WO2008097529A1

WO2008097529A1 - Tissue compatible material and methods using same

Info

Publication number: WO2008097529A1
Application number: PCT/US2008/001490
Authority: WO
Inventors: Bruno D.V. Marino; Israel M. Stein; Ben R. Bronstein
Original assignee: Dermal-Z Ltd.
Priority date: 2007-02-06
Filing date: 2008-02-05
Publication date: 2008-08-14
Also published as: WO2008097529B1

Abstract

A tissue compatible material having a prolamine protein derived from a plant, such as maize, commonly known as zein, which when sufficiently pure is capable of being provided alone or formulated with one or more agents or components for tissue implantation and augmentation applications and for 3-D laminar organ transplantation applications. Methods are described for using the tissue compatible material for such tissue applications such, as an injectable dermal filler, for soft tissue augmentation, tissue implants, grafts, as a substrate for skin replacement, or wound healing and in organ transplantation.

Description

TISSUE COMPATIBLE MATERIAL AND METHODS USING SAME

Priority is claimed to our Application Serial No. 60/899,807, filed February 6, 2007, and Application Serial No. 61/01 1 ,876, filed January 22, 2008.

Field of the Invention

The present invention relates to tissue compatible material provided by a prolamine protein derived from a plant, such as maize (corn), commonly known as zein, and in particular to such prolamine protein which when sufficiently pure is capable of being provided alone or formulated with one or more agents or components for tissue applications, such as dermal fillers, cutaneous augmentation, soft tissue augmentation, soft tissue implants, as a substrate for skin replacement or regeneration, or wound healing and in organ transplantation and surgical replacements. As such, zein may replace collagen currently used in such applications.

Background of the Invention

Dermal augmentation with fillers or implants to improve cosmetic appearance of various parts of the body including the naso-labial folds, lips, and other facial features is of increasing popularity around the world. The major forces responsible for facial aging are related to exposure of the skin to external environments (e.g., UV), gravity, individual skin characteristics and overall health as well as aging. Changes in structural proteins, primarily a reduction in collagen, are seen as aging occurs resulting in defects such as wrinkles and fine lines around the eyes and mouth and sagging around the jaw line and eyelids. Collagen as a dermal replacement filler to restore volume has been seen as a natural choice for decades employing animal and human-derived collagen (see, Baumann, L., Kaufman J., Sghari S. Dermatologic Therapy (2006) 19: 134-140). In addition to the collagens, a variety of new non-collagen dermal implant constituents have been introduced, such as various formulations of hyaluronic acid, poly-L-lactic acid (PLA), calcium hydroxylapatite, polymethylmethacrylate microspheres and combinations of these components, as well as formulations designed for variable volume and implant half life. A summary and comparison of collagen implants is provided below in Table 1 from the Baumann et al., 2006 paper. Table 1. Comparison of different collagen products

Product Components Pretest Storage Type of graft Duration

Zyderm Bovine collagen, Yes Refrigeration Xenograft 3 months

3% hdocaine

Zyplast Bovine collagen, Yes Refrigeration Xenograft 3 months

3% hdocaine

Cosmoderm Human-based collagen, No Refrigeration Homograft 4-7 months

3% lidocaine

Cosmoplast Human-based collagen, No Refrigeration Homograft 4-7 months

3% lidocaine

Cymetra Human dermal matrix No Refrigeration Homograft 4-18 months

Artecoll Bovine collagen, PMMA Yes Refrigeration Xenograft Semi-permanent

Resoplast Bovine collagen, PMMA Yes Refrigeration Xenograft Semi-permanent

Isologen Cultured autologous fibroblasts No Cryo-preserved Autograft < 18 months

Autologen Autologous collagen No No longer available Autograft No longer available

Dermologen Human-based collagen, No No longer available Homograft No longer available gags, elastin

While collagen based dermal fillers are used extensively today they suffer from a range of adverse reactions, in some cases due to immunologic responses. In addition, collagen derived from bovine sources may carry traces of prions and other bovine specific diseases and pathogens. In cases of human derived collagen, fear of disease transmission, including the possibility of HIV transfection, is of concern, also. Thus, it would be of value to utilize a non-animal and non-human, non-synthetic source for dermal implants that offers certainty of sterilization and has a long history of safe human use.

Despite the diversity of non-collagen dermal fillers (e.g., hyaluronic acid, poly-L- lactic acid and calcium hydroxylapatite microspheres) with respect to clinical indications and toxicity, non-collagen based dermal fillers do not offer the sheet like supramolecular structure that is intrinsic to collagen which offers superior lift and volume replacement. Smaller moieties are increasingly cross-linked to create bulk and ideally a planar orientation with optimized volume to reduce the amount of filler needed and to ease the injection process. Thus, a non-animal, non-human source of dermal filler material that has conformational characteristics similar to collagen is desirable.

Gels are increasingly used in carrier formulations to facilitate injection and provide a smooth and/or malleable implant that can be more naturally incorporated within individual facial features. Gels are typically carriers for micro-spheres, small molecules and cross- linked moieties, all amenable to smooth gel formulations in contrast to collagen based implants that do not form gels as easily. Thus a tissue implant possessing collagen like supramolecular structure and formulated as a smooth, easily injected gel would also be desirable.

The length of time a tissue implant remains in the body is a critical feature of the implant related to cost of replacement, enzymatic degradation and absorption of degradation products as well as their biocompatibility. Thus, in the event of an idiosyncratic or other adverse reaction to a tissue implant it is further desirable that the implant be degradable over a shorter period rather than a longer period of time. The majority of implants do not offer a spectrum of persistence in the body due to the natural characteristics of the materials and reactivity under physiologic conditions. Thus it would be desirable to employ a substrate for a dermal implant that could be easily prepared across a range of in vivo half-lives including permanent implants while still retaining the optimal characteristics for a dermal implant.

Finally, a tissue implant that offers options for use as temporary wound covering or a substrate for the reconstruction of the cutaneous surface, either by in-growth of skin cells or placement of skin grafts or other skin substitutes is of value. In addition, augmentation of soft tissues in the musculoskeletal, urological, and gynecological systems using safe and effective biosynthetic materials also is highly desirable. Currently, collagen— based products are used extensively for implants and as substrates for "bio-artificial skin", but suffer the risk of adverse reactions or disease transmission described earlier.

It is a feature of the present invention to use a prolamine protein, derived from a plant, such as zein, which is derived from maize (corn) in dermal fillers and tissue implants, rather than collagen or non-collagen material used heretofore. Biocompatibility of zein used for the aforesaid purposes is a critical feature of use and acceptance. Zein as a component of corn has been consumed for several thousand years and has been used in a wide variety of foodstuffs, edible film packaging (e.g., Lawton, J.W. Cereal Chemistry (2002) 79(1): 1-18), chewing gums and cosmetics as well as numerous applications for drug delivery (e.g., O'Donnell, P.B., Wu, C. Wang, J. Wang, Lisa, Oshlack, B., Chasin, M., Bodmeier, R., McGinty, J.W. European J. Pharmaceutics and Biopharmaceutics 43: 83-89). Maize is generally recognized as safe, however zein alone in pure form has been less extensively tested specifically for adverse reactions in the human tissue environment. The fatty acid component of zein known as the 9kd lipid-transfer protein (LTP) is a recognized allergen of -A-

raw maize and zein (see Pastorello el. al., Journal of Allergy and Clinical Immunology (2003), vol, 112, no. 4: 775-783) but can be quantitatively removed with routine protein purification methods.

A number of studies support bio- and cyto- compatibility of zein in vitro. Dong, J., Sun, Q, Wang, Jin- WE, Biomaterials (2004) 25: 56591-4697, reported good biocompatibility using zein films by growing cultured liver cells on a zein substrate that also appeared to support cell proliferation. Sun, Q-S,. Dong, J., Lin, Z-X, Yang, B., Wang, J-Y. Biopolymers (2005) 78, issue 5, 268-274, compared zein prepared as particles and as a film for cytocompatibility with that for polyhdydroxybutyrate (PHB), polylactic acid (PLA) and collagen using HL-7702 cells. Results from Sun et al., 2005, indicated that cell adhesion and viability were higher with the zein formulations than for PHB, PLA and collagen. Zein has been used in a number of microsphere applications for controlled release of drugs with moderate success demonstrating good hemocompatibility and no reported adverse reaction (e.g., Wang, HJ, Zx, Liu, Sy Sheng, Wang, JY. Control Release (2005) 105(1-2): 1290- 131). Although the above studies show the possible beneficial use of zein they are limited to their particular application, and do not describe use of zein in applications, such as dermal fillers, soft tissue augmentation, or a substrate of skin regeneration or composite skin grafts.

Summary of the Invention

Accordingly, it is an object of the present invention to provide a prolamine protein derived from a plant, such as maize, commonly known as zein, for use in tissue applications, such as dermal fillers, soft tissue augmentation, tissue implants, or as a substrate for grafts for any soft tissue or organ, whereby zein provides a non-animal, non-human, non-synthetic material that has a supramolecular structure having conformational characteristics similar to collagen, but does not have the drawbacks of collagen or non-collagen materials.

Zein may thus be used instead of collagen or non-collagen material in dermal fillers, tissue implants, and bioartificial skin, but which does not risk immunologic response or disease transmission of collagen, as it is derived from a plant source, and further zein's supramolecular structure allows the material to be incorporated into a variety of formulations, such as liquid polymers, gels, substrates, and films. Briefly described the present invention embodies a tissue compatible material having a prolamine protein derived from a plant, which when sufficiently pure is capable of being provided alone or formulated with one or more agents or components for tissue applications in patients. Such applications include, but are not limited to, an injectable dermal filler, cutaneous augmentation, soft tissue augmentation, skin replacement, or wound healing. Although the prolamine protein described herein is derived from maize (corn), known as zein, other plant derived proteins may similarly be used in tissue applications. The invention further embodies methods for using a tissue compatible material derived from maize in applications such as a dermal filler, cutaneous augmentation, soft tissue augmentation, skin replacement, or wound healing. Such as in formulations as a dermal filler or tissue implant for cosmetic or reconstructive skin augmentation applications and as a substrate for grafts for any soft tissue or organ or to cover denuded skin or gingival surfaces and to promote re- growth of skin in damaged tissue. Preferred embodiments of zein are based on repeating peptide segments and the intrinsic amphipathic nature of the protein resulting from variable hydrophilic and hydrophobic conformations. Zein may be injected in native helical and sheet conformations utilizing gel formulations or in other carriers, as micro-spheres, nanofibrils and other macromolecular forms and/or in conjunction with other tissue implant components. The tissue lifetime of zein implants can be made to range from months to years. Pure isoforms of zein, which are processed to be free from fatty acid components known to be allergenic and derived from organic and non-genetically modified zein offer a plant-based biopolymer for cosmetic dermal filler or soft tissue implantation or augmentation use.

Zein tissue implants can be modified to reflect localized skin tissue properties as well as be prepared and sealed as bulk agents for large pliable implants that may be used for breast augmentation. Zein-based tissue implants offer numerous advantages over currently marketed collagen and non-collagen based products for the spectrum of tissue applications offering a suitable replacement of all collagen based dermal fillers and tissue augmentation or implant applications.

The invention, based on the zein protein from Zea mays, fills the gap between the nearly ideal "natural" template for collagen tissue implants and the extensive cross-linking required to provide structure for non-collagen implants. Zein, in its processed form as a tissue implant, is a unique natural biomolecule with the following features:

1) it is absolutely free from pathogens;

2) it is of very low adverse reaction probability;

3) it is of a supportive supramolecular nature;

4) it can be formulated in many different forms to be easily injectable and recoverable;

5) it can be formulated over a range of in vivo longevity to adjust the period of degradation up to a permanent time period of years;

6) it is non-immunogenic; and

7) it can be re-formulated to provide highly specific zein films suitable for use as bioartificial skin or gingival graft.

Further, in accordance with the invention, zein formulations are also useful in the creation of three dimensional laminar structures used in the fields of organ transplantation and reconstructive surgery. Such three dimensional structures may be scaffolds, matrixes, beads, or lattices. The requirements in the aforesaid use are more demanding than for simple non-structured and/or subcutaneous implantation and/or non-organ tissue engineering. Such non-organ applications are found in Wang, H.J., Gong, S. J., Lin, Z.X., Fu, J.X, Xue, S.T., Huang, J.C., Wang, J.Y., In vivo biocompatibility and mechanical properties of porous zein scaffolds, Biomaterials 28 (2007) 3952-3964 (Wang et al. (2007)) and Gong, S., Wang, H., Sun, W. Sue, S. T., Wang, J. Y., Mechanical properties and in vitro biocompatibility of porous zein scaffolds, Biomaterials 27 (2006) 3793-3799.

The scaffolds for specific organ replacement are reviewed in Yang, S., Leong, K.F., Zhaohudi, M.E., Chua, C.K., The design of scaffolds for use in tissue engineering, Tissue Engineering, 7(6), (2001) 679-689, (Yang et al. (2001)) and Angelova, N., and Hunkeler, D., Rationalizing the design of polymeric and biomaterials, TIBECH, vol. 17 (1999) 409-421. 3 -D laminar structures incorporate zein and used for organ transplantation have a structured matrix designed as a template to induce formation of organ-like macrostructure with blood vessel supply and other complex components such as a nerve supply, drainage system and lymphatic system. These zein structures are flexible to accommodate mechanical compliance of soft tissue and blood vessel growth. In preferred embodiments described below the laminar structure used to recruit cells and subsequently transplanted to the subject is biodegradable leaving, ultimately, the cells and associated structures enabling organ function.

3-D structures in general (but not constructed of zein) are described in Vacanti, Fabrication of Vascularized Tissue, US 6,455,311, Sept. 24, 2002, and in Vacanti, et al., Fabrication of Vascularized Tissue Using Microfabricated Two-dimensional Molds. U.S. Patent Application Publication No. 2007/0148139, of Jun 28, 2007. Both disclosures describe the creation of a 3-D laminar structure based on the combination of multiple layers of a polymer on which specific cells have been cultured. The cell culture process requires a planar, flexible and porous yet rigid plate that has been etched according to specifications to produce channels and sites for cell proliferation. Once appropriate cells are recruited in the lab the device is implanted in the subject allowing immediate blood flow and operation. This approach to organ replacement is also disclosed by Mikos et al., Porous Biodegradable Polymeric Materials for Cell Transplantation, U.S. Patent No. 6,689,608, issued Feb. 10, 2004, in which the biodegradable features of PLA/PLGA composites are the preferred embodiments. Additionally, MacLaughlin et al., Delivery of Therapeutic Biologicals from Implantable tissue matrices. U.S. Patent No. 7,078,032, Issued JuI 18, 2006, utilize PLA/PLGA composites to recruit genetically engineered cells to express biologically active therapeutic agents in cell culture.

Zein formulations provided by these inventions represent a novel and advantageous polymer particularly for these 3-D laminar organ transplantation applications.

In addition to the novel structural features of zein accommodating the physical matrix requirements for organ replacement the zein molecule offers a key chemical advantage over PLA/PLGA (poly (lactic acid))/(poly (lactic-co-glycolic acid)) moieties. The creation of a 3- D laminar structure imposes constraints on diffusion according to the final macroscopic shape. In the case of a sphere the volume increases as a cubed function (4/3 pi r³) while the surface area increases as squared function (4 pi r²) . Thus, in the limit of increasing total volume cellular flow to the device outlet and or to the outer surfaces of the implanted device is impaired particularly for cells deep within the device. The rate of diffusion thus may determine the viability of cell recruitment according to the removal rate of cell by products and by products of the degradation of the matrix itself. In the case of the PLA/PLGA moieties both enzymatic and hydrolytic degradation produces acidic by products (e.g., lactic acid) that can be detrimental to cell recruitment and proliferation as well as lifetime of the matrix.

In summary, even though degradation products of PLA/PLGA polymers are known to be largely non-cytotoxic little is known about the degradation rate-dependent acidic by product effect on cells at the micro-sites of degradation or on the scaffold via autocatalytic effects. And, as described above, such effects may be crucial for success in large scale organ replacement devices of significant volume.

In this regard, Sung, H.K., Meredith, C, Johnson, C, Galis, Z., The effect of scaffold degradation rate on three-dimensional cell growth and angiogenesis. Biomaterials 25 (2004) 5735-5742, found that cell viability was inversely related to degradation rate of a PLA/PLGA scaffold. There was also a time-dependent decrease in pH corresponding to scaffold degradation and reduction in cell viability. Additionally, Murty, S.B., Hee, N.D., Thanoo, B.C., Deluca, P.P., Impurity formation studies with peptide-loaded polymeric microspheres, Part II, In vitro evaluation, International Journal of Pharmaceuticals 297 (2005) 62-72, concluded that the formation of glycolic and/or lactic acid monomers due to hydrolytic cleavage of the core polymer formed an acidic microenvironment in PLA/PLGA microspheres loaded with octreotide acetate peptide producing undesirable degradation of that moiety. Yang et al. (2001) reiterate these concerns with PLA/PLGA moieties.

In the case of zein the by products of degradation either via hydrolysis or enzymes (e.g., trypsin) will consist of amino acids or peptides, neither of which should result in the levels of acidic micro-sites seen with PLA/PLGA moieties. Furthermore, such by products may be carried away from the cells sites or used metabolically. It is not expected that any of the amino acids released nor peptide fragments will be cytotoxic to the cells remaining. Brief Description of the Drawings

FIG. 1 is a perspective view of a core element of zein; more specifically, the accessible surface of a single octamer molecule modeled adopting a polar protonated histidine (PPH) conformation, the hydrophobic residues (leucine and valine) and neutral residues (praline) are represented at (a) in the view, and PPH residues are represented at (b); and

FIG. 2 is a schematic-molecular structure diagram of zein; more specifically, the structure shown is a coiled-coil triple superhelix constructed from helical segments 7-9 looking down the superhelix axis.

Detailed Description of the Invention

Zein is the major storage protein of corn and comprises approximately 45 % of the protein in corn (see Lawton, J. W. Cereal Chemistry (2002) 79(1): 1-18). Zein isolates are not suitable for human consumption due to the hydrophobic nature of the molecule and low nitrogen content. There are four main types of zein (alpha, beta, gamma, delta) classified according to solubility. The embodiments disclosed here rely upon the alpha zeins, although any of the known zeins with molecular masses of 19 to 22 kDa or variations thereof are also suitable. Studies suggest that zein in solution exhibits a 3-D structure with high axial ratio resulting in a helical and coiled conformation with an oblong shape (see Momany, F.A., Sessa, DJ., Lawton, J.W., Selling, G.W., Hamaker, S.A.H., Willett, J.L., J. Agric. Food Chem. (2006) 54:543-547). Aggregates appear to self assemble and form sheet-like structures with super helical cores (see Momany et al., 2006). It is the macromolecular conformation that is of importance for dermal or tissue implants as well as for bioartiiicial skin. The helical and sheet conformation requires a hydrophobic core and a polar exterior. A visualization of a repeating core element has been published by Kogan, M. J., Dallcol, L, Gorostiza, P., Lopez-Iglesias, C, Pons, R., Pons, M., Sanz, F., Giralt, E. Biophysical Journal (2002) 83: 1 194-1204, illustrating the surface organization of repeating octamers with hydrophobic and neutral residues as core with polar residues presenting to the exterior is shown in FIG. 1. This model although for gamma zein represents intrinsic characteristics for all zeins for the purposes of the use described herein. The dual or amphipathic nature of the zein molecule provides an excellent bio-physical entity for tissue implants and bioartificial skin substrates, and thus enables zein to replace collagen in dermal or other tissue related uses.

Additional data for the coiled triple super-helical conformation has been described by Momany et al., 2006, with each of the helices representing a repeating unit of eight (8) residues, as shown in FIG. 2. The presence of sulfur containing amino acids result in strong intermolecular disulfide bonds which together with hydrophobic interactions provide for a large scale aggregate formation on the order of nanometers. For example, see Guo, Y., Liu, Z., An, H., Li, Minqian LI, HU, Jun, J. Cereal Sci. (2005) 41 :277-281.

The special nature of zein aggregates supports the use of zein as tissue implants in an aqueous physiological environment according to its polar or hydrophilic component, as well as offering an insoluble, hydrophobic core protecting the primary bulk mass from enzymatic degradation.

Preferably, formulations having zein have finely tuned ratios of hydrophilic to hydrophobic components, and therefore varying amphipathic properties, thus allowing the full spectrum of implant lifetime as well as offering new and unique options for individualized uses. Implants in one aspect could be essentially permanent (very low overall solubility) and in another aspect be transient (very high solubility). This unique feature of zein lies at the core of the invention and offers a unique platform for improved tissue implants and products. The coiled coil and larger conformational ensembles of zein while well studied and naturally abundant have only recently been characterized with respect to self assembly (see Kogan et al., 2002) over a range of molecular conformations and, as a result, have not been previously recognized as a non-animal/non-human substitute for collagen as tissue implant, tissue augmentation, skin substrate, graft or other component of soft tissue organs or any part of the human anatomy. The only other polyproline structure of a similar native state is collagen as a well known and long used component for tissue implants, skin augmentation and bio-artificial skin substrate.

The overall architectural features of the bulk agent are not expected to differ in a practical sense over the spectrum of compositions intended. The invention provides for several methods to create from pure zein a spectrum of tissue zein implants with unique characteristics relative to currently available products. In addition to zein of pure composition, a number of additions to zein are feasible and could extend the utility of zein to finer applications for facial dermal implants as well as augmentation for skin or tissue in a variety of organs and physiological systems. Sources of USP zein, decolorized and free from fatty acid residues can be obtained commercially or can be purified according to standard protein purification methods and purity assessed by standard chromatographic and electrophoretic methods.

A wide variety of approaches and techniques may be used to prepare zein tissue implants for cosmetic applications and for preparation of substrates for skin, tissue and organ augmentation, repair and scaffolding. The zein referred to herein is comprised of any of the zein family proteins (alpha, beta, gamma, or delta). Examples for tissue applications for zein are:

[I] as a dermal filler;

[2] for cutaneous augmentation (skin and underlying soft tissue);

[3] as a skin replacement product;

[4] as a scaffold/matrix to facilitate and/ or expedite cutaneous wound healing;

[5] for (non-cutaneous) cell therapy/tissue engineering uses such as:

[6] as a scaffold for repair/regeneration of tissues/organs, either directly or in combination with cells;

[7] hard and soft tissue regeneration and repair;

[8] as a bulking agent for augmentation of organs/tissues (e.g., bladder neck in patients with incontinence);

[9] as a substrate for cell/tissue culture;

[10] as bulk agent for sealing in pouches for breast augmentation;

II 1] a tissue implant or graft. However, zein may be used in any other tissue applications, including those currently involving synthetic non-collagen materials and collagen.

In order to use zein as a source material for the above tissue applications, the zein needs to be sufficiently pure. Pure zein for all formulations can be prepared as follows by specific example. Other methods with slightly different proportions of solvents and bench top equipment can also be used. Examples of purification of zein can be found in Cabra, V., Arrguin, R., Galvez, A, Quirasco, M, Vazquez-Duhalt, Farres, A. J. Agric. Food Chem (2005) 53: 725-729, and Dickey, L C, Parris, N, Craig, J C, Kurantz, M J. Industrial Crops and Products (2001) 13: 67-76.

Maize kernels of any variety are fine cut to approximately 2 mm particles with commercial feed mill (e.g., Davis Feed Mill, Perkasie, PA, USA) or similar with a counter- rotating ribbed disc mill. Prior to milling, the maize kernels are cracked with a roller mill and the pericarp removed by aspiration. The resulting powder is passed through a sieve of 3mm to ensure consistency. The particles are then grounded to a flour consistency in a disk mill (e.g., Weber Bros. & White, Metal Works, Inc., USA, or similar).

In 3 kilogram batches, the flour is mixed with hexane to remove lipids by agitation and decantation of supernatant after settling in 6 liter Erlynmyer flasks and air dried. A similar procedure on each batch is performed to remove carotenes and xanthophylls by extraction with chloroform-ethanol mixtures (2: 1). The defatted, decolored flour is then air dried.

The resulting flour can be treated in two ways:

Treatment 1 :

First, 26 kg of treated maize flour as above is added to 91 kg of ethanol water mixture (70% ethanol) in a 300 liter fermentor, such as a model Biostat UD (e.g., B. Braun Biotech, Inc., Allentown, PA, USA) and the suspension temperature raised to 50 degrees C over a 2 minute period and then held at 50 degrees C for 1 hour with stirring by turbine paddles at 200 φm. The fermentor slurry is then pumped to a decanter centrifuge (e.g., Sharpies, division of Alfa Laval, model PF-743, Warminster, PA or similar) running at 6000 rpm. The outgoing solid streams are rinsed by pumping liquid of the same composition as the original extraction liquid into the centrifuge through a separate inlet which results in a damp extracted maize and liquid fractions at separate centrifuge outlets.

The liquid fraction is cooled overnight at ambient temperature and then pumped into a 10.5 cm tubular-bowl centrifuge (e.g., Sharpies, division of Alfa Laval, model M-312-H-16, Warminster, PA, USA or similar) rotating at 15,000 rpm. The centrifuge generated 13 200 x g. The liquid fraction is then collected for analysis and concentration by evaporation in a circulating water bath held at 40 degrees C or by microfiltration through a ceramic micro- filter (e.g., Ceramem LMA 0.2 um, Waltham, MA, USA). The resulting liquid was centrifuged at 12 00Ox g for 30 minutes at 4 degrees C.

Treatment 2:

The defatted, decolorized maize flour is mixed with 95% ethanol (5: 1 solvent- flour (v/w))) using orbital agitation for 12 h at 25 degrees C (e.g., New Brunswick Scientific Model R76, Edison, NJ, USA) in batches as required. The solution is then centrifuged at 12,00Ox g for 30 min at 3 degrees C. The supernatant is recovered.

Supernatants from both treatments are then subjected to cationic ion-exchange chromatography using SP-spharose 2.6 cm x 11 cm (e.g., Amersham Biotech, Uppsala, Sweden) using citrate buffer, 0.02 M with 70% methanol, ph 3.5, as mobile phase. The resulting sample is solubilized in buffer to approx. 0.5 mg per liter and then filtered with 0.22 uM membrane (Millipore, Ireland) and injected into a high pressure liquid chromatograph ,„ (e.g., AKTA prim, Amersham Pharmacia Biotech, Uppsala, Sweden). The elution buffer had 0.7 M NaCl and a gradient from 0 to 1.2 mN NaCl is applied at a flow rate of 0.5 ml per min and eluant monitored at 280 nm through a UV detector. The peak is collected and concentrated by ultrafiltration using YMlO membranes (Amicon, Millipore, USA).

The resultant liquid fraction from one of the above treatments serves as initial component for all zein preparations. It is understood that any number of other purification approaches and methods could also be used to produce high purity zein that remains in the native state free from aggregation and denaturation.

Examples of zein preparations for use in tissue applications, such as dermal fillers, soft tissue augmentation, bulk soft tissue filler, bio-artificial skin, or as a substrate for grafts for any soft tissue or organ, will now follow:

Example 1.1

A flame dried, 250 mL, round bottom single neck flask is charged with 60 grams of purified zein as above, 4.00 mL of distilled glycerol, and 0.1 ml triple distilled water. The flask is fitted with a flame dried mechanical stirrer. The reactor is then purged with nitrogen three times before venting with nitrogen. The reaction mixture is heated to 40 degree C. and maintained at this temperature for about 18-20 hours. The resulting viscous liquid is dried under vacuum (0.1 mm Hg) at 40 degree C for about 16 hours to remove any traces of solvent to produce a zein polymer. The zein prolamine polymer is a stable liquid at room temperature injectable with a needle, such as a 28 to 30 gauge needle, into the body of a patient as a dermal filler or for soft tissue augmentation or implantation.

Example 1.2

The procedure in Example 1.1 is substantially repeated except that 1.0 ml triple distilled water is used instead of 0.1 ml. The reaction mixture is heated to 40 degree C. and maintained at this temperature for about 18-20 hours. The resulting viscous liquid is dried under vacuum (0.1 mm Hg) at 40 degree C for about 16 hours to remove any traces of solvent to produce a zein polymer. The zein prolamine polymer is a stable liquid at room temperature injectable with a needle, such as a 28 to 30 gauge needle, into the body of a patient as a dermal filler or for soft tissue augmentation or implantation.

Example 1.3

The procedure in Example 1.2 is substantially repeated except that 2.0 ml triple distilled water is used instead of 1.0 ml. The reaction mixture is heated to 40 degree C. and maintained at this temperature for about 18-20 hours. The resulting viscous liquid in then dried under vacuum (0.1 mm Hg) at 40 degree C for about 16 hours to remove any traces of solvent to produce a zein polymer. The zein prolamine polymer is a stable liquid at room temperature injectable with a needle, such as a 28 to 30 gauge needle, into the body of a patient as a dermal filler or for soft tissue augmentation or implantation.

Example 1.4

The procedure in Example 1.3 is substantially repeated except that 3.0 ml triple distilled water is used instead of 1.0 ml. The reaction mixture is heated to 40 degree C. and maintained at this temperature for about 18-20 hours. The resulting viscous liquid is then dried under vacuum (0.1 mm Hg) at 40 degree C for about 16 hours to remove any traces of solvent to produce a zein polymer. The zein prolamine polymer is a stable liquid at room temperature injectable with a needle, such as a 28 to 30 gauge needle, into the body of a patient as a dermal filler or tissue implant. As Examples 1.1.-1.4 show, pure zein formulations may be used as dermal filler with carriers or for soft tissue augmentation or implantation.

Example 2

Zein mixed with components, such as excipients and complexed with carriers, to provide a zein formulation for:

[1] as a dermal filler;

[2] for cutaneous augmentation (skin and underlying soft tissue);

[3] as a skin replacement product;

[4] as a scaffold/matrix to facilitate and/or expedite cutaneous wound healing;

[5] for (non-cutaneous) cell therapy/tissue engineering uses such as:

[6] as a scaffold for repair/regeneration of tissues/organs, either directly or in combination with cells,

[7] hard and soft tissue regeneration and repair;

[8] as a bulking agent for augmentation of organs/tissues (e.g., bladder neck in patients with incontinence); [9] as a substrate for cell/tissue culture.

[10] as bulk agent for sealing in pouches for breast augmentation.

[11] a tissue implant or graft

Example 3

Chemically modified zein by reaction with any individual or group of amino acid residues anywhere within the zein molecule, to provide a zein formulation for uses as described in Example 2 above by reference.

Example 4

Zein in combination with other agents for local and/or systemic therapy. Example 5

Coacervate of zein with any compatible agent. Example 6

Zein film comprised of any components in Examples 1 through 5, including nano- engineered films, folded and rolled films to achieve specific surface characteristics, such as for use as described in Example 2 above by reference.

Example 7

Zein plus a mimetic, such as botulinum toxin, for cosmetic dermal use and any of the uses described in Example 2 above by reference.

Example 8

Zein composition of micelles comprised of any components in Examples 1 through 5, for uses as described in Example 2 above by reference.

Example 9

Zein composition of microspheres in pure zein composition or comprised of any components in Examples 1 through 5, such as for uses as described in Example 2 by reference. Example 10

Zein microspheres with beneficial inclusions, drugs, antimicrobial compounds or other agents, such as for uses as described above in Example 2 by reference.

Example 1 1

Zein formulations prepared in aqueous solutions of water and ethanol ranging from 5 to 80% v/v water/ethanol with zein from 5 to 80% w/v, such as for use as described above in Example 2 by reference.

Example 12

Zein emulsions comprised of any compatible compounds produced by evaporation until desired viscosity is attained, such as for use as described above in Example 2 by reference.

Example 13

Zein preparation dispersed in a carrier of compatible glycols (e.g., polyethylene glycol, glycerol and similar), such as for use as described above in Example 2 by reference.

Example 14

Zein preparation dispersed in a carrier of poloxamer (Pl 88) or similar compounds, such as for use as described above in Example 2 by reference.

Example 15

Zein preparation dispersed in a carrier of an ionic liquid, such as for use as described above in Example 2 by reference.

Example 16

Zein prepared with varying ratios of water and ethanol and air dried on a smooth or rough surface, such as for use as described above in Example 2 by reference. Example 17

Zein prepared with varying ratios of water and ethanol and subjected to molecular combing or any other physical force to shape and orient zein molecular components to optimal aggregate configurations for use as described above in Example 2 by reference.

Example 18

Zein prepared in any number of hydrogel formulations, including polyvinyl alcohol and related compounds, such as for use as described above in Example 2 by reference.

Example 19

Zein formulations of any type with collagens (animal or human) used for dermal or tissue implants, skin augmentation, grafts and tissue or organ augmentation.

Example 20

Zein formulations cross linked with itself, such as for use as described above in Example 2 by reference.

Example 21

Zein formulation cross-linked with simple and/or complex polysaccharides for the use of dermal implants, tissue implants, skin augmentation, grafts, and tissue or organ augmentation.

Example 22

Zein formulations containing hylauronic acid, such as for use as described above in Example 2 by reference.

Example 23

Zein formulations containing silicon, such as for use as described above in Example 2 by reference. Example 24

Zein formulations containing hydroxyappetite, such as for use as described above in Example 2 by reference.

Example 25

Zein formulations containing poly (L-lactic acid) oligomers, such as for use as described above in Example 2 by reference.

Example 26

Zein formulations containing lidocaine or any other anesthetic, such as for use as described above in Example 2 by reference.

Example 27

Zein formulations with any other plant biopolymer including homologous polyprolamines such as for use as described above in Example 2 by reference.

Example 28

Zein formulations with any type of tissue matrix, such as for use as described above in Example 2 by reference.

Example 29

Zein formulations with polymethyl-metacrylate (PMMA), such as for use as described above in Example 2 by reference.

Example 30

Any of the above formulations provided in a kit form with zein supplied in one vial either as lyophilized solid, ethanol solution or in combination with any other carrier as needed. A second and third vial may be provided with additional components to effect the proper mixtures and preparation of a solution for injection. Example 31

Any zein formulation resulting in a gel consistency suitable for use in breast or other tissue implants.

Example 32

Any zein formulation incorporating an argentiferous agent or any other metallic or related agent or any effective compound for the purposes of inhibiting infection or controlling infections or in any application where germicidal control is desired for any of the applications referenced in Example 2 above.

For tissue augmentation or bulking, zein in solution or incorporated in a carrier may be directly injected into soft tissues such as the subcutaneous tissue or into tissue surrounding structures for example into the tissues surrounding the urethra or the ureterovesicle junction of the bladder.

For use as a substrate for tissue in-growth or re-surfacing, a film or formed zein structure may be placed at the treatment site and the native tissue allowed to regenerate upon or into, respectively, the zein. As a scaffold for skin regeneration in acute or chronic wounds, films or sheets of zein, other in its native state or as a composite with another biocompatible material, may be placed in the wound bed and the surround tissue allowed to re-surface the film or sheet.

For use as a scaffolding for the in vitro creation of composite tissues or organs, zein in its native configuration in gel formulations or as a composite with a biocompatible material is used as a scaffolding into or upon which cultured cells or tissues, respectively, are placed. The zein - cell/tissue composite is maintained in vitro until the composite is ready to implanted or applied to the animal.

For use as a temporary wound covering zein is used in its native form in a gel form or as a carrier for micro-sphere or nano-spheres of zein. The zein in gel form or as a film is placed on the wound bed to provide coverage and protection for the healing process. The zein composition may contain various medications including antibiotics and other agents such as silver useful to prevent or treat local infection and anti-inflammatory agents. Although plant protein derived from zein has been described above, other plant prolamines may similarly be used, which may be derived from wheat, rye, oats, amaranth and other plants (terrestrial, aquatic and marine).

Three types of matrices can be used in tissue engineering to create 3D laminar structures for organ transplantation: 1) planar high strength polymers that can be micro- fabricated (e.g., micro-molding, micro-machining, stereo-lithography, solid free form manufacturing) and stacked in laminar configurations, 2) fibrous polymeric scaffolds (i.e., non-machined) and, 3) hydrogels representing an amporphous cell-polymer matrix.

In the case of microfabrication, techniques are based on well established methods used to make integrated circuits having dimensions as small as a few nanometers and which can be mass produced at low per-unit costs. The scale of machining is critical to provide channels and structure for recruiting cells in an organized manner and meeting the aforesaid requirements specific to organ transplantation.

In the case of hydrogels composed typically of polyamides, alginates and methylcellulose (among many others) the polymer and cross linking agents are pre-mixed with cells for implantation and permitted to crosslink with cells in situ. Thereafter the hydrogel containing cells is placed in the body. Ma, US 6,872,387, Three-dimensional Hydrogel/Cell System, March 29, 2005, discloses the use of hydrogel in an organ replacement application.

Formulations of zein as disclosed herein can be used to produce all three types of tissue matrices and 3-D scaffolds as described previously. In this respect zein is novel and unique offering potentially a combination of macro-matrix characteristics to accommodate organ replacements for the lung, kidney and liver organs. Also, zein formulations as described herein exhibit a wide range of physical characteristics including amorphous structure and controllable degradation. Pore size, porosity, channel geometry and the ability to mold thin zein membranes for etching and stacking in laminar structures are controllable mechanical features of the zein formulations.

While numerous moieties for tissue scaffolds have been the subject of biocompatibility and mechanical properties, in vitro and in vivo applications as a basis for large scale organ replacement scaffolds have not been described except in the case of PLA/PLGA polymers. The studies of Wang et al. (2007) report zein scaffolds that are simple aggregations of purified and lyophilized zein but do not apply, disclose, teach or make obvious how to create 3-D scaffolds of zein for the highly demanding application of organ replacement. The work of Wang et al. (2007) does not address key and non-obvious features of such a device including:

[1] Specific scaffold architecture must be matched to the development of specific biological functions in tissues thus providing appropriate nutritional conditions and spatial organization for specific cell growth to the organ being replaced. The work of Wang et al. (2007) does not disclose or teach this feature.

[2] Mass transport characteristics of large and dense organ replacements are key to deliver adequate nutrients to cells and require a high degree of interconnectedness. The work of Wang et al. (2007) does not disclose or teach this feature.

[3] Processability and reproducibility to accommodate a variety of final shapes while retaining key physical features. The work of Wang et al. (2007) does not disclose or teach this feature.

In summary, the requirements of scaffolds for tissue engineering are complex and specific to the structure and function of the tissue of interest. With the use of Zein in accordance with this invention, the scaffold fabrication has the desired characteristics such as the degradation rate, porosity, pore size, shape, distribution, and mechanical properties. Factors such as pore size, shape, and tortuosity can all affect tissue ingrowth and are difficult to control precisely. However, in the case of zein and according to treatments 1 and 2 disclosed herein as well as by way of the examples provided the aforementioned variables may be controlled and thus are suitable for zein scaffolds for application in organ replacement. Further Examples

The following example illustrates fabrication of a zein scaffold as described above. The preferred embodiment employs zein purified by a process which produces the purity and quality of zein required for successful application of zein for tissue scaffold, microspheres, laminar sheets, injectable fillers and other uses described herein. The process utilizes zeolite adsorbents to remove color and odor from zein. Specifically, a solution of a zein containing product is placed in an aqueous alcohol solvent and then contacted with a zeolite adsorbent under conditions effective for purification. The zein eluant from the aforesaid process may then be further purified by contact with an activated carbon adsorbent or a mixture of activated carbon and zeolite adsorbents.

Further Example 1

High purity zein (as described above) is combined with a porogen in a process to produce a three dimensional tissue scaffold with the following approximate properties:

Young's modulus of between 30 and 120 MPa;

Compressive strength of between 5 and 15 MPa;

Pore size of between 50 and 450 um;

Porosity of between 60 and 90%;

Dimensions of approximately 1 cubic mm to 50 cubic cm;

Zein formulations consisting of pure zein, any type of cross-linked zein and zein microspheres of any composition.

The zein, solubilized in ethanol, is mixed with the porogen using a hand held homogenizer. The mixture is then dried under vacuum for 12 hours leaving a solid residue. The solid residue is mixed with double distilled water ten times and centrifuged each time to solubilize and remove the porogen. The material is lyophilized and then packed into molds as desired. The final shape and dimensions of the tissue scaffold will depend on the specific use. In the case of skin replacement a laminar sheet may be prepared. In the case of a replacement organ a three dimensional cylindrical shape can be fashioned by stacking laminar sheets within a suitable outer sheath or cover or additional laminar sheets may be used to both enclose the stacked sheets, wrap the stacked sheets and allow for a primary inlet and outlet for the stacked and contained assembly.

The scaffold is then placed in appropriate medium with cells allowing cell growth and vascularization within the scaffold. The cell perfused scaffold is then removed from culture and placed in the appropriate location of the individual allowing immediate perfusion of oxygenated blood. The scaffold made of zein degrades over time leaving an intact replacement organ.

Further Example 2

As described in Further Example 1 and incorporating laminar sheets that have been finely etched (approximately 0.1 micron resolution) on their surfaces to direct cell recruitment and vasculature architecture. Any number of techniques may be used to obtain this embodiment including but not limited to, micro-machining, solid free form fabrication, micro-electromechanical systems (MEMS), laser etching, and related etching processes. The etching may be effected directly on the surface of the zein sheets or the zein may be poured or packed onto a surface with etchings made using typical semiconductor etching processes.

Further Example 3

As described in Further Examples 1 and 2 and incorporating laminar sheets that are planar, folded accordion style, curvilinear or with multiple projections.

Further Example 4

As described in Further Examples 1, 2 and 3 and being of an architecture permitting removal of fabricated tissue in culture which can be further manipulated, folded and compacted allowing direct implantation into the individual without the scaffold.

Further Example 5

As described in Examples 1 through 4 and containing an additive to the zein scaffold that specifically promotes a desired feature of the newly vascularized tissue including enhanced cell adhesion with agarose, gelatin, glycosaminoglycans and other materials known to those skilled in the art of cell culture.

Further Example 6

As described in Further Examples 1 through 5 and using a zein hydrogel, or any form of crosslinked zein, allowing already formed replacement organ or any fabricated tissue to be further cultured in a gel like envelope offering macrostructure and support allowing a less rigid and architecturally defined mass for implantation as well as increased flow within the mass.

From the foregoing description, it will be apparent that an improved tissue compatible material and methods of use have been provided. Variations and modifications in the herein material and methods in accordance with the invention will undoubtedly suggest themselves to those skilled in the art. Accordingly, the foregoing description should be taken as illustrative and not in a limiting sense.

Claims

Claims:

1. A tissue compatible material comprising:

a prolamine protein derived from a plant which when sufficiently pure is capable of being provided alone or formulated with one or more agents or components for biological tissue applications.

2. The tissue compatible material according to Claim 1 wherein said tissue applications comprise one or more of dermal fillers, soft tissue augmentation, tissue implants, substrate for grafts for any soft tissue or organ, bioartificial skin replacement, or wound healing dressings or substrates, or three dimensional laminar structures or scaffolds for engineered tissue and in organ transplants or surgical reconstructions.

3. The tissue compatible material according to Claim 1 wherein said prolamine protein is zein.

4. The tissue compatible material according to Claim 1 wherein said prolamine protein is utilized to provide one of a liquid polymer, gel, substrate, film, or three-dimensional laminar structure.

5. The tissue compatible material according to Claim 1 wherein said prolamine protein is combined with a carrier to provide a polymer injectable into tissue, such as the dermus.

6. A method of using the tissue compatible material according to Claim 1 for one or more tissue applications.

7. The method of Claim 1 wherein said tissue applications are selected from the group consisting of dermal fillers, soft tissue augmentation, tissue implants, substrate for grafts for any soft tissue or organ, bioartificial skin replacement, wound healing dressings, substrates, three dimensional laminar structures, scaffolds for engineered tissue and in organ transplants and surgical reconstructions.