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Title:
CELLULOSE MEMBRANES EXHIBITING HIGH TRANSPARENCY AND TENSILE ELASTICITY
Document Type and Number:
WIPO Patent Application WO/2019/084357
Kind Code:
A1
Abstract:
Cellulosic membranes including, but not limited to, cellulose membranes exhibiting a high degree of transparency and tensile elasticity. Exemplary membranes include those derived from methods of processing bacterial cellulose pellicles that exclude the use of solvents, synthetic additives, and caustic materials.

Inventors:
SHERMAN, Ethan (9152 Woodjack Court, Jacksonville, Florida, 32256, US)
OLIVER, Dana (9152 Woodjack Court, Jacksonville, Florida, 32256, US)
Application Number:
US2018/057639
Publication Date:
May 02, 2019
Filing Date:
October 26, 2018
Export Citation:
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Assignee:
BACTERIAL CELLULOSE SOLUTIONS, INC. (9152 Woodjack Court, Jacksonville, Florida, 32256, US)
International Classes:
A61L15/28; C08L1/02; D21H17/25
Domestic Patent References:
WO2012100315A12012-08-02
Foreign References:
US20150368408A12015-12-24
US20110264119A12011-10-27
US20120231038A12012-09-13
US20130011385A12013-01-10
US5962277A1999-10-05
US5846213A1998-12-08
Other References:
WANG ET AL.: "Preparation and in vitro characterization of BC/PVA hydrogel composite for its potential use as artificial cornea biomaterial", MATERIALS SCIENCE AND ENGINEERING: C, vol. 30, no. 1, 1 January 2010 (2010-01-01), pages 214 - 218, XP026783545, DOI: doi:10.1016/j.msec.2009.10.006
Attorney, Agent or Firm:
FOREST, Jeremy (MANOA LAW GROUP, 4 Sand Hill RoadCroydon, New Hampshire, 03773, US)
Download PDF:
Claims:
CLAIMS:

1. A membrane comprising a bacterial cellulose material, wherein the membrane exhibits a transmittance of at least 85% when measured at 450 nm.

2. The membrane according to claim 1, wherein the membrane exhibits a transmittance of at least 85% when measured at 450 nm, an optical density equal to or less than 0.500, and an elastic modulus equal to or less than 0.10 MPa.

3. The membrane according to claim 1, wherein the the membrane exhibits an elastic modulus equal to or less than 0.10 MPa.

4. The membrane of claim 1, wherein the bacterial cellulose is derived from G. Xylinum.

5. The membrane of claim 1, wherein the membrane exhibits a transmittance of at least 90% when measured at 450 nm.

6. The membrane of claim 1, wherein the membrane exhibits an elastic modulus of about 0.01 to about 0.10 MPa.

7. The membrane of claim 1, wherein the membrane exhibits an optical density of 0.300 or less.

8. The membrane according to claim 1, wherein the membrane comprises about 0 to about 10 wt. % water.

9. The membrane of claim 1, wherein the membrane further comprises at least one therapeutic agent.

10. The membrane according to claim 9, wherein the at least one therapeutic agent comprises one or more antimicrobial agents.

11. The membrane according to claim 10, wherein the one or more antimicrobial agents are selected from at least one of a biguanide, a quartenary ammonium compound, and a chlorinated phenol.

12. The membrane according to claim 10, wherein the one or more antimicrobial agents are selected from at least one of chlorhexidine (CHX), benzalkonium chloride (BZK), parachlorometaxylenol (PCMX), or polyhexamethylene biguanide (PHMB).

13. The membrane of claim 1, wherein the membrane consists essentially of the bacterial cellulose material.

14. The membrane of claim 9, wherein the membrane consists essentially of the bacterial cellulose material and the therapeutic agent.

15. The membrane of claim 9, wherein the membrane consists essentially of the bacterial cellulose material, the therapeutic agent, and water.

16. The membrane of claim 1, wherein the membrane has a thickness of about 5 μιη to about 250 μιη.

17. The membrane of claim 1, wherein the membrane excludes non-cellulosic structural materials.

18. The membrane of claim 1, wherein the membrane comprises a flat or planar shape.

19. The membrane of claim 1, wherein the membrane is produced by a process that includes the static incubation of a media culture.

20. A kit comprising: a membrane comprising a cellulose material, wherein the membrane exhibits a transmittance of at least 85% when measured at 450 nm; a package containing the membrane; and instructions for applying the membrane to a target site in or on a patient, wherein the target site comprises a wound or a site of a medical procedure.

Description:
CELLULOSE MEMBRANES EXHIBITING

HIGH TRANSPARENCY AND TENSILE ELASTICITY

FIELD

[001] The present disclosure relates to the preparation and use of cellulose membranes having a high degree of transparency and tensile elasticity, including bacterial cellulose-based membranes having certain biomedical and industrial applications.

BACKGROUND

[002] Membrane materials derived from plant and animal sources have important uses in many medical and industrial applications. However, these types of membranes can pose some challenges, particularly in the medical industry: autologous-sourced materials, which are derived from the human patient, have limited availability, create discomfort for the patient during tissue harvest, and increase a patient's infection risk; xenograft materials sourced from animals, which inherently provide limited control over composition, behavior of final product, morphology, concern with cross contamination, and sterility; and synthetic materials, which historically lack either biocompatibility, strength, or elasticity.

[003] Plant and bacterial-sourced cellulose can provide a basis for creating membrane materials suitable for certain end-use applications. However, the challenge with traditionally-produced cellulosic membranes has been the inability to make them extremely thin and transparent, while retaining sufficient strength and elasticity. Moreover, many of the existing cellulosic membranes require the use of complex formulations supplemented with synthetic materials, and/or must be synthesized using solvents and harsh/caustic chemicals. Thus, there remains a need to develop new, environmentally-friendly production processes that permit strict control of the desired physical properties without sacrificing transparency and/or strength of the resulting membrane material.

SUMMARY

[004] Described herein are cellulosic membrane materials having superior transparency, tensile strength, and/or elasticity. In certain embodiments, the membrane exhibits a transparency of at least 85% when measured at 490 nm, an optical density of 0.500 or less, and/or elastic modulus equal to or less than 0.10 MPa. In certain embodiments, the membrane comprises a bacterial cellulose material. [005] Also described herein are kits comprising cellulose membranes. In certain embodiments, the kit comprises: a cellulose membrane exhibiting a transmittance of at least 85% when measured at 450 nm, an optical density of 0.500 or less, and/or an elastic modulus equal to or less than 0.10 MPa; a package containing the membrane; and instructions for applying the membrane to a target site in or on a patient, wherein the target site comprises a wound or a site of a medical procedure.

[006] Further described herein are methods of making a bacterial cellulose membrane. In certain embodiments, the method comprises: providing a cellulose pellicle; treating the cellulose pellicle with an alkaline solution; cleansing the cellulose pellicle in heated water at least one time; and rinsing the cellulose pellicle with water at least one time.

[007] Further described herein are methods of treating patients having a medical condition. In certain embodiments, the method comprises: identifying a patient having a medical condition, wherein the medical condition comprises a target site for treatment; and applying a cellulosic membrane to the target site, wherein the membrane exhibits a transparency of at least 85% when measured at 450 nm, an optical density equal to or less than 0.500, and/or elastic modulus equal to or less than 0.10 MPa.

BRIEF DESCRIPTION OF THE DRAWINGS

[008] FIG. 1 is a graph depicting the optical density of dry (DBC) and wet (WBC) bacterial cellulose membranes prepared according to the examples described herein against comparative examples of dry materials, Dermafill™ (DF) membrane and paper.

[009] FIG. 2 is a graph depicting the opacity of dry (DBC) and wet (WBC) bacterial cellulose membranes prepared according to the methods described herein against comparative examples of dry materials, Dermafill™ (DF) membrane and paper.

[010] FIG. 3 is a graph depicting the opacity of dry materials after wetting, including dry (DBC) bacterial cellulose and comparative dry materials, Dermafill™ (DF) membrane and paper.

[011] FIG. 4 is a graph depicting tensile force vs. displacement for a dry bacterial cellulose membrane (BCS BC) and a wetted version of the dry membrane (BCS BC2) prepared according to the examples decribed herein, plotted against the results for a dry Dermafill™ Xylinum Cellulose Membrane (Dermafill) and a wetted version of the dry Dermafill™ Xylinum Cellulose Membrane (Dermafill2). [012] FIG. 5 is a linear graph depicting tensile force vs. displacement (before failure) for a dry bacterial cellulose membrane (BCS BC) prepared according to the examples decribed herein, plotted against the results for a dry Dermafill™ Xylinum Cellulose Membrane (Dermafill).

DETAILED DESCRIPTION

[013] Applicant has surprisingly discovered processes that permit production of extremely thin cellulose membranes (e.g., bacterial cellulose) with superior transparency, mechanical strength, and tensile elasticity. In certain embodiments are described biosynthetic bacterial cellulose membrane materials exhibiting morphological and mechanical characteristics that may be reliably controlled by tuning chosen parameters of physical and chemical processes during their fabrication. The result is a novel cellulosic membrane material that is biocompatible, tunable for particular purposes, sterilizable and is storage stable.

[014] In certain embodiments, the membrane materials described herein have a variety of potential animal and human medical applications, including the use of membranes for treating the target site of a patient having a medical condition, such as: surgical or transcatheter heart valve tissue (e.g., aortic, mitral and pulmonary); vascular patch tissue, vascular graft conduit and anastomotic seals (e.g., small diameter coronary bypass graft, hydrocephalus catheter seal), vascular shunts; organ and other fluid-type patches and seals (e.g., cerebrospinal, spinal, pituitary, orbital, and

endo/perilymphatic fluid patches), surgical graft, patch or mesh (e.g., hernia, dental, dural, breast reconstruction, pelvic floor reconstruction, tympanic membrane replacement/repair, artificial tonsil scab, liver, kidney, lung - internal organs); temporary dermal tissue coverings and pre-forms for various conditions (e.g., cosmetic procedures, atopic dermatitis, psoriasis, radiation-induced dermatitis, implant coverings, tattoos, animal de-gloving) and general surgical and non-surgical wound care (e.g., surgically-active bandages coated or impregnated with antibiotics or other therapeutic agents, biofilm-active dressings with antibacterial agents active against sessile bacteria, geriatric bandages for treating delicate skin by facilitating re-epithelialization and angiogenesis, disposable pH-sensitive bandages that indicate the health of the wound as it heals, adaptive bandages that can be applied to initially deliver therapeutic agents, then once released, transform to promote healing or tissue in-growth) and antimicrobial prostheses weaves, coverings, and pouches.

[015] In certain embodiments, the membrane materials described herein have a variety of potential industrial applications including but not limited to: materials for solar cells, components of dielectric compositions, electroacoustic transducers, light-emitting diodes, and aerogels. [016] The following definitions shall apply to terms used herein:

[017] "Dehydrate" or "Dehydration" shall mean the removal of water from a cellulose matrix material, such that the resulting cellulose material comprises less than 10% water by weight.

[018] "Membrane" shall mean a thin membrane (e.g., flat/planar or shaped) of a cellulose matrix derived from the processing of a cellulose material (e.g., cellulose material sourced from a bacterial cellulose pellicle).

[019] "Non-cellulosic structural material" shall mean any non-cellulosic material that may typically be integrated into a cellulosic matrix to augment structure of the matrix such that the tensile strength and/or elasticity of a cellulosic membrane is materially affected. Exemplary non-cellulosic structural materials may include synthetic/nonsynthetic fibers, polymers, or fillers, such as wood, metal, fibers, and polymeric materials (e.g., rubbers, elastomers, etc.).

[020] "Pellicle" shall mean an unprocessed cellulose-based mass or matrix produced between a culture media and an air/oxygen rich interface from cellulose-producing bacteria in varying thicknesses as is disclosed.

[021] "Rehydrate" or "Rehydration" shall mean the addition of water to a cellulose matrix, subsequent to the intial dehydration of the cellulose matrix.

[022] "Tensile elasticity" or "Young' s modulus" or "elastic modulus" shall mean the tendency of an object to deform along an axis or axes when opposing forces are applied along that axis.

[023] "Transmittance" shall mean the fraction of incident electromagnetic radiation that is transmitted through a sample. As used herein, transmittance represents the inverse of "opacity," which is a measure of the inpenetrability of said electromagnetic radiation. "Optical density" shall represent the -log T, wherein "T" is tranmittance.

[024] In certain embodiments, the methods and compositions described herein may comprise the use of plant and/or bacterial (microbial)-based cellulose material. For example, bacterial microorganisms produce cellulose nanofibers at the interface of a medium of fluid and air in unique layers which together form pellicles. In the right conditions, this phenomenon is ideal to produce intricately interconnected, woven constructs beyond the technological capability currently used for processing woven fabrics. These fibers produced from bacteria are inherently pure and highly crystalline. In certain embodiments, woven fibrous structures may play an integral role in the ability of a matrix to hold sutures, seal holes or perforations, and resist tearing when used in certain medical applications. In certain circumstances, in vitro and in vivo research on microbial cellulose confirms that due to its biological and physical characteristics, it may be implemented as a "medical quality" material. Thus, microbial (biosynthetic bacterial) cellulose may be able to eliminate problems involving the use of certain synthetic materials in medical applications.

[025] Bacterial cellulose consists of β-1,4 glucan chains, and is chemically identical to plant cellulose. Microbial cellulose is a highly-crystalline cellulose rich in the la fraction and is synthesized in a reaction catalyzed by the cellulose synthase in the active UDPG form and the allosteric activator c-di-GMP. The cellulose synthase operon is known, as are the functions of the proteins encoded by the genes contained therein. It is a nanoproduct, since it consists of microfibrils about 3 nm in diameter, which form a fibril or strand, which is about 100 nm in length. In certain embodiments and depending on how the material is processed, bactierial cellulose - in contrast to plant-based cellulose - is of very high purity and is essentially accompanied by no other substances. Thus, in certain embodiments, the materials described herein comprise bacterial cellulose. In certain embodiment, the materials described herein consist essentially of bacterial cellulose.

[026] Cellulose strands made by many bacterial cells can form an intricately intertwined web, which forms an elastic, highly hydrated pellicle. The pellicle gathers on the surface of the medium in stationary or hydrodynamic culture. The texture of this material can be reminiscent of the fibrous structure of muscle. The efficiency of the biosynthesis process is dependent on the activity of the producing strain, the composition of the growth medium, and the culture conditions.

[027] In certain embodiments, described herein are methods of making a cellulose membrane. In certain embodiments, the cellulose membrane is derived from a bacterial cellulose pellicle. In certain embodiments, the process includes culturing the desired bacteria under appropriate conditions to produce cellulose in the form of a pellicle. Exemplary bacteria that may be employed include Komagataeibacter-xylinus, Komagataeibacter-europaeus-T, and Komagataeibacter-hansenii- T. Other exemplary bacteria include Acetobacter xylinum, Acetobacter pasturianus, Acetobacter aceti, Acetobacter ransens, Sarcina ventriculi, Bacterium xyloides, bacteria belonging to the genus Pseudomonas, bacteria belonging to the genus Agrobacterium, and bacteria belonging to Rhizobium. In certain embodiments, a strain of Acetobacter xylinum (also designated Gluconacetobacter xylinus) is used, such as, but not limited to, Acetobacter xylinum NCIB 8246 ATCC (American Type Culture Collection) number 23769, Acetobacter xylinum NQ5 ATCC number 53582, or Acetobacter xylinum BPR2001 ATCC number 7000178. [028] In certain embodiments, Applicant has surprisingly discovered that certain steps carried out to produce and process bacterial cellulose pellicles will provide cellulose membranes having unexpected physical properties, including a high degree of transparency, tensile strength, and elasticity. For example, the media composition for pellicle production may produce a cellulose matrix material having advantageous properties. Exemplary media may include glucose-containing media, mannitol-containing media, and other sugar-containing media. It is understood that other culture media, however, may also be useful in culturing cellulose-producing bacteria.

[029] In certain embodiments, media cultures may be incubated statically or hydrodynamically at about 20°C to 35°C with a starting pH of approximately 5 to 8, and from 1 day to 30 days or more. Following culturing, the cellulose pellicles can be physically removed from the culture and treated with a mild alkaline solution, such as an aqueous 1 N sodium hydroxide solution, to remove any viable bacteria or other microbes. The treated pellicle may then be cleansed with one or more successive washes of heated (e.g., boiling) water to remove any residual color bodies (e.g., yellows, oranges, depending on thickness of pellicle) or residual bacteria, growth media, or alkaline solution trapped in the matrix. For example, in certain embodiments, the pellicle can be removed from the alkaline solution and rinsed with fresh water. The rinsed pellicle may then be: (i) cleansed in boiling water for 15-20 minutes; (ii) removed and rinsed with distilled water; and (iii) subject to repeated cleanses/rinses following steps (i) - (ii) until the pellicle is no longer yellow, and/or the desired level of clarity/transparency is achieved.

[030] Following the cleansing/rinsing steps, the pellicle may then be optionally treated with a preservative solution, prior to dehydration, to evenly disperse desirable preservative or therapeutic agents throughout the cellulose matrix. Certain therapeutic antimicrobial agents can help maintain the sterility of the membrane over time, as well as prevent/treat against planktonic or sessile microbial infections during use in medical applications. In addition (or in the alternative), other therapeutic-type agents may be added to the matrix, such as drug agents, to help effect delivery of desired agents to the patient. For example, the pellicle may be soaked in an aqueous solution of PUMB for 1 hr prior to dehydration to produce the dry cellulose membrane. Alternatively, wet cellulose pellicles may be stored in the preservative solution for extended periods of time if wet cellulose membrane material is the desired end product. Accordingly, in certain embodiments the preservative solution comprises one or more therapeutic agents, such as drugs, hormones, growth factors, or antimicrobial agents. In certain embodiments, the one or more antimicrobial agents are selected from at least one of a biguanide, a quartenary ammonium compound, or a chlorinated phenol. Exemplary antimicrobial agents include, but are not limited to, chlorhexidine (CHX), benzalkonium chloride (BZK), parachlorometaxylenol (PCMX), polyhexamethylene biguanide (PHMB), silver, iodine, chitosan, gallium, or acetic acid.

[031] Thus, in certain embodiments is described a method of making a cellulose membrane, comprising: providing a cellulose pellicle; treating the cellulose pellicle with an alkaline solution; cleansing the cellulose pellicle at least one time with heated water; and rinsing the cellulose pellicle with water. In certain embodiments, the cellulose pellicle is cleansed at least 2 times, such as 2-6 times, with heated water (e.g., boiling water). In certain embodiments, the cellulose pellicle is rinsed at least 2 times, such as 2-6 times, with sterile water. In certain embodiments, the method further comprises soaking the cellulose pellicle in a preservative solution. In certain embodiments, the method further comprises dehydrating the cellulose pellicle to provide the membrane in a moistened, semi-moistend, or dry condition. In certain embodiments, the dehydrating comprises drying the cellulose pellicle on a substrate. In certain embodiments, the substrate imparts a textured surface to the membrane. In certain embodiments, the substrate imparts a grooved surface to the membrane. In certain embodiments, the substrate imparts a crosshatched surface to the membrane. Depending on the manner and extent to which the membrane material is dried, the resulting membrane may comprise a thickness of about 1 μιη to about 2 mm, such as about 5 μιη to about 250 μιτι, about 15 μιη to about 100 μιτι, about 20 μιη to about 80 μιτι, about 25 μιη to about 50 μιη or about 28 μιη to about 32 μιη. This textured surface may be used to promote/inhibit growth of cells/tissue/bacteria on the surface of the membrane.

[032] In certain embodiments, the resulting membrane may comprise a dry, semi-dry, or wet membrane. For example, in certain embodiments the dry membrane will comprise a moisture content of about 0 to about 10 wt. %, such as about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 wt %. In certain embodiments, the membrane will comprise a moisture content of less than 5, less than 4, less than 3, less than 2, or less than 1 wt %. In certain embodiments, the semi-dry membrane will comprise a moisture content of about 1 1 to about 25 wt. %, such as about 12, about 15, about 18, about 20, about 22, or about 25 wt %. In certain

embodiments, the membrane will comprise a moisture content of about 1 1 to about 15, about 16 to about 20, or about 21 to about 25 wt %. In certain embodiments, the wet membrane will comprise a moisture content of about 26 to about 99 wt. %, such as about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95 wt %. In certain embodiments, the membrane will comprise a moisture content of about 26 to about 35, about 35 to about 50, about 50 to about 85, or about 85 to about 99 wt %.

[033] Optionally, in certain embodiments the membranes may be prepared for sterile or non- sterile application. Exemplary sterilization techniques include, but are not limited to, radiation, heat, or treatment with ethylene oxide.

[034] The proprietary membrane production techniques described herein provide cellulose membranes exhibiting suprising physical properties, including high transmittance, tensile strength, and tensile elasticity. For example, Applicant has surprisingly discovered that the pellicle production and dehydration techniques described herein can provide a cellulose membrane having exceptional transparency (e.g., transmittance of at least 85%) and/or tensile elasticity (e.g., 0.10 MPa or less), making the membranes suitable for a variety of medical and industrial applications. As described herein, a reference to "transmittance" shall mean the inverse to a measurement of

"opacity". For example, a membrane exhibiting a measured opacity of 75% would be considered to have 25% transmittance.

[035] In certain embodiments, the cellulose membranes described herein exhibit a

transmittance of at least 80%, 82%, 85%, 88%, 90%, 92%, 94%, 96%, or 98% when measures at 450 nanometers (nm). In certain embodiments, the transmittance ranges from about 80% to about 100%, such as about 85% to about 99.9%, about 88% to 96% or about 90% to about 95%, each when measured at 450 nm. In certain embodiments, the membrane exhibits an elastic modulus of about 0.001 to about 0.10 MPa, such as about 0.01 to about 0.10 MPa, or about 0.02 to about 0.08 MPa. In certain embodiments, the membrane exhibits an optical density of about 0.500 or less, such as about 0.400 or less, about 0.300 or less, about 0.200 or less, about 0.100 or less, or in the range of about 0.100 to about 0.500. In certain embodiments, membranes having a high degree of

transparency/transmittance, and a low optical density, provide a high level of utility to practitioners and patients by allowing them to accurately place/locate membrane dressings, and evaluate the progress of wound healing without removing the dressing.

[036] Without being bound to any particular scientific theory, it is believed that the surprising, superior physical properties exhibited by the membrane materials described herein are a function of the non-caustic, environmentally-friendly production techniques developed and implemented by Applicant. Specifically, it is believed that the production techniques described herein take advantage of the high degree of polymerization and crystallinity exhibited in the cellulosic matrix of the pellicle, which is ultimately retained by and integrated into the resulting membrane. Contrariwise, it is believed that certain "amalgamation" techniques used to produce membranes from reconstituted or partially-reconstituted cellulose can reduce the level of polymerization in the cellulose matrix, resulting in membrane materials having reduced tensile strength and/or elasticity. Methods for effecting amalgamation often include the use of caustic materials, including strong oxidizers such as halogenated oxidizers (e.g., bleach (sodium hypochlorite), chlorine, etc.), permanganates, or peroxides, and/or acids such as acetic acid, sulfuric acid, nitric acid, hydrochloric acid, or phosphoric acid. Accordingly, in certain embodiment the methods described herein for producing cellulose membranes exclude the use of oxidizers, such as halogenated oxidizers. In certain embodiments, the methods described herein for producing cellulose membranes excludes the use of acids, such as acetic acid.

[037] In certain embodiments, the cellulose membranes described herein may be used to form shaped materials for use and/or application with respect to imperfect surfaces. For example, in certain emodiments the membranes may be used to form cylindrical structures for use in certain vascular applications. The membrane may take the cylindrical shape during the use of a cylindrical drying mesh during dehydration. In addition, cylindrical and shaped membrane materials can be substantially formed during the pellicle stage, wherein growth of a pellicle takes place on an oxygen- permeable substrate/mold (e.g., polymeric materials such as porous PTFE and non-woven textiles) to produce a tubular pellicle. Alternatively, the cylindrical structures can be formed from flat cellulose membranes after dehydration. That is, due to the uniquely elastic and flexible nature of the membrane materials described herein, shaped materials (e.g., cylinders and other pre-forms) may be nonetheless be achieved using flat and substantially dehydrated materials.

[038] In certain embodiments, the growth or drying of the pellicle may take place on a previously-prepared cellulose membrane. Without being bound to any particular theory, using a previously-prepared cellulose membrane as a growth or drying substrate can help provide desirable matrix formation by using the prior-made membrane as a "template". Also, such a method can be used to prepare a cellulose "laminate" comprising multiple membrane layers, which may be useful depending on the product' s desired end use. [039] In certain embodiments, the use of micropatterned molds/substrates may be used during pellicle growth and/or dehydration to allow for controlled orientation of surface-cellulose fibers on cellulose membrane surfaces, which may render them more conducive to endothelial cell attachment and growth in certain applications. Depending on the end-use of the membrane material, the ability to to align cellulose formation which will ultimately impact endothelial/epithelial cell attachment and growth. For example, during pellicle formation, the utilization of microfabrication technologies (e.g., micro-patterned substrate which acts as a mold) may be used to generate a surface pattern that impacts bacteria growth and movement, which leads to either a patterned cellulose surface layer or uniquely orientated fibers on the cellulose surface. Subsequently, when cells grow on this patterned or fiber oriented surface, they interact with the cellulose and dimensionally align, a desired feature for enhanced biocompatibility. Patterning has the ability to target cellular infiltration and

proliferation of endothelial cells, smooth muscles cells or myofibroblasts. Cellulose producing bacterial cells can also be grown under flow conditions (laminar flow) to orient the cellulose fibers in one direction.

[040] Also described herein are kits comprising cellulose membranes. In certain embodiments, the kit comprises: a cellulose membrane exhibiting a transmittance of at least 85% when measured at 450 nm, an optical density of 0.500 or less, and/or an elastic modulus equal to or less than 0.10 MPa; a package containing the membrane; and instructions for applying the membrane to a target site in or on a patient, wherein the target site comprises a wound or a site of a medical procedure. Exemplary packaging materials include, but are not limited to, moisture-proof plastic and foil packaging, including resealable and hermetically-sealed bags and containers. Optionally, the instructions may be included within - or printed on - the packaging. In certain embodiments, the instructions comprise directing a user to remove and/or replace the membrane after a period of time. For example, for kits prepared for use in certain wound-treatment applications, the instructions may direct the user (e.g., patient) to replace the membrane after about 24 hrs, about 48 hrs, after about 72 hours, or as otherwise directed by a medical practitioner.

[041] Methods for preparing and using the cellulosic membranes described herein will be apparent to those of ordinary skill in the art, with exemplary procedures being described below. It is understood that while most of the processes described herein were conducted by hand, any portion of the processes may be replaced via machines to provide a fully- or partially-automated process. EXAMPLES

Example 1

Ex. 1A: Growth Media

[042] 1 gallon of ozonized, de-ionized water was poured into a 2-gallon tank on a stove. 200g of organic, non-bleached crystal sugar (glucose) was added to the water, and the solution was allowed to boil for 15-20 minutes. The resulting growth media solution was then allowed to cool to room temperature prior to use.

Ex. IB: Seeding

[043] 0.5 gallons of growth media prepared according to the method set forth in Ex. 1 A was poured into a growth tray, to which was transferred 12 ml of G. Xylinum Spc. growth inoculum (growth inoculum obtained from previous batches of cellulose that have been grown for 30 days - media remaining in tray after removal of pellicle contains bacteria used for inoculation of subsequent trays). The inoculated tray was then covered with a paper towel to prevent contamination and allow oxygen transfer, and bacterial growth/pellicle formation was allowed to continue for 30 days under static conditions.

Ex. 1C: Pellicle Removal & Cleaning

[044] The pellicle prepared according to the procedure set forth in Ex. IB was separated from the growth tray by passing a gloved finger along the edges of the tray to loosen the edges and separate the pellicle from the walls of the tray. The pellicle was then gently removed from tray, with the liquid and living cells remaining in the pellicle being drained into the remaining media by wringing out the pellicle by hand. The removed pellicle was then rinsed with fresh tap water.

[045] The rinsed pellicle was then added to a solution of IN NaOH (200 mL) heated to 60°C, and allowed to soak for 15-20 minutes. The pellicle (having a yellow/orange color) was then removed from the NaOH soak, and rinsed with fresh tap water. The rinsed pellicle was placed into clean boiling water for 15-20 mins, removed, and then rinsed with clean room temperature water (this boiling/rinsing can be repeated as necessary until pellicle is yellow/orange color dissipates and desired clarity is achieved) until the pH is approximately neutral 6-8.

Example 2

Ex. 2A: Preservative Soak ("wet" membrane)

[046] An aqueous 0.002% PHMB solution was prepared by diluting a 2% PHMB stock solution with water. The pellicle prepared according to Ex. 1 was then added to the PHMB solution and permitted to soak until use. The pellicle can be stored under soaking for extended periods of time in a sterile, sealed container or package until use.

Ex. 2B: Preservative Soak ("dry" membrane)

[047] A 0.002% PHMB solution was prepared according to the method set forth in Ex. 2A. The pellicle prepared according to Ex. 1 was then added to the PHMB solution and permitted to soak for 1 hr prior to dehydration.

Example 3

Ex. 3A: Dehydration (dry membrane (about 0-10% moisture))

[048] Pellicle prepared accoding to the method set forth in Ex. 1 was placed onto

polypropylene mesh sheet, and laid out completely flat (no folds) on the mesh. The mesh was then placed over a drying table and suspended above the flat surface with plastic/metal standoffs to keep the mesh raised about the table so air can flow both under and over the cellulose sheet during drying, allowing for even drying and a final moisture content of about 0-10% after drying at room

temperature after 24-48 hr.

Ex. 3B: Dehydration (semi-dry membrane (about 11-25% moisture))

[049] Pellicle prepared accoding to the method set forth in Ex. 1 was placed onto

polypropylene mesh sheet, and laid out completely flat (no folds) on mesh. The mesh was then placed directly in contact with the drying table, such that moisture remained partially trapped within the cellulose matrix. The moisture content in final membrane product was about 11-25% after drying at room temperature after 24-48 hr.

Example 4

Ex. 4A: Storage (dry membrane)

[050] The dry cellulose sheet prepared according to the method of Ex. 3 A was carefully removed from the polypropylene mesh by stretching the polypropylene mesh under the cellulose sheet in order to release the attachment to the cellulose without breaking (the polypropylene mesh is elastic and can stretch easily allowing the stiffer cellulose sheet on top to detach - the mesh was pulled at a diagonal (-45°) angle to the cellulose sheet to remove sheet from mesh). As desired, the resulting membrane sheet can then be cut to size. Full sheets (e.g., ~6in x lOin) can be folded into thirds and placed into a Tyvek® pouch or foil pouch, followed by heat sealing. Ex. 4B: Storage (wet pellicle)

[051] The wet cellulose pellicle prepared according to the method of Ex. 1 was placed in a sealable container. About 1-2 mL of sterile water was then transferred to container, followed by sealing of the container/pouch.

Example 5

[052] The cellulose materials prepared according to the methods described herein were subjected to several comparative mechanical and spectrophotometric tests.

Ex. 5 A: Optical Properties

[053] A sheet of wet (not previously dried) bacterial cellulose prepared and stored according to Ex. 4B, and having a moisture level of about 90-95 wt %, was placed on top of the 96 well plate covering several of the wells, such that only those wells completely covered with the cellulose were used for evaluation.

[054] Dry cellulose membranes prepared according to Ex. 3 A were cut into circles that fit exactly into the bottom of the well. After evaluation in the dry configuration, the same cellulose circles shall be hydrated with ΙΟΟμΙ of distilled water and evaluated again after hydration

[055] Dermafill™ (dry; US Food & Drug Administration 510(k) approved) cellulose sheets shall be cut into circles and fit exactly into the bottom of the 96 well plate. After evaluation in the dry configuration, the same cellulose circles shall be hydrated with ΙΟΟμΙ of distilled water and evaluated again after hydration.

[056] For purposes of calibration, white printer paper was cut into circles and placed into the bottom of the well as a dry sheet. After evaluation in the dry configuration, the same paper circles shall be hydrated with ΙΟΟμΙ of distilled water and evaluated again after hydration.

[057] Each of the aforementioned samples was placed into the UV plate reader (BioTek® ELx808 absorbance reader). Measurements were obtained at 450 nm, with each measurement being repeated twice and averaged. The resulting opacity/transmittance and optical density data are charted in Figs. 1-3, and are reported below in Table 1.

Table 1 Dermafill™ Xylinum about 38% about 42% about 0.75 Cellulose Membrane

Dressing (DF)

Paper about 100% about 0% about 1.95

Opacity/transmittance data for dry membranes hydrated with ΙΟΟμΙ of distilled water are reported below in Table 2:

Table 2

Ex. 5B: Tensile Properties

[058] Procedure for determining Young's Modulus: Cellulose strips were cut ~lcm wide by ~5 cm long. The samples were placed into the jaws of the force gauge (Mark-10® MG50 Series) to measure the stress/strain to failure. The curve (stress vs. strain) was plotted in Excel and the slope of the line was calculated by fitting a linear trend line with an R 2 value >95%. The slope was taken from the x coefficient "m" of the trend line equation (Y = mx +b). The resulting tensile force vs. displacement for dry Ex. 3 A membrane (BCS BC) and dry Dermafill™ Xylinum Cellulose

Membrane (Dermafill) are plotted in Figs. 4-5, along with the results for wet Ex. 3 A membrane (BCS BC2) and wet Dermafill™ Xylinum Cellulose Membrane (Dermafill2) wetted with distilled water. These results were converted into megapascals (MPa), which are reported below in Table 3: Table 3

Additional Embodiments

[059] 1 . A membrane comprising a cellulose material, wherein the membrane exhibits at least one of: a transparency (transmittance) of at least 85% when measured at 450 nm; an optical density equal to or less than 0.500; or an elastic modulus equal to or less than 0.10 MPa.2. The membrane according to embodiment 1, wherein the membrane exhibits a transparency of at least 85% when measured at 450 nm, an optical density equal to or less than 0.500, and an elastic modulus equal to or less than 0.10 MPa.

[060] 3. The membrane according to embodiment 1, wherein the membrane exhibits a transparency of at least 85% when measured at 450 nm.

[061] 4. The membrane according to embodiment 1, wherein the membrane exhibits an optical density equal to or less than 0.500.

[062] 5. The membrane according to embodiment 1, wherein the the membrane exhibits an elastic modulus equal to or less than 0.10 MPa.

[063] 6. The membrane of any one of embodiments 1-5, wherein the cellulose material comprises bacterial cellulose.

[064] 7. The membrane of embodiment 6, wherein the bacterial cellulose is derived from G. Xylinum.

[065] 8. The membrane according to any one of embodiments 1-7, wherein the membrane exhibits a transmittance of at least 88% when measured at 450 nm.

[066] 9. The membrane according to any one of embodiments 1-7, wherein the membrane exhibits a transmittance of at least 90% when measured at 450 nm.

[067] 10. The membrane according to any one of embodiments 1-7, wherein the membrane exhibits a transmittance of at least 92% when measured at 450 nm.

[068] 11. The membrane according to any one of embodiments 1-7, wherein the membrane exhibits a transmittance of at least 94% when measured at 450 nm.

[069] 12. The membrane according to any one of embodiments 1-11, wherein the membrane exhibits an elastic modulus of about 0.001 to about 0.10 MPa.

[070] 13. The membrane according to any one of embodiments 1-11, wherein the membrane exhibits an elastic modulus of about 0.01 to about 0.10 MPa.

[071] 14. The membrane according to any one of embodiments 1-11, wherein the membrane exhibits an elastic modulus of about 0.02 to about 0.08 MPa. [072] 15. The membrane according to any one of embodiments 1-14, wherein the membrane exhibits an optical density of 0.500 or less.

[073] 16. The membrane according to any one of embodiments 1-14, wherein the membrane exhibits an optical density of 0.400 or less.

[074] 17. The membrane according to any one of embodiments 1-14, wherein the membrane exhibits an optical density of 0.300 or less.

[075] 18. The membrane according to any one of embodiments 1-14, wherein the membrane exhibits an optical density of 0.200 or less.

[076] 19. The membrane according to any one of embodiments 1-14, wherein the membrane exhibits an optical density of about 0.100 to about 0.500.

[077] 20. The membrane according to any one of embodiments 1-19, wherein the membrane comprises about 80 to about 99 wt. % water.

[078] 21. The membrane according to any one of embodiments 1-19, wherein the membrane comprises less than 2 wt. % water.

[079] 22. The membrane according to any one of embodiments 1-19, wherein the membrane comprises less than 1 wt. % water.

[080] 23. The membrane according to any one of embodiments 1-22, further comprising at least one therapeutic agent.

[081] 24. The membrane according to embodiment 23, wherein the at least one therapeutic agent comprises one or more antimicrobial agents.

[082] 25. The membrane according to embodiment 24, wherein the one or more antimicrobial agents are selected from at least one of a biguanide, a quartenary ammonium compound, and a chlorinated phenol.

[083] 26. The membrane according to any one of embodiments 24-25, wherein the one or more antimicrobial agents are selected from at least one of chlorhexidine (CHX), benzalkonium chloride (BZK), parachlorometaxylenol (PCMX), or polyhexamethylene biguanide (PHMB).

[084] 27. The membrane according to any one of embodiments 1-26, wherein the membrane consists essentially of the cellulose material.

[085] 28. The membrane according to any one of embodiments 1-26, wherein the membrane consists essentially of the cellulose material and the therapeutic agent.

[086] 29. The membrane according to any one of embodiments 1-26, wherein the membrane consists essentially of the cellulose material, the therapeutic agent, and water. [087] 30. The membrane according to any one of embodiments 1-29, wherein the membrane has a thickness of about 5 μιη to about 250 μιη.

[088] 31. The membrane according to any one of embodiments 1-30, wherein the membrane excludes non-cellulosic structural materials.

[089] 32. A kit compri sing :

a membrane comprising a cellulose material, wherein the membrane exhibits a transmittance of at least 85% when measured at 450 nm, an optical density equal to or less than 0.500, and/or elastic modulus equal to or less than 0.10 MPa;

a package containing the membrane; and

instructions for applying the membrane to a target site in or on a patient, wherein the target site comprises a wound or a site of a medical procedure.

[090] 33. The kit of embodiment 32, wherein the cellulose material comprises bacterial cellulose.

[091] 34. The kit of embodiment 33, wherein the bacterial cellulose is derived from G.

Xylinum.

[092] 35. The kit according to any one of embodiments 32-34, wherein the membrane exhibits a transmittance of at least 88% when measured at 450 nm.

[093] 36. The kit according to any one of embodiments 32-34, wherein the membrane exhibits a transmittance of at least 90% when measured at 450 nm.

[094] 37. The kit according to any one of embodiments 32-34, wherein the membrane exhibits a transmittance of at least 92% when measured at 450 nm.

[095] 38. The kit according to any one of embodiments 32-34, wherein the membrane exhibits a transmittance of at least 94% when measured at 450 nm.

[096] 39. The kit according to any one of embodiments 32-38, wherein the membrane exhibits an elastic modulus of about 0.001 to about 0.10 MPa.

[097] 40. The kit according to any one of embodiments 32-38, wherein the membrane exhibits an elastic modulus of about 0.01 to about 0.10 MPa.

[098] 41. The kit according to any one of embodiments 32-38, wherein the membrane exhibits an elastic modulus of about 0.02 to about 0.08 MPa.

[099] 42. The kit according to any one of embodiments 32-41, wherein the membrane exhibits an optical density of 0.400 or less. [0100] 43. The kit according to any one of embodiments 32-41, wherein the membrane exhibits an optical density of 0.300 or less.

[0101] 44. The kit according to any one of embodiments 32-41, wherein the membrane exhibits an optical density of 0.200 or less.

[0102] 45. The kit according to any one of embodiments 32-41, wherein the membrane exhibits an optical density of about 0.100 to about 0.500.

[0103] 46. The kit according to any one of embodiments 32-45, wherein the membrane comprises about 80 to about 99 wt. % water.

[0104] 47. The kit according to any one of embodiments 32-45, wherein the membrane comprises less than 2 wt. % water.

[0105] 48. The kit according to any one of embodiments 32-45, wherein the membrane comprises less than 1 wt. % water.

[0106] 49. The kit according to any one of embodiments 32-48, wherein the membrane further comprises at least one therapeutic agent.

[0107] 50. The kit according to embodiment 49, wherein the at least one therapeutic agent comprises one or more antimicrobial agents.

[0108] 51. The kit according to embodiment 50, wherein the one or more antimicrobial agents are selected from at least one of a biguanide, a quartenary ammonium compound, and a chlorinated phenol.

[0109] 52. The kit according to any one of embodiments 50-51, wherein the one or more antimicrobial agents are selected from at least one of chlorhexidine (CHX), benzalkonium chloride (BZK), parachlorometaxylenol (PCMX), or polyhexamethylene biguanide (PHMB).

[0110] 53. The kit according to any one of embodiments 32-52, wherein the membrane consists essentially of the cellulose material.

[0111] 54. The kit according to any one of embodiments 32-52, wherein the membrane consists essentially of the cellulose material and the therapeutic agent.

[0112] 55. The kit according to any one of embodiments 32-52, wherein the membrane consists essentially of the cellulose material, the therapeutic agent, and water.

[0113] 56. The kit according to any of embodiments 32-55, wherein the package is moisture proof.

[0114] 57. The kit according to any one of embodiments 32-56, wherein the instructions comprise directing a user to remove and/or replace the membrane after a period of time. [0115] 58. The kit according to any one of embodiments 32-57, wherein the membrane has a thickness of about 5 μιη to about 250 μιη.

[0116] 59. The kit according to any one of embodiments 32-58, wherein the membrane excludes non-cellulosic structural materials.

[0117] 60. A method of preparing a cellulose membrane comprising:

providing a cellulose pellicle, said cellulose pellicle comprising a cellulosic material;

treating the cellulose pellicle with an alkaline solution;

cleansing the cellulose pellicle in heated water at least one time; and

rinsing the cellulose pellicle with water at least one time.

[0118] 61. The method of embodiment 60, wherein the heated water is boiling.

[0119] 62. The method of any one of embodiments 60-61, wherein the cellulose pellicle is cleansed in boiling water at least 2 times.

[0120] 63. The method of any one of embodiments 60-61, wherein the cellulose pellicle cleansed in boiling water least 2 to 6 times.

[0121] 64. The method of any one of embodiments 60-63, the cellulose pellicle is rinsed with water at least 2 times.

[0122] 65. The method of any one of embodiments 60-63, the cellulose pellicle is rinsed with water at least 2 to 6 times.

[0123] 66. The method any one of embodiments 60-65, further comprising treating the cellulose pellicle in a preservative solution.

[0124] 67. The method any one of embodiments 60-66, further comprising dehydrating the cellulose pellicle.

[0125] 68. The method of embodiment 67, wherein the dehydrating comprises drying the cellulose pellicle on a substrate.

[0126] 69. The method of embodiment 68, wherein the substrate imparts a textured surface to the membrane.

[0127] 70. The method of embodiment 68, wherein the substrate imparts a grooved surface to the membrane.

[0128] 71. The method of embodiment 68, wherein the substrate imparts a crosshatched surface to the membrane.

[0129] 72. The method of any one of embodiments 60-71, wherein the membrane exhibits a transmittance of at least 84% when measured at 450 nm. [0130] 73. The method of any one of embodiments 60-71, wherein the membrane exhibits a transmittance of at least 88% when measured at 450 nm.

[0131] 74. The method of any one of embodiments 60-71, wherein the membrane exhibits a transmittance of at least 90% when measured at 450 nm.

[0132] 75. The method of any one of embodiments 60-71, wherein the membrane exhibits a transmittance of at least 92% when measured at 450 nm.

[0133] 76. The method of any one of embodiments 60-71, wherein the membrane exhibits a transmittance of at least 94% when measured at 450 nm.

[0134] 77. The method of any one of embodiments 60-76, wherein the membrane exhibits an elastic modulus equal to or less than 0.10 MPa.

[0135] 78. The method of any one of embodiments 60-76, wherein the membrane exhibits an elastic modulus of about 0.001 to about 0.10 MPa.

[0136] 79. The method of any one of embodiments 60-76, wherein the membrane exhibits an elastic modulus of about 0.01 to about 0.10 MPa.

[0137] 80. The method of any one of embodiments 60-76, wherein the membrane exhibits an elastic modulus of about 0.02 to about 0.08 MPa.

[0138] 81. The method of any one of embodiments 60-80, wherein the membrane exhibits an optical density of 0.500 or less.

[0139] 82. The method of any one of embodiments 60-80, wherein the membrane exhibits an optical density of 0.400 or less.

[0140] 83. The method of any one of embodiments 60-80, wherein the membrane exhibits an optical density of 0.300 or less.

[0141] 84. The method of any one of embodiments 60-80, wherein the membrane exhibits an optical density of 0.200 or less.

[0142] 85. The method of any one of embodiments 60-80, wherein the membrane exhibits an optical density of about 0.100 to about 0.500.

[0143] 86. The method of any one of embodiments 60-85, wherein the membrane comprises about 80 to about 99 wt. % water.

[0144] 87. The method of any one of embodiments 60-85, wherein the membrane comprises less than 2 wt. % water.

[0145] 88. The method of any one of embodiments 60-85, wherein the membrane comprises less than 1 wt. % water. [0146] 89. The method of any one of embodiments 60-88, wherein the preservative solution comprises at least one therapeutic agent.

[0147] 90. The method according to embodiment 89, wherein the at least one therapeutic agent comprises one or more antimicrobial agents.

[0148] 91. The method according to embodiment 90, wherein the one or more antimicrobial agents are selected from at least one of a biguanide, a quartenary ammonium compound, and a chlorinated phenol.

[0149] 92. The method according to embodiment 91, wherein the one or more antimicrobial agents are selected from at least one of chlorhexidine (CHX), benzalkonium chloride (BZK), parachlorometaxylenol (PCMX), or polyhexamethylene biguanide (PHMB).

[0150] 93. The method according to any one of embodiments 60-92, wherein the membrane consists essentially of the cellulose material.

[0151] 94. The method according to any one of embodiments 89-92, wherein the membrane consists essentially of the cellulose material and the therapeutic agent.

[0152] 95. The method according to any one of embodiments 89-92, wherein the membrane consists essentially of the cellulose material, the antimicrobial agent, and water.

[0153] 96. The method according to any one of embodiments 80-95, wherein the membrane has a thickness of about 5 μιη to about 250 μιη.

[0154] 97. The method according to any one of embodiments 80-96, wherein the method excludes the use of an amalgamation step.

[0155] 98. The method according to any one of embodiments 80-97, wherein the method excludes the use of an oxidizing agent.

[0156] 99. The method according to any one of embodiments 80-98, wherein the method excludes the use of a halogenated oxidizing agent.

[0157] 100. The method according to any one of embodiments 80-99, wherein the method excludes the use of an acid.

[0158] 101. The method according to any one of embodiments 80-100, further comprising producing a cellulose pellicle by culturing cellulose-producing bacteria in a culture media.

[0159] 102. The method according to embodiment 101, wherein the culture media comprises the use of a sugar.

[0160] 103. The method according to embodiment 102, wherein the sugar is glucose. [0161] 104. The method according to any one of embodiments 80-103, wherein the membrane excludes non-cellulosic structural materials.

[0162] 105. A method comprising:

identifying a patient having a medical condition, wherein the medical condition comprises a target site for treatment; and applying a cellulosic membrane to the target site, wherein the membrane exhibits a transparency of at least 85% when measured at 450 nm, an optical density equal to or less than 0.500, and/or elastic modulus equal to or less than 0.10 MPa.

[0163] 106. The method of embodiment 105, wherein the medical condition is at least one of the conditions described in paragraph [014].

[0164] 107. The method of embodiment 106, wherein the medical condition comprises radiation- induced dermatitis.

[0165] 106. The method according to embodiments 105-107, wherein the cellulosic membrane comprises a membrane according to any one of embodiments 2-31.