models.

[0209] Improved in vivo study protocols:

[0210] 1. Cut the apple slices into the desired shape and size

[0211] a. Cut the apple in half

[0212] b. Soak half of the apple in PBS, cut side down

[0213] c. Adjust the mandoline slicer to obtain the appropriate thickness (1.2mm in this example)

[0214] d. Take an even slice of the apple without the core visible and place it on a metric cutting board

[0215] e. Cut one side of the apple to further process into cubes and keep the other piece in PBS

[0216] f. Cut the apple tissue into cubes using the guide wire (5mm X 5mm).

[0217] g. Place the cut slices into a 1.5 mL microcentrifuge tube

[0218] h. Measure the cut surface of unused sections at least 10X and record in the laboratory manual.

[0219] 2. Add 1 mL of 0.1% SDS (in autoclaved dH2O) and incubate on a shaker at 180 RPM RT for 2 days (room temperature).

[0220] a. Check that the apple block is not floating.

[0221] b. If the apple still floats, continue with SDS processing

[0222] 3. Place the processed apples in the microcentrifuge tube into the biosafety cabinet.

[0223] 4. Remove 0.1% SDS solution (room temperature) with a Pasteur pipette

[0224] 5. Wash the apple slices 4 times with autoclaved dH2O (room temperature)

[0225] a. During washing, try to place the Pasteur pipette as close to the apple as possible without touching it. This is to allow the water to flow over it.

[0226] b. When there is no liquid in the tube, continue to use the Pasteur pipette to draw liquid solution from the apple

[0227] c. As you perform more washes, you will see a decrease in the amount of "soap foam" remaining when pipetting

[0228] d. Do not stop washing until you see no more "soap bubbles" coming out of the apples

[0229] e. Apples should not float

[0230] 6. Add 100 mM CaCl2 (in autoclaved dH2O) and leave overnight (room temperature)

[0231] 7. Remove CaCl2 solution (room temperature)

[0232] 8. Place the required samples in sterile microcentrifuge tubes in relative positions.

[0233] 9. Remove the final water wash from the microcentrifuge tube and replace it with 70% ethanol.

[0234] 10. Place in 70% ethanol for 30 minutes to 1 hour

[0235] 11. Remove 70% ethanol

[0236] 12. Continue washing the apple slices with water using the same technique mentioned above.

[0237] a. Make sure to replace the Pasteur pipette

[0238] 14. Continue washing with PBS until the apple slices stop floating (at least 4 times)

[0239] 15. Remove PBS and replace with 1% P / S PBS

[0240] 16. Implantation into animal models.

[0241] Example 2 - Mouse Implantation

[0242] In vivo mouse implantation studies were performed to investigate the in vivo effects of the scaffold biomaterial embodiments as described herein.

[0243] The results showed that complete cellular infiltration was observed after subcutaneous implantation in a mouse model (see Figure 7 ; 1, 4 and 8 weeks after implantation), collagen deposition ( Figure 4 A), and importantly, angiogenesis with functional vessel formation within 4 weeks after implantation ( Figure 4 B and 8). When implanted in vivo, the minimal footprint promotes cell infiltration, angiogenesis, and tissue repair, with minimal inflammatory response (primarily caused by the procedure itself rather than the stent). In these studies, the plant / fungal derived scaffolds were fully biocompatible in vivo. These scaffolds were also fully compatible with in vitro studies, such as ( Figure 5 ) as shown.

[0244] Non-biodegradable biomaterials:

[0245] The field has primarily focused on biodegradable materials; however, this approach presents many problems in practice. Unlike many commercial biomaterials, in certain embodiments, the biomaterials of the present invention may be considered non-resorbable (i.e., may not be completely broken down and absorbed by the body) (see Figure 9 ).

[0246] The non-resorbable nature of such scaffolds can provide certain advantages over competing commercial products. For example, they may be (i) more resistant to deformation and / or maintain their intended geometry for extended periods of time; (ii) have a minimal footprint compared to competing products, making them virtually invisible to the body and unlikely to provoke an immune response; (iii) compared to resorbable materials, they may avoid the production of byproducts that, upon decomposition, may produce an adverse immune response; and / or (iv) when resorbable biomaterials decompose, newly regenerated tissue may be damaged and subsequently eliminated; in some instances, the biomaterials described herein can avoid this situation.

[0247] In vitro studies:

[0248] The in vitro experiments described herein were performed to demonstrate cell invasion and proliferation within the cellulose scaffolds. When the first protocol (described in Example 1 above) was used, complete cell infiltration required several weeks. A modified protocol (also described in Example 1 above) was subsequently developed that included the addition of a calcium chloride wash (CaCl ) that gave similar results, but only within one week (see Figure 9 ).

[0249] In vivo studies:

[0250] Preclinical trials were conducted in a mouse model to investigate responses to subcutaneous implantation of 5×5×1 mm scaffolds over 1, 4, and 8 weeks. Cellulose-based scaffolds were derived from apple, fennel, and asparagus, and chitin-based scaffolds were derived from white mushrooms (see Figure 6 ).

[0251] In these studies, all scaffolds had similar biocompatibility, with no rejection and observed cell invasion and angiogenesis (the formation of blood vessels).

[0252] Example 3 - In vivo biocompatibility of scaffold biomaterials

[0253] To address the in vivo biocompatibility of scaffold biomaterials, the body's response to apple-derived cellulose scaffolds has been characterized. 3) and subcutaneously implanted them in a mouse model for 1, 4, and 8 weeks. Here, the immune response of immunocompetent mice, the deposition of extracellular matrix on the scaffold, and evidence of angiogenesis (blood vessel formation) in the implanted cellulose biomaterial were assessed. Notably, although a foreign body reaction was observed immediately after implantation, as expected from a surgical procedure, only a low immune response was observed in all animal groups at the completion of the study, without any mortality or overt infection. It was also found that surrounding cells invaded the scaffold, primarily activated fibroblasts, and deposited new extracellular matrix. Similarly, the scaffold itself was able to retain most of its original shape and structure over the 8-week study. Importantly, the scaffold clearly had an angiogenic effect, causing functional blood vessels to grow throughout the implanted biomaterial. In summary, this work demonstrates that there is a relatively simple method to produce a biocompatible 3D cellulose scaffold that can become vascularized and integrated into surrounding healthy tissue.

[0254] In these studies, natural apple inflorescence tissue and a convenient preparation method for producing implantable cellulose scaffolds were used. To examine biocompatibility, the scaffolds were subcutaneously implanted in wild-type immunocompetent mice (male and female; 6-9 weeks old). After implantation, the scaffolds were excised and histologically analyzed (H&E, Masson trichrome, anti-CD31 and anti-CD45 antibodies) at 1, 4, and 8 weeks. Histological analysis showed a characteristic foreign body reaction to the scaffold 1 week after implantation. However, the immune response was observed to gradually disappear 8 weeks after implantation. By 8 weeks, there was no immune response in the surrounding dermal tissue, and active fibroblast migration was present within the cellulose scaffold. This was accompanied by the deposition of new collagen extracellular matrix. In addition, active blood vessel formation within the scaffold was observed throughout the study period, indicating the pro-angiogenic properties of the natural scaffold. Finally, although the scaffolds retained most of their original shape, they did undergo slow deformation during the 8-week study period. In summary, these results indicate that natural cellulose scaffolds are biocompatible and may exhibit potential as surgical biomaterials.

[0255] Materials and Methods

[0256] All experimental procedures were approved by the Animal Care and Use Committee of the University of Ottawa. Wild-type C57BL / 10ScSnJ mice (male and female; 6-9 weeks old; n = 7 mice per group) were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and bred in our facility. All animals were kept at a constant room temperature (± 22°C) and humidity (~ 52%). They were fed a normal chow diet and maintained on a controlled 12-hour light / dark cycle.

[0257] Cellulose scaffold preparation McIntosh Red apples (Canada Fancy) were stored in the dark at 4°C for up to two weeks as previously described

[27] . To prepare apple slices, the fruit was sliced with a mandoline slicer to a uniform thickness of 1.14 ± 0.08 mm, measured with a vernier caliper. Only the outer (cephalic) tissue of the apple was used. Slices containing visible core tissue were not used. Slices were then cut parallel to the direction of the apple pedicel to a length of 5.14 ± 0.21 mm and an area of 26.14 ± 1.76 mm. 2 The apple tissue was then decellularized using a protocol related to reference

[14] for removing cellular material and DNA from the tissue sample while leaving an intact and three-dimensional scaffold. Individual apple tissue samples were placed in sterile 2.5 ml microcentrifuge tubes, and 2 ml of 0.1% sodium dodecyl sulfate (SDS; Sigma-Aldrich) solution was added to each tube. The samples were shaken at 180 RPM for 48 hours at room temperature. The resulting cellulose scaffolds were then transferred to new sterile microcentrifuge tubes, washed, and incubated in PBS (Sigma-Aldrich) for 12 hours. To sterilize the cellulose scaffolds, they were incubated in 70% ethanol for 1 hour and then washed 12 times with PBS. The samples were then kept in PBS containing 1% streptomycin / penicillin (HyClone) and 1% amphotericin B (Wisent, QC, Canada). At this point, the samples were used immediately or stored at 4°C for no more than 2 weeks.

[0258] Cellulose implantation Mice were anesthetized using 2% isoflurane USP-PPC (Pharmaceutical partners of Canada, Richmond, ON, Canada) and eye liquid gel (Alco Canada In., ON, Canada) was applied to protect their eyes. To prepare the surgical site, the back of the mouse was shaved and the skin was cleaned and disinfected using ENDURE 400Scrub-Stat4 surgical scrub (chlorhexidine gluconate, 4% solution; Ecolab Inc., Minnesota, USA) and Soluprep (2% w / v chlorhexidine and 70% v / v isopropyl alcohol; 3M Canada, London, ON, Canada). To keep the animals hydrated, 1 ml of 0.9% sodium chloride solution was administered subcutaneously (sc) (Hospira, Montréal, QC, Canada). During the operation, all sterilization measures required for survival surgery were applied. To implant the scaffold, two 8 mm incisions were made on the back (upper and lower) of each mouse. Two cellulose scaffold samples were implanted separately and independently in each mouse. The incision was then sutured using Surgipro II monofilament polypropylene 6-0 (Covidien, Massachusetts, USA), and 2% transdermal bupivacaine (as monohydrate; Chiron Compounding Pharmacy Inc., Guelph, ON, Canada) was applied topically to the surgical site to prevent infection. In addition, buprenorphine (as HCl) (0.03 mg / ml; Chiron Compounding Pharmacy Inc., Guelph, ON, Canada) was administered subcutaneously as an analgesic. All animals were then carefully monitored by animal care services for the next 3 days and the same pharmacological treatment was repeated.

[0259] Stent Resection: Mice were euthanized using CO₂ inhalation at 1, 4, and 8 weeks after stent implantation. After blood collection, dorsal skin was carefully excised and immediately immersed in PBS. Skin sections containing the cellulose scaffold were then photographed, sectioned, and fixed in 10% formalin for at least 48 hours. The samples were then maintained in 70% ethanol before being embedded in paraffin by the PALM Histology Center at the University of Ottawa.

[0260] Histological Analysis: Serial 5-μm-thick sections were cut starting 1 mm from the inner portion of the cellulose scaffold and stained with hematoxylin and eosin (H&E) and Masson's trichrome. For immunocytochemistry, heat-induced epitope retrieval was performed with citrate buffer (pH 6.0) at 110°C for 12 minutes. Primary antibodies anti-CD31 / PECAM1 (1:100; Novus Biologicals, NB100-2284, Oakville, ON, Canada), anti-α-smooth muscle actin (1:1000, ab5694, abcam, Toronto, ON, Canada), and anti-CD45 (1:3000; ab10558, abcam, Toronto, ON, Canada) were incubated for 1 hour at room temperature. Blocking reagents (Background Sniper, Biocare Medical, Concorde, CA, USA) and the detection system MACH 4 (Biocare Medical, Concord, CA, USA) were used according to company protocols. To assess cell infiltration, extracellular matrix deposition, and angiogenesis (angiogenesis), micrographs were captured using a Zeiss MIRAX MIDI Slide Scanner (Zeiss, Toronto, Canada) equipped with a 40x objective lens and analyzed using Pannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) and ImageJ software. Inflammation scores were assessed by a pathologist. Scores were subjectively assigned based on a qualitative analysis of the overall external response and the proportion of cell populations in the external response.

[0261] Scanning electron microscopy (SEM) was used to study the structure of cellulose. In general, the scaffolds were dehydrated by a continuous ethanol gradient (50%, 70%, 95% and 100%). The samples were then gold coated for 3 minutes using a Hitachi E-1010 ion sputtering device with a current of 15 mA. SEM imaging was performed on a JSM-7500F Field Emission SEM (JEOL, Peabody, MA, USA) at a voltage of 2.00-10.0 kV.

[0262] Statistical analysis All values reported here are mean ± SD. Statistical analysis was performed by one-way ANOVA using SigmaStat 3.5 software (Dundas Software Ltd, Germany). Values of p < 0.05 were considered statistically significant.

[0263] result

[0264] Scaffold preparation Cellulose scaffolds were prepared from apple tissue using a decellularization technique related to that described previously

[27] . All scaffolds were cut into the size of 5.14 ± 0.21 × 5.14 ± 0.21 × 1.14 ± 0.08 mm ( Figure 11 A), decellularized and prepared for implantation ( Figure 11 B) The scaffold appeared translucent after decellularization due to the loss of all plant cell material and debris. Figure 11 C) and scanning electron microscopy ( Figure 11 D) also confirmed the removal of apple cells. Analysis of histological images and measurement of average wall thickness (4.04 ± 1.4 μm) indicated that under this experimental condition, the cellulose scaffold was highly porous and able to be invaded by nearby cells, resulting in an acellular cellulose scaffold that maintained its shape.

[0265] Implantation of Cellulose Scaffolds Two separate skin incisions (8 mm) were made on the back of each mouse to create a small pocket for biomaterial implantation ( Figure 12 A). Place a cellulose scaffold ( Figure 12 B) was implanted in each subcutaneous pouch. Throughout the study, no mouse case exhibited any pain behavior that could be induced by the cellulose scaffold implantation, and none of them showed any visible signs of inflammation or infection. The cellulose scaffolds were excised and photographed at 1, 4, and 8 weeks after implantation to measure changes in scaffold size ( Figure 12 DF). At all time points, healthy tissue could be observed around the cellulose stent, with proximal or directly contacting blood vessels, and the stent maintained its square shape. The stent had a diameter of 26.3 ± 1.98 mm before implantation. 2 and observed that they slowly decreased as a function of their implantation time, based on the area of the scaffold visible to the naked eye on the skin ( Figure 12 G). Eight weeks after implantation, the stent size reached 13.82 ± 3.88 mm. 2 The near-plateau measurements showed that approximately 12 mm 2 (48%) changes.

[0266] Biocompatibility and cell infiltration in plant-derived cellulose scaffolds The biocompatibility and cell infiltration of the scaffolds were examined by H&E staining of fixed cellulose scaffolds at 1, 4, and 8 weeks after implantation ( Figure 13 ). A global view of a longitudinal cross-section of a representative cellulose scaffold is shown in Figure 13 In AC, the stent was implanted under the muscular layer of the dermis. Compared with the unimplanted stent, pink interstitial fluid was observed throughout the implanted stent (see Figure 11 C), highlighting their high porosity and permeability. In a global view, the scaffolds were observed to maintain their general shape throughout the study. Figure 13 In DF, magnified sections of the scaffold periphery are shown at each post-implantation time point. At 1 week, the dermal tissue surrounding the implant showed signs of an acute moderate to severe immune response (qualitative study performed by a pathologist) ( Figure 13 D). A dense cell layer can also be seen infiltrating the cellulose scaffold. At 1 week, the cell population within the scaffold is mainly composed of granulocytes, in particular; polymorphonuclear leukocytes (PMNs) and eosinophils ( Figure 13 D). There was also a population of dead cells and significant cellular debris. Importantly, all of these observations were consistent with the expected acute foreign body reaction after implantation [84-86]. At 4 weeks, significant differences were observed in the surrounding epidermal tissue as well as in the cell population that had migrated into the cellulose scaffold ( Figure 13 E). The epidermal tissue surrounding the cellulose scaffold had a reduced immune response, which was scored as mild to low. The cell population within the epidermis surrounding the scaffold now contained higher levels of macrophages and lymphocytes ( Figure 13 E). This is an expected feature of a foreign body response to implanted biomaterials and demonstrates the process of stent clearance [84-86]. As part of the inflammatory response, the multinuclear cell population inside the stent also increases ( Figure 13 E). Finally, 8 weeks after implantation, the immune responses evident at weeks 1 and 4 had completely disappeared ( Figure 13 F), the epidermal tissue now looks normal. In fact, the epidermal tissue in contact with the cellulose scaffold contains the same structure as normal epidermal tissue. In the periphery of the cellulose scaffold, due to reduced inflammation, there is a lower density of cells at this time, and in particular, there are no fragmented dead cells. On the contrary, the cell population now contains a high level of macrophages, multinuclear cells and active fibroblasts. Active fibroblasts (in the form of spindles) can be observed migrating from the surrounding epidermis into the cellulose scaffold. In fact, fibroblasts were found throughout the cellulose scaffold. These results indicate that the cellulose scaffold has been accepted by the host 8 weeks after implantation. At the same time as the H&E inflammation analysis, anti-CD45 staining was performed to assess the level of inflammation of the entire scaffold and surrounding dermal tissue ( Figure 13 G-13I). Clearly, inflammation throughout the dermis and within the scaffold increased after 1 week. However, the number of leukocytes in the surrounding dermis and scaffold decreased significantly over time, reaching near-basal levels at 8 weeks.

[0267] Extracellular Matrix Deposition in Cellulose Scaffolds The presence of active fibroblasts led us to wonder whether the cellulose scaffolds served as a substrate for new extracellular matrix deposition. This was determined using Masson's trichrome staining of fixed cellulose scaffold slides at each time point after implantation ( Figure 14 ). One week after implantation, histological studies showed a lack of collagen structure within the collagen scaffold ( Figure 14A, D, and G). As fibroblasts invaded the scaffolds, as seen by H&E staining and confirmed by anti-α-smooth muscle actin staining (data not shown), collagen deposition within the cellulose scaffolds was observed after 4 weeks ( Figure 14 B, E and H). At 8 weeks ( Figure 14 C, F and I), the collagen network is clearly visible in the lumen of the cellulose scaffold. The complexity of the deposited collagen network is Figure 14 This is highlighted in Figure 1, where we can detect individual collagen fibers within the collagen matrix. This contrasts with the characteristic high-density, thick, cable-like collagen organization found in scar tissue.

[0268] Vascularization of the cellulose scaffolds was observed as early as 1 week after implantation, with capillaries ranging in diameter from 8 to 25 μm identified within the scaffolds. At 4 and 8 weeks after implantation, blood vessels and capillaries were extensively observed within the scaffolds and surrounding dermis. We observed the presence of blood vessels in the cellulose scaffolds and surrounding dermis in macroscopic photographs taken during the resection procedure ( Figure 15 A). Multiple cross-sections of vessels with red blood cells (RBCs) were identified within 4 weeks of stent implantation ( Figure 15 B; H&E staining). The same results were obtained 8 weeks after implantation, where capillaries with RBCs and endothelial cells were clearly seen ( Figure 15 C; Masson trichrome staining. Anti-CD31 staining was used to identify endothelial cells in the scaffold, and all the results of angiogenesis were confirmed ( Figure 15 D).

[0269] analyze

[0270] In this study, the in vivo biocompatibility of acellular cellulose scaffolds derived from apple cypripedium tissue was evaluated. To this end, acellular cellulose scaffolds were implanted subcutaneously in immunocompetent mice to establish their biocompatibility. The data showed that the implanted scaffolds exhibited a low-grade inflammatory response, promoted cell invasion and extracellular matrix deposition, and served as a pro-angiogenic environment. Notably, none of the mice in this study died or showed any symptoms of implant rejection, such as edema, exudate, or discomfort, during the course of the study, indicating that the cellulose scaffolds were successfully implanted. These implanted scaffolds consisted of a porous network of cavities in which the original host plant cells resided

[69] . This structure effectively facilitated the transfer of nutrients throughout the plant tissue. As shown here and in previous studies, apple tissue can be decellularized

[27] . This simple treatment altered the appearance of the cypripedium tissue, rendering it transparent due to the removal of cellular material.

[0271] Following implantation, the results demonstrated rapid infiltration of the scaffold by host cells, initially consisting of inflammatory cells. Consistent with previous findings, the host animal's immune response followed a well-known timeline [84-88], ultimately demonstrating biocompatibility. As expected, the cell population within the scaffold at 1 week post-implantation was primarily composed of granulocytes, specifically polymorphonuclear leukocytes (PMNs) and eosinophils, constituting a pronounced inflammatory response. Production of a provisional matrix surrounding the scaffold was also observed, contributing to the inflammatory appearance of the peri-scaffold tissue [84-88]. This was not unexpected, being a result of the foreign material and a response to the surgical procedure [84-88]. By 4 weeks post-implantation, the scaffold's cell population had evolved to include lymphocytes, monocytes, macrophages, foreign polymorphonuclear cells, and scattered eosinophils. Typical of chronic inflammation, the cellular debris present in the provisional matrix at 1 week was now cleared by the host immune system [84-88]. At 8 weeks, the cellulose scaffold was now free of all provisional matrix and cellular debris, and low levels of macrophages and foreign polymorphonuclear cells were still visible within the scaffold. Consistent with the immune response within the cellulose scaffold, the surrounding tissue was observed to recover its original physiology. In fact, at 8 weeks post-implantation, the surrounding tissue was almost similar to the control tissue. Although the immune response and inflammation were low at 8 weeks, low levels of macrophages were observed within the scaffold. Although macrophages are traditionally associated with inflammation, they have beneficial effects consistent with our findings. Specifically, macrophages are also known to secrete growth and pro-angiogenic factors, ECM proteins, and pro-fibrotic factors that actively regulate fibroblast proliferation and angiogenesis in tissue repair and regeneration

[86] . Regardless, the majority of cells within the scaffold after 8 weeks were now reactive fibroblasts. These cells altered the microenvironment of the scaffold by secreting a new collagenous extracellular matrix. The new matrix showed a rather low density, suggestive of regeneration, rather than the characteristic high-density, cable-like collagenous tissue found in scar tissue

[89] .

[0272] These data also suggest that the scaffolds are pro-angiogenic, which may promote blood transport from the surrounding tissue

[90] . As with native tissue, the limited blood supply of the scaffold may lead to ischemia and potential necrosis. Interestingly, bioceramics with pore sizes less than 400 μm have been shown to result in reduced blood vessel growth and limit the diameter of blood vessels implanted in vivo. The porous structure of the cell wall structure consists of overlapping cell wall lumens with diameters ranging from 100 to 300 μm and a manual interconnection distance of 4.04 ± 1.4 μm. Therefore, the high porosity and low volume fraction of the cellulose scaffold are consistent with the promotion of vascularization. In summary, the cellulose scaffold appears to be free of a temporary matrix at this time and is fully accepted as a subcutaneous implant.

[0273] We also observed a decrease in scaffold area over time, but the cellulose scaffolds did not appear to be degrading. Instead, the change in area appeared to be due to the collapse of the cell wall cavities at the periphery of the scaffolds caused by the active movement of the mice. Active biodegradation is not expected to occur in mammals, as they lack the appropriate enzymes to digest plant-synthesized cellulose [91, 92]. Furthermore, the highly crystalline forms of cellulose found in plant tissues are known to be resistant to degradation in mammals

[92] . Alternatively, it has been demonstrated that cellulose implants can be chemically activated in vivo to make them more degradable

[93] . However, highly crystalline forms of cellulose have some of the lowest reported immune responses

[92] .

[0274] A large number of clinically approved biomaterials are used to treat specific patient conditions [1]. These biomaterials can be derived from human and animal tissues, synthetic polymers, and materials such as titanium and ceramics [1,2,26,49,50,53,54,56,74,76,94-106]. However, these approaches are not without drawbacks arising from concerns about sourcing, production costs, and / or widespread availability

[48] . Currently, there is significant interest in developing resorbable biomaterials that will degrade in vivo and serve only as temporary scaffolds to promote and support the repair or regeneration of damaged / diseased tissue

[49] . While this is an attractive scenario, it has also been found that as the scaffold degrades, the newly formed structure also collapses [53,64,107-109]. Furthermore, degradation products have been found to have toxic or adverse side effects [53,110,111]. For example, ear reconstruction has been a well-known challenge in tissue engineering. Early studies employed ear-shaped scaffolds produced from cartilage derived from animals or humans [53,58,59,61,63,64]. However, after implantation and eventual scaffold degradation, the ear is often found to collapse or deform.[60–62] Recent strategies have now opted to generate biocomposites consisting of a titanium framework embedded in a biological matrix.

[53]

[0275] The results presented herein suggest that plant-derived cellulose biomaterials may provide a potential approach for producing implantable scaffolds. This approach may be complementary to bacterial cellulose strategies [66, 69-71, 73, 80, 83, 102, 106, 112-115]. However, the results presented herein suggest that plant-derived materials may be cost-effective, readily ready for implantation, exhibit significant biocompatibility, possess the ability to maintain shape while supporting natural host cell extracellular matrix production, and / or may promote angiogenesis. In previous work, the present inventors have shown that scaffolds can be functionalized with proteins prior to in vitro culture. It is contemplated herein that functionalizing the scaffold surface with, for example, growth factors and matrix proteins could be used to promote invasion of specific cell types, further minimizing early immune responses, and / or promoting angiogenesis. In addition, cellulose scaffolds can be readily formed into specific shapes and sizes, providing the opportunity to generate new tissues with specific geometric properties. As shown herein, acellular cellulose scaffolds were biocompatible in immunocompetent mice under the conditions tested and may be considered as a new strategy for, for example, tissue regeneration.

[0276] Example 4 - Additional Decellularization Protocol Examples

[0277] This article describes an alternative decellularization isolation protocol. In this example, the plant was cooled in a -20°C freezer for 5 minutes to firm the soft tissue. The cooled plant tissue was sliced into uniform thicknesses measured with a vernier caliper using a mandolin slicer. The slices were then cut into segments, followed by decellularization, using a modified mammalian tissue protocol to remove cellular material and DNA from the tissue sample while leaving an intact and three-dimensional scaffold. This protocol was modified from a mammalian tissue protocol (Ott et al., 2008). Each tissue sample was placed in a sterile 2.5 mL microcentrifuge tube, and 2 mL of 0.5% sodium dodecyl sulfate (SDS; Sigma-Aldrich) solution was added to each tube. The samples were shaken at 160 RPM at room temperature for 12 hours. The resulting cellulose scaffold was then transferred to a new sterile microcentrifuge tube, washed, and incubated in PBS (Sigma-Aldrich) containing 1% streptomycin / penicillin (HyClone) and 1% amphotericin B (Wisent) for 6 hours. At this point, the sample was used immediately or stored in PBS at 4°C for no more than 2 weeks. The obtained decellularized cellulose scaffold can be Figure 1 Observed in A and B.

[0278] Example 5 - In vitro two-dimensional (2D) and three-dimensional (3D) cell culture - scaffold implantation, cell adhesion and cell proliferation

[0279] In this study, C2C12 mouse myoblasts, NIH3T3 mouse fibroblasts and HeLa human epithelial cell lines (all from American Type Culture Collection (ATCC)) were used. These cells were selected because they represent the most commonly used cell types in cell biology laboratories. 2D conventional cells were used to culture the above-mentioned cells for scaffold implantation. In the standard cell culture medium (high glucose DMEM (HyClone)) supplemented with 10% fetal bovine serum (HyClone), 1% penicillin / streptomycin (HyClone) and 1% amphotericin B (Wisent), cells were cultured in a T75 flask (Thermo Scientific) at 37°C and 5% CO . Culture medium was changed every other day, and cells were passaged when 80% confluent.

[0280] The scaffold seeding procedure was performed in a 24-well tissue culture plate. The wells were individually coated with polydimethylsiloxane (PDMS) to create a hydrophobic surface, leaving the cellulose scaffold as the only surface to adhere to. A 1:10 curing agent solution: elastomer (Sylgard 184, Ellsworth Adhesives) was applied to the surface of each well. The PDMS was allowed to cure at 80°C for 2 hours. The PDMS-24-well plate was allowed to cool to room temperature and then rinsed with sterile PBS. The scaffold was cut into 0.5×0.5 cm pieces and placed in each well. C2C12, NIH3T3, and HeLa were adhered and aliquoted to their correct concentrations. A 6×10 6 A 40 μL droplet of cells was prepared. The sample was placed in an incubator for 6 hours to allow the cells to adhere to the scaffold. Subsequently, 2 mL of DMEM was added to each well, and the sample was incubated for 48 hours. At this point, the sample containing mammalian cells was carefully transferred to a new 24-well PDMS-coated tissue culture plate. To continue cell proliferation, the culture medium was changed daily, and the scaffold was moved to a new 24-well plate every 2 weeks.

[0281] Adhesion and proliferation of mammalian cells were monitored and measured using immunofluorescence microscopy. Figure 5 AC, 16, and 17 show the adhesion and continued proliferation of the cell lines used.

[0282] Example 6 - Effects of Salt Pretreatment and Scaffold Biomaterial Functionalization

[0283] Decellularization is used to obtain 3D cellulose scaffolds that are free of native cells and nucleic acids. Decellularization is achieved using the surfactant sodium dodecyl sulfate (SDS). The SDS is removed before repopulating the scaffold with new cells; otherwise, the cells will disappear. For small scaffolds, the concentration of SDS can be low; however, for larger objects, higher concentrations of SDS can be used for complete decellularization. Residual SDS can be removed by extensive washing, especially when using low concentrations of SDS. In some cases, removing higher concentrations of SDS by washing alone can become difficult and time-consuming. As described herein, the addition of CaCl2 can allow residual SDS to be easily removed from decellularized scaffolds. Without wishing to be bound by theory, it is believed that the principle behind this concept is to use a salt buffer to force SDS into micelles. Sufficiently high salt concentrations can be used to stimulate sufficient micelle formation, and excessively high salt concentrations can cause salting out onto the biomaterial. Salt residues can be removed by several techniques, such as incubation with dH2O, acetic acid, or DMSO. Sonication can also be used to remove tightly bound debris. The concentration of CaCl2 can depend on the amount of residual SDS. In this study, decellularization was accomplished by using a 0.1% SDS aqueous solution. The concentration of CaCl2 may depend on the amount of SDS used for decellularization, as Figure 18 At a concentration of 100 mM, a moderate amount of salt / micelles precipitated onto the scaffold ( Figure 19 A). Salt residues were effectively removed by incubating the scaffold in dH2O ( Figure 19 B).

[0284] Cell growth was improved after removal of residual SDS and salts ( Figure 20 The addition of salt allows for easy removal of residual SDS; however, salt that has precipitated onto the biological material should also be removed to avoid tension problems. After the salt has forced the SDS into the micelles, the next step is to remove the salt. Salt residues can be removed using various techniques, such as sonication, incubation in water, incubation in acetic acid, and incubation in DMSO ( Figure 20 ).

[0285] In addition to CaCl2, other salts can be used to remove residual SDS from biological materials ( Figure 21 Washing biomaterials with salts having divalent cations caused greater cell growth than their monovalent counterparts, probably because divalent cations promote the formation of tighter SDS micelles ( Figure 21 ).

[0286] In certain embodiments, the addition of salt can change the critical micelle concentration (CMC) of the surfactant. At a certain concentration, called the cloud point, a phase transition can occur and the micelles become insoluble and can be easily washed away.

[0287] Different salt compounds can be used to accomplish the task of removing residual SDS from biological materials. PBS, KCl, CaCl2, MgCl2, CuSO4, KH2PO4, MgSO4, Na2CO3, and sodium ibuprofen (all 100 mM) were used as salt washes to clean biological materials and remove residual SDS. Figure 21 Each salt treatment shown in allowed cell growth; however, salts with divalent cations (CaCl2 and MgCl2) and carbonate anion groups promoted greater cell growth.

[0288] Biomaterial functionalization

[0289] Depending on the intended use of the biomaterial, the cellulose structure can be biochemically functionalized. As will be appreciated, this modification can expand potential uses and applications. For example, cellulose has free hydroxyl groups that can be used to combine the material with different molecules.

[0290] Two common reaction types used for this type of modification are acylation and alkylation. These reactions can connect hydrocarbon chains of various lengths to the cellulose structure through free hydroxyl groups. For example, when steric hindrance is a factor, different chain lengths and shapes may be useful. Using larger chains can reduce steric hindrance, and vice versa. Acylation using dicarboxylic acids can provide the possibility of functionalizing biomaterials. Some classes of dicarboxylic acids that can be used can include, but are not limited to, straight-chain saturated dicarboxylic acids, branched dicarboxylic acids, unsaturated dicarboxylic acids, substituted dicarboxylic acids and aromatic dicarboxylic acids. In addition to acylation and alkylation, other compounds can be used to mediate the connection between the functional group and the cellulose, such as compounds containing boron, sulfur, nitrogen and / or phosphorus.

[0291] Different functional groups can be added to the other end of the chain to achieve a certain function. These functional groups can include, but are not limited to, groups containing hydrocarbons, oxygen, nitrogen, sulfur, phosphorus, boron and / or halogens. The functional groups can be selected based on the intended application. For example, if the intended application is to prevent cell growth in certain areas, a sterically non-polar hydrocarbon functional group can be used; conversely, if the intended application is to promote cell growth, a carboxylic acid can be selected so that extracellular matrix proteins such as collagen can bind to cellulose. Different components of the cell wall can allow for the enhancement of certain structural properties of the biomaterial. The secondary cell wall structure of asparagus and apple tissue may contain, for example, pectin and lignin ( Figure 22 ), to give strength to the biomaterial.

[0292] As will be appreciated, the scaffold biomaterial is not limited to cellulose. Many other cell wall structures can be used for the biomaterial. Figure 22 In addition to the cellulose shown, there are also cinnamaldehyde, pectin and lignin. These additional secondary cell wall structures can also be functionalized.

[0293] Chemical modification of cellulose can allow control of the chemical and physical properties of biomaterials. As a result, biomaterials can be dedicated to specific purposes. For example, patterned cell growth can be achieved by inhibiting cell growth in certain areas (temporarily or permanently) and promoting cell growth in other areas. In addition, cell type-specific molecules can be introduced into biomaterials by these functionalization methods to promote the growth / invasion / differentiation of specific cell types. The functionalization of biomaterials can also allow the regeneration of biologically relevant microenvironments, which are related to appropriate cell function and tissue engineering.

[0294] Example 7 - Surface Biomodification

[0295] Natural cellulose can support mammalian cells, including C2C12 myoblasts, 3T3 fibroblasts, and human epithelial HeLa cells. However, functionalized biomaterials can also be chemically and mechanically tuned to suit specific intended uses. In these experiments, two different techniques were used to modify the stiffness of decellularized cellulose scaffolds. Additionally, phase-contrast images demonstrated that the biomaterials continued to support mammalian cell culture after chemical and physical modifications.

[0296] In order to functionalize the scaffold with collagen, the samples were incubated in a solution of 10% acetic acid and 1 mg / mL type I rat tail collagen (Invitrogen) for 6 hours and then washed in PBS before use. In order to chemically crosslink the scaffold, the samples were incubated in a 1% EM grade glutaraldehyde solution (Sigma-Aldrich) for 6 hours. The scaffold was then rinsed in PBS and incubated overnight in a solution of 1% sodium borohydride (Sigma-Aldrich) to reduce any unreacted glutaraldehyde. Before cells were seeded into the scaffold, all samples (native, collagen coated or cross-linked) were incubated in mammalian cell culture medium (as described below) in a standard tissue culture incubator maintained at 37°C and 5% CO2 for 12 hours. The results are shown in Figure 5. Figure 23 In AD, native tissue and unmodified scaffolds did not show any significant differences in mechanical properties. Compared with DMEM scaffolds, both collagen-functionalized and chemically cross-linked scaffolds showed a significant increase in elasticity. Under this experimental condition, decellularized (SDS), collagen-functionalized (SDS+Coll), and glutaraldehyde-cross-linked (SDS+GA) scaffolds all supported the growth of C2C12 cells.

[0297] Example 8 - Cellulose Scaffolds and Molding Techniques, Coatings

[0298] We have previously demonstrated how cellulose scaffolds can function as free-standing 3D biomaterials. Here, we show how decellularized cellulose can be cut into different macroscopic shapes ( Figure 24: ring). C2C12 mouse myoblasts were seeded onto the biomaterial and allowed to proliferate and invade the scaffold for two weeks. After two weeks, the structure was filled with cells ( Figure 24 The biomaterial can also be used in combination with traditional molding techniques. In this study, we demonstrate how cellulose constructs can be used for temporary and permanent reverse molding using gelatin and collagen, respectively ( Figure 24 BC). The melting temperature of gelatin is 32°C. For a temporary reverse mold, the cells are resuspended in a cell culture medium containing a 10% gelatin solution at 37°C. Shortly after the cells are seeded on the biomaterial, the gelatin solution cools below its melting temperature and solidifies. The formation of the gelatin gel gives the cells time to attach to the matrix. Once the gelatin gel is placed in an incubator and heated to 37°C, the gelatin melts while the cells remain on the biomaterial. Conversely, cellulose can also serve as a reverse mold for permanent gels when a gel is required. For a permanent reverse mold, cellulose is included in the collagen solution containing the cells ( Figure 24 C). The collagen solution quickly solidifies and forms a permanent gel containing the biomaterial and cells.

[0299] Molding technology can be further applied to other hydrogels, rather than just gelatin and collagen. Other possible gels can include, but are not limited to, for example, agarose, polyurethane, polyethylene glycol, xanthan gum, methylcellulose, alginate, hyaluronan, carboxymethyl cellulose, chitosan, polyacrylic acid, polyvinyl alcohol, polyester, hydrocolloid, gum arabic, pectin, and / or dextran. Hydrogels can also be impregnated with other compounds, such as growth factors, drugs, etc. This gel can also be functionalized with active side chains. As a result, it is expected that for example cellulose can have a function, and hydrogel can have a second function. In addition, in certain embodiments, a variety of hydrogels with multiple functions can be used in combination. Finally, these gels can be temporary and melt over time, and / or can be cross-linked with original cellulose or chitin scaffold to produce a multifunctional material with two or more mechanical / chemical properties, and the property can be time-dependent or time-independent.

[0300] Additional elements / compounds can be used to coat the surface, or they can be incorporated into the biomaterial by functionalization. The selection of additional elements depends on the intended application. For example, if the biomaterial is used to promote nerve regeneration, nerve growth factor (NGF) protein can be added. On the contrary, if the biomaterial is used for drug delivery, a viral capsule containing the drug can be used. In addition, if the immune response is problematic, the biomaterial can be coated with, for example, ibuprofen salts. It is expected that various elements can be added to the biomaterial. These elements may include, but are not limited to, proteins (e.g., collagen, elastin, and integrins), nucleic acids (e.g., DNA, RNA, and siRNA), fatty acids (e.g., stearic acid, palmitic acid, and linoleic acid), metabolites (e.g., aspartic acid, vitamin B2, and glycerol), ligands (e.g., vitamin D, testosterone, and insulin), antigens (e.g., peptides, polysaccharides, and lipids), antibodies (e.g., IgA, IgE, and IgG), viruses (e.g., HIV, HEP C, and vaccinia), synthetic polymers (e.g., nylon, polyester, and polytetrafluoroethylene), functional groups (carboxylic acids, esters, and imides), drugs (e.g., hydrocodone, amoxicillin, Plavix, etc.), vesicles (e.g., vacuoles, transport vesicles, and secretory vesicles), organic molecules (e.g., carbohydrates, ligases, and vitamins), and / or inorganic molecules (e.g., iron, titanium, and gold). In addition, bacteria (e.g., but not limited to, bifidobacteria) may be added to alter / control the microbiome. Where cell specificity is desired, cell recruitment factors may be included, for example.

[0301] Support structures for biomaterials

[0302] Other elements / compounds can be used as the supporting structure of biomaterial. The selection of element in addition can depend on the application of expectation. For example, if biomaterial will bear constant load or keep its shape, titanium structure can be included. For example, these elements / compositions can include titanium, low C steel, aluminum, Co-Cr alloy, 316 stainless steel, PMMA cement, ultra-high MW PE etc. In certain embodiments, these elements can be added to biomaterial interior (interior), biomaterial exterior, or both. In certain embodiments, such elements / compounds can include those approved by FDA.

[0303] Example 9 - Cell invasion and proliferation

[0304] Confocal laser scanning microscopy was used to image ~300 μm z sections of the top and bottom of the cellulose construct. Both sides were imaged because the depth of field was smaller than the ~1.2 mm thick ring. Figure 25 The xy and zy projections of cells on cellulose biomaterial are shown. The nucleus (blue) is found along the cellulose cell wall (red) ( Figure 25 xy projection). Orthogonal views of confocal scans showing cell invasion into the scaffold ( Figure 25Confocal imaging allows quantification of cell invasion and proliferation ( Figure 26 The cell nucleus images were thresholded using the ImageJ Adaptive Threshold plugin, and the total nuclear area was determined using the Analyze Particles plugin. Initially, cells were seeded on the top of the sample. Cell invasion was determined as the ratio of the nuclear area covering the top and bottom of the biomaterial. There was no statistical difference in cell invasion between the three different conditions ( Figure 26 In practice, the top:bottom ratio is close to 1 ( Figure 26 The total nuclear area of the imaged sections was calculated to compare cell proliferation under each condition. No significant differences in total nuclear area were found among the three conditions. Consequently, temporary and permanent reverse molding did not affect cell proliferation under the conditions tested.

[0305] Molding techniques as well as functionalization techniques can be used to connect different structures together. As a result, in certain embodiments, for example, large complex structures can be produced to mimic in vivo tissues.

[0306] Example 10 - Artificially fabricated structures within plant-derived decellularized cellulose scaffolds

[0307] Artificial fabrication of structures within plant cellulose scaffolds was performed to demonstrate the feasibility of generating different structures for specific purposes, such as increasing host cell migration into the cellulose scaffold. Figure 27 In a new study, researchers from the University of California, Berkeley, created an artificial structure based on a scaffold made of cellulose derived from apple.

[0308] In these studies, mice were anesthetized with 2% isoflurane USP-PPC (Pharmaceutical partners of Canada, Richmond, ON, Canada) and eye liquid gel (Alco Canada In., ON, Canada) was applied to protect their eyes. The back hair of the mice was shaved and the skin was cleaned and disinfected using ENDURE 400 Scrub-Stat4 surgical scrub (chlorhexidine gluconate, 4% solution; Ecolab Inc., Minnesota, USA) and Soluprep (2% w / v chlorhexidine and 70% v / v isopropyl alcohol; 3M Canada, London, ON, Canada). 1 ml of 0.9% sodium chloride solution was injected subcutaneously (sc) (Hospira, Montréal, QC, Canada) to keep the animals hydrated. Throughout the operation, all strict aseptic measures supported survival surgery. In order to implant the scaffold, two 8 mm incisions were made on the back (upper and lower) of each mouse. Two cellulose scaffold samples were implanted separately and independently in each mouse. The incision was then sutured using Surgipro II monofilament polypropylene 6-0 (Covidien, Massachusetts, USA), and 2% transdermal bupivacaine (as monohydrate; Chiron Compounding Pharmacy Inc., Guelph, ON, Canada) was applied topically to the surgical site to prevent infection. In addition, buprenorphine (as HCl) (0.03 mg / ml; Chiron Compounding Pharmacy Inc., Guelph, ON, Canada) was administered subcutaneously as an analgesic. All animals were then carefully monitored for the next 3 days by Animal Care Services and the same additional pharmacological treatments were repeated. One and four weeks after stent implantation, mice were euthanized using CO2 inhalation. The dorsal skin was carefully excised and immediately immersed in PBS solution. Skin sections containing the cellulose scaffold were then taken, sliced, and fixed in 10% formalin for at least 48 hours. The samples were then kept in 70% ethanol and subsequently embedded in paraffin by the PALM Histology Center at the University of Ottawa.

[0309] The results are shown in Figure 27 Two different structures were generated within a decellularized cellulose scaffold to demonstrate the feasibility of generating different structures from biomaterials for specific purposes, such as increasing host cell migration into the cellulose scaffold. Figure 27 In A, a 1 mm biopsy punch was used to create five negative cylindrical spaces within the cellulose scaffold. Figure 27In B, a 3 mm biopsy punch was used to create a single central negative space. Increased vascularization directly from the artificially created negative space was observed only after 4 weeks of implantation ( Figure 27 C and D). In 28C, blood vessels are located in each of the four corners of the biomaterial, suggesting increased vascularization within the artificially created negative space. Similarly, in 27D, blood vessels can be observed at the top of the cellulose scaffold, suggesting that blood vessels penetrate the cellulose scaffold. Cross-section of a representative cellulose scaffold stained with H&E ( Figure 27 EF).

[0310] Example 11 - Various Examples of Cellulose-Based Primitive Tissues and Structures in the Plant Kingdom

[0311] Figure 28 Various examples of cellulose-based primitive tissues and structures selected from the plant kingdom are provided, shown at 4 weeks and / or 8 weeks. The figure shows pictures depicting cellulose scaffolds from various sources, their resections and histology after 4 weeks and / or 8 weeks, as indicated.

[0312] In these studies, the cellulose scaffolds derived from various plants were implanted subcutaneously in mice to assess the biocompatibility of 4 weeks and / or 8 weeks. The selective tissues of various plants were implanted for 4 or 8 weeks to assess the biocompatibility of plant-derived cellulose and plant structures to host cell migration in vivo. In all embodiments, cell migration and proliferation were observed in the cellulose scaffolds, highlighting the biocompatibility of the cellulose scaffolds derived from plants in these experiments. Subcutaneous implantation of cellulose scaffold biomaterials was performed on the back area of the C57BL / 10ScSnJ mouse model through a small skin incision (8mm). Each implant was measured before implantation for support area comparison (first column: cellulose scaffold). As shown in the figure, the cellulose grafts were excised at 4 or 8 weeks (second column: resection). Continuous 5 μm thick sections were cut from 1mm inside the cellulose scaffold and stained with hematoxylin-eosin (H&E) (third column: histology). To assess cell infiltration, micrographs were captured using a Zeiss MIRAX MIDI Slide Scanner (Zeiss, Toronto, Canada) equipped with a 40× objective and analyzed using Pannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) and ImageJ software.

[0313] Example 12 - Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials (Prosthetics-Aesthetics)

[0314] Based on Example 3 above, cellulose scaffold implantation and resection were performed to evaluate subcutaneous implants. Figure 29Cellulose scaffold biomaterials were subcutaneously implanted on the back region of C57BL / 10ScSnJ mouse model through a small skin incision (8 mm). Figure 29 A). Each implant was measured before implantation for scaffold area comparison ( Figure 29 B). One week after surgery ( Figure 29 D), 4 weeks ( Figure 29 E) and 8 weeks ( Figure 29 F) The cellulose scaffold was removed and macroscopic photographs were taken (control skin Figure 29 C). At each time point, the blood vessels clearly integrated with the cellulose implant, demonstrating biocompatibility. Similarly, no acute or chronic inflammation was detected in the tissue surrounding the implant. Figure 29 G. The area of the stent before implantation was 26.30±1.98mm 2 After implantation, the stent area decreased to 20.74±1.80mm after 1 week. 2 , after 4 weeks, it decreased to 16.41±2.44mm 2 , and decreased to 13.82±3.88mm after 8 weeks 2 After 8 weeks of implantation, the surface area of the cellulose scaffold was significantly reduced by approximately 12 mm 2 (48%) (*=P<0.001; n=12-14).

[0315] For histological analysis, the following experiments were performed.

[0316] Serial 5-μm-thick sections were cut starting 1 mm from within the cellulose scaffold and stained with hematoxylin-eosin (H&E) and Masson's trichrome. For immunocytochemistry, heat-induced epitope retrieval was performed with citrate buffer (pH 6.0) at 110°C for 12 minutes. Primary antibodies anti-CD31 / PECAM1 (1:100; Novus Biologicals, NB100-2284, Oakville, ON, Canada), anti-α-smooth muscle actin (1:1000, ab5694, abcam, Toronto, ON, Canada), and anti-CD45 (1:3000; ab10558, abcam, Toronto, ON, Canada) were incubated for 1 hour at room temperature. Blocking reagents (Background Sniper, Biocare Medical, Concorde, CA, USA) and the detection system MACH 4 (Biocare Medical, Concord, CA, USA) were used according to company protocols. To assess cell infiltration, extracellular matrix deposition, and blood vessel formation (angiogenesis), micrographs were captured using a Zeiss MIRAX MIDI Slide Scanner (Zeiss, Toronto, Canada) equipped with a 40× objective and analyzed using Pannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) and ImageJ software.

[0317] Figure 30 Results of biocompatibility and cell infiltration are shown. Cross sections of representative cellulose scaffolds were stained with H&E and anti-CD45. These global views show an acute moderate-severe expected foreign body reaction at 1 week ( Figure 30 A), Mild chronic immunity at 4 weeks and subsequent clearance ( Figure 30 B), and finally, the cellulose scaffold assimilated into native mouse tissue at 8 weeks ( Figure 30 C). Higher magnification of the region of interest ( Figure 30 DF), see illustration ( Figure 30 AC), allowing observation of cell type populations during biomaterial assimilation. At 1 week, we could observe granulocyte populations, specifically; polymorphonuclear leukocytes (PMNs) and eosinophils, characteristic of an acute moderate to severe immune response, which is a normal response to the implantation procedure ( Figure 30 D) At 4 weeks, a reduced immune response was observed (mild to low immune response), and the cell population within the epidermis surrounding the scaffold now contained higher levels of monocytes and lymphocytes indicative of a chronic response ( Figure 30E). Finally, at 8 weeks, the immune response has completely resorbed and the epidermis now appears normal ( Figure 30 F). The immune response observed with H&E staining was confirmed using anti-CD45 antibody, a well-known leukocyte marker ( Figure 30 GI). The cell population within the scaffold is now mainly macrophages, multinuclear cells and activated fibroblasts.

[0318] The presence of active fibroblasts raised the question of whether the cellulose scaffolds served as a substrate for new extracellular matrix deposition. This was determined using Masson's trichrome staining of fixed cellulose scaffold slides at each time point after implantation ( Figure 31 ). One week after implantation, histological studies showed a lack of collagen structure within the collagen scaffold ( Figure 31 A, D, G). After 4 weeks, a small amount of collagen began to deposit in the scaffold ( Figure 31 B, E, H), and at 8 weeks, a large amount of collagen was clearly visible in many scaffold cavities ( Figure 31 C, F, I). The presence of active fibroblasts identified by morphology (H&E staining, spindle shape) and anti-α smooth muscle actin staining (data not shown) is completely consistent with the extensive collagen deposition observed at 8 weeks. The complexity of the deposited collagen network is Figure 31 Individual collagen fibers within the collagen matrix are visible, as highlighted in Figure 1. This contrasts with the characteristic high-density, thick, cable-like collagen tissue found in scar tissue.

[0319] As early as 1 week after implantation, capillaries ranging from 8 to 25 μm were identified within the scaffold. At 4 and 8 weeks after implantation, blood vessels and capillaries could be extensively observed within the scaffold and surrounding dermal tissue. We observed the presence of blood vessels in the cellulose scaffold and surrounding dermis in macroscopic photographs taken during resection ( Figure 32 A). Multiple cross-sections of vessels with red blood cells (RBCs) were identified within 4 weeks of stent implantation ( Figure 32 B; H&E staining). The same results were obtained 8 weeks after implantation, where capillaries with RBCs and endothelial cells were clearly seen ( Figure 32 C; Masson trichromatic method).

[0320] Example 13 - Bioinspired and biofunctional grafts for repairing spinal cord injuries

[0321] Sterile cellulose grafts that retain their shape and mechanical strength can be produced using the methods described herein. Using our in-house volumetric mechanical testing equipment, the elastic modulus of our native cellulose grafts was recorded at ~2 MPa when the grafts were compressed in a direction parallel to the through-microchannels. When the grafts were compressed perpendicular to the microchannels, the modulus was observed to be approximately an order of magnitude smaller. These values are highly consistent with the elastic moduli of the dura and pia mater, meaning that these grafts fall within the range of mechanical properties of most surrounding spinal cord tissues. Figure 33 Images of decellularized asparagus xylem structures and microchannels are shown.

[0322] Craniotomy and brain resection of adult rats allow the derivation of primary rat neurospheres. The dorsal area is cleaned to expose the medulla. Using malleable forceps, the posterior skull is removed, up to the frontal lobe, and a portion of the skull is removed to expose the brain. Finally, the olfactory bulb is cut and the brain is gently removed from the skull. The removed brain is immersed in a culture dish filled with dissection medium (MEM Alpha medium (Life Technologies Inc) 1% L-glutamine (Life Technologies Inc) and 1% penicillin (Life Technologies Inc) on ice. The brain is then sliced in brain matrix and the slices contain the hippocampus. The gray matter of the lateral side of the third ventricle is collected in a test tube with dissection medium, and the gray matter tissue in the dissection medium is continuously centrifuged and the supernatant is collected. Once all the supernatant has been removed, the last tube is centrifuged and the pellet is resuspended in 2 mL of culture medium (Advanced DMEM / F12 medium (Life Technologies Inc), 1% L-glutamine (Life Technologies Inc) and 1% N2 supplement (CEDARLANE LABORATORIES LTD)). The resuspended cell solution was aliquoted into 6-well ultra-low attachment plates containing 0.001% human epidermal growth factor and basic fibroblast growth factor (PEPROTECH) to allow proliferation of primary rat neurospheres. Neurospheres were seeded locally on top of each transplant in a custom-made cell culture chamber. Neurospheres were cultured and maintained in an incubator containing 5% CO2 for 2 weeks. The culture medium was changed every day. The scaffold samples were fixed with 4% paraformaldehyde. Cellulose cells were stained using the previously used protocol. Neurospheres were stained with wheat germ agglutinin (WGA) 488 (Invitrogen) and examined by confocal fluorescence microscopy ( Figure 34 A).

[0323] Decellularized vascular plants were implanted subcutaneously into mice following a protocol similar to that discussed in the study of Example 3. Histological results showed that vascular structures remained intact and evident throughout the scaffolds 4 weeks after implantation ( Figure 34B). Consistent with the structure, host cells could be observed through the entire 5 mm span of the cellulose scaffold. Following successful initial results in vitro and in vivo, the decellularized plant scaffolds were fabricated into spinal cord injury grafts. Female Sprague Dawley rats were anesthetized with isoflurane. The overlying skin was shaved and prepared with polyvinyl iodine. Under aseptic conditions and using sterile instruments, vertebrae T7 to T10 were exposed. After dissecting the dorsal and intercostal muscles, a laminectomy was performed at T8 and T9. The dura mater was exposed using micro scissors. The T8 spinal cord was transected in one clean cutting motion using micro scissors. Surgical foam was used to control any bleeding caused by the transection. The spinal cord was allowed to retract and the cellulose scaffold was moved and placed to connect the tail and skull ( Figure 34 C). After stent placement, the cellulose graft was secured using Tisseel fibrin glue (Baxter). The muscle layer of the incision was closed with 3-0 Vicryl suture material, and the epidermis and dermis were closed with Michel clips. Buprenorphine was administered before closure to ensure that it was actively working while the rat recovered from the anesthesia.

[0324] An increase in BBB score was observed over the course of 8 weeks.

[0325] Eight weeks after implantation, rats (n=7) showed improved motor activity (BBB=9.2±2.5), demonstrating coordinated stepping and weight bearing abilities ( Figure 35 ). In addition, at 8 weeks, a second spinal cord transection was performed (below the graft), resulting in a BBB score conversion to 0. Control rats (n=7, fibrin only) showed BBB scores in the range of 0 to 1. This result suggests that motor recovery may not be due to reflexes. The spinal cord was then dissected at 8 weeks and sectioned at the graft site. To show myelinated neurons, the slides were stained with a combination of hematoxylin, eosin, and luxol fast blue (H&E-LFB). The data showed that host cells passing through the graft microchannels stained positive, consistent with improved motor function ( Figure 35 D). Furthermore, we were able to demonstrate and optimize an MRI protocol that allows visualization of the spinal cord continuity and graft collapse without sacrificing the animal. Cranial and caudal interfaces ( Figure 35 Ai, 3A-iii) can be clearly distinguished from stent grafts ( Figure 35 A-ii). Figure 36 and 37 Global views of the spinal cord graft implanted in the T8-T9 region of the spine and the ventral portion of the surrounding transection site are shown. Figure 37As shown in (B), green filaments (red arrows) can be observed around the spinal graft extending in the ventral direction. These filaments represent mature neurons in the rat transect site after 12 weeks in vivo. In contrast, in the control (B), no organized neurofilaments can be observed, indicating that mature filaments are lacking in the control transect site. In addition, Hoechst staining showed a significant increase in the number of nuclei compared to the control, and such cells surrounded the spinal graft in the transect site.

[0326] In these studies, a scaffold biomaterial was inserted between the transected spinal cord stumps, after which fibrin glue wound repair was applied, showing that after only 8 weeks of study, control rats (n=4, no graft) did not show improvement in motor function and remained completely paralyzed (BBB between 0-1). It is noteworthy that rats (n=7) with implants derived from asparagus showed a BBB of 9.2±2.5, indicating that motor function was significantly improved in these studies. These animals showed coordinated stepping and the ability to bear weight. Therefore, implants derived from asparagus show promise for treating SCI in rat models. In certain embodiments, scaffold biomaterials as described herein can be used to recruit neural progenitor cells in damaged spinal cord tissue to improve motor function.

[0327] Example 14 - Plant decellularized scaffold for skin transplantation

[0328] Mice were anesthetized using 2% isoflurane USP-PPC (Pharmaceutical partners of Canada, Richmond, ON, Canada) and eye liquid gel (Alco Canada In., ON, Canada) was applied to protect their eyes. The back hair of the mice was shaved. The shaved skin was then treated with Nair gel for 2 minutes. Nair was carefully removed from the skin and the skin was cleaned and disinfected using ENDURE 400 Scrub-Stat4 surgical scrub (chlorhexidine gluconate, 4% solution; Ecolab Inc., Minnesota, USA) and Soluprep (2% w / v chlorhexidine and 70% v / v isopropyl alcohol; 3M Canada, London, ON, Canada). Animals were hydrated by subcutaneous injection (sc) of 1 ml of 0.9% sodium chloride solution (Hospira, Montréal, QC, Canada). Throughout the operation, strict aseptic measures supported survival surgery. A 5 mm circular skin biopsy was taken. A rubber insulating pad with gel superglue was carefully placed on the biopsy tissue while still exposing the skin biopsy tissue. The 8 points of the rubber pad were then sutured to the mice using Surgipro II monofilament polypropylene 6-0 (Covidien, Massachusetts, USA). The skin graft was then placed to replace the removed skin and sealed with two layers of absorbable transparent adhesive tape. Transdermal 2% bupivacaine (as monohydrate; Chiron Compounding Pharmacy Inc., Guelph, ON, Canada) was topically applied to the surgical site to prevent infection. Furthermore, subcutaneous administration of buprenorphine (as HCL) (0.03 mg / ml; Chiron Compounding Pharmacy Inc., Guelph, ON, Canada) was used as an analgesic. All animals were then carefully monitored for the next 3 days by animal care services, and identical additional pharmacological treatments were repeated. Transparent adhesive tape was changed every day and the skin graft was photographed.

[0329] Figure 38 Decellularized apple inflorescence tissue used for skin grafting is shown. Photos were taken 4 days later to measure the extent of host cell infiltration during wound healing ( Figure 38 C); Two weeks after scaffold implantation, mice were euthanized by CO₂ inhalation. Dorsal skin was carefully excised and immediately immersed in PBS. Skin sections containing the cellulose scaffold were then photographed, cut, and fixed in 10% formalin for at least 48 hours. The samples were then maintained in 70% ethanol before being embedded in paraffin by the PALM Histology Core Facility at the University of Ottawa.

[0330] Example 15 - Plant Decellularized Scaffold for Bone Graft

[0331] This study was performed to show the efficiency of the biomaterial as described herein for bone regeneration.Here, critical size calvarial defects in rats were used to demonstrate that a cellulose scaffold can successfully support bone regeneration in a 5 mm circular defect.

[0332] Sprague Dawley rats were anesthetized with oxygen containing isoflurane and received a subcutaneous injection of buprenorphine and sterile saline before the surgical procedure. The rats were cut from the bridge of the nose between the eyes to the tail end of the skull and ophthalmic liquid gel was applied to protect the eyes. The rats were placed in a stereotaxic frame and fixed on a water-heated warm pad by ear bars. An incision (1.5 cm) was made in the periosteum above the scalp from the nasal bone to the tail end of the middle sagittal crest (anterior bregma). The periosteum was divided into the sagittal midline and dissected. The skull was drilled using a 5 mm trephine drill and a surgical drill on the right (or left) side parietal bone. The scoring bone was separated from the dura mater, leaving a 5 mm circular defect in the rat skull. The defect was carefully washed with sterile saline and a 5 mm diameter cylindrical (1 mm thick) cellulose scaffold ( Figure 39 A) implanted into the defect ( Figure 39 B). The skin layers were sutured to close the skin. Rats were euthanized 4 weeks after surgery using carbon dioxide inhalation and exsanguination, and the cellulose scaffolds were recovered along with the surrounding bone tissue ( Figure 39 C) Histological analysis Figure 39 D). The tissue was fixed in a buffered formalin solution and dehydrated in ethanol before embedding in methyl methacrylate. Each 5 μm thick sample was stained with hematoxylin / eosin to highlight the presence of cellular components (nuclei and cytoplasm). Figure 40 D) To quantitatively determine the efficiency of the cellulose scaffolds, we used the scoring method shown in Table 1 - using quantitative histological scoring parameters (Kretlow et al., 2010).

[0333]

[0334] Table 1: Quantitative histological scoring parameters (Kretlow et al., 2010)

[0335] exist Figure 39 In the experiments shown, plant-derived cellulose scaffolds were evaluated for bone grafting. Each cylindrical (5 mm diameter, 1 mm thick) implant was measured prior to implantation for scaffold area comparison ( Figure 39 A). A cellulose scaffold implant was implanted into a rat skull defect and positioned to remain within the skull defect. The skin was then positioned over the graft and sutured to hold the scaffold in place ( Figure 39B). The scaffold and surrounding bone tissue were separated 4 weeks after implantation, and macroscopic photographs were taken ( Figure 39 C). The isolated tissue was then decalcified and processed / embedded in paraffin. Serial 5 μm thick sections were cut starting from 1 mm inside the cellulose scaffold and stained with hematoxylin-eosin (H&E). Figure 39 D) To evaluate bone regeneration, micrographs were captured using a Zeiss MIRAX MIDI Slide Scanner (Zeiss, Toronto, Canada) equipped with a 40× objective and analyzed using Panoramic Viewer (3DHISTECH Ltd., Budapest, Hungary) and ImageJ software.

[0336] Histological results showed direct contact between bone and scaffold at the defect and biomaterial scaffold interface.

[0337] Example 16 - Exemplary Forms of Scaffold Biomaterials

[0338] Figure 40 A to 40F show different example configurations, physical properties, and functionalization of cellulose-based scaffold biomaterials. Figure 40 A shows that cellulose can be used as blocks and cut into different shapes. Figure 40 B shows that cellulose can be dehydrated and ground into a powder form. Figure 40 B also shows that if the cellulose contains carboxymethyl cellulose, it can be easily cross-linked with citric acid and heat. Figure 40 C shows that cellulose in powder form can be rehydrated to the desired consistency to produce a gel ( Figure 40 D) or paste ( Figure 40 E, 40F).

[0339] Example 17 - Survival Rate After Implantation

[0340] Figure 41 A is a graph showing experimental survival of mice (n=190) and rats (n=12) 1 week, 4 weeks, and 8 weeks after implantation of biomaterials (from various sources). Figure 41 B shows the Figure 41 Biomaterial rejection rates at these same time points in A. In these experiments, all animals (mice and rats) survived following biomaterial implantation and for the entire duration of each trial, and none showed signs of implant rejection.

[0341] Example 18 - Examples of Plant and Fungal Tissues

[0342] Different plant classification systems are used in plant classification, and there are several versions of these systems (for example: Cronquist system and APG system).

[0343] By using a variety of plants classified in different plant categories, families, genera, and species in the experiments described herein, our data demonstrate that a variety of plants can be used to prepare scaffold biomaterials.

[0344] Generally speaking, the plant kingdom is divided into four major categories:

[0345] - flowering plants (angiosperms);

[0346] - conifers, cycads and similar species (gymnosperms);

[0347] - ferns and their relatives (ferns);

[0348] - Mosses and liverworts (bryophytes).

[0349] These four main categories contain many plant families, which are divided into many genera, which are divided into many species. The following is a list of the main plant families that can produce cellulose scaffolds:

[0350] Acanthaceae, Campanulaceae, Agate Fruit Family, Acorus Family, Acrophyceae, Actinidiaceae, Crustaceae, Wufu Flower Family, Poison Sheep Tree Family, Aizoaceae, Schizonepeta Family, Alismataceae, Bracteaceae, Pseudo-Pittosporaceae, Alstroemeriaceae, Liquidambaraceae, Amaranthaceae, Amaryllidaceae, Onychophyllaceae, Cinnamomum family, Anacardiaceae, Bracteaceae, Anastrophyllaceae, Uncaria family, Black Moss family, Black True Moss family, Anemiaceae, Leaf Moss family, Tetracolumnaceae, Annonaceae, Rabbit Ear Moss family, Horn Moss family, Gum Vine family, Monocarpaceae, Apiaceae, Aspleniaceae, Apocynaceae, Lithops family, Water Ivy family, Aquifoliaceae, Araceae, Araliaceae, Araucariaceae, Axis family, Palm family, Snow Leaf family, Aristolochiaceae, Cross-leaf Moss family, Asparagaceae , Asplenium family, Aromatic grass family, Wing-calyx tea family, Fragrant tea family, Hoof ferns family, Wrinkled moss family, Magnoliaceae, Tuberous crown moss family, Quercus family, Herba Viburnum family, Straight gourd family, Impatiens family, Arthralgia family, Hookwood family, Bead family, Basella family, Chenopodiaceae, Begonia family, Berberidaceae, Chilean vine family, Betulaceae, Fumotau family, Bignoniaceae, Rosewood family, Perfumery family, Pot-shaped moss family, Black hair fern family, Zecha family, Boraginaceae, Australian orchid family, Moss family, Cruciferae, Brevianthaceae, Bromeliaceae, Bruchiaceae, Petal split fruit family, Lepidoptera family, True moss family, Bryobartramiaceae, Shrimp moss family, Water hosta family, Oleaceae, Iris family, Boxwood family, Tobacco rod moss family, Glandular hair grass family, Brassicaceae, Immortelle family Palmaceae, Calceolariaceae, Calomniaceae, Calophyllaceae, Celastraceae, Celastraceae, Celastraceae, Cyperaceae, Cyperaceae, Cyperaceae, Campanulaceae, Cyperaceae, Cinnamonaceae, Cannaceae, Caprifoliaceae, Caprifoliaceae, Caricaparaceae, Caryophyllaceae, Nectariaceae, Caryophyllaceae, Casuarinaceae, Catagonaceae, Catoscopiaceae, Celastraceae, Cyperace ... Flower family, Salicaceae, Klein family, Dipsaci family, Garcinia family, Colchicum family, Croton family, Combretaceae, Commelinaceae, Asteraceae, Cistanche family, Herba Lycopodii family, Convolvulaceae, Moraceae, Cornaceae, Hosta family, Flos ruticaceae, Cistanche family, Crassulaceae, Flintaceae, Cryptocarpa family, Pendulaceae, Cucurbitaceae, Pteridaceae, Torch family, Cupressaceae, South Africa Cornaceae, Alsophilaceae, Cycadaceae, Cyperaceae, Cyperaceae, Cyperiaceae, Cyrtopodaceae, Cystodiaceae, Pteridaceae, Cyperiaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae, Pteridaceae,Dicnemonaceae, Dicnemonaceae, Dracaenae, Dicnemonaceae, Dioscoreaceae, Dioscoreaceae, Dioscoreaceae, Dioscoreaceae, Dioscoreaceae, Dioscoreaceae, Dioscoreaceae, Disceliaceae, Droseraceae, Droseraceae, Drosophila, Dryopteri dacae, Dryopteris family, Dioscoreaceae, Echinodiaceae, Elaeagnaceae, Elaeocarpus family, Stellaria family, Australian Polygonaceae, Big Cap Moss family, Silk Moss family, Ephedra, Short-lived Moss family, Equisetum family, Ericaceae, Coleus family, Southern Rhizoma family, Eucommia family, Euphorbiaceae, Synthetic Flower family, Cap Flower family, Collared Spring Moss family, Eustichiaceae, Short-suspensa family, Cardamine family, Fagaceae, Pteris family, Big Wind Moss family, Leaf Vine family, Sphagaceae, Microphylla family, Osmanthus family, Petalophyceae, Cucurbitaceae, Twist The family of genus is Glechoma, ... Elwingiaceae, Viburnum, Nelumbo, Glossopsaceae, Oil Moss, Alliaceae, Balsamiaceae, Monostomaceae, Fungus, Hydrangeaceae, Hydrocharitaceae, Field Hemp, Water Spike, Tower Moss, Hymenophytaceae, Hymenophytaceae, Hypericaceae, Gray Moss, Tree Gray Moss, Swollen Foot Pteridaceae, Peacock Moss, Curculigo, Cornaceae, Iridaceae, Bud Tree, Isoeceae, Theaceae, Iridaceae, Glutinous Wood, Crustaceae, Reed Moss, Hairy Ear Moss, Jubulopsaceae, Juglandaceae, Juncus, Water Lily Family, leaf moss family, Angustiaceae, spiny branch tree family, spiny cone family, split aphid family, short stamen family, Lamiaceae, hair amaryllidaceae, Akebia family, Lauraceae, Barringtaceae, Leguminosae, fine scale moss family, boat leaf moss family, bladderwort family, complex fork moss family, Oxalidaceae, polycystic moss family, finger leaf moss family, thin tooth moss family, Lepyrodontaceae, thin Luo Luo moss family, white tooth moss family, white moss family, Liliaceae, millet grass family, pond flower family, linaceae, mother grass family, Lepidoptera family, spiny lotus family, logan family, vine fern family, Lonchitidaceae, Lophiocarpa ceae, tooth calyx moss family, five-winged fruit family, split leaf moss family, moss parasitic family, orchid banana family, ring fern family, half moon moss family, lycopodiaceae, Lycopodiaceae, Lythrum family, Magnoliaceae, Nanxi moss family, chloris family, Malvaceae, Marantaceae, cystaceae, honey sac flower family, liverwort family, apple family, horn sesame family, beard moss family, Matong fern family, Carex family, cold moss family, black medicaceae, Melastomataceae, Meliaceae, honey flower family,Menispermaceae, Menispermaceae, Mesoptychiaceae, Fern family, Myrtaceae, Tubeflower family, Fork moss family, Microtheciellaceae, Feather fruit family, Cap stamen family, Mizutaniaceae, Lantern moss family, Corn grass family, Cup axis flower family, Monocarpaceae, Single leaf moss family, Single moon moss family, Chicken grass family, Mountain vinegar plum family, Moraceae, Moringa family, Wen Ding fruit family, Musaceae, Cracking fruit red family, Myricaceae, Myriniaceae, Myristicaceae, Fragrant shrub family, Myrtaceae, Golden hair moss family, Nasi family, Neckeraceae, Nelumbo family, Neotrichocoleaceae, Nepenthes family, Nephrolep Idaceae, sandberry family, white thorn family, southern cycad family, short horn moss family, Mirabilis family, Nymphaeaceae, Nasturtium family, eight-tooth moss, long table moss family, iron tree family, Oleaceae, striped fern family, onagraceae, five-stalk tea family, ball fern family, bottle herb family, mountain grapefruit family, orchid family, Orobanchaceae, Orthorrhynchiaceae, wood spirit moss family, osmanthus family, Oxymitraceae, Paeoniaceae, Pallaviciniaceae, small disk wood family, Pandan family, Galea family, Passifloraceae, Paulownia family, Pedunculaceae, stream moss family, tube calyx family, Pentadiplandraceae, five membrane grass family, five column wood family, root vegetable family, Peraceae, peripan Tree family, Petenaeaceae, Acanthaceae, Aphyllaceae, Ilexaceae, Pendulaceae, Allium family, Pendulaceae, Phyllostachys family, Phyllostachys family, Phyllodrepaniaceae, Ribes family, African Amaranth family, Phytolacca family, American Simulaceae, Juglandaceae, Pilotrichaceae, Pinaceae, Piperaceae, Pittosporum family, Feather moss family, Knot-footed fern family, Cotton moss family, Plantaginaceae, Platanus, Siderococo family, Purple leaf moss family, Pleuroziopsaceae, Ring seed family, Blue snow family, Poaceae, Podocarp family, Sichuan Carex family, Flower family, Polygonaceae, Polygonaceae, Polypodium family, Golden hair moss family, Rain long flower family, Light calyx moss family, Portulacaceae, Boxidan family, Potamogeton family, Assemblage family, Primulaceae, Trichodermaceae, Proteaceae, Trichodermaceae, Pseudo-complexaceae, Pteridaceae, Pteridaceae, Pterigynandraceae, Pteridaceae, Trichodermaceae, Pteridaceae, Putranjivaceae, Quillajaceae, Selaginellaceae, Flat-calyx moss, Rhaponticaceae, Ranunculaceae, Spikeweed, Regmatodontaceae, Oleaceae, Broomrapeaceae, Rhabdoweisiaceae, Axicarpaceae, Rhacocarpaceae, Rhamnaceae, Smilaxaceae, Juniperus, Rhombaceae, Moneywort, Newmoss, Rigodiaceae, Polidoraceae, Rosaceae, Rousseau family, Rubiaceae, Rampaceae,Rutaceae, Rutenbergiaceae, Qingfengtengceae, Pteridaceae, Salicaceae, Jasmineaceae, Sophoraceae, Santalaceae, Sapindaceae, Sapotaceae, Chenopodiaceae, Convolvulaceae, Sarraceniaceae, Saxifragaceae, Synechococcus, Chimonanthus, Schisandraceae, Dichroaceae, Glossy Moss, Cyperaceae, Idiomycetes, Vaticaceae, Pinaceae, Scorpidiaceae, Scrophulariaceae, Cupressaceae, Fine-leaved Moss, Moss, Serpotortellaceae aceae, Ileaceae, Simulaceae, Waxwood, Alcanthaceae, Rib-fruit Tea, Smilaxaceae, Solanaceae, Sorapillaceae, Capsulaceae, Globaceae, Sphagaceae, Sphenantheraceae, Wood Hair Moss, Potassium, Splachnobryaceae, Flamingocarpaceae, Province Oil Oil, Cleistaceae, Stemonaceae, Golden Sandalwood, Sclerophyllaceae, Stylocarpaceae, Cork Fruit, Traveler's Banana, Stylocarpaceae, Styracaceae, Sea Mangifera, Alumaceae, Algae Moss, Ginseng Family , Tamarixaceae, Gall pepper family, leather leaf moss family, Taxaceae, Blue star family, Trifoliaceae, Tetrasodium family, Tetrasodium family, Tetrasodium family, Tetrasodium family, Wood moss family, Theaceae, Lepidoptera, Venus fern family, Thomandersiaceae, Feather family, Suspension family, Thymelaeaceae, Umbrella family, Taigu family, Meizi family, Rock Acorus family, Coleoptera family, Strong flavor Trifoliaceae, Twist leaf moss family, Tao family, Velvet moss family, Trichotemnomataceae, Trigonaceae, Glandular tooth Aquilegiaceae, Acanthaceae, Acanthaceae, Nasturtiumaceae, Typhaceae, Ulmaceae, Urticaceae, Dichroaceae, Vandiemeniaceae, Pteridaceae, Verbenaceae, Vetaformaceae, Violaceae, Viridivelleraceae, Vitaceae, Acanthaceae, Wardiaceae, Welwitschiaceae, Welwitschiaceae, Acanthaceae, Acanthaceae, Acanthaceae, Acanthaceae, Acanthaceae, Acanthaceae, Acanthaceae, Azalea, Zymphaea, Zosteraceae, Terrestris.

[0351] Due to the new classification, some algae are no longer classified in the kingdom Plantae. However, these algae are candidates for the production of cellulose scaffolds as described herein. The kingdom Fungi has members that contain cell walls composed, for example, of cellulose. Algae are now classified in the kingdom Protista; however, it should be understood that in this disclosure, algae are intended to be included within the term "plants" as used herein. Suitable algae may include:

[0352] -Algae: plant-like single or multicellular organisms;

[0353] - Green algae: Spirogyra, Ulva, Chlamydomonas, Volvox;

[0354] -Red algae: Porphyra, Prototheca;

[0355] - Brown algae: kelp, sea cyst;

[0356] - Algae: Saprolegnia; and / or

[0357] -Ciliophora: Paramecium, Campanula.

[0358] Experiments have also demonstrated that chitin is a suitable scaffold that can be used in the scaffold biomaterials described herein using the protocols described herein. The kingdom Fungi is classified as follows:

[0359] - Ascomycetes: Agaricus (mushrooms), Ustilago (smut fungi), and Puccinia (rust fungi);

[0360] - Fertilized egg-forming fungi: Mucor, Rhizopus (bread mold), and Albugo;

[0361] - Coral fungi: Agaricus (mushroom), Ustilago (smut fungus), and Puccinia (rust fungus); and

[0362] - Fungi deuteroideum: Alternaria, Colletotrichum and Trichoderma.

[0363] As mentioned above, this fungus also represents a suitable candidate for obtaining the decellularized fungal tissue described herein above.

[0364] One or more illustrative embodiments have been described by way of example. It will be apparent to those skilled in the art that numerous changes and modifications may be made without departing from the scope of the invention as defined in the claims.

[0365] References

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[0493] All references cited in this section and elsewhere in this specification are hereby incorporated by reference in their entirety.

Claims

1. A scaffold biomaterial comprising a decellularized plant or fungal tissue from which cellular material and nucleic acids of the tissue have been removed, wherein the decellularized plant or fungal tissue comprises a three-dimensional porous structure based on cellulose or chitin.

2. The scaffold biomaterial of claim 1, wherein the decellularized plant or fungal tissue comprises a plant or fungal tissue that has been decellularized by heat shock, treatment with detergents, osmotic shock, freeze drying, physical lysis, electrical disruption, or enzymatic digestion, or any combination thereof.

3. The scaffold biomaterial according to claim 1 or 2, wherein the decellularized plant or fungal tissue comprises a plant or fungal tissue that has been decellularized by treatment with sodium dodecyl sulfate (SDS).

4. The scaffold biomaterial according to claim 3, wherein residual SDS has been removed by precipitating salt residues containing SDS micelles from the scaffold using a divalent saline solution.

5. The scaffold biomaterial according to claim 4, wherein dH2O, acetic acid, DMSO or ultrasonic treatment, or any combination thereof has been used to remove the divalent saline solution, salt residues and / or SDS micelles. The scaffold biomaterial according to claim 5 , wherein the divalent salt of the divalent salt solution comprises MgCl 2 or CaCl 2 .

7. The scaffold biomaterial of claim 6, wherein the plant or fungal tissue has been decellularized by treatment with an SDS solution containing about 1% or about 0.1% SDS in water, and residual SDS has been removed using an aqueous solution of CaCl2 at a concentration of about 100 mM followed by incubation in dH2O.

8. The scaffold biomaterial according to any one of claims 1 to 7, wherein the decellularized plant or fungal tissue is treated to introduce further structure and / or is functionalized at at least one free hydroxyl function by acylation, alkylation or other covalent modification to provide a functionalized scaffold biomaterial.

9. The scaffold biomaterial according to claim 8, wherein the decellularized plant or fungal tissue is processed to introduce microchannels, and / or functionalized with collagen, factors promoting cell specificity, cell growth factors or pharmaceutical agents.

10. The scaffold biomaterial according to any one of claims 1 to 9, wherein the plant or fungal tissue is apple inflorescence (Malus pumila) tissue, fern (Monilophytes) tissue, radish (Brassica rapa) root tissue, ginkgo branch tissue, horsetail (equisetum) tissue, hermocallis hybrid leaf tissue, kale (Brassica oleracea) stem tissue, conifer Douglas fir (Pseudotsuga menziesii) tissue, cactus fruit (pitaya) flesh tissue, periwinkle rose (Maculata Vinca) tissue, water lotus (Nelumbo nucifera) tissue, tulip (Tulipa gesneriana) petal tissue, plantain (Musa paradisiaca) tissue, broccoli (Brassica oleracea) stem tissue, maple leaf (Acer psuedoplatanus) stem tissue, beet (Beta vulgaris) primary root tissue, onion (Allium cepa) tissue, Orchidaceae tissue, Radish (Brassica rapa) stem tissue, Leek (Allium ampeloprasum) tissue, Maple (Acer) branch tissue, Celery (Apium graveolens) tissue, Onion (Allium cepa) stem tissue, Pine tissue, Aloe vera tissue, Watermelon (Citrullus lanatus var.lanatus) tissue, Meadowgrass (Lysimachia nummularia) tissue, Cactae tissue, Arctic Campion (Lychnis Alpina) tissue, Rhubarb (Rheum rhabarbarum) tissue, Pumpkin (Cucurbita pepo) tissue, Dracena (Asparagaceae) stem tissue, Tradescantia virginiana stem tissue, Asparagus (Asparagus officinalis) stem tissue, Mushroom (Fungi) tissue, Fennel (Foeniculum vulgare) tissue, Rose (Rosa) tissue, Carrot (Daucus carota) tissue or pear (Pomaceous) tissue, or genetically altered tissue prepared as follows, by direct genomic modification or by selective breeding, to produce additional plant or fungal structures that are configured to physiologically mimic the tissue and / or functionally promote the effects of the target tissue.

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