EP4651915A1 - Hydrogels and uses thereof - Google Patents

Abstract

The invention generally discloses a hydrogel material formed of nanocelluloses acrylamide.

Description

HYDROGELS AND USES THEREOF

TECHNOLOGICAL FIELD

The invention generally contemplates hydrogel materials, devices and products implementing same.

BACKGROUND OF THE INVENTION

Hydrogels are highly absorbent, three-dimensional hydrophilic crosslinked polymer networks. Hydrogels have been proposed as potential candidates for many different applications and fields, ranging from artificial muscles, vertebral disk replacements, absorbents, and electroconductive materials. However, many hydrogels exhibit poor mechanical properties, reflected in low extensibility, brittleness, low resilience, irreversible deformation, and lack of biocompatibility, which limit their applications. Many approaches have been attempted to endow hydrogels with these properties. Some of these approaches include utilizing topological polymers, nanocomposites, microgels and double network-based reinforcements.

Hydrogels with nanocomposite reinforcements have generated much attention. Nanomaterials such as carbon nanotubes (CNTs), silica, clay and other inorganic particles have been employed, producing some encouraging results, though mostly at a cost of reduced biocompatibility and potential cytotoxicity.

Recently, application of various cellulose-derived nano particles have emerged as attractive nanocomposite reinforcing agents due to their biocompatibility, abundance and relative ease of its chemical modification. Cellulose is the most abundant biopolymer on earth, and usually functions as a load-bearing component in plants’ cell walls, imparting the cell wall with mechanical strength and robustness. Thus, the main sources of cellulose are plants and plant-based waste such as paper, cotton, cardboard, and agricultural waste. In some cases, it can also be produced by bacteria or animals such as tunicates. Cellulose is composed of linear chains of 1-4 D-glucose, arranged in an ordered fibril structure, which is comprised of ordered crystalline regions linked together by less ordered amorphous areas. Cellulose may be degraded using mechanical shearing methods to Cellulose nano Fibers (CNF), which are comprised of both the crystalline and amorphous regions of the fiber, resulting in particles which are 10-40nm in width and span several microns in length. Further degradation, usually by acid hydrolysis, leads to extraction of the crystalline regions alone, known as Cellulose Nano Crystals (CNC). CNCs are 5-10nm in width and a few micrometers in length, depending on the cellulose source. CNC displays amazing mechanical properties, akin to materials such as carbon fibers and Kevlar, with single fibers displaying a Young’s modulus of 150GPa and tensile strength of 7.5GPa.

Among the possible ways to integrate cellulose-derived nanoparticles as reinforcing components to hydrogels, polymer grafting onto the cellulose back-bone has proven a versatile and effective option. Due to the abundance of hydroxyl groups throughout the cellulose chain, cellulose-derived nano particles are ideal candidates for these types of chemical modifications. A graft copolymer broadly consists of a backbone polymer (main chain) from which one or more monomers of a different polymer branch out, forming long polymeric chains (grafts), and may enable the combination of the leading properties of the materials used in one physical unit. Generally, grafting techniques may be divided into three approaches, which may be used individually or combined: First, “grafting to” in which the end of a pre-formed polymer is coupled to the functional groups of the backbone polymer. Second, “grafting from” in which the propagation of the grafted polymer chains occurs from initiating sites on the backbone polymer. Third, “grafting through” in which the macromonomer of backbone and grafted polymer are copolymerized using a co-monomer, usually by radical polymerization.

The modification of cellulose particles by polymer grafting has been studied extensively and has been used to grant many different properties onto cellulose particles such as increased dispersibility, fabrication of bio-degradable materials and increased processability. Among the various techniques investigated, the formation of free radicals on the cellulose-based particles’ backbone surface through direct oxidation, using oxidizers such as Fenton reagents (Fe^/FFCh) or ceric ions (Ce⁴⁺), have proven the most common and effective. In this method water soluble oxidizers form free radicals on the cellulose backbone in the presence of acid in an aqueous medium, without requiring additional pre-treatments. When employing oxidizing salts such as cerium ammonium nitrate ([NH4]2[Ce(NO3)6], CAN), the radicals formed can initiate the graft polymerization of vinyl and acryl monomers. In addition, many acrylic monomers such as acrylamide (AM) may also readily polymerize by radical methods, yielding a graft polymerization system of covalently-bound cellulose-acrylamide complexes, from which long polyacrylamide (PAAM) chains begin propagating from the particle’s surface, utilizing the principles of both the “grafting from” and “grafting through” approaches.

BACKGROUND PUBLICATIONS

[1] Littunen, K. et al. Free radical graft copolymerization of nanofibrillated cellulose with acrylic monomers. Carbohydr. Polym. 84, 1039-1047 (2011).

[2] Wang, B., Peng, Q., Yan, Y., Ding, Y. & Wang, Z. Biomimetic, strong, and tough hydrogels by integrating cellulose nanocrystals into polymer networks. Ind. Crops Prod. 158, (2020).

[3] Yang, J. et al. Tough nanocomposite hydrogels from cellulose nanocrystals/poly(acrylamide) clusters: Influence of the charge density, aspect ratio and surface coating with PEG. Cellulose 21, 541-551 (2014).

GENERAL DESCRIPTION

Cellulose-based materials have been proposed as reinforcing agents for various nanocomposites. Despite a wide range of compositions, forms and chemistries, many of the reported hydrogel systems suffer from a lack of sufficient mechanical strength and biocompatibility. Fibrillar cellulose-based materials have been proposed as reinforcing agents that may aid in overcoming these challenges. Unlike hydrogel systems reported, and other systems reinforced with cellulose-based materials, a composite of polyacrylamide reinforced with modified cellulose-derived nanofibers, namely cellulose nano crystals (CNC) and cellulose nano fibers (CNF), exhibited superior biocompatibility and mechanical properties. While CNC and CNF display different and sometimes opposing properties, by introducing mixtures of differing material ratios, composites of superior mechanical and rheological properties may be manufactured, opening the door for a greater gamut of uses and fabrication technologies.

As demonstrated herein, hydrogels of the invention have proven biocompatible when seeded with GFP-transfected mouse fibroblasts (3T3s), showing a significant increase in cell viability and proliferation as compared to samples comprised of polyacrylamide alone. Hydrogels of the invention have been manufactured by a metal-initiated, e.g., cerium-initiated graft polymerization of CNC and CNF to a polyacrylamide matrix. Propagation of acrylamide chains grafted on cellulose-derived nanoparticle’s surface is then initiated by radical polymerization and heat resulting in the polyacrylamide matrix depicted in Fig. 1. The long polymer chains were further crosslinked e.g., by adding N,N’ -methylene bisacrylamide (MBA) to form a uniform chemical network of long flexible chains interconnected by homogeneously dispersed cellulose-derived nanomaterials (e.g., in a form of nanoparticles). These nanomaterials act both as multifunctional crosslinkers and as reinforcing agents- resulting in highly extendable, resilient soft polymer hydrogels.

Thus, in a first of its aspects, there is provided a material comprising or consisting of a cellulose nanomaterial grafted with acrylamide, wherein the cellulose material consisting a mixture of both cellulose nano crystals (CNC) and cellulose nano fibers (CNF).

The invention further provides a matrix material comprising or consisting of a polymeric material comprising units of acrylamide, CNC and CNF, wherein said units are polymerized therebetween to provide the matrix material.

Further provided is a material derived or formed by grafting a cellulose nanomaterial with acrylamide monomers, wherein the cellulose nanomaterial is CNC and CNF.

Also provided is a hydrophilic or amphiphilic polymeric network composed of monomers derived from acrylamide, CNC and CNF, wherein each of the monomers is crosslinked to another monomer to provide a covalently-associated polymeric network.

The material of the invention is typically a three-dimensional polymeric structure that is substantially insoluble in water. The material is nevertheless capable of absorbing and retaining large amounts of water and may thus be characterized as hydrogel. In the context of the present invention, the material refers both to a hydrogel in the hydrated state (when it contains water when formed in an aqueous solution, or after hydration) and to a dry hydrogel (when it has been formed in a non-aqueous medium or when it has been dried) containing residual amounts of water, e.g., up to 1% (by weight) or no water at all.

When in a dry form, or in any form capable of absorbing other or additional materials, the hydrogel may absorb a material to be contained or entrapped therein. Typically, entrapment of a material(s) occurs within pores present in the hydrogel. The pores may be of non-uniform or uniform diameters (for example in the nm or micrometric range). In some embodiments, the pores are non-uniform in diameter and may be of diameter between 0.2 and 2 microns. The amount of water or any other material may be absorbed or permeated into the hydrogel, thus acting as a sorbent material.

Thus, in another of its aspects there is provided a hydrogel comprising or consisting of a cellulose nanomaterial grafted with acrylamide, wherein the cellulose material consisting cellulose nano crystals (CNC) and cellulose nano fibers (CNF). In some embodiments, the hydrogel is capable of absorbing at least 10 % by weight of water. In some embodiments, at least 10% of the hydrogel weight is water. In some embodiments, the water content (by weight relative to the total weight of the hydrogel) is 10, 15, 20, 25, 30, 35, 40, 45 or 50%. In some embodiments, the water content is greater than 50%.

In some embodiments, upon hydration, the hydrogel swelled to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 % its dry form. In some embodiments, the hydrogel swelled to between 1000 and 2000 or 1000 and 1500 % of its dry form.

In some cases, the weight of the hydrated hydrogel is 7 to 10 times its dry weight.

As known in the art, CNC and CNF are fibers produced from cellulose. Cellulose is composed of linear chains of 1-4 D-glucose, arranged in an ordered fibril structure, which is comprised of ordered crystalline regions linked together by less ordered amorphous areas. Cellulose may be degraded using mechanical shearing methods to CNF, which are comprised of both the crystalline and amorphous regions of the fiber, resulting in particles which are 10-40 nm in width and span several microns in length and may be further degraded, e.g., by acid hydrolysis, to yield the crystalline regions alone, known as CNC. Unlike CNF fibers, CNC fibers are 5-10 nm in width and a few micrometers in length, depending on the cellulose source. When either or both of CNC and CNF are reacted under metal-induced ring opening reaction and in the presence of acrylic or vinylic monomers (i.e., acrylamide, acrylic acid, vinyl acetate) at least one of the glucose units forming the CNC or CNF chain undergoes radical ring opening and reacts with the acrylamide to obtain the material of the invention. Under the polymerization conditions, it is not necessary for each and every one of the glucose units to undergo ring opening. Additionally, each of the CNC and CNF molecules may undergo different ring opening reactions, at different glucose units along the chain, yielding a mixture of different radical species that are reactive with the acrylamide monomers. In other words, each of the CNC and CNF units may have or comprise one or more glucose ring units that undergoes radical ring opening.

As used herein, each of the terms acrylamide unit”, CNC unit' and CNF unit' means a component of the polymeric material that is derived from acrylamide, CNC and CNF, respectively, following a chemical reaction as disclosed herein. The acrylamide unit is derived from acrylamide and thus in a polymer of the invention may have the structure , wherein each of the dashed lines designates a bond connecting to other units of the polymeric material. CNC and CNF, being different in lengths and structural compositions, similarly contain a plurality of sugar units of the structure . Thus, the CNC unit and CNF unit in a polymer of the invention may be of the form the dashed lines designates a bond connecting the sugar units and wherein the bond extending to AA designates a bond associating to a unit derived from acrylamide (AA). Thus, a polymeric material of the invention generally has a structure of a polyacrylamide that is grafted or formed in the presence of CNC and CNF molecules.

The polymeric material of the invention may be structurally depicted as ed lines designates a bond connecting the other sugar units of the CNC and/or CNF and to other acrylamide units of the material.

The mechanical and rheological properties of polymers of the invention are superior to those observed in other similar systems. Surprisingly, this may be so due to the structural difference existing between the CNC and CNF. While CNF chains are of greater lengths as compared to chains of CNC, and their inherent flexibility act as strong backbones, enabling acrylamide chains to propagate to form a strongly interconnected fiber, the CNC chains act as small crosslinking joints, which enable efficient crosslinking. Putting it differently, it is believed that CNF acts as the main load-baring component which grants the impressive mechanical properties observed for the resulting hydrogels, while CNC also grants some mechanical strength, but mainly contributes to a homogenous dispersion while still being compatible with the system. In other words, while CNF composites exhibit the "strongest" structures, due to the inherent entangling and aggregation of CNF, it forms clumps and inhomogeneous structures, leading to inherent defects. CNC (probably due to its colloidal stability and smaller size) enables the integration of relatively high loadings of CNF to form better homogenous structures that are capable of impressive mechanical performance. The contribution of CNC in aiding CNFs' integration is evident by the rheological measurements shown and discussed below.

As used herein, the term "grafted' or any lingual variation thereof refers to covalently attaching acrylamide monomers to the CNC and CNF backbones, which polymerize, to produce a grafted polymer of polyacrylamide, CNC and CNF. The grafted polymer chains making up the material of the invention may be further crosslinked to each other by using a bridging material such as N,N’ -methylene bisacrylamide (BAM) to form a uniform chemical network of long flexible chains.

All polymeric materials of the invention are polyacrylamide grafted on a nanocellulose consisting of CNC and CNF, wherein the ratio between the acrylamide units to the nanocellulose units (CNC+CNF) and the ratio between the two nanocelluloses (CNC and CNF) may be varied in order to vary or modulate or improve one or more of mechanical, rheological and biocompatible properties of the final polymeric material.

The ratio between the nanocellulose material (CNC and CNF combined) and the acrylamide used in processes of the invention may vary, wherein generally the amount of the acrylamide is greater than the amount of each and both nanocelluloses. Typically, the ratio between the two nanocelluloses (CNC and CNF combined) and acrylamide (designated NCAA ratio) is between 1: 100 and 1:5 NCAA. In other words, the amount of the acrylamide may at least 5 times and at most 100 times greater than the amount of the nanocelluloses combined. In some embodiments, the NCAA ratio is 1: 100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1 :30, 1:20, 1: 15, 1: 10 or 1:5.

The ratio between the CNC and the CNF may also vary. In some embodiments, the amount of the CNC may be increased relative to the amount of CNF in order to increase dispersibility and shear-thinning properties, leading to better homogeneity of the pre-polymerized solution. Higher amounts of CNC relative to NFC lead to increased stretchability and resilience. In such configurations, the ratio amount of CNC to CNF may be between 100: 1 and 10: 1. In some embodiments, the ratio CNCCNF is 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, or 10: 1. In some embodiments, the ratio is 5: 1, 4: 1, 3: 1 or 2: 1 CNCCNF.

In other embodiments, for improving toughness of the polymers, e.g., for increasing polymer capabilities of withstanding harsher mechanical demands at the cost of decreased homogeneity, CNF amounts may be increased relative to CNC. In such configurations, the ratio amount of CNF to CNC may be between 100: 1 and 10: 1. In some embodiments, the ratio CNF:CNC is 100:1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, or 10: 1. In some embodiments, the ratio is 5: 1, 4: 1, 3: 1 or 2: 1 CNF:CNC.

Thus, in most general terms, the ratio CNC to CNF may vary between 1: 100 to 100: 1 CNCCNF. In some embodiments, the ratio is between 5: 1 and 1:5 CNCCNF.

In some embodiments, in a material of the invention comprising acrylamide units, CNC units and CNF units, or in a material formed by metal-mediated radical copolymerization of acrylamide, CNC and CNF, having a ratio NCAA of between 1: 100 and 1:5, the CNC: CNF ratio is between l: 10 to 10: 1 CNCCNF.

In some embodiments, in a material of the invention comprising acrylamide units, CNC units and CNF units, or in a material formed by metal-mediated radical copolymerization of acrylamide, CNC and CNF, wherein the CNC:CNF ratio is between l: 10 to 10: 1 CNC:CNF, the ratio NCAA is between 1: 100 and 1:5.

In some embodiments, in a material of the invention the nanocelluloses are provided at a ratio amount NC: A A of between 1: 100 and 1:5, and the CNCCNF ratio is any one of between 1 : 100 to 100: 1; between 1: 10 to 10: 1; between 1:5 and 5: 1; between 100: 1 and 10: 1; between 1: 100 and 1:5; 1: 100; 1:90; 1:80; 1:70; 1:60; 1:50; 1:40; 1:30; 1:20; 1: 10; 1:5; 100: 1; 90: 1; 80: 1; 70: 1; 60: 1; 50: 1; 40: 1; 30: 1; 20: 1; 10: 1; 5: 1; 4: 1; 3: 1; or 2: 1 CNCCNF.

The invention further provides a process for manufacturing a material of the invention, the process comprising reacting a combination of CNC and CNF, at a predetermined CNC: CNF ratio, in presence of acrylamide under conditions allowing radical grafting and polymerization of the CNC, CNF and acrylamide.

In some embodiments, the process comprises treating a mixture of CNC, CNF and acrylamide, optionally in the presence of N,N’ -methylene bisacrylamide (MBA), and under conditions permitting co-polymerization.

In some embodiments, the process comprises forming a mixture of CNC and CNF at a desired ratio, wherein the mixture is formed by homogenization, e.g., by sonication.

In some embodiments, the homogenized CNC and CNF mixture is treated with acrylamide and optionally with MBA and thereafter treated with a metal ion precursor, such as a metal salt or a metal complex, optionally a cerium metal salt or complex, e.g., cerium(iv) ammonium nitrate ([NH4]2[Ce(NO3)6], CAN), or Fe²⁺ salts in the presence of hydrogen peroxide, under conditions permitting polymerization between the monomeric units (e.g., the acryl units, CNC units and CNF units, as defined) in the mixture.

As demonstrated herein, fabrication of hydrogels of the invention has proven highly tunable across a wide range of mechanical properties and rheological performances, expressing high resilience (~92 %) and elastic recovery (-100 %), high tensile strength (-0.5 MPa) and toughness (-1.9 MJ/m3), and high extensibility. When comparing the different ratios of the nanocellulose materials incorporated there seems to be a mediation between the nanocellulose involved. Incorporation of CNC provided dispersibility and shear-thinning properties leading to better homogeneity of the prepolymerized solution and throughout the fabricated samples, resulting in increased stretchability and resilience. Incorporation of CNF on the other hand resulted in tougher samples, capable of withstanding harsher mechanical demands at the cost of decreased homogeneity. The combination of both nanocellulose materials led to a decrease in the overall solution viscosity which aided in the dispersibility of the nanofibers throughout the matrix before polymerization, properties that may be further tuned to cater to different fabrication approaches such as mold-casting, injection molding and others.

Polymeric materials of the invention may be used as hydrogels. The hydrogels may be provided in a variety of forms, including beads, solid matrixes, blocks, sheets or may be provided as shaped objects of any form. The hydrogels may be useful in a variety of applications and may thus contain one or more additional active or non-active components. The materials are highly biocompatible with a living tissue and provide advantageous effects when positioned in contact with a subject’s tissue, e.g., within a subject’s body or topically on the subject’s skin or external tissues. The hydrogels may also be used as drug release platforms for controllably and effectively delivering drugs and other therapeutic or cosmetic agents to a subject’s tissue.

The hydrogels may be used in vivo, ex vivo or in vitro for a variety of purposes, such as biomaterials, supplying a suitable environment for growth, viability and proliferation of live cells. Such cells may be derived from a variety of tissues such as lung, liver, kidney, thymus, thyroid, heart, brain, pancreas, and the like, and cultured cell populations. Non-limiting examples of cells include fibroblasts, human umbilical vein endothelial cells (HUVECs), chondrocytes and osteoblasts. A surprising feature of the hydrogel or matrix material is that the use of the hydrogel or matrix allows growing and proliferation of cells without the necessity of adding cell attachment promoting elements such as the RGD peptide.

A biomaterial is also provided which is or comprises a hydrogel of the invention. The biomaterial may be used as a substrate for cells and tissue generation. The biomaterial may be used for forming a transplant for implanting in a mammal. The transplant may be configured for transplantation into the mammal. Transplants of the invention may be any implant that comprises a hydrogel of the invention and may be useful in inducing or improving therapeutic conditions benefiting from implantable delivery systems for biologically active and gene therapy products for the treatment of a variety of diseases or medical conditions. The hydrogel may be provided with cells to form a transplant for injection into a subject at a site where the transplant is intended to be placed. This allows a non-invasive transplantation method of cells. The hydrogel may be used as a matrix material for holding and delivering and biomaterials, including without limitation, cells secreting human nerve growth factors, satellite cells, striatal brain tissue, liver cells, bone marrow cells, dopamine-rich brain tissue and cells, cholinergic-rich nervous system cells, adrenal chromaffin cells, cultured epithelium, and cells releasing ciliary neurotropic factors, blood cells, hepatocytes, pancreatic tissue, hemopoietic stem cells, bone marrow, Leydig cells, thyroid cells, pituitary cells, cardiac cells, renal cells and others as disclosed herein (including specific examples of cells such as fibroblasts, human umbilical vein endothelial cells (HUVECs), chondrocytes and osteoblasts).

The hydrogel matrix of the invention may be used for long-term storage and proliferation of cellular tissues. Cells may be frozen in the hydrogel matrix without loss of viability.

Thus, the invention further provides a transplant for implanting in a subject, the transplant comprising cells contained within and/or a surface region of a hydrogel according to the invention.

Also provided is a method of protecting cells, the method comprising adding or contacting or immersing a hydrogel of the invention in a cell culture medium and allowing the cells to be absorbed by the hydrogel.

Further provided is a cell delivery system comprising a hydrogel of the invention comprising a plurality of cells to be delivered or transplanted.

A method is provided for delivery of viable cells to a mammal, the method comprising positioning a cell delivery system at a desired location for cell delivery to a mammal, the cell delivery system comprising a hydrogel according to the invention (used as the delivery system) and cells; wherein the hydrogel containing the cells inside pores formed therein and/or its surface.

Also provided is a device for releasing a material in a recipient subject, in vivo, ex vivo or in vitro, the device comprising a matrix containing the material and having a porosity permitting passage therethrough of the material.

Whether as a device, a drug delivery matrix, a biomaterial or any matrix material, hydrogels of the invention may be loaded to contain (or made to absorb) an active material, a biological sample, a solution, or any material whatsoever by any means suitable in the art. In some cases, the hydrogel may be immersed in a solution containing the material to be absorbed or precursors thereof. In other cases, the hydrogel may be treated under pressure conditions to cause effective absorbance of the material. Additionally or alternatively, the hydrogel may be treated by, e.g., plasma spray or other deposition means to cause the hydrogel to at least partially absorb the material. Surface coating of the hydrogel may also occur. Irrespective of the means, the material may be selected from any of the materials disclosed herein and also amongst active ingredients, diagnostic materials, drugs, coloring materials, amino acids, nucleic acids, peptides, polymers, taggant materials, biomaterials, cells, biological or chemical media, disinfectants, antioxidants, minerals, biominerals, and others.

In some embodiments, the material is selected from hydroxyapatite (HA), cells such as fibroblasts, human umbilical vein endothelial cells (HUVECs), chondrocytes and osteoblasts; and other active materials.

The combination of impressive mechanical performance, tunability of these mechanical properties and biocompatible properties may point to the fabricated composite as a promising candidate for biomedical and scaffolding applications.

Thus, hydrogels of the invention may be generally utilized in:

1. Manufacturing of medical device and implants, such as synthetic heart valves, vascular grafts, cartilage and bone grafts, tendons and ligaments, soft tissue replacement materials, tissue replacement materials, fibers, and others;

2. Manufacturing of material delivery devices for therapeutic or cosmetic agents and uses in vivo, ex vivo or in vitro,'

3. Tissue engineering as substrates for growth of cells and tissues in vivo and in vitro;

4. Biomaterials for use in vivo, ex vivo or in vitro;

5. Wound dressings;

6. Shock absorbing materials;

7. Energy storage devices; and

8. others.

The invention further provides a method of modulating mechanical or rheological properties of a hydrogel formed of CNF grafted with acrylamide monomers, the method comprising:

-in a method of manufacturing the hydrogel of CNF grafted with acrylamide, mixing the CNF with an amount of CNC, wherein the ratio amount of CNC to CNF is between 1:5 and 5: 1. The invention further provides a method of modulating mechanical or rheological properties of a hydrogel formed of CNC grafted with acrylamide monomers, the method comprising:

-in a method of manufacturing the hydrogel of CNC grafted with acrylamide, mixing the CNC with an amount of CNF, wherein the ratio amount of CNC to CNF is between 1:5 and 5: 1.

As used herein, the expression 'modulating mechanical or rheological properties " refers to increasing or decreasing, enhancing or diminishing at least one mechanical or rheological property selected from resilience; elasticity; tensile strength; toughness; compressive strength; extensibility; stretchability; pore size; surface texture or surface roughness; hardness; and Young’s modulus.

In some embodiments, in a method of manufacturing a hydrogel of CNF grafted with acrylamide, wherein the CNF is mixed with an amount of CNC, the improvement is the hydrogel stretchability and resilience.

In some embodiments, in a method of manufacturing a hydrogel of CNC grafted with acrylamide, wherein the CNC is mixed with an amount of CNF, the improvement is the hydrogel toughness.

The invention further provides a medical device or an implant comprising or consisting of a hydrogel of the invention.

The invention further provides:

A material comprising or consisting a cellulose nanomaterial grafted with acrylamide, wherein the cellulose material consisting a mixture of cellulose nano crystals (CNC) and cellulose nano fibers (CNF).

In some embodiments of any of the materials disclosed herein, the material is in a form of a matrix material comprising or consisting a polymeric material comprising units of acrylamide, cellulose nano crystals (CNC) and cellulose nano fibers (CNF), wherein said units are polymerized therebetween to provide the matrix material.

In some embodiments of any of the materials disclosed herein, the material is formed by grafting the cellulose nanomaterial with acrylamide monomers.

In some embodiments of any of the materials disclosed herein, the material is a hydrogel in a hydrated or dry hydrogel form.

In some embodiments of any of the materials disclosed herein, the material is in a dry form and the hydrogel is capable of absorbing at least 10 % by weight water. In some embodiments of any of the materials disclosed herein, the material is in the hydrated form and the hydrogel having a weight that is 7 to 10 times its dry weight.

In some embodiments of any of the materials disclosed herein, the material is in a form of beads, blocks, sheets or an amorphous form.

In some embodiments of any of the materials disclosed herein, the material comprising at least one active or non-active material.

In some embodiments of any of the materials disclosed herein, the material is biocompatible with a living tissue.

In some embodiments of any of the materials disclosed herein, the material is for delivery of an active material to a living tissue in vivo or ex vivo.

In some embodiments of any of the materials disclosed herein, the material is configured for positioning in contact with a tissue in a subject’s body or topically on a subject’s skin.

In some embodiments of any of the materials disclosed herein, the material is for use as a drug release platform for controllably and effectively delivering the drug to a subject’s tissue.

In some embodiments of any of the materials disclosed herein, the material is for causing proliferation of live cells in vivo.

In some embodiments of any of the materials disclosed herein, wherein the cells are fibroblasts.

In some embodiments of any of the materials disclosed herein, the material is for use in a method of manufacturing a medical device or an implant.

In some embodiments of any of the materials disclosed herein, the material is used as an implant that is selected from synthetic heart valves, vascular grafts, cartilage and bone grafts, tendons and ligaments, soft tissue replacement materials, tissue replacement materials, and fibers.

In some embodiments of any of the materials disclosed herein, the material is for use in a method of manufacturing a material delivery device for delivery of a therapeutic or a cosmetic agent in vivo or in vitro.

A process for manufacturing a hydrogel material comprising or consisting a cellulose nanomaterial grafted with acrylamide, wherein the cellulose material consisting a mixture of cellulose nano crystals (CNC) and cellulose nano fibers (CNF), the process comprising reacting a combination of CNC and CNF, at a predetermined CNC:CNF ratio, in presence of acrylamide or grafted acrylamide under conditions causing radical grafting and polymerization < )f the CNC, CNF and acrylamide.

In some embodiments of any of the methods disclosed herein, the ratio CNC:CNF is between 1: 100 to 100: 1.

In some embodiments of any of the methods disclosed herein, the ratio CNC:CNF is between 100: 1 and 10: 1.

In some embodiments of any of the methods disclosed herein, the ratio CNC:CNF is between 5: 1 and 1:5.

In some embodiments of any of the methods disclosed herein, the ratio CNC:CNF is 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 53: 1, 40: 1, 30: 1, 20: 1, or 10: 1.

In some embodiments of any of the methods disclosed herein, the ratio

CNC:CNF is 5: 1, 4: 1, 3: 1 or 2: 1.

In some embodiments of any of the methods disclosed herein, the CNC, CNF and acrylamide are provided at a ratio CNC and CNF, combined, to acrylamide (CNC+CNF:acrylamide) of between 1: 100 and 1:5.

In some embodiments of any of the methods disclosed herein, the ratio CNC+CNF: acrylamide is between 1: 100 and 1:5, and wherein the ratio CNC:CNF is between 1: 10 to 10: 1.

In some embodiments of any of the methods disclosed herein, the ratio CNC+CNF: acrylamide is 1: 100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1: 15, l: 10 or 1:5.

In some embodiments of any of the methods disclosed herein, the method is for increasing the hydrogel toughness, wherein the ratio CNF: CNC is between 100: 1 and 10: 1.

In some embodiments of any of the methods disclosed herein, the method is for increasing the hydrogel toughness, wherein the ratio CNF:CNC is 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, or 10: 1.

In some embodiments of any of the methods disclosed herein, the method is for increasing the hydrogel toughness, wherein the ratio CNF:CNC is 5: 1, 4: 1, 3: 1 or 2: 1.

In some embodiments of any of the methods disclosed herein, the method is for increasing hydrogel stretchability and resilience, wherein the ratio CNC:CNF is between 100: 1 and 10: 1. In some embodiments of any of the methods disclosed herein, the method is for increasing hydrogel stretchability and resilience, wherein the ratio CNC:CNF is 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, or 10: 1.

In some embodiments of any of the methods disclosed herein, the method is for increasing hydrogel stretchability and resilience, wherein the ratio CNC:CNF, is 5: 1, 4: 1, 3: 1 or 2: 1.

In some embodiments of any of the methods disclosed herein, the method comprising treating a mixture of CNC, CNF and acrylamide with N,N’ -methylene bisacrylamide (MBA).

In some embodiments of any of the methods disclosed herein, the method comprising forming a mixture of CNC and CNF at a CNC: CNF ratio, wherein the mixture is formed by homogenization.

In some embodiments of any of the methods disclosed herein, the method comprising homogenizing a CNC and CNF mixture and treating same with acrylamide and MBA, followed by addition of a metal ion precursor.

In some embodiments of any of the methods disclosed herein, the metal ion precursor is a metal salt or a metal complex.

In some embodiments of any of the methods disclosed herein, the metal ion precursor is a cerium metal salt or complex.

In some embodiments of any of the methods disclosed herein, he cerium metal salt is cerium(iv) ammonium nitrate ([NH4]2[Ce(NO3)6], CAN).

A process for modulating a mechanical or a rheological property of a hydrogel formed of CNF grafted with acrylamide monomers, the method comprising:

-in a process of manufacturing a hydrogel of CNF grafted with acrylamide, mixing the CNF with an amount of CNC, wherein the ratio amount CNC: CNF is between 1:5 and 5: 1.

A process for modulating a mechanical or a rheological property of a hydrogel formed of CNC grafted with acrylamide monomers, the method comprising:

-in a process of manufacturing a hydrogel of CNC grafted with acrylamide, mixing the CNC with an amount of CNF, wherein the ratio amount of CNC to CNF is between 1:5 and 5: 1. In some embodiments of any of the methods disclosed herein, the modulating a mechanical or a rheological property comprises improving the hydrogel stretchability and resilience.

In some embodiments of any of the methods disclosed herein, the modulating a mechanical or a rheological property comprises improving the hydrogel toughness.

A hydrogel comprising or consisting a material as disclosed herein as a matrix material.

In some embodiments of any of the hydrogels disclosed herein, the hydrogel comprising an active or a non-active material.

In some embodiments of any of the hydrogels disclosed herein, the hydrogel is for use as a drug release platform for controllably and effectively delivering a drug to a subject’s tissue.

In some embodiments of any of the hydrogels disclosed herein, the hydrogel is for supplying live cells to a tissue.

In some embodiments of any of the hydrogels disclosed herein, the hydrogel is for use in a method of forming a wound dressing, a shock absorbing material, or an energy storage device.

A medical device formed of a hydrogel according to the invention.

An implant formed of a hydrogel according to the invention.

In some embodiments of any of the hydrogels disclosed herein, the hydrogel is for use or preparation of an implant selected from synthetic heart valves, vascular grafts, cartilage and bone grafts, tendons and ligaments, soft tissue replacement materials, tissue replacement materials, and fibers.

A substrate for tissue engineering for growing cells and tissues in vivo or in vitro, the substrate being formed of or comprising a material according to any the invention.

A biomaterial formed of, or being, or comprising a material according to the invention. The biomaterial may comprise a mineral, e.g., hydroxyapatite.

Also provided is a biomineralized material being or comprising a material according to the invention, wherein optionally the biomineralized material comprises hydroxyapatite. The biomineralized material may further comprise at least one active ingredient or cells, as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 provides a depiction of a CNC/CNF grafted with acrylamide.

Fig. 2 is an illustrated overview of a fabricated hydrogel and its opacity in variable compositions. C1F0 fabricated with the addition of CNC alone, C0F1 fabricated with the addition of CNF alone, and C3F1, C1F1 and C1F3 fabricated with CNC and CNF at 3: l, 1: 1 and 1:3, respectively.

Figs. 3A-E demonstrate mechanical properties (tensile) of fabricated cellulose- based hydrogels. (A) Tensile stress-strain curves, (B) elongation at break, (C) ultimate tensile stress UTS, (D) toughness and (E) Young’s modulus of different hydrogels fabricated with variable composition of CNC and CNF.

Figs. 4A-E demonstrate cyclic tensile loading. Cyclic tensile tests of different fabricated hydrogel samples namely (A) C1F3, (B) C3F1, (C) C1F1, (D) C0F1 and (E) C1F0, with CNC and CNF in variable compositions.

Fig. 5 demonstrates viscosity as a function of shear rate for pre-polymerized solutions with varying ratios between CNC and CNF before polymerization.

Figs. 6A-B provide rheological measurements of the fabricated hydrogels composites represented in loss modulus G" and storage modulus G' with respect to frequency for samples C1F0 and C0F1 (A) and C1F3, C1F1, C3F1 (B).

Figs. 7A-E provide rheological measurements of the fabricated hydrogels composites represented in loss modulus G" and storage modulus G' with respect to amplitude for different fabricated hydrogel samples namely (A) C1F3, (B) C1F1, (C) C3F1, (D) C1F0 and (E) C0F1, with CNC and CNF in variable compositions.

Figs. 8A-B show swelling and degradation profiles of fabricated hydrogels in physiological conditions of pH 7.4.

Figs. 9A-D provide cytocompatibility of CNC-CNF and acrylamide-based hydrogels with mouse fibroblasts, 3T3s (transfected with GFP). (A) Confocal micrographs taken after culturing 3T3s-GFP onto the hydrogels for a week. The images are representative images of overlapped z-sections. (B) Confocal image for sample C1F3 represented in 3D view depicting the depth coverage by the cells. (C) Area covered by cells in individual hydrogels calculated from the confocal images using Image J. Statistical analysis performed by Tukey test, where ** represents p<0.001 (n=3). (D) Estimation of cellular proliferation and viability determined by the MTT assay with cell laden hydrogels after day 1 and day 7 of culture with mouse fibroblasts. Statistical analysis performed by 2-way ANOVA, where *** and ns represent p<0.001, and p=not significant, respectively (n=3).

Fig. 10 depicts representative compressive properties of fabricated hydrogel compositions.

Fig. 11 depicts representative compressive properties of fabricated hydrogel compositions with different MBA content.

Figs. 12A-B depict proliferation and cytotoxicity of endothelial cells cultured onto the fabricated hydrogels as determined by MTT assay (A) and under confocal microscopy (B).

Figs. 13A-C depict sterilization method comparison. (A) Relative cellular metabolic activity evaluated by alamarBlue at set time intervals of 1-, 7-, 14- and 21- days post cell-seeding for samples sterilized by EtOH, Autoclave and EtO. (B) Comparison of sterilization methods on cellular growth on sample C1F1. (C) confocal microscope images of (GFP) transfected mouse fibroblasts (3T3s) on the seeded C1F1 scaffolds following different sterilization techniques. Scale bar 100 pm.

Fig. 14 provides sterilization method comparison. Relative cellular metabolic activity evaluated by alamarBlue at set time intervals of 1-, 7-, 14- and 21 -days post cell-seeding for samples sterilized by EtOH and y-irradiation.

Figs. 15A-B provide a representative tensile (A) and compressive (B) properties of sun-, UV-, and heat-induced polymerization.

Figs. 16A-C provide a representation of biomineralized samples. (A) comparison between an unmineralized (left) and mineralized (right) samples. (B) Biomineralized sample cut in half showing HA penetration and distribution. (C) Representative compressive of Biomineralized samples.

Figs. 17A-D provide Cryo-SEM and EDS-mapping of biomineralized samples. (A) Cryo-SEM, 5K magnification scale bar 1 pm. (B) Cryo-SEM, 10K magnification, scale bar 1 pm. (C) Visual representation of sections I, II and III evaluated for EDS under SEM, scale bar 200 pm. (D) Visual representation of sections I, II and III evaluated for EDS, scale bar 1 mm. The table provides EDS elemental distribution of carbon (C), oxygen (0), calcium (Ca) and phosphorus (P) withing the three designated sections within the hydrogel.

Figs. 18A-B demonstrate: Fig. 18A- Pre-polymerized samples with increased cerium content. From left to right lx (control), 2x, lOx and 50x CAN content. Fig. 18B- a lOx CAN polymerized sample- resulting in a soft, pasty, and easy to break matrix.

Fig. 19 provides representative tensile tests of increased CNP content samples.

DETAILED DESCRIPTION OF EMBODIMENTS

Materials and Methods

Materials

Cellulose nanocrystals were supplied by Melodea Ltd. (Rehovot, Israel); cellulose nanofibrils were supplied by Cellulose Lab (Fredericton, New Brunswick, Canada). Acrylamide (AM), N,N’ -methylene bisacrylamide (MBA), cerium(IV) ammonium nitrate ([NH4]2[Ce(NO3)6],CAN), calcium chloride (CaCh), and dipotassium hydrogen phosphate (K2HPO4) were purchased from Sigma-Aldrich. Hydrochloric acid (HC1) was purchased from Romical (Beer Sheva, Israel).

Hydrogel Fabrication

Hydrogels were fabricated based on cerium-initiated graft copolymerization by using ceric ions to initiate polymerization of acrylamide grafted from the cellulose particle backbone. CNC:CNF mixture suspensions were prepared by mixing CNC and CNF suspensions (3 wt% each) at 1:3, 1 : 1, and 3: 1 (wt/wt) CNC to CNF and homogenized by sonication using a Q500 ultrasonic processor (Qsonica, Newtown, CT, USA) at 80% amplitude 1 s on/1 s off pulse regime for a total of 30 min. The CNC:CNF solutions were then diluted to 1.6 wt% with AM (0.07 mol) and MBA (0.03 mmol) to a total volume of 20 mL. Next, an initiator solution was prepared by dissolving CAN (0.036 mmol) in 5 mL double distilled water and adjusting the pH to 1 using 1 M HC1. The initiator solution was added dropwise to the cellulose -acrylamide mixture by stirring and poured into molds. Polymerization occurred at 55 °C for 12 hr. Fabricated hydrogels were carefully removed for further testing. The resulting series of compositions were labeled according to their CNC to CNF content ratio, and categorized, namely C1F3 (1:3), C1F1 (1: 1), C3F1(3: 1), C1F0 (1:0), and C0F1 (0: 1), where C and F represent CNC and CNF, respectively. Samples were immersed in All samples were immersed in phosphate buffer saline (PBS, (150 mM NaCl, 2.5 mM KC1, 10 mM K2HPO4, 2 mM KH2PO4, pH7.4) until reaching equilibrium prior to testing.

Mechanical testing (tensile)

Tensile tests were performed using a tensiometer (Instron 3345 tester, Norwood, MA, USA) equipped with a 10 ON load cell, using a crosshead speed of 10 mm/min on dumbbell samples (length=65 mm; width=10 mm; thickness=2 mm; gauge length=10mm; inner width=4 mm). Dimension measurements were conducted by a digital caliper (0.1 mm resolution). Each sample was elongated until failure. Stressstrain curves were plotted based on 4 repeats of each composition. Sample elongation and ultimate tensile stress (UTS) were recorded, toughness was defined as the area under the extension curve and the Young’s modulus was calculated by the slope of the linear elastic region.

Cyclic tensile tests were performed using a tensiometer (Instron 3345 tester, Norwood, MA, USA) equipped with a 100 N load cell, using a crosshead speed of 100 mm/min on dumbbell samples (length= 65 mm; width=10 mm; thickness=2 mm; gauge length=10 mm; inner width=4 mm). Dimension measurements were conducted by a digital caliper (0.1 mm resolution). Each sample was subjected to a 4-cycle extension at 100 % increments until reaching mechanical failure. Resilience was calculated from the second, third and fourth extensions of each cycle, by the ratio between the areas under the unloading and loading curves.

Rheology

The rheological properties of fabricated hydrogels were evaluated using a Kinexus PRO+ rheometer (Netzsch, Selb, Germany). Samples were tested using parallel plates that were 40mm in diameter at 25 °C. Solutions composed of different CNC:CNF ratios before polymerization were evaluated by viscosity as a function of shear-rate measurements, performed by ramping up the shear rate from y =0. 1 1/s to y = 1000 1/s at 25 °C, using 40 mm plates with a plate gap of 0.5 mm. Polymerized hydrogel samples were evaluated by frequency and amplitude sweeps. Frequency sweeps were preformed from 0.1 to 100 Hz at a fixed strain of 1%, and the storage (G’) and loss (G”) moduli were recorded. Amplitude sweeps were performed at a constant 1.33Hz, at a sheer strain (y(%)) in ranges between 0.1 and 100%. The linear viscoelastic region (EVER), yield point (xy), flow point (if) and the flow transition index (rf/ry) have been calculated in all samples.

Compression

Compression tests were performed using a tensiometer (Instron 3345 tester, Norwood, MA, USA) equipped with a 100 N load cell, using parallel compressive plates on cylindrical samples (diameter= 10 mm; height = 10 mm). Dimension measurements were conducted by a digital caliper (0.1 mm resolution). Each sample was compressed by 50% its original height 4 consecutive times. Stress-strain curves were plotted based on 3 repeats of each composition. Ultimate compressive stress (UCS) was recorded, Resilience was calculated from the second, third, and fourth extensions of each cycle, by the ratio between the areas under the unloading and loading curves.

Swelling and degradation studies

Swelling and degradation tests were conducted in physiological conditions by submerging hydrogel specimens in phosphate buffered saline (PBS, 150 mM NaCl, 2.5 mM KC1, 10 mM K2HPO4, 2 mM KH2PO4, pH7.4). For swelling analysis, samples were incubated in PBS for a fixed duration and weighed after gently removing the excess liquid from the surface. The samples were returned to PBS until the next reading. The procedure was repeated till a plateau was achieved for the swelling.

Degradation of the samples was determined by incubating the samples in PBS for 6 weeks with regular change of PBS and calculating the dry weight of the samples once per week. Prior to weighing each sample was washed using DI water to remove excess salts from the material. Both experimentations were performed in triplicates.

Cell culture

Maintenance of animal cells and sample preparation for biocompatibility studies

Green fluorescent protein (GFP) transfected mouse fibroblasts (3T3s) were generously donated by Professor Dr. Sharon Elizur-Schlesinger (The Hebrew University of Jerusalem). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing L-glutamine, glucose, sodium pyruvate, and sodium bicarbonate. Media was supplemented with fetal bovine serum to a final concentration of 10 % and antibiotics i.e., penicillin (10 Units/mL) and streptomycin (10 pg/mL) to minimize possible contamination. The cells were maintained in a humidified environment of a sterile cell culture incubator maintained with 5 % carbon dioxide at 37 °C. The media of the cultured cells was regularly changed, and cells were passaged after achieving 70 % confluency.

The hydrogels were sterilized with 70 % ethanol before cell seeding. In brief, the fabricated hydrogels were incubated in 70 % ethanol for 30 min and irradiated with UV simultaneously in a sterile cell culture hood. Samples were subsequently washed thrice with sterile phosphate buffer saline (PBS, calcium, and magnesium free, pH 7.4) with a 20 min incubation in each wash to remove any traces of ethanol. Then, samples were acclimatized with DMEM media overnight. The media was removed, and hydrogels were dried for 4 hours in a sterile cell culture incubator before adding the cells to enhance the efficiency of static cell seeding (5x l0⁴ cells/hydrogel) onto the porous hydrogel.

Analysis of cellular toxicity and proliferation

Proliferation and morphology of the cells onto the fabricated hydrogels was evaluated using a confocal microscope. As the 3T3s were transfected with GFP, no additional staining was conducted. A week post cell seeding, the cell laden hydrogels were fixed with 4 % paraformaldehyde, washed thrice with PBS for 10 min each and observed under a confocal microscope (exact specifications will be added). The area covered (%) by the cells with respect to the total area in each hydrogel composition was calculated by ImageJ using the confocal images obtained post culturing the cells for a week. Cellular migration was measured for composition C1F3 through the 3D view observed in confocal microscopy. Cellular proliferation and viability were determined for the cell laden hydrogels by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay that calculates the metabolic activity of the cells and provides an indicator for cellular viability. The procedure followed for the assay was as per the manufacturer’s protocol. In brief, the hydrogels were transferred to fresh wells to avoid any reading from cells that were not attached to the hydrogels. These cell laden hydrogels were then incubated in MTT dye for 4 h. The purple-colored product formed upon the reduction of MTT dye by cellular oxidoreductase is dissolved in dimethyl sulfoxide (DMSO), and the colorimetric reading was obtained in a spectrophotometer (exact specifications will be added) at 595 nm.

Human umbilical vein endothelial cells (HUVECs) were grown in endothelial cell growth medium supplemented with fetal calf serum (0.02 mL/mL), epidermal growth factor (5 ng/mL), basic fibroblast growth factor (10 ng/mL), insulin-like growth factor (20 ng/mL), vascular endothelial growth factor (0.5 ng/mL), ascorbic acid 1 pg/mL, heparin 22.5 pg/mL and hydrocortisone 0.2 pg/mL. HUVECs were cultured in sterile humidified environment of cell culture incubator maintained at 37 °C and 5 % carbon dioxide. Media was replenished every alternate day and cells were passaged at 70 % confluency. Passage 4-6 were used in all experiments. A combination of ethanol and UV irradiation was used to sterilize the fabricated hydrogels. Briefly, the hydrogels were treated with 70 % ethanol for 30 min followed by three washes with sterile phosphate buffer saline (PBS, pH 7.0) for 20 min each in a sterile environment of cell culture hood. The hydrogels were incubated in the media overnight for acclimatization, dried for 12 h, and irradiated with UV for 30 min before cell seeding (1 x 105 cells/ hydrogel).

A preliminary in vitro screening with human endothelial cells was conducted. The proliferation and viability test by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was carried out as per the manufacturer’s protocol. After pre-determined days of cell seeding (day 1, day 7, day 14 and day 21), the cell laden hydrogels were transferred to fresh wells to avoid any reading from the unattached cells and incubated in MTT dye for 4 h. The purple-colored product formed upon the reduction of MTT dye by cellular oxidoreductase is dissolved in dimethyl sulfoxide (DMSO) and the colorimetric reading was obtained in a spectrophotometer at 595 nm. The morphology of HUVECs cultured on the hydrogels for 2 weeks was observed by staining the F-actin filaments and nuclei with high-affinity phalloidin conjugated to tetramethyl rhodamine (TRITC) dye (Actin-555) and 4',6-diamidino-2- phenylindole (DAPI), respectively as per the manufacturer’s protocol. The stained samples were observed under the confocal microscope performed using a Leica DMI8 Stellaris 5 STED confocal system (Leica, Wetzlar, Germany) with the LAS X life science software (LAS X 4.4.0.24861; Leica, Wetzlar, Germany). Cell culture maintenance and sample preparation for biocompatibility studies (GFP-3T3)

Green fluorescent protein (GFP) transfected mouse fibroblasts (3T3s) were donated by Professor Dr. Sharon Elizur-Schlesinger (The Hebrew University of Jerusalem). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing L-glutamine, glucose, sodium pyruvate, and sodium bicarbonate. Media was supplemented with fetal bovine serum to a final concentration of 10 % and antibiotics i.e., penicillin (10 Units/mL) and streptomycin (10 pg/mL) to minimize possible contamination. The cells were maintained in a humidified environment of a sterile cell culture incubator maintained with 5 % carbon dioxide at 37 °C. The media of the cultured cells was regularly changed, and cells were passaged after achieving 70 % confluency.

Sterilization techniques

Ethanol solution

Sterilization with ethanol (EtOH) were performed by immersing hydrogel samples in 70% v/v EtOH at room temperature (RT, 25 °C). Next, samples were washed thrice with sterile phosphate-buffered saline (PBS, calcium, and magnesium free, pH 7.4) with a 20 min incubation period in each wash to remove traces of EtOH.

Ethylene oxide

Sterilization with ethylene oxide (EtO) was performed in accordance with ISO- 11135, for 8 h at 45°C and 40%-55% relative humidity. Samples were purged with N2 several times to remove residual EtO and stored at room temperature for at least 1 week. EtO sterilization was carried out by Mediplast Israel Ltd., Yavne, Israel.

Heat sterilization (autoclave)

Sterilization by heat was conducted by autoclaving. Samples were immersed in PBS (150 mM NaCl, 2.5 mM KC1, 10 mM K2HPO4, 2 mM KH2PO4, pH7.4) and exposed to 121 °C for 15 min. y-irradiation

Sterilization by y-radiation was performed by irradiation at 40°C with an average dose of 28 kGy and a nominal dose rate 7 kGy/h. 60Co gamma-ray source was used. Sterilization was carried out by Sor-Van Ltd. Soreq, Yavne, Israel.

Cellular metabolic activity assay (alamarBlue)

Cellular metabolic activity was evaluated using alamarBlue (Biorad) at set time intervals (1-, 7-, 14- and 21-days post cell-seeding) using 4 biological repeats. Prior to the assay, the scaffolds were placed in a 48-well plate to reduce the chance of detecting the metabolic activity of detached cells that may have adhered to the bottom of the wells. Next, alamarBlue was used according to the manufacturer's protocol. Scaffolds were incubated in 1 ml of 10% alamarBlue reagent in growth medium for 4h, at 37°C, 5% CO2. Following incubation, lOOul samples were transferred into a 96-well plate and fluorescence was measured by a 535 nm excitation and a 595 emission, using a plate reader. Results are reported as the fold change in dye reduction at each time point after normalization to blank (scaffolds without cells).

Confocal microscopy

Proliferation and morphology of the cells on the fabricated hydrogels was evaluated using a confocal microscope. Imaging was performed using a Leica DMI8 Stellaris 5 STED confocal system (Leica, Wetzlar, Germany) with the LAS X life science software (LAS X 4.4.0.24861; Leica, Wetzlar, Germany). 21 days post cell seeding, the cell-laden hydrogels were fixed with 4% paraformaldehyde (PFA), washed thrice with PBS for 10 min, and imaged.

Results and discussion

Hydrogel fabrication

Composite hydrogels were fabricated by redox-initiated free radical graft polymerization of AM and cellulose-derived particles. First, grafting of the AM onto the particles’ surface was initiated using Ce⁴⁺ as an oxidizer, which generates free radicals form the surface of the cellulose-derived particle, leading simultaneously to the formation of grafted cellulose particles and to the propagation of polyacrylamide (PAAM) chains from dissolved AM monomers. Propagating chains are then further crosslinked with BAM, which led to the formation of the uniform hydrogels. The resulting hydrogels ranged from completely transparent when integrating CNC alone (C1F0) to completely opaque when comprised of CNF only (C0F1) (Fig. 2).

Tensile testing

Mechanical properties of the fabricated hydrogels were evaluated by tensile tests. Typical tensile stress-strain curves are presented in (Fig. 3) and measured mechanical properties are summarized in (Table 1). Composites fabricated with the integration of CNC alone namely C1F0, expressed moderate UTS, recorded at 107 kPa, and an elongation at break of 441 %, while displaying a young’s modulus of 23 kPa. Conversely, composites fabricated with the integration of CNF alone namely C0F1, resulted in a hard and non-uniform gel, displaying a three-fold increase in young’s modulus, recorded at 307 kPa, and a high UTS of 390 kPa, but extend by only 105 %, the lowest value recorded. As these cellulose-derived particles differ in aspect ratio, rheological properties, and colloidal stability, these results demonstrate the manner of which the addition of either of these particles, using this proposed grafting system, may affect the range of mechanical performance achievable by these types of composites. Samples comprised of CNC:CNF mixtures seem to benefit from each of the cellulose- based components. In this manner, the incorporation of a 1:3 ratio of CNC to CNF namely sample C1F3, expresses an increase in elongation, UTS and toughness as compared to C0F1, recorded at 229 %, 527 kPa and 624 kJ/m³ respectively. Of note, out of the three CNC: CNF ratios evaluated, C3F1 results in a highly extendable gel, capable of reaching 687 % extension and achieving a UTS of 483 kPa. Notably, this composition yields the toughest hydrogel of all samples tested, reaching a calculated toughness of 1927 kJ/m³. These results suggest that by tailoring the ratios of added CNC or CNF a range of mechanical performance is displayed indicating the tunable mechanical nature of the fabricated hydrogels. A possible explanation can be attributed to the physical properties of both constituents; CNC displays strong colloidal stability and shear-thinning properties, which aid in creating a homogeneous dispersion aiding in the formation of a uniform network. CNF possesses higher aspect ratio (i.e., particle length) which grant increased mechanical performance, but tend to clump together and aggregate, leading to non-uniform densities throughout the network and increased viscosity which hinder processing and the loss of the nano-dimensional properties of the material.

Sample name Elongation at Ultimate Toughness Young's

(CI C:CHF) break (%) tensile stress (kJ/m³) modulus

(kPa) (kPa)

Table 1. Calculated mechanical properties attained by tensile tests of fabricated hydrogels.

Cyclic tensile testing

Resilience, elastic recovery, and extension profile were evaluated by cyclic tensile tests for 4 consecutive cycles. All samples evaluated followed roughly the same extension profile, where the extension behavior is systematically divided in two regimes. The first cycle shows a plastic deformation region, and the following 3 cycles display near perfect elasticity. As summarized in (Table 2), the resilience of each sample was calculated at two points for all samples; First following 200 % extension, which was the lowest common denominator reached by all samples tested. Second, the resilience was evaluated at the maximum extension cycle reached by each individual sample. All resilience calculations were conducted on the second, third and fourth extensions of each cycle, disregarding the first extension. At 200 % extension (Table 2, column A), all samples show impressive resilient characteristics, reaching close to or upwards of 90 %, with low error between repeating cycles. When comparing resilience at maximum extension reached by each individual sample (Table 2, column B), all samples continue to display a slight reduction in resilience compared to the values calculated at 200 %, possibly due to an accumulation of more permanent damage which leads to increased energy loss. However, a few key differences between the ratios tested can be surmised. First, C1F0 (Fig. 4) begins with expressing high resilience, 91.5 %, at 200 % extension, but experience a large decrease upon reaching their maximum recorded extension of 1000 % of 10.3 %, down to 82.2 %- pointing to material fatigue over the course of repeating extension cycles at increasing strains. C0F1 (Fig. 4E) exhibits the lowest resilience recorded at 200 % extension, reaching 87.3 %. This was also the maximum extension reached by this composition, thus resulting in a material expressing relatively low extensibility and resilience. When comparing samples composited with both CNC and CNF, all samples show exceptional resilience at 200 % extension, with C1F3, C1F1 and C3F1 (Figs. 4B-D) exhibiting 89 %, 92.5 % and 93.6 % resilience respectively. All these compositions express about a 2 % reduction in resilience between the 200% extension cycle and their respective maximum extension, reaching 87 %, 90 % and 90.5 % respectively. To emphasize the elastic nature of the material, C3F1 exhibits 90.5 % resilience at an 800 % extension and following 32 extension cycles at increasing strains. This points to the resulting composites possessing a highly resilient nature at a wide range of mechanical strains and that the maximum extendibility and ultimate stress can be fine-tuned by incorporating different compositions of CNC and CNF. Elastic recovery was used to further elucidate the elastic nature of the composites. Samples comprised of high contents of CNC, namely C1F0, C1F1 and C3F1, exhibit high elastic recovery (>95 %), which is further increased at higher strains, reaching close to 100 % recovery. Conversely, samples comprised of high CNF content, namely C0F1 and C1F3, exhibit lower elastic recovery following their initial stretch cycle to 100 % elongation, recovering by 90 %. This may arise from CNC enabling better dispersion throughout the matrix, resulting in a more uniform and better-defined network upon polymerization as compared to CNF. It is also possible that the use of CNF leads to an increased network density.

Table 2. Calculated resilience values of fabricated hydrogels at 200 % extension (A) and at maximum extension achieved by each composition individually (B)

Rheological properties

Viscosity measurements

Viscosity as a function of shear rate was performed to quantify the viscous behavior of the different solutions when incorporating varying ratios between CNC and CNF. As cellulose-derived nanoparticles generally and CNC specifically possess known shear-thinning properties, it was expected that by optimizing an effective mixture between CNC and CNF a reduction in overall viscosity will be achieved compared to CNF mixtures. Reduced viscosity may aid in the homogenization, handling and future molding of the pre-polymerized solutions and the homogeneity of resulting samples- leading to consistent mechanical performance and sample reproducibility. As the resulting flow curves suggest (Fig. 5), a reduction in viscosity occurred in all samples as shear rates increased. However, mixtures composed with the incorporation of CNF alone, C0F1, express both the highest viscosity measured and a visible plateau in viscosity between the shear rates of y= 10¹ (s/1) and y=10² (s/1), at which point the CNF particles aggregated and the solution appeared to separate into denser and thinner portions. On the other hand, solutions prepared by the incorporation of CNC alone, C1F0, express lower initial viscosity and shear-thinning behavior expected from CNC. When observing mixtures of both CNC and CNF, sample C1F3, composed mainly of CNF with a relatively low amount of CNC, expresses a reduction in viscosity compared to sample C0F1 while no phase separation was observed throughout the measurement. Samples C1F1 and C3F1 express similar viscosities to C1F0, showing the ability to incorporate CNF without compromising the solution’s viscosity, allowing effective mixture and dispersion, and resulting in homogeneous samples upon polymerization.

Frequency sweeps

Frequency sweeps were conducted to evaluate the structural integrity of the fabricated hydrogels (Fig. 6). Samples demonstrate a largely frequency-independent modulus with G’>G”, where the storage (G’) and loss (G”) moduli follow a similar trend, comparable with permanently crosslinked gels. Furthermore, there is an increase in storage modulus with increasing ratios of CNF. When comparing samples composed both of CNC and CNF to those incorporating CNC or CNF alone the following can be ascertained- first, C1F0 samples result in the softest composite, expressing the lowest storage modulus recorded (G’=3 kPa). On the other hand, C0F1 expresses a relatively high storage modulus (G’=24 kPa) (Fig. 6A), although only half the storage modulus measured for C1F3 (G’=50 kPa) (Fig. 6B). This may arise from the resulting inhomogeneity of composites fabricated by the incorporation of CNF alone, which leads to inconsistent densities throughout the composite, forming to a less stable structure overall. These results further validate the advantage in compositing these hydrogels with blends of both CNC and CNF- CNC eases the formation of a homogeneous dispersion and increases uniformity throughout the fabricated hydrogel, which benefits the structural integrity of the sample. CNF enables the formation of tougher, rigid structures as compared to CNC-based composites, indicated by increased storage moduli achieved in samples C1F3. Even by the addition of relatively small amounts of CNF in sample C3F1, comprise of three parts CNC and one part CNF, a higher storage modulus is reached compared to samples incorporating CNC alone (C1F0), recorded at lOkPa as opposed to 3 kPa respectively.

Amplitude sweeps

Amplitude sweeps were employed to describe the rheological stability of the composites. The linear viscoelastic region (LVER) of samples incorporating compositions of CNC and CNF (C1F3, C1F1 and C3F1) is increased compared to samples comprised of CNC or CNF alone (Table 3), indicating increased structural stability overall. Next, the flow points (rf) of all samples follow similar trends to the LVERs calculated. Here C1F3 (Fig. 7A) and C3F1 (Fig. 7C) outperform others, reaching the flow transition point at y= 31.6 %, indicating an ability to withstand a wider range of deformations compared to other compositions. Finally, the flow transition index, which is derived from the values calculated above, points to the area in which the LVER has passed but the amount of deformation has yet to arrive at a critical point where the micro-structure begins to crack, where G”> G’. It can be determined that C0F1 (Fig. 7E) shows the poorest structural integrity, expressing the lowest flow transition index measured at 10. C1F0 (Fig. 7D) on the other hand shows a relatively high flow transition index of 59.9. This may show that by employing CNC the material is able to withstand a higher amount of deformation, possibly due to the small size of the CNC particles, which act as efficient, small crosslinking joints, acting as stress recovery mechanisms. C1F3 (Fig. 7A) and C1F1 (Fig. 7B) expresses a higher flow transition index compared to C0F1 but lower than C1F0, recorded at 39.8 and 31.6 respectively. This may point to CNF still governing much of the mechanical structure of the samples in these compositions. The role of CNC, which we theorize aids as a rheological modifier, appears to be more pronounced in C3F1 (Fig. 7C), which exhibits the highest flow transition index of all samples evaluated, recorded at 63.06.

Sample Linear Flow Flow name viscoelasti point transition

(CNCCNF) c region index

(tVE(%)} (T_f/T_y)

Table 3. Calculated linear viscoelastic region (LVER), flow point and flow transition index of fabricated hydrogel compositions. Swelling and degradation profiles

Swelling analysis

The swelling properties of hydrogels may affect their mechanical performance and aid in determining the material’s potential applications and roles. As many potential functions for hydrogels are intended for biomechanical applications, PBS at physiological pH was chosen as a swelling buffer. All compositions tested retain their original shape indefinitely when submerged in excess PBS without dissolving or rupturing, implying a strong crosslinked network. C1F0 samples swell the most, swelling to 1150 % their original weight over a 5-day period. C0F1 samples swell the least, reaching 750 % their original weight over a 5-day period, possibly pointing to increased network density resulting from the longer, fibrillated CNF particles. The swelling properties of CNC:CNF blends then generally relate to the ratio of its constituents- increased CNC ratio leads to increased swelling ratio, as can be seen in sample C3F1 while increased CNF ratio leads to a decrease in swelling capabilities (Fig. 8A). Furthermore, Samples which incorporate higher loadings of CNF reach their swelling equilibrium faster, as can be seen in samples C0F1 and C1F3, equilibrating after 10-15 hours as opposed to samples with higher CNC content, C1F0 and C3F1, which reach equilibration after 60 hours.

Degradation analysis

As stated above, many possible applications for hydrogels are intended to function in aqueous environments or, more importantly, in biologically active environments such as the human body, and may become exposed to several degrading factors. Thus, it is important to determine possible degradation of the material. Degradation may lead to residual material circulating in the body, which if uncontrolled may lead to undesired effect such as clotting, creating blockage and inflammation. Results suggests most tested samples closely keep their original weight (>95 %) throughout the 6-week period evaluated. Composites fabricated by the incorporation of CNF alone, C0F1, lost some weight (-10 %) following the first week tested, which may result from sample inhomogeneity, leading to un-crosslinked residues leeching out of the sample (Fig. 8B). Biocompatibility and cytotoxicity studies

Preliminary screening of the fabricated hydrogels for their potential as a biomaterial was conducted by evaluating their cytocompatibility and toxicity with mammalian fibroblasts. The GFP transfected 3T3s (GFP-3T3s) were statically seeded on the hydrogels, while media was added to the wells containing the hydrogels after an hour to allow time for maximum cellular adhesion. It is vital for any biomaterial to be non-toxic and biocompatible. A preliminary evaluation of CNC, CNF, CNC: CNF blends and a control group comprised of acrylamide alone without the addition of cellulose-derived nanoparticles (AM) was conducted. Hydrogels seeded with the GFP- 3T3s cells indicate that all samples fabricated with the addition of the cellulose-derived nanoparticles support the adhesion and proliferation of mammalian cells, as can be seen in the confocal microscope images (Fig. 9A). MTT analysis indicated a significant difference for cellular adhesion in all hydrogels when compared with the control AM hydrogel. No significant difference was observed amongst the test samples (C1F1, C3F1 and C1F3) for cellular adhesion as observed on day 1 post cell seeding, indicating that all hydrogels fabricated based on mixtures of CNC and CNF exhibit similar properties suitable for cell adhesion (Fig. 9B). However, post 7 days of cell seeding, sample C1F3 shows significant growth of cells as compared to C1F1 and C3F1 (Fig. 9B). CNF appears to contribute to cellular proliferation compared to the addition of CNC, which is indicated by a significant difference in cellular growth when comparing samples C0F1 and C1F0 following incubation for 7 days (Fig. 9B). The area covered by the fibroblasts calculated from the confocal images also reveals that C1F3 has significantly higher cell coverage compared to samples C1F1 and C3F1 (Fig. 9C). Finally, Morphological observations reveal that sample C1F3 exhibits a more uniform cellular growth and deeper cell migration (~ 80 pm) with wide-spread spindle shaped morphology (Fig. 9D). These preliminary evaluations indicate that all hydrogels fabricated based on the incorporation of mixtures of CNC: CNF (C1F1, C1F3 and C3F1) show improved cellular behavior compared to samples fabricated with the incorporation of one of the cellulose-derived nanoparticles (C1F0 and C0F1) or without the addition of any cellulose nanoparticles (AM), and show promise as a biomaterial, with C1F3 displaying the most suitable properties for cellular adhesion and proliferation compared to the other composites based on CNC: CNF mixtures evaluated. Compression

Compressive mechanical properties of hydrated hydrogels were evaluated. Fig. 10 presents typical stress-strain curves obtained by compressing samples to 50 % their original height over 4 loading-unloading cycles. The calculated compressive properties are shown in Table 4. All sample composed with various loadings of CNC evaluated (C1F3, C1F1, C3F1, and C1F0) express impressive resilient properties (>90%) and, following a first conditioning cycle, are able to withstand repeated deformation cycles with negligible hysteresis. Sample C0F1, prepared with the addition of CNF alone, expresses a significant reduction in resilience, calculated at an average of 79.4%, possibly due to reduced sample homogeneity. While most samples showed impressive resilience, samples composed of more CNC content (C3F1 and C1F0) expressed moderately better resilience compared to samples composed of more CNF (C1F3 and C0F1). Generally, ultimate compressive stress (UCS) is more affected by addition of CNF into the matrix. Sample C0F1 expressed the highest UCS measured, at an average of 280 kPa. However, if comparing between samples expressing higher resilient properties, a composition with a relatively high loading of CNF but with the addition of CNC, sample C1F3, composed of one part CNC and three parts CNF, expresses high UCS (189 kPa), a 5-fold increase compared to the addition of CNC alone, sample C1F0, which expressed the lowest average UCS measured (38 kPa), expressing a good mediation between compressive strength and resilient properties.

Table 4. Calculated ultimate compressive stress (A) and resilience (B) values of fabricated hydrogels. The effect of changing the crosslinker’s, N,N’ -methylene bisacrylamide (MBA), content was evaluated. A series of hydrogels with MBA content ranging from 0.2 mg/ml (control) to 1 mg/ml were fabricated using compositions fabricated with the addition of CNC alone (C1F0). Fig. 11 depicts typical stress-strain curves obtained by compressing samples by 50 % their original height over 4 loading-unloading cycles. The calculated compressive properties are shown in Table 5. Generally, the increase in MBA content significantly increases the UCS at 50% compression, increasing nearly 3- fold between the control group (0.2 mg/ml) and a 1 mg/ml MBA concentration, reaching 42 kPa and 116 kPa respectively. Resilience of all samples remains high (-90%) and does not seem to be significantly affected by the change in MBA content.

Table 5. Calculated ultimate compressive stress (A) and resilience (B) values of fabricated hydrogels.

MTT analysis indicates that samples composed of mixtures between CNC and CNF (C1F1, C3F1 and C1F3) do not exhibit any significant difference in cellular adhesion after 1 day post cell seeding. However, following a week post cell seeding, C1F1 exhibits significantly higher proliferation of human umbilical vein endothelial cells (HUVECs) as compared to the other two CNC:CNF mixture samples tested (C3F1 and C1F3), and continues to be significantly more suitable for cell growth until the end of study (day 21) (Fig. 12A). Similar observation is also made on evaluating the morphological appearances of endothelial cells onto the hydrogels 14 days after cell seeding (Fig. 12B). HUVECs observed on this hydrogel composition, C1F1, exhibit characteristic three-dimensional tubular network, characteristic of endothelial cells, indicating an optimal environment for their sustainable growth with intercellular interactions. All hydrogels appear to promote improved cellular adhesion and growth as compared to samples composed of acrylamide alone (AM). The ability of these hydrogels to promote the growth of HUVECs and supporting the aforementioned tubular structures without the need of any additional growth factors or cellular adhesion motifs emphasizes the potential of the fabricated hydrogels as potential scaffolds for tissue engineering.

All materials intended for tissue engineering applications must undergo some form of terminal sterilization prior to implementation or contact with cells. The type of sterilization method used may affect the material’s characteristics and alter its ability to promote cell growth and proliferation. Comparative cellular growth was compared on hydrogel samples (C1F0, C0F1, C1F1 and AM) treated with different terminal sterilization methods- ethanol solution (EtOH, 70%), Autoclave and Ethylene oxide (EtO), with a separate experiment conducted to compare samples sterilized by y- irradiation versus EtOH-sterilized samples. Cell proliferation was compared by evaluating the cellular metabolic activity using alamarBlue, while the fluorescent emission of green fluorescent protein (GFP) transfected mouse fibroblasts (3T3s) was viewed under confocal microscopy to compare cell morphology and proliferation. Comparative cellular growth following 21 days, evaluated by alamarBlue and set time intervals (1-, 7-, 14- and 21-days post cell-seeding) and seen in Figs. 4A and B, reveals that the best cellular growth was obtained in samples sterilized with EtOH with all samples composed with either CNC or CNF (C1F0, C0F1, and C1F1) exhibiting significant higher cellular growth compared to acrylamide alone (AM). Samples sterilized by EtO seem to facilitate cellular growth in a slightly reduced manner, with only sample C1F1 achieving comparable growth with the similar composition sterilized by EtOH. Autoclaved samples facilitate the least amount of cellular growth in all samples, pointing to this sterilization method being the least suitable as a terminal sterilization method of this material. Confocal microscopy imagery was used to compare the cell morphology, as seen in Fig. 13. EtOH and EtO treated samples show uniform cellular growth with widespread spindle-shaped morphology and clearly visible nuclei. Cells seeded onto autoclaved samples seem to form small clusters and appear to be much smaller and rounder, without forming a visible intracellular network, further suggesting suboptimal conditions for cell growth and proliferation onto samples terminally sterilized in this manner. In the second experiment conduced, comparing samples sterilized by y-irradiation versus EtOH-sterilized samples, both terminal sterilization methods seem to facilitate excellent cellular growth. All samples composed with either CNC or CNF (C1F0, C0F1, and C1F1) exhibiting significant higher cellular growth compared to acrylamide alone (AM). A similar trend is observed during this experiment and the previous one, where sample C1F1 exhibits the highest cellular growth. These results indicate that EtOH, EtO and y-irradiation are well as terminal sterilization methods for this material (Fig. 14).

Literature describing photopolymerization of cellulose/acrylic monomer/CAN systems can be found at (Zhu, Qiu, Sakai, and Ito 2017; Zhu, Qiu, Sakai, Zang, et al. 2017). C1F0 samples were prepared as mentioned above (General methods, hydrogel fabrication). Three polymerization methods were compared. First, a control group polymerized by placing the pre-polymerized hydrogel mixture at 55 °C for 12 h. Second, by exposing the pre-polymerized hydrogel mixture to the sun (full-spectrum light source) for 40 min. Third, by exposing the pre-polymerized hydrogel mixture to an ultra-violet (UV) light source emitting at 365nm for 40 min.

Alternative polymerization methods may be used to cater to different applications. While polymerization by heat may be suited for molding the hydrogel into complex shapes, photopolymerization may be used to cater to 3D printed applications. Exposing the pre-polymerized hydrogel mixtures to either direct sunlight or the UV light source resulted in relatively rapid polymerization, in a matter of minutes, as compared to classic heat-induced polymerization, which occurs over several hours. Once polymerized, the tensile and compressive mechanical properties of the hydrogels were evaluated, as shown in Fig. 15 and summarized in Table 6. Generally, both samples polymerized by light (either sunlight or UV) formed cohesive networks in a matter of minutes but were significantly softer and less extendable than heat polymerized samples. The compressive properties of sun- and UV-polymerized samples were expressing roughly half the UCS of the heat-polymerized samples, measured at 23, 20 and 42 kPa respectively, yet retaining excellent resilience, measured at 93.2% for both light-polymerized samples. In terms of tensile properties, sun- and UV- polymerized samples could stretch roughly half the length of the heat-polymerized samples before breaking and expressed a third of the UTS, measured at 32, 33.2 and 92.7 kPa respectively.

Table 6. Calculated mechanical properties of photopolymerized hydrogels.

C1F0 samples were prepared as mentioned above (General methods, hydrogel fabrication). Biomineralization hydroxyapatite onto the scaffold template was performed by immersing samples in alternating solutions of calcium (CaCh) and phosphate (K2HPO4). As such, hydrogel samples were immersed in solutions composed of 250 mmol/L CaCh (27.75 mg/ml) and 150 mmol/L K2HPO4 (26.13 mg/ml) in alternating cycles, 24 hr. at a time for 8 consecutive days, beginning with sonication (30 min, 01/01, 25%) to facilitate solution penetration.

Accumulation of white-colored HA formed throughout the hydrogel network, as was evident by visual comparison between an unmineralized and mineralized samples (Fig. 16A). HA seemed to penetrate and distribute well throughout the hydrogel as seen when cutting the sample in half and holding it open with tweezers (Fig. 16B). Comparing the compressive properties of mineralized and unmineralized samples revealed that mineralizing the samples increases their compressive properties significantly as compared to unmineralized samples, reaching an UCS of 93 and 42 kPa respectively, as seen in Fig. 16C and summarized in Table 7. Mineralized samples form a less resilient structure, expressing a resilience of 82% as opposed to 91% expressed by unmineralized samples, though they are still able to withstand repeated cycles of deformation, able to return to their original form immediately. Morphological analysis, evaluated by Cryo-SEM and shown in Fig. 17A and B, suggest that mineralized samples are made up of a uniform porous network, composed of pores roughly 1-0.5 pm in diameter. EDS-mapping was used to evaluate the presence and distribution of HA throughout the gel, determined by the presence of calcium and phosphate (Ca and P respectively). Mappings were obtained from three visually distinct sections of the gel (I, II and III) where a gradient of penetration was expected, as seen in Figs. 17C and D. Indeed, as seen in the Table of Fig. 17D, while Ca and P account for roughly 37% of the composition in the outer layer I, Ca and P account for only 13.7% of the composition in the inner layer III, suggesting a gradient forming throughout the gel.

Table 7. Calculated ultimate compressive stress (A) and resilience (B) values of biomineralized hydrogels.

Fabrication of composite with macro-filaments (gauze)

Integrating macro-sized reinforcing filaments, particularly filaments composed of cellulose, was tested as means to strengthen the tensile properties of the hydrogel. To that end, cotton fibers taken from a gauze pad and Viscose fibers, both composed of high (>90%) amounts of cellulose, were used as a template onto which a C1F1 hydrogel composition was cast. Both sample the gauze and viscose filaments were treated with consecutive washes of acetone, NaOH (0.2M) and HC1 (0.2M), and DDW in that order, to remove any residual substances and dried. Hydrogels were cast on a polystyrene plate along with a control without the addition of any filaments to ensure a successful reaction. After casting samples were placed in an oven at 55°C for 12 hr.

While the control samples (without filaments) polymerized, all samples in which filaments were integrated did not fully polymerize. Moreover, the macro-sized filaments could be easily separated from the hydrogel matrix, suggesting that even though these filaments are composed of cellulose, they do not react with the system in the same manner the nano-sized filaments (CNC and CNF) react.

Without wishing to be bound by theory, the metal salt, e.g., cerium ammonium nitrate (CAN), is the driving force of the polymerization reaction. Attempting to use longer, macro-sized cellulosic filaments such as cotton and Viscose fibers, as described above, led to uneven or complete lack of polymerization of the system. Theorizing the properties of CAN enabling polymerization are “socked up” by the interaction with the macro-sized cellulosic filaments, an attempt to increase the amount of CAN in the system was evaluated.

In the original preparation protocol, cellulose nano particles (CNPs, either CNC or CNF) and CAN were provided at a concentration of 15mg and 0.8mg per ml respectively. To that end the following amounts and calculations were applied:

• CNC: CNF content was kept at a constant 15mg/ml

• Macro-sized cellulosic filaments - 150 mg of cotton fibers were added to each 1 ml of cast gel, resulting in a X10 increase in overall cellulose content.

• CAN-The original amount of 0.8 mg/ml CAN was used as a control. A series of solutions with a 2x (1.6mg/ml), lOx (8 mg/ml) and 50x (40mg/ml) CAN were used.

Each CAN concentration was cast in 3 1 ml repeats- 2 samples with cotton fibers, and the other without, as a control evaluating the respective CAN concentration’s effect on the polymerization progress.

While the control (lx) and 2x CAN concertation were able to polymerize into a cohesive hydrogel, a lOx and 50x increase formed a soft, pasty, and easy to break matrix, as can be seen in Fig. 18. In all samples the macro-sized cellulosic cotton filaments were not integrated into the matrix and could be easily separated and pulled out, suggesting CAN increase may not be the limiting factor in successful integration of these macro-sized filaments as a strengthening component to the hydrogels.

Increased cellulose nanoparticle content

The fabrication protocol disclosed above was adjusted to increase the CNP:PAAM ratio to XI.5 and X2. A 1: 1 CNC:CNF solution was used to demonstrate the mixture of both cellulose-derived nanoparticles (CNPs) in one, and cast into Eppendorf tubes to form cylindrical samples. The ratio of CNP to CAN was constant, under the assumption that CANs’ reactive potential was dictated by the cellulose content (as previously theorized when trying to integrate macro-cellulose fibers into the matrix). Amounts of AM and BAM were reduced as shown in Table 8:

Table 8: Tested combinations

Increasing the CNP ratio resulted in increased viscosity of the pre-polymerized solutions, which hindered homogeneous mixture and casting. The polymerized samples seemed less homogeneous as well, with uneven dispersion of the CNPs as their content increased. Tensile tests were performed to evaluate the UTS of the new compositions. As seen in Fig. 19, the increase in CNP loading resulted in samples able to reach higher stress levels, where a X2 increase in CNP content led to nearly doubling the UTS compared to the control (XI), reaching 0.4 MPa compared to 0.26 MPa respectively. However, the same composition was able to be elongated significantly less, able to be extended roughly 200% as opposed to the 275% achieved by the control sample.

Claims

CLAIMS:

1. A material comprising or consisting a cellulose nanomaterial grafted with acrylamide, wherein the cellulose material consisting a mixture of cellulose nano crystals (CNC) and cellulose nano fibers (CNF).

2. The material according to claim 1, in a form of a matrix material comprising or consisting a polymeric material comprising units of acrylamide, cellulose nano crystals (CNC) and cellulose nano fibers (CNF), wherein said units are polymerized therebetween to provide the matrix material.

3. The material according to claim 1 or 2, formed by grafting the cellulose nanomaterial with acrylamide monomers.

4. The material according to any one of the preceding claims, being a hydrogel in a hydrated or dry hydrogel form.

5. The material according to claim 4, wherein in a dry form the hydrogel is capable of absorbing at least 10 % by weight water.

6. The material according to claim 4, wherein in the hydrated form, the hydrogel having a weight that is 7 to 10 times its dry weight.

7. The material according to any one of the preceding claims, in a form of beads, blocks, sheets or an amorphous form.

8. The material according to any one of the preceding claims, comprising at least one active or non-active material.

9. The material according to any one of the preceding claims, biocompatible with a living tissue.

10. The material according to claim 8 or 9, for delivery of an active material to a living tissue in vivo or ex vivo.

11. The material according to any one of the preceding claims, configured for positioning in contact with a tissue in a subject’s body or topically on a subject’s skin.

12. The material according to any one of the preceding claims, for use as a drug release platform for controllably and effectively delivering the drug to a subject’s tissue.

13. The material according to any one of the preceding claims for causing proliferation of live cells in vivo.

14. The material according to claim 13, wherein the cells are fibroblasts, human umbilical vein endothelial cells (HUVECs), chondrocytes and osteoblasts.

15. The material according to any one of claims 1 to 8, for use in a method of manufacturing a medical device or an implant.

16. The material according to claim 15, wherein the implant is selected from synthetic heart valves, vascular grafts, cartilage and bone grafts, tendons and ligaments, soft tissue replacement materials, tissue replacement materials, and fibers.

17. The material according to any one of claims 1 to 8, for use in a method of manufacturing a material delivery device for delivery of a therapeutic or a cosmetic agent in vivo or in vitro.

18. A process for manufacturing a hydrogel material comprising or consisting a cellulose nanomaterial grafted with acrylamide, wherein the cellulose material consisting a mixture of cellulose nano crystals (CNC) and cellulose nano fibers (CNF), the process comprising reacting a combination of CNC and CNF, at a predetermined CNC:CNF ratio, in presence of acrylamide or grafted acrylamide under conditions causing radical grafting and polymerization of the CNC, CNF and acrylamide.

19. The process according to claim 18, wherein the ratio CNC:CNF is between 1: 100 to 100: 1.

20. The process according to claim 18, wherein the ratio CNC:CNF is between 100: 1 and 10: 1.

21. The process according to claim 19, wherein the ratio CNC: CNF is between 5: 1 and 1:5.

22. The process according to claim 18, wherein the ratio CNC:CNF is 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, or 10: 1.

23. The process according to claim 18, wherein the ratio CNC:CNF is 5: 1, 4: 1, 3: 1 or 2: 1.

24. The process according to claim 18, wherein the CNC, CNF and acrylamide are provided at a ratio CNC and CNF, combined, to acrylamide (CNC+CNF: acrylamide) of between 1: 100 and 1:5.

25. The process according to claim 24, wherein the ratio CNC+CNF: acrylamide is between 1: 100 and 1:5, and wherein the ratio CNC:CNF is between l: 10 to 10: 1.

26. The process according to claim 25, wherein the ratio CNC+CNF: acrylamide is 1: 100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1: 15, 1: 10 or 1:5.

27. The process according to claim 18, for increasing the hydrogel toughness, wherein the ratio CNF:CNC is between 100: 1 and 10: 1.

28. The process according to claim 18 or 27, for increasing the hydrogel toughness, wherein the ratio CNF:CNC is 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, or 10: 1.

29. The process according to claim 18 or 27, for increasing the hydrogel toughness, wherein the ratio CNF:CNC is 5: 1, 4: 1, 3: 1 or 2: 1.

30. The process according to claim 18, for increasing hydrogel stretchability and resilience, wherein the ratio CNC:CNF is between 100: 1 and 10: 1.

31. The process according to claim 18 or 30, for increasing hydrogel stretchability and resilience, wherein the ratio CNC:CNF is 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, or 10: 1.

32. The process according to claim 18 or 31, for increasing hydrogel stretchability and resilience, wherein the ratio CNC:CNF, is 5: 1, 4: 1, 3: 1 or 2: 1.

33. The process according to any one of claims 18 to 32, comprising treating a mixture of CNC, CNF and acrylamide with N,N’ -methylene bisacrylamide (MBA).

34. The process according to any one of claims 18 to 33, comprising forming a mixture of CNC and CNF at a CNC:CNF ratio, wherein the mixture is formed by homogenization.

35. The process according to any one of claims 18 to 34, comprising homogenizing a CNC and CNF mixture and treating same with acrylamide and MBA, followed by addition of a metal ion precursor.

36. The process according to claim 35, wherein the metal ion precursor is a metal salt or a metal complex.

37. The process according to claim 35 or 36, wherein the metal ion precursor is a cerium metal salt or complex.

38. The process according to claim 37, wherein the cerium metal salt is cerium (iv) ammonium nitrate ([NH4]2[Ce(NO3)6], CAN).

39. A process for modulating a mechanical or a rheological property of a hydrogel formed of CNF grafted with acrylamide monomers, the method comprising:

-in a process of manufacturing a hydrogel of CNF grafted with acrylamide, mixing the CNF with an amount of CNC, wherein the ratio amount CNC: CNF is between 1:5 and 5: 1.

40. A process for modulating a mechanical or a rheological property of a hydrogel formed of CNC grafted with acrylamide monomers, the method comprising: -in a process of manufacturing a hydrogel of CNC grafted with acrylamide, mixing the CNC with an amount of CNF, wherein the ratio amount of CNC to CNF is between 1:5 and 5: 1.

41. The process according to claim 39, wherein the modulating a mechanical or a rheological property comprises improving the hydrogel stretchability and resilience.

42. The process according to claim 40, wherein the modulating a mechanical or a rheological property comprises improving the hydrogel toughness.

43. A hydrogel comprising or consisting a material according to any one of claims 1 to 8 as a matrix material.

44. The hydrogel according to claim 43, comprising an active or a non-active material.

45. The hydrogel according to claim 43 or 44, for use as a drug release platform for controllably and effectively delivering a drug to a subject’s tissue.

46. The hydrogel according to claim 43 or 44, for supplying live cells to a tissue.

47. The hydrogen according to claim 43 or 44, for use in a method of forming a wound dressing, a shock absorbing material, or an energy storage device.

48. A medical device formed of a hydrogel according to claim 43 or 44.

49. An implant formed of a hydrogel according to claim 43 or 44.

50. The implant according to claim 49, being selected from synthetic heart valves, vascular grafts, cartilage and bone grafts, tendons and ligaments, soft tissue replacement materials, tissue replacement materials, and fibers.

51. A substrate for tissue engineering for growing cells and tissues in vivo or in vitro, the substrate being formed of or comprising a material according to any and of claims 1 to 8.

52. A biomaterial being or comprising a material according to any one of claims 1 to 8.

53. The biomaterial according to claim 52, comprising a mineral.

54. The biomaterial according to claim 52, wherein the mineral is hydroxyapatite.

55. A biomineralized material being or comprising a material according to any one of claims 1 to 8.

56. The biomineralized material according to claim 55, comprising hydroxyapatite.

57. A transplant for implanting in a subject, the transplant comprising cells contained within and/or on a surface region of a material according to any one of claims 1 to 8.

58. A method of protecting cells, the method comprising adding or contacting or immersing a material according to any one of claims 1 to 8 in a cell culture medium and allowing the cells to be absorbed by the hydrogel.

59. A cell delivery system comprising a material according to any one of claims 1 to 8 and a plurality of cells to be delivered or transplanted.

60. A method for delivery of viable cells to a mammal, the method comprising positioning a cell delivery system at a desired location for cell delivery to a mammal, the cell delivery system comprising a material according to any one of claims 1 to 8 and cells; wherein the material containing the cells inside pores formed therein and/or on its surface.

61. The device according to claim 48 for releasing a material, in vivo, ex vivo or in vitro, the device comprising a matrix containing the material and having a porosity permitting passage therethrough of the material.