WO2013063602A1 - Design and fabrication of biomimetic soft tissue scaffolds based on controlled assembly of highly crystalline beta-glucan nanofibrils and use in reconstructive surgery - Google Patents

Abstract

The present invention relates to the field of medical devices and surgical implants. Embodiments of the invention provide scaffolds, including biomimetic soft tissue scaffolds, grafts, and prostheses. A nanoporous scaffold is provided comprising a plurality of pores ranging in size from 300 micron to 3 mm, a porosity ranging from 20-80%, and a tensile strength ranging from about 60 N/cm to about 200 N/cm. In embodiments the scaffolds are grown in a manner to comprise at least some of the pores, for example, by delivering oxygen through an array of porous capillaries contacting a culture medium inoculated with a cellulose generating bacteria. Specific embodiments of the invention can be used as biocompatible materials for tissue engineering and regenerative medicine, implants, surgical meshes, biomedical devices and health care products and, more particularly, to biomimetic honeycomb structured soft tissue scaffolds for use in reconstructive surgery.

Description

DESIGN AND FABRICATION OF BIOMIMETIC SOFT TISSUE SCAFFOLDS BASED ON CONTROLLED ASSEMBLY OF HIGHLY CRYSTALLINE BETA-GLUCAN

NANOFIBRILS AND USE IN RECONSTRUCTIVE SURGERY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application relies on the disclosure of and claims priority to and the benefit of the filing date of U.S. Provisional Application No. 61/552,376, filed October 27, 2011, the disclosure of which is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] Field of the Invention

[0003] The present invention relates to the field of medical devices and surgical implants. More particularly, embodiments of the invention provide scaffolds, including biomimetic soft tissue scaffolds, grafts, and prostheses. In embodiments of systems and methods of the invention, by controlling the assembly of β-glucan nano fibrils the morphology of biomaterials can be controlled during design and production resulting in scaffolds that mimic the

characteristics of soft tissues. Specific embodiments of the invention relate to biocompatible materials, tissue engineering and regenerative medicine, implants, surgical meshes, biomedical devices and health care products and, more particularly, to biomimetic honeycomb structured soft tissue scaffolds for use in reconstructive surgery.

Description of Related Art

[0004] There are more than 800,000 inguinal hernia repairs performed annually in the United States at the total cost of several billion dollars. Surgical meshes made of synthetic polymers such as polypropylene have been used for years. These meshes are integrated in tissues by a rapid process, however, which causes scar tissue formation and which can result in patient discomfort and pain.

[0005] Since 1999 several biological prostheses have been approved by the FDA for use for hernia repair, including those derived from human, porcine, fetal bovine skin, porcine intestine submucosa, and bovine pericardium. Such prostheses promote tissue in-growth with less contraction than observed with synthetic meshes. They also have ability to remodel tissue and act thus as scaffold. Available biological prostheses, however, are very expensive, suffer from variability in structure, and most importantly, because they are derived from human or animals, provide the risk of disease transfer and immunological response. There is a huge unmet medical need to provide soft tissue grafts which perform in the manner as biological prostheses thus providing controlled tissue integration but are not derived from human or animal sources.

[0006] Tissue scaffolds based on biosynthetic cellulose are known and include those provided by International Published Patent Application No. WO 2011/038373 entitled "Three- dimensional Bioprinting of Biosynthetic Cellulose (BC) Implants and Scaffolds for Tissue Engineering." Described are nano-cellulose based structures comprising a network of multiple layers of interconnected biosynthetic cellulose, which are produced by non-mechanical or static means and are characterized as having higher density and/or tensile strength as compared with BC structures produced by mechanical or static methods. Such materials are typically produced from bacteria and thus are often referred to as bacterial cellulose (BC).

[0007] Bacterial cellulose based products have been known for external uses, including those described in U.S. Patent No. 4,912,049 entitled "Process for the Preparation of Cellulose Film, Cellulose Film Produced Thereby, Artificial Skin Graft and Its Use." More particularly, described is a liquid gas permeable cellulose film produced from Acetobacter xylinum, which is dehydrated while stretched. The cellulose film is used as a temporary wound dressing and is capable of absorbing exudate at the surface of the injury into the microscopic spaces existing between the films fibers where the exudate coagulates within the film to cause temporary adhesion of the film to the wound surface. As the wound heals, the film is released.

[0008] Implants comprising bacterial cellulose are provided by U.S. Published Patent Application No. 2007/0128243 entitled "Implantable Microbial Cellulose Materials for Various Medical Applications," which describes a method for preparing an implantable device for medical and surgical applications comprising: incorporating a material comprising microbial cellulose into an implantable device for repair or replacement of soft tissue. Also provided is an implantable composition comprising microbial cellulose, such as a tissue scaffold.

[0009] Even further, U.S. Published Patent Application No. 2009/0017506 entitled

"Continuous Fermentation Process to Produce Bacterial Cellulosic Sheets" describes a method of producing cellulosic sheets from bacteria in a continuous fermentation process to produce multiple sheets having about the same cellulose content without replacement of nutrients consumed by the bacteria. [0010] U.S. Published Patent Application No. 2006/0134758 entitled "Process for Obtaining a Cellulosic Wet Sheet and a Membrane, the Equipment Used Obtain the Membrane and the Membrane Obtained" describes a method of producing a cellulosic wet sheet of 0.25 to 200 mm in thickness, which is whitened, washed, and rinsed prior to packaging.

[001 1 ] What these existing films and methods of making them lack, however, is the ability to control morphology of the mesh structure during growth of the film. Further, because they lack the requisite morphology conducive to implants, such scaffolds lack the ability to control or direct tissue integration into the implant once implanted. What is needed are biomimetic materials, more accurately mimicking soft tissue, engineered with a structure that encourages tissue growth, especially in a desired, more controlled manner.

[0012] Soft tissue is composed of collagen nano fibrils which provide strength and stiffness, elastin fibrils which provide elasticity, and a proteoglycan matrix which provides viscoelastic properties, which allow diffusion and nutrition through the tissue. Alternatively, plant tissue has a similar composition to soft tissue but collagen is replaced by nanocellulose fibrils. There is no elastin and proteoglycans present in plant tissue, as these are replaced by pectins, and a hemicellulose and lignin matrix. The morphology of plant tissue is characterized by a high degree of organization of building blocks such as cellulose nanofibrils which provide unique mechanical properties but also the ability to control diffusion of liquids. When selecting materials capable of mimicking properties of human or animal tissue, materials comprising cellulose nanofibrils are highly desired substitutes for materials comprising collagen nanofibrils.

[0013] Cellulose can also be generated by certain cellulose producing micro-organisms. Bacterial cellulose (BC) is a unique and promising material for use as implants and scaffolds in tissue engineering. It comprises a pure cellulose nanofiber mesh and is remarkable for its strength and ability to be engineered structurally and chemically at all length scales. High water content and purity render the material biocompatible and thus suitable for medical applications.

[0014] WO 2008/079034 entitled "A Biomaterial Composed of Microbiological Cellulose for Internal Use, a Method of Producing the Biomaterial and the Use of the Biomaterial

Composed of Microbiological Cellulose in Soft Tissue Surgery and Bone Surgery" describes a composite biomaterial of microbiological cellulose and synthetic polymers for internal use, which when introduced into an organism becomes covered with a structure of connective tissue, and retains the characteristics of a native membrane and biocompatibility with surrounding tissue. Such biomaterial is produced by shaking a culture of bacteria with the mesh of another polymer to have cellulose fibers pervade the mesh, then a stationary culture is carried out where the mesh is penetrated and the cellulose accretes on its surface. This composite biomaterial is purported to address issues of scarring and mesh shrinkage.

[0015] In some applications, where absorbability or resorbability is important for assimilation of a mesh, bacterial cellulose has been used. U.S. Patent No. 7,709,631 entitled "Oxidized Microbial Cellulose and Use Thereof," for example, provides a method of making a bioresorbable oxidized biocellulose which entails producing microbial cellulose (from for example, Acetobacter xylinum), soaking the biocellulose in NaCl, and oxidizing the microbial cellulose with sodium meta-periodate.

[0016] U.S. Patent No. 7,645,874, "Cellulose Oxidation by Nitrogen Dioxide in a

Perfluorinated Tertiary Amine Solvent," describes a method of making bioabsorbable oxidized cellulose by combining cellulose from Acetobacter xylinum, nitrogen dioxide, and a

perfluorinated tertiary amine to obtain an oxidized cellulose with a lower tensile strength and greater elongation than an oxidized cellulose produced using a perfluorinated hydrocarbon.

[0017] U.S. Published Patent Application No. 20070213522 entitled "Oxidized Microbial Cellulose and Use Thereof describes a method of making a bioresorbable oxidized biocellulose comprising (i) producing microbial cellulose and (ii) oxidizing the microbial cellulose with a solution of sodium meta-periodate.

[0018] Indeed it is well known for example from JP 3 165 774 Al to use cellulose produced by a microorganism (also referred to as microbial, microbiological, or bacterial cellulose) as biomaterial in surgical applications, such as tissue implants for the abdominal wall, the skin, subcutaneous tissue, organs, for the digestive tract, as well as for cartilaginous tissue and for Hpoplastics. Further, for example, it is known from JP 8 126 697 A2, EP 186 495 A2, JP 63 205 109 Al, and JP 3 165 774 Al that microbial cellulose can be specifically shaped for its respective application in its production process, for example, in the shape of lamina, rods, cylinders and strips.

[0019] U.S. Published Patent Application No. 20040142019 entitled "Microbial-Derived Cellulose Amorphous Hydrogel Wound Dressing" describes a microbial-derived cellulose amorphous gel wound dressing with a cellulose content by weight ranging from 1.0% to about 99%, about 2.5% to 65%, about 3.0% to 50%, 3.5% to about 12%, 4% and 7%.

[0020] U.S. Published Patent Application No. 20070286884 entitled "Implantable Microbial Cellulose Materials for Hard Tissue Repair and Regeneration" describes an implantable composition comprising microbial cellulose and an agent for promoting hard tissue growth (such as a protein, a growth factor, or a drug).

[0021 ] WO 2005/003366 entitled "A Method for the Production of Bacterial Cellulose" describes a method of producing bacterial cellulose by culturing Acetobacter xylinum bacteria for the production of a surface cellulose membrane, which is isolated from the culture liquid and further purified to provide wound dressings.

[0022] EP 2371401 entitled "A Method of Production of a Cartilage-Like Biomaterial Designed for Reconstructive Surgery" describes a method of preparing a cartilage-like biomaterial for reconstructive surgery implants by culturing microbial cellulose in a flat bioreactor or inside polyethylene tubes (a stationary culture of bacterium Gluconacetobacter xylinus), then purifying, rinsing, and modeling the material into a desired shape.

[0023] The nanostructured network and morphological similarities with collagen make BC very attractive for cell attachment, cell migration, and the production of additional extracellular matrices. In vitro and in vivo studies have shown that BC implants do not show foreign-body reaction, fibrosis, or capsule formation. Inflammatory giant cells have not been detected around BC networks and connective tissue integrates well with BC structures. The biocompatibility of bacterial cellulose (BC) has been evaluated in a rat model. The study was done on Wistar rats by placing squared pieces (lxl cm) subcutaneously for 1, 4 and 12 weeks. There were no macroscopic signs of inflammation, such as redness or exudates around the implanted BC pieces or in the incision at any time point. Overall, there were no histological signs of inflammation in the specimens, i.e. an abnormally high number of small cells in the connective tissue and especially around the blood vessels in the connective tissue (Helenius et al. 2006).

[0024] BC hydrogel materials, which are highly absorbent and used as scaffolds in tissue engineering, can be dried by methods such as critical point drying, freeze-drying, dewatering by organic solvents such as ethanol or acetone, air drying under normal or higher pressure, as well as hot-press drying. By drying under pressure, very flat foils (loss of 99%> water) of high density and high mechanical stability can be obtained. In such materials, the nanofibril network collapses, resulting in a dense physical cross-linking of the cellulose chains ("hornification"). The material only absorbs a small amount of water and it is thus not attractive as biomaterial.

[0025] For example, U.S. Published Patent Application No. 20050042263 entitled "Dura Substitute and a Process for Producing the Same" describes a method of producing a dural substitute comprising: producing a polysaccharide sheet; removing contaminants in said sheet; and dehydrating said sheet. Additionally, U.S. Patent No. 6,599,518 entitled "Solvent

Dehydrated Microbially-Derived Cellulose for in vivo Implantation" describes a method of making an implantable material for medical and surgical applications by treating a microbially- derived cellulose to render it non-pyrogenic, then dehydrating it with methanol, ethanol, propanol, isopropanol, or acetone.

[0026] U.S. Published Patent Application No. 2005/0042250 entitled "Thermally Modified Microbial-Derived Cellulose for in vivo Implantation" which is incorporated by reference herein in its entirety, describes a method for preparing an implantable or topical material for medical or surgical applications comprising: a) providing a microbial-derived cellulose; b) treating said microbial-derived cellulose to render said cellulose non-pyrogenic; c) partially dehydrating said microbial-derived cellulose by exposing it to temperatures below 0°C, then exposing said microbial cellulose to temperatures above 0°C; and d) discarding liquid that was removed.

[0027] U.S. Patent Nos. 7,510,725 and 7,374,775 entitled "Process for Producing a Dura Substitute" describe a method for producing a dural substitute, comprising: (a) producing a cellulosic material from a culture of Acetobacter xylinum; (b) removing contaminants from the cellulosic material by contacting the cellulosic material with a caustic solution; and (c) removing at least a portion of the water from the cellulosic material by freezing, melting, and removing at least a portion of the melted moisture from said cellulosic material, thereby providing a cellulosic dural substitute which is a cellulosic sheet, mesh, or film.

[0028] Bacterial cellulose is, from a biocompatibility point of view, very attractive since it can be completely integrated with rat tissue and very few macrophages have detected at studied time intervals. It has even been difficult to determine where the interface between the material and the rat tissue is located. This integration has been seen especially on the porous side of the BC: the rat tissue was totally integrated with the BC and a mature, organized tissue containing BC, fibroblasts, newly synthesized collagen and blood vessels. It appeared as though the loose nano fibrils at the porous side of the BC were ideal for integration with the surrounding tissue.

[0029] Other favorable characteristics of bacterial cellulose material include that it exhibits a unique water holding capacity caused by the presence of hydroxyl groups on the surface of the cellulose fibrils. This may be one of the reasons for good biocompatibility of this material (Esguerra et al. 2010). The state of water has been studied using dielectric spectroscopy (Gelin et al. 2007). Hydroxyl groups on the surface of cellulose nanofibrils offers the possibility for physical cross-linking.

[0030] U.S. Patent No. 4,588,400 entitled "Liquid Loaded Pad for Medical Applications" describes a liquid loaded pad for medical applications. The pad generally comprises a fibrous mass of microbially-produced cellulose fibrils and a sterile, physiologically-acceptable liquid retained within the fibrous mass, which has a coherent and dimensionally stable structure.

[0031 ] U.S. Patent No. 6,071,727 entitled "Production of Microbial Cellulose" describes pellicular microbial cellulose having a cellulose:water absorbed weight ratio ranging from about 1 : 178 to about 1 :226, which can be prepared by aerobically incubating a medium containing a cellulose producing microorganism in a rotating disk bioreactor or on a linear conveyor reactor using Acetobacter xylinum. U.S. Patent No. 5,955,326 entitled "Production of Microbial Cellulose Using a Rotating Disk Film Bioreactor" is similar.

[0032] Further, U.S. Patent No. 7,709,021 entitled "Microbial Cellulose Wound Dressing for Treating Chronic Wounds" describes a microbial cellulose dressing consisting essentially of from 1.5% to 4.5% microbial cellulose by weight and water, and wherein the wound dressing is capable of donating greater than 75% of its liquid weight to a dry or necrotic portion of said chronic wound and absorbing liquid in an amount effective for treatment of a chronic wound.

[0033] U.S. Patent Nos. 7,704,523 and 7,390,499 entitled "Microbial Cellulose Wound Dressing for Treating Chronic Wounds" describe a nonpyrogenic microbial cellulose wound dressing consisting essentially of water and from 1.5 to 4.3 wt. % of microbial cellulose, and in some embodiments also polyhexamethylene biguanide (PHMB), wherein the wound dressing absorbs fluid exudate in an amount that is about 20% to about 200% of its weight from an exuding chronic wound and donates moisture in an amount that is greater than 75% of its weight to a dry or necrotic portion of a chronic wound. U.S. Published Application Nos. 2005/0019380, 2004/0161453, 2003/0203013 are similar.

[0034] A recent study using the dorsal skinfold chamber model showed good tissue integration and production of extracellular matrix around BC implant (Esguerra et al. 2010). One of the concerns when using materials produced in microbial processes is the potential presence of endotoxins which might be residues from bacterial cell wall. The endotoxin levels in the water incubated with the porous cellulose scaffold were determined to be less than

0.1 endotoxin units (EU)/ml (Bodin et al 2010), below the limit for endotoxin set by the FDA for medical devices of 0.5 EU/ml.

[0035] Furthermore, it is known from JP 3 272 772 A2 and EP 396 344 A2 to use shaped bio-material as micro-luminal blood vessel substitutes, whereby the vessel prosthesis is cultivated on a hollow support which is permeable to oxygen (for example cellophane, Teflon, silicon, ceramic material, non-woven texture, fibers). The described process for producing the hollow microbial cellulose comprises the culturing of a cellulose synthesizing microorganism on the inner and/or outer surface of a hollow support permeable to oxygen, said support being made of cellophane, Teflon, silicon, ceramic material, or of a non- woven and woven material, respectively. Said hollow support permeable to oxygen is inserted into a culture solution. A cellulose synthesizing microorganism and a culture medium are added to the inner side and/or to the outer side of the hollow support. The culturing takes place under addition of an oxygenous gas (or liquid) also to said inner side and/or to the outer side of the hollow support. A gelatinous cellulose of a thickness of 0.01 to 20 mm forms on the surface of the support.

[0036] Another process for producing hollow microbial cellulose is described in EP 396 344 A2, which provides a method of manufacturing by way of two glass tubes of different diameter. The glass tubes are inserted into one another and culturing of the microorganisms is carried out in the space between the two tube walls within 30 days. The result is microbial cellulose of a hollow cylindrical shape and evaluated for its blood compatibility, antithrombogenic property by a blood vessel substitute test in a dog.

[0037] Even further, WO 01/610 26 Al and (Klemm et al. Prog. Polymer Sci. 26 (2001) 1561-1603) describe a method for producing shaped biomaterial by means of culturing cellulose producing bacteria in a cylindrical glass matrix, in particular for microsurgical applications as blood vessel substitutes of 1-3 mm diameter and smaller.

[0038] U.S. Published Patent Application No. 20100042197 entitled "Preparation of Hollow Cellulose Vessels" describes hollow cellulose vessels, tubes, artificial blood vessels, and patches prepared by culturing cellulose-producing microorganisms on the outer surface of a hollow carrier, and providing an oxygen containing gas on the inner side of the hollow carrier, wherein the oxygen containing gas has an oxygen level higher than atmospheric oxygen.

[0039] WO 89/12107 describes various methods for producing microbial cellulose at a gas/liquid interface, where the yield of cellulose can be improved by increasing the concentration of oxygen available to the bacteria by bubbling, agitation or increasing the pressure or concentration of oxygen in the ambient gas environment.

[0040] Oxygen is reported to be a limiting factor for the yield of microbial cellulose produced (Schramm & Hestrin J. Gen. Microbiol. 11 (1954) 123-129). On the other hand Watanabe et al. (Biosci. Biotechnol. Biochem. 59 (1995) 65-68) reported that a higher oxygen tension in the gaseous phase than atmospheric air inhibits BC production.

[0041 ] U.S. Pat. No. 6,017,740 and corresponding EP 0792935 describe a process for the production of bacterial cellulose in an aerated and agitated fermentation tank and increased oxygen pressure and content are used to increase the yield of microbial cellulose.

[0042] U.S. Published Patent Application No. 20100297239 entitled "Osseointegrative Meniscus and Cartilage Implants Based on Beta-Glucan Nanocomposites" describes medical implants to treat meniscus and cartilage damage produced by a method of culturing a

microorganism on a solid substrate by providing to the microorganism a first level of oxygen to cause the microorganism to produce a first type of glucan units resulting in deposition of cellulosic fibrils on the solid substrate, and providing to the microorganism a second level of oxygen to cause the microorganism to produce a second type of glucan units resulting in production of a hydrogel.

[0043] Notwithstanding various attempts in the art to design implantable biomaterials from bacterial cellulose, there remains still the need to be able to grow BC materials having a desired form and morphology which allows for cells to pass through or into the implant and integrate while retaining other desired properties such as strength, elasticity, and high porosity. SUMMARY OF THE INVENTION

[0044] Objects of the invention provide nanoporous biomaterials that mimic structure and properties of human and animal soft tissue. Such materials can be used to replace or fortify tissue in a body and provide biomimetic characteristics in that the materials perform in a manner similar to the tissue they substitute or are implanted to strengthen. Nanoporous biomaterials of embodiments of the invention can be designed and produced with any desired architecture by controlling the assembly of highly crystalline β-glucan nano fibrils.

[0045] Examples of such nanofibrils are cellulose nanofibrils which belong to β-glucan biopolymer family. Cellulose nanofibrils can be isolated for example from wood material, annual plants, animals such as tunicates, or can be produced by fungi or bacteria. Preferred embodiments include cellulose nanofibrils produced by bacteria, also referred to as bacterial cellulose. A preferred bacteria used to produce nanocellulose materials according to the invention is Acetobacter Xylinus . Biomimetic and biocompatibility characteristics associated with cellulose nanoporous biomaterials make them ideal candidates for surgical applications.

[0046] Objects of the invention provide for the design and manufacture of biomimetic soft tissue scaffolds using assembly of highly crystalline β-glucan nanofibrils. These scaffolds, or cellulose meshes, are suitable for applications such as soft tissue replacement implants, and the repair of soft tissue defects, particularly for hernia repair and reconstructive surgery.

[0047] Biological prostheses according to embodiments of the invention can be used in applications where tissue integration is desired. While synthetic meshes tend to result in scarring, biological prostheses, such as collagen-based materials, have instead been shown to support tissue integration. Such existing biological materials because they are collagen-based, however, are typically associated with a risk of disease transfer. More particularly, one disadvantage of using collagen as reinforcing nanofibril is a risk of transferring diseases (when it comes from animal) or inducing immunological reaction when it comes from another individual.

[0048] Implants and scaffolds of embodiments of the invention provide a replacement for soft tissue comprising collagen nanofibrils which provide strength and stiffness, elastin fibrils which provide elasticity, and a proteoglycan matrix which provides viscoelastic properties and allows diffusion and nutrition through the tissue. Biomimicking of soft tissue as described in this specification refers to the design and fabrication of a biomaterial scaffold which resemble soft tissue architecture and shows similar properties.

[0049] Instead of collagen, embodiments of the invention comprise P-l→4-glucan based polymers such as cellulose, chitin (and partially deacetylated chitin which is called chitosan), hyaluronic acid, and so on, as the basis for the scaffold. These polymers assemble by physical interactions including hydrogen bonding and Van der Waals interactions into nanofibrils of the same dimensions as collagen nanofibrils. Likewise, the inventive materials exhibit a similar mechanical behavior.

[0050] The P-l→4-glucan nanofibrils are used by the current invention as building blocks to provide architecture similar to soft tissue. The elasticity of soft tissue is provided by design and production of "crimp," a result of waviness of the fibrils within fascicles, into the structure. More particularly, soft tissue such as ligaments and tendons are made up of small components known as fascicles. Within the fascicles are fibrils. The network and "crimp" in the

embodiments is achieved by mechanical deformation and locking of the structure by developing hydrogen bonding and Van der Waals interactions. These can be achieved during sublimation of ice formed in the material by freezing or by critical point drying. The process can be performed by freezing the deformed sample and freeze drying or by critical point drying or by liquid carbon dioxide treatment or by any type of controlled dehydration.

[0051 ] One aspect of scaffolds of the invention is their ability to provide for and encourage cell in-growth when implanted into the body of a human or animal. In preferred embodiments, this cell in-growth is provided by macroporosity in the structure, for example, provided by a unique honeycomb structure with channels which can vary from 500 micrometers to 3mm in width. The macropores or channels can be provided by any means, such as for example by laser or water jet. In preferred embodiments, the macroporous structure of the scaffolds is produced by growing bacterial cellulose in a bioreactor. In embodiments, porous capillaries can be provided as support elements in the bioreactor, which deliver oxygen enriched air to the bacteria in a growth medium capable of supporting and enhancing the growth of bacterial cellulose. In specific embodiments, the channels are produced by inserting porous capillaries or oxygen permeable material during preparation of the biomaterial and delivering air or oxygen enriched air through the capillaries. By growing the scaffold in this manner, a high density of a cellulose network is obtained in the outer wall of the channels formed around the support elements, which imparts unique mechanical properties to the scaffold material.

[0052] The P-l→4-glucan nano fibrils can be produced by a variety of biological species including bacteria and fungi, or can be isolated from annual plants or trees. In preferred embodiments, the control of production of P-l→4-glucan by bacteria is achieved by targeted air or oxygen delivery. In embodiments high oxygen tension Acetobacter xylinum produces a high concentration of P-l→4-glucan (cellulose) in the form of entangled nano fibrils.

[0053] The biomaterial described by this innovation can have many characteristics or attributes including but not limited to, an anisotropic structure, a nanopourous structure with or without macroporosity for cell in-growth, a continuous fibrous network, highly crystalline nanofibrils, biocompatibility, high tensile and burst strength, and high suture pull out values.

[0054] One characteristic of the current invention is the anisotropic structure, that is, there is a difference in the biomaterial 's mechanical or physical properties when measured along different axes. This structure is a result of the dense cellulose network aided by channels going through the cellulose. The channels comprise a very dense cellulose layer as the channel wall, allowing the scaffold to be porous and maintain strength. The varying characteristics overall allow the scaffold to be both durable and flexible.

[0055] The continuous network and highly crystalline structure of the P-l→4-glucan nanofibrils of embodiments are what provide the high strength of the mesh. The network refers to the array of overlapping and entangled biocellulose nanofibrils which collectively form the biosynthetic mesh. With a tensile strength similar to aluminum, the crystalline cellulose strength is further improved by the entanglement of the nanofibrils. Though the overall mesh is flexible, the highly crystalline nanofibrils provide great strength and durability.

[0056] Another beneficial characteristic of embodiments is the structure of the mesh that has a porosity between 0% and 100%, with preferred embodiments ranging from 75-90% and from about 15-60%. In addition to a nanoporous structure, the scaffold can also have a macroporous (pores larger than 30 nanometers) characteristic. The macroporosity allows cells to pass through the mesh and to integrate with the mesh or implant. In embodiments, the macroporosity of the scaffolds and nanocellulose materials can range from 0-25%, 25-50%), 50-75%), 75-100%), and more specifically range from 0-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-65%, 65- 70%, 70-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95% based on surface area. In embodiments, macroporosity is determined by the amount of surface area taken up by the pores or voids in the material relative to the amount of cellulose material in the surface. For example, for a sheet of nanocellulose material having a surface area of 10 cm x 10 cm, the macroporosity of the sample would be 50%> when there are 50, 1 cm x 1cm square voids or channels punched or grown into that face of the material.

[0057] In the context of this specification, the biocompatibility of the mesh refers to the ability of the biosynthetic cellulose to integrate with the surrounding tissue to which the mesh is applied. Synthetic meshes can be rejected or have other negative immunological and histological effects such as disease transfer and severe inflammation. By using a naturally occurring material, such as cellulose, and one that accurately mimics collagen, it is less likely for the mesh to be rejected and cause complications such as inflammatory reactions. Embodiments may also comprise a soft, gel like layer on the surface of the scaffold to aid tissue integration.

[0058] For certain applications, strength of the scaffold is highly desirable as it ensures the scaffold will be maintained throughout the healing process. Scaffold and mesh embodiments of the invention are very strong with a maximum burst force above 60N/cm and with high suture pull out values, such as more than 30 N. In preferred embodiments, scaffolds can be prepared having a maximum burst force above 150 N/cm, such as above 160 N/cm, or even 200 N/cm. In addition, current embodiments are also highly elastic (extension to break above 20%). Such strength and elasticity allow for increased ability for healing and increased biomimicry of soft tissue.

[0059] Additionally, embodiments may allow formation and diffusion of proteoglycans within the structure to provide viscoelastic properties. Nutrients, oxygen, proteins, growth factors and proteoglycans diffuse through the space between the fibrils, i.e., the nanoporous features of the bacterial cellulose. Some embodiments are designed to allow cells to penetrate and pass through the material (multichannel or macroporosity). Such embodiments are designed to accommodate cells in the channels and support extracellular matrix production which results in tissue formation without contraction.

[0060] Another advantageous characteristic of embodiments is that the scaffolds can be prepared as non-degradable scaffolds. In contrast, most biologically occurring materials are degradable, meaning they will break down or deteriorate over time, which would be problematic for soft tissue repair. A non-degradable biological material provides a biologically compatible scaffold that will maintain structure and function. Moreover, embodiments provide meshes with good memory. Good memory effect on a macro scale renders the scaffolds suitable for laparoscopic and open surgery due to optimization of surgical handling of the mesh.

[0061 ] Particular embodiments of the invention provide methods of making a nanoporous scaffold comprising delivering oxygen through an array of porous capillaries contacting a culture medium inoculated with a cellulose generating bacteria to obtain a nanoporous scaffold comprising channels.

[0062] Such methods can comprise channels that are honeycomb shaped and comprise β-glucan nano fibers oriented perpendicular to channel walls. Objects of the invention provide for such methods and products, with channels disposed only partially through the scaffold.

[0063] A preferred cellulose generating bacteria is chosen from Gluconacetobacter xylinus or Acetobacter xylinum for preparing the scaffolds and for preparing them according to the methods disclosed herein.

[0064] A method of making a nanoporous scaffold comprising dense nanocellulose layers by delivering on demand nutrients in dynamic culture is also provided. Scaffolds can comprise space between nanocellulose layers for allowing diffusion of nutrients, oxygen, proteins, growth factors and proteoglycans but not cells into and through the scaffold.

[0065] Also provided is a method of making a cellulose sheet comprising an amount of cellulose up to about 40% using vacuum and pressure driven dehydration.

[0066] Objects further provide a method of making a β-glucan nanocomposite where targeted oxygen delivery of oxygen tension above 50% provides high concentration of cellulose nanofibrils.

[0067] A nanoporous honeycomb channeled scaffold comprising β-glucan nanofibers is provided by the invention. In embodiments, the scaffold comprises honeycomb channels have a width ranging from 300 micron to 3 mm, a porosity ranging from 20-80%, and a tensile strength ranging from about 60 N/cm to about 200 N/cm. At least some of the pores or channels of scaffolds of the invention can be introduced into the bacterial cellulose material in a mechanical or non-mechanical manner. [0068] Embodiments provide a nanoporous scaffold prepared by any of the methods disclosed in this specification, such as those provided just above.

[0069] Such nanoporous scaffolds can comprise honeycomb shape channels.

[0070] A method of physical crosslinking of the scaffolds can comprise forming hydrogen and Van der Waals bonds through sublimation of a frozen scaffold.

[0071 ] A biosynthetic cellulose material having pores sized and shaped to allow for invasion by fibroblasts and other cells when implanted into a human or animal is also included within the scope of the invention.

[0072] Objects of the invention further provide a biosynthetic cellulose material in which fibroblasts and other cells produce an extracellular matrix when implanted into a human or animal.

[0073] Methods of treating animal and humans which suffer from a soft tissue defect are also provided, which comprise implantation of a scaffold provided by this specification, or one which could be prepared by a method provided in this specification.

[0074] Specific methods of treating animal and humans to reinforce soft tissue can be chosen from one or more of defects of the abdominal and thoracic wall, muscle flap

reinforcement, prolapsed repair, reconstruction of the pelvic floor, hernias, suture-line reinforcement and reconstructive surgery by implantation of β-glucan scaffold.

[0075] Preferred embodiments of the invention provide a nanoporous scaffold comprising a plurality of pores ranging in size from 300 micron to 3 mm, a porosity ranging from 20-80%, and a tensile strength ranging from about 60 N/cm to about 200 N/cm, wherein the scaffold is grown in a manner to comprise at least some of the pores.

[0076] Such scaffolds can be prepared by delivering oxygen through an array of porous capillaries contacting a culture medium inoculated with a cellulose generating bacteria.

[0077] Such scaffolds can comprise pores having a honeycomb cross section.

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The accompanying drawings illustrate certain aspects of some of the embodiments of the present invention, and should not be used to limit or define the invention. Together with the written description the drawings serve to explain certain principles of the invention. [0079] FIGS. 1 A-B are images of an embodiment of a nanocellulose scaffold for soft tissue replacement according to the invention.

[0080] FIGS. 2A-B are schematic diagrams of a representative honeycomb type nanoporous structure of a soft tissue scaffold of the invention.

[0081 ] FIG. 3 is an image from a Scanning Electron Micrograph illustrating the nanoporous structure of a representative bacterial nanocellulose scaffold with a honeycomb configuration according to an embodiment of the invention.

[0082] FIG. 4 is an image from a Scanning Electron Micrograph of nanocellulose illustrating a representative bacteria produced compact horizontal middle layer according to an embodiment of the invention.

[0083] FIG. 5 is an image from a Scanning Electron Micrograph illustrating a channel with compact nanocellulose walls in a representative bacterial nanocellulose scaffold according to an embodiment of the invention.

[0084] FIG. 6 is a graph illustrating the effect of cellulose content on strength of the nanocellulose soft tissue graft according to an embodiment of the invention.

[0085] FIG. 7 is a graph illustrating a suture pull out test performed on nanocellulose soft tissue grafts with various nanocellulose content according to an embodiment of the invention.

[0086] FIGS. 8A-C are images of various channel patterns and sizes in the mesh, illustrating the variety of macroporosity configurations, including shape, size, and spacing of pores, that can be achieved through embodiments of the invention.

[0087] FIG. 9 is a graph of burst testing results illustrating the effect of porosity versus strength of a nanocellulose scaffold according to an embodiment of the invention.

[0088] FIGS. 10A-B are photographs of a prototype of a Biosynthetic Cellulose mesh comprising a honeycomb configuration with an enhanced view of the honeycomb shape and size according to an embodiment of the invention.

[0089] FIGS. 11 A-B are photographs of a representative bioreactor for preparing bacterial cellulose mesh according to embodiments of the invention and an exemplary BC mesh grown in such a bioreactor, respectively. [0090] FIGS. 12A-B are photographs of a prototype bioreactor comprising a honeycomb structure and an exemplary BC mesh material comprising the resultant honeycomb type structure/channels, which material was grown in such a bioreactor, respectively.

[0091 ] FIGS. 13A-B are schematic computer model images of a full scale bioreactor for cultivation of honeycomb type structure BC mesh and an enhanced view of said bioreactor, respectively, showing hexagonal supports with 5mm spacing and the hexagonal tubes closed at the top but open through the plate, according to an embodiment of the invention.

[0092] FIG. 14A is a CAD drawing for a honeycomb type bioreactor.

[0093] FIGS. 14B-D are photographs of a 3D printed bioreactor for cultivation of honeycomb mesh made from a 3D printing process by sintering of PP particles. The bioreactor is oxygen and air permeable. The hexagonal support elements are hollow.

[0094] FIG. 15 is confocal microscopy image illustrating the distribution of fibroblasts within a honeycomb type structure of a nanocellulose soft tissue scaffold according to an embodiment of the invention.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

[0095] Reference will now be made in detail to various exemplary embodiments of the invention. It is to be understood that the following discussion of exemplary embodiments is not intended as a limitation on the invention. Rather, the following discussion is provided to give the reader a more detailed understanding of certain aspects and features of the invention. More particularly, to facilitate a better understanding of the present invention, the following examples of certain aspects of some embodiments are given. In no way should the following examples be read to limit, or define, the scope of the invention.

[0096] FIGS. 1 A-B illustrate a representative embodiment of a scaffold of the invention that is a mesh type bacterial cellulose-based product. As shown, the soft tissue scaffold is a thin, flexible material with the ability to withstand stretching. The scaffold is rigid enough in order to retain shape when handled, yet flexible enough to take a variety of shapes depending on the area to which it is applied. These characteristics make the nanocellulose soft tissue scaffold ideal for soft tissue repair, such as hernia repair, as these characteristics are shared by real soft tissue, accurately mimicking natural results. [0097] Embodiments of the present invention relate to biomimetic soft tissue scaffolds, grafts, and prostheses, as well as methods and bioreactors for making them. Such scaffolds can be prepared according to methods of the invention, which include growing bacterial cellulose to a desired thickness and having desired properties, including strength and suture pull out characteristics. As shown, the mesh can be pliable and flexible, with elastic properties as well. Depending on the end-use application, the characteristics can be altered to impart specific properties into the resultant material. For example, when using the scaffold for pelvic floor repair, a material with higher strength and less elasticity may be preferred than when using the product for other soft tissue repair.

[0098] Scaffolds of the current invention can be prepared for any surgical procedure, including any tissue and muscle repair or transplant procedure, whether open or laparoscopic, including, but not limited to, reinforcement of soft tissue, hernia repair or reinforcement, reconstructive procedures, suture line reinforcement, skin grafts, prolapse, treatment of urinary incontinence, cardiac wall repair or reinforcement, abdominal or thoracic wall repair or reinforcement, and pelvic floor repair or reinforcement. Preferred embodiments include a mesh designed with strength and elasticity characteristics desired for hernia repairs, such as a material capable of mimicking the characteristics of abdominal muscle tissue. Even further, embodiments of the invention can be used for any internal or external procedure.

[0099] Biocellulose for scaffolds of the invention can be generated from any cellulose- containing material. Examples include annual plants, trees, fungi or bacteria, with preferred embodiments generated from bacteria such as the genera Aerobacter, Acetobacter,

Achromobacter, Agrobacterium, Alacaligenes , Azotobacter, Pseudomonas, Rhizobium, and Sarcina, specifically Gluconacetobacter xylinus, Acetobacter xylinum, Lactobacillus mali, Agrobacterium tumefaciens, Rhizobium leguminosarum bv.trifolii, Sarcina ventriculi , enterobacteriaceae Salmonella spp., Escherichia coli, Klebsiella pneu-moniae and several species of cyanobacteria to name a few. Cellulose can be generated from any vascular plant species, which include those within the groups Tracheophyta and Tracheobionta.

[00100] Biocellulose nano fibrils formed from cellulose producing bacteria most closely mimic the characteristics of collagen found in human and animal soft tissue. The array of fibrils provide a porous yet durable and flexible material. The network of nanofibrils allow nutrients, oxygen, proteins, growth factors and proteoglycans to pass through the space between the fibrils (i.e., the nanoporous structure) as well as through the macropores, if present, allowing the scaffold to integrate with the implant and surrounding tissue. The nanofibrils also provide the elasticity and strength need to replace natural collagen. The channels formed within the BC, comprise dense nanocellulose walls that allow cell infiltration through the scaffold, which is crucial to the success of any surgical mesh.

[00101 ] Embodiments of the invention are an improvement over existing scaffolds which typically have one or more of deficient mechanical properties, insufficient cellulose content, lack of dimensional stability, poor cell infiltration and poor tissue integration.

[00102] Design and Architecture of Bacterial Cellulose Scaffolds.

[00103] FIGS. 2A-B provide schematic diagrams of a representative embodiment of a scaffold of the invention. As illustrated, the scaffolds can be prepared to have any shape and configuration of macroporosity, such as a honeycomb structure. This honeycomb structure, or any desired structure, is prepared by using a complementary configured bioreactor. For example, a bioreactor comprising a plurality of honeycomb shaped supports spaced a desired distance from one another can be used to grow the bacterial cellulose scaffold having the desired honeycomb pattern. In preferred embodiments, the scaffold comprises at least a portion of the material that is a soft, gel-like material to promote cell adhesion and tissue integration. This can be accomplished at surfaces of the bacterial cellulose material that are exposed to an atmosphere having an oxygen deficiency. In embodiments, a surface of the scaffold, such as the upper or lower surface, can have a soft, gel-like consistency. One way to achieve a soft, gel-like characteristic in the material is by reducing the amount of oxygen present near the end point of cultivation such that there is an oxygen deficiency.

[00104] The macroporous channels are designed to increase cell in-growth which promotes cell integration. In embodiments, it may be crucial for the mechanical performance of the honeycomb, or hexagonal, structure to have a very dense cellulose layer as the wall of the channel. This is achieved by delivery of oxygen through an array of permeable capillaries. The middle part of the honeycomb structure is composed of the dense layer of nanocellulose. This is achieved by use of an enriched oxygen atmosphere in the bioreactor tray and enriched medium particularly with an increased nitrogen source. [00105] It is within the skill of the art to configure a bioreactor according to any corresponding desired scaffold configuration. In preferred embodiments, the size of the channels or holes can be in the range of microns to millimeters, or even nanometers. Generally, when referring to nanoporosity of the scaffold material in the context of this specification, the spacing between cellulose fibers is what contributes to the random nanoporosity. In embodiments, nanoporous channels, holes, or voids can also be introduced into the material by growing the cellulose around supports that will leave a pore or hole in the material when the material is removed from the bioreactor. Typically, however, this type of porosity purposefully introduced into the scaffolds will be in the micron to millimeter range and thus may generally be referred to herein as macroporosity even if the actual size of the channels is in the nanometer range.

[00106] The shape of the purposefully introduced pores can be any shape as well as any size desired for a particular application. For example, the channels can comprise a cross sectional shape that is triangular, square, rectangular, rhombus, hexagonal or honeycomb, pentagonal, heptagonal, and round or circular, or substantially triangular, square, rectangular, rhombus shaped, pentagonal, heptagonal, and round. The honeycomb pattern in the context of this specification can be used to describe the shape of the individual channels or the scattered organization of the channels within the scaffold. In specific embodiments, the introduced holes or channels can be any size ranging in diameter from 0-5mm, with specific ranges including but not limited to 0-lmm, l-2mm, 2-3mm, 3-4mm and 4-5mm. Smaller diameter channels can also be used, including from about 0-900 micron, such as from 10-800 micron, or 20-700 micron, or 30-600 micron, or from about 40-500 micron, such as 50-400 micron, or from 60-300 micron, or from 75-350 micron, or from 100-200 micron, and so on. It is also possible to fabricate scaffolds having channels or voids in the material that are 0-10,000 nm, such as 100-200 nm, or from about 300-400 nm, or 500-600 nm, or 700-800 nm, or 900-1,000 nm, or 1,100 to 1 ,200 nm, or from about 1,300-1,400 nm, or 1,500-2,000 nm, or 3,000-4,000 nm, or 5,000-7,500 nm, or from about 8,000-9,000 nm and so on, or even on the order of from 10-90 nm.

[00107] Likewise, the distance between channels when measured center to center can range from 0-lOmm, with specific ranges including but not limited to, 0-lmm, l-2mm, 2-3mm, 3- 4mm, 4-5mm, 5-6mm, 6-7mm, 7-8mm, 8-9mm, and 9- 10mm, or even smaller. Indeed, any of the specific dimensions provided for the cross sectional diameter of the channels can be used as the dimension of the spacing between channels. Examples of particular embodiments of the invention include a bacterial cellulose scaffold comprising cylindrical channels having a circular or hexagonal cross section ranging in diameter from 50 micron to 3 mm, with spacing between channels ranging from 0.5 mm to 2 mm, or from 500 micron to 5 mm measured center to center. Especially preferred are bacterial cellulose scaffolds comprising any one or more of 0.5 mm, 0.6 mm, 1 mm, 2 mm, 3 mm, or 4 mm voids spaced apart by any one or more of 0.5 mm, 1 mm, 5 mm, or 1.5 cm.

[00108] The pattern of channels disposed in the material can be random or ordered. Thus, the spacing between channels need not be consistent from channel to channel throughout the material. For example, two channels in the material may be spaced apart by 1 mm, while two other channels may be spaced apart by 0.5mm. Similarly, the size of the channels need not be the same size for every channel in the material. For example, the material may comprise one or more channel having a width of 3 mm and also comprise one or more channel having a width of 1 mm. Even further, the shape of the channels may also be varied throughout the material. For example, it may be desired to obtain a scaffold material having channels with a circular cross section disposed over a majority of the surface area of the material but also have channels with a honeycomb cross section around the perimeter or close to the perimeter of the material, or reversed if desired. Any combination of size, shape, or spacing of channels can be used. Even further, the channels can be oriented in any manner relative to one another within the scaffold. For example, when the cross section of the channel is hexagonal or square, for example, the sides of the channels can be oriented parallel with one another such that the shape and width of the bacterial cellulose material disposed between the channels is consistent throughout the material. Alternatively, the channels can be oriented corner to corner, such that the bacterial cellulose comprises an hour glass shape between each channel and thus the width of the material is wider at some portions of the material than others. Combinations of these configurations are also possible, as well as the channels being oriented corner to side, and so forth. Endless

embodiments are possible according to the invention, and the scaffold material can be configured to have a uniform width between channels or varying widths. [00109] Characterization by SEM of Nanoporous BC Soft Tissue Scaffold.

[001 10] The architecture of the BC soft tissue scaffolds with honeycomb structure was characterized by scanning electron microscopy (SEM). Preparation for analysis by SEM can be performed in any manner acceptable in the art. For example, samples of the invention were quenched in liquid nitrogen, thereafter lyophilized overnight (Heto PowerDry PL3000, Thermo Fisher Scientific), then mounted on SEM studs, sputtered with a gold film, and finally analyzed using a Leo Ultra 55 field emission gun (FEG) scanning electron microscope. SEM images were taken at various magnifications.

[001 1 1 ] As shown in FIG. 3, a representative Scanning Electron Micrograph (SEM) of nanocellulose honeycomb structure produced by bacteria shows details of the architecture that can be prepared according to embodiments of the invention. This image shows a perspective view of the bacterial cellulose sheet comprising a dense layer of nanocellulose fibrils. The nanofibrils within the scaffold as shown are interconnected, providing strength and durability to the mesh. While dense, it can still be seen that the nanofibrils provide space within the middle layer to allow for porosity. This nanoporous structure provides for the passage of nutrients into and through the scaffold when implanted to allow for increased biocompatibility.

[001 12] FIG. 4 shows a top view the middle dense nanocellulose layer. It is clearly seen that there is high density and orientation of nanocellulose fibrils. Spacing between the fibrils provides for diffusion of nutrients, oxygen, proteins, growth factors and proteoglycans.

[001 13] FIG. 5 shows the structure of single channel. Channels according to the invention are introduced to the material during the growth process. Columnar supports, such as permeable pipettes or capillary tubes, are provided in the bioreactor in a manner to allow for growth of the bacterial cellulose on and around the outside of the supports. Once the BC material is formed to the desired shape, size, and/or thickness, the supports are removed which leaves voids in the shape of channels through the BC material. In preferred embodiments, the columnar supports are hollow cylinders with an oxygen permeable surface through which oxygen can pass to enrich the growth of the bacterial cellulose on and around the outside of the supports. Due to the high level of oxygen introduced at this surface, the bacterial cellulose grows more rapidly and denser than in other areas of the material. As a result, a high density of nanocellulose is formed at the wall of the channel, i.e., the material that grows on and around the support. In preferred embodiments, the channels are formed into the scaffold material, but not all the way through. In other embodiments, the channels can pass completely through the scaffold, such that holes or passageways, i.e., through holes, are formed into and through the material.

[001 14] Biomechanical Evaluation of Nanocellulose Soft Tissue Scaffolds.

[001 15] Bacterial cellulose specimens were prepared for tensile strength testing by cutting out between five and ten rectangular strips, about 10 mm wide, from each scaffold. Scaffolds having different cellulose contents were prepared by compression dehydration of BC hydrogel. The scaffolds were weighed before compression, placed between two absorbent pads, and pressed between two platens of a universal testing machine until the weight that would give the desired cellulose content was attained. The samples were lyophilized and rewetted.

[001 16] The different cellulose contents used for tensile strength testing were 1 % (native BC), 10 %, 20 %, and 30 %. Cellulose content can be any amount ranging from about 0.1% to about 99.0% for example based on weight, such as from about 5%> to 75%, about 8% to 50%, 15%) to about 40%), or from about 25% to about 35%. For the suture pull out tests the cellulose contents used were 1 %, 10 % and 20%. To achieve these, scaffolds with a thickness of 10 mm and 20 mm, respectively, were pressed to a thickness of 1 mm using confined compression in an Instron 5565 A. For each cellulose content, three scaffold sheets were pressed and cut into two rectangular strips generating six specimens for each. In each strip two holes were punched, 5 mm apart and 5 mm from the edge following the method described by Barber et al. A suture thread (No. 2 Fiber Wire; Arthrex) was used to make a horizontal mattress suture and the ends were tied together.

[001 17] The grips of the tensile testing machine were covered with fine sandpaper and silicone at the edge, to prevent the specimens from slipping or breaking at the jaw. A 100 N load cell was used. The specimens were then extended at a rate of 10 mm/min until failure. The tests were performed using Instron 5565 A with Biobath.

[001 18] FIG. 6 is a graph showing the effect of cellulose content on strength of the nanocellulose soft tissue scaffold. Above 10% cellulose content, the scaffold has strength to break above 60 N/cm. The strength of the scaffolds according to embodiments of the invention can range from 0-100 N/cm, with preferred embodiments ranging from 30-70 N/cm. The strength can also include, but is not limited to, the follow ranges, 0-10 N/cm, 10-20 N/cm, 20-30 N/cm, 30-40 N/cm, 40-50 N/cm, 50-60 N/cm, 60-70 N/cm, 70-80 N/cm, 80-90 N/cm, and 90-100 N/cm. All samples showed extension to break at about 20%. For hernia devices, CTQ's (Critical to Quality) values for Tear Force are greater than approximately 6.4 N, and for suture pull out force greater than 16 N. CTQ's (Critical to Quality) for Burst small plunger test (5 cm diameter) Bursting Force are greater than 200N and Burst elongation is less than 40%.

[001 19] During biomechanical evaluation BC meshes were tested for tensile properties using a 6 cm width sample folded 3 times. BC meshes fulfill biomechanical requirements (plunger and tensile properties) and have similar performance to currently marketed meshes. They are however stiffer. Additionally, it is preferred that the cut tear force for such scaffolds be greater than 6.4 N.

[00120] FIG. 7 shows results from suture pull-out tests of scaffold embodiments of the invention. As illustrated, the nanocellulose content and scaffold architecture have a dramatic effect on suture pull-out force as can be seen with a force above 30 N for scaffolds with more than 10% nanocellulose. Preferably, scaffolds of the invention comprise at least 10% bacterial cellulose and have a pull out force above 10 N, such as a cellulose content of at least 10-15% and a pull out force above 15 N, such as a cellulose content of at least 20% and a pull out force of greater than 20 N. Other embodiments can comprise scaffolds with a cellulose content ranging from 10-45% and having a pull out force ranging from 10-60 N. Especially preferred

embodiments comprise a cellulose content ranging from about 30% to about 60% and have a pull out force of about 20 N to 40 N or higher.

[00121 ] FIGS. 8A-C are photographs of various exemplary scaffolds according to

embodiments of the invention. FIG. 8A provides several examples of a bacterial cellulose sheet comprising circular holes disposed in and through the sheet. The bacterial cellulose sheet, as shown here, can be compressed according to techniques described in this specification. The holes or pores can be punched into the material after the growth and/or compression process, for example, by using laser ablation or water jet techniques or the holes can be formed into the bacterial cellulose material during the growth process. As illustrated, a number of holes can be introduced into the bacterial cellulose material having any shape or size. FIG. 8A from top to bottom illustrates small (about 2 mm), medium (about 3 mm), and large (about 4 mm) size holes, respectively. The holes can be spaced apart by about 5 mm, or by about 4 mm, or by about 3 mm, by about 2 mm, or by about 1 mm. As can be seen, with small holes a porosity of about 20% can be obtained and if the same number of holes is used but slightly larger, then a porosity of about 35% can be obtained, and even slightly larger holes can result in a porosity of about 50%. The greater number of holes and/or the larger the holes may reduce the mechanical strength of the product but increase the cellular and/or tissue in-growth capability of the material.

[00122] As shown in FIG. 8B, the holes can be of any shape as well. Here, hexagonal or honeycomb shape holes of about 2 mm, 3 mm, or 3.5 mm widths are provided and are spaced apart by about 6 mm, or by about 5 mm, or by about 4 mm, or by about 3 mm, or by about 2 mm, or by about 1 mm. Various porous bacterial cellulose sheets can be prepared, for example, sheets comprising 20% or 40%, or 50% porosity. In this context, porosity refers to the amount of space the voids occupy in the two-dimension surface of the sheet, i.e., the surface area for example of the top surface of the bacterial cellulose sheet. The honeycomb shape holes can be oriented in any manner relative to one another in the sheet. Here, the holes are oriented in horizontal rows with the holes disposed with their edges parallel to the adjacent holes in the row. The shape of the resultant sheet reveals that the width between holes with this type of

configuration provides for a uniform or substantially uniform width throughout the material.

[00123] FIG. 8C is another variation of a bacterial cellulose sheet according to an

embodiment of the invention. Here, the holes ranging in widths from about 2-4 mm are honeycomb shaped and are oriented in horizontal rows corner to corner relative to the adjacent holes, while being spaced apart from one another by about 4 mm, or 3 mm, or 2 mm, or 1 mm, or even 0.5 mm. The width of the honeycomb shaped holes can be measured edge to edge or corner to corner. This provides for a varied width of the sheet throughout the sheet. It is important to note that although examples are shown of the bacterial cellulose scaffolds comprising holes on the order of millimeters, it is within the skill of the art to enlarge or reduce the size of the holes to obtain a sheet for a particular application. As illustrated, a macroporosity ranging from 20-50% is obtained in the scaffold embodiments exemplified.

[00124] FIG. 9 shows the effect of different channel or hole patterns on the reduction of Burst Force (Strength in %). The hexagon pattern 1 (face to face, or edge to edge) performs best because it gives the lowest reduction of burst force at the same porosity for all patterns compared. There is however a large drop in burst strength between samples with medium porosity (porosity 37.8%) compared with samples with high porosity (porosity 52.2%). Samples with medium porosity have hexagonal channels of 2.9 mm and are separated by 5mm whereas high porosity has hexagonal channels of 3.49 mm separated with 1.5mm.

[00125] FIGS. 10A-B are photographs of a representative bacterial cellulose mesh according to an embodiment of the invention. FIG. 10A shows the mesh as a sheet of bacterial cellulose comprise a plurality of holes. FIG. 10B shows the configuration of the holes in this embodiment is a hexagonal shape, although any shape can be used. The holes are about 3 mm in width and are spaced apart by about 5 mm. The mesh sheet can be cut or grown to any desired size or thickness. In some applications a sheet of about 10 cm x 10 cm may be desired or smaller sheets of about 3 cm x 3 cm may be needed for other applications.

[00126] Materials of the invention can be prepared for any desired application and having any desired morphological or mechanical characteristics. The following examples of particular scaffolds that can be prepared provide guidance on the different combinations of mechanical properties of the materials that can be achieved. The combinations provided in Table I below can be re-arranged to obtain materials having one or more of the properties listed. Indeed the characteristics of the materials of the invention can comprise any feature or combinations of features present in other existing products, such as those disclosed in U.S. Application

Publication No. 20100158985 entitled "Porous Structures of Microbial-Derived Cellulose for in vivo Implantation" or any synthetic polypropylene product, or composites thereof.

[00127] Table I. Examples of Bacterial Cellulose Mesh

[00128] Preparation of Nanoporous BC Soft Tissue Scaffold.

[00129] An object of the invention provides methods of making a nanoporous scaffold comprising delivering oxygen through an array of porous capillaries contacting a culture medium inoculated with a cellulose generating bacteria, such as Gluconacetobacter xylinus, to obtain a nanoporous scaffold with honey comb channeled structure comprising β-glucan nanofibers perpendicular to channel walls.

[00130] Embodiments of the invention also provide increased cellulose production and growth in layers in dynamic culture. Methods of the invention provide for the control of media concentration and oxygen delivery. In embodiments, oxygen delivery having a concentration above that found in air can be provided to prevent or eliminate the problem of oxygen

deficiency, which often leads to poor growth of bacterial cellulose. Bioreactors can be configured to provide any number of scaffold configurations, including scaffolds having a macroporous honeycomb-type structure. Using such methods and bioreactors, multichannels (i.e., macroporosity) can be introduced into the bacterial cellulose material during the production step (i.e., during growth of the material or scaffold). Channels introduced into the material in this manner will be characterized by having a high density of cellulose material on the inner wall of the channels due to the increased cellulose production resulting from targeted oxygen delivery using porous capillaries. That is, the bacterial cellulose is encouraged to grow around the porous capillary supports because an increased amount of oxygen is provided through the capillary material. Method embodiments of the invention are also capable of controlling nanoporosity during growth and by post treatment. An increased cellulose content can also be obtained in the materials by compression and physical crosslinking. A soft gelly-like, tissue integrative top and bottom layer of the material can be produced by oxygen deficient cultivation conditions.

[00131 ] In preferred embodiments, the scaffold comprises at least a portion of the material that is a soft, gel-like material to promote cell adhesion and tissue integration. In embodiments, a surface of the scaffold, such as the upper or lower surface, can have a soft, gel-like consistency. One way to achieve a soft, gel-like characteristic in the material is by reducing the amount of oxygen present near the end point of cultivation such that there is an oxygen deficiency. The channels are designed to increase cell in-growth which promotes cell integration. It is crucial for mechanical performance of honeycomb structure to have very dense cellulose layer as the wall of the channel. This is achieved by delivery of oxygen through the capillaries. The middle part of the honeycomb structure is composed of the dense layer of nanocellulose. This is achieved by use of enriched oxygen atmosphere in tray bioreactor and enriched medium particularly with increased nitrogen source.

[00132] Although particular bioreactors and preparation methods may be designed to obtain specific scaffolds having desired characteristics, the following general manufacturing method is provided for illustration and general guidance. To obtain various scaffolds and bacterial cellulose sheets according to the invention tray bioreactors were inoculated with

Gluconacetobacter xylinus ATCC^®700178. It is not critical the type of organism used, and any desired bacteria or organism can be used, including genetically modified organisms. A suspension of 4x 10⁶ bacteria per ml and 25 ml of sterile culture media (described below) was added to each tray. The controlled volumes of sterilized media were added each 6 hours to the top of the tray in such matter that bacteria cultivation was not disturbed. The preferential addition is to use microspray, where media is added with a low pressure spray, mist, sprinkle or drip. The amount of the added media is calculated to be consumed by bacteria during 6 hour time period. The composition of the medium can be varied in order to control production rate of cellulose and network density. In order to increase nanocellulose density the air above the tray surface is exchanged with humidified oxygen enriched atmosphere. The last two hours of cultivation was performed with oxygen deficiency by replacing air above tray with mixture of air with nitrogen. The trays were placed in a bacteriology cabinet and the bacteria were allowed to grow under these semi-dynamic conditions for 7 days at 30 °C. The bacteria were removed by immersing the pellicles in 0.1 sodium carbonate overnight, followed by 24 h in fresh 0.1M NaOH heated in a 60 °C water bath. The samples were then carefully rinsed with large amounts of 60 °C deionized water to remove bacterial residues and neutralize the pH using acetic acid. After cleaning, the BC scaffolds were cut in rectangular scaffolds (1 x6 cm).

[00133] Examples of suitable media for growing bacteria include but are not limited to: Schramm-Hestrin-medium which contains, per liter distilled water, 20 g of glucose, 5 g of bactopeptone, 5 g of yeast extract, 3.4 g of disodium-hydrogenphosphate dehydrate and 1.15 g of citric acid monohydrate and which exhibits a pH value of between 6.0 and 6.3; 0.3 wt% green tea powder and 5wt% sucrose with pH adjusted to 4.5 with acetic acid; Medium composed of (fructose [4 % w/vl], yeast extract [0.5 % w/v], (NH4)2S04 [0.33 % w/v], KH₂P0₄ [0.1 % w/v], MgS0₄ ^»7H₂0 [0.025 % w/v], corn steep liquor [2 % v/v], trace metal solution [1 % v/v, (30 mg EDTA, 14.7 mg CaCl₂ ^«2H20, 3.6 mg FeS0₄ ^«7H₂0, 2.42 mg Na₂Mo0₄ ^«2H₂0, 1.73 mg

ZnS0₄ ^«7H₂0, 1.39 mg MnS0₄ ^«5H₂0 and 0.05 mg CuS0₄ ^«5H₂0 in 1 liter distilled water)] and vitamin solution [1 % v/v (2 mg inositol, 0.4 mg pyridoxine HC1, 0.4 mg niacin, 0.4 mg thiamine HC1, 0.2 mg para-aminobenzoic acid, 0.2 mg D-panthothenic acid calcium, 0.2 mg riboflavin, 0.0002 mg folic acid and 0.0002 mg D-biotin in 1 liter distilled water)]). Any medium comprised of sugar source, nitrogen source and vitamins can be successfully used. Bacteria grow even on apple or pineapple juice, or coconut milk or beer waste or wine.

[00134] Once grown, BC scaffolds can be compressed to further increase the mechanical strength of the materials. For example, according to embodiments, BC scaffolds were compressed using vacuum press to 10 % of initial thickness, frozen, and lyophilized. The compressed scaffolds were then rewetted with DI water. The dimensional stable scaffolds with 10% cellulose content and 0.6 mm thickness were produced by this process. An example of a nanocellulose soft tissue scaffold prepared using compression is shown in FIGS. 1 A-B.

[00135] Preparation of Honeycomb Nanoporous BC Soft Tissue Scaffold.

[00136] A honeycomb type scaffold of the invention was prepared comprising channels in the material having a controlled shape and size. A general method for comprising such scaffolds include equipping tray bioreactors (similar to those described previously) with array of oxygen permeable capillaries (optical fibers) with outer diameters varying between 1 micron and 5 mm, with a preferred size in the range of about 10 to 3,000 micron, such as 50-500 micrometers. The distance between the capillaries can vary for example from 1 micron to about 5 mm, such as from 50 micrometers to 500 micrometers, including 50-100 micron, 100-150 micron, 150-200 micron, 200-250 micron, 250-300 micron, 300-350 micron, 350-400 micron, 400-450 micron, and 450-500 micrometers. The length of the capillaries can vary from 1 mm to 3 mm, depending on the thickness of the resultant bacterial cellulose sheet that is desired. All capillaries are attached to a permeable plate which can deliver oxygen. In embodiments, the capillaries are parallel to each other, but can be oriented in any manner to achieve a particular channel pattern in a desired bacterial cellulose sheet. The array looks like a brush in embodiments. [00137] Tray bioreactors were inoculated with Gluconacetobacter xylinus ATCC^R700178, although any equivalent bacteria or organism can be used. A suspension of 4x 10⁶ bacteria per ml and 25 ml of sterile culture media (described below) was added to each tray. The controlled volumes of sterilized media were added each 6 hours to the top of the tray in such matter that bacteria cultivation was not disturbed. The preferential addition is to use microspray. The amount of the added media is calculated to be consumed by bacteria during a 6 hour time period. The composition of the medium can be varied in order to control production rate of cellulose and network density. For example, in order to increase nanocellulose density the air above the tray surface can be exchanged with humidified oxygen enriched atmosphere. The oxygen is continuously delivered through the capillaries with a starting point varying between 3 and 5 hours. After 1 to 3 days of cultivation (depending on the length of the capillaries, the growth of BC is about 1 mm per 24 hours) the middle layer is produced. In embodiments, the thickness of middle horizontal layer can be controlled between 1 and 2 mm depending on requirements on mechanical performance. Indeed, any thickness of material can be produced including from 0.5 mm to about 10 mm.

[00138] After 1 to 2 days the new capillary array is placed on the top side of the tray bioreactor and oxygen is delivered. The trays were placed in a bacteriology cabinet and the bacteria were allowed to grow under these semi-dynamic conditions for 7 days at 30 °C. The bacteria were removed by immersing the resultant pellicles in 0.1 sodium carbonate overnight, followed by 24 h in fresh 0.1M NaOH heated in a 60 °C water bath. The samples were then carefully rinsed with large amounts of 60 °C deionized water to remove bacterial residues and neutralize the pH using acetic acid. After cleaning, the BC scaffolds were cut in rectangular scaffolds (1 x6 cm). BC scaffolds were compressed using vacuum press to 10 % of initial thickness, frozen, and lyophilized. They were then rewetted with DI water. The dimensional stable scaffolds with 10% cellulose content and honeycomb structure with 0.5-0.7 mm thickness were produced by this process.

[00139] In addition to creating a honeycomb structure nanocellulose scaffold by growing such a scaffold in a bioreactor, other methods such as laser ablation, water jet cutting, or other mechanical methods (such as needles or pins or a microarray of needles/pins) of introducing holes into the material, may be used to achieve the same result or end product. Such techniques disclosed in U.S. Application Publication Nos. 20110262696 and 20110262706 using punch- type machines can for example be used to introduce porosity into the material. These techniques allow for different size and patterns of channels within the scaffold than can be created by growing the scaffold in a bioreactor. However, although the method of achieving the honeycomb structure is different, desired mechanical properties are still maintained.

Embodiments of the invention include any method of making a nanoporous biocellulose honeycomb structure scaffold. In embodiments for example a sheet of bacterial cellulose can be grown to a desired thickness and compressed using techniques illustrated herein. Then a desired amount of holes can be introduced into the material to achieve a desired porosity. Alternatively, the BC materials can be grown to include a desired porosity and then processed using laser ablation or water jet cutting to introduce additional pores where needed in a particular material.

[00140] In preferred embodiments, a bioreactor with a hole or pore separation distance of 5mm or less can be used, such as from about 1-3 mm. The pores themselves can be about 600 micron, in the range of about 1-6 mm. One way of producing this type of material is to employ oxygen permeable polypropylene pipettes (hollow inside, not rods, capped with silicon film), held in place with a sheet of metal comprising holes for individual pipettes, and allowing bacterial cellulose to grow around the outside of the pipettes.

[00141 ] FIGS. 11 A-B, respectively, show an example of such a bioreactor and a

corresponding mesh product grown in the bioreactor. As shown in FIG. 11 A, the pipettes are held in a particular pattern or array, in this case a honeycomb pattern, so that as the bacterial cellulose grows around the outside of the pipettes, the desired shape for the scaffold will be formed. Here, the honeycomb pattern refers to the orientation of the pipettes relative to one another but can also refer to the cross sectional shape of the pipettes themselves. FIG. 1 IB shows the resulting BC scaffold grown in the FIG. 11 A bioreactor. The BC material can be produced with the pipettes going completely or only partially through the resulting scaffold.

[00142] FIGS. 12A-B are photographs of an additional embodiment of a honeycomb structure bioreactor and BC mesh with honeycomb type structure grown in such bioreactor, respectively. As shown, this bioreactor is smaller than the one previously mentioned. According to embodiments of the invention, bioreactors can vary in size in order to obtain a desired size of BC material for the scaffold. In the embodiment shown in FIG. 12A, the bioreactor is comprised of pipettes and a base. As shown in FIG. 12B, the resultant mesh can comprise channels, or holes, that go completely through the material. Additionally, it can be seen that each hexagonal channel has a uniform amount of biocellulose material surrounding it. Although hexagonal shaped pipettes were used in this embodiment it is understood that within the context of this invention any shape supports can be used, such as supports having a circular, square, rectangular, or triangular cross section to name a few.

[00143] Additional embodiments of bioreactors are shown in FIGS. 13A-B, which provide schematic drawings of a full scale bioreactor that can be used for cultivation of honeycomb structure BC mesh and an enhanced view of said bioreactor, respectively. Each image shows hexagonal support elements with 5 mm spacing between the supports. Any spacing between supports can be used, including for example from 100 micron to 3 mm. The tubular supports, here hexagonal tubes, can either be open or closed at the top, but are usually open at the bottom near the plate or base. In this embodiment the bacterial cellulose may grow around the hexagonal elements by allowing oxygen to permeate through the supports to where the nanocellulose is growing thereby creating a dense nanocellulose layer at the interface of the outer surface of the permeable support and the nanocellulose as a result of the oxygen enriched environment.

[00144] In many embodiments, a bioreactor may be created by comprising two or parts. In additional embodiments a bioreactor may be created through use of 3D printed templates.

FIGS. 14A-D show respectively an image of a CAD drawing for a honeycomb structure bioreactor, images of fragments of a 3D printed bioreactor from a CAD drawing, and a full scale 3-D printed bioreactor from a CAD drawing. In this embodiment, the hexagonal support elements are hollow and oxygen and air permeable. Producing a bioreactor by this method allows for precise measurements in a bioreactor of any size and shape as well as the resulting scaffold, including a bioreactor that is rigid or flexible. Without a 3D printer hexagonal element size is limited to the available size of pipettes.

[00145] Tissue Integration into Nanocellulose Channels.

[00146] Nanocellulose scaffolds with honeycomb structure were evaluated with an in vitro study using fibroblasts. 3T6-Swiss Albino cells ATCC^® CCL-96 in passage 3 were expanded in T-75 flasks using growth medium containing Dulbecco's Modified Eagle Medium (DMEM) high glucose 4.5g/L (Invitrogen, Gaithersburg, MD), 10% fetal bovine serum (Gemini Bio- Products, Calabasas, CA) and 1% antibiotic/antimycotic solution (Invitrogen). Nanocellulose scaffolds of size 1 x6 cm were placed in deionized water and steam sterilized (1 bar, 121 °C) for 20 min. After cooling, the scaffolds were immersed in growth medium overnight and 3T6 mouse fibroblasts (passage 5) were seeded into the micro-channels using a cell density of 10⁶ cells/cm² (surface area: lcm²/channel, 0 0.53mm) resulting in a total of 3x l0⁶ cells/scaffold. After allowing the cells to attach to the scaffolds (1 h), constructs were cut to dimensions of 1 ^x 1 cm using a scalpel. The cut scaffolds (approximately 0.5 ^x 10⁶ cells/scaffold) were transferred to 48-well plates and growth medium was added. The following day, denoted as day 1, growth medium was replaced with differentiation medium, consisting of growth medium supplemented with 50 μg/ml L-ascorbic acid (Sigma-Aldrich, St. Louis, MO), to stimulate the production of collagen. Cell/scaffold constructs were cultured in an incubator with an atmosphere containing 5% C0₂ at 37 °C and at 95% relative humidity. The differentiation medium was changed every second or third day for a period of 4 weeks.

[00147] CellTiter 96^® Aqueous Non-Radioactive Cell Proliferation also known as MTS assay, is a colorimetric method used to determine the number of viable cells in proliferation. Constructs collected for MTS analysis (n=3 per group) at day 1, 14, 21 and 28 were transferred to new 48-well plates to avoid false positive signals from cells attached to the bottom of the old wells rather than the scaffolds. An Epoch microplate spectrophotometer controlled using the Gen5 Data Analysis software (BioTek Instruments, Winooski, VT) was used to measure the absorbance at 490 nm. The study showed that cell migrated, proliferated and produced extracellular matrix in the channels of nanocellulose honeycomb scaffolds. The collagen production was verified. FIG. 15 shows an exemplary confocal microscopy image of cells which are aligned in the channel of a honeycomb structured nanocellulose soft tissue scaffold. This shows that the scaffolds are very suitable for soft tissue repair and have excellent design for cell in-growth and tissue integration.

[00148] In a pilot rat study conducted for three months, the chronic subcutaneous tolerability (biocompatibility) of high content bacterial cellulose samples was evaluated with respect to various sample composition. The study showed that the cellulose implants were well tolerated and associated with a minimal inflammatory response and good biocompatibility at all time points. In some areas around the implants, it was difficult to discern a distinct interface between the cellulose and the animals' subcutaneous tissue, which may or may not represent true tissue integration into the implant. Encapsulation was also seen around the implants.

[00149] In embodiments, implants of the present invention can have porosity on a

macroscopic level as well as on a microscopic level. Due to the way the material is grown, the bacterial cellulose material comprises a nanoporous structure. In addition to this nanoporous structure, the material is grown around oxygen permeable supports to form macroscopic pores in the material. It is not critical how many pores are disposed in the material and various materials can be prepared having a high number of macroscopic pores or a low number depending on the particular mechanical properties desired and/or depending on a particular application for which the material will be used. Cells cannot enter the nano-sized pores of the BC material. Therefore, to allow for tissue integration into the BC material once implanted, at least some macroscopic pores would be desired. For greater tissue integration, it may be desired to construct a material comprising a great number of macroscopic holes or pores.

[00150] BC mesh comprising cylindrical holes with a 1.5 mm diameter cross section and spaced 1.5 cm between the holes is an example of a type of BC material that would allow for tissue integration once the BC material is implanted into a patient. Another type of material that can be constructed for tissue integration could have 600 micron diameter holes. Other preferred embodiments include 2 mm, 3 mm, or 4 mm holes spaced apart by 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm. It is within the skill of the art to construct BC materials with any other shape holes, such as hexagonal or square, and with any amount of holes or orientation of the holes relative to one another and/or the overall sheet of BC material that may be desired for a particular application. Preferred embodiments comprise a BC sheet material having a nanoporous structure and macroporosity defined by hexagonal or rhombic shape pores ranging in diameter from about 0.1 mm to about 5 mm and spaced apart a distance ranging from about 0.5 mm to about 5 mm. It is not critical how the spacing distance between holes is measured and in embodiments can be measured edge to edge, or center to center, or corner to corner, if applicable. Macroporosity can also be defined in embodiments of the invention by a pore area of about 0 up to 100% relative to the overall surface area of the BC sheet, such as for example comprising marcroscopic pores that consume from about 5-95% of the area of one surface of the sheet, or from about 10-90%, or from about 20-80%, or from about 25-75%, or from about 30-70%), or from about 40-60%), or even 50%. Expressed another way, the BC materials can comprise a porosity of 1 pore per cm² up to 100 pores per cm², such as about 5-90 pores/cm², or about 10-75 pores/cm², or from about 15-60 pores/cm², or about 20-50 pores/cm², or about 25-40 pores/cm², such as 30 pores/cm².

[00151 ] As shown and described in this specification, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular embodiments disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below.

[00152] The present invention has been described with reference to particular embodiments having various features. One skilled in the art will recognize that these features may be used singularly or in any combination based on the requirements and specifications of a given application or design. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. Where a range of values is provided in this specification, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range as well. All numbers and ranges disclosed above may vary by some amount. As used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. While compositions and methods are described in terms of "comprising," "containing," or "including" various components or steps, the compositions and methods can also "consist essentially of or "consist of the various components and steps. It is intended that the specification and examples be considered as exemplary in nature and that variations that do not depart from the essence of the invention are intended to be within the scope of the invention.

[00153] Further, each of the references cited in this disclosure are incorporated by reference herein in their entireties. If there is any conflict in the usages of a word or term in this specification and one or more patent or other documents that may be incorporated herein by reference, the definitions that are consistent with this specification should be adopted.

Claims

1. A method of making a nanoporous scaffold comprising delivering air or oxygen enriched air through an array of porous capillaries or permeable structures contacting a culture medium inoculated with a cellulose generating bacteria to obtain a nanoporous scaffold comprising channels.

2. The method of claim 1, wherein the channels are honeycomb shaped and comprise β-glucan nanofibers oriented perpendicular to channel walls.

3. The method of claim 1, wherein the channels are disposed only partially through the scaffold.

4. The method of claim 1, wherein the cellulose generating bacteria is chosen from Gluconacetobacter xylinus or Acetobacter xylinum.

5. A method of making a nanoporous scaffold comprising dense nanocellulose layers by delivering on demand nutrients in dynamic culture.

6. The method of claim 5, wherein space between nanocellulose layers allows diffusion of nutrients, oxygen, proteins, growth factors and proteoglycans but not cells.

7. A method of making a cellulose sheet comprising an amount of cellulose up to about 40% using vacuum and pressure driven dehydration.

8. A method of making a β-glucan nanocomposite where targeted oxygen delivery of oxygen tension above 50% provides high concentration of cellulose nanofibrils.

9. A nanoporous honeycomb channeled scaffold comprising β-glucan nanofibers.

10. The scaffold of claim 9, wherein the honeycomb channels have a width ranging from 300 micron to 3 mm, and wherein the scaffold has a porosity ranging from 20-80% and a tensile strength ranging from about 60 N/cm to about 200 N/cm.

11. The scaffold of claim 10, wherein the channels are not prepared in a mechanical manner.

12. A nanoporous scaffold prepared by the method of any of claims 1-8.

13. The nanoporous scaffold of claim 12 comprising honeycomb shape channels.

14. A method of physical crosslinking of the scaffold of claim 9 comprising forming hydrogen and Van der Waals bonds through sublimation of a frozen scaffold.

15. A biosynthetic cellulose material having pores sized and shaped to allow for invasion by fibroblasts and other cells when implanted into a human or animal.

16. A biosynthetic cellulose material in which fibroblasts and other cells produce an extracellular matrix when implanted into a human or animal.

17. A method of treating animal and humans which suffer from soft tissue defect by implantation of a scaffold according to claim 9.

18. A method of treating animal and humans to reinforce soft tissue chosen from one or more of defects of the abdominal and thoracic wall, muscle flap reinforcement, prolapsed repair, reconstruction of the pelvic floor, hernias, suture-line reinforcement and reconstructive surgery by implantation of β-glucan scaffold.

19. A nanoporous scaffold comprising a plurality of pores ranging in size from

300 micron to 3 mm, a porosity ranging from 20-80%, and a tensile strength ranging from about 60 N/cm to about 200 N/cm, wherein the scaffold is grown in a manner to comprise at least some of the pores.

20. The nanoporous scaffold of claim 19 prepared by delivering oxygen through an array of porous capillaries contacting a culture medium inoculated with a cellulose generating bacteria.

21. The scaffold of claim 19 or 20, wherein the pores have a honeycomb cross section.