WO2007120840A2

WO2007120840A2 - Methods and compositions for printing biologically compatible nanotube composites

Info

Publication number: WO2007120840A2
Application number: PCT/US2007/009161
Authority: WO
Inventors: Nicole Levi; Faith Coldren; David Carroll
Original assignee: Wake Forest University Health Sciences; Wake Forest University
Priority date: 2006-04-14
Filing date: 2007-04-13
Publication date: 2007-10-25
Also published as: WO2007120840A3

Abstract

A method for forming a cell scaffold is carried out by providing an ink composition comprising, in combination, a solvent, nanoparticles, and a physiologically acceptable polymer; and then ink-jet printing said composition on a solid support to form said cell scaffold therefrom. Viable cells may subsequently be deposited on the scaffold, for example by ink-jet printing.

Description

METHODS AND COMPOSITIONS FOR PRINTING BIOLOGICALLY COMPATIBLE NANOTUBE COMPOSITES

Nicole Levi, Faith Coldren, and David Carroll

Government Funding

This invention was made with Government support under grant number FA9550-04-1-0161 from the Air Force AFOSR. The US Government has certain rights to this invention.

Related Applications

This application claims the benefit of United States Provisional Patent Application Serial No. 60/744,855, filed April 14, 2006, the disclosure of which is incorporated by reference herein in its entirety.

Field of the Invention

The present invention concerns methods and compositions useful for the production of three-dimensional constructs of viable cells.

Background of the Invention

Development of three-dimensional tissue scaffolds into which cells may be seeded to generate new tissue is a broad venture that incorporates biomaterial type, strength, and structure tailored for specific cell types. Well-designed scaffolds should consist of biopolymers which may slowly be reabsorbed into the body following implantation, while simultaneously promoting cell adhesion, proliferation, and production of extracellular matrix proteins.¹ Addition of single-wall carbon nanotubes into a polymer system may increase the strength and stiffness of the structure and also offers a means to apply electrical stimulus to cells seeded into the matrix.²"^2"7 Recent studies indicate that electrically stimulated cells cultured atop carbon nanotubes proliferate more rapidly than on control surfaces and further, secrete their own extracellular matrix proteins following attachment.^{3 8} There are many means for scaffold development, including lithography,⁹ leaching techniques,¹⁰ hydrogels,"'¹² electrospinning,¹³'¹⁴ and inkjet printing.^15"18

Each method has the ability to produce stable porous scaffolds for infiltration of cells.

However, current research indicates that cells proliferate best on nanostructured substrates as compared to smoother surfaces.⁶'⁷ In this regard, techniques such as electrospinning are promising because they generate biopolymer fibers in the nanometer regime. There are challenges to the utilization of techniques like electrospinning however, such as incompatibility with the formation of fully three dimensional scaffolds with architecture and difficulty with the use of nanocomposites which may be desired for further functionalities.

Summary of the Invention

We have developed a composition of printable, biocompatible, "inks" for use in the creation of tissue scaffolds in three dimensions. In general, and in some embodiments, this composition comprises, consists of or consists essentially of a host material (sometimes referred to as a physiologically acceptable polymer) such as; collagen, alginates, fibronectin, elastin, poly(lactide), poly(glycolide), etc., and mixtures or co-polymers, thereof, in some embodiments a bi-phasic dispersant agent such as PEG, and finally a nanophase dispersant. The function of the host is to provide a scaffolding surface for the growth of tissues, the dispersant can be used to mediate solvent drying, or to aid in the dispersion of the nanophase. Finally the nanophase is used to impart functionalities to the scaffolding such as stiffening, strengthening, etc.

A first aspect of the invention is, accordingly, a method for forming an array of viable cells by depositing, spraying, or printing a cellular composition of the cells on a substrate (e.g., under conditions in which at least a portion of the cells remain viable. The substrate employed is a scaffold that comprises, in combination, nanoparticles and a polymer.

A second aspect of the invention is an array (e.g., a tissue scaffold) comprising, in combination,

(a) a scaffold, said scaffold comprising nanoparticles and a polymer; and (b) viable cells deposited (e.g., by printing or ink-jet printing) on the scaffold.

A further aspect of the invention is a liquid composition useful for forming a scaffold for viable cells, comprising (a) nanoparticles; (b) polymer; and (c) solvent. A further aspect of the present invention is the use of a liquid composition as described herein for carrying out a method as described herein.

The present invention is explained in greater detail in the drawings herein and the specification set forth below.

Brief Description of the Drawings

Figure 1: Transmission electron micrograph of fibronectin/ SWNT composite printed directly onto formvar coated copper grid. Scalebars are lμm and 0.5μm.

Figure 2: Sodium alginate (A) without and (B) with SWNT. Figure 3: Collagen (A) in a water solution, (B,C) PEG solution, both without

SWNT. (D,E) Collagen in a PEG solution with SWNT; fibrous formations present in this sample.

Figure 4: A. AFM of PLGA printed with SWNT suspended in tetraglycol. B. SEM of the same sample which shows the fiber formation in the center of the printed drop.

Figure 5: AFM height morphology profiles of (A)decellularized blood vessel material, (B) collagen printed with PEG and SWNT, and (C) PLGA printed with tetraglycol and SWNT.

The present invention is explained in greater detail in the following non- limiting specification.

Detailed Description of the Invention

"Nanoparticles" for carrying out the present invention may be in any shape and include rods, ellipsoids, spheroids, tubes (single walled and multi-walled), and complex or combined shapes {e.g., as demonstrated by S. Chen, Z.L. Wang, J. Ballato, S. Foulger, and D.L. Carroll, "Monopod, Bipod, and Tetrapod Gold Nanocrystals", Journal of the American Chemical Society jaO38927. DEC (2003)). The nanoparticles may be composed of- any suitable material including carbon (doped and undoped) metals such as Ag and Au, ceramic (silicon, silica, alumina, calcite, hydroxyapatite, etc.) organic polymers (including stable polymers and bioabsorbable polymers), and composites and mixtures thereof. See, e.g., US Patents Nos. 6,942,897; 6,929,675; 6,913,825; 6,899,947; 6,888,862; 6,878,445; 6,838,486; 6,294,401 ; etc. The nanoparticles may be conductive, semiconductive, or nonconductive (insulating). Carbon nanoparticles (e.g., fullerenes) include nanotubes (including both single-wall and multi-wall nanotubes), buckyballs, fiillerenes of other configuration (e.g., ellipsoid), and combinations or mixtures thereof. The nanoparticles may be coupled to (e.g., covalently coupled to) other agents (e.g., proteins, peptides, antibodies) or ligands (e.g., to cell-surface proteins or peptides on the cells being delivered) depending upon the particular application thereof. Diameters of the nanoparticles can be from about 0.1 or 4 nanometers to about 1 micron. Lengths of the nanoparticles can be from 0.8 nm to 100, 200, or 500 microns or more.

"Viable cells" as used herein include prokaryotic and eukaryotic cells such as gram negative and gram positive bacterial cells, yeast cells, plant cells, and animal cells (e.g., reptile, amphibian, avian, mammalian, etc.). Mammalian cells (e.g., human, mouse, rat, monkey, dog, cat, etc.) are in some embodiments preferred. Cells may be of any type, including precursor, progenitor, or "stem" cells, or may be of any suitable tissue (e.g., liver, pancreas, muscle (e.g., smooth muscle), skin, bone (e.g., osteoblast), cartilage (e.g., chondrocytes), tendon, nerve, etc.). In some embodiments the cells are cancer cells (e.g., colon, lung, breast, prostate, brain, liver, or ovarian cancer cells, etc.).

"Polymers" that are used to carry out the present invention may be natural or synthetic and may be bioabsorbable or stable. In general the polymers are preferably physiologically acceptable or biocompatible. Suitable examples include but are not limited to alginate, collagen, fibronectin, polylactide, polyethylene glycol, polycaprolactone, polycolide, polydioxanone, polyacrylates, polysulfones, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates, polyanhydrides, polyamino acids carbohydrates, polysaccharides and modified polysaccharides, and derivatives and copolymers thereof See, e.g., US Patent Nos. 6,991,652 and 6,969,480.

"Solvent" as used herein may be any suitable solvent or combination thereof as is known in the art, including but not limited to water, acids such as acetic acid or phosphoric acid, N-methyl-2-pyrrolidone, 2-pyrrolidone, C₂-C₈ aliphatic alcohol, glycerol, tetraglycol, glycerol formal, 2,2-dimethyl-l,3-dioxolone-4-methanol, ethyl acetate, ethyl lactate, ethyl butyrate, dibutyl malonate, tributyl citrate, tri-n-hexyl acetylcitrate, diethyl succinate, diethyl glutarate, diethyl malonate, triethyl citrate, triacetin, tributyrin, diethyl carbonate, propylene carbonate, acetone, methyl ethyl ketone, dimethylacetamide, caprolactam, dimethyl sulfoxide, dimethyl sulfone, caprolactam, N,N-diethyl-m-toluamide, 1 -dodecylazacycloheptan-2-one, 1,3- dimethyl-3,4,5,6-tetτahydro-2-(lH)-pyrirnidinone, and combinations thereof (see, e.g., US Patent No. 5,759,563); and/or acetone, benzyl alcohol, benzyl benzoate, N- (betahydromethyl) lactamide, butylene glycol, caprolactam, caprolactone, corn oil, decylmethylsulfoxide, dimethyl ether, dimethyl sulfoxide, 1 -dodecylazacycloheptan- 2-one, ethanol, ethyl acetate, ethyl lactate, ethyl oleate, glycerol, glycofurol (tetraglycol), isopropyl myri state, methyl acetate, methyl ethyl ketone, N-methyl-2- pyrrolidone, esters of caprylic and/or capric acids with glycerol or alkylene glycols, oleic acid, peanut oil, polyethylene glycol, propylene carbonate, 2-pyrrolidone, ^• sesame oil, [-t-/-]-2,2-dimethyl-l,3-dioxolane-4-methanol, carbitol, triacetin, triethyl citrate, and combinations thereof {see, e.g., US Patent No. 6,413,536). Preferred solvents include, but are not limited to, water, tetraglycol, polyethylene glycol, acetic acid, dimethyl sulfoxide, C₂-Cs aliphatic alcohol, vegetable oil such as corn oil, isopropyl myristate, 1 -dodecylazacycloheptan-2-one, N-methyl-2-pyrrolidone, and combinations thereof.

"Support" as used herein may be an article of any suitable shape (flat, curved, formed, etc.) and may be made of any suitable material, including metals, glass, ceramics, organic polymers, and composites thereof. ' Subjects that may be implanted with constructs or arrays of the present invention include both human subjects and animal subjects (particularly mammalian subjects such as dogs, cats, horses, pigs, sheep, cows, etc.) for veterinary purposes.

The disclosures of all United States patent references cited herein are to be incorporated herein by reference in their entirety.

1. Compositions.

As noted above, the present invention provides compositions (sometimes referred to as "ink" compositions) useful for making scaffolds upon which viable cells may be deposited. In general the composition comprises: (a) nanoparticles (e.g., from 0.1, 0.5 or 1 percent by weight up to 10,

20 or 50 percent by weight);

(b) polymer (e.g., from 1, 2 or 3 percent by weight up to 40, 50 or 60 percent by weight); (c) a solvent {e.g., from 1 or 5 percent by weight up to 60 or 80 percent by weight, or more); and

(d) optionally, live cells as described herein (e.g., 0, or from 0.01 or 0.1 percent by weight up to 50 or 80 percent by weight of live cells). In some embodiments the polymer is preferably physiologically acceptable or biocompatible (that is, suitable for implant in a human or animal subject without unduly excessive adverse reaction).

In some embodiments the scaffold is printed separately from the printing or deposition of live cells; in other embodiments the live cells are formulated in and printed with the scaffold ink described herein.

In some embodiments the polymer comprises a single polymer; in other embodiments the polymer comprises a combination of different polymers. Where a combination of different polymers is employed, each polymer in the combination — if charged — can be of the same charge or a different charge. For some embodiments the composition is preferably in a form suitable for spraying or ink-jet printing (discussed further below), and hence preferably has a viscosity of from about 1 or 2 centipoise (and in some embodiments at least 20, 30 or 50 centipoise) up to 60, 80, 100, or 200 centipoise or more. Preferably the nanoparticles in the composition are stably suspended therein (that is, the composition is stable at room temperature without settling of the nanoparticles for at least two weeks, or more preferably at least one month).

2. Methods of making and using.

The compositions described above are applied to a solid support by any suitable means, including spraying or printing. Application may be uniformly or in patterns. In one embodiment, ink-jet printing (e.g., thermal ink-jet printing) is preferred. Thermal ink-jet printing may be carried out with apparatus such as described in US Patent No. 7,051,654 to Boland, but with the scaffold ink compositions described herein, rather than the compositions described therein. The compositions may be applied in a single layer or multiple layers, depending upon the particular end structure or array being produced. Such application forms a "substrate" or "scaffold" on the solid support to which cells may then be applied. The scaffold so formed generally comprises, in combination, nanoparticles (e.g., from 0.01, 0.1, or 1 or 5 to 10, 20 or 50 percent by weight of said scaffold) and a polymer {e.g., from 99 or 95 to 50, 40 or 20 percent by weight of said scaffold).

Cells are then applied to the scaffold. As with application of the polymer/nanoparticle compositions to the support, the cells may be applied by any suitable means, such as spraying or printing, with ink-jet printing being (in one embodiment) preferred. The cells may be applied as a single application or multiple applications (uniformly or in patterns) to create three dimensional arrays. In some embodiments cells may be sandwiched between multiple layers of nanotube/polymer scaffold layers. Indeed, multiple layers (e.g., 3, 4, 5, 6, 10, 20, 30 or more) of scaffold and cells, in any order or combination, may be carried out to produce the desired structures or arrays such as three-dimensional, contoured, or shaped arrays.

Methods and compositions for forming three-dimensional structures by deposition of viable cells are described in W. Warren et al., US Patent No. 6,986,739 (Sciperio Inc.). Methods and compositions for the ink-jet printing of viable cells are described in T. Boland et al., US Patent No. 7,051 ,654.

In some embodiments, the polymers within the scaffold are cross-linked after they are ink-jet printed. Such cross-linking can be carried out by any suitable technique, such as separately applying (e.g., by ink-jet printing through a different orifice) a cross-linking agent (e.g., a carbodiimide, an aldose sugar, D-I- glyceraldehyde, genipin, etc.) onto the scaffold, by utilizing polymers that are cross- linked upon exposure to light (e.g., UV light) or heat, etc. An advantage of cross- linking is, in some embodiments, to maintain or enhance the physical integrity of the scaffold.

Once the arrays are formed by the methods described above, the arrays or constructs may be cultured further in vitro in accordance with known techniques to grow the cells (e.g., for subsequent implantation as a prosthesis or the like in a subject, or for the commercial production of a desired compound such as naturally occurring or transgenic protein or peptide from the cells in a fermentation process).

In some embodiments, the growth or proliferation of the viable cells can be enhanced while they are growing in vitro by subjecting the viable cells to an electric field or current sufficient to enhance the proliferation thereof of said viable cells. The electrical field or current may be achieved by any suitable means, such as by connecting the scaffold (directly or indirectly) to a power supply, and/or connecting culture media in which the cells are cultured to a power supply. 3. Applications.

By making possible the printing of cell scaffolds with functional characteristics that can be enhanced, modified or adjusted in a variety of different ways (depending on, among other things, the selection of nanoparticles used), the present invention has a number of applications. Particular applications include, but are not limited to, the following:

A. Electrically conductive scaffolds. By including electrically conductive nanoparticles, the scaffolds can be operatively associated with a current source (such as a battery or voltage regulator) and used to electrically stimulate cells thereon (e.g., muscle cells, nerve cells, skin cells, or any other cell type for which electrical stimulation stimulates growth or enhances proliferation thereof). Particular electrically conductive nanoparticles include, but are not limited to, metal and carbon nanoparticles and nanotubes, including nanowires. Such scaffolds can also be used for applying heat to the scaffolding.

B. Stiffened scaffolds. By including nanoparticles in the scaffold in an appropriate amount (e.g., from 0.001 or 0.01 percent by weight, up to 10 or 20 percent by weight of the ink composition), the elastic modulus of the scaffold can be increased by at least 20 or 50 percent, up to 200 or 500 percent or more, as compared to a scaffold of the same configuration and composition without nanoparticles.

C. Patterned scaffolds. By including nanoparticles in the scaffold in an appropriate amount (e.g., from 0.001 or 0.01 percent by weight, up to 10 or 20 percent by weight of the ink composition), cell scaffolds with improved definition of topographical features (such as lines, ridges, wells, vias, composite shapes, etc.) are obtained. For such features, aspect ratios (A/B) of topographical features on the printed scaffold (which may be printed as a single layer or multiple layers as described above) are in some embodiments preferably at least 1, 2, or 3 (where A is the heighth (or depth) and B is the width of the topographical feature, when the topographical feature is measured in cross-section. D. Contrast agents. Nanoparticles used to carry out the present invention can comprise or contain a contrast or imaging agent to provide detectability of the scaffold in an imaging system such as NMR, X-ray, or the like. Such contrast or imaging agents can comprise Gd complexes, metals such as Fe, and Fe3θ<j, encapsulated contrast agents such as fullerene and encapsulated Gd complexes. See, e.g., US Patent No. 6,797,380.

E. Antimicrobial nanoparticles. Nanoparticles used to carry out the present invention can comprise or contain an antimicrobial {e.g., antibacterial) agent, such as when the scaffolds are used as a tissue implant scaffold to grow cells for tissue implantation. Antimicrobial metal (including metal alloy) particles can comprise any suitable metal materials {e.g., silver) or bi-, tri- or multicomponent or alloyed metals, typically of a size of from 2 ran to 1000 nm).

F. Active agents. Nanopartic.es can be formed of a polymer such as a biodegradable polymer {e.g., PLGA) that contain an active agent to be released into the scaffold. Any suitable active agent beneficial to the cells on the scaffold (or tissue surrounding a region into which the scaffold is implanted) including but not limited to, protein growth factors, cytokines, antibodies, nucleic acids, carbohydrates, antibiotics, etc. See, e.g., PCT Application WO 2006/099333 to Atala et al.. F. Free radical scavengers. Nanoparticles comprised, consisting of, or consisting essentially of a free-radical scavenger can be utilized to produce a scaffold that scavenges such free radicals and reduces their deleterious effects on cells grown thereon. Examples include, but are not limited to, fullerene and transition metal oxides. H. Others. Other applications of the present invention include quantum dot nanoparticles {e.g., CdSe QD from Evident Technologies) for tracking of targeted or tagged agents within the scaffold, transition metal oxides for catalytic crosslinking. etc.

The present invention is explained in greater detail in the following non- limiting Examples.

EXPERIMENTAL

In this work we demonstrate a unique compatibility between biopolymer/ nanotube composites and thermal inkjet printing that allows for the development of ideal fibrous scaffolds similar in nature to both electrospun material and native tissues.

Common biomaterials for scaffold development, which we have used here, include alginates, collagen I, fibronectin, and polylactic co-glycolic acid (PLGA) variations. Collagen I and fibronectin are natural biopolymers found in vivo and alginates have been shown to act as viable artificial replacements similar to glycoaminoglycosans which naturally occur in the body. PLGA is a material used in sutures and as additional material in tissue scaffolds, which hydrolyses into glycolic and lactic acids which are reabsorbed by the body. Collagen and other extracellular matrix proteins are typically reincorporated into the tissues following implantation. Likewise, a variety of cell types are known to have increased proliferation on nanofibrous materials such as collagen fibrils or carbon nanotubes.³'⁷"¹⁹ Single-wall carbon nanotubes (SWNT) have been shown to act as a viable matrices which do not illicit immune response and are cleared from the body over time.^20"24 Ideally, incorporation of such nanostructuring into three dimensional biomaterials could provide an added functionality to the scaffold allowing for the potential of creating fully filled organs; one of the primary goals of regenerative medicine.

In our approach, the factors that influence a "bio-ink¹ and can lead to clogging of printheads include viscosity and concentration; also the solvents for the polymers and nanotubes must be compatible with one another and with the printhead.¹⁸ As cited in the literature typical limits for viscosity of print solutions are about 2OcP.¹⁸ However, we have found that some solutions with PLGA, which are well above the accepted limits of viscosity, allow for fine structure printing, see Table 1.

Table 1 : Viscosities for biopolymer/ carbon nanotube composites using a cone on late viscometer.¹

Solutions were measured at a shear rate of 229.45 s^" except * at 28.68 s and ** at 54.36 s^" Thermal inkjet printers heat a small quantity of solution to about 300⁰C which vaporizes the bubble and forces nanoliter volumes of the ink through the nozzles onto the waiting substrate. We found little difficulty with nanotube aggregation due to temperature gradients or shearing of the surrounding fluid. As shown in Figure 1, printed fibronectin and nanotube composites reveal that nanotube bundles are randomly oriented and uniformly dispersed.

Atomic force (AFM) and scanning electron microscopy. (SEM) analysis of the printed composites reveals morphology similar to electrospun material and native vessels and also the formation of fibers in a variety of samples. For example, alginate samples have fiber structures either with or without the addition of SWNT and there appears to be no significant change in fiber morphology as may be seen in Figure 2a. However, a striking difference was observed in printed samples of collagen I when printed with polyethylene glycol (PEG) with and without SWNT as seen in Figure 2b. Composites of collagen hydrated with a PEG solution were found to be very globular in nature whereas a well-defined, aligned fibrous formation was observed when SWNTs dispersed using a PEG solution was added to the collagen 'ink'. See Figures 3a-d. Fibrous structures were also present in PLGA sample printed with SWNT as compared to samples without. Fibers were observed in the printed PLGA samples as seen using AFM and SEM, see Figure 4a,b. InkJet printing of tissue scaffold biopolymers is possible with a wide variety of water soluble and insoluble polymers as evidenced in this work. The addition of carbon nanotubes was found to have a beneficial effect on the morphology of the printed polymers. The printed materials which form fibers upon addition of nanotubes indicates that specific structures could be printed into scaffolds; it is known that specific cell types favor certain morphologies and sizes of the structures they are seeded into. AFM comparison with decellularized blood vessel material shows that similar morphologies exist for the real tissue material and materials generated by printing nanotube/ biopolymer composites, see Figure 5.

InkJet printing offers a viable alternative for polymer scaffold development in tissue engineering as well as for other device manufacturing needs. We have shown that not only can carbon nanotubes be printed in polymeric systems, but they generate the formation of fibers within the matrix which could be valuable in allowing cellular penetration and fluid flow into the designed scaffold. In addition, the fibrous structures that form using the inkjet printing system are similar to the surface features of real tissue. Techniques like inkjet printing allow placement of cells directly into the scaffolds to form a complete material. Our technique allows fibrous structures to form directly from the printed material without the need for added materials or coatings onto the waiting substrates, which decreases the need to manipulate the 5 printed system. Supplementation to the properties of the scaffold by carbon nanotubes include increased strength and compressibility as shown in non-printed polymeric systems and further offer the advantage to employ the conductive nature of the SWNT for electrical stimulation of the seeded cells. Overall, we have developed new materials for use in an inkjet printing system which incorporate carbon nanotubes 0 for their beneficial properties while also adjusting the polymer morphology toward a more preferred cell substrate.

Materials and Methods

Hardware for our print setup is removed from a Hewlett Packard DeskJet 660c 5 printer while the body and other components are custom-built in house. Print cartridges are prepared by first removing residual ink, sonicating the entire cartridge

: in water, and finally rinsing the cartridge with ethanol. The desired "inks" can then be supplied directly to the cartridges, placed in the printer, and printed onto our substrate. 0 Collagen I lyophilized from calf skin was used (Elastin Products Co.) with

0.05% acetic acid and magnetically stirred until completely dissolved and was then diluted to lmg/ml in water in accordance with previous protocols. A solution of PLGA from Purac Corp. was stirred until dissolved in 100% tetraglycol solution (Sigma Aldrich) at concentrations of 20mg/ml and lOOmg/ml. Alternatively, 5 lOOmg/ml PLGA was dissolved in dimethyl sulfoxide (Sigma Aldrich). Equal amounts of each PLGA solution were found best for printing. Sodium alginate

(Dharma Trading Co.) solution was prepared at a concentration of lmg/ml and shaken until dissolved. Print preparation of 0.01% Fibronectin (Sigma Aldrich) was prepared ^v in water. A composition of PLGA and collagen was made with the final 0 concentrations of collagen, 2.86mg/ml, and PLGA, 14.29mg/ml in a 1:2.5 acetic acid to tetraglycol solvent ratio.

Initially, a 10,000MW polyethylene glycol (PEG) solution consisting of Ig PEG, lmg HiPCo carbon single-wall nanotubes (Carbon Nanotechnologies Inc.) in 10ml water was horn sonicated (Branson) on 20%duty cycle at 40% power for ten minutes. ImI of this solution was suspended in a 3000 MW PEG solution prepared by adding 100mg/ml PEG in water and sonicating in a water bath for 10 minutes to obtain a uniform solution. This dispersion of nanotubes was uniform and printed repeatedly without any clogging. We refer to this solution as nanotube stock A. Since the nanotube/ PEG solutions are not compatible with PLGA as PLGA is very hydrophobic we made A stock of O.lmg/ml HiPCo tubes in tetraglycol was sonicated with a horn sonicator on duty cycle 40% and power of 20% for ten minutes and a uniform solution was obtained. We refer to this solution as nanotube stock B.

Biopolymer/ nanotube solutions were prepared using nanotube stock A with sodium alginate and collagen I. Nanotube stock B was used with PLGA and fibronectin stocks. To prepare the solutions, equal amounts of the above-described biopolymer and nanotube stocks were pipetted together and immediately printed. All solutions retained a uniform dispersion of nanotubes following mixing of the polymer and tubes. Printing of the solutions followed immediately and all solutions were : printed onto clean glass slides, or copper grids for electron microscopy observation.

Reference List

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(2006). 6. Dersch,R. et al., Nanoprocessing of polymers: applications in medicine, sensors, catalysis, photonics. Polymers for Advanced Technologies 16, 276-282 (2005).

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The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

That which is claimed is:

1. A method for forming a cell scaffold, comprising: providing an ink composition comprising, in combination, a solvent, nanoparticles, and a physiologically acceptable polymer; and then ink-jet printing said composition on a solid support to form said cell scaffold therefrom.

2. The method of claim 1, wherein said solvent is selected from the group consisting of water, organic solvents, and combinations thereof.

3. The method of claim 1, wherein said solvent is selected from the group consisting of water, tetraglycol, polyethylene glycol, acetic acid, dimethyl sulfoxide, C₂-Cg aliphatic alcohol, vegetable oil, isppropyl myristate, 1-dodecylazacycloheptan- 2-one, N-methyl-2-pyrrolidone, and combinations thereof.

4. The method of claim 1, wherein said nanoparticles comprise carbon nanoparticles, metal nanoparticles, ceramic nanoparticles, organic polymer nanoparticles, or composites or mixtures thereof.

5. The method of claim 1, wherein said nanoparticles comprise nanotubes.

6. The method of claim 1, wherein said nanoparticles comprise carbon nanotubes, metal nanotubes, ceramic nanotubes, organic polymer nanotubes, or composites or mixtures thereof.

7. The method of claim 1, wherein said polymer is selected from the group consisting of alginates, collagen I, fibronectin, and polylactic co-glycolic acids.

8. The method of claim 1, wherein:

(a) said nanoparticles are included in said ink composition in an amount of from 0.1 to 50 percent by weight;

(b) said a polymer is included in said ink composition in an amount of from 1 to 60 percent by weight; and (c) said solvent is included in said composition in an amount of from 1 to 80 percent by weight; and said ink composition having a viscosity of from 1 to 200 centipoise.

9. The method of claim 8, wherein said ink composition has a viscosity of at least 20 centipoise.

10. The method of claim 1, wherein said nanoparticles are electrically conductive.

11. The method of claim 1, wherein said nanoparticles are included in said composition in an amount of from 0.001: to 20 percent by weight, and said scaffold has an elastic modulus that is at least 20 percent greater than a scaffold of the same configuration and composition without said nanoparticles.

12. The method of claim 1, wherein said nanoparticles are included in said composition in an amount of from 0.001 to 20 percent by weight, wherein said scaffold is a patterned having topographical features formed thereon by said ink-jet printing, and wherein said topographical features have an aspect ratio of at least 1.

13. The method of claim 1, wherein said nanoparticles comprise or contain a contrast agent.

14. The method of claim 1, wherein said nanoparticles are antimicrobial.

15. The method of claim 1, wherein said nanoparticles have an active agent encapsulated therein.

16. The method of claim 1, wherein said nanoparticles comprise or contain a free radical scavenger.

17. The method of claim 1, wherein said ink-jet printing step is repeated at least once to form a three-dimensional array.

18. In a method for forming an array of viable cells by ink-jet printing a cellular composition of said cells on a substrate, the improvement comprising: employing as said substrate a scaffold, said scaffold comprising nanoparticles and a physiologically acceptable polymer; wherein said scaffold is formed by ink-jet printing said scaffold on a solid support.

19. The method of claim 18, wherein said cells are mammalian cells.

20. The method of claim 18, wherein said mammalian cells are selected from the group consisting of stem, liver, pancreas, muscle skin, bone, cartilage, tendon, nerve cells, and cancer cells.

21. The method of claim 18, wherein said step of ink-jet printing said scaffold on a solid support is carried out by: providing an ink composition comprising, in combination, a solvent, nanoparticles, and a physiologically acceptable polymer; and then ink-jet printing said composition on a solid support to form said cell scaffold therefrom.

22. The method of claim 21, wherein said solvent is selected from the group consisting of water, tetraglycol, polyethylene glycol, acetic acid, dimethyl sulfoxide, C₂-C₈ aliphatic alcohol, vegetable oil, isopropyl myristate, 1-dodecylazacycloheptan- 2-one, N-methyl-2-pyrrolidone, and combinations thereof.

23. The method of claim 21, wherein said nanoparticles comprise carbon nanoparticles, metal nanoparticles, ceramic nanoparticles, organic polymer nanoparticles, or composites or mixtures thereof.

24. The method of claim 21, wherein said nanoparticles comprise nanotubes.

25. The method of claim 21, wherein said solvent is selected from the group consisting of water, tetraglycol, polyethylene glycol, acetic acid, dimethyl sulfoxide, C₂-Ce aliphatic alcohol, vegetable oil, isopropyl myristate, 1-dodecylazacycloheptan- 2-one, N-methyl-2-pyrrolidone, and combinations thereof.

26. The method of claim 18, wherein:

(a) said nanoparticles nanoparticles are included in said ink composition in an amount of from 0.1 to 50 percent by weight;

(b) said a polymer is included in said ink composition in an amount of from 1 to 60 percent by weight; and

(c) said solvent is included in said composition in an amount of from 1 to 80 percent by weight; and said ink composition having a viscosity of from 1 to 200 centipoise.

27. An array of viable cells produced by the method of claim 18.

28. The array of claim 27, wherein said array is a three-dimensional array.

29. An array comprising, in combination,

(a) a scaffold, said scaffold comprising nanoparticles and a physiologically acceptable polymer.

(b) viable cells deposited on said scaffold.

30. The array of claim 29, wherein said array is a three-dimensional array.

31. A method of culturing viable cells, comprising:

(a) providing an array of claim 29, and

(b) culturing said viable cells on said scaffold in vitro.

32. A method of enhancing proliferation of viable cells, comprising:

(a) providing an array of claim 29,

(b) culturing said cells on said scaffold in vitro; and

(c) subjecting said viable cells to an electric field or current sufficient during said culturing on said scaffold to enhance the proliferation of said viable cells.

33. A liquid composition useful for forming a scaffold for viable cells, comprising:

(a) 0.1 to 50 percent by weight of nanoparticles;

(b) 1 to 60 percent by weight of a physiologically acceptable polymer, said polymer having a molecular weight of 500 to 50,000 Daltons; and

(c) 1 to 80 percent by weight of a solvent.

34. The composition of claim 33, wherein: said composition has a viscosity of from about 1 centipoise to 200 centipoise; and said composition is a stable suspension for at least one month at room temperature.