WO2017062417A1 - Échafaudages pour tissus neuronaux et leurs utilisations - Google Patents

Échafaudages pour tissus neuronaux et leurs utilisations Download PDF

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Publication number
WO2017062417A1
WO2017062417A1 PCT/US2016/055438 US2016055438W WO2017062417A1 WO 2017062417 A1 WO2017062417 A1 WO 2017062417A1 US 2016055438 W US2016055438 W US 2016055438W WO 2017062417 A1 WO2017062417 A1 WO 2017062417A1
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WO
WIPO (PCT)
Prior art keywords
cells
patterned
tissue scaffold
tissue
μιη
Prior art date
Application number
PCT/US2016/055438
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English (en)
Inventor
Jeffrey Schwartz
Jean E. Schwarzbauer
Casey M. JONES
Patrick E. DONNELLY
Stephen B. BANDINI
Shivani Singh
Original Assignee
The Trustees Of Princetion University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US14/875,168 external-priority patent/US10675138B2/en
Application filed by The Trustees Of Princetion University filed Critical The Trustees Of Princetion University
Priority to US15/767,616 priority Critical patent/US11364105B2/en
Publication of WO2017062417A1 publication Critical patent/WO2017062417A1/fr

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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Definitions

  • ECM extracellular matrix
  • a goal of regenerative medicine is to promote formation of new tissue that closely resembles the normal tissue in organization and function. Controlling cell growth in a spatially defined way enables regeneration of damaged or diseased tissues having the proper alignment of constituent cells and/or alignment of molecular complexes that the cells produce.
  • cells direct the arrangement of ECM fibrils to correspond to their actin filaments by using cell surface receptors that are indirectly connected to the actin cytoskeleton. Therefore, a major challenge in regenerative medicine is to promote cells to assemble ECM fibrils, such as collagen, into particular orientations or alignments on a scaffold device in order to generate tissues with the required functional properties.
  • the present invention is based, at least in part, on the discovery of methods for generating cell-adhesive chemical (i.e., non -biologic) patterns in nano- and micro-scale dimensions using a technique for surface-modifying solids or polymers to generate devices that have utility as bio-scaffold materials and other devices including electrodes and sensors.
  • cell-adhesive chemical i.e., non -biologic
  • the devices of the present invention can be used in, for example, regenerative medicine, wound repair, and transplant biology, as well as in screening assays to determine the effects of a test compound on living tissue by examining the effect of the test compound on various biological responses, such as for example, cell viability, cell growth, migration, differentiation and maintenance of cell phenotype.
  • the present invention provides patterned scaffolds for tissue.
  • the tissue scaffold comprises a base layer comprising a pattern of stripes, and an oxide layer comprising the pattern of stripes.
  • the base layer and the oxide layer comprise a pattern of oxide layer stripes on the base layer.
  • the present invention provides a tissue scaffold comprising a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; and an extracellular matrix component aligned in parallel on the stripes.
  • the tissue scaffold comprises a base layer comprising a pattern of stripes, an oxide layer comprising the pattern of stripes, and a non-biologic cell adhesive layer disposed on the oxide layer.
  • the present invention provides a tissue scaffold comprising a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; a non-biologic cell adhesive layer disposed on the oxide layer; and an extracellular matrix component aligned in parallel with the stripes.
  • the present invention provides an artificial tissue comprising living cells attached to a tissue scaffold comprising a base layer comprising a pattern of stripes, and an oxide layer comprising the pattern of stripes.
  • the present invention provides an artificial tissue comprising living cells attached to a tissue scaffold comprising a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; and a non-biologic cell adhesive layer disposed on the oxide layer.
  • the present invention provides an artificial tissue comprising living cells attached to a tissue scaffold comprising a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; and an extracellular matrix component aligned in parallel with the stripes.
  • the present invention provides an artificial tissue comprising living cells attached to a tissue scaffold comprising a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; a non-biologic cell adhesive layer disposed on the oxide layer; and an extracellular matrix component aligned in parallel with the stripes.
  • the present invention provides a method for making a tissue scaffold comprising: generating a pattern of stripes on a base layer by
  • the method for making a tissue scaffold further comprises contacting the patterned oxide layer with a non-biologic cell adhesive compound to generate a patterned cell adhesive layer.
  • the present invention provides a method for making a tissue scaffold comprising: generating a pattern of stripes on a base layer by
  • photolithography to form a substrate having a patterned base layer; depositing an oxide layer onto the patterned base layer to form a substrate having a patterned oxide layer; contact the substrate having the patterned oxide layer with cells and culturing under conditions suitable for the production of extracellular matrix components; and removing the cells from the substrate to provide a tissue scaffold comprising an extracellular matrix component aligned in parallel with the stripes.
  • the present invention provides a method for making a tissue scaffold comprising: generating a pattern of stripes on a base layer by photo- lithography to form a substrate having a patterned base layer; depositing an oxide layer onto the patterned base layer to form a substrate having a patterned oxide layer; contacting the patterned oxide layer with a non-biologic cell adhesive compound to generate a substrate having a patterned cell adhesive layer; contacting the substrate having the patterned cell adhesive layer with cells and culturing under conditions suitable for the production of extracellular matrix components; and removing the cells from the substrate to provide a tissue scaffold comprising an extracellular matrix component aligned in parallel with the stripes.
  • the present invention provides a method for making an artificial tissue comprising living cells attached to a tissue scaffold comprising contacting a tissue scaffold of the present invention with cells and culturing under conditions suitable for cell growth and/or differentiation.
  • the present invention provides medical devices comprising the artificial tissues or tissue scaffolds of the invention.
  • the present invention provides methods of tissue repair and regeneration comprising implanting the artificial tissues of the present invention in a subject in need of such tissue repair or regeneration.
  • the present invention provides methods for identifying a compound that modulates a tissue function.
  • the methods include providing a patterned scaffold for tissue as described herein, contacting the tissue with a test compound, and measuring the effect of the test compound on a tissue function in the presence and absence of the test compound, wherein a modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates a tissue function, thereby identifying a compound that modulates a tissue function.
  • the present invention provides methods for identifying a compound useful for treating or preventing a tissue disease.
  • the methods include providing a patterned scaffold for tissue as described herein, contacting the tissue with a test compound, and measuring the effect of the test compound on a tissue function in the presence and absence of the test compound, wherein a modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates a tissue function, thereby identifying a compound useful for treating or preventing a tissue disease.
  • scaffolds for nerve tissue regeneration and methods of use thereof are provided that overcome the limitations of previous systems and methods for controlling cell alignment on polymer substrates by using cell-assembled, aligned ECM patterned polymer scaffolding that controls neurite outgrowth as opposed to the unpatterned scaffolding systems of previous inventions.
  • the presence of aligned ECM patterns in the scaffolding systems of the present invention helps promote and coordinate neurite growth in the directions of the aligned ECM patterns.
  • the cells are neural cells.
  • the neural cells can be selected from the group consisting of neural stem cells, oligopotent stem cells, differentiated neurons, differentiated glial cells and neural crest cells.
  • the tissue scaffold containing neural cells is co-cultured with glial support cells.
  • Figs. 1 A and IB are schematics of the preparation of a nanoscale-patterned surface using a self-assembled monolayer of phosphonate (SAMP).
  • SAMP phosphonate
  • 1 A (A) Spin-cast with hexamethyldisilazane (HMDS); (B) spin-cast AZ-5412E photoresist; (C) expose to UV through a photomask; (D) develop in AZ-312 MIF; (E) vapor deposition of zirconium tetra-t-butoxide (1), then heat to form adhesion layer; (F) assembly of the SAMP.
  • HMDS hexamethyldisilazane
  • C expose to UV through a photomask
  • D develop in AZ-312 MIF
  • E vapor deposition of zirconium tetra-t-butoxide (1), then heat to form adhesion layer
  • F assembly of the SAMP.
  • IB The photolithographically patterned surface (E)
  • Fig. 4 illustrates intensities of the radial sums as a function of angle, which represents cell conformity with the SAMP/Zr02/Si02/Si pattern. These plots correspond to the images in Figs. 3A-D. Full-width at half-maximum value (FWUM) for 20 X 10, 32°; 30 X 30, 29°; 100 X 40, 49°; FWUM for the control is not applicable. The peaks narrow as the dimensions decrease, indicating greater conformity of the cell to the pattern.
  • FWUM Full-width at half-maximum value
  • Figs. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H and 51 show 3T3 fibroblasts on 30X30 (top),
  • Fig. 8 shows phase contrast images of NIH3T3 cells growing on patterned and unpatterned PET.
  • Fig. 9 shows fibronectin alignment on PET surfaces and results of fast Fourier transformation (FFT) analysis of the images of the cells confirming fibronectin alignment on the patterned PET surfaces.
  • FFT fast Fourier transformation
  • Fig. 10 shows the results of fast Fourier transformation analysis of fibronectin alignment on patterns of different dimensions.
  • Fig. 11 shows collagen alignment on PET surfaces and results of fast Fourier transformation analysis of the images of the cells confirming collagen alignment on the patterned PET surfaces.
  • Fig. 12 shows extracellular matrix on patterned PET surfaces before and after decellularization, fibronectin (FN) alignment and collagen type I (Col-I) alignment after decellularization, and a scanning electron micrograph (SEM) of the entire matrix.
  • the bar graph and graph show that angular alignment of collagen on the pattern is good, and that there is essentially no directionality of collagen on the unpatterned surface.
  • Fig. 13 shows neurites from plated PC12 cells on decellularized matrix on unpatterned (top) and patterned (bottom) surfaces. As shown, neurites aligned in the direction of collagen fibril orientation on patterned surfaces, and there was no alignment on the unpatterned control.
  • Fig. 14 shows fibroblast alignment after one day on nylon, PET and PEEK surfaces treated with Zr02 and Zr02/SAMP.
  • Fig. 15 shows fibroblast alignment after three days on nylon, PET and PEEK surfaces treated with Zr02 and Zr02/SAMP.
  • Fig. 16 represents the process by which the micropatterned polymer scaffolding base is generated, utilizing basic photolithographic techniques.
  • Figs. 17 A, 17B and 17C represent fibroblast alignment on the micropatterned polymer scaffolding base.
  • NIH3T3 cells cultured on unpatterned (a) or a 10 x 10 ⁇ striped pattern (b, c) were visualized by phase contrast microscopy after 4 hours (a, b) and 10 days (c).
  • Scale bars ⁇ . Double arrow indicates the orientation of the
  • Figs. 18 A, 18B, 18C, 18D, 18E and 18F represent oriented assembly of ECM comprised of fibronectin and collagen fibrils.
  • Col -I type I collagen
  • FN fibronectin. ** p ⁇ 0.0005.
  • Figs. 19 A, 19B, 19C, 19D, 19E and 19F represent the characterization of decellularized ECM. After decellularization matrices were stained with anti-fibronectin (a) or anti-collagen type I antibodies (b) and the FFT pixel intensity distribution plots for fibronectin (FN) and type I collagen (Col-I) in this matrix are shown in (c). (d, e) Images of type I collagen fibrils in decellularized matrix on unpatterned or 10 x 10 ⁇
  • Figs. 20 A, 20B, 20C, 20D, 20E and 20F represent directional neurite outgrowth on decellularized ECM.
  • PC12 cells were induced to extend neurites with NGF for 72 hr on unpatterned (a, c) or 10 x 10 ⁇ patterned (b, d) decellularized matrix PET substrates.
  • Cells were visualized by staining the actin cytoskeleton with rhodamine-phalloidin (a-d; red in c, d).
  • Fig. 21 represents directional radial glial cell alignment and growth.
  • GFP-tagged radial glial cells were cultured for 2 and 4 days on ECM and was then plotted as a histogram to observe angular alignment with the ECM.
  • Fig. 22 represents how PC12 cells and glial cells align with ECM PC12 cells were differentiated with neural growth factor (NGF) and cultured for 3 days on decellularized ECM and stained for F-actin to observe neurite alignment. Radial glial cells were initially cultured for 2 days on ECM and PC 12 cells were then seeded onto substrate and co- cultured for 3 days. FFTs were used to determine alignment of cells with FWFDVI highlighted for cells on unpatterned and patterned ECM.
  • NGF neural growth factor
  • Figs. 23 A and 23B demonstrate neurite outgrowth and directionality using sympathetic cervical ganglia.
  • Substrates included a glass coverslip coated with a 10 ⁇ g/ml solution of laminin (Laminin), decellularized fibroblast ECM on an unpatterned glass coverslip (Unpatterned ECM), and decellularized aligned ECM on a 20 ⁇ x 20 ⁇ patterned PET surface (Patterned ECM).
  • neurite outgrowth was fitted with an ellipse (lines on images) to determine if neurite extension deviated from circular.
  • the aspect ratios (long axis divided by the short axis) displayed in the graph were calculated and averaged for a minimum of 3 samples per condition.
  • the asterisk (*) indicates that the aspect ratio on patterned ECM is significantly different from the other substrates.
  • the present invention is based, at least in part, on the discovery of methods for generating cell-adhesive chemical (i.e., non -biologic) patterns in nano- and micro-scale dimensions using a technique for surface-modifying solids or polymers to prepare devices that have utility as bio-scaffold materials, electrodes, sensors or other devices.
  • cell-adhesive chemical i.e., non -biologic
  • the devices and methods of the present invention are simpler than previously described methods and devices in that it is not necessary to provide a biologic cell adhesion molecule, such as an RGD peptide, to mimic an in vivo environment to properly align and/or orient cells to form a functional tissue in vitro with appropriate cellular organization and biological activities.
  • a biologic cell adhesion molecule such as an RGD peptide
  • the lack of biologic cell -adhesive materials prevents encumbrance of cell receptors that may be involved in cell spreading and extracellular interactions affecting cellular organization and tissue alignment.
  • the patterns do not affect the physical properties of the substrate.
  • the devices and methods of making the devices of the present invention are also simpler than previously described methods and devices in that they can be performed in about one hour and the base layer used to generate the scaffolds does not need to comprise a reactive side-chain-containing species.
  • the patterns are more cell- adhesive than the substrates on which the patterns are developed. Cells attach to the patterns but are not constrained into the patterns by physical means. As the cells proliferate, they align into unmodified areas of the patterns to form confluent monolayers of cells while maintaining pattern alignment across the substrate surface.
  • described herein are methods and devices which may be used in a broad range of applications, for example, in regenerative medicine, wound repair, transplant biology, drug delivery, testing the effect of substances upon cells, tissue formation, cell actuation, and developmental biology.
  • the present invention provides patterned scaffolds for tissue.
  • the present invention provides a tissue scaffold comprising a base layer comprising a pattern of stripes, and an oxide layer comprising the pattern of stripes.
  • the tissue scaffold comprises a base layer comprising a pattern of stripes, an oxide layer comprising the pattern of stripes, and a non-biologic cell adhesive layer disposed on the oxide layer.
  • a base layer for use in the present invention may be a solid, rigid, or hard polymeric surface, a semi-rigid polymeric surface, a soft polymeric surface, a hard non- polymeric surface, a semi-rigid non-polymeric surface, or a soft non-polymeric surface.
  • a base layer is biologically inert.
  • a base layer comprises two or more surfaces.
  • a base layer comprising a soft polymeric or non-polymeric surface may be placed temporarily on a solid or semi-rigid polymeric or non-polymeric surface.
  • a base layer for use in the compositions and methods of the invention may have a Young's modulus of about 0.001-0.1, 0.005-0.2, 0.005-0.5, 0.05-1.0, 0.075-1.0, 0.1-2.0, 1.0-2.0, 1.5-5.0, 2.0-5.0, 3.0-7.0, 3.0-10, 5.0-15, 5.0-20, 10-20, 15-30, 20-30, 25-50, 30-50, 50-75, 50-100, 75-125, 100-150, 125-150, 150-200, 175-200, 200- 250, or about 250-300 gigapascals (GPa). Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention. For example, a Young's modulus of about 6.5-9.8, or 5.2-7.8 GPa is intended to be encompassed by the present invention.
  • a base layer for use in the compositions and methods of the invention may have a Young's modulus of about 0.001-0.1, 0.005-0.2, 0.005-0.5, 0.05-1.0, 0.075-1.0, 0.1-2.0, 1.0-2.0, 1.5-5.0, 2.0-5.0, 3.0-7.0, 3.0-10, 5.0-15, 5.0-20, 10-20, 15-30, 20-30, 25-50, 30-50, 50-75, 50-100, 75-125, 100-150, 125-150, 150-200, 175-200, 200- 250, or about 250-300 kilopascals (kPa). Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention. For example, a Young's modulus of about 6.5-9.8, or 5.2-7.8 kPa is intended to be encompassed by the present invention. For nerve regeneration, a lower Young's modulus may be used.
  • the base layer is selected from the group consisting of a hard polymeric surface, a semi-rigid polymeric surface, and a soft polymeric surface, a hard non-polymeric surface, a semi-rigid non-polymeric surface, and a soft non-polymeric surface.
  • the base layer comprises a polyamide, a polyamide hydrogel, a polyurethane, a polyurea, a polyester, a polyester hydrogel, a polyketone, a polyimide, a polysulfide, a polysulfoxide, a polysulfone, a polythiophene, a polypyridine, a
  • polypyrrole polypyrrole
  • polyethers silicone (polysiloxane), polysaccharides, fluoropolymers, epoxies, aramides, amides, imides, polypeptides, polyethylene, polystyrene, polypropylene, glass reinforced epoxies, liquid crystal polymers, thermoplastics, bismaleimide-triazine (BT) resins, benzocyclobutene ABFGxl3, low coefficient of thermal expansion (CTE) films of glass and epoxies, polyvinyls, polyacrylics, polyacrylates, polycarbonates,
  • polytetrafluoroethylene PTFE
  • PET polyethylene terephthalate
  • quartz silicon (e.g., silicon wafers)
  • glass ceramic, metals and metal alloys including titanium, titanium alloys, tantalum, zirconium, stainless steel and cobalt-chromium alloys, metal oxides, poly(vinyl pyrrolidone), poly(2-hydroxyethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, polyacrylamide hydrogels, polyrethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polylactides (PLA), polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA), polylactones, polylactone hydrogels, polyanhydrides, polyphosphazenes, polygermanes, polyorthoesters, polyolefin
  • polyacrylamide hydrophilic polyester, polyacrylamide hydrogel, polyester hydrogel or polylactone hydrogel.
  • the base layer is silk or collagen.
  • the base layer is titanium or stainless steel.
  • the base layer is a support structure.
  • the support structure is selected from the group consisting of a Petri dish, a cover-slip, a glass slide, a multi-well plate, a microfluidic chamber, an implant, and a medical device.
  • Medical devices for use as base layers in the present invention include, for example, diagnostic implant devices, biosensors, stimulators, neural stimulators, neural activity recorders, diabetic implants such as glucose monitoring devices, external fixation devices, external fixation implants, orthopedic trauma implants, implants for use in joint and spinal disorders/reconstruction such as plates, screws, rods, plugs, cages, scaffolds, artificial joints (e.g., hand, wrist, elbow, shoulder, spine, hip, knee, ankle), wires and the like, oncology related bone and soft tissue replacement devices, dental and oral/maxillo- facial devices, cardiovascular implants such as stents, catheters, valves, rings, implantable defibrillators, and the like, contact lenses, ocular implants, keratoprostheses, dermatologic implants, cosmetic implants, implantable medication delivery pumps; general surgery devices and implants such as but not limited to drainage catheters, shunts, tapes, meshes, ropes, cables, wires, sutures, skin staples, burn sheets, and vascular patches; and temporary or non-
  • the base layer comprises a pattern of stripes having at least two parallel stripes wherein adjacent stripes are separated by a space.
  • the width and spacing of the stripes is about 0.1 ⁇ to about 1000 ⁇ .
  • the width and spacing of the stripes may be varied over the range from about 0.1 ⁇ to about 1000 ⁇ , from about 1 ⁇ to about 500 ⁇ , from about 1 ⁇ to 250 ⁇ , from about 1 ⁇ to 160 ⁇ , from about 1 ⁇ to 100 ⁇ , from about 1 ⁇ to 90 ⁇ , from about 1 ⁇ to 80 ⁇ , from about 1 ⁇ to 70 ⁇ , from about 1 ⁇ to 60 ⁇ , from about 1 ⁇ to 50 ⁇ , from about 1 ⁇ to 40 ⁇ , from about 1 ⁇ to 30 ⁇ , from about 1 ⁇ to 20 ⁇ , from about 1 ⁇ to 10 ⁇ , from about 2 ⁇ to 100 ⁇ , from about 2 ⁇ to 90 ⁇ , from about 2 ⁇ to 80 ⁇ , from about 2 ⁇ to 70 ⁇ , from about 2 ⁇ to 60 ⁇ , from about 2 ⁇ to 50 ⁇ , from about 2 ⁇ to 40 ⁇ , from about 2 ⁇ to 30 ⁇ , from about 2 ⁇ to 20 ⁇ , from about 2 ⁇ m to 10
  • the width and spacing of the stripes can be equivalent or different.
  • both the width and spacing can be about 0.1, about 0.2, about 0.25, about 0.5, about 0.75, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 5, about 16, about 17, about 18, about 19, or about 20 ⁇ .
  • the width can be about 0.1, about 0.2, about 0.25, about 0.5, about 0.75, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 ⁇ , about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 ⁇ , and the spacing can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 ⁇ . Values intermediate to the above recited values are also con- templated to be part of the invention. For example, a width and spacing of about a width and spacing
  • the stripes are about 5 ⁇ to 100 ⁇ wide and spaced about 5 ⁇ to 100 ⁇ apart. In another preferred embodiment, the stripes are about 5 ⁇ wide and spaced about 5 ⁇ apart. In another preferred embodiment, the stripes are about 10 ⁇ wide and spaced about 10 ⁇ apart. In another preferred embodiment, the stripes are about 20 ⁇ wide and spaced about 20 ⁇ apart. In yet another preferred embodiment, the stripes are about 30 ⁇ wide and spaced about 30 ⁇ apart. In yet another preferred embodiment, the stripes are about 20 ⁇ wide and spaced about 10 ⁇ apart.
  • An oxide for use in the oxide layer of the scaffold on the present invention includes any compound(s) that when contacted with the patterned base layer forms a continuous layer on the patterned base layer.
  • the oxide is a metal oxide, e.g., formed from an alkoxide precursor.
  • the alkoxide is of a transition metal.
  • Periodic Table Group 3-6 and 13-14 metals are transition metals suitable for use in the present invention. Such metals include Zr, Al, Ti, HE, Ta, Nb, V and Sn. Depending upon the position of the transition metal on the Periodic Table, a transition metal alkoxide will have from three to six alkoxide groups or a mixture of oxo and alkoxide groups.
  • an alkoxide group has from 2 to 4 carbon atoms, and includes, for example, ethoxide, propoxide, zso-propoxide, butoxide, zso-butoxide, tert-butoxide and fluoronated alkoxide.
  • a metal alkoxide for use in the present invention is zirconium tetra(tert-butoxide).
  • a metal alkoxide for use in the present invention is tantalum pentaethoxide. Suitable oxides for use in the present invention also include those described in U.S. Patent Publication No. 2009/0104474 and PCT Publication No.
  • the oxide layer comprises a metal oxide.
  • the metal oxide is formed from the precursor zirconium tetra(tert-butoxide).
  • the cell adhesive layer comprises an adhesive chemical compound.
  • a cell adhesive chemical compound is any organic compound that is sufficiently reactive to react with the oxide layer, e.g., sufficiently reactive with a metal oxide or alkoxide, such as, for example, an organic compound comprising a phosphonic, carboxylic, sulfonic, phosphinic, phosphoric, sulfinic, or hydroxamic acid group.
  • the cell adhesive layer comprises a phosphonate.
  • the scaffolds further comprise living cells. It has been discovered in accordance with the present invention that cells can adhere to the patterned oxide layers in the absence of a cell adhesive layer disposed on the oxide layer.
  • the present invention provides an artificial tissue comprising living cells attached to a tissue scaffold comprising a base layer comprising a pattern of stripes, and an oxide layer comprising the pattern of stripes.
  • the present invention provides an artificial tissue comprising living cells attached to a tissue scaffold comprising: a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; and a non-biologic cell adhesive layer disposed on the oxide layer.
  • the type of cells is not limited, and includes, for example, fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts and chondrocytes, hepatocytes, macrophages, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, tendon cells, ligament cells, neural cells, epithelial cells, and stem cells.
  • Stem cells include embryonic stem cells, adult stem cells, and induced pluripotent stem cells.
  • the cells are mesenchymal stem cells.
  • the cells are human cells.
  • the present invention provides a tissue scaffold
  • the present invention provides a tissue scaffold comprising: a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; a non-biologic cell adhesive layer disposed on the oxide layer; and an extracellular matrix component aligned in parallel with the stripes.
  • Extracellular matrix components are known in the art and include for example, fibronectin and collagens.
  • the scaffolds comprising an extracellular matrix component may further comprise living cells attached to the matrix component.
  • the present invention provides an artificial tissue comprising living cells attached to a tissue scaffold comprising: a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; and an extracellular matrix component aligned in parallel with the stripes.
  • the present invention provides an artificial tissue comprising living cells attached to a tissue scaffold comprising: a base layer comprising a pattern of stripes; an oxide layer comprising the pattern of stripes; a non-biologic cell adhesive layer disposed on the oxide layer; and an extracellular matrix component aligned in parallel with the stripes.
  • the type of cells is not limited, and includes, for example, fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts and chondrocytes, hepatocytes, macrophages, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, tendon cells, ligament cells, neural cells, epithelial cells, and stem cells.
  • Stem cells include embryonic stem cells, adult stem cells, and induced pluripotent stem cells.
  • the cells are mesenchymal stem cells.
  • the cells are human cells. The cells need not be the same cells used to produce the extracellular matrix component on the substrate.
  • the cells may be obtained from a subject to be treated with the artificial tissue generated on the substrate.
  • the present invention provides a method for making a tissue scaffold comprising: generating a pattern of stripes on a base layer by
  • the method for making a tissue scaffold further comprises contacting the patterned oxide layer with a non-biologic cell adhesive compound to generate a patterned cell adhesive layer.
  • the method may comprise depositing a photoresist onto the base layer, thereby generating a photoresist layer, placing a mask on top of the photoresist layer and exposing the photoresist layer to ultraviolet radiation, thereby generating a patterned base layer.
  • depositing refers to a process of placing or applying an item or substance onto another item or substance (which may be identical to, similar to, or dissimilar to the first item or substance). Depositing may include, but is not limited to, methods of using spraying, dip casting, spin coating, evaporative methods, sputter methods, immersion methods, extractive deposition methods, or other methods to associate the items or substances.
  • depositing includes applying the item or substance to substantially the entire surface as well as applying the item or substance to a portion of the surface.
  • spin coating is used to apply a photoresist layer onto the base layer.
  • Spin coating is a process wherein the base layer is, for example, mounted to a chuck under vacuum and is rotated to spin the base layer about its axis of symmetry and a liquid or semi-liquid substance, e.g,. a photoresist, is dripped onto the base layer, with the centrifugal force generated by the spin causing the liquid or semi-liquid substance to spread substantially evenly across the surface of the base layer.
  • a liquid or semi-liquid substance e.g,. a photoresist
  • Photoresist is any substance that is sensitive to ultraviolet radiation, e.g., wavelengths of light in the ultraviolet or shorter spectrum ( ⁇ 400 nm).
  • a photoresist may be positive or negative.
  • Suitable photoresists include, without limitation, AZ-5412E, AZ- 701, AZ-1505, AZ-1518, and AZ-4330 (all are available from AZ Electronic Materials).
  • the photoresist is AZ-5412E.
  • a base layer comprising a photoresist may be patterned by providing a mask comprising the desired shape and/or pattern, i.e., a striped pattern.
  • the mask may be a solid mask such as a photolithographic mask. The mask is provided and placed on top of the photoresist layer. Subsequently, a portion of the photoresist layer (i.e., the portion of the photoresist not covered by the mask) is exposed to ultraviolet radiation.
  • the mask placed on top of the photoresist layer is typically fabricated by standard photolithographic procedure, e.g., by means of electron beam lithography.
  • Other methods for creating such masks include focused energy for ablation (micromachining) including lasers, electron beams and focused ion beams.
  • chemical etchants may be used to erode materials through the photoresist when using an alternative mask material.
  • Examples of chemical etchants include hydrofluoric acid and hydrochloric acid. Photolithographic masks are also commercially available.
  • Any suitable material e.g., a material that has a flat surface, e.g., a metal (gold, silver, platinum, tantalum, or aluminum), a ceramic (alumina, titanium oxide, silica, or silicon nitride), may be used for making the mask.
  • a metal gold, silver, platinum, tantalum, or aluminum
  • a ceramic alumina, titanium oxide, silica, or silicon nitride
  • a combination of positive and negative photoresists can be used.
  • a positive photoresist is deposited on a base layer in a particular pattern and subsequently a negative photoresist in a complementary pattern is applied. This results in a patterned tissue scaffold that comprises a pattern that comprises regions that are more cell adhesive next to regions that are less cell-adhesive.
  • the mask is removed and an oxide is deposited to the patterned base layer to form a patterned oxide layer.
  • the oxide binds directly onto the base layer and does not depend on the introduction of reactive side chain-containing species into the polymeric base layer.
  • One embodiment of the invention encompasses a method for making a tissue scaffold comprising the steps of generating a pattern on a base layer of protective photoresist compound stripes and unprotected base layer stripes using photolithography, to form a patterned base layer; and depositing an oxide layer onto the unprotected stripes to form a patterned oxide layer.
  • a thin oxide layer e.g., a metal oxide
  • a continuous layer is a layer that is formed by a matrix of individual molecules that are chemically bonded and linked to each other, as opposed to individual molecules covering the surface.
  • oxide molecules e.g., metal oxide molecules
  • a thin oxide layer is deposited onto the patterned base layer as a non-continuous layer, i.e., a pattern of individual molecules covering the surface.
  • An oxide for use in the oxide layer of the scaffold on the present invention includes any compound(s) that when contacted with the patterned base layer forms a continuous layer on the patterned base layer.
  • the oxide is a metal oxide, e.g., formed from an alkoxide precursor.
  • the alkoxide is of a transition metal.
  • Periodic Table Group 3-6 and 13-14 metals are transition metals suitable for use in the present invention. Such metals include Zr, Al, Ti, HE, Ta, Nb, V and Sn. Depending upon the position of the transition metal on the Periodic Table, a transition metal alkoxide will have from three to six alkoxide groups or a mixture of oxo and alkoxide groups.
  • an alkoxide group has from 2 to 4 carbon atoms, and includes, for example, ethoxide, propoxide, iso-propoxide, butoxide, iso-butoxide, tert-butoxide and fluoronated alkoxide.
  • a metal alkoxide for use in the present invention is zirconium tetra(tert-butoxide).
  • a metal alkoxide for use in the present invention is tantalum pentaethoxide.
  • Suitable oxides for use in the present invention also include those described in U.S. Patent Publication No. 2009/0104474 and WO 09/052352, the entire contents of each of which are incorporated herein by reference.
  • the oxide layer comprises a metal oxide.
  • the metal oxide is formed from the precursor zirconium tetra(tert- butoxide).
  • the oxide is deposited onto the patterned base layer under conditions suitable to form an oxide layer on the patterned base layer. This may be achieved using any suitable technique known to one of ordinary skill in the art and includes, for example, vapor or immersion deposition and sol-gel processes.
  • the step of forming a patterned oxide layer may include subjecting the oxide to pyrolysis, microwaving, complete hydrolysis or partial hydrolysis. In one embodiment, when heating conditions are employed, the oxide is heated to between about 50°C and about the melting point of the polymer, e.g., not at or above the melting point of the polymer. In another embodiment, when heating conditions are employed, the oxide is heated to between about 50°C and about the glass transition temperature of the polymer, e.g., not at or above the glass transition temperature of the polymer.
  • the oxide is deposited by vapor phase deposition of a metal alkoxide.
  • the metal alkoxide is zirconium tetra(tert-butoxi de) .
  • the thickness of the patterned chemical layer is controlled by the deposition and heating times. Shorter exposure times for deposition (about 5 minutes) and heat (about 10 minutes) generally produce about a 1 nm layer (about 2 monolayers).
  • the thickness of the layer can be determined by, for example, quartz crystal microgravimetry (QCM).
  • the patterned chemical layer is about 0.1 to about 100 nm, 0.1 to about 70 nm, about 0.1 to about 50 nm, about 0.1 to about 30 nm, 0.1 to about 20 nm, about 0.1 to about 10 nm, is about 0.1 to about 10 nm, 0.1 to about 7 nm, about 0.1 to about 5 nm, about 0.1 to about 3 nm, 0.1 to about 2 nm, about 0.1 to about 1 nm, about 0.5 to about 2 nm , about 1 to about 2 nm, about 1 to about 1.5 nm, about 1.5 to about 2 nm, or about 0.1 nm, 0.5 nm, 1 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, 2 nm, 2.1 nm, 2.2 nm,
  • the patterned chemical layer is 2 nm or less in thickness. In an embodiment, the patterned chemical layer is about 1 to about 1.5 nm in thickness. In an embodiment, the patterned chemical layer is about 10 to about 70 nm in thickness. In another embodiment, multiple layers of a semi-rigid or soft polymer are coated on the base layer so long as the polymer can still flex. It should be understood that a range between any two figures listed above is specifically contemplated to be encompassed within the metes and bounds of the present invention.
  • the patterned oxide layer can be about 1 to about 50, about 1 to about 45, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 2 to about 50, about 2 to about 45, about 2 to about 40, about 2 to about 35, about 2 to about 30, about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 5, about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 5 to about 25, about 5 to about 20, about 5 to about 15, about 5 to about 10, about 10 to about 50, about 10 to about 45, about 10 to about 40, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 10 to about 15, about 20 to about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, or about 45 to about 50 monolayers thick. Ranges and values intermediate to be,
  • the patterned oxide layer is about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2 monolayers thick. In one embodiment, the patterned oxide layer is more than about 2 monolayers thick. In another embodiment, the patterned oxide layer less than about 2 monolayers thick. In another embodiment, the patterned oxide layer about 1 monolayer thick.
  • a cell adhesive chemical compound is deposited onto the patterned oxide layer, to form a patterned cell adhesive layer.
  • the cell adhesive chemical compound is any organic compound that is sufficiently reactive with the oxide layer, e.g., sufficiently reactive with a metal oxide or alkoxide, such as, for example, an organic compound comprising a phosphonic, carboxylic, sulfonic, phosphinic, phosphoric, sulfinic, or hydroxamic group. Any suitable method may be used to deposit the organic layer onto the patterned oxide layer. In one embodiment, the cell adhesive chemical compound is deposited onto the patterned oxide layer by a dipping method.
  • the patterned oxide layer is contacted with a phosphonic acid to generate a patterned cell adhesive layer comprising a phosphonate.
  • the acid used to treat the patterned oxide layer comprises any alipha-tic or aromatic moiety and contains two acidic groups.
  • the acidic groups are carboxylic acid groups (-C02H).
  • the acidic groups are both phosphonic and car-boxylic acid groups.
  • the acid used to treat the patterned oxide layer compris-es an alkyl chain.
  • the alkyl chain is 4-18 carbons in length.
  • the alkyl chain is optionally substituted with any aromatic or aliphatic moiety, e.g. an alkyl, aryl, alkenyl or alkynyl group.
  • Non-limiting examples of phosphonic acids include ⁇ , ⁇ -bisphosphonic acids.
  • Non-limiting examples of carboxylic acids include ⁇ , ⁇ -biscarboxylic acids.
  • Nonlimiting examples of mixed phosphonic/carboxylic acids include a-carboxylic-co-phosphonic acids.
  • Non-limiting examples of mixed phosphonic/carboxylic acids include a-phosphonic-co- carboxylic acids.
  • the bisphosphonic acid is an octadecylphosphonic acid (ODPA) derivative.
  • the phosponic acid is 1,4-butane- diphosphonic acid.
  • a cell-avoidance layer is deposited on all or a portion of the patterned oxide layer. In one embodiment, a cell-avoidance layer may be deposited in a pattern that is complementary to a cell adhesive layer. A cell-avoidance layer is a layer that inhibits the adhesion of cells.
  • Exemplary compounds suitable for use as a cell-avoidance layer include, for example, compounds with terminal pegylated groups and those comprising an alkyl terminal group.
  • the scaffolds of the present invention further comprise living cells.
  • the present invention provides a method for making an artificial tissue comprising living cells attached to a tissue scaffold comprising contacting a tissue scaffold of the present invention with cells and culturing under conditions suitable for cell growth and/or differentiation.
  • the type of cells is not limited, and includes, for example, fibro-blasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts and chondrocytes, hepatocytes, macrophages, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, tendon cells, ligament cells, neural cells, epithelial cells, and stem cells.
  • Stem cells include embryonic stem cells, adult stem cells, and induced pluripotent stem cells. In one preferred embodiment, the cells are
  • mesenchymal stem cells In another preferred embodiment, the cells are human cells. Culture conditions for cell growth and/or differentiation are known to those of skill in the art.
  • one embodiment of the invention encompasses a method for making a tissue scaffold comprising the steps of (1) generating a pattern on a base layer of protective photoresist compound stripes and unprotected base layer stripes using photolithography, to form a patterned base layer; and (2) depositing an oxide layer onto the unprotected stripes to form a patterned oxide layer.
  • One embodiment further comprises contacting the patterned oxide layer of the substrate with cells; culturing the cells under conditions suitable for the production of extracellular matrix components; and removing the cells from the substrate to provide a tissue scaffold comprising an extracellular matrix component aligned on the stripes.
  • the scaffolds of the present invention further comprise an extracellular matrix component aligned in parallel with the pattern of stripes.
  • the present invention provides a method for making a tissue scaffold comprising generating a pattern of stripes on a base layer by photolithography to form a substrate having patterned base layer; depositing an oxide layer onto the patterned base layer to form a substrate having a patterned oxide layer; contacting the substrate having the patterned oxide layer with cells and culturing under conditions suitable for the production of extracellular matrix components; and removing the cells from the substrate to provide a tissue scaffold comprising an extracellular matrix component aligned in parallel with the stripes.
  • the present invention provides a method for making a tissue scaffold comprising generating a pattern of stripes on a base layer by photolithography to form a substrate having patterned base layer; depositing an oxide layer onto the patterned base layer to form a substrate having a patterned oxide layer; contacting the patterned oxide layer with a non-biologic cell adhesive compound to generate a substrate having a patterned cell adhesive layer; contacting the substrate having the patterned cell adhesive layer with cells and culturing under conditions suitable for the production of extracellular matrix components; and removing the cells from the substrate to provide a tissue scaffold comprising an extracellular matrix component aligned in parallel with the stripes.
  • one embodiment of the method further comprises contacting the patterned cell adhesive layer of the substrate with cells; culturing the cells under conditions suitable for the production of extracellular matrix components; and removing the cells from the substrate to provide a tissue scaffold comprising an extracellular matrix component aligned on the stripes.
  • the patterned substrates comprising cells may be made by the methods described hereinabove, and then cultured under conditions suitable for the production of extracellular matrix components.
  • Extracellular matrix components include, for example, fibronectin and collagens. Such conditions are known to those of skill in the art. These conditions include culturing cells at sufficient density in the presence of growth factors or serum. The skilled artisan can optimize conditions for different cell types. Additives may be used to stimulate the production of certain proteins. For example, type I collagen production may be stimulated by adding ascorbic acid to the culture medium.
  • the cells may be removed from the substrate, i.e., the substrate is decellularized.
  • Methods for decellul arizing are known in the art and include, for example, methods to loosen cell attachments from the extracellular matrix followed by lysis of cell membranes and solubilization of intracellular components under conditions that maintain the integrity and activity of the matrix.
  • decellularization may be accomplished by treatment to remove calcium by chelation to loosen cell attachments, followed by incubation with non-ionic detergent in a hypotonic buffer at alkaline pH to lyse cell membranes and solubilize intracellular components.
  • the scaffolds comprising an extracellular matrix component may further comprise living cells attached to the matrix component.
  • the present invention provides a method for making an artificial tissue comprising living cells attached to a tissue scaffold comprising the step of contacting a tissue scaffold of the present invention with cells and culturing under conditions suitable for cell growth and/or differentiation.
  • the type of cells is not limited, and includes, for example, fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts and chondrocytes, hepatocytes, macrophages, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, tendon cells, ligament cells, neural cells, epithelial cells, and stem cells.
  • Stem cells include embryonic stem cells, adult stem cells, and induced pluripotent stem cells.
  • the cells are mesenchymal stem cells.
  • the cells are human cells.
  • the cells need not be the same cells used to produce the extracellular matrix component on the substrate.
  • the cells may be obtained from a subject to be treated with the artificial tissue generated on the substrate. Culture conditions for cell growth and/or differentiation are known to those of skill in the art.
  • cells may be attached by placing the scaffold in culture with a cell suspension and allowing the cells to settle and adhere to the surface.
  • Cells respond to the patterning in terms of adherence and in terms of assembling ECM proteins in the pattern on the scaffold.
  • Cells also respond to the patterning in terms of maturation, growth and function.
  • the cells on the scaffold may be cultured in an incubator under physiologic conditions (e.g., at 37°C) until the cells form a two-dimensional (2D) tissue, the orientation of which is determined by the pattern provided on the tissue scaffold.
  • any appropriate cell culture method may be used to establish the tissue on the tissue scaffold.
  • the seeding density of the cells will vary depending on the cell size and cell type but can easily be determined by methods known in the art.
  • cells are seeded at a density of between about 1 x 10 3 to about 6 x 10 5 cells/cm 2 , or at a density of about 1 X 10 3 , about 2 X 10 3 , about 3 X 10 3 , about 4 X 10 3 , about 5 X 10 3 , about 6 X 10 3 , about 7 X 10 3 , about 8 X 10 3 , about 9 X 10 3 , about 1 X 10 4 , about 2 X 10 4 , about 3 X 10 4 , about 4 X 10 4 , about 5 X 10 4 , about 6 X 10 4 , about 7 X 10 4 , about 8 X 10 4 , about 9 X 10 4 , about 1 X 10 5 , about 1.5 X 10 5 , about 2 X 10 5
  • the patterned tissue scaffold is contacted with a plurality of cells and cultured such that a living tissue, e.g., a tissue having at least in part, in vivo biological activity, is produced.
  • a living tissue is removed from the tissue scaffold.
  • the patterned tissue scaffold comprises a plurality of cells on the stripes, and can be organized into a tissue structure.
  • the cells can comprise human cells.
  • the cells can consist essentially of a member selected from the group consisting of fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, tendon cells, ligament cells, neural cells, epithelial cells, and stem cells.
  • the patterned tissue scaffolds of the invention may be used in a broad range of applications, including, but not limited to, devices for use in tissue repair and support such as sutures, surgical and orthopedic screws, and surgical and orthopedic plates, natural coatings or components for synthetic implants, cosmetic implants and supports, repair or structural support for organs or tissues, substance delivery, bioengineering platforms, platforms for testing the effect of substances upon cells, cell culture, wound healing, neural regeneration and numerous other uses.
  • the living tissue is removed from the scaffold prior to use. In another embodiment, the living tissue is not removed from the scaffold prior to use.
  • the base layer of the patterned tissue scaffolds of the present invention may be tissue prepared on the tissue scaffolds of the invention or a medical device, such as an orthopedic screw or plate that comprises cells of the same tissue in which the devices will be used.
  • medical devices suitable for use in the present invention include, diagnostic implant devices, biosensors, stimulators, diabetic implants such as glucose monitoring devices, external fixation devices, external fixation implants, orthopedic trauma implants, implants for use in joint and spinal disorders/reconstruction such as plates, screws, rods, plugs, cages, scaffolds, artificial joints (e.g., hand, wrist, elbow, shoulder, spine, hip, knee, ankle), wires and the like, oncology related bone and soft tissue replacement devices, dental and oral/maxillofacial devices, cardiovascular implants such as stents, catheters, valves, rings, implantable defibrillators, and the like, contact lenses, ocular implants, keratoprostheses, dermatologic implants, cosmetic implants, implantable medication delivery pumps; general surgery devices and implants such as but not limited to
  • flexible membranes comprising a patterned tissue scaffold may be used to support or connect tissue or structures that have experienced injury, surgery, or
  • a patterned tissue scaffold comprising a soft polymer may be used as a graft to connect and/or bind tissue and provide a platform for tissue regeneration, internally or externally.
  • the soft polymer may be biodegradable or nonbiodegradable.
  • patterned soft tissue scaffolds of the invention is as a barrier for the prevention of post-operative induced adhesion(s).
  • the patterned tissue scaffolds of the invention may be used to organize ECM deposition and prevent the formation of adhesions.
  • the patterned tissue scaffolds of the invention is as templates for nerve growth.
  • the patterned tissue scaffolds may be used to culture neural cells in a pattern mimicking the in vivo environment such that suitable neural connections form rather than the unorganized array of neural cells that are produced without use of a patterned scaffold.
  • the patterned scaffolds are prepared according to Fig. 16.
  • the components of the nervous system are categorized as belonging to either the peripheral nervous system (PNS) or the central nervous system (CNS).
  • PNS peripheral nervous system
  • CNS central nervous system
  • the basic anatomy of the nervous system at a cellular level is defined by the presence of neurons, specialized cells which are electrically excitable and serve to transmit information through electrical and chemical signaling. Signaling between neurons occurs via synapses, which are membrane-to-membrane junctions between neuron cells, or between neurons and other cell tissues, for example between efferent nerve fibers and muscle fibers at the
  • Glial cells are non-neuronal cells which provide support for neurons in a variety of manners, including maintaining homeostasis, providing nutrients and oxygen, providing structural integrity to the neural network, providing and maintaining a myelin sheath, and to combat pathogens and remove dead neurons. It is also possible that glial cells may assist neurons to form synaptic connections with other neurons.
  • the CNS is the part of the nervous system that is comprised of the brain and the spinal cord.
  • the PNS is the part of the nervous system that is comprised of nerves that are outside the CNS, in that the PNS excludes the brain and the spinal cord.
  • the PNS is primarily comprised of ganglia, which are clusters of neurons found in the PNS. Ganglia in the PNS are comprised mainly of somata and dendritic structures which are interconnected to other ganglia to form a system known as a plexus. Ganglia primarily function to provide intermediate connections between different neurological structures in the body, including different structures in the PNS, and between the PNS and the CNS. There are three major groups of ganglia found among vertebrate animals, which include the dorsal root ganglia, the cranial nerve ganglia, and the autonomic ganglia.
  • Neurons are comprised of a number of different structure features. At a basic level, neurons are comprised of dendrites, a soma (cell body) containing the nucleus and other essential cellular machinery, an axon, a long thin nerve fiber that conducts electrical impulses away from the dendrites to the axon terminal, which is oriented to another neuron's dendrites or to a target cellular tissue.
  • a major characteristic of neurons is the presence or absence of a myelin sheath along the axon of the neuron. Neurons may be either myelinated or unmyelinated, depending on the location of the neuron in the nervous system. Myelin is an electrically insulating, i.e.
  • dielectric material primarily comprised of lipids, proteins, cholesterol and water, the precise compositions of each varying by its location in the nervous system.
  • the myelin sheath serves to increase the speed at which electrical impulses are conducted along the axon.
  • Schwann cells glial cells
  • oligodendrocytes provide the sheath.
  • a defining characteristic of neurons is that once neural progenitor cells have fully differentiated into neurons, the neurons do not undergo any further cellular division.
  • this invention relates to scaffolds for nerve tissue regeneration.
  • this invention relates to methods for regenerating nerve tissue.
  • the present invention provides methods of tissue repair and regeneration comprising implanting the artificial tissues of the present invention in a subject in need of such tissue repair or regeneration.
  • Another embodiment is directed to a method of tissue repair or regeneration comprising implanting a tissue scaffold of invention into tissue in need of repair or regeneration in a subject in need thereof.
  • neural cells seeded onto the patterned polymer scaffold will grow in alignment with the pattern on the polymer.
  • neural cells may be seeded onto the scaffolding.
  • the neural cells will grow in alignment with the fibrillar matrix. Quantification of neurite expansion has shown enhanced alignment of neural cells when cultured on patterned/aligned ECM compared to an unpatterned matrix.
  • the scaffolding can be rendered bioactive to more precisely control and regulate nerve tissue growth, differentiation, and regeneration.
  • the neural cells which are seeded on the scaffolding can be induced to connect or reconnect with other neural cells, or potentially other cellular targets, for example, but not limited to, muscle tissue at the MJ. This has wide potential therapeutic application as a treatment for a number of neurodegenerative diseases as well as aging.
  • stem cells including but not limited to, omnipotent, pluripotent, and induced pluripotent stem cells, for example, embryonic stem cells and adult stem cells including, but not limited to, neural stem cells (NSCs), as well as oligopotent stem cells including, but not limited to, neural progenitor cells and
  • NSCs neural stem cells
  • oligopotent stem cells including, but not limited to, neural progenitor cells and
  • Co-culture with glial support cells is another possibility, including, but not limited to CNS macroglial cells, for example, astrocytes and astrocyte precursor cells, oligodendrocytes and oligodendrocyte precursor cells, and PNS macroglial cells, for example Schwann cells and Schwann precursor cells.
  • CNS macroglial cells for example, astrocytes and astrocyte precursor cells, oligodendrocytes and oligodendrocyte precursor cells, and PNS macroglial cells, for example Schwann cells and Schwann precursor cells.
  • Further possible cell types include already differentiated neurons and glial cells, neural crest cells, such as those that give rise to the PNS, including, but not limited to, the dorsal root ganglia, and neural tube cells, such as those that give rise to the CNS, including, but not limited to, the basal ganglia.
  • PC12/PC12 Adh cell line derived from pheochromocytoma of the rate adrenal medulla.
  • Other appropriate cell lines include, but are not limited to, those derived from neuroblasts, including, but not limited to, Neuro-2a, NlE-115, B41 A3, B104-1-1, which are cell lines derived from mice neuroblasts, and SK-N-AS, FMR-32, SK-N-DZ, SK-N-FI, BE(2)-M17, BE(2)-C, which are cell lines derived from human neuroblasts.
  • cell lines include, but are not limited to, those derived from Schwann cell lines, including immortalized Schwann cell lines, for example, but not limited to, SW10 (mouse), R3 and RSC96 (rat) neuronal Schwann cells.
  • immortalized Schwann cell lines for example, but not limited to, SW10 (mouse), R3 and RSC96 (rat) neuronal Schwann cells.
  • patterned tissue scaffolds contacted or seeded with living cells are combined with a drug such that the function of an implant or graft will improve.
  • a drug such that the function of an implant or graft will improve.
  • antibiotics, anti-inflammatories, local anesthetics or combinations thereof can be added to the cell-treated a patterned tissue scaffold to speed the healing process.
  • Other compounds, molecules, elements, or ions such as growth factors, trophic factors, cytokines, steroids, metabolites, and other signaling molecules and hormones such as those involved in growth and differentiation, for example, but not limited to, signaling molecules involved in neural development and differentiation, such as those signaling molecules involved in the Notch signaling pathway, may be added to the scaffolding in order to promote, direct, alter, or repress axon alignment, outgrowth, and neural regeneration and cellular growth.
  • the tissues of the present invention can be used to study functional differentiation of stem cells (e.g., pluripotent stem cells, multipotent stem cells, induced pluripotent stem cells, and progenitor cells of embryonic, fetal, neonatal, juvenile and adult origin).
  • stem cells e.g., pluripotent stem cells, multipotent stem cells, induced pluripotent stem cells, and progenitor cells of embryonic, fetal, neonatal, juvenile and adult origin.
  • the patterned tissue scaffolds of the invention are contacted with undifferentiated cells, e.g., stem cells, and differentiation is observed. Patterned tissue scaffolds containing cells are also useful for measuring tissue activities or functions, investigating tissue developmental biology and disease pathology, as well as in drug discovery.
  • the present invention also provides methods for identifying a compound that modulates a tissue function.
  • the methods include providing a tissue scaffold comprising a tissue produced according to the methods of the invention, contacting the tissue with a test compound; and measuring the effect of the test compound on a tissue function in the presence and absence of the test compound, wherein a modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates a tissue function, thereby identifying a compound that modulates a tissue function.
  • the present invention also provides methods for identifying a compound useful for treating or preventing a disease.
  • the methods include providing a tissue scaffold comprising a tissue produced according to the methods of the invention, contacting a tissue with a test compound; and measuring the effect of the test compound on a tissue function in the presence and absence of the test compound, wherein a modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates a tissue function, thereby identifying a compound useful for treating or preventing a disease.
  • tissue scaffold comprising a base layer and an oxide layer, where the base layer and the oxide layer comprise a pattern of oxide layer stripes on the base layer, and the tissue scaffold further comprises a plurality of neural cells on the stripes, where the neural cells are selected from the group consisting of neural stem cells, oligopotent stem cells, differentiated neurons, differentiated glial cells and neural crest cells.
  • the tissue scaffold further comprises a non- biologic cell adhesive layer disposed on the oxide layer.
  • Another embodiment further comprises an extracellular matrix component aligned on the stripes.
  • the base layer is selected from the group consisting of a semi-rigid polymeric surface and a soft polymeric surface.
  • the base layer is selected from the group consisting of polyesters, polyamides, polylactones,
  • the base layer is selected from the group consisting of silk and collagen.
  • the base layer is selected from the group consisting of nylon, polyethylene terephthalate,
  • polycaprolactone fumarate polyethylene glycol fumarate, and methylene bis acrylamide.
  • the stripes are about 5 ⁇ to 100 ⁇ wide and spaced about 5 ⁇ to 100 ⁇ apart. In some embodiments the stripes are about 5 ⁇ wide and spaced about 5 ⁇ apart, or about 10 ⁇ wide and spaced about 10 ⁇ apart, or about 20 ⁇ wide and spaced about 20 ⁇ apart, or about 30 ⁇ wide and spaced about 30 ⁇ apart, or about 20 ⁇ wide and spaced about 10 ⁇ apart.
  • the oxide is a metal oxide.
  • the metal oxide is zirconium oxide.
  • the cell adhesive layer comprises a phosphonate.
  • the oligopotent stem cells are selected from the group consisting of neural progenitor cells and neuroblasts.
  • the neural crest cells are selected from the group consisting of dorsal root ganglia cells, neural tube cells, and basal ganglia cells.
  • the tissue scaffold is co-cultured with glial support cells.
  • the glial support cells are selected from the group consisting of CNS macroglial cells and PNS macroglial cells.
  • the CNS macroglial cells are selected from the group consisting of astrocytes, astrocyte precursor cells, oligodendrocytes and oligodendrocyte precursor cells.
  • the PNS macroglial cells are selected from the group consisting of Schwann cells and Schwann precursor cells.
  • modulate are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
  • contacting e.g., contacting a tissue with a test compound
  • any form of interaction e.g., direct or indirect interaction
  • the term “contacting” includes incubating a compound and a tissue (e.g., adding the test compound to a tissue).
  • Test compounds may be any agents including chemical agents (such as toxins), small molecules, pharmaceuticals, peptides, proteins (such as antibodies, cytokines, enzymes, and the like), and nucleic acids, including gene medicines and introduced genes, which may encode therapeutic agents, such as proteins, antisense agents (i.e., nucleic acids comprising a sequence complementary to a target RNA expressed in a target cell type, such as RNAi or siRNA), ribozymes, and the like.
  • chemical agents such as toxins
  • small molecules such as pharmaceuticals, peptides, proteins (such as antibodies, cytokines, enzymes, and the like)
  • nucleic acids including gene medicines and introduced genes, which may encode therapeutic agents, such as proteins, antisense agents (i.e., nucleic acids comprising a sequence complementary to a target RNA expressed in a target cell type, such as RNAi or siRNA), ribozymes, and the like.
  • antisense agents i.e., nucleic acids comprising
  • test compound may be added to a tissue by any suitable means.
  • the test compound may be added drop-wise onto the surface of a tissue of the invention and allowed to diffuse into or otherwise enter the tissue, or it can be added to the nutrient medium and allowed to diffuse through the medium.
  • thermally grown oxide layer Silicon Quest, Inc.
  • polyetheretherketone polyetheretherketone
  • nylon 6,6, and polyethylene terephthalate films of 0.05 mm thickness (Goodfellow, Corp.), glass cover slips (12 mm, No. 2; VWR) were obtained from commercial sources.
  • the dimensions of the photolithographic patterns used were (width of stripe X space between stripes; all nominal dimensions are in ⁇ ): 10 X 10, 20 X 20, 10 X 20, 20 X 10, 20 X 30, 30 X 10, 30 X 30, 40 X 30, 50 X 30, 60 X 30, and 100 X 40. Some minor shadowing was observed with stripes ⁇ 20 ⁇ .
  • Hexanes, toluene, methanol, 2-propanol, hexamethyldisilazane (HMDS), formaldehyde, 4',6-diamidino-2-phenylindole (DAP I), and anti-vinculin antibodies Sigma-Aldrich
  • zirconium tetra(tert-butoxide) Strem Chemicals, Inc.
  • DMEM Dulbecco's modified eagle medium
  • hMSCs bone marrow derived human mesenchymal stem cells
  • PT-3001 serum containing "bullet” medium
  • trypsin/EDTA CC-3232
  • NP-40 nonyl phenoxypolyethoxylethanol
  • EMD Chemicals sulfuric and hydrochloric acids
  • 30 % hydrogen peroxide J.T. Baker
  • rhodamine phalloidin Molecular Probes
  • 1,4-butanediphosphonic acid (Acros Organics); AZ-5214E photoresist and AZ-312 MIF developer (Capitol Scientific, Inc.); absolute ethyl alcohol (Pharmco-Aaper) were used as received.
  • Photomasks were fabricated using a Heidelburg DWL 66 laser writer equipped with a 20 mm focal length writehead. NTH 3T3 mouse fibroblasts and bone marrow derived hMSCs were passaged bi-weekly and were stored at 37 °C until use.
  • Silicon wafers were cut into 1 cm x 1 cm coupons;
  • HMDS 2-propanol, then dried under nitrogen and finally heated (95 °C) for 10 min, with the exception of nylon and PET, which were not heated to avoid glass transition.
  • HMDS was spin cast onto the substrate surfaces (4000 rpm, 40 sec) followed by AZ-5214E photoresist (4000 rpm, 40 sec). Substrates were baked for 45 sec (95 °C), exposed to UV (365 nm, 4 W) through a photomask for 30 sec, and then developed in AZ-312 MIF for 30-34 sec. The substrates were rinsed vigorously in deionized water and examined by optical microscopy. All patterns were fabricated and analyzed at minimum in duplicate.
  • SAMP self-assembled monolayer of phosphonate
  • the chamber was then cooled to room temperature.
  • the chamber was back-filled with zero-grade nitrogen, and valves were closed to isolate the chamber prior to dismounting.
  • the chamber was opened, and the substrates were soaked in an ethanol solution of 1,4-butanediphosphonic acid (0.25 mg/mL) for 24 hr.
  • the preparation of the nanoscale-patterned surface is depicted schematically in Figs. 1 A and IB.
  • the substrates were then rinsed sequentially with ethanol and 2-propanol, dried under nitrogen, and then inspected by optical microscopy. This procedure removes the HMDS, the photoresist, and any Zr0 2 on the photoresist to leave a negative pattern in which the SAMP/Zr0 2 is directly attached to the substrate.
  • the ability to make multiple, patterned substrates is limited only by the size of the photolithographic mask, the UV source, and the deposition chamber.
  • SAMP/Zr0 2 was analyzed by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and energy dispersion spectroscopy (EDS).
  • XPS X-ray photoelectron spectroscopy
  • SEM scanning electron microscopy
  • EDS energy dispersion spectroscopy
  • a VG scientific ESCALab Mk II equipped with a Mg Ka (1253.6 keV) anode source operating at 15 keV accelerating voltage and 20 mA and a VG scientific hemispherical sector analyzer (HAS) detector were used.
  • a pass energy of 50 eV was used to collect survey (1000 to 0 eV) XPS data.
  • Detailed XPS data were collected at a pass energy of 20 eV with a dwell time of 500 and a step size of 0.05 eV.
  • Data analysis was carried out using CasaXPS software (Casa Software Ltd.). Spectra were calibrated against adventitious
  • Pattern stability studies Silicon and glass substrates were photolithographically patterned with 30 X 30 stripes of Zr0 2 as described above. The substrates were immersed in serum-containing medium (DMEM with 10% calf serum) for 18 days. Glass and Si substrates were removed on days 3, 6, 9, 12, and 18 and were rinsed gently with PBS. Optical microscopy determined if the Zr0 2 stripes remained intact, XPS analysis was used to identify elemental presence on the surface, and AFM investigated the surface morphology.
  • serum-containing medium DMEM with 10% calf serum
  • NTH 3T3 fibroblasts SAMP -Patterned and control (unpatterned SAMP) samples were placed in individual wells of 24-well plates and were rinsed twice with phosphate buffered saline (PBS).
  • NIH 3T3 fibroblasts were plated at 30,000 cells per well on the substrates in serum-free DMEM and were allowed to attach at 37 °C for 3 hr. The medium was changed to DMEM with 10% calf serum and the attached cells were allowed to spread for an additional 21 hr (24 hr total).
  • Cells were fixed using 3.7 % formaldehyde in PBS for 15 min, permeabilized with 0.5 % NP-40 in PBS for 15 min at room temperature, and stained with rhodamine- phalloidin and DAPI for cell shape and orientation studies. Images were captured using a Nikon TE2000U microscope.
  • Cell toxicity was studied using 3T3 cells plated on silicon surfaces completely coated with a SAMP of 1,4-butanediphosphonic acid bonded onto Zr0 2 as described above. Cells were plated as described above in duplicate, and were allowed to proliferate for 3 days; after 24 hrs and 3 days they were stained and analyzed as above. Cells were counted and cell-spreading areas were measured using ImageJ, 10 image fields were used for the measurements and field dimen-sions were 853 ⁇ x 683 ⁇ . Cell doubling time was calculated assuming constant growth rate.
  • 3T3 Cells were plated at 30,000 cells/well in serum-containing DMEM (10% calf serum). Bone marrow-derived MSCs were trypsinized with trypsin/EDTA and plated at 30,000 cells/ well in serum containing "bullet" medium. The cells were incubated at 37 °C as the cells grew to confluence. Time points on silicon were taken at 24 hr, 3 days, and 8 days at which time the cells were stained for imaging as described above. Polymer time points were 24 hr and 3 days. Cells were plated on all surfaces in duplicate.
  • Origin 8.5 software was used to generate box plots.
  • FDVIDS was followed by spin-casting AZ-5412E photoresist; the photoresist-coated material was exposed to UV light through a photomask of a negative of the desired striped pattern; the surface was developed in AZ-312 MIF to remove exposed photoresist; the substrate was exposed to vapor of zirconium tetra(tert-butoxide) (1) to yield the Zr0 2 layer when deposition was followed by mild thermolysis; the SAMP was then formed through immersion in an ethanol solution of 1,4-diphosphonobutane (this step also removed remaining photoresist exposing the underlying substrate).
  • SAMP/Zr0 2 /substrate (4) is outlined in Fig. IB. Eleven striped patterns defined as the width of the SAMP/Zr0 2 stripe (in ⁇ ) X spacing between stripes (in ⁇ ), e.g., 20 X 30 were used.
  • Spectroscopic analysis was used to determine elemental composition and distribution of photolithographic patterns of SAMP/Zr0 2 on oxide-terminated Si (Si0 2 /Si), PEEK, PET, and nylon 6,6.
  • AFM analysis of striped patterns of SAMP/Zr0 2 /Si0 2 /Si showed heights to be 10 - 70 nm; these nanoscale thick patterns should not affect physical properties of the substrate.
  • AFM images of the SAMP/Zr0 2 pattern on PET showed an average height of 70 nm. Variations in height measured on any substrate surface are likely due to small changes in vapor-phase deposition conditions for 1.
  • the height of the patterned Zr0 2 stripe on Si (relative to that of underivatized regions) remained nearly constant following an initial increase in height within 9 days of immersion.
  • the initial pattern height of Zr0 2 /Si0 2 /Si was 12 nm; after immersion in culture medium this height increased to 20-25 nm and remained at this level for the duration of the study from day 3 to day 18.
  • the Zr0 2 /glass pattern height (before adding protein) was also 12 nm; after immersion it increased from 26 nm (day 3) to 50 nm (day 18).
  • EDS analysis of the Zr0 2 /Si0 2 /Si pattern at day 3 showed, in addition to the striped pattern for Zr (as in Fig. 2 above), that nitrogen-containing material covered the entire surface.
  • NIH3T3 fibroblasts were plated on SAMP/Zr0 2 -patterned or unpatterned silicon surfaces and allowed to attach and spread for 3 hrs. Medium was replaced with
  • FWFDVI full-width at half- maximum values
  • cell aspect ratio the ratio of cell width to length; an aspect ratio of 1.0 means the cell is perfectly round
  • orientation of the cell long axis with regard to the pattern Fig. 2A-2C
  • the "box -plots" represent the distribution of cell aspect ratio measurements (Fig. 2B).
  • cells are more elongated on the narrower 20 X 10 and 30 X 30 patterns compared to unpatterned or 100 X 40 patterns.
  • Cell alignment with the pattern direction was determined by measuring the angles of cellular long axes relative to the patterns. Analysis of variance found a statistical difference among the individual groups (p ⁇ 0.001, a ⁇ 0.05), and pair-wise comparison tests found statistical differences between each of the patterned surfaces compared to the control (p ⁇ 0.0001, a ⁇ 0.0045) (Fig. 2C).
  • all of these SAMP/Zr0 2 stripe dimensions can influence the direction of the cellular long axis in such a way that attached cells are oriented in the pattern direction compared to cells on an unpatterned surface.
  • the 100 X 40 pattern can cause alignment of cells in the direction of the pattern but does not cause the cells to become more elongated than does an unpatterned surface.
  • the 20 X 20 and 30 X 30 dimensions had the narrowest distributions for cell long axis orientation and pattern correspondence.
  • SAMP/Zr0 2 -treated substrates had 34 ⁇ 6 cells. Cell numbers increased on these surfaces over the next 3 days, to 134 ⁇ 32 cells; this corresponds to doubling times of 1.0 day. This is typical of cell growth on standard culture materials. Cell-spreading areas followed the same trend: after 1 day (3017 ⁇ 1830 ⁇ 2 ), and at the end of the 3 -day study (2912 ⁇ 1643 ⁇ 2 ) for SAMP/Zr0 2 -treated substrates. These data show that SAMP/Zr0 2 -modified surfaces are not cytotoxic.
  • 3T3 fibroblasts were maintained over 8 days; the cells grew to confluence and covered the entire 1 cm x 1 cm silicon coupon.
  • 3T3 cells were plated on 10 X 10 and 30 X 30 stripes and on unpatterned control surfaces; time points were taken after 24 hr, 3 days, and 8 days with immunostaining of actin (Fig. 5A-5I).
  • Fig. 5A-5I immunostaining of actin
  • 3T3 cells were aligned on the 30 X 30 stripes, and were oriented in the direction of the 10 X 10 pattern in which a single stripe is too narrow to entirely contain a cell (Fig. 5A, D).
  • MSCs and fibroblasts displayed a similar response to a 30 X 30 patterned substrate.
  • Photolithographic patterning was extended to polymers that are representative of three classes of biomaterials, polyesters (PET), polyamides (nylon), and
  • PEEK polyetheretherketone
  • the polyethylene terephthalate (PET) sheet with thickness of 0.05 mm was used as received (Goodfellow Corp., Oakdale, PA).
  • PET polyethylene terephthalate
  • the sheet was cut into -0.5 cm x 0.5 cm square pieces with a notch in the upper right corner to identify the top side of the PET film.
  • the cut sheets were cleaned by sonication in isopropanol for 15 minutes, dried in a stream of nitrogen gas and warmed for 10 minutes before spin-coating at 3000 rpm with diazonaphthoquinone sulfonic ester positive photoresist (AZ5214-E).
  • the resist was cured for 45 seconds and exposed to UV light through a photolithographic mask.
  • tetram ethyl -ammonium hydroxide solution (AZ312 MIF) to dissolve away the photoresist from the UV-exposed regions to provide 10 X 10, 30 X 30 and 60 X 30 patterns
  • PET films micro-patterned with photoresist were placed in a glass chamber with inlets for vacuum and vapors of zirconium tetra(tert-butoxide), Zr(OBu l )4 (Sigma- Aldrich).
  • the vacuum inlet was opened to evacuate the chamber to 10 "3 torr. Both the inlets were opened for 5 minutes for vapor deposition under vacuum flow followed by closing the vacuum inlet for 5 minutes to allow deposition without external evacuation.
  • the samples were heated to 47 deg C using a heating tape to allow formation of surface- bound metal oxide Zr0 2 layer. The heating tape was removed and the chamber was cooled to room temperature.
  • the PET films were then immersed promptly in 1 mM solution of 1, 4-butane diphosphonic acid (Acros Organic) solution in ethanol for 17 hours.
  • the patterned films were sonicated for 3 minutes, rinsed with isopropanol and dried in a stream of nitrogen gas.
  • NIH3T3 (ATCC) cells were cultured in DMEM supplemented with 10% bovine calf serum. The cells were maintained at 37° C in a humidified incubator containing 5% carbon dioxide. NIH3T3s were trypsinized every 3-4 days and were re-plated for the next passage.
  • the PET sheet was disinfected using 70% ethanol for 20 minutes, washed three times with sterile phosphate buffered saline (PBS) and immersed in 1 ml DMEM in a 24-well plate.
  • NIH3T3s were lifted from the culture plate using trypsin and suspended in media supplemented with 10% serum to inactivate the trypsin.
  • the cell suspension was centrifuged and the cell pellet was re-suspended in media without added serum.
  • the cells were counted using a hemocytometer and the PET sheet was seeded with 50,000 NIH3T3s. The cells were allowed to adhere to PET substrate in absence of serum proteins for 4 hours at 37° C.
  • NIH3T3s were cultured in media with 10% serum and the media was refreshed on day 3, 6 and 8. The media was supplemented with 50 ⁇ g/ml ascorbic acid on day 6 and 8 to augment collagen type I synthesis.
  • the phase contrast images of NIH3T3 cells growing on PET were taken using a Nikon Eclipse TSlOO microscope and a Cooke SensiCam QE High Performance camera. Cell alignment on the patterned PET is depicted in Fig. 8. Fibronectin immunostaining and fast Fourier transformation analysis confirmed that cells and fibronectin aligned with the pattern. Fibronectin alignment and quantification of alignment of cells are shown in Fig. 9. As shown in Fig.
  • fibronectin alignment correlated with the pattern dimension, with decreasing conformity as the patterned stripes become wider.
  • collagen type I immunostaining and fast Fourier transformation analysis demonstrated that collagen also aligned with the patterns, demonstrating that collagen alignment can be controlled by a chemical pattern on the substrate.
  • the PET substrate with adhered cells was decellularized by treatment to remove calcium by chelation to loosen cell attachments followed by incubation with non-ionic detergent in a hypotonic buffer at alkaline pH to lyse cell membranes and solubilize intracellular components (Mao and Schwarzbauer (2005) Matrix Bio. 6:389-99).
  • the treatment leaves extracellular matrix aligned with the pattern.
  • Fig. 12 shows extracellular matrix on patterned PET surfaces before and after decellularization, fibronectin (FN) alignment and collagen type I (Col-I) alignment after decellularization, and a scanning electron micrograph (SEM) of the entire matrix. As shown in Fig. 12, the alignment of the matrix fibrils is maintained after decellularization.
  • the histogram and graph show that angular alignment of collagen on the pattern is good (a relatively narrow angular distribution), but that there is essentially no directionality of collagen on the unpatterned surface (the very broad "peak" on the lower portion of the graph).
  • Primed PC12 cells (surrogates for neurons) were seeded on the decellularized matrix and cultured in differentiating media for 72 hours. As shown in Fig. 13, neurites from plated PC12 cells were aligned with the pattern, while there was no alignment on the unpatterned control.
  • Striped patterns of 30 ⁇ in width spaced by 30 ⁇ (30 X 30) were prepared on nylon, PET and PEEK treated with Zr0 2 with or without further treatment with bisphos- phonate SAMP as described in the previous examples.
  • the surface was exposed to vapor of the zirconium alkoxide precursor of Zr0 2 for 5 minutes (Zr0 2 5 min) or 10 minutes (Zr0 2 10 min) before heating to make the oxide. The longer the time of initial exposure, the thicker the layer of oxide that formed.
  • the Zr0 2 5 min patterned substrate was used for further treatment with bisphosphonate SAMP.
  • NIH 3T3 fibroblasts (30,000 cells/well) were plated on the treated substrates and imaged after one day (Fig. 14) and 3 days (Fig. 15). As shown in Figs. 14 and 15, there was significant cell alignment using Zr0 2 alone and very good cell alignment on SAMP -treated substrates.
  • Fibroblasts plated on patterned substrates assemble a natural fibrillar matrix containing fibronectin.
  • a robust ECM was assembled.
  • Immunofluorescence detection with anti-fibronectin antibodies showed matrix fibrils that were oriented with the pattern (Fig. 18B) in contrast to the matrix on the unpatterned surface (Fig. 18 A).
  • the degree of matrix alignment was quantified by performing two-dimensional fast-Fourier transform analysis (FFT) of the images. The radial summation of pixel intensity in the FFT output for each angle was plotted between 0 to 180°.
  • FFT fast-Fourier transform analysis
  • a peak centered at 90° for the patterned substrates indicated alignment of matrix fibrils parallel to underlying patterned stripes (Fig. 18C). No peak was obvious for matrix on an unpat-terned substrate. The variation in height and shape of the major peak is represented quantitatively by full width-half maximum (FWHM) values. A significantly lower FWHM for the patterned sample indicates higher alignment of fibrils compared to the random fibrils on the unpatterned substrates (Fig. 18D).
  • the matrix also contained type I collagen fibrils (Fig. 18E) that were co-aligned with the fibronectin fibrils and the pattern (Fig. 18F).
  • PC12 cells as surrogates for neurons, were plated on this aligned ECM/PET material of Example 10 and were stimulated for neurite outgrowth that was quantitatively evaluated through directional analysis (Fig. 20).
  • the cells cultured on patterned or un- patterned, decellularized matrix extended neurites, which were visualized by staining the actin cytoskeleton (Figs. 20A, B).
  • Co-staining of matrix fibrils indicated correspondence between the direction of neurite outgrowth and the orientation of fibrils (Figs. 20C, D). Measurements of neurite projection angles relative to the patterned matrix showed the majority of neurites in line with the 10 ⁇ x 10 ⁇ pattern (90° peak, Fig.
  • Radial glial cells (rat radial glial cell line C6 modified to express GFP obtained from Dr. Martin Grumet at Rutgers University) were plated on the patterned PET material of Example 10, were attached to and were aligned to the patterns and proliferated in alignment with the patterns until reaching confluency on polymers.
  • Radial glial cells were also plated onto decellularized matrix and cultured for variable periods of time (1-7 days) while maintaining alignment with matrix fibrils.
  • PC12 cells were then plated onto radial glial cell/matrix substrate and co-cultured. Both cell types were observed to attach and grow in register with the aligned matrix on the patterned polymers. Analysis through FFT was done showing that all neurites and glial cells were highly aligned with pattern and ECM direction (Fig. 22). In co-culture with glial cells, neuron cells extend a dense network of aligned neurites spanning the decellularized matrix/polymer substrate.
  • Schwann cells (rat neonatal Schwann cells) were plated on the polymer patterns of Example 10 and attach and grow in alignment with patterned stripes. Cells were able to grow over a 10-day period on patterned polymers and retain alignment. Schwann cells plated on cell-assembled, aligned decellularized matrix were attached in alignment with matrix fibrils and proliferate to confluence.
  • Schwann cells were able to proliferate extensively on decellularized matrix while retaining alignment with matrix fibrils.
  • Schwann cells in co-culture with fibroblasts on the patterned PET of Example 10 grow aligned with patterns and assemble a dense ECM network of fibronectin, collagen, and other ECM proteins produced by fibroblasts and Schwann cells.
  • Sympathetic cervical ganglia were plated onto substrates to assess neurite outgrowth and directionality.
  • Substrates included a glass coverslip coated with a 10 ⁇ g/ml solution of laminin (Laminin), decellularized fibroblast ECM on an unpatterned glass coverslip (Unpatterned ECM), and decellularized aligned ECM on a 20 ⁇ x 20 ⁇ patterned PET surface prepared according to Example 10 (Patterned ECM).
  • Laminin laminin
  • Unpatterned ECM unpatterned glass coverslip
  • Decellularized aligned ECM on a 20 ⁇ x 20 ⁇ patterned PET surface prepared according to Example 10
  • Figs. 23 A and 23B A single ganglion with extended neurites is shown in each image. The substrate is indicated above the image.
  • Fig. 23 A and 23B A single ganglion with extended neurites is shown in each image. The substrate is indicated above the image.
  • Fig. 23 A and 23B A single ganglion with extended neurites is
  • 23B neurite outgrowth was fitted with an ellipse (lines on images) to determine if neurite extension deviated from circular, i.e., showed directionality.
  • Samples on unpatterned ECM, patterned ECM, and laminin were measured.
  • the aspect ratios (long axis divided by the short axis) displayed in the graph were calculated and averaged for a minimum of 3 samples per condition, and show that the neurites grown on patterned ECM substrates showed significant directionality, i.e., the aspect ratio is significantly different from 1.0 (which would be circular or round).

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Abstract

La présente invention concerne des échafaudages pour tissus, des procédés de génération de tels échafaudages et des procédés d'utilisation de tels échafaudages pour générer des tissus neuronaux alignés et fonctionnels destinés à être utilisés dans des procédés comprenant la médecine régénérative, la réparation des plaies et la transplantation.
PCT/US2016/055438 2011-12-07 2016-10-05 Échafaudages pour tissus neuronaux et leurs utilisations WO2017062417A1 (fr)

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US20020050220A1 (en) * 2000-08-14 2002-05-02 Olivier Schueller Deformable stamp for patterning three-dimensional surfaces
US20020095219A1 (en) * 2000-10-20 2002-07-18 Gabriele Nelles Method of forming a cell pattern on a surface, cellular networks and tissues based theron
US6645644B1 (en) * 1996-10-17 2003-11-11 The Trustees Of Princeton University Enhanced bonding of phosphoric and phosphoric acids to oxidized substrates
US20040096476A1 (en) * 2002-07-17 2004-05-20 Uhrich Kathryn E. Therapeutic devices for patterned cell growth
US20060085063A1 (en) * 2004-10-15 2006-04-20 Shastri V P Nano- and micro-scale engineering of polymeric scaffolds for vascular tissue engineering
US20080131709A1 (en) * 2006-09-28 2008-06-05 Aculon Inc. Composite structure with organophosphonate adherent layer and method of preparing
US7396594B2 (en) * 2002-06-24 2008-07-08 The Trustees Of Princeton University Carrier applied coating layers
US20080299169A1 (en) * 2005-08-12 2008-12-04 Diane Hoffman-Kim Topographical Templating of Polymeric Materials Using Cellular Morphology
US20090104474A1 (en) * 2007-10-17 2009-04-23 Princeton University Functionalized substrates and methods of making same
US20140330392A1 (en) * 2011-12-07 2014-11-06 The Trustees Of Princeton University Scaffolds for tissues and uses thereof
US9056154B2 (en) * 2006-02-21 2015-06-16 The Trustees Of Princeton University High-yield activation of polymer surfaces for covalent attachment of molecules

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6645644B1 (en) * 1996-10-17 2003-11-11 The Trustees Of Princeton University Enhanced bonding of phosphoric and phosphoric acids to oxidized substrates
US7879456B2 (en) * 1996-10-17 2011-02-01 Jeffrey Schwartz Carrier applied coating layers
US20020050220A1 (en) * 2000-08-14 2002-05-02 Olivier Schueller Deformable stamp for patterning three-dimensional surfaces
US20020095219A1 (en) * 2000-10-20 2002-07-18 Gabriele Nelles Method of forming a cell pattern on a surface, cellular networks and tissues based theron
US7396594B2 (en) * 2002-06-24 2008-07-08 The Trustees Of Princeton University Carrier applied coating layers
US20040096476A1 (en) * 2002-07-17 2004-05-20 Uhrich Kathryn E. Therapeutic devices for patterned cell growth
US20060085063A1 (en) * 2004-10-15 2006-04-20 Shastri V P Nano- and micro-scale engineering of polymeric scaffolds for vascular tissue engineering
US20080299169A1 (en) * 2005-08-12 2008-12-04 Diane Hoffman-Kim Topographical Templating of Polymeric Materials Using Cellular Morphology
US9056154B2 (en) * 2006-02-21 2015-06-16 The Trustees Of Princeton University High-yield activation of polymer surfaces for covalent attachment of molecules
US20080131709A1 (en) * 2006-09-28 2008-06-05 Aculon Inc. Composite structure with organophosphonate adherent layer and method of preparing
US20090104474A1 (en) * 2007-10-17 2009-04-23 Princeton University Functionalized substrates and methods of making same
US20140330392A1 (en) * 2011-12-07 2014-11-06 The Trustees Of Princeton University Scaffolds for tissues and uses thereof

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