Patent Application
20240307592
The present invention relates generally to aerogels, hydrogels, and foams. More specifically, the present invention relates to aerogels, hydrogels, and foams derived from and/or comprising decellularized plant or fungal tissue or structural cells thereof.
Scaffold materials are highly sought after in a number of different fields, especially those providing homogenous and/or reproducible 3-dimensional structures. In the pharmaceutical, medical device, therapy, and food spaces, biocompatible and/or edible scaffold materials are particularly sought after, and those capable of supporting cell growth are highly desirable.
Various scaffold materials have been developed, many of which are based on synthetic polymers or other such materials. Some of which are known to be biocompatible and/or bio-inert, but additional scaffold materials are still of significant interest for a variety of applications.
Scaffold biomaterials comprising decellularized plant or fungal tissue have been developed and described in PCT patent publication WO2017/136950, entitled “Decellularised Cell Wall Structures From Plants and Fungus and Use Thereof as Scaffold Materials”. Remarkable biocompatibility, and uses in a variety of therapeutic applications, are described. These scaffold biomaterials are of significant interest for a variety of different applications.
Nevertheless, additional scaffold materials, and particularly those providing aerogels, hydrogels, and/or foams, are desirable in a variety of fields. Aerogels, hydrogels, and/or foams providing tunable or customizable physical/mechanical properties and/or micro/macro-scale architectures are especially sought after.
Alternative, additional, and/or improved aerogels, hydrogels, and/or foams, as well as methods and/or uses thereof, are desirable.
Provided herein are aerogels, hydrogels, and foams derived from and/or comprising decellularized plant or fungal tissue or structural cells thereof. As described in detail hereinbelow, aerogels, hydrogels, and foams have now been developed which may be derived from and/or may comprise decellularized plant or fungal tissue or structural cells thereof, and which: may comprise plant or fungal microstructures and/or architectures of interest; may be produced by readily scalable production methods; may provide for a wide range of scaffold microstructures and/or macrostructures and/or biochemistry; may provide tunable mechanical properties; may provide tunable porosity; may be biocompatible in vitro and/or in vivo; may be stable to a variety of conditions (such as cooking conditions in the case of food products); or any combinations thereof. By using single structural cells, groups of structural cells, or both, derived from a plant or fungal tissue (the single structural cells or groups of structural cells having a decellularized 3-dimensional structure lacking cellular materials and nucleic acids of plant or fungal tissue), distributed within a carrier derived from one or more dehydrated, lyophilized, or freeze-dried hydrogels, a variety of aerogels, hydrogels, and foams have now been developed and prepared having desirable properties. In certain embodiments, the single structural cells, groups of structural cells, or both, may be derived from plant or fungal tissue (typically decellularized plant or fungal tissue) using mercerization treatment as described herein, which allows for reproducible and scalable production. Related methods and uses, as well as productions methods, are also described in detail herein.
In an embodiment, there is provided herein an aerogel or foam comprising:
In another embodiment of the above aerogel or foam, the aerogel or foam has been rehydrated.
In still another embodiment of any of the above aerogel or aerogels or foam or foams, the plant or fungal tissue from which the single structural cells or groups of structural cells are derived may comprise decellularized plant or fungal tissue.
In yet another embodiment of any of the above aerogel or aerogels or foam or foams, the plant or fungal tissue may be decellularized using SDS and optionally CaCl2).
In another embodiment of any of the above aerogel or aerogels or foam or foams, the single structural cells, groups of structural cells, or both, may be derived from the plant or fungal tissue by mercerization. In certain embodiments, the plant or fungal tissue may be decellularized plant or fungal tissue.
In still another embodiment of any of the above aerogel or aerogels or foam or foams, the mercerization may comprise treatment of the plant or fungal tissue using sodium hydroxide and hydrogen peroxide with heating.
In yet another embodiment of any of the above aerogel or aerogels or foam or foams, the aerogel or foam may comprise a particle size distribution of the single structural cells with an average feret diameter within a range of about 1 μm to about 1000 μm, such as about 100 to about 500 μm, for example about 100 to about 300 μm.
In another embodiment of any of the above aerogel or aerogels or foam or foams, the hydrogel may comprise alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, agar, pluronic acid, triblock PEO-PPO-PEO copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), dissolved or regenerated plant cellulose, dissolved cellulose-based hydrogel, hyaluronic acid, extracellular matrix proteins (e.g. collagen, gelatin, or fibronectin, or any combinations thereof), monoacrylated poly(ethylene glycol), poly(ethylene glycol) diacrylate, poly(ethylene glycol) diacrylate (PEGDA)-co-PEGMA, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(lactic-co-glycolic acid), chitosan, chitin, xanthan gum, elastin, fibrin, fibrinogen, cellulose derivatives, carrageenan, or microcrystalline cellulose, or any combinations thereof; wherein the hydrogel is optionally cross-linked.
In still another embodiment of any of the above aerogel or aerogels or foam or foams, the aerogel or foam may comprise templated or aligned microchannels created by directional freezing; or by molding using that possess microscale features; by punching, pressing, stamping, or otherwise forming geometric patterns, depressions, holes, channels, grooves, ridges, wells, or other structural features within and/or onto at least one surface; or any combinations thereof.
In yet another embodiment of any of the above aerogel or aerogels or foam or foams, the plant tissue may comprise apple tissue or a pear tissue.
In another embodiment of any of the above aerogel or aerogels or foam or foams, the aerogel or foam may comprise about 5% to about 95% m/m single structural cells, groups of structural cells, or both, when the aerogel or foam is in hydrated form.
In still another embodiment of any of the above aerogel or aerogels or foam or foams, the hydrogel may comprise alginate, pectin, or both, and the aerogel or foam may be rehydrated with a CaCl2) solution providing cross-linking.
In yet another embodiment of any of the above aerogel or aerogels or foam or foams, the aerogel or foam may have bulk modulus within a range of about 0.1 to about 500 kPa, such as about 1 to about 200 kPa.
In another embodiment of any of the above aerogel or aerogels or foam or foams, the aerogel or foam may be rehydrated and may further comprise one or more animal cells.
In still another embodiment of any of the above aerogel or aerogels or foam or foams, at least some cellulose and/or cellulose derivative(s) of the aerogel or foam may be cross-linked by physical cross-linking (e.g. using glycine) and/or chemical cross-linking (e.g. using citric acid in the presence of heat); wherein at least some cellulose and/or cellulose derivative(s) of the aerogel or foam is functionalized with a linker (e.g. succinic acid) to which one or more functional moieties are optionally attached (e.g. amine-containing groups, wherein cross-linking may further optionally be achieved with one or more protein cross-linkers such as formalin, formaldehyde, glutaraldehyde, and/or transglutaminase); or any combinations thereof.
In yet another embodiment, there is provided herein single structural cells, groups of structural cells, or both, derived from a decellularized plant or fungal tissue by mercerization of the decellularized plant or fungal tissue, the single structural cells or groups of structural cells having a decellularized 3-dimensional structure lacking cellular materials and nucleic acids of plant or fungal tissue, and lacking one or more base-soluble lignin components of the plant or fungal tissue.
In yet another embodiment, there is provided herein a method for preparing an aerogel or foam, comprising:
In yet another embodiment of the above method, the mercerization may comprise treatment of the decellularized plant or fungal tissue with a base and a peroxide, preferably sodium hydroxide or another hydroxide base as base, and hydrogen peroxide as peroxide.
In still another embodiment of any of the above method or methods, the mercerization may comprise treatment of the decellularized plant or fungal tissue with aqueous sodium hydroxide solution and hydrogen peroxide while heating.
In yet another embodiment of any of the above method or methods, the decellularized plant or fungal tissue may be treated with the aqueous sodium hydroxide solution for a first period of time before the hydrogen peroxide is added to the reaction.
In another embodiment of any of the above method or methods, the hydrogen peroxide may be added as a 30% aqueous hydrogen peroxide solution.
In still another embodiment of any of the above method or methods, the hydrogen peroxide for mercerization may be used in a ratio of:
In yet another embodiment of any of the above method or methods, the method may further comprise a step of neutralizing the pH with one or more neutralization treatments.
In another embodiment of any of the above method or methods, the neutralization treatment may comprise treatment with an acid solution, preferably an aqueous HCl solution.
In still another embodiment of any of the above method or methods, the mercerization may be performed with heating to about 80° C.
In yet another embodiment of any of the above method or methods, the mercerization may be performed using a ratio of decellularized plant or fungal tissue:aqueous sodium hydroxide solution (m:v, in g:L) of about 1:5 for a 1M aqueous sodium hydroxide solution, or an equivalent ratio for another aqueous sodium hydroxide solution concentration.
In another embodiment of any of the above method or methods, the mercerization may be performed for at least about 30 minutes, preferably for about 1 hour.
In still another embodiment of any of the above method or methods, the resultant single structural cells or groups of structural cells having a decellularized 3-dimensional structure may be collected by centrifugation.
In yet another embodiment of any of the above method or methods, the single structural cells, groups of structural cells, or both, may be mixed or distributed in a hydrogel comprising alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, agar, pluronic acid, triblock PEO-PPO-PEO copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), dissolved or regenerated plant cellulose, dissolved cellulose-based hydrogel, hyaluronic acid, extracellular matrix proteins (e.g. collagen, gelatin, or fibronectin, or any combinations thereof), monoacrylated poly(ethylene glycol), poly(ethylene glycol) diacrylate, poly(ethylene glycol) diacrylate (PEGDA)-co-PEGMA, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(lactic-co-glycolic acid), chitosan, chitin, xanthan gum, elastin, fibrin, fibrinogen, cellulose derivatives, carrageenan, or microcrystalline cellulose, or any combinations thereof; wherein the hydrogel is optionally cross-linked.
In another embodiment of any of the above method or methods, the method may further comprise a step of performing directional freezing of the mixture to introduce templated or aligned microchannels on a surface of the mixture, within the mixture, or both; a step of molding the mixture using molds having microscale features contacting one or more surfaces of the mixture and/or the aerogel or foam resulting from dehydrating, lyophilizing, or freeze-drying of the mixture, so as to introduce templated or aligned microchannels; a step of punching, pressing, stamping, or otherwise forming geometric patterns, depressions, holes, channels, grooves, ridges, wells, or other structural features within and/or onto at least one surface of the mixture and/or the aerogel or foam before, during, or after dehydrating, lyophilizing, or freeze-drying of the mixture; or any combinations thereof.
In still another embodiment of any of the above method or methods, the directional freezing may be performed by creating a thermal gradient across the mixture from one or more directions so as to form aligned ice crystals beginning from the cold side(s) of the thermal gradient.
In still another embodiment of any of the above method or methods, a microarchitecture of the microchannels produced from directional freezing may be controlled by creating the mixture including a solvent containing varying amounts of one or more other dissolved compounds such as Sucrose, Dextrose, Trehalose, Corn Starch, Glycerol, Ethanol, Mannitol, Sodium chloride, CaCl2, Gelatine, Citric acid, PVA, PEG, Dextran, NaF, NaBr, NaI, phosphate buffer, or another such agent, which alters the structural properties of aligned ice crystals which grow from the cold side of the thermal gradient.
In yet another embodiment of any of the above method or methods, the mixture may be directionally frozen over a period of at least about 30 minutes, preferably over a period of about 2 hours.
In another embodiment of any of the above method or methods, the mixture may be directionally frozen by cooling to a temperature between about −190° C. and about 0° C., such as at least about −15° C., preferably about −25° C.
In still another embodiment of any of the above method or methods, the step of dehydrating, lyophilizing, or freeze-drying the mixture to provide the aerogel or foam may comprise freezing the mixture followed by lyophilizing or freeze-drying the mixture.
In yet another embodiment of any of the above method or methods, the method may comprise a further step of cross-linking the hydrogel (such as cross-linking the hydrogel before or after freezing/lyophilisation, for example), rehydrating the aerogel or foam, or both; optionally using CaCl2) solution to provide cross-linking where alginate or pectin or agar hydrogel is present.
In another embodiment of any of the above method or methods, the method may comprise a further step of culturing animal cells on or in the aerogel or foam.
In another embodiment, there is provided herein an aerogel or foam produced by any of the method or methods as described herein.
In still another embodiment, there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein, for bone tissue engineering.
In yet another embodiment, there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein, for templating or aligning growth of cells.
In another embodiment of the above use, the cells may comprise muscle cells.
In still another embodiment of the above use, the cells may comprise nerve cells.
In yet another embodiment, there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein for repair of spinal cord injury.
In another embodiment, there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein as insulation or packaging foam.
In still another embodiment, there is provided herein a method for bone tissue engineering or repair in a subject in need thereof, comprising:
In yet another embodiment, there is provided herein a method for templating or aligning growth of cells, comprising:
In another embodiment of the above method, the cells may comprise muscle cells or nerve cells.
In still another embodiment, there is provided herein a method for repairing spinal cord injury in a subject in need thereof, comprising:
In another embodiment, there is provided herein a food product comprising any of the aerogel or aerogels or foam of foams as described herein.
In still another embodiment of the above food product, the food product may be created for a cell-based or plant-based meat industry, and may utilize cellular agriculture techniques to create cultured meat products or plant-based meat products comprising or using aerogels and/or foams as described herein such as those including materials derived from decellularized plant or fungal tissues. The Examples included hereinbelow support that aerogels and/or foams as described herein may be cooked, may support mammalian cell growth, may be coloured and formed into plant-based and/or cell-based meat products. The Examples set out hereinbelow include a detailed example of a plant-based tuna fish mimic as an illustrative example.
In still another embodiment of the above food product, the food product may comprise a dye or coloring agent.
In yet another embodiment of any of the above food product or food products, the food product may comprise two or more aerogel or foam subunits glued together.
In another embodiment of any of the above food product or food products, the glue may comprise agar.
In still another embodiment of any of the above food product or food products, the aerogel or foam may comprise templated or aligned microchannels optionally formed by directional freezing, and wherein the aerogel or foam comprises muscle cells, fibroblasts, myofibroblasts, neurons, dorsal root ganglion cells, neuronal structures such as axons, neural precursor cells, neural stem cells, glial cells, endogenous stem cells, neutrophils, mesenchymal stem cells, satellite cells, myoblasts, myotubes, muscle progenitor cells, adipocytes, preadipocytes, chondrocytes, osteoblasts, osteoclasts, pre-osteoblasts, tendon progenitor cells, tenocytes, periodontal ligament stem cells, or endothelial cells, or any combinations thereof, aligned along the templated or aligned microchannels; preferably wherein the aerogel or foam comprises templated or aligned microchannels optionally formed by directional freezing, and wherein the aerogel or foam comprises muscle cells, fat cells, connective tissue cells (e.g. fibroblasts), cartilage, bone, epithelial, or endothelial cells, or any combinations thereof, aligned along the templated or aligned microchannels.
In another embodiment, there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein in a food product.
In still another embodiment, there is provided herein a method for preparing single structural cells, groups of structural cells, or both, from decellularized plant or fungal tissue, comprising:
In yet another embodiment of the above method, the mercerization may comprise treatment of the decellularized plant or fungal tissue with a base and a peroxide, preferably sodium hydroxide or another hydroxide base as base, and hydrogen peroxide as peroxide.
In another embodiment of any of the above method or methods, the mercerization may comprise treatment of the decellularized plant or fungal tissue with aqueous sodium hydroxide solution and hydrogen peroxide while heating.
In still another embodiment of any of the above method or methods, the decellularized plant or fungal tissue may be treated with the aqueous sodium hydroxide solution for a first period of time before the hydrogen peroxide is added to the reaction.
In yet another embodiment of any of the above method or methods, the hydrogen peroxide may be added as a 30% aqueous hydrogen peroxide solution.
In still another embodiment of any of the above method or methods, the hydrogen peroxide for mercerization may be used in a ratio of:
In yet another embodiment of any of the above method or methods, the method may further comprise a step of neutralizing the pH with one or more neutralization treatments.
In another embodiment of any of the above method or methods, the neutralization treatment may comprise treatment with an acid solution, preferably an aqueous HCl solution.
In still another embodiment of any of the above method or methods, the mercerization may be performed with heating to about 80° C.
In yet another embodiment of any of the above method or methods, the mercerization may be performed using a ratio of decellularized plant or fungal tissue: aqueous sodium hydroxide solution (m:v, in g:L) of about 1:5 for a 1M aqueous sodium hydroxide solution, or an equivalent ratio for another aqueous sodium hydroxide solution concentration.
In still another embodiment of any of the above method or methods, the mercerization may be performed for at least about 30 minutes, preferably for about 1 hour.
In yet another embodiment of any of the above method or methods, the resultant single structural cells or groups of structural cells having a decellularized 3-dimensional structure may be collected by centrifugation.
In still another embodiment, there is provided herein single structural cells, groups of structural cells, or both, prepared by any of the method or methods as described herein.
In yet another embodiment, there is provided herein a cellulose-based hydrogel comprising cellulose derived from decellularized plant or fungal tissue via dissolution with dimethylacetamide and lithium chloride followed by regeneration with ethanol.
In still another embodiment, there is provided herein a method for preparing a cellulose-based hydrogel comprising:
In another embodiment of the above method, the solvent exchange with ethanol may be performed using a dialysis membrane, or by adding ethanol on top of the dissolved cellulose to promote solvent exchange.
In still another embodiment of any of the above method or methods, the method may further comprise bleaching the cellulose-based hydrogel with hydrogen peroxide.
In yet another embodiment, there is provided herein a cellulose-based hydrogel comprising cellulose derived from decellularized plant or fungal tissue via dissolution with: dimethylacetamide and lithium chloride, LiClO4, xanthate, EDA/KSCN, H3PO4, NaOH/urea, ZnCl2, TBAF/DMSO, NMMO, an ionic liquid (IL) (such as 1-butylpyridinium chloride and aluminum chloride, alkyl imidazolium in association with nitrate, preferably a room temperature ionic liquid), or any combinations thereof.
In still another embodiment, there is provided herein a method for preparing a cellulose-based hydrogel comprising:
In another embodiment, there is provided herein a cellulose-based hydrogel prepared by any of the method or methods as described herein.
In another embodiment of any of the aerogel or aerogels or foam or foams as described herein, the hydrogel may comprise any of the cellulose-based hydrogel or cellulose-based hydrogels as described herein.
In still another embodiment, there is provided herein a food product comprising any of the aerogel or aerogels or foam or foams or structural cell or structural cells as described herein, wherein the food product is a meat mimic and comprises a plurality of lines providing the appearance of fatty white lines found in tuna, salmon, or another fish-type meat.
In yet another embodiment of the above food product, the food product may be a mimic of tuna, salmon, or another fish meat.
In another embodiment of any of the above food product or food products, the food product may contain one or more dyes or colorants providing the color of tuna, salmon, or another fish meat.
In still another embodiment of any of the above food product or food products, the plurality of lines may be formed in cuts or channels formed in the aerogel or foam.
In yet another embodiment of any of the above food product or food products, the plurality of lines may comprise titanium dioxide, optionally combined with agar binding agent or another such binding agent.
In another embodiment of any of the above food product or food products, the titanium dioxide, optionally combined with agar binding agent, may be applied into cuts or channels formed in the aerogel or foam to provide the appearance of the fatty white lines found in tuna, salmon, or another fish-type meat.
In another embodiment, there is provided herein a method for preparing a food product, the food product being a tuna, salmon, or other fish meat mimic, said method comprising:
In another embodiment of the above method, the dye or coloring agent applied to the cuts or channels may comprise titanium dioxide.
In still another embodiment of any of the above method or methods, the dye or coloring agent applied to the cuts or channels may be combined with a binding agent.
In yet another embodiment of any of the above method or methods, the binding agent may comprise agar.
In another embodiment, there is provided herein a food product prepared by any of the method or methods described herein.
In another embodiment, there is provided herein a non-resorbable dermal filler comprising an aerogel, foam, structural cell, or cellulose-based hydrogel as described herein; or any combinations thereof.
In yet another embodiment, there is provided herein a dermal filler comprising single structural cells, groups of structural cells, or both, derived from a plant or fungal tissue, the single structural cells or groups of structural cells having a decellularized 3-dimensional structure lacking cellular materials and nucleic acids of plant or fungal tissue, the single structural cells, groups of structural cells, or both, being derived from the plant or fungal tissue by mercerization.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may further comprise a carrier fluid or gel.
In still another embodiment of any of the above dermal filler or dermal fillers, the carrier fluid or gel may comprise water, an aqueous solution, or a hydrogel.
In yet another embodiment of any of the above dermal filler or dermal fillers, the carrier fluid or gel may comprise a saline solution, or a collagen, hyaluronic acid, methylcellulose, and/or dissolved plant-derived decellularized cellulose-based hydrogel.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may further comprise an anesthetic agent.
In still another embodiment of any of the above dermal filler or dermal fillers, the anesthetic agent may comprise lidocaine, benzocaine, tetracaine, polocaine, epinephrine, or any combinations thereof.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may comprise PBS (saline), hyaluronic acid (cross-linked or non-crosslinked), alginate, collagen, pluronic acid (e.g. pluronic F 127), agar, agarose, or fibrin, calcium hydroxylapatite, Poly-L-lactic acid, autologous fat, silicone, dextran, methylcellulose, or any combinations thereof.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may comprise at least one of: 2% lidocaine gel; a triple anesthetic gel comprising 20% benzocaine, 6% lidocaine, and 4% tetracaine (BLTgel); 3% Polocaine; or a mixture of 2% lidocaine with epinephrine.
In another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a size, diameter, or minimum feret diameter of at least about 20 μm.
In another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a size, diameter, or maximum feret diameter of less than about 1000 μm.
In still another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a size, diameter, or feret diameter distribution within a range of about 20 μm to about 1000 μm.
In yet another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a particle size, diameter, or feret diameter distribution having a peak about 200-300 μm.
In another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a mean particle size, diameter, or feret diameter within a range of about 200 μm to about 300 μm.
In another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have an average projected particle area within a range of about 30,000 to about 75,000 μm2.
In still another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may be sterilized.
In yet another embodiment of any of the above dermal filler or dermal fillers, the sterilization may be by gamma sterilization.
In still another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may be formulated for subdermal injection, deep dermal injection, subcutaneous injection (e.g. subcutaneous fat injection), or any combinations thereof.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may be provided in a syringe or injection device.
In another embodiment, there is provided herein a use of any of the dermal filler or dermal fillers as described herein as a soft tissue filler, for reconstructive surgery, or both.
In another embodiment, there is provided herein a use of any of the dermal filler or dermal fillers as described herein for improving cosmetic appearance of a subject in need thereof.
In another embodiment, there is provided herein a use of any of the dermal filler or dermal fillers as described herein for increasing tissue volume, smoothing wrinkles, or both, in a subject in need thereof.
In another embodiment, there is provided herein a method for improving cosmetic appearance, increasing tissue volume, smoothing wrinkles, or any combinations thereof, in a subject in need thereof, said method comprising:
In another embodiment of the above use or uses or method or methods, native cells of the subject may infiltrate the dermal filler.
In yet another embodiment of the above use or uses or method or methods, the dermal filler may be non-resorbable such that the decellularized plant or fungal tissue remains substantially intact within the subject.
These and other features will become further understood having regard to the following Description and accompanying Drawings, wherein:
Described herein are aerogels, hydrogels, and foams derived from and/or comprising decellularized plant or fungal tissue or structural cells thereof. It will be appreciated that embodiments and examples are provided for illustrative purposes intended for those skilled in the art, and are not meant to be limiting in any way.
Provided herein are aerogels, hydrogels, and foams derived from and/or comprising decellularized plant or fungal tissue or structural cells thereof. As described in detail hereinbelow, aerogels, hydrogels, and foams have now been developed which may be derived from and/or may comprise decellularized plant or fungal tissue or structural cells thereof, and which: may comprise plant or fungal microstructures and/or architectures of interest; may be produced by readily scalable production methods; may provide for a wide range of scaffold microstructures and/or macrostructures and/or biochemistry; may provide tunable mechanical properties; may provide tunable porosity, density, architecture (amorphous, aligned, channeled, etc. . . . ), and/or alignment; may be biocompatible in vitro and/or in vivo; may be stable to a variety of conditions (such as cooking conditions in the case of food products); may be produced at scale with control over micro and/or macro structural properties; may allow for control over density, long range architecture, and/or mass manufacture; may be scalable in terms of quantity of material produced as well as product size and/or shape; may be produced with GRAS components to maintain edibility; may be freeze-dried to provide shelf stability and/or shippability; or any combinations thereof. By using single structural cells, groups of structural cells, or both, derived from a plant or fungal tissue (the single structural cells or groups of structural cells having a decellularized 3-dimensional structure lacking cellular materials and nucleic acids of plant or fungal tissue), distributed within a carrier derived from one or more dehydrated, lyophilized, or freeze-dried hydrogels, a variety of aerogels, hydrogels, and foams have now been developed and prepared having desirable properties. In certain embodiments, the single structural cells, groups of structural cells, or both, may be derived from plant or fungal tissue (typically decellularized plant or fungal tissue) using mercerization treatment as described herein, which allows for reproducible and scalable production. Related methods and uses, as well as productions methods (some or all of which may be automatable), are also described in detail herein.
In an embodiment, there is provided herein an aerogel or foam comprising:
As will be understood, an aerogel or foam may comprise generally any 3-dimensional scaffold or matrix. Typically, aerogels and foams as described herein are highly porous and lightweight (low density), although porosity and density may be adjusted as desired as is also described herein. The aerogels and foams are typically hydrophilic, and may be provided as either dry aerogels or foams, or rehydrated or wetted aerogels or foams (sometimes also referred to herein as hydrogels) additionally comprising water, an aqueous solution (such as a cell culture buffer, a salt solution, a buffer, or another aqueous solution), or another liquid (such as an alcohol, for example ethanol, or a non-aqueous liquid).
As will be understood, plant or fungal tissue may comprise a plurality of linked plant cells formed as an extended 3D structure. Such plant or fungal tissue may be decellularized (for example, by using the decellularization methods as described in WO2017/136950, entitled “Decellularised Cell Wall Structures From Plants and Fungus and Use Thereof as Scaffold Materials”, which is herein incorporated by reference in its entirety) so as to provide decellularized plant or fungal tissue lacking cellular materials and nucleic acids of plant or fungal cells, but maintaining 3 dimensional structure substantially intact. Such decellularized plant or fungal tissue (such as decellularized hypanthium or pulp tissue, or any other suitable plant or fungal tissues/structures/components of interest) may comprise an extended 3D structure (which may be comprised of any one or more of cellulose, hemicellulose, pectin, lignin, or the like; typically, the extended 3D structure may comprise a lignocellulosic structure/material), which may comprise a plurality of linked structural cells. As described herein, single structural cells, groups of structural cells (comprising a plurality of linked single structural cells), or both, may be derived from the extended 3D structure, the single structural cells or groups of structural cells having a decellularized 3-dimensional structure lacking cellular materials and nucleic acids of plant or fungal tissue. In certain embodiments, the single structural cells or groups of structural cells may comprise isolated structural cells, or small groups of clustered structural cells, the structural cells having a substantially intact 3-dimensional structure typically resembling a hollow cell or pocket as shown in
In certain embodiments of the aerogel or aerogels or foam or foams, the plant or fungal tissue from which the single structural cells or groups of structural cells are derived may comprise decellularized plant or fungal tissue.
As described herein, single structural cells, groups of structural cells, or both, may preferably be derived from a decellularized plant or fungal tissue, and may even more preferably be derived from a decellularized plant or fungal tissue using mercerization treatment as described in detail herein. However, it will be understood that in certain embodiments, single structural cells, groups of structural cells, or both, may instead be derived from plant or fungal tissue and then decellularized afterward, or may be derived from plant or fungal tissue in a manner that concurrently provides decellularization, for example. In certain embodiments, structural cells may comprise decellularized structural cells comprising the cell wall which previously contained one or more plant cells prior to decellularization.
In certain embodiments, the aerogels, foams, hydrogels, and other such materials as described herein may comprise cell wall architectures and/or vascular structures found in the plant and/or fungus kingdoms to create 3D scaffolds which may promote cell infiltration, cell growth, bone tissue repair, bone reconstruction, regenerative therapy, spinal cord repair, etc. As will be understood, biomaterials as described herein may be produced from any suitable part of plant or fungal organisms. Biomaterials may comprise, for example, one or more substances such as cellulose, chitin, lignin, lignan, hemicellulose, pectin, lignocellulose, and/or any other suitable biochemicals/biopolymers which are naturally found in these organisms.
As will be understood, unless otherwise indicated, the meaning/definition of plant and fungi kingdoms used herein is based on the Cavalier-Smith classification (1998).
In certain embodiments, the plant or fungal tissue may comprise generally any suitable plant or fungal tissue or part appropriate for the particular application. In certain embodiments, the plant or fungal tissue may comprise an apple hypanthium (Malus pumila) tissue, a fern (Monilophytes) tissue, a turnip (Brassica rapa) root tissue, a gingko branch tissue, a horsetail (equisetum) tissue, a hermocallis hybrid leaf tissue, a kale (Brassica oleracea) stem tissue, a conifers Douglas Fir (Pseudotsuga menziesii) tissue, a cactus fruit (pitaya) flesh tissue, a Maculata Vinca tissue, an Aquatic Lotus (Nelumbo nucifera) tissue, a Tulip (Tulipa gesneriana) petal tissue, a Plantain (Musa paradisiaca) tissue, a broccoli (Brassica oleracea) stem tissue, a maple leaf (Acer psuedoplatanus) stem tissue, a beet (Beta vulgaris) primary root tissue, a green onion (Allium cepa) tissue, a orchid (Orchidaceae) tissue, turnip (Brassica rapa) stem tissue, a leek (Allium ampeloprasum) tissue, a maple (Acer) tree branch tissue, a celery (Apium graveolens) tissue, a green onion (Allium cepa) stem tissue, a pine tissue, an aloe vera tissue, a watermelon (Citrullus lanatus var. lanatus) tissue, a Creeping Jenny (Lysimachia nummularia) tissue, a cactae tissue, a Lychnis Alpina tissue, rhubarb (Rheum rhabarbarum) tissue, a pumpkin flesh (Cucurbita pepo) tissue, a Dracena (Asparagaceae) stem tissue, a Spiderwort (Tradescantia virginiana) stem tissue, an Asparagus (Asparagus officinalis) stem tissue, a mushroom (Fungi) tissue, a fennel (Foeniculum vulgare) tissue, a rose (Rosa) tissue, a carrot (Daucus carota) tissue, or a pear (Pomaceous) tissue. Additional examples of plant and fungal tissues are described in Example 18 of WO2017/136950, entitled “Decellularised Cell Wall Structures from Plants and Fungus and Use Thereof as Scaffold Materials”, herein incorporated by reference in its entirety.
In certain embodiments, the decellularized plant or fungal tissue may be cellulose-based, chitin-based, chitosan-based, lignin-based, lignan-based, hemicellulose-based, or pectin-based, or any combination thereof. In certain embodiments, the plant or fungal tissue may comprise a tissue from apple hypanthium (Malus pumila) tissue, a fern (Monilophytes) tissue, a turnip (Brassica rapa) root tissue, a gingko branch tissue, a horsetail (equisetum) tissue, a hermocallis hybrid leaf tissue, a kale (Brassica oleracea) stem tissue, a conifers Douglas Fir (Pseudotsuga menziesii) tissue, a cactus fruit (pitaya) flesh tissue, a Maculata Vinca tissue, an Aquatic Lotus (Nelumbo nucifera) tissue, a Tulip (Tulipa gesneriana) petal tissue, a Plantain (Musa paradisiaca) tissue, a broccoli (Brassica oleracea) stem tissue, a maple leaf (Acer psuedoplatanus) stem tissue, a beet (Beta vulgaris) primary root tissue, a green onion (Allium cepa) tissue, a orchid (Orchidaceae) tissue, turnip (Brassica rapa) stem tissue, a leek (Allium ampeloprasum) tissue, a maple (Acer) tree branch tissue, a celery (Apium graveolens) tissue, a green onion (Allium cepa) stem tissue, a pine tissue, an aloe vera tissue, a watermelon (Citrullus lanatus var. lanatus) tissue, a Creeping Jenny (Lysimachia nummularia) tissue, a cactae tissue, a Lychnis Alpina tissue, a rhubarb (Rheum rhabarbarum) tissue, a pumpkin flesh (Cucurbita pepo) tissue, a Dracena (Asparagaceae) stem tissue, a Spiderwort (Tradescantia virginiana) stem tissue, an Asparagus (Asparagus officinalis) stem tissue, a mushroom (Fungi) tissue, a fennel (Foeniculum vulgare) tissue, a rose (Rosa) tissue, a carrot (Daucus carota) tissue, or a pear (Pomaceous) tissue, or a genetically altered tissue produced via direct genome modification or through selective breeding, or any combinations thereof.
As will also be understood, cellular materials and nucleic acids of the plant or fungal tissue may include intracellular contents such as cellular organelles (e.g. chloroplasts, mitochondria), cellular nuclei, cellular nucleic acids, and/or cellular proteins. These may be substantially removed, partially removed, or fully removed from the plant or fungal tissue, and/or from the structural cells. It will recognized that trace amounts of such components may still be present in the decellularised plant or fungal tissues and/or structural cells as described herein. As will also be understood, references to decellularized plant or fungal tissue herein are intended to reflect that such cellular materials found in the plant or fungal source of the tissues have been substantially removed—this does not preclude the possibility that the decellularized plant or fungal tissue or structural cells may in certain embodiments contain or comprise subsequently introduced, or reintroduced, cells, cellular materials, and/or nucleic acids of generally any kind, such as animal or human cells, such as bone or bone progenitor cells/tissues.
Various methods may be used for producing decellularized plant or fungal tissue as described herein. By way of example, in certain embodiments, the decellularised plant or fungal tissue may comprise plant or fungal tissue(s) which have been decellularised by thermal shock, treatment with detergent (e.g. SDS, Triton X, EDA, alkaline treatment, acid, ionic detergent, non-ionic detergents, and zwitterionic detergents), osmotic shock, lyophilisation, physical lysing (e.g. hydrostatic pressure), electrical disruption (e.g. non thermal irreversible electroporation), or enzymatic digestion, or any combination thereof. In certain embodiments, biomaterials as described herein may be obtained from plants and/or fungi by employing decellularization processes which may comprise any of several approaches (either individually or in combination) including, but not limited to, thermal shock (for example, rapid freeze thaw), chemical treatment (for example, detergents), osmotic shock (for example, distilled water), lyophilisation, physical lysing (for example, pressure treatment), electrical disruption and/or enzymatic digestion.
In certain embodiments, the decellularised plant or fungal tissue may comprise plant or fungal tissue which has been decellularised by treatment with a detergent or surfactant. Examples of detergents may include, but are not limited to sodium dodecyl sulphate (SDS), Triton X, EDA, alkyline treatment, acid, ionic detergent, non-ionic detergents, and zwitterionic detergents. In preferred embodiments, the plant or fungal tissue may be decellularized using SDS and CaCl2).
In still further embodiments, the decellularised plant or fungal tissue may comprise plant or fungal tissue which has been decellularised by treatment with SDS. In still another embodiment, residual SDS may be removed from the plant or fungal tissue by washing with an aqueous divalent salt solution. The aqueous divalent salt solution may be used to precipitate/crash a salt residue containing SDS micelles out of the solution/scaffold, and a dH2O, acetic acid or dimethylsulfoxide (DMSO) treatment, or sonication, may have been used to remove the salt residue or SDS micelles. In certain embodiments, the divalent salt of the aqueous divalent salt solution may comprise, for example, MgCl2 or CaCl2).
In another embodiment, the plant or fungal tissue may be decellularised by treatment with an SDS solution of between 0.01 to 10%, for example 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 another suitable organic solvent, and the residual SDS may have been removed using an aqueous CaCl2) solution at a concentration of about 100 mM followed by incubation in dH2O. In certain embodiments, the SDS solution may be at a higher concentration than 0.1%, which may facilitate decellularisation, and may be accompanied by increased washing to remove residual SDS. In particular embodiments, the plant or fungal tissue may be decellularised by treatment with an SDS solution of about 0.1% SDS in water, and the residual SDS may have been removed using an aqueous CaCl2) solution at a concentration of about 100 mM followed by incubation in dH2O.
Further examples of decellularization protocols which may be adapted for producing decellularized materials as described herein may be found in WO2017/136950, entitled “Decellularised Cell Wall Structures from Plants and Fungus and Use Thereof as Scaffold Materials”, herein incorporated by reference in its entirety.
In certain embodiments, aerogels, foams, and/or hydrogels as described herein may comprise the single structural cells, groups of structural cells, or both, distributed within a carrier. In certain embodiments, the carrier may be derived from a dehydrated, lyophilized, or freeze-dried hydrogel. As will be understood, the carrier may comprise generally any suitable carrier material, structure, or matrix, for providing support and/or structure to the aerogel, foam, and/or hydrogel, and may be used to support, carry, join, or hold the single structural cells, groups of structural cells, or both of the aerogel, foam, and/or hydrogel. The person of skill in the art having regard to the teachings herein will be aware of a variety of different carriers which may be used, and which may be selected to suit the particular application(s) of interest. In certain embodiments, the carrier may comprise a hydrogel into which the single structural cells, groups of structural cells, or both, are mixed, or the carrier may be derived from a dehydrated, lyophilized, or freeze-dried hydrogel, within which the single structural cells, groups of structural cells, or both are distributed/mixed. A variety of different hydrogels may be used for providing the carrier, such as but not limited to hydrogel(s) comprising any one or more of alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, agar, pluronic acid, triblock PEO-PPO-PEO copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), dissolved or regenerated plant cellulose, dissolved cellulose-based hydrogel, hyaluronic acid, extracellular matrix proteins (e.g. collagen, gelatin, or fibronectin, or any combinations thereof), monoacrylated poly(ethylene glycol), poly(ethylene glycol) diacrylate, poly(ethylene glycol) diacrylate (PEGDA)-co-PEGMA, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(lactic-co-glycolic acid), chitosan, chitin, xanthan gum, elastin, fibrin, fibrinogen, cellulose derivatives, carrageenan, or microcrystalline cellulose, or any combinations thereof. In certain embodiments, the hydrogel/carrier may optionally be cross-linked.
In certain embodiments of any of the aerogel or aerogels or foam or foams described herein, the hydrogel may comprise alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, agar, pluronic acid, triblock PEO-PPO-PEO copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), dissolved or regenerated plant cellulose, dissolved cellulose-based hydrogel, hyaluronic acid, extracellular matrix proteins (e.g. collagen, gelatin, or fibronectin, or any combinations thereof), monoacrylated poly(ethylene glycol), poly(ethylene glycol) diacrylate, poly(ethylene glycol) diacrylate (PEGDA)-co-PEGMA, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(lactic-co-glycolic acid), chitosan, chitin, xanthan gum, elastin, fibrin, fibrinogen, cellulose derivatives, carrageenan, or microcrystalline cellulose, or any combinations thereof; wherein the hydrogel is optionally cross-linked.
In certain embodiments of the aerogels or foams described herein, the aerogel or foam may be rehydrated, optionally with water, an aqueous solution, a buffer, a cell buffer, an alcohol (such as ethanol), or another aqueous or non-aqueous liquid or solution suitable of the application(s) of interest. Alternatively, the aerogels or foams may be provided in dry form
In preferred embodiments of the aerogel or aerogels or foam or foams described herein, the single structural cells, groups of structural cells, or both, may be derived from the plant or fungal tissue, preferably decellularized plant or fungal tissue, by mercerization. Other approaches for obtaining single structural cells, groups of structural cells, or both, such as maceration or other liquid-based extractions, are also contemplated; however, as described herein mercerization is preferred.
As will be understood, mercerization may comprise any suitable process for treating plant or fungal tissue (preferably, decellularized plant or fungal tissue) to obtain single structural cells, groups of structural cells, or both, typically using a liquid extraction solution employing base and preferably further employing a peroxide. Typically, mercerization of the plant or fungal tissue (preferably decellularized plant or fungal tissue), disassembles the plant or fungal tissue into tissue/cellular components (including single structural cells, groups of structural cells, or both). In certain embodiments, the mercerization may employ an alkaline/base solution and a peroxide. In certain embodiments, more than one treatment or solution may be used, either simultaneously or sequentially.
In certain embodiments, mercerization may comprise at least one treatment with a base solution. As will be understood, the base solution may comprise generally any suitable base, such as any suitable base capable of osmotic shock and/or disruption of hydrogen bonding and/or polymer crystal structure so as to extract intact tissue structures. As will be understood, particularly for food and/or medical applications, the base may be selected to be appropriate for the particular application and may, for example, be selected to be physiologically occurring, easily washed away, non-harmful, and/or selected accordingly to a variety of factors relevant to the particular application, as desired. In certain embodiments, the base may comprise NaOH, KOH, or a combination thereof. In an embodiment, the base may be dissolved/mixed in a suitable solvent, to form the base solution. Typically, the solvent may comprise water, although other solvents, or combinations of solvents (such as, for example, a mixture of water and ethanol), are also contemplated. The base concentration in the base solution may be tailored to suit the particular application of interest. Typically, the base solution may comprise a base concentration of about 0.1 to 10M, or any concentration therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these concentrations. In certain embodiments, the base concentration may be about 0.5M to 3M, or any value (optionally rounded to the nearest 0.1) therebetween, or any subrange spanning between any two of these concentrations. By way of example, in certain embodiments, the base solution may comprise an aqueous solution of NaOH, having a concentration of about 0.5M-3M. As will be understood, the base solution, as well as the treatment conditions (i.e. heating, stirring) may be tailored to suit the particular application, desired structures to be extracted, plant or fungal tissue being used, etc. . . . , as desired.
In certain embodiments, bases may include a base selected from the group consisting of: Carbonates; Nitrates; Phosphates; Sulfates; Ammonia; Sodium hydroxide; Calcium hydroxide; Magnesium hydroxide; Potassium hydroxide; Lithium hydroxide; Zinc hydroxide; Sodium carbonate; Sodium bicarbonate; Butyl lithium; Sodium azide; Sodium amide; Sodium hydride; Sodium borohydride; or Lithium diisopropylamine. Depending on the base and/or intended use of the products, neutralization and/or washing may be performed to remove residual base and other reagents so as to prevent undesirable contamination, for example.
In preferred embodiments, the mercerization may comprise treatment of the plant or fungal tissue (preferably decellularized plant or fungal tissue) using sodium hydroxide and hydrogen peroxide with heating.
In yet another embodiment of any of the aerogel or aerogels or foam or foams described herein, the aerogel or foam may comprise a particle size distribution of the single structural cells with an average feret diameter within a range of about 1 μm to about 1000 μm, such as about 100 to about 500 μm, for example about 100 to about 300 μm.
In yet another embodiment of any of the aerogel or aerogels or foam or foams described herein, the plant tissue may comprise apple tissue or pear tissue.
In another embodiment of any of the aerogel or aerogels or foam or foams described herein, the aerogel or foam may comprise about 5% to about 95% m/m, such as about 10-50% m/m (or more), single structural cells, groups of structural cells, or both, when the aerogel or foam is in hydrated form.
In still another embodiment of any of the aerogel or aerogels or foam or foams described herein, the hydrogel may comprise alginate, pectin, or both, and the aerogel or foam may be rehydrated with a CaCl2) solution, providing cross-linking.
In yet another embodiment of any of the aerogel or aerogels or foam or foams described herein, the aerogel or foam may have bulk modulus within a range of about 0.1 to about 500 kPa, such as about 1 to about 200 kPa.
In another embodiment of any of the aerogel or aerogels or foam or foams described herein, the aerogel or foam may be rehydrated and may further comprise one or more animal cells.
In another embodiment of any of the aerogel or aerogels or foam or foams described herein, the aerogel or foam may be rehydrated and may further comprise any one or more cells selected from fibroblasts, myofibroblasts, neurons, dorsal root ganglion cells, neuronal structures such as axons, neural precursor cells, neural stem cells, glial cells, endogenous stem cells, neutrophils, mesenchymal stem cells, satellite cells, myoblasts, myotubes, muscle progenitor cells, adipocytes, preadipocytes, chondrocytes, osteoblasts, osteoclasts, pre-osteoblasts, tendon progenitor cells, tenocytes, periodontal ligament stem cells, endothelial cells, or any combinations thereof. Cells may be selected to suit the particular application(s) of interest. In certain embodiments, where food product applications are of interest, the one or more cells may comprise muscle cells, fat cells, connective tissue cells (i.e. fibroblasts), cartilage, bone, epithelial, or endothelial cells, or any combinations thereof, for example.
In still another embodiment of any of the aerogel or aerogels or foam or foams described herein, at least some cellulose and/or cellulose derivative(s) of the aerogel or foam may be cross-linked by physical cross-linking (e.g. using glycine) and/or chemical cross-linking (e.g. using citric acid in the presence of heat); wherein at least some cellulose and/or cellulose derivative(s) of the aerogel or foam is functionalized with a linker (e.g. succinic acid) to which one or more functional moieties are optionally attached (e.g. amine-containing groups, wherein cross-linking may further optionally be achieved with one or more protein cross-linkers such as formalin, formaldehyde, glutaraldehyde, and/or transglutaminase); or any combinations thereof. In certain embodiments, it is contemplated that cross-linking may impart additional structural integrity to the aerogels, foams, and/or hydrogels as described herein, and the degree of cross-linking may be controlled to adjust physical properties of the resultant products. In certain embodiments, cellulose or cellulose derivatives or other materials of the structural cells of the aerogels or foams may be cross-linked; materials of the carrier (typically derived from a hydrogel) may be cross-linked; or combinations thereof. Example 8 below provides illustrative examples of physical and chemical cross-linking approaches, including those employing linkers. The person of skill in the art having the benefit of the teachings herein, and taking into consideration the structural cells and carrier/hydrogel being used in the particular aerogel/foam, will be aware of suitable approaches for achieving cross-linking.
In certain embodiments, the carrier of the aerogel or aerogels or foam or foams as described herein may, or may not, be cross-linked. In certain embodiments, the carrier may be cross-linked before dehydrating, lyophilizing, or freeze-drying; after dehydrating, lyophilizing, or freeze-drying; or both. In embodiments in which the carrier is cross-linked, the carrier may typically be cross-linked after mixing or distribution of the single structural cells, groups of structural cells, or both therein, and before or after dehydrating, lyophilizing, or freeze-drying of the mixture.
In still another embodiment of any of the aerogel or aerogels or foam or foams as described herein, the aerogel or foam may comprise templated or aligned microchannels created by directional freezing; by molding using molds having microscale and/or macroscale features; by punching, pressing, stamping, or otherwise forming geometric patterns, depressions, holes, channels, grooves, ridges, wells, or other structural features within and/or onto at least one surface; or any combinations thereof.
Approaches for directional freezing are described in detail in the Examples below. Briefly, by creating a larger thermal gradient on one side of a hydrogel, linear and highly aligned ice crystals may form from the cold side. This may force the surrounding hydrogel polymers to form around the ice crystals, creating aligned microscale channels. After lyophilizing the resulting material, a scaffold may be created with many microchannels. As will be understood, directional freezing may be used to template or form channels and/or other structural features interior to the aerogels and/or foams, on the surface of the aerogels and/or foams, or both.
In still another embodiment of any of the above method or methods, a microarchitecture of the microchannels produced from directional freezing may be controlled by creating the mixture including a solvent containing varying amounts of one or more other dissolved compounds such as Sucrose, Dextrose, Trehalose, Corn Starch, Glycerol, Ethanol, Mannitol, Sodium chloride, CaCl2, Gelatine, Citric acid, PVA, PEG, Dextran, NaF, NaBr, NaI, phosphate buffer, another sugar or salt, or another such agent, which may alter the structural properties of aligned ice crystals which grow from the cold side of the thermal gradient.
In certain embodiments, directional freezing (also referred to as freeze casting, ice-templating) may comprise a process through which well-controlled microscale features (channels, pores, etc) may be created in an aerogel comprising a hydrogel, polymer, biomacromolecules etc. In certain embodiments, the process may typically involve the controlled solidification of an aqueous solution, suspension or sol-gel followed by sublimation in a lyophilizer. The solution which undergoes controlled freezing may typically be placed on a cold plate which creates a non-uniform thermal gradient which typically starts on one side. Ice crystals form from the cold side and grow linearly away from the cold surface. As the ice crystals grow, they displace the solution components (polymer, hydrogel, colloids, single structural cells, etc.) and they collect between the growing ice crystals. After complete freezing, sublimation in a lyophilizer may typically be performed which may remove the ice crystals leaving behind an aerogel or foam with anisotropic templated nano to microscale features, such as aligned channels. The final structural properties of the features may therefore be dependent on the structure of the ice crystals which form in the solution. Therefore, other solutes which will impact ice crystallization may allow for control over the final architecture of the aerogel. In such scenarios it is contemplated that dissolving other salts, lipids, sugars and/or other additives into the aqueous solution may impact ice crystal formation. In certain embodiments, such compounds may include any of the following, alone or in combination: Sucrose, dextrose Trehalose, Corn Starch, Glycerol, Ethanol, Mannitol, Sodium chloride, CaCl2, Gelatine, Citric acid, PVA, PEG, Dextran, NaF, NaBr, NaI, phosphate buffer, etc. In addition to additives in the solution, it is also contemplated that in certain embodiments temperature and freezing rate may also impact ice crystal geometry. Temperatures from −195° C. to 0° C. may be used in certain embodiments, with operating temperatures between −30° C. to −10° C. being more typically employed to create the aligned, directionally frozen scaffolds in certain embodiments.
Molding of the aerogels, foams, and hydrogels as described herein is also described in detail in the Examples below. In certain embodiments, aerogel/foam/hydrogel precursor mixtures may be introduced into containers or molds, and subsequently dehydrated, lyophilized, or freeze-dried. In preferred embodiments, the aerogel/foam/hydrogel precursor mixture may be frozen within the container or mold prior to the dehydrating, lyophilisation, or freeze-drying. In certain embodiments, the mold or container may be designed so as to provide an aerogel/foam/hydrogel having a desired shape and/or size. In certain embodiments, the container or mold may be designed to present microscale and/or macroscale features to the surface of the aerogel/foam/hydrogel contained therein (for example, the mold may have structural features such as projections/depressions on it's interior walls to form structural on the surface and/or internal to the aerogels and/or foams), and/or may be designed to project microscale and/or macroscale features into the aerogel/foam/hydrogel contained therein, so as to impart desired structure to the aerogel/foam/hydrogel by molding. Examples of microscale and/or macroscale features may include geometric patterns, channels, depressions, tunnels, or holes, or any other microscale and/or macroscale features desired or suitable for the particular application(s) of interest.
In certain embodiments, macroscale and/or microscale structural features may be imparted to the aerogels/foams/hydrogels by mechanical processing such as by punching, pressing, stamping, drilling, or otherwise forming geometric patterns, depressions, holes, channels, grooves, ridges, wells, or other structural features within and/or onto at least one surface of the aerogels/foams/hydrogels as desired. In some embodiments, it is contemplated that such mechanical processing may be computer-guided (for example, by numerical control) using automated machinery, for example. Mechanical processing may be performed before, during, and/or after freezing and/or lyophilisation or freeze-drying, for example.
In yet another embodiment, there is provided herein single structural cells, groups of structural cells, or both, derived from a decellularized plant or fungal tissue by mercerization of the decellularized plant or fungal tissue, the single structural cells or groups of structural cells having a decellularized 3-dimensional structure lacking cellular materials and nucleic acids of plant or fungal tissue, and lacking one or more base-soluble lignin components of the plant or fungal tissue. As will be understood, mercization with base (such as NaOH, and optionally additionally in the presence of hydrogen peroxide, for example) may remove one or more base-soluble lignin components, however as shown in the Examples below overall 3-dimensional structure of the resultant single structural cells, groups of structural cells, or both, derived from the decellularized plant or fungal tissue may remain substantially intact. Resultant structural cells from mercerization may differ from structural cells obtained in an alternative manner such as an acid-employing maceration approach, which may remove certain acid-soluble lignin components rather than base-soluble lignin components and therefore provide structural cells having different lignin content, for example. In certain embodiments, the single structural cells, groups of structural cells, or both, may be provided in dried form, or suspended in an aqueous or non-aqueous liquid or solution such as, but not limited to, water, an aqueous buffer, or ethanol.
In certain embodiments, any of the aerogel, aerogels, foam, or foams as described herein may additionally comprise one or more cells cultured or located therein/thereon. In certain embodiments, the one or more cells may comprise any one or more of muscle cells, fat cells, connective tissue cells, fibroblasts, myofibroblasts, neurons, dorsal root ganglion cells, neuronal structures such as axons, neural precursor cells, neural stem cells, glial cells, endogenous stem cells, neutrophils, mesenchymal stem cells, satellite cells, myoblasts, myotubes, muscle progenitor cells, adipocytes, preadipocytes, cartilage cells, bone cells, epithelial cells, endothelial cells, chondrocytes, osteoblasts, osteoclasts, pre-osteoblasts, tendon progenitor cells, tenocytes, periodontal ligament stem cells, or endothelial cells, or any combinations thereof.
In certain embodiments, aerogels and foams as described herein may be generally biocompatible. By way of example, in certain embodiments it is contemplated that aerogels and foams as described herein may be compatible with a variety of different cell types relevant for tissue engineering and/or food applications, and may be biocompatible with cells from many different species and kingdoms including, but not limited to, human, rodent (e.g. mouse, rat, guinea pig), lagomorpha (e.g. rabbit, hare), carpine (goat), ovine (e.g. Sheep, lamb, mutton), porcine (e.g. pig, hog, boar), bovine (e.g. cow, bison, buffalo), feline, canine, fish (e.g. salmon, tuna, snapper, mackerel, cod, trout, carp, catfish and sardines), mullosca, crustacean (e.g. crabs, lobsters, crayfish, shrimps, prawns), avian (e.g. chicken, turkey, duck), reptile, amphibian, insect, and/or plant species. As will be understood, cells may be selected based on the particular application(s) of interest, which may include, but are not limited to, therapeutic (human or veterinary), food, or other such applications.
In yet another embodiment, there is provided herein a method for preparing an aerogel or foam, comprising:
Aerogels, foams, plant or fungal tissue, decellularization, structural cells and groups of structural cells, and hydrogels have already been described in detail hereinabove.
As will be understood, single structural cells, groups of structural cells, or both, may be obtained from plant or fungal tissue (preferably from decellularized plant or fungal tissue) by performing mercerization. Mercerization may comprise any suitable process for treating plant or fungal tissue (preferably, decellularized plant or fungal tissue) to obtain single structural cells, groups of structural cells, or both, typically using a liquid extraction solution employing base and preferably further employing a peroxide. Typically, mercerization of the plant or fungal tissue (preferably decellularized plant or fungal tissue), disassembles the plant or fungal tissue into tissue/cellular components (including single structural cells, groups of structural cells, or both). In certain embodiments, the mercerization may employ an alkaline/base solution and a peroxide. In certain embodiments, more than one treatment or solution may be used, either simultaneously or sequentially. In certain embodiments it is contemplated that mercerization may be performed on plant or fungal tissue and decellularization may be performed afterwards, or mercerization may be performed on plant or fungal tissue and mercerization conditions may be selected so as to simultaneously provide decellularization. However, as described herein, it is preferred that mercerization be performed on plant or fungal tissue that has already previously been decellularized.
In certain embodiments, mercerization may comprise at least one treatment with a base solution.
As will be understood, the base solution may comprise generally any suitable base, such as any suitable base capable of osmotic shock and/or disruption of hydrogen bonding and/or polymer crystal structure so as to extract intact tissue structures. As will be understood, particularly for food and/or medical applications, the base may be selected to be appropriate for the particular application and may, for example, be selected to be physiologically occurring, easily washed away, non-harmful, and/or selected accordingly to a variety of factors relevant to the particular application, as desired. In certain embodiments, the base may comprise NaOH, KOH, or a combination thereof. In an embodiment, the base may be dissolved/mixed in a suitable solvent, to form the base solution. Typically, the solvent may comprise water, although other solvents, or combinations of solvents (such as, for example, a mixture of water and ethanol), are also contemplated. The base concentration in the base solution may be tailored to suit the particular application of interest. Typically, the base solution may comprise a base concentration of about 0.1 to 10M, or any concentration therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these concentrations. In certain embodiments, the base concentration may be about 0.5M to 3M, or any value (optionally rounded to the nearest 0.1) therebetween, or any subrange spanning between any two of these concentrations. By way of example, in certain embodiments, the base solution may comprise an aqueous solution of NaOH, having a concentration of about 0.5M-3M. As will be understood, the base solution, as well as the treatment conditions (i.e. heating, stirring) may be tailored to suit the particular application, desired structures to be extracted, plant or fungal tissue being used, etc. . . . , as desired.
In certain embodiments, bases may include a base selected from the group consisting of: Carbonates; Nitrates; Phosphates; Sulfates; Ammonia; Sodium hydroxide; Calcium hydroxide; Magnesium hydroxide; Potassium hydroxide; Lithium hydroxide; Zinc hydroxide; Sodium carbonate; Sodium bicarbonate; Butyl lithium; Sodium azide; Sodium amide; Sodium hydride; Sodium borohydride; or Lithium diisopropylamine. Depending on the base and/or intended use of the products, neutralization and/or washing may be performed to remove residual base and other reagents so as to prevent undesirable contamination, for example.
In preferred embodiments, the mercerization may comprise treatment of the plant or fungal tissue (preferably decellularized plant or fungal tissue) using sodium hydroxide and hydrogen peroxide with heating.
The single structural cells or groups of structural cells (having a decellularized 3-dimensional structure) resulting from mercerization may be collected. The resultant single structural cells or groups of structural cells may be provided in dried form, or as a paste or gel, or in another suitable form as desired.
Mercerization processes in other industries, such as in the pulp and paper industry, strip down to cellulose polymers/fibres (i.e. complete destruction of plant structures). In contrast, mercerization processes as described herein (regardless of whether the plant or fungal tissue is decellularized before, during, or after the mercerization) may provide for retention of intact single structural cells or groups of structural cells with 3-dimensional structure. While mercerization may be performed before decellularization of the plant or fungal tissue, this was not preferred as it is expected to take longer, be less efficient, and may result in a less pure resultant material to be decellularized. Accordingly, mercerization of already decellularized plant or fungal tissue is preferred. Mercerization processes as described herein may be used to obtain decellularized but intact single structural cells and/or plant tissue structures of interest (e.g. parenchyma tissue, ground tissue, epidermal tissue, vascular bundles, sieve tubes, petioles, veins, roots, root hairs, etc. . . . ) as desired to suit the particular application(s) of interest.
In embodiments of the methods as described herein, the single structural cells or groups of structural cells (having a decellularized 3-dimensional structure) resulting from mercerization may be mixed or distributed in a hydrogel, to provide a mixture.
In certain embodiments, the hydrogel into which the single structural cells, groups of structural cells, or both, are mixed or distributed may comprise any one or more of alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, agar, pluronic acid, triblock PEO-PPO-PEO copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), dissolved or regenerated plant cellulose, dissolved cellulose-based hydrogel, hyaluronic acid, extracellular matrix proteins (e.g. collagen, gelatin, or fibronectin, or any combinations thereof), monoacrylated poly(ethylene glycol), poly(ethylene glycol) diacrylate, poly(ethylene glycol) diacrylate (PEGDA)-co-PEGMA, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(lactic-co-glycolic acid), chitosan, chitin, xanthan gum, elastin, fibrin, fibrinogen, cellulose derivatives, carrageenan, or microcrystalline cellulose hydrogel(s), or any combinations thereof. In certain embodiments, the hydrogel/carrier may optionally be cross-linked.
In further embodiments of the methods as described herein, the mixture of the single structural cells, groups of structural cells, or both, and the hydrogel, may be dehydrated, lyophilized, or freeze-dried to provide the aerogel or foam. The person of skill in the art having the benefit of the teachings set out herein, including the Examples provided below, will be aware of a variety of suitable techniques and apparatus for dehydrating, lyophilizing, or freeze-drying, or otherwise drying or removing at least some liquid/solvent from the mixture, to provide aerogels and/or foams as described herein.
In certain embodiments of the above method, the mercerization may comprise treatment of the decellularized plant or fungal tissue with a base and a peroxide, preferably sodium hydroxide or another hydroxide base as base, and hydrogen peroxide as peroxide. Mercerization may chemically disassemble the decellularized plant or fungal tissue into single structural cells, groups of structural cells, or both, without destroying lignocellulose structures contributing to the 3-dimensional structure of the single structural cells.
In still another embodiment of any of the above method or methods, the mercerization may comprise treatment of the decellularized plant or fungal tissue with aqueous sodium hydroxide solution and hydrogen peroxide while heating.
In still another embodiment of any of the above method or methods, the mercerization may be performed with heating to about 80° C. In certain embodiments, such heating may allow for reduced reaction time, particularly when using sodium hydroxide, for example.
In yet another embodiment of any of the above method or methods, the decellularized plant or fungal tissue may be treated with the aqueous sodium hydroxide solution for a first period of time before the hydrogen peroxide is added to the reaction. In certain embodiments, the first period of time may be about 1 minute to about 24 hours, or any time point therebetween, or any subrange spanning between any two such time points.
In certain embodiments, the peroxide may be added to the reaction in intervals, such as about 15 minute intervals. An illustrative non-limiting example of such peroxide interval approaches may proceed as follows:
In another embodiment of any of the above method or methods, the hydrogen peroxide may be added as a 30% aqueous hydrogen peroxide solution.
In still another embodiment of any of the above method or methods, the hydrogen peroxide for mercerization may be used in a ratio of:
As will be understood, these ratios may be scaled up or down to suit the particular application as desired—the recited quantities are provided for showing relative ratios, not absolute quantities.
In yet another embodiment of any of the above method or methods, the method may further comprise a step of neutralizing the pH with one or more neutralization treatments. In another embodiment of any of the above method or methods, the neutralization treatment may comprise treatment with an acid solution, preferably an aqueous HCl solution.
In yet another embodiment of any of the above method or methods, the mercerization may be performed using a ratio of decellularized plant or fungal tissue:aqueous sodium hydroxide solution (m:v, in g:L) of about 1:5 for a 1M aqueous sodium hydroxide solution, or an equivalent ratio for another aqueous sodium hydroxide solution concentration. As will be understood, these ratios may be scaled up or down to suit the particular application as desired—the recited quantities are provided for showing relative ratios, not absolute quantities.
In another embodiment of any of the above method or methods, the mercerization may be performed for at least about 30 minutes, preferably for about 1 hour.
In still another embodiment of any of the above method or methods, the resultant single structural cells or groups of structural cells having a decellularized 3-dimensional structure may be collected by centrifugation.
In yet another embodiment of any of the above method or methods, the single structural cells, groups of structural cells, or both, may be mixed or distributed in a hydrogel comprising alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, agar, pluronic acid, triblock PEO-PPO-PEO copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), dissolved or regenerated plant cellulose, dissolved cellulose-based hydrogel, hyaluronic acid, extracellular matrix proteins (e.g. collagen, gelatin, or fibronectin, or any combinations thereof), monoacrylated poly(ethylene glycol), poly(ethylene glycol) diacrylate, poly(ethylene glycol) diacrylate (PEGDA)-co-PEGMA, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(lactic-co-glycolic acid), chitosan, chitin, xanthan gum, elastin, fibrin, fibrinogen, cellulose derivatives, carrageenan, or microcrystalline cellulose hydrogel, or any combinations thereof; wherein the hydrogel is optionally cross-linked.
In another embodiment of any of the above method or methods, the method may further comprise a step of performing directional freezing of the mixture to introduce templated or aligned microchannels on a surface of the mixture, within the mixture, or both; a step of molding the mixture using molds having microscale features contacting one or more surfaces of the mixture and/or the aerogel or foam resulting from dehydrating, lyophilizing, or freeze-drying of the mixture, so as to introduce templated or aligned microchannels; a step of punching, pressing, stamping, or otherwise forming geometric patterns, depressions, holes, channels, grooves, ridges, wells, or other structural features within and/or onto at least one surface of the mixture and/or the aerogel or foam before, during, or after dehydrating, lyophilizing, or freeze-drying of the mixture; or any combinations thereof.
Directional freezing, molding, and mechanically processing for imparting microscale and/or macroscale structures to the aerogels, foams, and/or hydrogels have already been described in detail hereinabove, and are further described in the Examples set out below.
In still another embodiment of any of the above method or methods, the directional freezing may be performed by creating a thermal gradient across the mixture from one or more directions so as to form aligned ice crystals beginning from the cold side(s) of the thermal gradient.
In yet another embodiment of any of the above method or methods, the mixture may be directionally frozen over a period of at least about 30 minutes, preferably over a period of about 2 hours.
In another embodiment of any of the above method or methods, the mixture may be directionally frozen by cooling to a temperature of between about −190° C. and about 0° C., such as a temperature of at least about −15° C., preferably about −25° C.
In still another embodiment of any of the above method or methods, the step of dehydrating, lyophilizing, or freeze-drying the mixture to provide the aerogel or foam may comprise freezing the mixture followed by lyophilizing or freeze-drying the mixture.
In yet another embodiment of any of the above method or methods, the method may comprise a further step of cross-linking the hydrogel, rehydrating the aerogel or foam, or both; optionally using CaCl2) solution to provide cross-linking where alginate or pectin or agar hydrogel is present.
As will be understood, a variety of techniques may be used for cross-linking where desired, and may be selected based on the particular aerogel/foam/hydrogel and/or application(s) of interest. In certain embodiments, structural cells may be mixed with a single hydrogel, or a combination of hydrogels, and cross-linking may, or may not, be performed. In certain embodiments, the mixture may then be frozen, followed by lyophilisation or freeze drying to form an aerogel or foam.
In embodiments where the optional cross-linking is desired, an illustrative list of hydrogels, and a corresponding list of potential cross-linkers, is provided below for illustrative and non-limiting purposes for the person of skill in the art. In certain embodiments, such cross-linking approaches may be used if desired, but as will be understood cross-linking may be optional and the decision on whether or not to cross-link may be made to suit the application(s) of interest and specific details or demands thereof. In certain embodiments, combinations of hydrogels may be used, with or without extra crosslinking. Various illustrative examples are provided in the Examples below, particularly in Example 2.
In another embodiment of any of the above method or methods, the method may comprise a further step of culturing animal cells on or in the aerogel or foam. In certain embodiments, the method may comprise a further step of culturing muscle cells, fibroblasts, myofibroblasts, neurons, dorsal root ganglion cells, neuronal structures such as axons, neural precursor cells, neural stem cells, glial cells, endogenous stem cells, neutrophils, mesenchymal stem cells, satellite cells, myoblasts, myotubes, muscle progenitor cells, adipocytes, preadipocytes, chondrocytes, osteoblasts, osteoclasts, pre-osteoblasts, tendon progenitor cells, tenocytes, periodontal ligament stem cells, or endothelial cells, or any combinations thereof on or in the aerogel or foam.
In another embodiment, there is provided herein an aerogel or foam produced by any of the method or methods as described herein.
As will be understood, aerogels, foams, and/or hydrogels as described here may be configured and/or used for a wide variety of different applications. By way of non-limiting example, it is contemplated that in certain embodiments there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein for bone tissue engineering; for templating or aligning growth of cells; for regenerative medicine; for repair of spinal cord injury; for preparing a food product; or any combinations thereof.
In another embodiment, there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein for templating or aligning growth of cells. In certain embodiments, the cells may comprise muscle cells, nerve cells, or both.
In yet another embodiment, there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein for repair of spinal cord injury.
In another embodiment, there is provided herein a use of any of the aerogel or aerogels or foam or foams as described herein as insulation or packaging foam.
In still another embodiment, there is provided herein a method for bone tissue engineering or repair in a subject in need thereof, comprising:
In yet another embodiment, there is provided herein a method for templating or aligning growth of cells, comprising:
In another embodiment of the above method, the cells may comprise muscle cells or nerve cells or both.
In still another embodiment, there is provided herein a method for repairing spinal cord injury in a subject in need thereof, comprising:
In another embodiment, there is provided herein a food product comprising an aerogel or aerogels or foam of foams as described herein, the aerogel(s) or foam(s) being designed/selected so as to be food-safe and edible. In still another embodiment, the food product may additionally comprise a dye or coloring agent; a preservative; a flavoring agent; a salt; a marinade; or other food-related ingredient or agent of interest.
In yet another embodiment of any of the above food product or food products, the food product may comprise two or more aerogel or foam subunits glued together. In certain embodiments, the glue may comprise agar.
In certain embodiments, the food product may be designed or configured to mimic a traditional meat product. By way of example, tuna, salmon and similar fish are characterized by the lines found interspersed between the flakes of meat. These lines are due to the presence of fat (omega-3). Wild salmon typically have fewer and thinner white lines due to the fact that wild salmon typically burn more calories than farmed salmon. As well, their meat is redder from increased blood supply. Therefore, the presence of these white lines and their appearance, thickness, will depend on the desired look of the meat to be achieved. An illustrative and non-limiting example of a protocol to produce these lines in aerogel biomaterials as described herein may proceed as follows:
In still another embodiment of any of the food product or food products, the aerogel or foam may comprise templated or aligned microchannels optionally formed by directional freezing.
In certain embodiments, the aerogel or foam may comprise muscle cells, fibroblasts, myofibroblasts, neurons, dorsal root ganglion cells, neuronal structures such as axons, neural precursor cells, neural stem cells, glial cells, endogenous stem cells, neutrophils, mesenchymal stem cells, satellite cells, myoblasts, myotubes, muscle progenitor cells, adipocytes, preadipocytes, chondrocytes, osteoblasts, osteoclasts, pre-osteoblasts, tendon progenitor cells, tenocytes, periodontal ligament stem cells, or endothelial cells, or any combinations thereof, optionally aligned along templated or aligned microchannels; preferably wherein the aerogel or foam comprises templated or aligned microchannels optionally formed by directional freezing, and wherein the aerogel or foam comprises muscle cells, fat cells, connective tissue cells (e.g. fibroblasts), cartilage, bone, epithelial, or endothelial cells, or any combinations thereof, aligned along the templated or aligned microchannels.
In another embodiment, there is provided herein a use of an aerogel or aerogels or foam or foams as described herein in a food product, the aerogel(s) and/or foam(s) being designed/selected so as to be food-safe and edible.
In still another embodiment, there is provided herein a method for preparing single structural cells, groups of structural cells, or both, from decellularized plant or fungal tissue, comprising:
In yet another embodiment of the above method, the mercerization may comprise treatment of the decellularized plant or fungal tissue with a base and a peroxide, preferably sodium hydroxide or another hydroxide base as base, and hydrogen peroxide as peroxide.
In another embodiment of any of the above method or methods, the mercerization may comprise treatment of the decellularized plant or fungal tissue with aqueous sodium hydroxide solution and hydrogen peroxide while heating.
In still another embodiment of any of the above method or methods, the decellularized plant or fungal tissue may be treated with the aqueous sodium hydroxide solution for a first period of time before the hydrogen peroxide is added to the reaction.
In yet another embodiment of any of the above method or methods, the hydrogen peroxide may be added as a 30% aqueous hydrogen peroxide solution.
In still another embodiment of any of the above method or methods, the hydrogen peroxide for mercerization may be used in a ratio of:
In yet another embodiment of any of the above method or methods, the method may further comprise a step of neutralizing the pH with one or more neutralization treatments.
In another embodiment of any of the above method or methods, the neutralization treatment may comprise treatment with an acid solution, preferably an aqueous HCl solution.
In still another embodiment of any of the above method or methods, the mercerization may be performed with heating to about 80° C.
In yet another embodiment of any of the above method or methods, the mercerization may be performed using a ratio of decellularized plant or fungal tissue:aqueous sodium hydroxide solution (m:v, in g:L) of about 1:5 for a 1M aqueous sodium hydroxide solution, or an equivalent ratio for another aqueous sodium hydroxide solution concentration.
In still another embodiment of any of the above method or methods, the mercerization may be performed for at least about 30 minutes, preferably for about 1 hour.
In yet another embodiment of any of the above method or methods, the resultant single structural cells or groups of structural cells having a decellularized 3-dimensional structure may be collected by centrifugation.
In still another embodiment, there is provided herein single structural cells, groups of structural cells, or both, prepared by any of the method or methods as described herein.
Also provided herein are cellulose-based hydrogels that may have a variety of different applications. In certain embodiments, such cellulose-based hydrogels as described herein may be for use as hydrogel for preparing aerogels and/or foams as described herein, by way of non-limiting example.
In an embodiment, there is provided herein a cellulose-based hydrogel comprising cellulose derived from decellularized plant or fungal tissue via dissolution with dimethylacetamide and lithium chloride followed by regeneration with ethanol. Illustrative examples of such dissolution are described in further detail in Example 3 below.
In still another embodiment, there is provided herein a method for preparing a cellulose-based hydrogel comprising:
As will be understood, cellulose-based hydrogels may comprise a hydrogel containing one or more cellulose or cellulose derivatives. Typically, the cellulose and/or cellulose derivatives may be obtained by dissolution of the cellulose and/or cellulose derivatives from decellularized plant or fungal tissue. As will be understood, cellulose and/or cellulose derivatives may alternatively be obtained by dissolving plant of fungal tissue which has not been decellularized in certain embodiments, but as described herein dissolution of cellulose and/or cellulose derivatives from decellularized plant or fungal tissue is preferred. Preparation of decellularized plant or fungal tissue has already been described in detail hereinabove, and is further described in the Examples below.
Cellulose and/or cellulose derivatives of the decellularized plant or fungal tissue may be dissolved by treatment with DMAc and LiCl. Illustrative examples of such dissolution treatments are described in further detail in Example 3 below.
In another embodiment of the above methods, the solvent exchange with ethanol may be performed using a dialysis membrane, or by adding ethanol on top of the dissolved cellulose to promote solvent exchange.
In still another embodiment of any of the above method or methods, the method may further comprise bleaching the cellulose-based hydrogel with hydrogen peroxide.
In yet another embodiment, there is provided herein a cellulose-based hydrogel comprising cellulose derived from decellularized plant or fungal tissue via dissolution with: dimethylacetamide and lithium chloride, LiClO4, xanthate, EDA/KSCN, H3PO4, NaOH/urea, ZnCl2, TBAF/DMSO, NMMO, an ionic liquid (IL) (such as 1-butylpyridinium chloride and aluminum chloride, alkyl imidazolium in association with nitrate, preferably a room temperature ionic liquid), or any combinations thereof.
In still another embodiment, there is provided herein a method for preparing a cellulose-based hydrogel comprising:
Treatments for dissolving cellulose and/or cellulose derivatives of the decellularized plant or fungal tissue may be designed or selected to suit the particular application(s) of interest. Examples of agents that may be used for dissolving cellulose and/or cellulose derivatives of the decellularized plant or fungal tissue may include, but are not limited to, dimethylacetamide and lithium chloride, LiClO4, xanthate, EDA/KSCN, H3PO4, NaOH/urea, ZnCl2, TBAF/DMSO, NMMO, an ionic liquid (IL) (such as 1-butylpyridinium chloride and aluminum chloride, alkyl imidazolium in association with nitrate, preferably a room temperature ionic liquid), or any combinations thereof.
In another embodiment, there is provided herein a cellulose-based hydrogel prepared by any of the method or methods as described herein.
In another embodiment of any of the aerogel or aerogels or foam or foams as described herein, the hydrogel may comprise any of the cellulose-based hydrogel or cellulose-based hydrogels as described herein.
In still another embodiment, there is provided herein a food product comprising any of the aerogel or aerogels or foam or foams or structural cell or structural cells as described herein, wherein the food product is a meat mimic and comprises a plurality of lines providing the appearance of fatty white lines found in tuna, salmon, or another fish-type meat.
In yet another embodiment of the above food product, the food product may be a mimic of tuna, salmon, or another fish meat.
In still another embodiment, there is provided herein a food product comprising any of the aerogel or aerogels or foam or foams or structural cell or structural cells as described herein, wherein the food product is a meat mimic, and may optionally comprise a plurality of lines or other patterns providing the appearance of fatty materials or fatty deposits found in a natural meat.
In yet another embodiment of the above food product, the food product may be a mimic of a poultry, bovine, fish, or porcine meat, or any other suitable meat. In certain embodiments, the food product may mimic steak, chicken, pork, or another such meat, for example.
In another embodiment of any of the above food product or food products, the food product may contain one or more dyes or colorants providing the color of tuna, salmon, or another fish meat, or another meat.
In still another embodiment of any of the above food product or food products, the plurality of lines may be formed in cuts or channels formed in the aerogel or foam.
In yet another embodiment of any of the above food product or food products, the plurality of lines may comprise titanium dioxide, optionally combined with agar binding agent or another such binding agent.
In another embodiment of any of the above food product or food products, the titanium dioxide, optionally combined with agar binding agent, may be applied into cuts or channels formed in the aerogel or foam to provide the appearance of the fatty white lines found in tuna, salmon, or another fish-type meat, or another meat.
In another embodiment, there is provided herein a method for preparing a food product, the food product being a tuna, salmon, or other fish meat mimic, or another meat mimic, said method comprising:
In another embodiment of the above method, the dye or coloring agent applied to the cuts or channels may comprise titanium dioxide.
In still another embodiment of any of the above method or methods, the dye or coloring agent applied to the cuts or channels may be combined with a binding agent.
In yet another embodiment of any of the above method or methods, the binding agent may comprise agar.
In another embodiment, there is provided herein a food product prepared by any of the method or methods described herein.
In another embodiment, there is provided herein a non-resorbable dermal filler comprising an aerogel, foam, structural cell, or cellulose-based hydrogel as described herein; or any combinations thereof.
In yet another embodiment, there is provided herein a dermal filler comprising single structural cells, groups of structural cells, or both, derived from a plant or fungal tissue, the single structural cells or groups of structural cells having a decellularized 3-dimensional structure lacking cellular materials and nucleic acids of plant or fungal tissue, the single structural cells, groups of structural cells, or both, being derived from the plant or fungal tissue by mercerization.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may further comprise a carrier fluid or gel.
In still another embodiment of any of the above dermal filler or dermal fillers, the carrier fluid or gel may comprise water, an aqueous solution, or a hydrogel.
In yet another embodiment of any of the above dermal filler or dermal fillers, the carrier fluid or gel may comprise a saline solution, or a collagen, hyaluronic acid, methylcellulose, and/or dissolved plant-derived decellularized cellulose-based hydrogel.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may further comprise an anesthetic agent.
In still another embodiment of any of the above dermal filler or dermal fillers, the anesthetic agent may comprise lidocaine, benzocaine, tetracaine, polocaine, epinephrine, or any combinations thereof.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may comprise PBS (saline), hyaluronic acid (cross-linked or non-crosslinked), alginate, collagen, pluronic acid (e.g. pluronic F 127), agar, agarose, or fibrin, calcium hydroxylapatite, Poly-L-lactic acid, autologous fat, silicone, dextran, methylcellulose, or any combinations thereof.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may comprise at least one of: 2% lidocaine gel; a triple anesthetic gel comprising 20% benzocaine, 6% lidocaine, and 4% tetracaine (BLTgel); 3% Polocaine; or a mixture of 2% lidocaine with epinephrine.
In another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a size, diameter, or minimum feret diameter of at least about 20 μm.
In another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a size, diameter, or maximum feret diameter of less than about 1000 μm.
In still another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a size, diameter, or feret diameter distribution within a range of about 20 μm to about 1000 μm.
In yet another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a particle size, diameter, or feret diameter distribution having a peak about 200-300 μm.
In another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have a mean particle size, diameter, or feret diameter within a range of about 200 μm to about 300 μm.
In another embodiment of any of the above dermal filler or dermal fillers, the structural cells may have an average projected particle area within a range of about 30,000 to about 75,000 μm2.
In still another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may be sterilized.
In yet another embodiment of any of the above dermal filler or dermal fillers, the sterilization may be by gamma sterilization.
In still another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may be formulated for subdermal injection, deep dermal injection, subcutaneous injection (e.g. subcutaneous fat injection), or any combinations thereof.
In another embodiment of any of the above dermal filler or dermal fillers, the dermal filler may be provided in a syringe or injection device.
In another embodiment, there is provided herein a use of any of the dermal filler or dermal fillers as described herein as a soft tissue filler, for reconstructive surgery, or both.
In another embodiment, there is provided herein a use of any of the dermal filler or dermal fillers as described herein for improving cosmetic appearance of a subject in need thereof.
In another embodiment, there is provided herein a use of any of the dermal filler or dermal fillers as described herein for increasing tissue volume, smoothing wrinkles, or both, in a subject in need thereof.
In another embodiment, there is provided herein a method for improving cosmetic appearance, increasing tissue volume, smoothing wrinkles, or any combinations thereof, in a subject in need thereof, said method comprising:
In another embodiment of the above use or uses or method or methods, native cells of the subject may infiltrate the dermal filler.
In yet another embodiment of the above use or uses or method or methods, the dermal filler may be non-resorbable such that the decellularized plant or fungal tissue remains substantially intact within the subject.
Additional details relevant to plant-derived dermal fillers are described in US provisional patent application no. 63/036,126, entitled “Dermal Fillers”, which is herein incorporated by reference in its entirety.
Plant-derived scaffolds can provide desirable biological/physical properties (such as in vitro/in vivo biocompatibility), are readily producible, and can provide fixed mechanical/structural properties. However, many plant-derived scaffolds do not provide a significant level of control over parameters such as surface biochemistry, tuneable mechanical properties, tuneable micro/macro-scale architectures, and/or scalable production methods, for example.
This example describes the development of plant-derived scaffolds, with an emphasis toward providing one or more (or all) of the following characteristics: derived from decellularized plant materials; ability to retain desirable plant microarchitectures; amendable to scalable production method(s); ability to provide a wide range of scaffold biochemistry; able to provide tuneable mechanical properties; ability to provide tuneable porosity; in vitro biocompatibility; in vivo biocompatibility; and/or stable during cooking conditions (where food product applications are desired).
The present example describes development of scaffolds that meet all the characteristics above. These scaffold materials are prepared in this example by first decellularizing plant materials, followed by performing a mercerization treatment in which the decellularized materials are treated under basic conditions at high temperatures to separate the plant tissues into single intact decellularized plant structural cells (or groups of structural cells comprising small clusters of linked structural cells). A strong oxidizer is then introduced to make the resulting slurry of cells white in colour. The whitening is performed to produce a final product that provides a blank canvas for various applications in which colouring may be desired (for example, in food products, etc. . . . ). Then, the slurry is neutralized and centrifuged to result in a thick paste comprising a high concentration of decellularized plant structural cells. This resulting product may then be mixed with a wide variety of hydrogels with varying biochemical properties to produce composite hydrogel mixture(s). At this point, the hydrogels can be placed into large scale molds and lyophilized to produce a final product in the form of a lightweight, stable and large format aerogel or foam. A library of these aerogels or foams was created in this example with varying mechanical, structural and biochemical properties which may be useful for a variety of different applications. Moreover, upon (re)hydration, the aerogels and foams (also referred to herein as hydrogels upon rehydration in a liquid, most often water or aqueous solutions) may be further crosslinked and/or further modified for downstream use.
Processes used for producing aerogel formulations of this example are as follows:
Decellularization of plant tissue was performed as described in PCT patent publication WO2017/136950, entitled “Decellularised Cell Wall Structures From Plants and Fungus and Use Thereof as Scaffold Materials”, which is herein incorporated by reference in its entirety. A 5-day protocol was used as follows:
Day 1: Apples were peeled and then sliced on a mandolin to 1 mm thick. Slices were added to a 4 L beaker with a 0.1% SDS solution and shaken at 130 RPM. Appropriate Lot #'s were assigned and batch production records maintained.
Day 2: The 0.1% SDS solution was removed from the beakers and replaced with a fresh 0.1% SDS solution. Shaking resumed at 130 RPM.
Day 3: The 0.1% SDS solution was removed from the beakers and replaced with a fresh 0.1% SDS solution. Shaking resumed at 130 RPM.
Day 4: The 0.1% SDS solution was removed from the beakers and replaced with a fresh 0.1M CaCl2) solution. Shaking resumed at 130 RPM.
Day 5: The 0.1M CaCl2) solution was removed and the contents were washed 3× with 1 L of sterile water. After removing the water the beaker was filled with 1 L of 70% ethanol incubated for 30 mins. Finally, the ethanol was removed and the contents were washed 3× with 1 L of sterile water.
Mercerization was most often performed on Day 5 of the above protocol after the final washes with sterile water. However, the product obtained on the Day 5 step could alternatively be stored in the fridge until needed. Typically decellularized plant materials used in these studies were stored for no more than 2 weeks in the fridge; however, it is expected that such plant materials may be stored much longer if needed or desired. Freezing the decellularized plant materials, or lyophilizing them, are also contemplated steps for preservation of the Day 5 product; however, this was not normally performed in these studies.
In this example, mercerization was performed as follows:
Day 5 (cont'd): Mercerization was performed as follows:
Results are shown in
A study examining the yield of material after drying was performed, in which 7 samples were prepared from the mercerized materials prepared as described immediately above, which each had an initial mass of 1 g. After lyophilization, collecting and weighing the dry mass, the average mass was 0.052±0.005 g or ˜5%. This indicates that the mercerized paste was ˜95% water.
Several preparation steps in the procedure described above were determined/optimized based on a number of experiments and observations and analysis of results.
Firstly, in traditional mercerization/maceration protocols used for other applications, H2O2 is often added at the start of the process with the base/acid (mercerization/maceration, respectively). In the present studies, it is now found that the early use of peroxide may cause highly exothermic reaction which can make the solutions more difficult to handle and importantly, the reaction produces significantly more foam. By leaving the peroxide step to later in the mercerization process, it has now been found that less foam was produced. Moreover, during subsequent neutralization and centrifugation steps, less foam and gas build up would occur when peroxide was left to the end of the processing steps.
Secondly, studies were performed to investigate optimal amounts of NaOH to be used in the reaction, which is currently set at a 1:5 ratio of AA:NaOH (mass:volume, eg. 500 g:2.5 L). To evaluate these ratios, a series of experiments were performed to determine the minimum ratio of AA material to sodium hydroxide solution (NaOH) that will yield properly mercerized AA. Three different ratios were tested (i.e., 1:1, 1:2, and 1:5) using the mercerization protocol; all samples were mercerized for 60 minutes at 80° C., then neutralized and centrifuged until a stable pH was achieved. The protocol was as follows:
Although the 1:1 solution of AA:NaOH can process the most amount of decellularized AA, and is therefore the most efficient, it was not the optimal approach in the conditions tested. At a 1:1 dilution it was found that the solutions become very thick and demand more peroxide leading to foaming difficulties. Therefore, workable and safely scalable solutions were preferred and it was determined that the 1:5 dilution was the preferred approach for producing raw mercerized materials in these studies.
(Related Experimental Results for Preparation of Aerogels, Foams, and Hydrogels are also Described in Example 2 Below)
It is contemplated that processes of creating a mercerized apple “paste” as described herein may provide a variety of advantages. Firstly, this raw product may be entirely produced through liquid-based steps from start to finish. With the exception of initial apple peeling and preparation (a process to which automated industrial equipment is available) for decellularization, all further steps may be executed in liquid solutions at large scales if desired. This may provide for generating a large amount of validated raw product of decellularized plant tissue-derived structural cells with intact microarchitectures (single cell units) as opposed to fully dissolved cellulose. Secondly, protocols are developed and described herein to mix the raw product with other hydrogels to create composite biomaterials with controllable structural properties.
Once mixed or distributed into the hydrogel, freezing and lyophilization processes are developed and described herein allowing for control over the architecture of the materials which may be used to produce highly porous and ultralight dry “foams” and “aerogels”. The aerogel and foam formats are convenient and desirable, as they may be highly stable, may be stored under vacuum, may be very light, and may also possess mechanical properties relevant to tissue engineering (for example, 10's-100's of kPa). Moreover, for use in a biological context, it is contemplated that they may then be rehydrated into a hydrogel form, while maintaining their structural integrity.
Additional materials on the preparation of aerogels, foams, and hydrogels are provided in Example 2 below. Results of some such aerogel preparations, as prepared in this example, are shown in
Given the highly porous nature of the aerogels, foams, and hydrogels, and their resemblance to bone tissues, it is contemplated that aerogels, foams, and hydrogels as described herein may be used for bone tissue engineering. Ongoing small scale calvarial defect studies assessing the effectiveness of alginate- and pectin-based rehydrated aerogels (comprising the decellularized single structural cells and/or groups of structural cells as described) in bone tissue engineering support such applications. SEM and optical imaging of the aerogel scaffolds are further described in Example 2 below. Production and short-term stability of scaffolds for bone repair are also described in Example 12 below.
Various food formulations have also been developed, and cooking testing has been performed. Results of ongoing studies indicate that rehydrated aerogels may be coloured with beet juice and/or food dyes.
Results also indicate that rehydrated aerogels may be pan fried with butter. Formulations with Alginate have been tested (and additional testing is ongoing), and results obtained so far indicate that shape was stable, a crispy exterior was produced, and what visually appears like a roboust/solid interior was observed.
It is additionally contemplated that composite materials may be produced by “gluing” aerogel scaffolds together to make larger structures. Agar has been tested as a glue, and results have been favourable.
Modification of these materials to add amine groups to cellulose and/or cellulose derivatives is also contemplated. Initial glycine-based modification chemistry is described in Example 3 below. It is contemplated that one purpose of adding this functional group to the materials may be to employ the enzyme transglutaminase (aka “meat glue”), which may provide the possibility of using edible meat-glue (transglutaminase) to glue together aerogel scaffolds with each other or with sections of real meat in large formats, with possibility of controlling long range structure, mechanical properties, and/or other relevant properties.
Directional freezing approaches are described in further detail below. These approaches may provide for, for example, templating of muscle cells to grow into aligned myotubes on the aerogel scaffolds. It is contemplated that directional freezing may be used to produce structural features in aerogels, foams, and hydrogels, which may be useful for a variety of applications including in spinal cord repair, for example. Directional freezing is mainly described below in terms of directional freezing in one direction, however it will be understood that multi-direction directional freezing may also be used as desired to provide various arrangements of structural features. Typically, directional freezing may be achieved by placing a vessel containing the solution to be frozen on a cold plate to ensure that ice crystals form at one edge and grow linearly away from the cold edge. However, it is also contemplated to begin the freeze-casting process in this way and slowly move the vessel so that the location of the cold side changes in time. Conversely, the cold plate itself may be moved to a different location on the vessel to nucleate another group of ice crystals which grow in a different direction from the initial set. In yet another embodiment, the vessel may have two or more cold plates attached to it which can be turned on simultaneously, or at separate points during the freezing process in order to create highly complex, yet controlled, architectures in the resulting aerogel, for example.
Directional freezing approaches have been employed in polymer science applications, and is contemplated herein as a strategy to create aligned biomaterials for tissue engineering applications, for example. By creating a larger thermal gradient on one side of a hydrogel, linear and highly aligned ice crystals may form from the cold side. This may force the surrounding hydrogel polymers to form around the ice crystals, creating aligned microscale channels. After lyophilizing the resulting material, a scaffold may be created with many microchannels.
To achieve directional freezing in this Example, a custom-built apparatus was designed around a peltier module. Briefly, a Phanteks CPU Cooler (PH-TC14PE) with 140 mm fans was used to displace heat and oriented in an upside down configuration (any similar large CPU cooler and fans could be used). A peltier element (TEC-12706) was placed with the hot side down on the CPU block with the cold side facing up. Finally, a 4×4″ copper plate was then mounted on top of the peltier element to become an efficient cooling surface. In between each interface thermal compound (Arctic MX-4) was placed to ensure efficient heat transfer.
The peltier element was sourced from AEP's collection of parts. Based on its power usage (12V/4.2A) it is assumed that the element is a TEC-12706 element; however, there was no code on the element itself. Finally, a k-type thermocouple was embedded in the bottom side of the copper plate as close as possible to the peltier element to track temperatures and freezing rates.
To power the device, 12V was supplied directly to the peltier element and also fed to a voltage buck converter. The buck converter was used to supply 12V to the fans. This allows eventual use of higher voltages to drive the peltier while only supplying 12V to the fans.
Mixing or Distributing of single structural cells, groups of structural cells, or both (obtained from mercerized decellularized plant tissue) with a hydrogel to provide a mixture:
An alginate hydrogel was created by autoclaving alginate powder in dH2O at a concentration of 5% (w/v). The final concentration of alginate was 1%. A composite biomaterial gel was produced comprising 7.5 g of mercerated apple (i.e. single structural cells, groups of structural cells, or both, obtained from mercerized decellularized apple tissue), 3 mL of 1% alginate, and 4.5 ml of water. The alginate hydrogel and the composite biomaterial gel were mixed using two 50 mL syringes connected with an f/f luer lock connector. The mixture was passed back and forth 30 times. Syringe mixing is shown in
To create a vessel for directional freezing, the tips of 15 mL falcon tubes were cut off and the cap end was tightly sealed with a double layer of parafilm. This allowed the hydrogel mixture prepared above to be delivered from the top open end, while the bottom end is left on top of the copper plate. The parafilm layer ensured a very thin boundary between the cold surface and the gel. In this case ˜3-4 mL of hydrogel mixture was delivered into the tubes (˜3 cm high when standing vertical). The tubes were first allowed to cool in the fridge on the copper plate for at least an hour before the power was turned on. After cooling, the device was powered for two hours to allow time for the entire sample to freeze. Upon freezing, linear features could be seen in the frozen material; however, it was difficult to photograph them. After freezing, the tubes were immediately placed in a lyophilizer which operated for 36 hours. Upon removing the resultant aerogel samples, a porous biomaterial was observed. Multiple images were taken.
It was subsequently determined that an additional freezing was beneficial before lyophilization. Specifically, after freezing on the directional freezer, the samples were placed in a −20° C. freezer overnight to ensure the entire sample had frozen. The next day the samples were then lyophilized. This approach ensured that the resulting aerogels and foams did not collapse.
Optical microscopy and SEM was performed. The two scaffolds were sectioned either perpendicular or parallel to their long axis, and subsequently crosslinked/rehydrated in 0.1M CaCl2), and imaged with optical microscopy or SEM.
Other approaches to directional freezing were also attempted. Another device was custom-built in a manner almost identical to the device described above. However, the peltier element was removed along with all electronics and the fan. The passive directional freezer could then be placed in a styrofoam box and liquid nitrogen was poured into the box to cover the CPU cooler fins only. In this embodiment, the LN2 pulls heat away from the copper surface on which the samples are mounted through the heat fins and heat pipes of the CPU cooler. The plate temperature reaches about −130° C. within several minutes and the samples freeze in about 15 mins compared to 2 hours on the original device. The freezing apparatus is depicted in
Results indicate that the very rapid freezing due to the temperatures reached with LN2 prevented the creation of large scale, long range, ice crystals and therefore prevented the organization of the hydrogel mixture into channeled, aligned structures. The results are dense, highly uniform aerogel scaffolds. It is contemplated that the amorphous and uniform scaffolds created in this manner may be useful in tissue engineering and food applications, for example. These results further expand the catalog of potential scaffold architectures, and provides additional tunability options, and provides material properties which may be used in a variety of applications.
Based on the results optioned herein, it is contemplated that directional freezing may be used to impart microstructures to aerogels, foams, and hydrogels as described herein which may provide for a variety of beneficial properties for a variety of different applications.
By way of example, directional freezing may be used to provide aerogels, foams, and/or hydrogels for use in spinal cord repair. In certain embodiments, it is contemplated that the aligned structures produced by controlled directional freezing (as in the first method above using the Peltier) may result in a scaffold which may be particularly well-suited for in spinal cord repair by providing biocompatible scaffolds with directional microchannels for aligning/directing spinal cord cells following implantation of the aerogel to promote healing. Such aerogel scaffolds may be produced in a scalable and controllable fashion.
By way of another example, directional freezing may be used to provide aerogels, foams, and/or hydrogels for use in a variety of food applications. It has further been observed that when the same hydrogel mixtures described above are placed into larger format containers (ex. 60 mm diameter petri dishes) which are shallower and wider than the falcon tubes, the long range alignments tend to occur parallel to the surface of the freezing plate. This was an unexpected result, which may be desirable for a number of applications. By way of example, in this case the creation of large, flat “sheets” of material with long range alignment parallel with the plane of the sheet may be desirable for applications in cultured and plant-based meats, for example. In these applications, it is contemplated that cells will align with the structures in the aerogel/hydrogel scaffolds to create cultured muscle tissues that more closely resemble real tissues. In addition, these highly structured scaffolds may also possess structural and mechanical properties similar to real meat, and/or may have value in the plant-only based meats. Furthermore, in certain embodiments it is contemplated that cultured and plant-only based scaffolds may be generated in which they are combined with real meat to provide a new class of alternative meat products which are part plant-based and part animal-based, for example.
By way of yet another example, it is contemplated that fine tuning of formulations used in directional freezing may provide additional control over resultant structural features in the aerogels/foams. By way of example, it is contemplated that the inclusion of various salts in the formulations may be used alter and potentially control the microarchitecture of the aligned structural features by augmenting ice crystal formation. It is also contemplated that channeled molds may be used to form the aligned structures around pillars which may be later removed from the scaffold to impart an array of channels with larger sizes. Alternatively, or in addition, it is contemplated that pressing needle arrays through the scaffolds may be used to create alternative channel sizes which complement the aligned structures from directional freezing, for example.
In this example, decellularized AA (apple) was produced according to a 4-day process and used as starting material. Wet decellularized AA was then broken down into a slurry of single, intact, AA structural cells in a 1-day liquid-based mercerization process. After a final centrifugation step, a clean, moist paste was isolated and used in further processing steps. The paste was malleable, but will hold its own shape (does not easily settle or flow, highly viscous). Material was white in colour. 30 apples produced ˜150 g of moist paste (95% water). Chemicals at the concentrations used were considered GRAS (SDS, NaOH, HCL, H2O2, CaCl2), H2O). Final product was designed to contain zero, or only trace amounts of any of those chemicals due to extensive washing and neutralization steps. Although not as extensively analyzed as apple, Pear and Asparagus also responded in a similar fashion as decellularized AA in the above process. It is contemplated that many different plant types may be used.
A variety of different aerogel, foam, and hydrogel products, and precursors thereof, may be prepared according to methods as described herein.
By way of example, the resultant product obtained from mercerization of the decellularized plant or fungal tissue may be provided, the product comprising single structural cells, groups of structural cells, or both, as described herein, and the product may be provided as a paste or gel, or may be provided as a dry sticky powder (when lyophilized or otherwise dried without a carrier hydrogel). Such products may be generally stable, may be sterilized with EtOH, or it is contemplated that such products may in certain embodiments be sterilized by gamma sterilization. In certain embodiments, such products may be mixed with other liquids or gels. Generally, such products did not readily dissolve in aqueous or alcohol-based solutions.
Aerogel products, or aerogel precursors, may also be provided. By way of example, in certain embodiments, the paste, gel, dry sticky powder, or other such products as described in the paragraph immediately above may be mixed with one or more (optionally food grade) hydrogels such as, but not limited to, Gelatin, Agar, Pluronic Acid, Alginate, Pectin, Methylcellulose (MC) and/or Carboxymethylcellulose (CMC) hydrogels, providing an aerogel precursor. In certain embodiments, the aerogel precursor products may comprise about 10% to about 50% (such as about 10%, about 20%, or about 50%) (m/m) of the paste, gel, dry sticky powder, or other such products as described in the paragraph immediately above, but other concentrations are also contemplated as this is controllable over a full range. The aerogel precursor may then be placed into any suitable size of container or mold, which will dictate its final size and thickness. Aerogel precursor products may be frozen (typically at −20° C. overnight), and then lyophilized (typically for at least about 24 hours), resulting in a highly porous dry aerogel or foam product. Controlling freezing temperature (for example, −20° C., −80° C., −130° C.) and formulation % (m/m) may allow for control over porosity of the resultant aerogels and foams. Results indicate control over porosity may be achieved from a level equivalent to the original AA scaffold and down. The 10% (m/m) formulations were very low porosity but very fragile, and so may be reserved for applications where fragility is not a concern. The 50% formulation provided the best experience for the user for most applications. Freezing method (directional vs non-directional) may be used to provide control over microarchitecture geometry (aligned-porous vs homogenous-porous), as desired for the particular application or product. Initial studies indicate that that solvent (e.g. DMSO vs H2O) may also allow for control over porosity and microarchitecture, for example. Such products may be sterilized with EtOH, and it is contemplated that gamma sterilization may also be possible.
Additional products are also contemplated, such as rehydrated aerogels or foams as described herein to which liquid, such as water or an aqueous solution (such as a cell buffer) or another liquid or solution (such as an alcohol) have been introduced. In studies described herein, rehydration of aerogels and foams resulted in stable hydrogels with microarchitectures intact. Alginate and Pectin-based aerogels and foams could be rehydrated in CaCl2) in order to provide crosslinking. Rehydrated aerogels and foams were stable under shaking in aqueous and ethanol based solutions for hours/days. Pectin-based aerogels and foams were not stable in 0.9% saline and underwent rapid degradation, however these were stable in PBS, H2O and EtOH. Such rehydrated aerogels and foams have also been cell culture validated with NIH3T3 and C2Cl2 cells for up to at least 2 weeks in ongoing studies. Indeed, results indicate that rehydrated aerogels and foams as described herein are expected to behave similarly to decellularized plant-derived scaffold biomaterials as described in WO2017/136950 with respect to cell culture. Alginate and Gelatin based rehydrated aerogels and foams were superior to Pectin based aerogels and foams (which break down over time) under the conditions tested. Alginate and Pectin based rehydrated aerogels and foams are expected to be well-suited for implantation in vivo (for bone tissue engineering applications, for example). Such products may be sterilized with EtOH (for example, by 60 min shaking in EtOH), and it is contemplated that gamma sterilization may also be possible.
Results described herein indicate that aerogels and foams, and rehydrated aerogels and foams, as described herein may allow for control of surface biochemistry, particularly in that aerogels and foams, and rehydrated aerogels and foams, may be formulated with defined biochemistries (gelatin, alginate, pectin, MC, CMC, etc. . . . ) as desired. Various “plant-based” hydrocarbon polymers primarily composed of sugar may be used as hydrogel or carrier. Results also indicate that control over mechanics of aerogels, foams, rehydrated aerogels, and rehydrated foams may also be achieved. Aerogels, foams, rehydrated aerogels, and rehydrated foams as described herein may have controllable mechanical properties that may vary as a function of formulation %. In general the mechanical properties have been observed to vary from about 1 to about 200 kPa under the conditions tested. Exact values may depend on the hydrogel type and dry vs wet format/state of the aerogel or foam. An observed rule of thumb is that rehydrated aerogels and foams were about 10× softer than their dry aerogel or foam equivalent. Control over porosity may also be achieved. Results indicate that porosity may be controlled by altering the formulation %, freezing temperature, freezing method, and/or solvent used. An observed rule of thumb was that under the conditions tested porosity was either equivalent to original AA scaffolds (following decellularization, before further processing by mercerization, etc. . . . ), or may be decreased from there (i.e. higher density, lower pore sizes). Results indicate that control over size may also be achieved. The freezing container/mold for the hydrogel mixture prior to lyophilization and crosslinking dictated the final size of the resulting aerogel or foam product. It is additionally contemplated that sterilization may also be readily achieved. It is contemplated that generally all product types may be EtOH sterilized, and it is contemplated that gamma sterilization may also be possible. Results further indicate that rehydrated aerogels and foams as described herein may be suitable for in vitro cell culture. Cell culture was successful on alginate, pectin and gelatin based rehydrated aerogels and foams. Agar and pluronic acid based rehydrated aerogels and foams do not appear to be compatible with cell culture under the conditions tested so far, however this may also be beneficial for applications were cell growth is not desired, for example. Alginate based rehydrated aerogels and foams were the best performing products so far for in vitro cell culture applications under the conditions tested.
In this example a library of different aerogels and foams was prepared and tested, using various hydrogels and single structural cells and/or groups of structural cells obtained from decellularized plant tissue by mercerization treatment.
Stock solutions:
Final 5% alginate and pectin stock solutions are shown in
Pluronic stock solution preparation procedure is shown in
AA (apple) mercerization and neutralization:
Alginate aerogels:
In order to make uniform mixtures of the mercerized material and the base hydrogel, the components were loaded into syringes connected by F/F luer lock connectors. The solutions were passed across the syringe 30×. A syringe-based mixing apparatus is shown in
The sterilized and hydrated aerogels were placed in a 24-wells plate (1 sample per well) with 2 mL of DMEM. GFP 3T3 cells were cultured in 100 mm Petri dishes in DMEM media (10% FBS, 1% P/S) at 37° C., 5% CO2. The cells were washed with PBS and trypsinized with 0.25% trypsin. The cells were pelleted and resuspended in DMEM at a concentration of 2×106 cells/mL. 25 μL of the cell resuspension were pipetted on each sterilized and hydrated aerogel, meaning each aerogel was seeded with 50,000 cells. After a 4 hour incubation at 37° C., 5% CO2, 2 mL of DMEM were added to each well containing a seeded aerogel and the plates were placed back in the incubator.
The aerogels were seeded again with GFP 3T3 cells using the same method described above after 7 days of incubation. After a total of two weeks since the first seeding (there has been two seedings—on day 1 and day 7), the cells were fixed on the aerogels. The samples were washed twice with 1 mL of PBS. The cells were incubated 10 minutes in 3.5% paraformaldehyde for fixation. The samples were again washed twice with 1 mL of PBS and stained with 0.1% Congo Red for 10 minutes. Finally, the samples were washed with PBS and stored in 2 mL of PBS at 4° C.
10 mm biopsy punches of all the different aerogels formulations have been mechanically tested (dry samples) with CellScale's Univert instrument. Additionally, the formulations that were seeded with GFP 3T3 cells were also mechanically tested wet with the same settings/compression cycle.
The samples were compressed at 90% of their height (1 repetition) during 20 seconds (stretch duration). 5 or 6 samples were mechanically tested per formulation.
About 30-40 g of mercerized AA material are obtained from the maceration of 100 g of wet decellularized AA.
Using the hydrogen peroxide during the mercerization rather than separating the mercerization and discolouring steps saved time (the same amount of mercerized AA material was obtained in about 4× less time). When combining the two steps at the same time, it takes about one hour to obtain the off-white mercerized material.
Table 2 shows various aerogel formulations that were prepared for the library in this Example.
The agar and pectin (1.5) aerogels were very fragile once hydrated and they crumbled into smaller pieces.
Some formulations were not well suited for cell growth as they were either too fragile; they crumbled once hydrated; or they disintegrated during the sterilization step. This was the case for the pluronic, methylcellulose, and pluronic+pectin aerogels. Accordingly, these staples were not seeded with GFP 3T3 cells.
Results for mechanical testing of dry aerogel samples are shown in
Results in
To determine the significance of swelling on aerogel size, the approximate diameter of aerogels used for dry and wet mechanical testing were measured. The effects of liquid retention were observed after foam aerogels were stored in DMEM in an incubator for several days. Diameter data was not obtained for all aerogel formulations due to some aerogel types disintegrating during sterilization (i.e., methylcellulose and Pluronic). Dry and wet diameter measurements were obtained for agar, alginate, and pectin AA formulation aerogel. From these measurements, the approximate area, height and volume of AA aerogels was determined and shown below in Tables 4, 5, and 6.
For statistical analysis, t-tests were performed between each aerogel type's dry and wet measurements. With the exception of alginate and gelatin aerogels (both 1.5 g and 7.5 g conditions), all aerogel area measurements decreased in size once wet, likely due to the samples somewhat breaking down and losing some stability in liquid. An analysis of variance (ANOVA) was also performed to determine the significance of interactions between gel type and AA concentration (i.e., 1.5 g AA and 7.5 g AA). Overall, there was no significant difference observed between AA concentrations used for the aerogels. When comparing gel type, however, there was a significant difference.
For aerogel height, the alginate and gelatin formulations were the only AA aerogel types to demonstrate significant increase due to aerogel swelling after submersion in DMEM. An increase in volume was similarly observed with the alginate and gelatin aerogels as well after hydration; the remaining aerogel formulations all demonstrated a significant decrease in aerogel volume once wet, likely due to some aerogel degradation. Furthermore, an ANOVA for the height revealed a significant difference between the different concentrations, indicating that the freeze-drying process or the variability in the filling method may influence aerogel height in some way. Moreover the gel type and interaction effects were also significant at the 0.05 level. These findings can be observed below in Tables 4, 5, and 6.
The alginate aerogel with 7.5 g of AA was imaged with confocal microscopy.
This example provides data indicating that an array of different formulations for aerogels and foams with different properties may be prepared. The front-runners for aerogels and foams for a wide variety of applications are the 50% decellularized and mercerated AA in alginate and gelatin gels, followed by the pectin gels. Moreover, most of these gels were manually mixed by hand stirring, with the exception of the gelatin gels. The gelatin samples were more thoroughly mixed with a luer lock connection system with two syringes. It is contemplated that applying this technique to additional formulations would result in less sample variation and a more uniform gel.
This example describes a number of approaches for creating dissolved cellulose-based hydrogels from decellularized plant tissues and other synthetic cellulose sources. In the examples below, a goal was to combine mercerized plant cellulose materials such as the structural cells as described above with newly developed cellulose-based hydrogels to create composite aerogels, foams, and other scaffolds. This may be desirable in a number of different applications, as the resulting aerogels (e.g. both the structural cells and the carrier/hydrogel) will be entirely produced from decellularized plant tissues.
This example describes the dissolution of cellulose from decellularized apple scaffolds using dimethylacetamide and lithium chloride, and its regeneration by solvent exchange using 95% ethanol.
Contemplated mechanism for dissolution of cellulose in DMAc/LiCl as proposed by McCormic et al. (a) and Morgenstern et al. (b) is as follows:
Possible reaction scheme for cellulose dissolution with DMAc and LiCl may also proceed as follows (showing interaction among Li+ cation, Cl-anion, and DMAc when cellulose dissolves into DMAc/LiCl system):
Mercerized material (i.e. single structural cells, groups of structural cells, or both, obtained from mercerization treatment of a decellularized apple tissue as described hereinabove) was mixed with the regenerated cellulose obtained as described above in this example. In an attempt to avoid premature regeneration, mercerized material (1 g) was mixed with acetone and sonicated for 5 minutes. The material was then centrifuged at 5000 rpm for 7 min. The mercerized cellulose (water exchanged for acetone) was mixed with the DMAc LiCl dissolved cellulose mixture (5 mL). The combination was mixed in a dual syringe, luer lock connector set-up.
Lyophilized Composite Aerogels Produced from Mixtures of Regenerated Cellulose and Mercerized Materials:
After producing mixtures of regenerated cellulose and mercerized materials, the hydrogels were frozen and lyophilized (as described above) to produce aerogels/foams which could be left dry or rehydrated. Several formulations were prepared as follows:
Methylcellulose-based Gels were also prepared. In this next example, an all-cellulose material was prepared using methylcellulose and mercerized material (i.e. single structural cells, groups of structural cells, or both, obtained from mercerization treatment of a decellularized apple tissue as described hereinabove).
Decellularized apples were mercerized in 1 M NaOH for 1 h. In order to lighten the colour, hydrogen peroxide (stock 30%) was added to the mixture (5% v/v of the 30% stock) during the 1 h mercerization process. The solution was then neutralized with HCl and centrifuged to collect the material. To ensure the pH remained stable, the pellet was resuspended in dH2O, and the solution was neutralized again. This process was repeated until the pH remained between 6.8 and 7.2 for subsequent cycles.
The gelation process involved dissolving the methylcellulose in 10 mL of 2 M NaOH for 1 h with stirring on ice. A glycine solution was also prepared by dissolving glycine in 2 M NaOH. After 1 h, 5 mL of the glycine solution was added, and the mixture stirred on ice for an additional hour. The mercerized apple was introduced at one of two different stages. One method of introduction involved mixing in the mercerized apple with the viscous solution after the second hour of glycine treatment. This particular mixing method involved using syringes connected with an F/F luer lock system. For the higher methylcellulose concentration (1 g), the mixing with syringes was exceedingly difficult. As a result, a second preparation method was developed. In this approach, the mercerated apple was added directly to the 2 M NaOH with the methylcellulose at the start of the reaction. Mixing was accomplished with magnetic stirring. See Table 7 for the formulations tested. It was observed that when the glycine was added, the viscosity of the mixture increased; however, it appeared that this was largely due to physical crosslinking induced by temperature increases. The gels were left at room temperature overnight to crosslink.
It was found that neutralization for aerogels this size was a challenge as there was a slow release of NaOH or the neutralizing solution. The 5% acetic acid was insufficient to fully neutralize the NaOH (as shown above). This was surprising. Comparatively, the addition of 30% acetic acid had the opposite effect: the acid used for neutralization slowly released from the aerogel and required additional base treatment. It was found that 12-15% acetic acid was an appropriate concentration for neutralization without making the pH swing too far to one extreme.
In an attempt to control the swelling, it was sought to increase the concentration of glycine. It was found that the 30% glycine solution was approaching the saturation point. At 40%, heat was required to dissolve the glycine. As the gel is thermoresponsive, the glycine needed to be cooled prior to addition to the methyl cellulose and mercerized AA mixture; however, upon cooling the solution, the glycine crashed out of the solution.
This example describes use of aerogels and foams as described herein, such as those prepared in Examples 1 and 2, for bone tissue engineering.
This Example describes standard operating procedures for implantation and resection of decellularized biomaterials into trephinated calvarial defects. The study was conducted to evaluate the potential of aerogels and foams as described herein for bone regeneration applications, in a rat critical-size, bilateral defect model. The biomaterials (alginate and pectin based aerogels) were implanted in rats for periods of 4 and 8 weeks. 5 mm bilateral, circular defects were created in the rat calvarium. Once the bone defects were excised, the aerogel (alginate or pectin aerogel formulations, Table 2 provides formulations for the 5% alginate aerogel and the 5% pectin aerogel used in the bone tissue engineering example) biomaterials (5 mm diameter by 1 mm thickness) were placed within the defect. Overlying skin was sutured, and the rats were left to recover for a period of 4 to 8 weeks. Specimens were collected at each time points and computational tomography (CT scan), implant dislocation mechanical testing, and histology were performed.
Table 2 provides formulations for the 5% alginate aerogel and the 5% pectin aerogel used in this bone tissue engineering example.
After the rats were allowed to recover for the desired amount of time (e.g. 8 weeks), specimens were collected and scanned with computational tomography (CT). Histology was then also performed.
This example describes studies of peroxide ratios for mercerization processes as described herein, such as those used for preparing the structural cells of the aerogels and foams as described herein, such as those in Examples 1 and 2.
In order to test different ratios of peroxide, a constant ratio of decellularized apples to NaOH was used. This was 100 g of decellularized apple for 500 mL of 1 M NaOH. The amount of 30% stock hydrogen peroxide added to this constant ratio was 20 mL, 10 mL, and 5 mL. These amounts correspond to a final concentration of 1.15%, 0.58%, and 0.30% respectively (in regards to the concentration in the 1 M NaOH). Clearly, as shown in
It should be noted that the peroxide treatment may be done after the mercerization as well; however, it is observed that the high temperature and basic conditions of the mercerization speeds up the lightening process.
Results indicate that different peroxide ratios may be used to achieve a similar final product. Reducing the concentration of peroxide from 1.15% to 0.3% did not affect the bleaching of the final product after neutralization.
This example describes studies of mass proportions of structural cells of the aerogels and foams as described herein, such as those in Examples 1 and 2. As described herein, in certain embodiments the aerogel or foam may comprise about 10-50% m/m (or more) single structural cells, groups of structural cells, or both, when the aerogel or foam is in hydrated form.
As will be understood, the mass of the aerogels and foams when dry will be significantly different from the mass of the rehydrated (or wet) aerogels and foams. In some experimentally measured examples, the dry mass of prepared aerogels was about 5.18% of the wet mass of the same aerogels.
As will further be understood, the structural cells of the aerogels and foams as described herein may have a wide range of sizes, and may be substantially uniform of may be a mixture of different sizes. In certain embodiments, the structural cells may have a size ranging from about 20 μm to about 1000 μm. If desired, it is contemplated that larger particles may be obtained by reducing the mercerization time or changing the source material with a different size range of plant cells, for example. Typically, for apple decellularized tissue, the structural cells provide a mean particle size of ˜200 μm, however it will be understood that other sizes are also contemplated.
The images were thresholded and segmented using Fiji ImageJ. A watershed was applied to the image after it was converted to binary. The Feret diameter was calculated with the Analyze Particles plugin. The histograms of the particle sizes were very similar for each case (
To further investigate the particle sizes, an ANOVA was performed with a Tukey post-hoc analysis at a significance level of 0.05. The data is summarized in Table 8 and Table 9.
These results also show that different ratios of decellularized apples to NaOH can lead to similar end products. This flexibility in the production process allows for practical adjustments during scale-up, for example.
This example describes a protocol used for mechanical testing of aerogels and foams as described herein, such as those in Examples 1 and 2. In certain embodiments, the aerogels or foams as described herein may have a bulk modulus within a range of about 0.1 to about 500 kPa, such as about 1 to about 200 kPa, as determined by the protocol described in this example.
Sample preparation
For calculation of the bulk modulus, the raw data is provided as a force-extension curve which is converted into a stress-strain plot based on the measured dimensions of the sample. The linear portion of the compression curve is assessed and the slope determined in kPa.
This example describes approaches for cross-linking of hydrogels in aerogels and foams as described herein, such as those in Examples 1 and 2 and elsewhere herein. In certain embodiments, at least some cellulose and/or cellulose derivative(s) of the aerogel or foam may be cross-linked by:
The cellulose-based materials may be cross-linked in a variety of ways. Broadly, crosslinking may be by physical cross-linking or chemical cross-linking, or both.
An example of physical cross-linking is the use of glycine, which may, by way of illustrative example, be implemented as follows:
The gelation process may involve dissolving methylcellulose in 10 mL of 2 M NaOH for 1 h with stirring on ice. A glycine solution was also prepared by dissolving glycine in 2 M NaOH. After 1 h, 5 mL of the glycine solution was added, and the mixture stirred on ice for an additional hour. The mercerized apple structural cells were introduced at one of two different stages. One method of introduction involved mixing in the mercerized apple with the viscous solution after the second hour of glycine treatment. This particular mixing method involved using syringes connected with an F/F luer lock system. It should be noted that for the higher methylcellulose concentration (1 g), the mixing with syringes was exceedingly difficult. As a result, a second preparation method was developed. In this approach, the mercerated apple was added directly to the 2 M NaOH with the methylcellulose at the start of the reaction. Mixing was accomplished with magnetic stirring. See Table 7 for the formulations tested. It was observed that when the glycine was added, the viscosity of the mixture increased. The gels were left at room temperature overnight to crosslink.
Such physical cross-linking with glycine has already been described in detail in Example 3 above, with reference to Table 7 and
An example of chemical cross-linking is the use of citric acid and heat wherein the carboxylic acid groups may react with carboxymethylcellulose to form a chemically cross-linked matrix, which may, by way of illustrative example, be implemented as follows:
Preparation of carboxymethyl cellulose gels crosslinked with citric acid in combination with mercerized apple material (e.g. structural cells):
After 16 h, a hard “glass or epoxy” like material was obtained. This was rehydrated and formed membrane materials.
Expanding on chemical cross-linking, it is also contemplated herein that the cellulose structure may be functionalized with linker molecules that may be then used to add specific moieties to the cellulose chain for cross-linking purposes (among other purposes). For example, it is contemplated that succinic acid may be used to add a carboxylic acid group to the cellulose structure. The succinylated material may be used to add amine groups. It is contemplated that the amine groups may then be crosslinked with available protein cross-linkers such as formalin, formaldehyde, glutaraldehyde, and/or transglutaminase.
a) Solvent and sample preparation
After solvent preparation (DMA and LiCl) the cellulose pieces are immersed in DMA. The LiCl is then added. The mixture was stirred for 30 minutes. After 30 minutes, the succinic anhydride (AS) was added. The mixture is placed in the oven at 80° C. for 6 h. After this process, the solvent is removed, and the cellulose is washed intensely with water until the scaffold is clean and free of visible residue. The images in
Cellulose after reaction is complete is shown in
In order to confirm the chemical crosslinking was accomplished, Fourier Transform Infrared Spectroscopy was used. The spectral shifts reveal that the succinic anhydride was successfully chemically bonded to the scaffold. This linker molecule may be used to attach other molecules such as collagen. FTIR spectra is shown in
Methodology: Through homogenous succinylation using succinic anhydride
Cellulose suspended in DMAc/LiCl allowed to stir in the presence of CO2 (2-5 bar) to obtain a clear solution. Afterwards, succinic anhydride is added to the clear solution of cellulose and stirred at room temperature. The resulting reaction mixture is poured to vigorously stirring water (200 mL). Then, water was slightly acidified with a diluted (0.05 M) hydrochloric acid solution to obtain white precipitates. The crude product is collected and dissolved in DMAc/LiCl and reprecipitated in water (200 mL). The white product is further washed with water (200×3 mL) to remove DMAc/LiCl. The pure white product is dried under vacuum at 70° C. for further esterification process.
Cellulose succinylation using succinic anhydride, pyridine in dichloromethane in a static system. The reaction is performed under reflux. After the period of reaction, the reaction is quenched by adding of MeOH. Then, the material is washed several times to remove pyridine.
Chemical modifications of cellulose, such as esterification, etherification and grafting (from or onto) are among the most common techniques. Such derivatizations may be achieved via heterogenous or homogenous approaches. Heterogenous modifications on the surface of cellulose fibres have been more common due to the solubility challenge of cellulose. Homogenous modifications of cellulose, on the other hand, are desirable as the latter may enable to control the DS by adjusting the reaction conditions. Table 10 shows examples of esterification processes.
Esterification may involve high temperature and a crosslinker agent. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), citric acid, and fumaric acid are commonly used in an esterification reaction to form a hydrogel.
EDC promotes crosslinking between carboxyl groups and hydroxyl or amine groups with the formation of nontoxic, water-soluble urea derivative. EDC is preferable for crosslinking reaction because of high conversion efficiency, mild reaction conditions, nontoxicity, and easily separable byproducts and compatibility with materials. Also, during the esterification of cellulose and citric acid, anhydride intermediate is formed.
So, before transglutaminase application, the cellulose may be submitted to chemical modification using succinylation (acylation) followed by preparation of activated NHS-esters and their reaction with nucleophilic amino acid residues present in proteins.
The following procedure outlines illustrative examples of general usage for MooGloo (i.e., the transglutaminase enzyme) in food applications, such as for the binding and gluing of AA aerogels and other biomaterials.
In certain embodiments, aerogel and/or foam materials as described herein may be used for preparing cell-based and/or plant-based meat products, meat mimics, cell cultured meat, cell-based meat, other food applications, cellular agriculture applications, etc. Mercerized materials (such as structural cells) as described herein may be combined with a number of different hydrogels (as described above), for example, alginate, pectin, gelatin, methylcellulose, carboxymethylcellulose, dissolved and regenerated cellulose, etc. The mixtures may then be placed into a container of generally any suitable size, frozen and then lyophilized to remove all water. Once completely dehydrated, the material may be crosslinked with (for example) calcium chloride or another crosslinking agent. This step may be performed either before or after lyophilization, and may result in a desirable format of the material. This example shows use of the aerogel for the production of a plant-based meat product.
In certain embodiments, the aerogel may be used to produce a plant-based meat product by customizing its method of preparation, such as the vessel in which it is prepared. By way of example, to produce a piece of tuna sashimi, the mercerized material was molded into a 100 mm dish, then frozen for 24 hours at −20° C. The frozen material was then lyophilized for 48 hours, and then crosslinked with calcium chloride. In an embodiment, once crosslinked, the material may then be stained, dyed or cut to any desired shape such as for a piece of sashimi. The material was stained with food coloring by adding a few drops (2-6 drops, or desired amount for desired colour) to a container with water such that the water was covering the material entirely (e.g. a material used to form a 100 mm dish-10-20 mL of coloured water or food dye). Once stained, the material was cut into a rectangular shape with a scalpel, knife or another sharp blade. Vertical slices may be cut across the slice to mimic the white lines found in tuna or salmon. A food-grade gluing agent such as agar, agar-agar, gelatin or similar agents may be used to fill in the lines cut by the blade. In one embodiment, 1-5% agar may be used to glue pieces of material together, or fill in lines made by cutting the material, approximately half to ¾ of the way through. The agar may also be combined with food grade titanium dioxide (Pan Tai, PTR-630) (0.1-1 g per 100 mL of agar) to color the lines white.
The resulting biomaterial may be cooked once cut, stained and prepared to a desired appearance, and texture. The material may be pan-fried, baked, or prepared in another method (e.g. under vacuum, sous vide), or left uncooked. In one embodiment, the material may be pan-fried for about 1-10 minutes on each side with a small amount (¼ tsp) of butter on a cast-iron skillet heated to 200° C. (see
Once cooked, the material may be mechanically tested to determine its bulk compression modulus. In one embodiment, the material was cut to a 1 cm×1 cm size piece. It was placed atop a platen on a mechanical tester (such as a Univert by CellScale) and compressed. The rate (compression force or size per second), maximum load (depending on the load cell), displacement of compression (from 5% to 95% of its size), among other features may be customized. In one embodiment, a round sample of approximately 10 mm diameter and 5 mm thickness was mechanically tested by compression in both axial and radial directions. The biomaterial was cut to the specified size, and placed on the mechanical tester. Size measurements were recorded then the material was compressed three times to 50% of its size.
Measured bulk modulus from 10 cm cooked and uncooked biomaterials were as follows:
The present data shows that the mercerized aerogel may be used to produce a plant-based scaffold for the development of a meat-free alternative for a food product. The resulting material may be customized wherein its size, shape, appearance, texture and/or mechanical properties may all be tuned for the production of the desired material, such as a piece of fish (e.g. salmon or tuna) or a steak or piece of chicken, for example. Additionally, the data supports that cooking may result in an increase in the bulk modulus or a stiffening of the material. This may be a desirable property, as cell-based meat may also stiffen from cooking. These results are promising for the development of a variety of plant-based food products. Finally, as the aerogel may be compatible for cell culture (see
In another embodiment, mercerized materials, structural cells, aerogels, foams, dissolved celluloses, and/or other such materials as described herein may be used for dermal filler applications. By way of example, the mercerized material may be used on its own as a dermal filler hydrogel, or it may be combined with other carrier gels and/or fluids such as saline, collagen, hyaluronic acid, methylcellulose, and/or dissolved plant-derived decellularized cellulose, for example. Such formulations may be used in combination with lidocaine, for example, or another such agent relevant for dermal filler applications. This Example provides data for the use of the mercerized decellularized apple structural cells as particles for dermal fillers.
Needle Occlusion Test for merAA (Mercerized Apple):
The size analysis of the mercerized material revealed that the process (as already described in detail hereinabove) may separate the scaffolds into single structural cells. These apple cell walls have sizes that are comparable to HA particle sizes used in dermal fillers. Therefore, the mercerized material was selected as a candidate for dermal filler applications. This may be used as a material on its own, or in combination with other gels/formulations as discussed below. The first test was an occlusion test through a 27 G needle. It was found that the needle did not occlude, aside from large chunks that were filtered out. This is a significant result, as HA fillers typically use 27-30 G needles, whereas BellaFill uses 26 G needles.
The mixtures were made in 5 ml syringes and transferred to 1 cc syringes for a volume of 0.3 ml. With the use of interlock connectors on the 5 ml syringes, we could mix the mercerized AA with the saline solution thoroughly by mixing 30×.
Formulations were as Follows:
The maximum extrusion force was used to compare the three formulations. The descriptive statistics are displayed below, and visually compared in
Descriptive Statistics of the Maximum Extrusion Force from a 1 cc Syringe:
This data shows that the undiluted mercerized material had a significantly greater extrusion force than the diluted mercerized material and the water control. Moreover, the results reveal that there was no significant difference between the diluted mercerized material and the water control. This provides insight into the sensitivity of the measurement. The viscosity of the diluted material is different from distilled water; however, this slight difference was not discernable with the current method.
As the occlusion test was successful, the material was implicated as a dermal filler in a test rat study. The injection volumes were 600 μL, and 4 injections per animal were performed. All the formulations contained 0.3% lidocaine. Lidocaine was used in this case but a number of other anesthetics are also possible. Typical anesthetics may include, for example, 2% lidocaine gel and a triple anesthetic gel composed of 20% benzocaine, 6% lidocaine, and 4% tetracaine, (BLTgel). Dental blocks with 3% Polocaine are given with a 30 G needle to anesthetize the upper and lower lips and perioral region prior to injection. Also mixes of 2% lidocaine with epinephrine may be used.
The first formulation was mercerized AA alone. The second filler was mercerized material diluted with 0.9% saline to give a final concentration of 20% volume of the mercerized material. Similarly the third formulation comprised a 20% mercerized material and 3.5% collagen mixture. Lastly, the fourth filler was a 20% mercerized material and regenerated cellulose mixture. The regenerated cellulose was derived from decellularized apple tissue and was dissolved according to the methods previously described above with DMAc and LiCl. The formulations were coded as MER, MER20SAL80, MER20COL80, and MER20REG80 respectively.
Dermal Filler Formulation Components were as Follows:
Cellulose is a polymer that presents abundant hydroxyl groups and can be used to prepare hydrogels with fascinating structures and properties. On the basis of the cross-linking method, the hydrogels can be divided into chemical gels and physical gels. Physical gels are formed by molecular self-assembly through ionic or hydrogen bonds, while chemical gels are formed by covalent bonds.
Cellulose and cellulose derivatives define their extensive usage in different applications, and cellulose esters and cellulose ethers are two main groups of cellulose derivatives with different physicochemical and mechanical properties. Cellulose esters are water-insoluble polymers with good film-forming characteristics which find a variety of applications.
The attachment of the polyfunctional carboxylic acids via esterification with a cellulosic hydroxyl group and its esterification with another cellulosic hydroxyl group produce crosslinked cellulose chains. Attachment of the carboxylic acid moiety to cellulose's hydroxyl group via esterification reaction of the first cyclic anhydride would expose a new carboxylic acid unit in carboxylic acid, which has the proper chemical connectivity to form a new intramolecular anhydride moiety with the adjacent carboxylic acid unit.
Citric acid (CA) is regarded as a non-toxic and relatively inexpensive crosslinking agent that has been used to modify polysaccharides such as cellulose.
The suggested mechanism for the cross-linking process of cellulose with citric acid with regard to the conventional cross-linking of cellulose using citric acid in the presence of acid catalysts is shown below:
Aerogels Produced from Cellulose Crosslinked with Citric Acid
In this example, various characteristics of aerogels produced from cellulose crosslinked with citric acid were evaluated such as the pore size and alignment along with the stability of aerogels. The aerogels were prepared from regenerated cellulose or from mercerized cellulose crosslinked with citric acid to compare the effect of the two forms of cellulose in aerogel preparation for downstream applications. The goal was to produce an aerogel which can be used as a scaffolds for bone repair and/or spinal cord regeneration. The first aerogel was produced from a mixture of 1) regenerated cellulose (ADICLS) crosslinked with citric acid and 2) succinylated cellulose generated by unidirectional freezing. The second aerogel was produced from a mixture of 1) mercerized cellulose (Merc. AA) crosslinked with citric acid (S4) and 2) succinylated cellulose generated by unidirectional freezing. The stability of the two aerogels was then compared.
Crosslinking with Citric Acid
Four different samples were prepared by mixing mercerized cellulose (Merc. AA) (0.1 g/mL) and succinylated cellulose (0.1 g/mL) in different proportions according to the table below, and mixed with 2% citric acid to evaluate if 2% citric acid was enough to produce a stable aerogel in PBS. Briefly, the polymers were suspended in 50 mL of water to form a gel before citric acid was added and kept under vigorous stirring with a glass rod for 15 minutes. The reaction was performed at 100° C. for 20 minutes on a hot plate shaker and all surface moisture was removed. The material was removed from the oven and kept at room temperature overnight. The next day, the material was incubated in the oven for 2 h at 105° C. The reaction products were then slurried in water (60 ml) for 30 min, adjusted to pH 7, and washed three times by centrifuging at 4500 rpm for 10 minutes to remove the unreacted components.
Agarose (1%) was liquefied in hot water (70° C.) and mixed with 40% hydroxyapatite (HP). Crosslinked Mer. AA (20 g) and succinylated cellulose were loaded in a syringe together with 12 mL of the liquefied agarose and HP. The polymers were mixed using two 60 mL syringes with a Luer Lock connector. The amount of succinylated cellulose was adjusted according to the proportion described in Table 1.
The resulting material was placed in a 60 mm TC dish and incubated at −20° C. for at least 2 hours to completely freeze the material which was then lyophilized for 24 hours.
As shown in
Aerogels were prepared using regenerated cellulose to compare the porosity with the other aerogel prepared from Mer. AA CL.
The particle size of the aerogels was assessed by comparing aerogels produced from crosslinked regenerated cellulose (D1CL) and crosslinked mercerized cellulose (S4), both mixed with succinylated mercerized cellulose. These materials were evaluated with the goal to obtain more homogeneous suspension and further aligned porous formation under directional freezing. The samples were prepared as described in Table 2 below.
The aerogels were prepared as previously described above. Briefly, the polymers were mixed in the amount indicated in the table above using two 50 mL syringes connected with an f/f Luer Lock. The previously prepared sample (S4-crosslinked mercerized cellulose) was mixed with water (4 mL) and inserted into the syringes together with succinylated cellulose and the polymers were mixed at least 30×. The same procedure was realized for the crosslinked regenerated cellulose (D1). The samples were placed into steel tubes onto the directional freezer for 2 hrs.
After directional freezing the material was transferred to the freezer −20° C. and kept for 24 h, followed by lyophilization for at least 24 h.
The aerogel prepared from crosslinked regenerated cellulose (D1) was named “ADICLS” and presented two layers. Previous results have shown that aligned pores are usually only formed in the bottom portion, so the microscopy imaging was performed in the bottom portion (portion directly in contact with the copper plate used for directional freezing) and in the top surface of the bottom layer for analysis as indicated by the circled region in
The results demonstrate that sample S4 produced a more porous and organized structure with a region showing aligned porousness. However, the samples were highly unstable in water when the stability was assessed. Further optimisation in the crosslinking process was therefore performed.
The density (or the porosity) of the final material depends on the initial mass of mercerized cellulose. As such, the cellulose mass was increased and the effect of citric acid concentration on the aerogel during the crosslinking process was evaluated. Mercerized cellulose was selected to prepare these samples.
Mercerized cellulose (Merc. AA) was prepared in water as described above. Citric acid with different concentrations were dissolved in a minimal amount of water (3 mL) in a beaker. The citric acid was then added in the Merc. AA gel and mixed vigorously. The crosslinking process was performed as described above. The different concentrations of citric acid used to prepare samples S6-S10 is shown the table below.
Reaction products were slurried in water and the pH was adjusted to pH 7. The cellulose citrate product was air dried.
Hydrogels were then produced by mixing S6, S9 and S10 with succinylated mercerized cellulose. Since gels produced using S7 and S8 were relatively unstable, these samples were discarded and not used in subsequent experiments. Succinylated mercerized cellulose was selected because it has more hydrophilic characteristics which allows production of more homogeneous and uniform hydrogels which can improve alignment of pores under directional freezing.
The polymers were prepared as described above. Briefly, the two polymers (crosslinked Merc. AA+succinylated mercerized cellulose) were mixed using two 50 mL syringes connected with an f/f luer lock connector. The crosslinked mercerized cellulose (S6, S9 or S10) were mixed with water and the succinylated cellulose added thereafter and inserted into the syringes and the polymers were mixed. The samples were placed into the directional freezer. The aerogels were prepared and named according to the table below.
As shown in
The stability of each aerogel in PBS was then evaluated.
All previous samples were prepared by crosslinking the polymers with citric acid before aerogel production. The following aerogels were prepared by crosslinking the aerogel to assess the effect of crosslinking after the aerogels are formed. The crosslinking reaction was thus performed as the last step, after lyophilization.
Certain parameters were adjusted according to the findings described above. Briefly, the concentration of cellulose was raised, the citric acid concentration was adjusted to 10% according to the results described above. In addition, the viscous suspensions were dispersed using FisherBrand 850 Homogenizer in an attempt to improve homogeneity.
The hydrogels were prepared from either mercerized cellulose, regenerated cellulose, succinylated mercerized cellulose or combinations thereof according to the table below.
The hydrogels described in the table above were prepared as previously described but without crosslinking.
In contrast to the previous aerogels in which the polymers were crosslinked before preparation of the aerogels,
The crosslinking process was then performed as described above using a 10% citric acid solution with the resulting aerogels shown in
The microscopy images highlights the differences in morphology between each aerogel. The aerogel produced with Merc. AA presented some aligned structure which could not be observed in the other two aerogels (D1A and Merc. AA+D1A). The morphology of the aerogel produced with only regenerated cellulose showed larger pores and the material was less compacted compared to the aerogel prepared with Merc. AA+D1A, which showed a more compact and uniform structure.
The aerogel prepared with Merc.AA+succinylated cellulose was then examined.
The stability of each aerogel described in the table above was analyzed and images are shown in
Since succinylated material is more hydrophilic, a longer incubation time of 24 hours in PBS was performed to confirm the aerogel stability.
The images of the aerogels shows that all aerogels were stable in saline solution, PBS and water using the reticulation in the final step of the process.
In this example, needles were used to generate porous structures in the aerogels.
Four hydrogels were prepared from 1) Merc.AA, 2) D1A, 3) Merc.AA+D1A, and 4) Merc.AA+succinylated cellulose and pore size was assessed in
The samples were then placed into steel tubes and onto the directional freezer for 2 hrs.
Crosslinking with citric acid was then performed by incubating the samples in the oven at 110° C. for 2 h. The crosslinking was also performed for 1.5 hours and the stability of the resulting was examined. Since no significant differences could be observed in terms of morphology and stability of the aerogels PBS, the duration of the crosslinking reaction was standardized at 110° C. in the oven for 1.5 h. The needles were then used to obtain a porous structure. A circular 4 cm silicone mold was inverted and the 30 G needle tips were carefully inserted (perpendicularly) into its base; the needles were arranged into 3 groups of 4 to ensure that each aerogel would contain 4 ‘pores’. To create a base, the bottom of each HDMC mold/metal tube was wrapped with two layers of parafilm, then placed directly over each group of needles. The size of each needle is indicated in the table below.
The aerogels were examined by microscopy to evaluate the porous structures.
The microscopy images shows differences in the morphology of each aerogel. The aerogel produced with Merc. AA shows well defined pores produced by the needles which are readily observed in
In this example, needles (30 G/0.3 mm) were inserted in a silicone mold and the hydrogel was added in the mold. The pores within the hydrogels were then examined.
Four hydrogels were prepared from the same formulation as in example 13 and described in the table above. The four aerogels are: 1) Merc.AA, 2) D1A, 3) Merc.AA+D1A, and 4) Merc.AA+succinylated cellulose and are shown in
The aerogels prepared using the silicone molds and needles (30 G) are shown in
The aerogels were then examined by microscopy to evaluate the porous structures.
The microscopy images shows that the pores obtained in the aerogels prepared from Merc. AA and Merc. AA+regenerated cellulose (D1A) are well defined, demonstrating that the needles are stably positioned and yields improve pore formation. More specifically, the morphology of the aerogel produced from Merc. AA presented larger pores having a size of about 700 μm whereas the aerogel produced from AMerc. AA+regenerated cellulose displayed a different structure with smaller pore size of about 420 μm. These results highlight the effect of regenerated cellulose on the morphological organization of the aerogels.
Moreover, the images shows that larger pores are formed when using needles in the preparation of the aerogels.
In this example, various samples were examined by Fourier-transform infrared spectroscopy (FTIR) to evaluate the effect of citric acid concentration and if the conditions used were effective in getting cellulose which is covalently crosslinked by ester linkage.
The valent vibrations of CH and OH groups of organic acids are detected between 2800 cm-1 and 3500 cm-1. The complex absorption band in the region of 3500-3200 cm-1 originates from the valent vibrations of OH groups. The valent vibrations of the citric acid free OH group yields a band with a maximum at 3495 cm-1, while valent vibrations of the OH groups involved in intramolecular and intermolecular hydrogen bonds are observed at 3448 cm-1 and 3293 cm-1, respectively (2).
Valent vibrations of C═O group from acid group yield a band at about 1760 cm-1, which in the case of citric acid appears at 1753 cm-1 (accordance with the reference data (2,3)) and if C═O groups are involved in the formation of hydrogen bonds or molecules are dimerized, vibrations occur at lower frequencies, a bond at 1714 cm-1 (4).
There are mainly three sites of hydroxyls on a glucose ring of cellulose, i.e., O(2)-H(2)(the 2-hydroxyl group), O(3)-H(3) (the 3-hydroxyl group), and O(6)-H(6) (the 6-hydroxyl group). The region between 3700-3000 cm-1 is the most interesting and clearly visible changes related to development of intra- and intermolecular hydrogen bonds.
The samples S6-S10 analyzed are as previously described in example 12 and according to the table below.
The FTIR profile of all samples are similar but clearly can be observed that samples with low concentration of citric acid (2 and 4%) are similar and others with 5, 10 and 20% of citric acid originate very similar FTIR mainly S8 and S10. All samples show the band at 1730 cm-1 (ester carbonyl) that confirms the ester linkage. Additionally the band at 1587 cm-1 in samples (S8, S9 and S10) show the presence of carboxylate ion. In samples S6 and S7 the symmetric oscillations of O═C—OH groups are observed at the wave numbers 1630 cm-1 and neither asymmetric oscillations of O═C—OH groups at 1392 cm-1, nor valent vibrations of citric acid C—OH group at 1082 cm-1 are present.
Additional samples according to the formulations described in the table below were analyzed by FTIR. According to previous results described above, 10% citric acid was selected as the concentration for crosslinking, which provided the best relation of morphology/stability in PBS.
Mercerized cellulose (Merc.AA 151) presents a broad band which is reduced after crosslinking with citric acid at 110° C. As mentioned above, the valent vibrations of the citric acid free OH group yield a band with a maximum at 3495 cm-1, while valent vibrations of the OH groups involved in intramolecular are observed at lower wavenumber.
A narrower shift to 3411 cm-1 (
Referring to
Referring to
A change in the position of the absorption band of C—O group from citric acid (1753 cm-1) into the band with a maximum at 1729 cm-1 (Commercial sources-Literature data) and 1735 cm-1 (
The synthesis reaction of aerogels from cellulose and citric acid is based on the esterification reaction of carboxyl groups of citric acid with hydroxyl groups of cellulose a shown below.
Yang & Wang, 1996 (5) showed that citric acid (CA) first loses one molar of water to form aconitic acid (AA) isomers, which will then release a second molar of water to form AA anhydrides for crosslinking reactions under an elevated temperature. The band at 1630 cm-1, referring to C═C, also appears. This band was clearly observed in samples S6 and S7 (
Yang et al. (1996) analysed that pure CA does not form C═C bond and anhydride structure under a temperature at 150° C. The samples were heated from room temperature to 160° C. and measured by FT-IR again. New peaks at 1858 cm-1 and 1793 cm-1, representing the formation of anhydride groups can appear in the CA sample (5). These peaks are not present in any samples as only a band at 1630 cm-1 is observed, referring to C═C, appears (
Lu & Yang, (1999) (6) have proven that the yellowing problem on citric acid (CA)-treated cotton fabrics was caused by the formation of unsaturated acids, cis- or trans-aconitic acid (AA), under high temperature. In the analyzed samples, a yellowing was observed after the crosslinking and neutralization process. The materials were all yellow to some degree, but there was a transition from a less intense, pale, translucent yellow to a darker more vibrant yellow such that (S6=S9=S10) <AMerc.AA<AMerc.AA+succinylated <AMerc.AA+regenerated). This observation needed to be confirmed by preparing more samples.
In this example, mechanical testing was conducted using dry and wet samples from each aerogel formulation (Merc.AA, Merc.AA+Succinylated cellulose and Merc.AA+regenerated cellulose).
All samples were cutted using a 5 mm biopsy punch (n=16 per formulation) and the 5 mm wet samples were soaked in saline for 30 min prior to mechanical testing.
The mechanical analysis showed high Young′ moduli of all dried samples. The aerogel prepared with mercerized cellulose and regenerated cellulose (AMerAA+Regen.) presented the higher Young' modulus (504 kPa), followed by an aerogel prepared with only mercerized cellulose (AMerc.AA-306 kPa) and the aerogel with succinylated cellulose (AMerc. AA+succ.) showed Young's modulus 220 kPa. A possible explanation for higher Young's modulus with regenerated cellulose can be attributed to change of particle size of cellulose and also the modifications of crystalline conformation (that will be evaluated by NMR) could contribute to easier cross link reaction with citric acid and the production of more resistant compression structure.
After immersion for 30 minutes in saline solution the wet aerogels showed a significant decrease in Young's moduli (
The three-dimensional hydrogel networks formed by covalently linking cellulose chains using citric acid as a crosslinker can be an alternative to biomaterial prepared as preformed scaffolds for traditional surgical implantation. The biocompatibility was further evaluated in in the examples below which tested different post-treatment processes to remove any potential toxicity of the crosslinking agent.
In this example, the pH was examined to evaluate the biocompatibility of the samples for downstream uses. The samples examined are described in the table below.
The aerogels were prepared in a 24-well TC dish, then sterilized and seeded with MC3T3 cells. The samples were grown in culture for 4 weeks and stained for fluorescent microscopy imaging in order to measure cell adhesion and proliferation as described in example 18.
The aerogels were prepared and placed in 24-well plates, lyophilized and carefully removed from the plates for the crosslinking reaction with citric acid.
The aerogels were first sterilized and placed into growth media (GM) to assess the acidity before neutralization. As shown in
The lyophilized aerogels were incubated in a sodium bicarbonate solution (28.8 g/L) overnight at 4° C. in order to neutralize any residual citric acid.
Since the cell culture media contains both sodium bicarbonate (2.2 g/L) and phenol red, a pH indicator dye, is believed that the sodium bicarbonate reacted with residual citric acid and, thus, released it into solution. As a result, the overall pH of the media dropped below normal levels and this triggered a rapid change in colour.
The scaffolds were then incubated in DMEM overnight, at 4° C., and the pH of the solutions were measured the following morning. The pH of all 3 solutions were approximately 6.52.
The sodium bicarbonate solution was then removed and the tubes were filled with 30 mL of water. The samples were then placed onto a platform rocker and the water was changed every 5 min, until a total of 5 water washes were completed.
To sterilize the aerogels, each set of samples (n=5 per aerogel formulation) were incubated in 20 mL of 70% EtOH for 30 min.
The GFP-NIH3T3 cells were grown for 1 week prior to staining and imaging. The aerogels were seeded two additional times (on Days 4 and 6) after the initial seeding. Hoechst dye was used to stain the cell nuclei.
The aerogels with GFP-NIH3T3 cells were then stained with Hoechst and subjected to microscopy imaging.
For all samples, individual cells as well as cell clusters were observed indicating the aerogels support cell growth and are thus biocompatible in vitro.
Establishing a completely food-safe process was undertaken by performing the entire fabrication in a shared kitchen (apple processing, decellularization, mercerization, scaffold fabrication and cooking) utilizing all chemicals in the food-grade category to create biomaterials compatible with food industry. Mercerization is a key step in the process of creating the biomaterial. Currently, this step uses high temperatures along with concentrated acids and bases, which require a fume hood, with the utilization of different types of personal protective equipment (PPEs), and cannot be safely performed in the kitchen. In order to solve this hurdle, food-safe chemical alternatives like sodium bicarbonate (NaHCO3), commonly referred to as baking soda, acetic acid (CH3COOH), commonly referred to as vinegar (when diluted), and citric acid (C6H8O7) widely utilized on food product formulation, will be used to replace NaOH and HCl respectively. Mukhtar et al. (2018) demonstrated that the sodium bicarbonate has significant effect on the physicochemical properties of sugar palm fiber, thus this chemical could be an alternative in comparison with established alkaline chemicals for treating cellulose fibers, representing a cost-effective and environmentally friendly option for the food-safe biomaterial version.
Once crystalline cellulose chains are highly ordered and tightly packed in a microfibril these microfibrils must be disrupted or swollen to make cellulose chains more accessible to interact with dyes, flavours, proteins, fibers, oils, fatty acids, etc. Sodium bicarbonate (NaHCO3) is used in the pretreatment process of cellulose and showed to be effective not only to disintegrate the cellulose structure but also to facilitate the amorphization of the crystalline cellulose as well as the extended removal of integrated lignin (Morehead, 1950). The new method with sodium bicarbonate used to prepare our “base material” is a process applied to remove lignin and to disintegrate microfibrils with less degradation/loss of cellulose. Other research already showed that the pretreatment by sodium bicarbonate was effective to swell the fiber structure (Kahar et al., 2013) and the swelling of macro- and microfibrils is useful to make cellulose chains more accessible.
Also, the substitution of medical grade chemicals represents a key step in the development of a food-safe version of the biomaterial. Moreover, the utilization of ingredients or additives considered generally recognized as safe (GRAS), was necessary to produce a material considered safe as a food alternative. According to the FDA, “GRAS” is considered any substance intentionally added to food (food additive), that is subject to premarket review and approval by FDA, unless the substance is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use, or unless the use of the substance is otherwise excepted from the definition of a food additive. In addition, different trials were performed to efficiently substitute the equipment utilized in the laboratory with equipment from the kitchen to create a process for the food safe biomaterial fabrication, feasible to replicate in a conventional kitchen (FDA, 2019).
In this example, sodium bicarbonate was used to mercerize decellularized AA as described in Mukhtar et al., 2018 with modifications as described below in order to develop a kitchen-safe mercerization process. The mercerization step was adapted to kitchen-safe alternative by using 10% sodium bicarbonate (NaHCO3) at 80° C. instead of 1 M NaOH for the alkaline treatment. The goal was to create a mercerization step which would be completely food grade.
In this example, sodium bicarbonate was used to mercerize decellularized AA as described in Mukhtar et al., 2018 with modifications as described below in order to develop a kitchen-safe mercerization process. The mercerization step was adapted to kitchen-safe alternative by using 10% sodium bicarbonate (NaHCO3) at room temperature for five days instead of 1M NaOH for the alkaline treatment. The goal was to create a mercerization step which would be completely food grade.
In this example, three mercerization procedures were compared: 1) 1 h at 80° C. using sodium bicarbonate, 2) 5 days at room temperature using sodium bicarbonate, and 3) 1 h at 80° C. using NaOH. The objective was to develop a kitchen-compatible process of mercerization that achieves a similar product as the NaOH mercerized counterpart. Sodium bicarbonate, commonly known as baking soda, is a potential alternative to 1M NaOH that cannot be used in a kitchen. Acetic acid, also known for many applications in food, was used as the acid to neutralize the mercerized product.
The goal was to evaluate if 10% sodium bicarbonate is a viable alternative to 1M NaOH. Microscopy images of the three samples were compared to determine particle size. FTIR was also performed on the three samples.
The characterization of the products shown in
The treatment utilizing 10% Sodium bicarbonate for Mercerization during 1 h at 80° C. created a lot of carbonation after neutralization with acid, needing a decarbonization step before the centrifugation. The physical appearance was similar to previous NaOH mercerized AA samples with minor differences. Sodium bicarbonate mercerized AA was slightly more liquid, had a minor green/yellow colour, and was less opaque than NaOH mercerized AA.
The treatment utilizing 10% Sodium bicarbonate for Mercerization during 5 days at room temperature also demonstrated the need for pressure release before the centrifugation. On the fifth day, the mixture of decell apple and 10% sodium bicarbonate exhibited a more pronounced colour (red/brown) compared to the 10% Sodium bicarbonate for Mercerization during 1 h at 80° C. treatment. Also larger apple chunks were observed, and more precipitation than treatment B.
Taken together, the mercerization step utilizing 10% Sodium Bicarbonate resulted in a Mer AA having a similar structure to the NaOH treatment according to microscopy images and chemical structure analysis. In addition, no difference (P>0.05) could be observed between the two sodium bicarbonate treatments.
FT-IR analysis showed similar trends in all 3 samples, indicating that there are similarities in the functional groups present, meaning bicarbonate mercerization could be used as a viable alternative to NaOH mercerization in the kitchen. FTIR analysis presents the peaks in the range 3600 to 2925-cm-1 which are associated with free O—H stretching vibration of the OH groups in the cellulose molecules and hydrogen bonded OH stretching vibration. Peaks between 2925 to 2880 cm-1 correspond to aliphatic saturated C—H stretching associated with methylene groups in cellulose. Lignin can also be assigned a broad region including an interval 3300-3600 cm-1 (intramolecular hydrogen bond in phenolic groups, OH stretching of alcohols, phenols, acids and weakly bounded absorbed water). In addition, lignin is composed of three basic units, namely p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) [78]. Guaiacyl (G) and syringyl (S) are the main units of lignin, but the ratio of S/G varies from one to another plant.
The bands at the 1241 cm-1 and 1317 cm-1 can be assigned as G-ring stretching and S ring stretching respectively. The presence of the band at 1241 cm-1 (C—O stretching vibration in xyloglucan) only in raw and decellularized cellulose shows the process realized with bicarbonate was effective to remove lignin.
The absorption in FTIR spectra including an interval between 1750-1700 cm-1 (C—O stretching in unconjugated groups) reflects changes in various functional groups in lignin and hemicelluloses (carbonyls, ester groups, ketones, aldehydes, carboxylic acids). The absence of the band at 1740 cm-1 in mercerized and bicarbonate material also confirms that our process was effective to remove lignin and hemicellulose from the raw and decellularized material.
The band at 1628 cm-1 can be assigned to the absorbed water and the band at 897 cm-1 which is specific to glucose ring stretching vibration decreased slightly in samples obtained from both processes. It may be due to thermal degradation of β-(1,4) glycosidic bonds. Decreasing the absorption at this band indicates a decrease of the amorphous form of cellulose.
The Mer AA single cells Feret diameter was smaller (P<0.05) in NaOH-Control when compared to both bicarbonate mercerized samples. In addition, the bicarbonate treatments did not demonstrate a difference in feret diameter with each other (P>0.05). The NaOH control demonstrated a normal distribution, whereas a slight left skew was observed for the RT and heated bicarbonate. For all three mercerizations, every particle was under 500 μm.
In this example, the sample preparation was adapted to the kitchen (Corer and food processor) and the resulting Mer AA was characterized. The bleaching step was also adapted by comparing 15% H2O2 stock solution to 30% H2O2 stock solution.
The samples were then prepared and analyzed utilizing the following setting: Range-start 4000.0 and End: 400.0; scan: 32; Resolution:2.
The Reduction of H2O2 stock solution concentration in the bleaching step of the apple mercerization was attempted and the characterization of the final material was performed. Based on the results, it is possible to infer that the yield of the Mer AA aerogel prepared with 15% H2O2 stock solution was 25%. Also comparing 10% Bicarb with 15% H2O2 stock solution, 10% Bicarb with 30% H2O2 stock solution, and 1M NaOH with 30% H2O2 stock solution, the FTIR analysis demonstrated that the chemical structure of the Bicarb Mer AA treated with different stock solutions concentration of H2O2 was similar. Moreover, the particle cells analysis demonstrated that the Mer AA single particles Feret diameter was smaller (P<0.05) in NaOH-Control when compared to Bicarb 30% and Bicarb 15% hydrogen peroxide stock solutions. Also, the two bicarbonate treatments did not differ between each other with regards to feret diameter (P>0.05). Thus, the reduction of H2O2 final concentration for the bleaching step did not demonstrate any major differences compared to the control.
In this example, entire fabrication of a food grade biomaterial was performed in the kitchen with kitchen equipment and food grade chemicals. The steps for the edible biomaterial included:
A-Decellularization-Performed in a 5-day process
The container is labelled and identified with both the material and solution's Spiderwort lot number and expiry date, along with the researcher's initials, and the date.
The decellularized apple was mercerized in a large pot on a gas stove with a temperature probe to ensure minimal variance in mercerization temperature. The solution was mixed manually.
A solution of a 10% Sodium bicarbonate was freshly manufactured and added to a clean pot. The temperature was raised to 80° C., followed by the addition of the decellularized apple.
On this step, the mercerized apple was mixed with a texturizing agent, followed by a cross-linking.
In this example, different cooking methods on the biomaterial were tested to investigate the effect of physical and organoleptic properties including mass yield, visual appearance, and microscopy.
Different cooking methods on the scaffold were performed to investigate the effect on physical and organoleptic properties including mass yield, visual appearance, and microscopy. Three different cooking methods were tested (Sous vide, pan-fry, and baking) and the yield, and microscopy were analyzed. The mass yield had a large variation between methods with the following numbers: sous vide-74.29%, pan-cooking-59.94%, and baking-32.87%, with the Sous Vide demonstrating the greater yield (p<0.05). The results are summarized in the table below. Moreover, a browning was observed when fried with oil. Specifically, the highlights for each treatment considering the yield, microscopy and visual characteristics were for baking: samples began shrunk in size, no browning occurred, had the lowest average mass yield of the three after cooking was 32.87%, samples were opaque white, cooked puck was dry on the outside while the interior was still gel-like. Pan-cooking: non-uniform shape of pucks lead to uneven browning, average mass yield of 59.94%. Sous vide: little change in appearance after vacuum sealing and cooking, puck was translucent white in colour, browning occurred during searing, greatest average mass yield of 74.29%. All samples were gel-like internally after cooking.
In this example, the apple processing was performed in the kitchen and the decellularization, and mercerization in the lab. The goal was to evaluate the colour, odour, and tactile characteristics (texture) of the biomaterial produced with 10% sodium bicarbonate and 15% hydrogen peroxide stock solution.
A sensory analysis panel was conducted to gain insight into the sensory characteristics of the Spiderwort Inc. biomaterial to determine the characteristics of colour, odour, tactile parameters, flavour and texture. Also, analyze the potential presence of any residual flavours from processing such as SDS, CaCl2), sodium bicarbonate, and any acidic neutralizing agent (acetic acid, citric acid), as well as to understand how various cooking methods affect the flavour and mouthfeel of the product. For this objective two different sensory analyses were conducted. For the second analysis, the entire process for the biomaterial fabrication was performed in the kitchen.
The first sensory test (the biomaterial was fabricated utilizing the Mer AA 138) was composed of two different parts: First one called “trick station” where the panellists did not know which one of two samples displayed was the biomaterial and an adapted paired comparison test was performed. On the second part of the sensory analysis test, the panellists had the identification of the biomaterial, references (cod and squid), and the colour, odour, and tactiles characteristics were analyzed. The Mer AA 138 utilized for the first sensory analysis scaffold fabrication presented a yield of 25.13%, compared to raw apple and 45.94%, compared to decell apple. On the first test, 9 panelists participated (4 Female and 5 Male, ranging from <20 to 50 years old). From the panelists that participated on the adapted paired comparison test, over 50% of the total panelists were not able to get the perfect scoring combination for both cooking methods, with 28.54% of wrong answers in each cooking treatment. Also, it is possible to infer that the cooking treatment was not an effect (Deep fry and Sous Vide). In addition, for the characterization of colour, odour, and tactile parameters (raw and cooked), words were generated and the most frequent were selected to describe the parameter as follows:
Based on the results, the baseline formulation had a good starting point. Over 50% of the total panelists were not able to get the perfect scoring combination for both cooking methods, demonstrating the potential of the material to mimic the meat matrix. Also, analyzing the most common comments it is possible to infer that the addition of vegetable protein would be beneficial to the formulation, giving the material more elasticity, more natural shape, and a less translucent colour.
The results are summarized in the tables below.
In this example, gustatory sense was used to evaluate the taste, and texture characteristics of the biomaterial produced with 10% sodium bicarbonate and 15% H2O2. All steps of decellularization and mercerization were adapted to the kitchen. The goal was to evaluate not only the presence of any residual taste of sodium bicarbonate, H2O2, and citric acid, with the goal of developing new formulations.
On the second sensory test (the biomaterial was fabricated utilizing the Mer AA 139), the panellists had the identification of the biomaterial, and references (different references for flavour and texture). The objective was to use the references to help in the sensory analysis and characterization of taste and texture parameters. The Mer AA 139 utilized for the second sensory analysis scaffold fabrication presented a yield of 21.30%, compared to raw apple and 62.71%, compared to decell apple. On the second test, 7 panellists participated in the sensory analysis test (4 Females and 3 Males, ranging from <20 to 50 years old). The beef flavour was the most frequently flavour observed in the scaffold submitted to the Sous vide cooking method, potentially due to an association with the Maillard reaction which can occur after the searing using butter (reaction between the carbonyl group on a sugar and amino group) (Boekel et al., 2006). Also, a fish/scallop flavour was noticed in the Sous Vide and deep fry treatments. The oil/butter was the most noticeable in the deep fry treatment, while a residual sodium bicarbonate taste was noticed in the oven treatment potentially due to the concentration of the bicarbonate, demonstrating the necessity for more washing steps to avoid this taste. Based on the results, regarding flavour, the scaffold demonstrated the ability to efficiently absorb flavours.
Regarding texture, the texture characteristic most noticed in the Sous Vide and Deep fry treatment was the succulence whereas, in the oven method was the dryness. In addition, a texture parameter detected in all three treatments was the cohesiveness. The deep fried scallops and biomaterial were the most similar ones. Analyzing the most common comments it is possible to infer that the addition of vegetable protein would be beneficial to the formulation, giving the material more elasticity, more natural shape, and a less translucent colour. Also a deeper investigation regarding the “beef” flavour should be valuable for product development.
In this example, different techniques to create fibres in the scaffold resembling the fibres found in meat were used to develop a product with a unique texture.
The objective was to use the unidirectional freezing technique to create aligned porous resembling meat fibres.
The unidirectional freezing represents a technique to create aligned porous resembling meat fibres. The freeze alignment is a technique utilized to produce porous materials with orientation structure in an aqueous solution or slurry of proteins. The dispersion of inorganic particles or polymer in water allied to the growth speed control and ice crystals orientation creates unidirectional porous scaffolds after ice crystals sublimation, leaving pores (Zhang et al., 2005). The concentrations of the polymer, as well as the concentration of the cross-linker, are factors that can affect the alignment and the size of the porous (Wu et al., 2010). The time, pH, and mold apparatus were the factors tested to establish the best condition to acquire the aligned porous looking fibers in the Spiderwort Inc. formulation. All the conditions tested resulted in a sort of aligned porous. However, the best formulation up to the present moment was the mixture of Red beet (10%) in 2% Sodium alginate with Mer palm heart with acidic pH and 4 h in the Unidirectional freezing and placed in a inox cylindrical mold. The inox mold (mold material) clearly affected positively the creation of horizontal aligned porous. Both formulations placed in the inox mold during the UF, demonstrated horizontal alignment porous through all the biomaterial. In the coloured material the horizontal aligned porous like-fibres were more evident and were noticed on the surface and on the center of the biomaterial.
In this example, palm hearts were used to mimic meat.
Heart of palm is a vegetable harvested from the inner core and growing bud of certain palm trees and presents a natural fibrous appearance being broadly used as a vegan seafood option. The peach palm (Bactris gasipaes Kunth) is a tropical palm, source of fruit and heart of palm, the last one consisting of the edible inner core of the palm stem with the following characteristics: cylindrical, soft, tender, and slightly sweet. The peach palm heart is the central part of the palm heart, divided into three parts (basal, central, and apical), which differ hardness. The central portion, considered of higher quality and the most common sold in the market in canned versions, is rich in fibers. The total dietary fiber content of the central part is 45.62 (g 100 g-1), whereas cellulose, hemicellulose, and lignin are 37.76, 5.38, and 0.44 (g 100 g-1), respectively. In addition, a hardness of 2.21(N) and an elasticity of 8.47 (mm) are observed in the central part of the palm heart (Stevanato et al., 2020). Thus, canned decellularized fibres from palm heart (palm heart fibres) were utilized in a tentative way to mimic the fibres observed in the meat. Moreover, the mercerization of the palm heart (longitudinal) was also performed to observe if this process could fabricate a fibrous material which could be utilized in the future. The mercerized palm heart (PH) demonstrated a fibrous appearance with a milk colour, similar to the raw scallop. The fibers noticed in the Mer PH were not apparent in the scaffold.
Vegan products (fish fillet and scallop) were elaborated utilizing the mixture of decellularized PH fibres, Mercerized PH, and 2% Sodium alginate. The “fish fillet” and “scallop” made using the aforementioned demonstrated the appearance of biomaterial similar to the target conventional products and developed a flaky structure resembling fish meat. On the other hand, the biomaterial still presented a spongy texture in the cut potentially due to the concentration of alginate utilized. Alginate (Alg) is a linear copolymer of (1-+4)-linked ß-D mannuronic acid (M) and α-L-guluronic acid (G) residues in varying sequences, considered the main structural component in marine brown algae (Phaeophyceae). The alginate combined with a specific concentration of Ca2+ can create a cohesive or firm gel (Yang et al., 2020).
In this example, sodium alginate was used to glue different pieces and/or layers of scaffold to create a “whole muscle” and test the efficacy of the glue in different types of cooking.
The utilization of different types of GRAS “glue” represents a key step for different plant-based formulations to develop a complex structure mimicking the meat one. The alginates are widely utilized in different food products, due to their unique properties as food additives. Recently, a study at Colorado State University, utilized the alginate to “glue” beef pieces together and demonstrated the capacity of this texturizing to hold the pieces together at ordinary temperatures allowing irregularly shaped pieces of meat to be restructured into a whole muscle, resulting in a more profitable product (Yimin et al., 2018). For that, the 2% sodium alginate was utilized to glue different pieces and/or layers of scaffold to create a “whole muscle”. Also, the efficacy of the “glue” was tested in different types of cooking. The 2% Sodium alginate solution demonstrated efficacy to glue together pieces and/or layers of lyophilized biomaterial. In addition, the sodium alginate as a glue was able to maintain the scaffold glued during pan-cooking and boiling. No colour denaturation was observed on treatment B in the pan-cooking whereas, a heat denaturation was observed during the boiling. The excess of colour was lost during the cross-link on treatment B, but the scaffold did not lose the colour even during the boiling process, confirming previous trials.
In this example, a plant-based version of a fish fillet was developed using Mer AA.
After the Sous Vide, both treatments were pan-fried for 1 min each side.
The utilization of decellularized palm heart allows to reproduce vegan formulation with fibre-like texture such as fish fillet and fish products. The proximate composition of fish fillet and fish product can vary for Moisture from 66.30 to 82.30, for protein from 8.20 to 25.90, for fat from 0.1 to 21.0, and for ash from 0.96 to 2.85 (Reddy et al., 2012; Atanasoff, et al., 2013; Venugopal & Shahidi, 1996). In order to mimic fish fillet or fish product, decellularized palm heart was utilized to reproduce the fibres, the mercerized apple was utilized as the scaffold whereas an isolated vegetable protein (pea protein) was added in the formulation instead of animal protein, and sunflower oil substituting animal oil. The fish muscle is composed of sarcoplasmic proteins such as myoglobin, hemoglobin, globulins, albumins, and various enzymes, considered the most water soluble ones. In addition, other types of proteins constituting the fish muscle are stromal proteins, collagen and elastin, the least soluble fractions (Venugopal & Shahidi, 1996). The mercerized apple was incorporated into the formulation in order to provide a better texture to the final product. For the fish fillet project, one formulation was developed and subdivided into two treatments in which different approaches were performed to test the effect on the final texture. However, both treatments demonstrated similar texture. The utilization of the Lyophilization for the treatment B didn't demonstrate necessary. The texture of the two different treatments were similar to fish products but still not to a fish fillet. Thus, the formulation needs an ingredient to increase elasticity and hardness of the formulation such as the utilization of other types of vegetable proteins or Konjac Flour.
Canning is a widespread technique consisting of a combination of processes such as immersion in acid brine, heat treatment, exhaustion, and hermetic sealing which promotes food preservation and shelf life improvement. However, canning can affect the mechanical properties. Stevanato et al. (2020) demonstrated a decrease in the total fibre and cellulose contents. Also showed a decrease in the mechanical properties for the palm heart decreasing the hardness and elasticity (Stevanato et al., 2020).
A current challenge is to increase the scalability of the products to a commercially viable level. Here we present an alternative method for a continuous feed crosslinking, which involves extruding the material into a crosslinker bath to crosslink “on the go”. As the material exits the extruder, it can be crosslinked in that shape. The screen. mold, or perforated plate which the material passes through dictates the shape of the crosslinked material. The bulk material behind the crosslinked portions remain in the fluid, gel, or sol phase until they are pushed into the crosslinker. As such, the bulk material can be stored in syringes, or other configurations that can be delivered to the shape determiner and crosslinking bath such as pumps and platens. This bulk processing allows for high throughput material manufacturing. It is complementary to the inverse methods of molding and crosslinking around spacers.
Potential applications include, but are not limited to: packaging materials, insulations, sealants (vascular, pleural, gastro, muscular, fascia), nucleus pulposus, tissue fillers, wound repair, meniscus, designer tissues, neural scaffolds.
To test the biocompatibility of the aerogel materials, a study was conducted where the scaffolds were subcutaneously implanted under the skin of Sprague Dawley rats and resected after 4 and 12 weeks. The scaffolds were examined for cell penetrance as well as inflammation.
The results show significant cellular penetrance into the scaffold material, both around and into the centre of the implanted material. More cells can also be seen in the implants resected after 12 weeks, compared to 4. Additionally, no significant inflammation was noted thus suggesting graft acceptance.
The non-directionally frozen scaffolds were sectioned into 5 μm thick sections and stained with H&E and MT. Similarly to the directionally frozen scaffolds, staining revealed that the scaffolds remained at their implant sites throughout the duration of the study and revealed vascularization into the native tissue as early as 4 weeks. The fibrin sealant appears to have degraded and collagen deposition is also present.
In contrast to the directionally-frozen scaffolds, there is significantly more open space not occupied by scaffold or cells. Therefore the amount of cellular infiltration, although more is evident after 12 weeks compared to 4, is still significantly less than directionally frozen scaffolds. This suggests that the cellulose scaffold offers a significantly better support structure for cellular infiltration and migration into the tissue.
Given the highly porous nature and structural linearity of the aerogels, it is contemplated that the aerogels described herein would be suitable for spinal cord injury repair. Small scale transection injury studies assessing the effectiveness and biocompatibility of the scaffolds were performed. Here, directionally frozen aerogel scaffolds were implanted into the transected spinal cord of the rat. This was done to determine whether the material would provide a suitable support structure for axonal regeneration. The biomaterials were implanted in rats for 4 and 12 weeks. A complete spinal cord transection was induced between T9-T10 in Sprague Dawley rats and allowed to dieback for 10 minutes. The distance from the dieback was measured and an appropriately sized aerogel scaffold was cut to size then implanted between the cut ends.
Scaffolds were directionally frozen as previously described with the Pelletier apparatus, followed by lyophilization and crosslinking in calcium chloride solution. After sterilization, 4 mm pieces were punched out from the larger sample producing cylindrical samples. The scaffolds were brought to the surgical theatre in 1× sterile PBS solution, and washed in 0.9% saline solution prior to surgical implantation.
In this example, the aerogel scaffolds was also examined for its ability to serve as a support material for the regeneration of bone tissue, supporting recalcification of surrounding tissues in a rat critical-size bilateral defect model. Here, a trephine was used to generate two 5 mm diameter holes in the cranium of the Sprague Dawley Rat. Once the bone defects were excised, the aerogel formulations were placed within the defect. Overlying skin was sutured and the rat left to recover for a period of 4 to 8 weeks. Specimens were collected at each time point and computational tomography (CT scanning) was performed.
After the rats were allowed to recover for the desired amount of time (e.g. 8 weeks), specimens were collected and scanned with computational tomography (CT). Histology was then also performed.
One or more illustrative embodiments have been described by way of example. It will be understood to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051537 | 10/29/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63107226 | Oct 2020 | US |
