WO2007103135A2 - Active cell growth substrates and uses thereof - Google Patents

Active cell growth substrates and uses thereof Download PDF

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Publication number
WO2007103135A2
WO2007103135A2 PCT/US2007/005291 US2007005291W WO2007103135A2 WO 2007103135 A2 WO2007103135 A2 WO 2007103135A2 US 2007005291 W US2007005291 W US 2007005291W WO 2007103135 A2 WO2007103135 A2 WO 2007103135A2
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Prior art keywords
substrate
cell
kpa
scaffold
diameter
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PCT/US2007/005291
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French (fr)
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WO2007103135A3 (en
Inventor
Michael P. Sheetz
James C. Hone
Monica Tanase
Samuel J. Wind
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Columbia University in the City of New York
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Columbia University in the City of New York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides

Definitions

  • the shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms. [0005] Throughout the biological kingdom there is a wide diversity of shapes, and this phenomenon has interested physical biologists for a long time (see On Growth and Form. D'Arcy W. Thompson (Dover Publications, 1992)).
  • the invention is based, in part, on the finding that a cell growth substrate having a variable rigidity can be constructed and utilized to control cellular functions and responses.
  • the invention provides for a cell growth substrate comprising: a) a cross-linked polymer, wherein increased strain on the polymer does not substantially increase rigidity of the polymer, and wherein the polymer has an elastic modulus (E) of about 0.1 kPa to about 10.0 kPa; and b) a growth medium, wherein the substrate is permeable to liquids, gases, and cellular by-products.
  • the polymer is not collagen, not hyaluronic acid, not polydimethylsiloxane (PDMS), and not a carbohydrate based gel.
  • the polymer comprises an anionic polymer, a cationic polymer, an amphipathic polymer, a neutral polymer, a synthetic polymer, or any combination thereof.
  • the cationic polymer comprises chitosan or polylysine.
  • the amphipathic polymer comprises gelatin, fibrin, or carboxymethyl chitin.
  • the neutral polymer comprises dextran, agarose, or pullulan.
  • the synthetic polymer comprises a polyester or derivative thereof.
  • the polyester comprises polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, or polyacrylamide.
  • the polymer is functionalized with chemical groups.
  • the substrate further comprise a magnetic material, a Piezo actuator, or a combination thereof. The substrate contains such a material in order to provide a way to cause the substrate to be moved, strained or stretched.
  • the magnetic material comprises nickel or iron.
  • the magnetic material is in the form of a bead.
  • the bead is about 0.1 mm to about 5 mm in diameter, about 0.2 mm to about 4 mm in diameter, about 0.3 mm to about 3 mm in diameter, about 0.4 mm to about 2 mm in diameter, or about 0.5 mm to about 1 mm in diameter. In one embodiment, the bead is about 2.7 mm in diameter.
  • the magnetic material is in the form of a nanowire. In one embodiment, the nanowire is about 2-50 mm in length, about 3-40 mm in length, about 4-30 mm in length, about 5-20 mm in length, or about 6-10 mm in length.
  • the nanowire is about 30 mm in length, hi one embodiment, the nanowire is about 100-500 nm in diameter, about 150-450 nm in diameter, about 200-400 nm in diameter, or about 250-350 nm in diameter. In one embodiment, the nanowire is about 300 nm in diameter.
  • the elastic modulus is about 0.3 kPa to about 8 kPa, about 0.5 kPa to about 7 kPa, about 0.6 kPa to about 6 kPa, about 0.7 kPa to about 6 kPa, about 0.8 kPa to about 5 kPa, about 0.9 kPa to about 4 kPa, or about 1.0 kPa to about 3 kPa.
  • the invention also provides for an apparatus for applying a perturbation to a cell in vitro, the apparatus comprising: a) the substrate described herein; and b) an external force generator associated with the substrate.
  • the external force generator comprises an electrical force, a magnetic force, a mechanical force, or a combination thereof.
  • the external force generator comprises a pipet-assisted manipulation device, a laser tweezer, an optical trap, or a magnetic field generator.
  • the external force generator stretches the substrate.
  • the substrate is stretched intermittently.
  • the external force generator comprises a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate.
  • the apparatus further comprises a means for quantifying a cellular response to the perturbation.
  • the cellular response quantified is cell growth, cell differentiation, apoptosis, cell movement, cell proliferation, cell morphology changes, or a combination thereof.
  • the invention provides a scaffold for supporting cells, the scaffold comprising: a) portions of the substrate described herein; and b) fibers stretched over the substrate portions.
  • the portions are strips in the form of a fence.
  • the fence is about 0.5 ⁇ m to about 3 ⁇ m thick, about 0.6 ⁇ m to about 2.5 ⁇ m thick, about 0.7 ⁇ m to about 2 ⁇ m thick, about 0.75 ⁇ m to about 1.5 ⁇ m thick, or about 0.8 ⁇ m to about 1 ⁇ m thick. In one embodiment, the fence is about 2 ⁇ m thick.
  • the fence is about 3 ⁇ m to about 12 ⁇ m in height, about 4 ⁇ m to about 11 ⁇ m in height, about 5 ⁇ m to about 10 ⁇ m in height, about 5.5 ⁇ m to about 9 ⁇ m in height, or about 6 ⁇ m to about 8 um in height. In one embodiment, the fence is spaced about 10 ⁇ m to about 200 ⁇ m apart, about 15 ⁇ m to about 150 ⁇ m apart, about 20 ⁇ m to about 100 ⁇ m apart, or about 25 ⁇ m to about 50 ⁇ m apart.
  • the fence is spaced about 25 ⁇ m apart, hi one embodiment, the fence is less than about 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 ⁇ m in length. In one embodiment, the fiber is absorbable.
  • the fiber comprises polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide-lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester), catgut suture, collagen, silk, chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid, elastin, or any combination thereof.
  • PGA polyglycolic acid
  • PLA polylactic acid
  • PDA polyglycolide-lactide
  • polycaprolactone polydioxanone
  • polyoxalate polyoxalate
  • a polyanhydride a poly(phosphoester)
  • catgut suture collagen
  • silk chitin
  • chitosan hydroxyapatite
  • bioabsorbable calcium phosphate hyaluronic acid
  • elastin or any combination thereof.
  • the fiber is about 50 nm to about 1500 run in diameter, about 100 nm to about 1250 nm in diameter, about 200 nm to about 1000 nm in diameter, about 250 nm to about 900 nm in diameter, about 300 nm to about 800 nm in diameter, about 350 nm to about 750 nm in diameter, about 400 nm to about 700 nm in diameter, or about 500 nm to about 600 nm in diameter.
  • the fiber is coated with a growth factor, an extracellular matrix molecule, or a combination thereof.
  • the extracellular matrix molecule comprises hyaluronic acid, collagen, chondroitin, or a combination thereof.
  • the invention also provides a method for stimulating a cellular response in vitro, the method comprising: a) plating a cell on, or in, the substrate described herein or the scaffold described herein; b) perturbing the substrate or the scaffold containing the cell; and c) detecting the cellular response, the perturbation of the substrate or the scaffold stimulating the cellular response.
  • the perturbation comprises a mechanical perturbation, an electrical perturbation, a magnetic perturbation, or any combination thereof.
  • perturbing comprises subjecting the substrate or the scaffold to the external force generator.
  • the external force generator stretches the substrate or the scaffold.
  • the substrate or scaffold is perturbed intermittently.
  • the substrate or scaffold is perturbed up to 10 times per hour, up to 5 times per hour, up to 2 times per hour, or 1 time per hour. In one embodiment, the substrate or scaffold is perturbed up to 10 times per day, up to 5 times per day, up to 2 times per day, or 1 time per day. In one embodiment, the substrate or scaffold is perturbed up to 10 times per week, up to 5 times per week, up to 2 times per week, or 1 time per week. In one embodiment, the cellular response is cell growth, cell differentiation, apoptosis, cytokinetics, mo ⁇ hological changes, or a combination thereof.
  • the cellular response is detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, mRNA expression levels, a change in cell morphology or a combination thereof.
  • the substrate or the scaffold has an elastic modulus of about 3 kPa. In one embodiment, the substrate or the scaffold promotes a fibroblast cellular response. In one embodiment, the substrate or the scaffold has an elastic modulus of about 2 kPa. In one embodiment, the substrate or the scaffold promotes a myoblast cellular response. In one embodiment, the substrate or the scaffold has an elastic modulus of about 1 kPa. In one embodiment, the substrate or the scaffold promotes a neuronal cellular response. In one embodiment, the cell is obtained from a tissue. In one embodiment, the cell (a) is a primary cell, (b) is a cell from a cell culture that has been passaged, or (b) is a cell from a cell line.
  • the invention also provides a method for promoting growth of a cell in vivo, the method comprising: a) inserting the scaffold into a subject; and b) applying a magnetic field to the subject, wherein the application of the magnetic field promotes growth of the cell.
  • the scaffold has an elastic modulus of about 0.3 kPa to about 8 kPa, about 0.5 kPa to about 7 kPa, about 0.6 kPa to about 6 kPa, about 0.7 kPa to about 6 kPa, about 0.8 kPa to about 5 kPa, about 0.9 kPa to about 4 kPa, or about 1.0 kPa to about 3 kPa.
  • the scaffold has an elastic modulus of about 3 kPa. In one embodiment, the scaffold promotes a fibroblast cellular response. In one embodiment, the scaffold has an elastic modulus of about 2 kPa. In one embodiment, the scaffold promotes a myoblast cellular response. In one embodiment, the scaffold has an elastic modulus of about 1 kPa. In one embodiment, the scaffold promotes a neuronal cellular response. In one embodiment, cell growth comprises wound healing in a subject. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human, a dog, a cat, a mouse, a rat, a horse, a pig, a cow, or a bird.
  • One aspect of the present invention provides for a cell maintenance substrate permeable to liquids, gases, and cellular by-products, that is made up of a growth medium and a polymer with a constant elasticity through stretch and a rigidity where the elastic modulus (E) is in the range of about 0.1 kPa to about 10.0 kPa.
  • the elastic modulus of the substrate is about 0.3 kPa to about 8 kPa.
  • the polymer is cross-linked.
  • the polymer comprises natural polymers and their derivatives, synthetic polymers and their derivatives, or some combination.
  • the natural polymers may include anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers.
  • the anionic polymers comprise hyaluronic acid, alginic acid, carageenan, chondroitin sulfate, dextran sulfate, or pectin.
  • the cationic polymers may include chitosan or polylysine.
  • the amphipathic polymers may include of collagen, gelatin, fibrin, or carboxymethyl chitin.
  • the neutral polymers may inlcude dextran, agarose, or pullulan.
  • the synthetic polymers can be polyesters or derivatives thereof.
  • the polyesters comprise polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyacrylamide, or polydimethylsiloxane.
  • the cell maintenance substrate of the present invention further includes a magnetic material.
  • the magnetic material may comprise nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt.
  • the nanowire is made of nickel.
  • the magnetic material is in the form of a bead.
  • the diameter of the magnetic bead is greater than or equal to about 0.1, 0.2, 0.3, 0.5, 0.6, 0.75, 0.8, 0.9, 1.0, 2.0, 2.5, 3, 4, or 5 ⁇ m in diameter. In particular embodiments, the diameter of the
  • the magnetic material is in the form of a nanowire.
  • the diameter of the magnetic wire is greater than or equal to about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 run in diameter.
  • the diameter of the wire is about 300 nm in diameter.
  • the length of the magnetic wire is greater than or equal to about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ⁇ m in length. In specific embodiments, the length of the magnetic wire is about 30 ⁇ m in length.
  • the invention provides an apparatus for applying a mechanical perturbation to a cell in vitro, wherein the apparatus is made up of the cell maintenance substrate described above and an external force generator associated with the substrate.
  • the external force generator acts as a means for physically manipulating the substrate.
  • the external force generator may entail a pipet-assisted manipulation device.
  • the external force generator may be a laser tweezer.
  • the external force generator comprises an optical trap.
  • the external force generator stretches the substrate.
  • the external force generator may include a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate.
  • the external force generator can be a magnetic field generator.
  • the apparatus of the present invention may further encompass a means for quantitating a cellular response to the mechanical perturbation.
  • the cellular response quantitated is cell growth, cell differentiation, apoptosis, cell movement, cell morphology changes, or some combination of these responses.
  • Elastic modulus (E) refers to tensile elasticity and reflects the measure of the stiffness of a given material.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa.
  • the substrate promotes a fibroblast cellular response.
  • the substrate has an elastic modulus of about 2 kPa.
  • the substrate promotes a myoblast cellular response.
  • the substrate has an elastic modulus of about 1 IcPa.
  • the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention.
  • the cell is excised from a tissue.
  • the cell is excised from the tissue of an animal.
  • the cell is excised from the tissue of a mammal, hi some embodiments, the cell is excised from the tissue of a human.
  • the cell is obtained from a cultured cell line.
  • the invention provides a method for stimulating growth of a cell in vitro, wherein cells are plated on or in the cell maintenance substrate described above, the substrate containing the cells is perturbed (for example, mechanically, electrically, or magnetically), and growth of the cell is subsequently detected.
  • mechanically perturbing the substrate entails subjecting the substrate to an external force generator described above which results in stimulating growth of cells in the substrate.
  • growth of the cell is detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or some combination of these measurements.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
  • the invention provides a method for stimulating cell differentiation in vitro, wherein cells are plated on or in the cell maintenance substrate previously described above, the substrate containing the cells is mechanically perturbed, and differentiation of cells is detected. Mechanical perturbation of the substrate stimulates the differentiation of the cell and entails subjecting the substrate to an external force generator described above.
  • differentiation of the cell is detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or some combination of these measures.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa.
  • the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above. [0016] In another aspect, the invention provides for a method of stimulating a cellular response in vitro.
  • the method includes plating a cell on or in the cell maintenance substrate described above, mechanically perturbing the substrate containing the cell, and detecting a cellular response, wherein the mechanical perturbation of the substrate stimulates a cellular response.
  • mechanically perturbing the substrate entails subjecting the substrate to an external force generator described above.
  • the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination.
  • the cellular response can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or some combination.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response.
  • the current invention provides for a method of promoting growth of a cell in vivo, which includes creating a matrix ex vivo from the cell maintenance substrate described above, inserting the matrix into the body of a subject, and applying a magnetic field to the subject. Application of the magnetic field promotes the growth of the cell within the subject.
  • the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In particular embodiments, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
  • the invention also provides for a method of treating a wound, which entails creating a matrix ex vivo from the cell maintenance substrate described above, inserting the matrix into the body of a subject, and applying a magnetic field.
  • the exposure of cells to a magnetic field results in the proliferation of the cells.
  • the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination of the responses.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa.
  • the substrate has an elastic modulus of about 3 kPa.
  • the substrate promotes a fibroblast cellular response.
  • the substrate has an elastic modulus of about 2 IcPa.
  • the substrate promotes a myoblast cellular response.
  • the substrate has an elastic modulus of about 1 kPa.
  • the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
  • FIG. 1. is a schematic that emphasizes the steps involved in the responses of cells to their environment.
  • FIG. 2 illustrates the three basic mechanisms of force sensing.
  • FIG. 2A depicts the conversion of force into biochemical signals by partial protein unfolding (as shown for the fibronectin module III). This can result in the gain or loss of binding sites, increased separation between protein domains, or the gain or loss of enzyme function.
  • FIG. 2B depicts the opening of some mechanosensitive ion channels can be regulated by membrane tension (Ba), whereas the opening of others requires that their intra- and/or extracellular domains are physically connected to force-bearing filaments (Bb).
  • FIG. 2C shows the Stabilizing receptor-ligand bonds by switching them to a long-lived state by force (catch bonds). Yellow arrows indicate forces.
  • FIG. 2A depicts the conversion of force into biochemical signals by partial protein unfolding (as shown for the fibronectin module III). This can result in the gain or loss of binding sites, increased separation between protein domains, or the gain or loss of enzyme function.
  • FIG. 2B depicts the opening of
  • FIG. 3 are schematics that illustrate (a) how inward curvature of the plasma membrane could cause BAR-domain (Bin, amphiphysin, Rvs domain) proteins to release Rac and (b) how outward curvature could activate the opening of an ion channel.
  • FIG. 4 illustrates the position-dependent mechanism of rigidity sensing.
  • FIG. 4A depicts the crucial feature in such a model is that the enzyme, Fyn, and the substrate to be activated by stretch (kinase and substrate in this example) move relative to one another by actin rearward transport.
  • FIG. 4B if the surface is hard, the components would be close enough for modification to occur (causing small displacement).
  • FIG. 4A depicts the crucial feature in such a model is that the enzyme, Fyn, and the substrate to be activated by stretch (kinase and substrate in this example) move relative to one another by actin rearward transport.
  • FIG. 4B if the surface is hard, the components would be close enough for modification to occur (causing small displacement
  • ECM extracellular matrix
  • F applied force
  • RPTPot receptor-like protein tyrosine phosphatase- ⁇ .
  • FIG. 5 shows the steps in mechanosensing over time that involve periodic testing of the substrate, substrate modification and changes in cellular protein content.
  • FIGS. 6A-B represent a local force assay.
  • FIG. 6A depicts magnetic tweezers (tip at top left corner) exerting a force on 2.7 ⁇ m magnetic beads bound to a spreading MEF cell.
  • FIG. 6B is an image showing the rearward flow of actin that displaces the bead from the cell edge, towards the nucleus (purple trace) prior to application of force.
  • the bead moves under the competing action of the two forces, cellular and magnetic. Shown here is the case of a large magnetic force ( 1 - 1.2 nN) and the bead is temporarily pulled back to the cell edge (red trace).
  • FIG. 6C is a graph that demonstrates the cell responding to the local stress by pulling in a contractile manner (positive and negative rearward velocities) and reinforcing the cytoskeletal adhesion to the stress site during the adaptive phase.
  • the end of adaptive phase is marked by the recovery of the bead's constant rate of displacement.
  • FIG. 7 represents the spatio-temporal dynamics of ⁇ Actinin in response to local force.
  • FIG. 7A is an epifluorescent image of an MEF cell expressing ⁇ Actinin-GFP that shows an accumulation of the fluorophore around two 2.7 mm beads.
  • FIG. 7B represents the intensity of fluorescence vs. time and distance from center of the bead, r. Data at constant (r, t) was obtained by averaging over the arcsector shown in FIG. 7A. The frame of reference is centered on the bead and moves with it. Red and blue indicate the highest and respectively lowest levels of intensity. Maximum protein accumulation is observed ⁇ 50 sec from the application of large force. The duration of the adaptive phase is -105 sec.
  • FIG. 8 depicts active substrates.
  • FIG. 8A (left) is a diagram of magnetic nanowires (for example, nickel) that can be fabricated by electrochemical deposition into porous templates. Scanning electron micrographs (SEM) of nickel nanowires are shown in the right panel.
  • FIG 8B is a schematic of soft gels containing self-assembled arrays of magnetic nanowires that can serve as substrates for cells requiring rigid substrates for normal function.
  • FIG. 8C represents external magnets that can actuate the embedded wires and impart quasi-local stresses at the surface of the gels.
  • FIG. 8D demonstrates the restoration of spreading that can occur after 4 - 24 hrs, especially in the regions closer to the actuating magnet where wires deflect more and the local stresses are larger.
  • FIG. 9 represents a whole-cell dynamic environment.
  • FIG. 9A is a schematic of a stress gradient that can be established across the width of free floating elastic substrates by having a varying initial length (diagonal clamp) and constant elongation.
  • FIGS. 9B-E are representative experiments that were performed to test if the cells detect increased rigidity in the high-strain regions of polyacrylimide gels. Images are shown at same magnification.
  • FIGS. 10A-B show laser tweezers (represented by red circle) that were used to place silica beads coated with fibronectin (FN) or vitronectin (VN) at the edge of the active growth cones. The rearward movement of the beads was recorded and further analyzed for reinforcement.
  • FN fibronectin
  • VN vitronectin
  • FIGS. lOC-D are graphs that demonstrate that RPTP ⁇ is required for the reinforcement of FN-specific integrin-cytoskeleton bonds in the neuronal growth cones.
  • FIGS. 10E-F are schematics of the trajectories of individual beads that were generated in cases of non-reinforced (FIG. 10E) and reinforced of rearward movement (FIG. 10F). The laser trap is represented by a red circle. These trajectories are representative of an average bead behavior in both categories.
  • FIGS. 10G-H depict the mean square displacement (MSD) that was calculated for the initial 10s of rearward movement for each given condition. The results (represented as mean+standard error) were statistically significant as confirmed by t-test (p ⁇ 0.01).
  • FIGS. 1 IA-B are graphs showing that Ov ⁇ integrins are required for the reinforcement of the FN-cytoskeleton bonds at the leading edge of the growth cones. Function blocking antibodies to the ots ⁇ i and ⁇ v ⁇ 3 integrins had no effect on binding and the reinforcement of FN-coated beads, but function blocking of the ⁇ v and ⁇ v ⁇ 6 integrins reduced binding and reinforcement to the background levels.
  • FIGS. 1 IC-F are microscopy images representing that localization of RPTP ⁇ and cCv ⁇ integrin in growth cones were ECM-specific. Both RPTP ⁇ and ⁇ v ⁇ integrins were localized to the leading edge of active growth cones in neurons plated on FN-coated glass (FIG. 11C, FIG. 1 IE). On laminin (LN), RPTP ⁇ was localized to the growth cones, while ⁇ v ⁇ integrin was expressed at a very low level along the axons and at even lower levels in the growth cones (FIG. 1 ID, FIG. 1 IF). The scale bar is 5 ⁇ m. Insets provide image of the entire neuron. [0035] FIGS.
  • FIGS. 12A-B represent Hippocampal neurons isolated from Pl brains of wild type and RPTP ⁇ knockout mice that were plated on FN-coated polyacrylamide gels of decreasing rigidities, incubated for 36h in serum-free medium, fixed and visualized by anti-Tau immunofluorescence. Neuronal stages of differentiation were observed on ECM-coated polyacrylamide gels, wherein stage 2 was characterized by many neurites of approximately equal length (FIG. 12A). At stage 3, -one significantly longer axon was differentiated (FIG. 12B). The Scale bar represents approximately lO ⁇ m. [0036] FIGS.
  • FIG. 12C-D are graphs that represent quantification of differentiation stages and neurite lengths, which revealed an effect of rigidity and a role for RPTP ⁇ .
  • Control neurons RPTPa+/+
  • RPTP ⁇ -/- neurons showed a high level of differentiation irrespective of matrix rigidity (FIG. 12C).
  • control neurons extended longer axons on soft than on rigid surfaces; whereas, RPTP ⁇ -/- neurons extended longer axons on both soft and rigid surfaces (FIG. 12D).
  • the results, in both FIG. 12C and 12D, (represented as mean + standard error) were statistically significant as confirmed by t-test (p ⁇ .01).
  • FIGS. 12E-F represent Hippocampal neurons isolated from Pl brains of wild type and RPTP ⁇ knockout mice that were plated on FN-coated polyacrylamide gels of decreasing rigidities, incubated for 36h in serum-free medium, fixed and visualized by anti-Tau immunofluorescence.
  • the axons in RPTP ⁇ -/- neurons appeared wavier than the axons in wt neurons. No other morphological differences between RPTP ⁇ -/- and wild type neurons were observed.
  • the scale bar represents approximately 15 ⁇ m.
  • FIG. 13 are graphs depicting that the laminin (LN) rigidity response is RPTP ⁇ - independant in neurons.
  • FIG. 13A is a graph that shows that axon differentiation is inhibited by increasing rigidities of LN -coated substrates in both contol and knockout neurons.
  • FIG. 13B is a graph that shows neurite extension is stimulated by soft LN-coated substrates, and loss of RPTP ⁇ had no effect on this behavior
  • FIGS. 14A-B are graphs that show rigidity response in the growth cones is SFK- dependent.
  • Primary wild type neurons were plated on FN-coated substrates of varying rigidities, and a SFK inhibitor (lO ⁇ M SU6656) was added after cell adhered to the substrate. After 36h incubation, there was no difference in wild type neurons on rigid versus soft surfaces. However, axon elongation and differentiation were inhibited compared to controls.
  • FIGS. 14C-D are fluorescent micrographs that depict immunofluorescent staining of
  • FIGS. 14E-F are fluorescent micrographs that demonstrate phosphorylation of pl30Cas, a known substrate for SFKs, requires RPTP ⁇ activity and rigid matrix.
  • Immunofluorescent staining of phosphorylated pl30Cas showed high levels of phospho-pl30Cas in the presence of RPTP ⁇ and rigid matrices. In RPTPa+/+ neurons plated on soft matrix, and in RPTP ⁇ -/- neurons regardless of the matrix rigidity, the observed levels of phopho-pl30Cas were significantly lower.
  • FIG. 15 represents a proposed model for the molecular mechanism of the FN-specific reinforcement and rigidity response in hippocampal neurons.
  • FIG. 16 is a photographic representation of 3D substrates depicting coated silk fibers
  • Cell-cell contacts are dynamic, and cells seem to evaluate the level of force and make adjustments, as the cytoskeleton filaments and their linkages to transmembrane proteins assemble, break down and reassemble.
  • Sensing of geometry at the sub-cellular level is a crucial component of the cellular sensing of two-dimensional (2D) versus 3D matrices (FIG. 1).
  • 2D two-dimensional
  • 3D matrices FIG. 1
  • Recent studies have determined that the same matrix protein will elicit a different response when it is organized in filaments from when it is displayed on a flat surface (Cukierman, E. , Pankov, R. , Stevens, D. R. & Yamada, K. M. Science 294, 1708-1712 (2001); Cukierman, E. , Pankov, R. & Yamada, K. M. Curr. Opin. Cell Biol. 14, 633-639 (2002); Katz, B. Z. et al. MoI Biol.
  • Complicated transduction processes such as rigidity responses involve several steps that can combine the transduction of force and/or geometry sensing with time (reviewed in Refs 24,25) (Discher, D. E. , Janmey, P. & Wang, Y. L. Science 310, 1139-1 143 (2005); Giannone, G. & Sheetz, M. P. Trends Cell Biol, (in the press). Guanine nucleotide-exchange factors, Ca 2+ ion channels, receptor-like protein tyrosine phosphatases, Src-family kinases and membrane receptors have all been invoked as early steps in force or geometry transduction (von Wichert, G. et al. J.
  • FIG. 2 These include the force-induced exposure of otherwise cryptic peptide sequences (FIG. 2A), the opening of mechanosensitive ion channels (FIG.
  • the isometric ATPase activity of motors keeps the tension in the cytoskeleton constant, and the recruitment of other motors will then increase the overall tension in the cytoskeleton. Because the cytoskeleton filaments are dynamic and undergo assembly— disassembly cycles on the timescale of seconds to minutes, filament tension will be rapidly lost and must constantly be maintained by motor activity.
  • Conformational strain may be capable of increasing the activity of some enzymes.
  • the question of whether the activity of some enzymes can be upregulated by strain if they or their substrates are physically integrated into a force-bearing structure is relatively unexplored in the context of mechanosensing. Force might open up enzymatic cleavage sites through partial unravelling.
  • Fibronectin for example, has a partially cryptic disulphide-isomerase (Langenbach, K. J. & Sottile, J. J. Biol. Chem. 11$, 7032-7038 (1999) and a cryptic metalloprotease activity (Schnepel, J. & Tschesche, H J. Protein Chem.
  • Titin contains a module that can show kinase activity and computational studies indicated a mechanical opening of its active site (Grater, F., Shen, J., Jiang, H., Gautel, M. & Grubmuller, H. Biophys. J. 88, 790-804 (2005)). Furthermore, it is well known that enzymes exert strain on their substrates on binding — thereby catalysing the reaction (reviewed in Ref. 7) (Bustamante, C, Chemla, Y.R., Forde, N.R. & Izhaky, D. Annu. Rev. Biochem.
  • Mechanosensitive ion channels The structural diversity of mechanosensitive channels seems to have been driven by the physiological necessity to detect mechanical stimuli — from thermal energy to high pressures — as changes in conductive state (FIG. 2B) (reviewed in Sukharev, S. & Anishkin, A. Trends Neurosci. 27, 345—351 (2004)). Some channels respond to stress in the lipid bilayer, whereas others must be physically connected to the cytoskeleton and/or the extracellular matrix to transmit forces to the channel.
  • the bacterial mechanosensitive K + channel, MscL represents the first case and has been structurally analysed.
  • Membrane-tension forces are mainly concentrated in the interfacial polar headgroup regions of MscL, thereby inducing helix tilting that opens and wets the pore interior (Kung, C. Nature 436, 647-654 (2005); Sotomayor, M. & Schulten, K. Biophys. J. 87, 3050-3065 (2004))
  • Bacterial channels are relatively force-insensitive and only open at high tensions that are approaching the lytic tensions for the lipid bilayer.
  • Plant channels seem to operate at tensions that are of an order of magnitude lower than bacterial channels, whereas typical membrane tensions in animal cells are a thousandfold lower than the activating bacterial tensions (Sheetz, M. P. Nature Rev. MoI. Cell Biol. 2, 392-396 (2001)).
  • the bond-survival time of the strongest non-covalent bond is diminished from more than a day under static conditions to about 1 minute or 0.001 second at bond stresses of 5 pN or 170 pN, respectively (Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Nature 397, 50-53 (1999)); whereas 5 pN can be exerted onto a protein complex by a single motor protein. Therefore, non-covalent bonds will fail under any level of tensile stress if held for sufficient time periods (Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E.
  • Adhesion sites form in response to tensile forces acting on the membrane, and one consequence of the shortened bond lifetimes under force must be high turnover rates of the constituents in newly formed adhesion sites. Turnover rates on the timescale of seconds have been observed for acti ⁇ filaments and the two focal-contact proteins, paxillin and vinculin, which might serve as signalling molecules after their release from the cell contacts (Vallotton, P., Gupton, S.L., Waterman- Storer, C. M. & Danuser, G. Proc. Natl Acad. ScL USA 101, 9660-9665 (2004); Von Wichert, G. , Haimovich, B. , Feng, G. S.
  • Cells may be able to form force-sustaining adhesion sites. Because force is transmitted across the membrane, not by single integrins, but by integrin clusters that increase in size with time in a force-dependent manner (Riveline, D. et al. J. Cell Biol. 153, 1175—1186 (2001); Bershadsky, A. D. et al. Eur. J. Cell Biol. 14 Dec 2005 (10.1016/j.ejcb.2005.11.001); Wehrle-Haller, B.
  • ECM of cells is a complex 3D fibrous meshwork with a wide distribution of fibres and gaps that provide complex biochemical and physical cues, which are very different from uniformly coated 2D surfaces. Cell reactions to 3D matrices are altered from their reaction to 2D matrices of the same material (Cukierman, E. , Pankov, R. , Stevens, D. R. & Yamada, K. M.
  • Geometry sensing can refer to the formation of signalling complexes by changes in the spacing of molecular-recognition sites the geometrical shape of the substrates. For example, surface steps as small as 11 run can lead to contact guidance (Curtis, A. & Wilkinson, C. Biochem. Soc. Symp. 65, 15—26 (1999)). Restricting cells to spreading on adhesive micropatterns of various shapes can regulate cell proliferation and cell death (Chen, C. S. , Mrksich, M. , Huang, S., Whitesides, G. M. & Ingber, D.E.
  • micropatterns also regulates whether mesenchymal stem cells differentiate into adipocytes or osteoblasts (McBeath, R. , Pirone, D. M. , Nelson, C. M. , Bhadriraju, K. & Chen, C. S. Dev. Cell 6, 483-495 (2004)). Because micropatterns confine cell shape, thereby causing an integrated cellular response, the ability of cells to sense nanoscale surface features, including the size of nanoscale fibres and topographies, as well as the spatial presentation of molecular- recognition sites will be considered.
  • Fibroblasts move on collagen fibres by a specific mode of motility that involves one myosin isoform (Meshel, A. S., Wei, Q., Adelstein, R. S. & Sheetz, M. P. Nature Cell Biol. 1, 157-164 (2005).
  • the same myosin isoform was shown to be involved in in vivo-like organization and morphogenesis of fibroblasts grown on electrospun nanofibrous matrices (Schindler, M. et al. Biomaterials 26, 5624—5631 (2005). It has also been shown that 3D networks of nanofibres presenting the neurite-promoting laminin epitope promoted the selective differentiation of neural progenitor cells (Silva, G. A.
  • the BAR domains of arfaptins were shown to bind to the small GTPases Rac, adenosine-ribosylation factor- 1 (ARFl), ARF3 and ARF6, as well as to the ARF-like protein- 1 (ARLl ; Refs 93-96) (Lu, L., Horstmann, H., Ng, C. & Hong, W. J. Cell ScL 114, 4543-4555 (2001); Ta ⁇ cone, C. et al. Nature 41 1, 215-219 (2001); Van Aelst, L. , Joneson, T. & Bar- Sagi, D. EMBO J, 15, 3778-3786 (1996); Williger, B. T. , Ostermann, J.
  • BAP2 ⁇ /IRSp53 insulin-receptor substrate protein of 53 IcDa
  • WAVE Wiskott-Aldrich syndrome protein
  • K + channels are opened by a convex curvature of the membrane (Patel, A. J. , Lazdunski, M. & Honore, E. Curr. Opin. Cell Biol. 13, 422-428 (2001)).
  • Concave and convex membrane curvatures might be sensed by two different mechanisms.
  • BAR domains might sense the concave membrane curvature that is formed in contact with external posts and fibres, whereas membrane channels might selectively be opened if membranes come into contact with surface indentations.
  • BAR-domain proteins do indeed bind preferentially to concave surfaces, such as posts or fibres, the local release of Rac and its subsequent activation, for example by integrin-linked kinase (Filipenko, N. R. , Attwell, S. , Roskelley, C. & Dedhar, S. Oncogene 24, 5837-5849 (2005)), might lead to a local enhancement of traction forces.
  • Rhen activation is known to enhance focal- complex assembly (Filipenko, N.R., Attwell, S., Roskelley, C. & Dedhar, S. Oncogene 24, 5837-5849 (2005); Burridge, K. & Wennerberg, K Cell 116, 167-179 (2004); Civelekoglu- Scholey, G. et al. J. Theor. Biol. 232, 569-585 (2005); Machacek, M. & Danuser, G. Biophys. J. 90, 1439—1452 (2006)) and cells adhere more weakly on surfaces with nanopits than on those with nanoposts (Dalby, M. J., Riehle, M. O., Sutherland, D. S.
  • the rigidity of the external ligand linkage will determine how far the cytoskeleton-linked complexes will be displaced in a given time period before reaching isometric conditions and whether the force will be sufficient to activate any of the force sensors through the exposure of otherwise cryptic peptide sequences.
  • the separation between the fixed and moving components is greater, and might be sufficient to prevent the sensor from interacting with the stationary component (FIG. 4).
  • Extracellular signal-regulated kinases (ERKs) and Rho constitute part of an integrated mechanoregulatory circuit that links matrix stiffness, through integrin clustering, to cytoskeletal tension and, ultimately, regulation of tissue phenotype (Silver, F. H. & Siperko, L. M. Crit. Rev. Biomed. Eng. 31, 255-331 (2003); Ingber, D. E. Proc. Natl Acad. Sd. USA 102, 11571-11572 (2005); McBeath, R., Pirone, D.M., Nelson, C.
  • Transcription factors that are recruited to the adhesion sites could have an important role in translating the physical stimulus that is sensed at the periphery into biochemical signals that alter gene expression. Transcription factors might be modified in a force-dependent manner and transported to the nucleus; for example, paxillin is modified at focal-contact sites and is then transported to the nucleus (Woods, A. J. et al. J. Biol. Chem. 277, 6428-6437 (2002)). This mechanism provides an obvious way to transform force on specific intracellular-adhesion sites to a change in protein expression.
  • Tyrosine kinases that interact with G proteins of the Ras family to stimulate cell proliferation and differentiation are also downregulated, as is ⁇ -actinin (Dalby, M.J., Riehle, M.O., Sutherland, D. S., Agheli, H. & Curtis, A. S. Eur. Cell Mater. 9, 1-8 (2005)).
  • Many of the tyrosine kinases and phosphatases that have been linked to changes in cell and tissue shape are also linked to the early events of force and rigidity sensing (reviewed in Ref. 25) (Giannone, G. & Sheetz, M. P. Trends Cell Biol. (in the press)).
  • Cell behavior may be determined by cell dynamics and the state of the cellular environment. The latter parameters can allow a better understanding of the feedback mechanism between cell contractility and mechanosensing, as well as the role of pulsatory activity in cell function and replication. Such studies can also be motivated by the need for better tissue scaffolds, and implantable materials (Vogel, V. & Baneyx, G. Annu Rev Biomed Eng 5, 441-63 (2003)). Elucidating the dynamic nature of the interactions of the cell with its environment can clarify the requirements for better biomedical devices and drive the subsequent phases of research in engineered materials and technologies.
  • the mechanical perturbations can be generated using one of the following non-limiting examples of devices: pipet-assisted manipulation device, laser tweezer, optical trap, magnetic field generator, or a substrate stretching device, wherein a stationary clamp is attached to a first end of the substrate and a mobile clamp is attached to a second end of the substrate.
  • these devices can physically manipulate an engineered environment, such as a cell growth substrate or scaffold of the invention.
  • cell growth can be assessed via measuring a gross change in cell size, an increase in cell proliferation, an increase in focal adhesion assembly, or a combination thereof.
  • cell differentiation can be determined via examining the expression of target genes utilizing standard molecular biology methods practiced in the art.
  • apoptosis can be investigated via examining cell viability or the expression and/or downregulation of target genes.
  • cell movement can be assessed via motility assays utilized in the art.
  • cell morphology changes can be examined via light, fluorescent, or electron microscopy methods utilized by one skilled in the art.
  • a cell growth substrate of the invention can be permeable to liquids, gases, and cellular by-products. It can comprise a growth medium in addition to a cross-linked polymer, wherein increased strain on the polymer does not substantially increase rigidity of the polymer, and wherein the polymer has an elastic modulus (E) of about 0.1 kPa to about 10.0 kPa (E is described in Engler at al., (2004) Biophys J S6: 617-28, which is hereby incorporated by reference). Polymers are long chain organic molecules that are assembled from smaller molecules called monomers.
  • Polymers comprise many repeating monomer units in long chains, and can be classified as synthetic or natural polymers (for example, those of biological in nature that can comprise proteins, carbohydrates, and nucleic acids).
  • the polymer is cross-linked and can comprise natural polymers and their derivatives, synthetic polymers and their derivatives, or a combination thereof.
  • These natural polymers can be anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers.
  • Non-limiting examples of anionic polymers can include hyaluronic acid, alginic acid, carageenan, chondroitin sulfate, dextran sulfate, and pectin.
  • Some examples of cationic polymers include but are not limited to, chitosan or polylysine. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).
  • amphipathic polymers can include, but are not limited to collagen, gelatin, fibrin, and carboxymethyl chitin.
  • Non-limiting examples of neutral polymers can include dextran, agarose, or pullulan.
  • polyesters can also be used to generate the cell growth substrate of the invention.
  • polyesters can include polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyacrylamide, and polydimethylsiloxane.
  • a magnetic tweezers apparatus uses expertise in condensed matter physics with a focus in magnetism and magnetic materials, as well as interdisciplinary training in materials science, microfabrication, and cell biology, systems can be engineered for measuring the limits of cellular responses to mechanical perturbations.
  • a magnetic tweezers apparatus generates forces as large as 20 nN with frequencies from 0 to 3 kHz, values that match and exceed those found in tissues.
  • This system allows application of local forces at the position of interest: lamellipodium, lamella, and perinuclear region, via beads (for example, magnetic beads) attached to specific receptors on the cellular dorsal surface.
  • the bead can be a magnetic bead.
  • the diameter of the magnetic bead can be greater than or equal to about 0.1, 0.2, 0.3, 0.5, 0.6, 0.75, 0.8, 0.9, 1.0, 2.0, 2.5, 3, 4, or 5 ⁇ m in diameter.
  • the diameter of the bead is about 2.7 ⁇ m in diameter.
  • the cell growth substrate described above also comprises a magnetic material, such as a magnetic bead, wire and the like.
  • Non- limiting examples of magnetic material include nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt.
  • the trajectory of the beads in the example above is the result of the interplay between the magnetic force and the force exerted by the cell on the bead.
  • f ⁇ bronectin-coated beads were placed on laminin coated glass substrates and mouse embryonic fibroblasts (MEFs) were subsequently allowed to spread.
  • MEFs mouse embryonic fibroblasts
  • the adaptive phase is defined by the presence of fluctuations in the bead's velocity and direction of movement, and can last between 20 and 140 seconds. Since the magnetic force is constant, the variations in bead velocity indicate variations in the cell force. This can be interpreted as a cyclic testing of the site of mechanical stimulation. As the cell adapts to the local stress, it generates pulsatory traction forces at the site of the mechanical signal, i.e. the bead. This is reminiscent of the periodic lamellipodial contractions in spreading and migrating cells (Dobereiner, H. G., et al.,. J Appl Physiol 98, 1542-6 (2005); Dubin-Thaler, B.
  • Whole cell assays can allow the cellular adaptive response to be studied when the environment (adhesive substrate) has different rigidities or is pulsating.
  • two assays can be utilized: active and stretchable substrates.
  • Active substrates such as cell growth substrates
  • embedded magnetic materials such as magnetic
  • the nanowires comprise a magnetic material.
  • Some non- limiting examples of magnetic material include nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt.
  • the magnetic nanowires were nickel nanowires.
  • the diameter of the magnetic wire can be greater than or equal to about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm in diameter.
  • the diameter of the wire is about 300 nm in diameter.
  • the length of the magnetic wire (for example, the nanowire) can be greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ⁇ m in length, hi another embodiment, the length of the magnetic wire is about 30 ⁇ m in length.
  • nanowire is about 300 nm in diameter, 30 ⁇ m long, and is comprised of nickel.
  • Synthetic biodegradable, matrixes such as the cell grwoth substrate of the invention described in Example 1 or the microfence scaffold described in Example 2, can be manufactured using synthetic polymers (such as those described above), in addition to naturally occurring, absorbable materials, such as polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide- lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester), catgut suture, collagen, silk, chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid, elastin, and the like.
  • synthetic polymers such as those described above
  • absorbable materials such as polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide- lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester
  • An active substrate can be fabricated via suspending the wires in unpolymerized cell growth substrates (for example those made of polyacrylimide gels) and aligning them by a uniform magnetic field. Ferromagnetic nanowires are highly responsive to magnetic fields (Tanase, M. et al., (2001) Nano Letters 1, 155-158) and can be directed to self-assemble in arrays perpendicular to the gels' surface. Polymerization is then induced, and the wires remain in this configuration even after the aligning field is removed.
  • the active substrate can be fabricated via suspending magnetic beads in unpolymerized cell growth substrates (for example those made of polyacrylimide gels) and aligning them by a uniform magnetic field.
  • soft gels can be employed that do not support normal cell growth (FIG. 8B).
  • An external oscillating magnetic field will tilt the embedded magnetic nanowires, and transmit local oscillatory displacements at the surface of the gels and at the same time to the basal side of the cells (FIG. 8C).
  • the displacements achieved at the surface of the gels were on the order of hundreds of nanometers.
  • the cells may be able to sense the local displacements as sites of increased rigidity (FIG. 8D). This assay may allow the spatial correlation of mechano-response, and its controlled initiation.
  • the stress at the surface of the gels can be reliably modulated, and the density of nanowires in the array determines the distance between the wires and ultimately the number of oscillating sites per cell.
  • the mechano-induced cellular functions can be controlled.
  • substrates can be developed (such as those described above) that support many cells while being stretched. Cells can be plated on soft gels that will be subject to oscillatory stresses. A system that provides a stress gradient within the same substrate can therefore generate parallel data for cells in different mechanical environments.
  • the invention provides an apparatus for applying a mechanical perturbation to a cell (for example, a cell in vitro), wherein the apparatus comprises the cell growth substrate described above in addition to an external force generator associated with the substrate.
  • the apparatus can further comprise a means for quantitating a cellular response to the mechanical perturbation.
  • cell responses can include cell growth; cell differentiation; apoptosis; cell movement/cytokinetics; and cell morphology changes.
  • cell growth can be assessed via measuring a gross change in cell size, an increase in cell proliferation, an increase in focal adhesion assembly, or a combination thereof.
  • gross changes in cell size can be determined with microscopy methods (light, fluorescence, electron, and the like) by obtaining cell size measurements.
  • Cell proliferation can be examined via FACS analysis, cell density readings (i.e., OD ⁇ oo absorption readings), or other methods commonly used in the art.
  • cell differentiation can be determined via examining the expression of target genes (for example, via examining mRNA or protein levels) utilizing standard molecular biology methods practiced in the art.
  • apoptosis can be investigated via examining cell viability or the expression and/or downregulation of target genes (for example, via examining mRNA or protein levels).
  • cell movement can be assessed via motility assays utilized in " the art.
  • cell morphology changes can be examined via light, fluorescent, or electron microscopy methods utilized by one skilled in the art.
  • the external force generator is a means for physically manipulating the cell growth substrate.
  • external force generating devices include a pipet-assisted manipulation device, laser tweezers, a magnetic twisting cytometry device, an optical trap, a magnetic field generator, and a substrate-stretching device (for example, a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate) (Sniadecki et al., (2006) Annals Biomed Eng 34(1): 59-74). . . .
  • a purpose of the whole-cell assay consisting of stretchable substrates is to investigate the long-term effects of pulsatory signal in soft environments on cell proliferation and migration.
  • the invention provides a method for stimulating growth of a cell in vitro.
  • cells can be plated on or in the cell growth substrate described above, the substrate containing the cell is perturbed (for example, mechanically, electrically, or magnetically), and the growth of the cell of the cell is detected, wherein the perturbation of the substrate stimulates the growth of the cell.
  • the invention also provides a method for stimulating cell differentiation in vitro where cells can be plated on or in the cell growth substrate described above, the substrate containing the cell is perturbed (for example, mechanically, electrically, or magnetically), and the differentiation of the cell of the cell is detected, wherein the perturbation of the substrate stimulates the differentiation of the cell.
  • an elastic modulus is the mathematical description of an object or substance's tendency to be deformed along an axis when an opposing force is applied along that axis.
  • (E) describes tensile elasticity and thus reflects the measure of the stiffness of a given material. It is defined as the ratio of tensile stress to tensile strain and can be experimentally determined from the slope of a stress-strain curve created during tensile tests conducted on a sample of the material (described in Engler et al., (2004) Biophys J 86: 617-28, which is hereby incorporated by reference).
  • the cell growth substrate can have an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa.
  • the cell growth substrate can promote a fibroblast cellular response (such as growth, differentiation, apoptosis, cytokinetics, morphological changes, and the like).
  • the substrate can promote a myoblast cellular response, such as those previously described.
  • the cell growth substrate can promote a neuronal cellular response, such as those described above.
  • the growth of the cell can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or a combination thereof. Examples of methods used to detect cell growth have been described above.
  • differentiation of the cell can be detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or a combination thereof.
  • the -expression of target genes can be ascertained by examining mRNA or protein-levels utilizing standard molecular biology and biochemical methods practiced in the art. Cell morphology changes in addition to changes in size can be examined via light, fluorescence, or electron microscopy methods utilized by one skilled in the art.
  • perturbing the cell growth substrate entails subjecting the substrate harboring cells to an external force generator previously described.
  • the cell can be excised from a tissue (for example muscular tissue, such as skeletal, cardiac, or smooth; neuronal; connective; epithelial; or haemopoietic).
  • the tissue can come from a cultured cell line or an animal (for example a mammal, such as a dog, cat, human, bird, and the like).
  • the invention also provides for a method of promoting the growth of a cell in vivo.
  • the method can comprise the ex vivo creation of a matrix from the cell growth substrate described above followed by insertion of the matrix into the body of a subject and subsequently applying a magnetic field to the subject, wherein the application of the magnetic field can promote the growth of the cell.
  • the invention provides for a method of treating a wound, where the method can comprise the ex vivo creation of a matrix from the cell growth substrate described.above followed by insertion of the matrix into the body of a subject and subsequently applying a magnetic field to the subject, wherein exposure to the magnetic field can result in the proliferation of cells.
  • the subject can be an animal (for example a mammal, such as a dog, cat, human, horse, cow, sheep, rabbit, bird, and the like).
  • the cell growth substrate can have an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa.
  • the substrate can have an elastic modulus of about 3 kPa, wherein the cell substrate can promote a fibroblast cellular response.
  • the substrate can have an elastic modulus of about 2 kPa, wherein the substrate can promote a myoblast cellular response.
  • the substrate can have an elastic modulus of about 1 kPa, wherein the cell substrate can promote a neuronal cellular response.
  • Mechanical perturbations can stimulate a cellular response.
  • perturbing the cell substrate entails subjecting the substrate harboring cells to an external force generator previously described.
  • the growth of the cell can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or a combination thereof. Examples of methods used to detect cell growth have been described above.
  • Cell differentiation can be detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or a combination thereof.
  • the expression of target genes can be ascertained by examining mRNA or protein levels utilizing standard molecular biology and biochemical methods practiced in the art.
  • Cell morphology changes in addition to changes in size can be examined via light, fluorescence, or electron microscopy methods utilized by one skilled in the art.
  • EXAMPLE 1 - RPTP ⁇ is required for the fibroncctin-specific rigidity response in hippocampal neurons
  • RPTP ⁇ receptor-like protein tyrosine phosphatase alpha
  • RPTP ⁇ was shown to mediate fibronectin-specific rigidity responses in hippocampal neurons in an SFK-dependent process that leads to the recruitment of fibronectin and pl30Cas phosphorylation at the leading edge of the growth cone.
  • the results show that the rigidity of a cell growth substrate can be manipulated to affect a cellular response, such as cell growth and differentiation (for example, as shown with neurons).
  • RPTP ⁇ is reported to be required for reinforcement of fibronectin-cytoskeleton bonds and the rigidity response in hippocampal neurons.
  • Neurons were plated on coverglass or gels that were pre-incubated with 20 ⁇ g/mL polylysine and subsequently coated with 50 ⁇ g/mL laminin (BD Biosciences) or 50 ⁇ g/mL fibronectin (Roche).
  • Optical gradient laser trap set at 10OmW (40pN/ ⁇ m) (Axiovert TV 100; Carl Zeiss Microimaging, Inc) was equipped with a 10Ox objective, and calibrated as described previously (Choquet, D., et al., (1997). Cell 88, 39-48).
  • the beads were held at the smooth lamelipodium-like edges of the growth cones for approximately 3 -5 s, and then, the laser trap was turned off to check for binding. If the bead was bound, the trap was turned back on and the rearward movement of the bead was recorded using a cooled CCD camera.
  • the fraction of the beads bound and moving rearward was calculated as mean + standard error for at least three independent experiments and statistical significance of the results confirmed by t-test (p ⁇ .01).
  • the total number of beads included in analysis was at least 35 beads for each condition.
  • the rearward movements of the beads were further analyzed using Nanotracker plug-in, with a tracking accuracy of 3-5nm for 0.64 ⁇ m beads.
  • the MSD values were calculated using an algorithm modified from (Qian, H., et al., (1991). Biophys J 60, 910-921).
  • the uniformity of coating on the substrate surface was examined by coating the gels with proteins conjugated to Cy5 fluorophore (Amersham Biosciences) according to manufacturer's instructions and visualized by confocal microscopy. Experiments were performed 36h after the neurons were plated on the polyacrylamide gels. Neurite extension was quantified for at least 50 neurites for each condition and statistical significance of the results confirmed by t-test (p ⁇ .01). Data is presented as mean+standard error of at least two independent experiments. In experiments with SFK inhibitor, lO ⁇ M SU6656 (Calbiochem) was added after neurons were adhered to the substrate, and further incubated for total time of 36h.
  • BD Transduction Laboratories BD Transduction Laboratories
  • X v ⁇ antibody USBiological
  • a mouse monoclonal anti-Tau antibody Biosource
  • an affinity purified polyclonal rabbit anti-phoshoY165Cas antibody Cell Signaling Technology
  • anti-cts ⁇ i Chemicon
  • anti- ⁇ v BD Pharmingen
  • anti- ⁇ v ⁇ USBiological antibodies
  • the fraction of breaking events was reciprocally proportional to the rigidity of the trap and hence to the rigidity response (Jiang, G., et al., (2003). Nature 424, 334-337). As predicted, the number of breaking events was significantly higher in RPTP ⁇ -/- neurons than in controls (Fig. 10C). Therefore, the reinforcement of FN-clustered integrin-cytoskeleton bonds was impaired in RPTP ⁇ -/- growth cones, indicating that the rigidity response was impaired as well. [00141] The effect of RPTP ⁇ deletion on reinforcement was further determined by quantifying the diffusivity of the beads through calculation of the mean square displacement (MSD).
  • MSD is used as a measure of the stiffness of the bead-cytoskeleton contact and is inversely proportional to the bead reinforcement (Choquet, D., et al., (1997). Cell 88, 39-48; Qian, H., et al., (1991). Biophys J 60, 910-921). Individual trajectories of the beads were generated (FIG. 1OE, 10F), and MSD was determined as described previously (Qian, H., et al., (1991). Biophys J 60, 910-921). The average MSD of the beads moving rearwards was determined during the initial period of time after the beads moved outside of the trap.
  • the average MSD of the beads was two to threefold higher during early rearward movement on RPTP ⁇ -/- growth cones compared to RPTPa+/+ growth cones (FIG. 10G). MSD was on average higher for FN-coated beads bound to RPTP ⁇ -/- growth cones compared to RPTPa+/+ growth cones (FIG. 10G); however, no significant difference was observed for VN-coated beads (FIG. 10H).Thus, the greater bead diffusion along perpendicular axis in RPTP ⁇ -/- growth cones supports the hypothesis that RPTP ⁇ is required for reinforcement of integrin-cytoskeleton bonds.
  • VN vitronectin
  • RPTP ⁇ may be involved in a signaling pathway upregulated by the activation of (a) FN-specific integrin(s).
  • FN-stimulated RPTPa signaling is activated through a v ⁇ 6 integrin
  • the particular integrin involved in the RPTP ⁇ -mediated reinforcement in neurons was investigated next. The expression of a variety of FN-specific integrin subunits was reported in the hippocampus (Pinkstaff, J. K., et al., (1999). J Neurosci 19, 1541-1556), including ⁇ v ⁇ e integrins (Chan, C. S., et al., (2003). J Neurosci 23, 7107-7116), whose expression was previously believed to be limited to epithelial cells.
  • RPTP ⁇ was abundantly present in the growth cones of neurons plated both on FN and LN.
  • FIG. 1 1C, 1 ID an increased fraction of OC v ⁇ integrins accumulated at the edge of the growth cone upon interaction with FN may cause RPTP ⁇ activation, leading to the upregulation of the downstream rigidity response pathway.
  • RPTPcc ⁇ neurons are deficient in FN-specific rigidity response
  • Fibronectin and laminin show differential distribution in the mammalian brain during development (Chun, J. J., and Shatz, C. J. (1988). J Cell Biol 106, 857-872; Hagg, T., et al., (1989). Neuron 3, 721-732).
  • stage 1 is characterized by the absence of neurites; at stage 2, neurites of approximately equal lengths are extended (FIG. 12A); and at stage 3, the significantly longer axons are differentiated (FIG. 12B).
  • Neurons were isolated from the brains of neonate mice (Pl) and plated on FN -coated polyacrylamide gels of varying rigidities. After 48h of incubation in serum-free medium, the lengths of extended neurites were measured and the differentiation stages of the neurons were determined (FIG. 12C).
  • RPTPa+/+ neurons differentiated faster on soft than stiffer FN-coated substrates (38.7%+4.8% neurons at stage 3 on rigid, 39.5%+3.7% on intermediate, and 62.7%+2.3% on soft)
  • RPTP ⁇ -/- showed no preference for soft substrates (62.0%+6.5% neurons at stage 3 on rigid, 64.1%+6.6% on intermediate, and 66.3%+5.6% on soft) and differentiated at a rate similar to wild type neurons plated on soft FN-coated gels.
  • the average lengths of the neurites both axons and dendrites were also reflective of the absence of a FN rigidity response in the absence of RPTP ⁇ .
  • the soft substrate stimulated neurite extension in RPTPa+/+ neurons
  • SFKs and in particular Fyn, have been previously implicated as RPTP ⁇ substrates in a variety of processes including the FN rigidity response in fibroblasts, SFKs may be involved in this process in neurons as well. Therefore, the effect of a broad SFK inhibitor (10 ⁇ M SU6656) on the neurite extension and rigidity response to FN-coated substrates was examined. Similar to RPTP ⁇ -/- neurons, neurons cultured in the presence of the SFK inhibitor, showed no preference for the soft FN matrices, and there was no difference in neurite extension between substrates of different rigidities in the presence of inhibitor (FIG. 14A).
  • pl30Cas was known as an indispensable component in the regulation of actin cytoskeleton organization, focal contact formation, and migration of fibroblasts (Cary et al., (1998). J Cell Biol. 140(l):211-21; Cho and Klemke (2000). J Cell Biol. 149(l):223-36; Honda et al., (1999.) Biochem Biophys Res Commun. 262(l):25-30), its role in neuronal motility has been poorly understood.
  • dissociated hippocampal neuronal cultures displayed different growth properties on polyacrylamide gels of different rigidities.
  • RPTP ⁇ -/- mice display a severe hippocampal phenotype (Petrone, A., et al., (2003). Embo J 22, 4121-4131), and that RPTP ⁇ was implicated in force transduction and the rigidity response in fibroblasts (Jiang, G., et al., (2006). Biophys J 90, 1804-1809; Von Wichert, G., et al., (2003). J Cell Biol 161, 143- 153), RPTP ⁇ ablation may affect the rigidity response in neurons as well.
  • RPTP ⁇ -/- neurons lacked the ability to distinguish between FN-coated substrates of varying rigidities, unlike RPTPa+/+ neurons which differentiated faster and grew longer neurites on softer than on stiffer substrates, hi contrast, the LN rigidity response was not affected by the absence of RPTP ⁇ , indicating FN specificity of the integrin(s) that activate RPTP ⁇ .
  • the correlation between the rigidity response and the reinforcement of integrin-cytoskeleton bonds was confirmed (Choquet, D., et al., (1997). Cell 88, 39-48). Motile growth cones were logical candidates to test for the impairment of reinforcement.
  • the RPTP ⁇ -mediated rigidity response in hippocampal neurons was shown to be SFK-dependent and that Fyn, as well as its direct substrate pl30Cas localize to the leading edge of the growth cones in a rigidity-dependant manner.
  • the role of Fyn in the neuronal rigidity response might seem contradictory, since the broad inhibitor of SFKs had not only inhibited the rigidity response, but also overall neurite extension. This can be explained by a specific and non-redundant role that Fyn plays in the rigidity response. Therefore, the molecular mechanism of the FN rigidity response in neurons may be mediated through a pathway similar to the one proposed in fibroblasts (Jiang, G., et al., (2006).
  • FIG. 15 shows that rigidity could be sensed by the relative displacement of RPTP ⁇ -immobilized Fyn and a liganded integrin complex with pl30Cas that depends upon the rigidity of the surrounding matrix.
  • the rigidity of the matrix triggers force-dependent activation of the RPTP ⁇ , followed by activation of Fyn, that consequently phosphorylates stretch-sensitive pl30Cas. This results in the further recruitment of the focal contact proteins causing the reinforcement of the interaction between the growth cone and the substrate. This reinforcement has a negative effect on the neurite extension.
  • the force exerted by the actin-myosin network in response to the substrate rigidity does not reach critical threshold necessary for the reinforcement of the FN-cytoskeleton bonds and subsequent focal contact formation. Therefore, the neurite extension is stimulated on soft matrices.
  • This model proposes that Fyn plays a critical role through its immobilization by palmitate groups to lipid domains near the leading edge. This is speculative but the phenotype of the Fyn knockout mice is similar to that of the RPTP ⁇ -/- in several respects (Grant et al., (1992) Science 258(5090): 1903-10). Similarly, pl30Cas is an important component in many motility pathways including rigidity response (Kostic and Sheetz, (2006) MoI Biol Cell. 17(6):2684-95; Tamada, M., et al., (2004). Dev Cell 7, 709-718; Vuori and Ruoslahti (1995) J Biol Chem.
  • pl30Cas-/- mice die in utero before the brain has functionally developed making it difficult to determine the effect of pl30Cas ablation on brain development and function (Honda et al., (1998) Nat Genet. 19(4):309-l 1). Nevertheless, studies in dissociated cerebellar neurons showed that pl30Cas is required for neurite extension (Huang, J., et al., (2006). MoI Biol Cell 17, 3187-3196). Further, phosphorylation of p 130Cas has been recently shown to be directly related to force transduction (Sawada et al., (2006). Cell. 127(5):1015-26).
  • pl30Cas could signal to a variety of different pathways that normally promote growth and not differentiation.
  • the model is plausible but much more is needed to prove the exact roles of the components.
  • the same molecular components would be implicated in the rigidity responses of such different cells as fibroblasts and neurons, particularly since in the two cases the cells have different rigidity responses.
  • the response to the rigid matrix is needed for growth and motility, whereas, in neurons, the response to rigid matrix inhibits differentiation and mobility of the growth cones.
  • the response to rigid matrix appears to promote proliferation, and not differentiation.
  • the response to soft matrices is apoptosis in the case of the fibroblasts and increased differentiation in the case of neurons.
  • Neuronal growth cones are smaller than lamellae of the fibroblasts and they are known to pull the neurites forward. Reinforcement of the integrin-cytoskeleton bonds and focal contact formation stabilizes the lamellipodia and supports cell spreading in fibroblasts (Choquet, D., et al., (1997). Cell 88, 39-48; Giannone, G. et al. (2004). Cell 116, 431-43). Growth cones distinguish FN from LN by forming contacts on FN similar to focal contacts characterized in fibroblasts (Gomez, T. M., et al., (1996). J Neurobiol 29, 18-34).
  • the stiff substrates may support formation of these focal contacts, which in turn stabilize interactions between growth cones and the substrate. This might result in reduced velocity of the growth cone progression and eventually in shorter neurites (FIG. 15). Similar rigidity responses could lead to different cellular responses in different cell backgrounds.
  • the FN-rigidity response in neurons may require RPTP ⁇ activation through CC v ⁇ ⁇ integrins, and subsequent recruitment of Fyn and its substrate pi 30Cas to the leading edge.
  • This pathway appears to be critical for matrix rigidity-dependent regulation of neurite extension and axon differentiation, which can explain at least some of the abnormal aspects of hippocampal structure and function in RPTPot-/- mice. Since FN plays an important regulatory role in both normal development and variety of pathological processes in the brain, one could use these findings as a basis for further understanding of the basis for those diseases.
  • fences are 1--2 micrometers in thickness, 5-10 micrometers in height, and up to lmm in length with spacing of 20 to 100 micrometers (see FIG. 16).
  • Piezo actuators P ⁇ ezo Systems, Cambridge, MA
  • magnetic wires in the fences that will move them maximally 1-2 micrometers.
  • Fibers will be electrospun of silk or other polymers and stretched over the fences in one-dimensional arrays (FIG. 16). Fiber diameters of 100 to 1000 nm will be used and fibers will be coated with growth factors, adhesion proteins, extracellular matrix molecules or fragments, or any combination thereof. In a typical experiment, cells will be applied to the fibers and followed over time to determine growth and differentiation patterns along with forces and morphology. [00165]

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Abstract

The present invention provides for a cell growth substrate, wherein the substrate comprises a growth medium and a cross-linked polymer, wherein increased strain on the polymer does not substantially increase rigidity of the polymer, and wherein the polymer has an elastic modulus (E) of about 0.1 kPa to about 10.0 kPa. An apparatus for applying a perturbation to a cell in vitro is also provided, wherein the apparatus comprises the cell growth substrate and an external force generator associated with the substrate. The invention further provides methods for stimulating growth of a cell in vitro, cell differentiation in vitro, and a cellular response in vitro, for promoting growth of a cell in vivo, for treating a wound.

Description

ACTIVE CELL GROWTH SUBSTRATES AND USES THEREOF
[0001] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. [0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
[0003] The work described herein was supported in whole, or in part, by National Institute of Health Grant No. GM036277-20 A2. Thus, the United States Government has certain rights to the invention.
BACKGROUND OF THE INVENTION
[0004] The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms. [0005] Throughout the biological kingdom there is a wide diversity of shapes, and this phenomenon has interested physical biologists for a long time (see On Growth and Form. D'Arcy W. Thompson (Dover Publications, 1992)). It is still unknown how cells of 10-40 μm in diameter can assemble and reproducibly shape an organism that is metres in size, nor is it known how cells recognize their spatial position within such multicellular systems. During development cellular monolayers curve and contort to create the complex morphology of the different tissues. Extracellular matrices and their neighbouring cells constitute the main signals that any individual cell uses to establish and maintain its shape. However, there is a wonderful diversity in the form of biological systems that is often intimately linked to cellular function. Therefore, cells must sense physical aspects of their environment and respond appropriately over time for proper cell function.
SUMMARY OF THE INVENTION
[0006] The invention is based, in part, on the finding that a cell growth substrate having a variable rigidity can be constructed and utilized to control cellular functions and responses. [0007] The invention provides for a cell growth substrate comprising: a) a cross-linked polymer, wherein increased strain on the polymer does not substantially increase rigidity of the polymer, and wherein the polymer has an elastic modulus (E) of about 0.1 kPa to about 10.0 kPa; and b) a growth medium, wherein the substrate is permeable to liquids, gases, and cellular by-products. In one embodiment, the polymer is not collagen, not hyaluronic acid, not polydimethylsiloxane (PDMS), and not a carbohydrate based gel. In one embodiment, the polymer comprises an anionic polymer, a cationic polymer, an amphipathic polymer, a neutral polymer, a synthetic polymer, or any combination thereof. In one embodiment, the cationic polymer comprises chitosan or polylysine. In one embodiment, the amphipathic polymer comprises gelatin, fibrin, or carboxymethyl chitin. In one embodiment, the neutral polymer comprises dextran, agarose, or pullulan. In one embodiment, the synthetic polymer comprises a polyester or derivative thereof. In one embodiment, the polyester comprises polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, or polyacrylamide. In one embodiment, the polymer is functionalized with chemical groups. In another embodiment, the substrate further comprise a magnetic material, a Piezo actuator, or a combination thereof. The substrate contains such a material in order to provide a way to cause the substrate to be moved, strained or stretched. In one embodiment, the magnetic material comprises nickel or iron. In one embodiment, the magnetic material is in the form of a bead. In one embodiment, the bead is about 0.1 mm to about 5 mm in diameter, about 0.2 mm to about 4 mm in diameter, about 0.3 mm to about 3 mm in diameter, about 0.4 mm to about 2 mm in diameter, or about 0.5 mm to about 1 mm in diameter. In one embodiment, the bead is about 2.7 mm in diameter. In one embodiment, the magnetic material is in the form of a nanowire. In one embodiment, the nanowire is about 2-50 mm in length, about 3-40 mm in length, about 4-30 mm in length, about 5-20 mm in length, or about 6-10 mm in length. In one embodiment, the nanowire is about 30 mm in length, hi one embodiment, the nanowire is about 100-500 nm in diameter, about 150-450 nm in diameter, about 200-400 nm in diameter, or about 250-350 nm in diameter. In one embodiment, the nanowire is about 300 nm in diameter. In one embodiment, the elastic modulus is about 0.3 kPa to about 8 kPa, about 0.5 kPa to about 7 kPa, about 0.6 kPa to about 6 kPa, about 0.7 kPa to about 6 kPa, about 0.8 kPa to about 5 kPa, about 0.9 kPa to about 4 kPa, or about 1.0 kPa to about 3 kPa.
[0008] The invention also provides for an apparatus for applying a perturbation to a cell in vitro, the apparatus comprising: a) the substrate described herein; and b) an external force generator associated with the substrate. In one embodiment, the external force generator comprises an electrical force, a magnetic force, a mechanical force, or a combination thereof. In one embodiment, the external force generator comprises a pipet-assisted manipulation device, a laser tweezer, an optical trap, or a magnetic field generator. In one embodiment, the external force generator stretches the substrate. In one embodiment, the substrate is stretched intermittently. In one embodiment, the external force generator comprises a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate. In one embodiment, the apparatus further comprises a means for quantifying a cellular response to the perturbation. In one embodiment, the cellular response quantified is cell growth, cell differentiation, apoptosis, cell movement, cell proliferation, cell morphology changes, or a combination thereof.
[0009] The invention provides a scaffold for supporting cells, the scaffold comprising: a) portions of the substrate described herein; and b) fibers stretched over the substrate portions. In one embodiment, the portions are strips in the form of a fence. In one embodiment, the fence is about 0.5 μm to about 3 μm thick, about 0.6 μm to about 2.5 μm thick, about 0.7 μm to about 2 μm thick, about 0.75 μm to about 1.5 μm thick, or about 0.8 μm to about 1 μm thick. In one embodiment, the fence is about 2 μm thick. In one embodiment, the fence is about 3 μm to about 12 μm in height, about 4 μm to about 11 μm in height, about 5 μm to about 10 μm in height, about 5.5 μm to about 9 μm in height, or about 6 μm to about 8 um in height. In one embodiment, the fence is spaced about 10 μm to about 200 μm apart, about 15 μm to about 150 μm apart, about 20 μm to about 100 μm apart, or about 25 μm to about 50 μm apart. In one embodiment, the fence is spaced about 25 μm apart, hi one embodiment, the fence is less than about 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 μm in length. In one embodiment, the fiber is absorbable. In one embodiment, the fiber comprises polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide-lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester), catgut suture, collagen, silk, chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid, elastin, or any combination thereof. In one embodiment, the fiber is about 50 nm to about 1500 run in diameter, about 100 nm to about 1250 nm in diameter, about 200 nm to about 1000 nm in diameter, about 250 nm to about 900 nm in diameter, about 300 nm to about 800 nm in diameter, about 350 nm to about 750 nm in diameter, about 400 nm to about 700 nm in diameter, or about 500 nm to about 600 nm in diameter. In one embodiment, the fiber is coated with a growth factor, an extracellular matrix molecule, or a combination thereof. In one embodiment, the extracellular matrix molecule comprises hyaluronic acid, collagen, chondroitin, or a combination thereof. [0010] The invention also provides a method for stimulating a cellular response in vitro, the method comprising: a) plating a cell on, or in, the substrate described herein or the scaffold described herein; b) perturbing the substrate or the scaffold containing the cell; and c) detecting the cellular response, the perturbation of the substrate or the scaffold stimulating the cellular response. In one embodiment, the perturbation comprises a mechanical perturbation, an electrical perturbation, a magnetic perturbation, or any combination thereof. In one embodiment, perturbing comprises subjecting the substrate or the scaffold to the external force generator. In one embodiment, the external force generator stretches the substrate or the scaffold. In one embodiment, the substrate or scaffold is perturbed intermittently. In one embodiment, the substrate or scaffold is perturbed up to 10 times per hour, up to 5 times per hour, up to 2 times per hour, or 1 time per hour. In one embodiment, the substrate or scaffold is perturbed up to 10 times per day, up to 5 times per day, up to 2 times per day, or 1 time per day. In one embodiment, the substrate or scaffold is perturbed up to 10 times per week, up to 5 times per week, up to 2 times per week, or 1 time per week. In one embodiment, the cellular response is cell growth, cell differentiation, apoptosis, cytokinetics, moφhological changes, or a combination thereof. In one embodiment, the cellular response is detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, mRNA expression levels, a change in cell morphology or a combination thereof. In one embodiment, the substrate or the scaffold has an elastic modulus of about 3 kPa. In one embodiment, the substrate or the scaffold promotes a fibroblast cellular response. In one embodiment, the substrate or the scaffold has an elastic modulus of about 2 kPa. In one embodiment, the substrate or the scaffold promotes a myoblast cellular response. In one embodiment, the substrate or the scaffold has an elastic modulus of about 1 kPa. In one embodiment, the substrate or the scaffold promotes a neuronal cellular response. In one embodiment, the cell is obtained from a tissue. In one embodiment, the cell (a) is a primary cell, (b) is a cell from a cell culture that has been passaged, or (b) is a cell from a cell line.
[0011] The invention also provides a method for promoting growth of a cell in vivo, the method comprising: a) inserting the scaffold into a subject; and b) applying a magnetic field to the subject, wherein the application of the magnetic field promotes growth of the cell. In one embodiment, the scaffold has an elastic modulus of about 0.3 kPa to about 8 kPa, about 0.5 kPa to about 7 kPa, about 0.6 kPa to about 6 kPa, about 0.7 kPa to about 6 kPa, about 0.8 kPa to about 5 kPa, about 0.9 kPa to about 4 kPa, or about 1.0 kPa to about 3 kPa. In one embodiment, the scaffold has an elastic modulus of about 3 kPa. In one embodiment, the scaffold promotes a fibroblast cellular response. In one embodiment, the scaffold has an elastic modulus of about 2 kPa. In one embodiment, the scaffold promotes a myoblast cellular response. In one embodiment, the scaffold has an elastic modulus of about 1 kPa. In one embodiment, the scaffold promotes a neuronal cellular response. In one embodiment, cell growth comprises wound healing in a subject. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human, a dog, a cat, a mouse, a rat, a horse, a pig, a cow, or a bird. [0012] One aspect of the present invention provides for a cell maintenance substrate permeable to liquids, gases, and cellular by-products, that is made up of a growth medium and a polymer with a constant elasticity through stretch and a rigidity where the elastic modulus (E) is in the range of about 0.1 kPa to about 10.0 kPa. In one embodiment, the elastic modulus of the substrate is about 0.3 kPa to about 8 kPa. In another embodiment, the polymer is cross-linked. In other embodiments, the polymer comprises natural polymers and their derivatives, synthetic polymers and their derivatives, or some combination. In some embodiments, the natural polymers may include anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers. In particular embodiments, the anionic polymers comprise hyaluronic acid, alginic acid, carageenan, chondroitin sulfate, dextran sulfate, or pectin. In another embodiment, the cationic polymers may include chitosan or polylysine. In a further embodiment, the amphipathic polymers may include of collagen, gelatin, fibrin, or carboxymethyl chitin. In further embodiments, the neutral polymers may inlcude dextran, agarose, or pullulan. In some embodiments, the synthetic polymers can be polyesters or derivatives thereof. In particular embodiments, the polyesters comprise polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyacrylamide, or polydimethylsiloxane. In further embodiments, the cell maintenance substrate of the present invention further includes a magnetic material. In some embodiments, the magnetic material may comprise nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt. In a specific embodiment, the nanowire is made of nickel. In another embodiment, the magnetic material is in the form of a bead. In a further embodiment, the diameter of the magnetic bead is greater than or equal to about 0.1, 0.2, 0.3, 0.5, 0.6, 0.75, 0.8, 0.9, 1.0, 2.0, 2.5, 3, 4, or 5 μm in diameter. In particular embodiments, the diameter of the
bead is about 2.7 μm in diameter. In some embodiments, the magnetic material is in the form of a nanowire. In other embodiments, the diameter of the magnetic wire is greater than or equal to about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 run in diameter. In further embodiments, the diameter of the wire is about 300 nm in diameter. In particular embodiments, the length of the magnetic wire is greater than or equal to about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μm in length. In specific embodiments, the length of the magnetic wire is about 30 μm in length.
[0013] In another aspect, the invention provides an apparatus for applying a mechanical perturbation to a cell in vitro, wherein the apparatus is made up of the cell maintenance substrate described above and an external force generator associated with the substrate. In particularly useful embodiments, the external force generator acts as a means for physically manipulating the substrate. In one embodiment, the external force generator may entail a pipet-assisted manipulation device. In another embodiment, the external force generator may be a laser tweezer. In a further embodiment, the external force generator comprises an optical trap. In a particular embodiment, the external force generator stretches the substrate. In specific embodiments of the invention, the external force generator may include a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate. In some embodiments, the external force generator can be a magnetic field generator. The apparatus of the present invention may further encompass a means for quantitating a cellular response to the mechanical perturbation. In one embodiment, the cellular response quantitated is cell growth, cell differentiation, apoptosis, cell movement, cell morphology changes, or some combination of these responses. Elastic modulus (E) refers to tensile elasticity and reflects the measure of the stiffness of a given material. In one embodiment, the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 IcPa. In specific embodiments, the substrate promotes a neuronal cellular response. Various cell types can be sustained on the cell maintenance substrate of the invention. In one embodiment, the cell is excised from a tissue. In another embodiment, the cell is excised from the tissue of an animal. In a further embodiment, the cell is excised from the tissue of a mammal, hi some embodiments, the cell is excised from the tissue of a human. In additional embodiments, the cell is obtained from a cultured cell line.
[0014] In other aspects, the invention provides a method for stimulating growth of a cell in vitro, wherein cells are plated on or in the cell maintenance substrate described above, the substrate containing the cells is perturbed (for example, mechanically, electrically, or magnetically), and growth of the cell is subsequently detected. In one embodiment, mechanically perturbing the substrate entails subjecting the substrate to an external force generator described above which results in stimulating growth of cells in the substrate. In another embodiment, growth of the cell is detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or some combination of these measurements. hi one embodiment, the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response. Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
[0015] In additional aspects, the invention provides a method for stimulating cell differentiation in vitro, wherein cells are plated on or in the cell maintenance substrate previously described above, the substrate containing the cells is mechanically perturbed, and differentiation of cells is detected. Mechanical perturbation of the substrate stimulates the differentiation of the cell and entails subjecting the substrate to an external force generator described above. In one embodiment, differentiation of the cell is detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or some combination of these measures. In one embodiment, the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response. Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above. [0016] In another aspect, the invention provides for a method of stimulating a cellular response in vitro. The method includes plating a cell on or in the cell maintenance substrate described above, mechanically perturbing the substrate containing the cell, and detecting a cellular response, wherein the mechanical perturbation of the substrate stimulates a cellular response. In one embodiment, mechanically perturbing the substrate entails subjecting the substrate to an external force generator described above. In another embodiment, the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination. The cellular response can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or some combination. In one embodiment, the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response. Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above. [0017] In further aspects, the current invention provides for a method of promoting growth of a cell in vivo, which includes creating a matrix ex vivo from the cell maintenance substrate described above, inserting the matrix into the body of a subject, and applying a magnetic field to the subject. Application of the magnetic field promotes the growth of the cell within the subject. In one embodiment, the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination. In one embodiment, the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In particular embodiments, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response. Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
[0018] In another aspect, the invention also provides for a method of treating a wound, which entails creating a matrix ex vivo from the cell maintenance substrate described above, inserting the matrix into the body of a subject, and applying a magnetic field. The exposure of cells to a magnetic field results in the proliferation of the cells. In one embodiment, the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination of the responses. In some embodiments, the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 IcPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response. Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
BRIEF DESCRIPTION OF THE DRAWINGS [0019] FIG. 1. is a schematic that emphasizes the steps involved in the responses of cells to their environment.
[0020] FIG. 2 illustrates the three basic mechanisms of force sensing. FIG. 2A depicts the conversion of force into biochemical signals by partial protein unfolding (as shown for the fibronectin module III). This can result in the gain or loss of binding sites, increased separation between protein domains, or the gain or loss of enzyme function. FIG. 2B depicts the opening of some mechanosensitive ion channels can be regulated by membrane tension (Ba), whereas the opening of others requires that their intra- and/or extracellular domains are physically connected to force-bearing filaments (Bb). FIG. 2C shows the Stabilizing receptor-ligand bonds by switching them to a long-lived state by force (catch bonds). Yellow arrows indicate forces. [0021] FIG. 3 are schematics that illustrate (a) how inward curvature of the plasma membrane could cause BAR-domain (Bin, amphiphysin, Rvs domain) proteins to release Rac and (b) how outward curvature could activate the opening of an ion channel. [0022] FIG. 4 illustrates the position-dependent mechanism of rigidity sensing. FIG. 4A depicts the crucial feature in such a model is that the enzyme, Fyn, and the substrate to be activated by stretch (kinase and substrate in this example) move relative to one another by actin rearward transport. In FIG. 4B, if the surface is hard, the components would be close enough for modification to occur (causing small displacement). In FIG. 4C, if the surface is soft, then enzyme and substrate would be separated before force could activate the reaction (in this case phophorylation of the linker; causing large displacement). ECM, extracellular matrix; F, applied force; RPTPot, receptor-like protein tyrosine phosphatase-α.
[0023] FIG. 5 shows the steps in mechanosensing over time that involve periodic testing of the substrate, substrate modification and changes in cellular protein content. [0024] FIGS. 6A-B represent a local force assay. FIG. 6A depicts magnetic tweezers (tip at top left corner) exerting a force on 2.7 μm magnetic beads bound to a spreading MEF cell. FIG. 6B is an image showing the rearward flow of actin that displaces the bead from the cell edge, towards the nucleus (purple trace) prior to application of force. When the external force is applied, the bead moves under the competing action of the two forces, cellular and magnetic. Shown here is the case of a large magnetic force ( 1 - 1.2 nN) and the bead is temporarily pulled back to the cell edge (red trace).
[0025] FIG. 6C is a graph that demonstrates the cell responding to the local stress by pulling in a contractile manner (positive and negative rearward velocities) and reinforcing the cytoskeletal adhesion to the stress site during the adaptive phase. The end of adaptive phase is marked by the recovery of the bead's constant rate of displacement.
[0026] FIG. 7 represents the spatio-temporal dynamics of αActinin in response to local force. FIG. 7A is an epifluorescent image of an MEF cell expressing αActinin-GFP that shows an accumulation of the fluorophore around two 2.7 mm beads. FIG. 7B represents the intensity of fluorescence vs. time and distance from center of the bead, r. Data at constant (r, t) was obtained by averaging over the arcsector shown in FIG. 7A. The frame of reference is centered on the bead and moves with it. Red and blue indicate the highest and respectively lowest levels of intensity. Maximum protein accumulation is observed ~50 sec from the application of large force. The duration of the adaptive phase is -105 sec.
[0027] FIG. 8 depicts active substrates. FIG. 8A (left) is a diagram of magnetic nanowires (for example, nickel) that can be fabricated by electrochemical deposition into porous templates. Scanning electron micrographs (SEM) of nickel nanowires are shown in the right panel. FIG 8B is a schematic of soft gels containing self-assembled arrays of magnetic nanowires that can serve as substrates for cells requiring rigid substrates for normal function. FIG. 8C represents external magnets that can actuate the embedded wires and impart quasi-local stresses at the surface of the gels. FIG. 8D demonstrates the restoration of spreading that can occur after 4 - 24 hrs, especially in the regions closer to the actuating magnet where wires deflect more and the local stresses are larger.
[0028] FIG. 9 represents a whole-cell dynamic environment. FIG. 9A is a schematic of a stress gradient that can be established across the width of free floating elastic substrates by having a varying initial length (diagonal clamp) and constant elongation. FIGS. 9B-E are representative experiments that were performed to test if the cells detect increased rigidity in the high-strain regions of polyacrylimide gels. Images are shown at same magnification. [0029] FIGS. 10A-B show laser tweezers (represented by red circle) that were used to place silica beads coated with fibronectin (FN) or vitronectin (VN) at the edge of the active growth cones. The rearward movement of the beads was recorded and further analyzed for reinforcement. In non-reinforced beads (FIG. 10A), the linkage between the bead and the cytoskeleton was broken by the force of the trap and the beads were pulled back into the trap after initial rearward movement (breaking event). If reinforcement occurred (FIG. 10B), the linkage was not broken and beads moved out of the optical trap towards the axon hillock. Scale bar represents 4 μm.
[0030] FIGS. lOC-D are graphs that demonstrate that RPTPα is required for the reinforcement of FN-specific integrin-cytoskeleton bonds in the neuronal growth cones. [0031] FIGS. 10E-F are schematics of the trajectories of individual beads that were generated in cases of non-reinforced (FIG. 10E) and reinforced of rearward movement (FIG. 10F). The laser trap is represented by a red circle. These trajectories are representative of an average bead behavior in both categories.
[0032] FIGS. 10G-H depict the mean square displacement (MSD) that was calculated for the initial 10s of rearward movement for each given condition. The results (represented as mean+standard error) were statistically significant as confirmed by t-test (p < 0.01). [0033] FIGS. 1 IA-B are graphs showing that Ovβό integrins are required for the reinforcement of the FN-cytoskeleton bonds at the leading edge of the growth cones. Function blocking antibodies to the otsβi and αvβ3 integrins had no effect on binding and the reinforcement of FN-coated beads, but function blocking of the αv and αvβ6 integrins reduced binding and reinforcement to the background levels.
[0034] FIGS. 1 IC-F are microscopy images representing that localization of RPTPα and cCvββ integrin in growth cones were ECM-specific. Both RPTPα and αvββ integrins were localized to the leading edge of active growth cones in neurons plated on FN-coated glass (FIG. 11C, FIG. 1 IE). On laminin (LN), RPTPα was localized to the growth cones, while αvβδ integrin was expressed at a very low level along the axons and at even lower levels in the growth cones (FIG. 1 ID, FIG. 1 IF). The scale bar is 5μm. Insets provide image of the entire neuron. [0035] FIGS. 12A-B represent Hippocampal neurons isolated from Pl brains of wild type and RPTPα knockout mice that were plated on FN-coated polyacrylamide gels of decreasing rigidities, incubated for 36h in serum-free medium, fixed and visualized by anti-Tau immunofluorescence. Neuronal stages of differentiation were observed on ECM-coated polyacrylamide gels, wherein stage 2 was characterized by many neurites of approximately equal length (FIG. 12A). At stage 3, -one significantly longer axon was differentiated (FIG. 12B). The Scale bar represents approximately lOμm. [0036] FIGS. 12C-D are graphs that represent quantification of differentiation stages and neurite lengths, which revealed an effect of rigidity and a role for RPTPα. Control neurons (RPTPa+/+) showed faster differentiation on soft than on rigid substrates; whereas, RPTPα-/- neurons showed a high level of differentiation irrespective of matrix rigidity (FIG. 12C). Similarly, control neurons extended longer axons on soft than on rigid surfaces; whereas, RPTPα-/- neurons extended longer axons on both soft and rigid surfaces (FIG. 12D). The results, in both FIG. 12C and 12D, (represented as mean + standard error) were statistically significant as confirmed by t-test (p<.01).
[0037] FIGS. 12E-F represent Hippocampal neurons isolated from Pl brains of wild type and RPTPα knockout mice that were plated on FN-coated polyacrylamide gels of decreasing rigidities, incubated for 36h in serum-free medium, fixed and visualized by anti-Tau immunofluorescence. The axons in RPTPα-/- neurons appeared wavier than the axons in wt neurons. No other morphological differences between RPTPα-/- and wild type neurons were observed. The scale bar represents approximately 15μm.
[0038] FIG. 13 are graphs depicting that the laminin (LN) rigidity response is RPTPα- independant in neurons. FIG. 13A is a graph that shows that axon differentiation is inhibited by increasing rigidities of LN -coated substrates in both contol and knockout neurons. FIG. 13B is a graph that shows neurite extension is stimulated by soft LN-coated substrates, and loss of RPTPα had no effect on this behavior
[0039] FIGS. 14A-B are graphs that show rigidity response in the growth cones is SFK- dependent. Primary wild type neurons were plated on FN-coated substrates of varying rigidities, and a SFK inhibitor (lOμM SU6656) was added after cell adhered to the substrate. After 36h incubation, there was no difference in wild type neurons on rigid versus soft surfaces. However, axon elongation and differentiation were inhibited compared to controls.
[0040]v FIGS. 14C-D are fluorescent micrographs that depict immunofluorescent staining of
Fyn occurring at lower levels of edge accumulation on soft than on rigid matrices in RPTPa+/+ neurons (FIG. 14C). In RPTPa-/- growth cones, there was decreased edge accumulation regardless of rigidity (FIG. 14D), indicating that RPTPα-mediated rigidity response required Fyn recruitment to the leading edge.
[0041] FIGS. 14E-F are fluorescent micrographs that demonstrate phosphorylation of pl30Cas, a known substrate for SFKs, requires RPTPα activity and rigid matrix.
Immunofluorescent staining of phosphorylated pl30Cas (anti-phospho-Y615-SFK) showed high levels of phospho-pl30Cas in the presence of RPTPα and rigid matrices. In RPTPa+/+ neurons plated on soft matrix, and in RPTPα-/- neurons regardless of the matrix rigidity, the observed levels of phopho-pl30Cas were significantly lower.
[0042] FIG. 15 represents a proposed model for the molecular mechanism of the FN-specific reinforcement and rigidity response in hippocampal neurons.
[0043] FIG. 16 is a photographic representation of 3D substrates depicting coated silk fibers
(red) suspended on microfences (black).
DETAILED DESCRIPTION OF THE INVENTION.
[0044] On the basis of extensive analyses, the physical aspects of the cellular environment that are sensed by cells are force and geometry at the nano-to-micrometre level. A multitude of design principles are emerging to describe the mechanosensory elements that are integrated into the structural motifs of various proteins that can be mechanically switched between conformations (reviewed in Silver, F. H. & Siperko, L. M. Crit. Rev. Biomed. Eng. 31, 255—331 (2003); Bershadsky, A. D. , Balaban, N. Q. & Geiger, B. Annu. Rev. CellDev. Biol. 19, 677-695 (2003); Martinac, B. J. Cell ScL 117, 2449-2460 (2004); Kung, C. Nature 436, 647-654 (2005); Shemesh, T. , Geiger, B. , Bershadsky, A. D. & Kozlov, M. M. Proc. Natl Acad. ScL USA 102, 12383-12388 (2005); Bustamante, C. , et al., Annu. Rev. Biochem. 73, 705-748 (2004); Vogel, V. Annu. Rev. Biophys. Biomol. Struct. 35, (2006)). Cellular mechanotransduction systems can then transduce the physical signals into biochemical responses. More complex physical parameters such as matrix rigidity or micrometre-level geometry can be measured by integrated force- and geometry-dependent transduction processes (FIG. 1). Local mechanosensing is transduced into biochemical signals that result in cell responses. Force-induced changes in protein conformation and geometry-dependent interactions are locally transduced into biochemical signals that activate various mechanosensitive signalling pathways, ultimately regulating cellular mechanoresponses. As an example, the dramatic difference in morphology that the same cells will assume after only 4 hours in different matrices is shown. Human fibroblasts project a dendritic network of extensions in collagen matrices but not on collagen- coated coverslips. Fibroblasts were incubated for 4 hours on collagen-coated surfaces (FIG. 1; bottom panel) or in collagen matrices (FIG. 1; top panel).
[0045] Therefore, it is important to differentiate between the primary sensory processes, the transduction processes and the downstream mechanoresponsive pathways that integrate the multiple biochemical signals that are derived from sensing and transduction events over space and time (FIG. 1).
[0046] It has also been postulated that cytoskeletal filaments can propagate stresses over long distances (Ingber, D. E. Proc. Natl Acad. ScL USA 102, 11571-1 1572 (2005); Wang, N. & Suo, Z. Biochem. Biophys. Res. Commun. 328, 1133-1138 (2005)), which would require the existence of other distant mechanosensory and signalling components. Considering the diversity of mechanosensory proteins, it is possible that transduction of mechanosensing into biochemical processes activates many signalling pathways that might interact to produce controlled functional responses. Subsequent cellular-motility responses can elicit additional mechanical signals that will lead to a second cycle of responses. The primary cellular responses to mechanical signals occur in seconds to minutes, therefore hundreds of thousands of stimulus-response cycles might occur over the days that a cell is maintained in culture.
[0047] Cell-cell, cell— matrix and flow forces are all sensed in different contexts, from the high forces that are sensed by chondrocytes in the cartilage matrix to the relatively low flow forces that are sensed in kidney tubules (Chen, C. S. , Tan, J. & Tien, J. Annu. Rev. Biomed. Eng. 6, 275-302 (2004); Praetorius, H. A. & Spring, K. R. Annu. Rev. Physiol. 67, 515-529 (2005); Tschumperlin, D. J. et al. Nature 429, 83-86 (2004)). The forces that are developed by the cells themselves on matrix or cellular contacts are also important (Zaidel-Bar, R. , et al., Biochem. Soc. Trans. 32, 416—420 (2004). Cell-cell contacts are dynamic, and cells seem to evaluate the level of force and make adjustments, as the cytoskeleton filaments and their linkages to transmembrane proteins assemble, break down and reassemble.
[0048] Sensing of geometry at the sub-cellular level is a crucial component of the cellular sensing of two-dimensional (2D) versus 3D matrices (FIG. 1). Recent studies have determined that the same matrix protein will elicit a different response when it is organized in filaments from when it is displayed on a flat surface (Cukierman, E. , Pankov, R. , Stevens, D. R. & Yamada, K. M. Science 294, 1708-1712 (2001); Cukierman, E. , Pankov, R. & Yamada, K. M. Curr. Opin. Cell Biol. 14, 633-639 (2002); Katz, B. Z. et al. MoI Biol. Cell 11, 1047-1060 (2000); Grinnell, F., Ho, C.H., Tamariz, E., Lee, D. J. & Skuta, G MoI. Biol. Cell 14, 384-395 (2003)), and that cells can sense nanoscale surface topographies (reviewed in Ref. 19) (Dalby, M. J., Riehle, M.O., Sutherland, D. S., Agheli, H. & Curtis, A. S. Eur. Cell Mater. 9, 1-8 (2005). In the context of a tissue, the cells will need to discriminate formed matrix fibres from the same matrix molecules that are present in a soluble form or individually attached to a neighbouring cell or a foreign body. Although there are many examples of local geometries that are imposed by the environment, adjacent cells or matrices, fibre curvature having the ability to cause membrane curvature and how the spatial distribution of cytoplasmic adhesion proteins could conform to those curved membranes all appear to be a role.
[0049] The transduction of local mechanostimuli into biochemical signals occurs through several signalling pathways, but many recent examples show that transduction occurs locally at the cell periphery, even though the forces and the biochemical signals propagate throughout the cell (Choquet, D. , Felsenfeld, D. P. & Sheetz, M. P. Cell 88, 39-48 (1997); Galbraith, C. G. , .Yamada, K. M. & Sheetz, M. P. J. Cell Biol. 159, 695-705 (2002); Riveline, D. et al. J. Cell Biol. 153, 1175-1186 (2001);. von Wichert, G. et al. J. Cell Biol. 161, 143-153 (2003)). Complicated transduction processes such as rigidity responses involve several steps that can combine the transduction of force and/or geometry sensing with time (reviewed in Refs 24,25) (Discher, D. E. , Janmey, P. & Wang, Y. L. Science 310, 1139-1 143 (2005); Giannone, G. & Sheetz, M. P. Trends Cell Biol, (in the press). Guanine nucleotide-exchange factors, Ca2+ ion channels, receptor-like protein tyrosine phosphatases, Src-family kinases and membrane receptors have all been invoked as early steps in force or geometry transduction (von Wichert, G. et al. J. Cell Biol. 161, 143-153 (2003); Katsumi, A. et al. J. Cell Biol. 158, 153-164 (2002); Katsumi, A., Naoe, T., Matsushita, T., Kaibuchi, K. & Schwartz, M. A. J. Biol. Chem. 280, 16546-16549 (2005); Munevar, S. , Wang, Y. L. & Dembo, M. J. Cell ScL 117, 85-92 (2004)). The downstream signals can then involve complex signalling pathways that intersect and modify each other to produce reliable cell responses. One system of interest is the cellular response to the rigidity of the environment (Discher, D.E., Janmey, P. & Wang, Y.L. Science 310, 1139— 1143 (2005)), which seems to be important in cancerous growth (Paszek, M. J. et al. Cancer Cell 8, 241—254 (2005)). Recent studies indicate that the loading rate of the force onto the cell can be crucial (Jiang, G., Huang, A.H., Cai, Y., Tanase, M. & Sheetz, M. P. Biophys. J. 9 Dec 2005 (6/opλy.s/.105.072462 v I)), in addition to the possible matrix stiffening that is caused by the physical activity of cells (Yeung, T. etal. Cytoskeleton 60, 24-34 (2005); Storm, C, Pastore, J.J., MacKintosh, F. C, Lubensky, T.C. & Janmey, P.A. Nature 435, 191-194 (2005)). Integration of different mechanical signals, at different locations and times, occurs through target proteins and signalling pathways that elicit a programmed response to properly shape cells and tissues.
[0050J Through nanofabrication and other current technologies, the mechanisms of mechanosensing can now be addressed systematically (Table 1). These techniques can be applied to cells with altered protein content through molecular-biological manipulations to address the molecular bases of cellular responses. [0051] Table 1. Techniques for evaluating mechano- and form-sensing mechanisms
Cell-generated force measurement
Deformable substrates (Munevar, S. , Wang, Y. & Dembo, M. Biophys. J. 80, 1744-1757 (2001)).
Microfabricated surfaces and cantilevers (Balaban, N. Q. et al. Nature Cell Biol. 3, 466-472 (2001); Galbraith, C. G. & Sheetz, M. P. Proc. Natl Acad. ScL USA 94, 9114-9118 (1997); Saif, M. T. , et al., Ann. Biomed. Eng. 31, 950-961 (2003)).
Laser tweezers (Jiang, G., et al., Nature 424, 334-337 (2003)).
Figure imgf000024_0001
[0052] Mechanical force plays a fundamental role in organism development and function. The presence or absence of mechanical stimuli coupled with the ability of cells to correctly sense and interpret stresses, are crucial elements in tissue development. Biological systems are dynamic entities, they respond and adapt to their environment in a time-dependent manner. In the living organisms, tissues are subject to oscillatory stimuli during actions such as walking, breathing, and cardiac activity. When underutilized, certain tissue becomes non-viable, and in extreme cases necrotic (muscles, bones, heart). A number of studies have shown that cells respond preferentially to specific frequencies depending on their function (Mack, P. J., et al., Am J Physiol Cell Physiol 287, C954-62 (2004); Ito, S. et al. Am J Physiol Lung Cell MoI Physiol (2006); Murfee, W. L. et al., J Appl Physiol 98, 2376-80 (2005)). Cellular time-dependent mechanotransduction is critical for activation of intracellular signal and the manner in which this signaling cascade regulates cell function is currently not well understood (Tamada, M., et al., Dev Cell 7, 709-18 (2004); Matthews, B. D., Overby, D. R., Mannix, R. & Ingber, D. E. J Cell Sd 119, 508-18 (2006); Vogel, V. Annu Rev Biophys Biomol Struct. (2006) 35:459-88; Chen, C. S., Tan, J. & Tien, J. Annu Rev Biomed Eng 6, 275-302 (2004)).
[0053] In vivo, other cells and the extracellular matrix (ECM) constitute the substrate on which cells grow. It has been shown that the rigidity of the environment is one of the key factors that determine cell behavior (FIG. 4) (Discher, D. E., et al.,. Science 310, 1 139-43 (2005); Engler, A. J. et al. J Cell Biol 166, 877-87 (2004); Georges, P. C. & Janmey, P. A. J Appl Physiol 98, 1547-53 (2005); Yeung, T. et al., Cell Motil Cytoskeleton 60, 24-34 (2005)). Contractile activity in a soft tissue could make it seem hard, because an external pull on the integrin before it moves significantly will cause unfolding while the substrate and enzyme are still close together. A time-dependent mechanism would be similar except that activity, rather than position, would be force-dependent with a biphasic response. The velocity of the cytoskeleton contraction would define the time window of the relevant activity in that model. Position-dependent mechanisms are likely more robust. (Kostic, A. & Sheetz, M. P. MoI Biol Cell. 2006 Jun;17(6):2684-95; Jiang, G., et al., Biophys J 90, 1804-9 (2006)). However, all these studies were performed in passive conditions not representative of the host environments in vivo, where oscillations occur due to the contractile activity of cells or due to cyclical activities of the host organism. Preliminary observations indicate that oscillatory mechanosignaling can stimulate contact assembly and override the cells' sensitivity to substrate rigidity. [0054] Conversion of force into biochemical signals
[0055] Studies of the mechanical properties of single molecules using nanotools (Kellermayer, M. S. , Smith, S. B. , Granzier, H. L. & Bustamante, C. Science 276, 1 1 12-1 116 (1997); Rief, M., Gautel, M., Oesterhelt, F. , Fernandez, J. M. & Gaub, H. E. Science 276, 1 109- 1 112 (1997); Tskhovrebova, L. , Trinick, J. , Sleep, J. A. & Simmons, R. M. Nature 387, 308- 312 (1997)) revealed a diverse set of structural motifs that could change conformation over a range of mechanical forces and could potentially serve mechanosensory functions (reviewed in Silver, F. H. & Siperko, L. M. Crit. Rev. Biomed. Eng. 31, 255-331 (2003); Bershadsky, A. D., Balaban, N.Q. & Geiger, B. Annu. Rev. Cell Dev. Biol. 19, 677-695 (2003); Martinac, B. J. Cell Sd. 1 17, 2449-2460 (2004); Kung, C. Nature 436, 647-654 (2005); Shemesh, T., Geiger, B., Bershadsky, A.D. & Kozlov, M.M. Proc. Natl Acad. ScL USA 102, 12383-12388 (2005); Bustamante, C. , Chemla, Y. R. , Forde, N. R. & Izhaky, D. Annu. Rev. Biochem. 73, 705-748 (2004); Vogel, V. Annu. Rev. Biophys. Biomol. Struct. 35, June 2006 (doi: \0Λ i46/annιιrev.biophys.35.102013)). (FIG. 2). These include the force-induced exposure of otherwise cryptic peptide sequences (FIG. 2A), the opening of mechanosensitive ion channels (FIG. 2B), and receptor— ligand interactions that strengthen if strained (FIG. 2C). It is important to note that such force-induced changes are only effective if they produce a change at the biochemical level. Here, a few selected proteins that are part of the physical network through which force is transmitted bidirectionally from the cell exterior to the interior and vice versa are discussed.
[0056] Exposure of cryptic peptide sequences. Most extracellular matrix (ECM) proteins, as well as many proteins that link the integrins to the cytoskeleton, consist of tandem-repeat sequences (these include spectrin-family members, such as α-actinin and dystrophin, as well as talin, titin, fibronectin, cadherins and others; FIG. 2A). Forces can induce the unravelling of modules, which results in alterations in molecular-recognition sites, or the exposure of peptide sequences that are otherwise hidden in the folded modules (reviewed in Ref. 8) (Vogel, V. Annu. Rev. Biophys. Biomol. Struct. 35, June 2006 (doi: 10.1146/annurev.biophys35.102013)). Differences in the mechanical stability between such repeats can define the sequence in which they unravel (Craig, D., Gao, M., Schulten, K. & Vogel, V. Structure 12, 21-30 (2004); Craig, D. , Krammer, A. , Schulten, K. & Vogel, V. Proc. Natl Acad. ScL USA 98, 5590-5595 (2001); Oberhauser, A. F., Badilla-Fernandez, C, Carrion- Vazquez, M. & Fernandez, J. M. J. MoI. Biol. 319, 433^47 (2002)).
[0057] The passing of energy barriers to unravel proteins typically coincides with the breakage of force-bearing hydrogen bonds that stabilize their tertiary structure39"40. The mechanical stability of the modules therefore depends on how frequently the force-bearing hydrogen bonds can be attacked by free water molecules. An important role of amino-acid side chains in regulating mechanical stability is whether they shield or expose those underlying bonds from electrophilic attack by water molecules (Craig, D. , Gao, M. , Schulten, K. & Vogel, V. Structure 12, 21-30 (2004); Craig, D., Gao, M., Schulten, K. & Vogel, V. Structure 12, 1-10 (2004)). Local charges also affect the shielding efficiency of amino-acid side chains and, consequently, pH and ionic strength can both further tune the mechanical stability of proteins (Craig, D. , Gao, M. , Schulten, K. & Vogel, V. Structure 12, 21-30 (2004); Rounsevell, R. W., Steward, A. & Clarke, J. Biophys. J. 88, 2022-2029 (2005)) Finally, many modules are mechanically stabilized by disulphide bonds and their redox state can be force-sensitive if the disulphide bonds are buried deep in the module (Bhasin, N. et al. J. Biol. Chem. 279, 45865— 45874 (2004)).
[0058] Exploiting hierarchical mechanical stabilities for mechanosensing is particularly powerful if the tandem repeats of a protein have different molecular- recognition sites, which can be functionally switched in response to partial unravelling. Sequential unfolding can signal the magnitude of the stress that is acting on multimodular proteins. The cell-adhesion protein fibronectin, for example, consists of more than 50 modular repeats — which are of special interest with respect to mechanosensing — and is abundant in serum as well as in many matrices. It physically links the ECM to the contractile cytoskeleton through its tripeptide sequence, RGD, which is recognized by integrins. Cell contractility is sufficient to partially unfold fibronectin in matrix fibres (Baneyx, G., Baugh, L. & Vogel, V. Proc. Natl Acad. Sci. USA 99, 5139-5143 (2002); Barker, T.H. et al. J. Biol. Chem. 280, 36483-36493 (2005)) (FIG. 2A). Although many molecular-recognition sites are present in the loop regions of these β-sandwich motifs, many cryptic binding sites have been identified that are buried within individual fibronectin modules in the folded state (reviewed in Refs 8,46). (Vogel, V. Annu. Rev. Biophys. Biomol. Struct. 35, June 2006 (doi: 10.1146/ annurev. biophys.2, 5.102013); Pankov, R. & Yamada, K. M. J. Cell Sci. 1 15, 3861-3863 (2002)).
[0059] Force regulation of protein activities. One way of transducing force into biochemical signals is to alter enzyme activity by directly applying force to either the enzyme or its substrate, or to an activator/inhibitor of the enzyme (reviewed in Refs 47,48). (Khan, S. & Sheetz, M.P. Annu. Rev. Biochem. 66, 785-805 (1997); Bustamante, C. Protein Sci. 13, 3061-3065 (2004)). Force is a potent regulator of enzyme activity, as many enzymes can undergo conformational changes during activity (Min, W. et al. Ace. Chem. Res. 38, 923-931 (2005)), which often also involves conformational changes of their substrates (Forde, N. R., Izhaky, D., Woodcock, G.R, Wuite, GJ. & Bustamante, C. Proc. Natl Acad. Sci. USA 99, 11682-1 1687 (2002)). In addition, many motor proteins will experience a decrease in turnover — for example, ATP hydrolysis — with increasing force (reviewed in Khan, S. & Sheetz, M.P. Annu. Rev. Biochem. 66, 785-805 (1997); Purcell, T. J., Sweeney, H. L. & Spudich, J. A. Proc. Natl Acad. Sci. USA 102, 13873- 13878 (2005)). As tension in the cytoskeleton increases, motor activity will decrease, until isometric conditions are reached. The isometric ATPase activity of motors keeps the tension in the cytoskeleton constant, and the recruitment of other motors will then increase the overall tension in the cytoskeleton. Because the cytoskeleton filaments are dynamic and undergo assembly— disassembly cycles on the timescale of seconds to minutes, filament tension will be rapidly lost and must constantly be maintained by motor activity.
[0060] Conformational strain may be capable of increasing the activity of some enzymes. The question of whether the activity of some enzymes can be upregulated by strain if they or their substrates are physically integrated into a force-bearing structure is relatively unexplored in the context of mechanosensing. Force might open up enzymatic cleavage sites through partial unravelling. Fibronectin, for example, has a partially cryptic disulphide-isomerase (Langenbach, K. J. & Sottile, J. J. Biol. Chem. 11$, 7032-7038 (1999) and a cryptic metalloprotease activity (Schnepel, J. & Tschesche, H J. Protein Chem. 19, 685-692 (2000)), but it is not known whether these activities can be regulated by force. Titin contains a module that can show kinase activity and computational studies indicated a mechanical opening of its active site (Grater, F., Shen, J., Jiang, H., Gautel, M. & Grubmuller, H. Biophys. J. 88, 790-804 (2005)). Furthermore, it is well known that enzymes exert strain on their substrates on binding — thereby catalysing the reaction (reviewed in Ref. 7) (Bustamante, C, Chemla, Y.R., Forde, N.R. & Izhaky, D. Annu. Rev. Biochem. 73, 705—748 (2004)) — and straining the enzymatic substrate could therefore potentially regulate enzyme activity. Recent studies show that the Src substrate is primed (activated) for phosphorylation by mechanical unfolding. Finally, some cytoplasmic proteins, including vinculin (Bakolitsa, C. et al. Nature 430, 583-586 (2004); Johnson, R. P. & Craig, S. W. J. Biol. Chem. 269, 12611-12619 (1994)) and Src (Harrison, S.C. Cell 1 12, 737-740 (2003)), are allosterically activated when their regulators bind to or unmask sites, thereby opening otherwise autoinhibited conformations. Force may also induce a similar opening of sites in those molecules. The mechanisms by which force can regulate protein function are only gradually emerging because new tools and assays are required to investigate force-induced functional > changes.
[0061] Mechanosensitive ion channels. The structural diversity of mechanosensitive channels seems to have been driven by the physiological necessity to detect mechanical stimuli — from thermal energy to high pressures — as changes in conductive state (FIG. 2B) (reviewed in Sukharev, S. & Anishkin, A. Trends Neurosci. 27, 345—351 (2004)). Some channels respond to stress in the lipid bilayer, whereas others must be physically connected to the cytoskeleton and/or the extracellular matrix to transmit forces to the channel.
[0062] The bacterial mechanosensitive K+ channel, MscL, represents the first case and has been structurally analysed. Membrane-tension forces are mainly concentrated in the interfacial polar headgroup regions of MscL, thereby inducing helix tilting that opens and wets the pore interior (Kung, C. Nature 436, 647-654 (2005); Sotomayor, M. & Schulten, K. Biophys. J. 87, 3050-3065 (2004)) Bacterial channels are relatively force-insensitive and only open at high tensions that are approaching the lytic tensions for the lipid bilayer. Plant channels seem to operate at tensions that are of an order of magnitude lower than bacterial channels, whereas typical membrane tensions in animal cells are a thousandfold lower than the activating bacterial tensions (Sheetz, M. P. Nature Rev. MoI. Cell Biol. 2, 392-396 (2001)).
[0063] Although clearly demonstrated for bacteria, it has not been shown whether either in- plane membrane tension or forces that are normal to the membrane are the primary cause of channel opening in animal cells, but most of the osmotic and other mechanosensitive responses require tethering to force-bearing filaments (Martinac, B. J. Cell ScL 117, 2449-2460 (2004). In the case of out-of-plane force-sensing channels, the effects of force are partially understood at the molecular level in only a few specialized cases. In the case of the outer-hair-cell bundle of the ear, the architecture of the linkage is known, but the molecular components have not been identified (Hudspeth, A. J. C. R. Biol. 328, 155-162 (2005)). In the case of the Caenorhabditis elegans touch sensor, the molecular components have been identified through genetic screens, but the architecture of the linkage is not known (Emstrom, G. G. & Chalfie, M. Annu. Rev. Genet. 36, 411-453 (2002)). Other channels are implicated in the regulation of the mechanically induced propagation of Ca2+ waves — probably through tethering to cytoskeletal components (Bao, L., Locovei, S- & Dahl, G. FEBS Lett. 572, 65-68 (2004)). Similarly, ion-dependent mechanosensory phenomena in bone and cartilage (Haut Donahue, T. L., Genetos, D. C, Jacobs, C. R., Donahue, H. J. & Yellowley, C. E. Bone 35, 656-663 (2004)) are probably responding to out-of-plane forces on channel-cytoskeleton linkages.
[0064] Stabilizing adhesion sites against force-induced breakage. In the protein networks that couple the ECM to the contractile cytoskeleton, the physical connections are mediated by weak non-covalent bonds and the weakest link will fail first if the protein network is under tension. Because of thermal activation, these bonds have modest lifetimes (Evans, E. Annu. Rev. Biophys. Biomol. Struct. 30, 105—128 (2001)). In the case of slip bonds, bond lifetimes are progressively shortened when forces act on a protein— protein or protein— ligand complex, as force tilts the energy landscape, thereby accelerating dissociation of the complex. The bond-survival time of the strongest non-covalent bond, which is formed between biotin and streptavidin, is diminished from more than a day under static conditions to about 1 minute or 0.001 second at bond stresses of 5 pN or 170 pN, respectively (Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Nature 397, 50-53 (1999)); whereas 5 pN can be exerted onto a protein complex by a single motor protein. Therefore, non-covalent bonds will fail under any level of tensile stress if held for sufficient time periods (Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Nature 397, 50—53 (1999)). Adhesion sites form in response to tensile forces acting on the membrane, and one consequence of the shortened bond lifetimes under force must be high turnover rates of the constituents in newly formed adhesion sites. Turnover rates on the timescale of seconds have been observed for actiή filaments and the two focal-contact proteins, paxillin and vinculin, which might serve as signalling molecules after their release from the cell contacts (Vallotton, P., Gupton, S.L., Waterman- Storer, C. M. & Danuser, G. Proc. Natl Acad. ScL USA 101, 9660-9665 (2004); Von Wichert, G. , Haimovich, B. , Feng, G. S. & Sheetz, M. P. EMBOJ. 22, 5023-5035 (2003); Woods, A. J. et al. J. Biol Chem. 277, 6428-6437 (2002). [0065] Cells may be able to form force-sustaining adhesion sites. Because force is transmitted across the membrane, not by single integrins, but by integrin clusters that increase in size with time in a force-dependent manner (Riveline, D. et al. J. Cell Biol. 153, 1175—1186 (2001); Bershadsky, A. D. et al. Eur. J. Cell Biol. 14 Dec 2005 (10.1016/j.ejcb.2005.11.001); Wehrle-Haller, B. & Imhof, B. Trends Cell Biol. 12, 382-389 (2002)), a single bond-rupture event does not necessarily lead to a failure of attachment. Assuming N identical bonds are loaded in parallel, and the force is distributed equally among the bonds, the force needed to break a cluster of parallel bonds is about a factor of N larger than it is for a single bond under the same rate of loading. And, most importantly, this value has a nonlinear and even steeper dependency if rebinding events of single broken bonds can occur (Evans, E. Annu. Rev. Biophys. Biomol. Struct. 30,. 105-128 (2001); Tees, D. F. , Waugh, R. E. & Hammer, D. A. Biophys. J. 80, 668- 682 (2001)).
[0066] To counteract the exponentially decreasing lifetime of bonds with force, mechanisms may exist wherein the bond lifetime can be increased by force. Although this has been theoretically predicted (Dembo, M. , Torney, D. C. , Saxman, K. & Hammer, D. Proc. R. Soc. Lond. B 234, 55—83 (1988)) evidence exists only for two types of receptor-ligand complex that are switched by force from a short-lived to a long-lived state. These include adhesion molecules, such as the bacterial adhesin FimH (Thomas, W. E. , Nilsson, L. M. , Forero, M. , Sokurenko, E. V. & Vogel, V. MoI. Microbiol. 53, 1545-1557 (2004); (Thomas, W. E. , Trintchina, E. , Forero, M. , Vogel, V. & Sokurenko, Cell 109, 913-923 (2002)), and P- and potentially L-selectins (Evans, E., Leung, A. , Heinrich, V. & Zhu, C. Proc. Natl Acad. Sci. USA 101, 11281-11286 (2004); Marshall, B. T. et al. Nature 423, 190-193 (2003); Yago, T. et al. J. Cell Biol. 166, 913- 923 (2004)). Other adhesion proteins, such as integrins or proteins that anchor the cytoskeleton to integrins, may also be capable of forming these catch bonds that strengthen under the influence of force (FIG. 2C), or they may rely on the formation of multiple linked bonds. [0067] Geometry sensing [0068] The natural ECM of cells is a complex 3D fibrous meshwork with a wide distribution of fibres and gaps that provide complex biochemical and physical cues, which are very different from uniformly coated 2D surfaces. Cell reactions to 3D matrices are altered from their reaction to 2D matrices of the same material (Cukierman, E. , Pankov, R. , Stevens, D. R. & Yamada, K. M. Science 294, 1708-1712 (2001)). How do these different physical and chemical properties co-regulate local cell form and ultimately global cell function? Most relationships between cell form and function have been deduced from 2D-cell-culture experiments, but tools have recently been developed to fabricate patterns, topographies and fibres at the micro- and nanoscale. It is therefore possible to learn more about the differential contributions of various physical and chemical properties that are involved in the regulation of cell function.
[0069] Geometry sensing can refer to the formation of signalling complexes by changes in the spacing of molecular-recognition sites the geometrical shape of the substrates. For example, surface steps as small as 11 run can lead to contact guidance (Curtis, A. & Wilkinson, C. Biochem. Soc. Symp. 65, 15—26 (1999)). Restricting cells to spreading on adhesive micropatterns of various shapes can regulate cell proliferation and cell death (Chen, C. S. , Mrksich, M. , Huang, S., Whitesides, G. M. & Ingber, D.E. Science 276, 1425-1428 (1997), or the direction of polarization can control the direction in which the cells move (Jiang, X., Bruzewicz, D.A., Wong, A.P., Piel, M. & Whitesides, G. M. Proc. Natl Acad. ScL USA 102, 975-978 (2005)). The size of micropatterns also regulates whether mesenchymal stem cells differentiate into adipocytes or osteoblasts (McBeath, R. , Pirone, D. M. , Nelson, C. M. , Bhadriraju, K. & Chen, C. S. Dev. Cell 6, 483-495 (2004)). Because micropatterns confine cell shape, thereby causing an integrated cellular response, the ability of cells to sense nanoscale surface features, including the size of nanoscale fibres and topographies, as well as the spatial presentation of molecular- recognition sites will be considered.
[0070] Sensing substrate curvature. 3D matrix fibres are sensed by cells differently from the same matrix molecules in 2D5 perhaps because of the membrane curvature that the fibres induce. (FIG. 3). To artificially produce replacement matrices, tissue engineers have fabricated many microporous scaffolds from a large range of materials with limited success. Micro- and nanofibrous scaffolds are now under investigation for bone and soft-tissue engineering applications. It has been shown that astrocytes preferentially adhere and proliferate on carbon fibres that have a diameter above 100 run and low surface energies (McKenzie, J.L., Waid, M.C., Shi, R. & Webster, T. J. Biomaterials 25, 1309-1317 (2004). Fibroblasts move on collagen fibres by a specific mode of motility that involves one myosin isoform (Meshel, A. S., Wei, Q., Adelstein, R. S. & Sheetz, M. P. Nature Cell Biol. 1, 157-164 (2005). The same myosin isoform was shown to be involved in in vivo-like organization and morphogenesis of fibroblasts grown on electrospun nanofibrous matrices (Schindler, M. et al. Biomaterials 26, 5624—5631 (2005). It has also been shown that 3D networks of nanofibres presenting the neurite-promoting laminin epitope promoted the selective differentiation of neural progenitor cells (Silva, G. A. et al. Science 303, 1352-1355 (2004)). Furthermore, the nanofibre alignment and direction of mechanical strain affect the ECM production (Lee, C. H. et al. Biomaterials 26, 1261-1270 (2005)). As the complexity of the natural ECM is appreciated, in part through the fabrication of analogous model systems that mimic subsets of selected properties, new biomaterials and scaffolds for tissue engineering can be better designed.
10071] But how can cells differentiate between a concave and a convex membrane curvature? It has been found that cells adhere differently to surfaces that are structured with nanoposts compared with nanopit-covered surfaces. Although some 13-nm high nanoposts increased cell spreading, proliferation and cytoskeletal formation (Dalby, M.J., Riehle, M. O., Sutherland, D.S., Agheli, H. & Curtis, A.S. Eur. J. Cell Biol. 83, 159-169 (2004)), a nanopitted pattern that was embossed into polymethylmethacrylate decreased adhesion relative to flat substrates (Martines, E. , McGhee, K. , Wilkinson, C. & Curtis, A. IEEE Trans Nanobioscience 3, 90-95 (2004)). In fact, fibroblasts seem to endocytose nanocolumns (Dalby, M. J. et al. Exp. Cell Res. 295, 387-394 (2004)). As the cell forms adhesions on substrates with micro- or nanoscale surface features, the membrane is forced to follow the external surface contours. [0072] Recent studies of endocytosis show that the BAR-domain (Bin, amphiphysin, Rvs domain) proteins can induce or recognize a concave membrane curvature and recruit small G proteins. The BAR domains of arfaptins were shown to bind to the small GTPases Rac, adenosine-ribosylation factor- 1 (ARFl), ARF3 and ARF6, as well as to the ARF-like protein- 1 (ARLl ; Refs 93-96) (Lu, L., Horstmann, H., Ng, C. & Hong, W. J. Cell ScL 114, 4543-4555 (2001); Taπϊcone, C. et al. Nature 41 1, 215-219 (2001); Van Aelst, L. , Joneson, T. & Bar- Sagi, D. EMBO J, 15, 3778-3786 (1996); Williger, B. T. , Ostermann, J. & Exton, J. H. FEBS Lett. 443, 197-200 ( 1999)). Another member of the BAR-domain family, BAP2α/IRSp53 (insulin-receptor substrate protein of 53 IcDa) has been shown to be the link between Rac and WAVE (the Wiskott-Aldrich syndrome protein (WASP)-related protein) in regulating membrane ruffling (Miki, H. , Yamaguchi, H. , Suetsugu, S. & Takenawa, T. Nature 408, 732— 735 (2000)). However, it is still unclear whether GTPase binding and membrane-curvature sensing and induction are common features of all BAR-domain family members (FIG. 3A) (Habermann, B. EMBO Rep. 5, 250-255 (2004); Zimmerberg, J. & McLaughlin, S. Curr. Biol. 14, R250-R252 (2004)). The soluble form of the BAR-domain protein has bound Rac and would be expected to release it on binding to curved membrane regions, which could stimulate further motility in those regions of the cell. Outward curvature could be induced by filopodial extension or the formation of retraction fibres by the cell pulling away from contacts (FIG. 3). Several of the BAR-domain proteins have effects on motility, which is consistent with a role in sensing the form of concave matrix-induced membrane curvature (Carstanjen, D. et al. J. Immunol. 174, 4613-1620 (2005); Krugmann, S. et al. Curr. Biol. 11, 1645-1655 (2001)). Therefore, the recruitment of BAR proteins to membranes that are bound to curved surfaces can have profound effects on cell function, because the BAR proteins that would assemble on membranes with a concave curvature could locally increase small-G-protein activity. [0073] Another implication of membrane curvature is that the inner and outer membrane leaflets, which have an equal tension in a planar configuration, exhibit differential tensions on bending. Beyond affecting the local lipid composition and, potentially, the redistribution, spatial clustering or segregation of transmembrane proteins, this could also lead to the opening of mechanogated ion channels (FIG. 3B). Certain K+ channels, for example, are opened by a convex curvature of the membrane (Patel, A. J. , Lazdunski, M. & Honore, E. Curr. Opin. Cell Biol. 13, 422-428 (2001)).
[0074] Concave and convex membrane curvatures might be sensed by two different mechanisms. BAR domains might sense the concave membrane curvature that is formed in contact with external posts and fibres, whereas membrane channels might selectively be opened if membranes come into contact with surface indentations. If BAR-domain proteins do indeed bind preferentially to concave surfaces, such as posts or fibres, the local release of Rac and its subsequent activation, for example by integrin-linked kinase (Filipenko, N. R. , Attwell, S. , Roskelley, C. & Dedhar, S. Oncogene 24, 5837-5849 (2005)), might lead to a local enhancement of traction forces. In support of this proposition, Rac activation is known to enhance focal- complex assembly (Filipenko, N.R., Attwell, S., Roskelley, C. & Dedhar, S. Oncogene 24, 5837-5849 (2005); Burridge, K. & Wennerberg, K Cell 116, 167-179 (2004); Civelekoglu- Scholey, G. et al. J. Theor. Biol. 232, 569-585 (2005); Machacek, M. & Danuser, G. Biophys. J. 90, 1439—1452 (2006)) and cells adhere more weakly on surfaces with nanopits than on those with nanoposts (Dalby, M. J., Riehle, M. O., Sutherland, D. S. , Agheli, H. & Curtis, A. S. Eur. J. Cell Biol. 83, 159-169 (2004); Martines, E. , McGhee, K., Wilkinson, C. & Curtis, A. A. IEEE Trans Nanobioscience 3, 90-95 (2004)).
[0075] Protein clustering and spacing. Because membranes are fluid, there can be mechanical or diffusional separation of components over micron distances that can have biochemical consequences (Sheetz, M. P. Annu. Rev. Biophys. Biomol. Struct. 22, 417—431 (1993). The most dramatic examples are in the formation of immune synapses (Bromley, S. K. et al. Annu. Rev. Immunol. 19, 375—396 (2001)), where there is good evidence that the physical separation of different transmembrane receptors and their spatial patterning regulates T-cell activation (Mossman, K. D., Campi, G., Groves, J. T. & Dustin, M. L. Science 310, 1191-1193 (2005); Campi, G., Varma, R. & Dustin, M.L. J. Exp. Med. 202, 1031-1036 (2005)). [0076] Many protein aggregates form at sites of cell— matrix or cell— cell contacts and the size and shape of those aggregates is influenced by the spacing of the extracellular ligands to which they bind. The formation of cell-adhesion sites requires that the RGD peptides are clustered (Koo, L. Y. , Irvine, D. J. , Mayes, A. M. , Lauffenburger, D. A. & Griffith, L. G. J. Cell Sci. 1 15, 1423-1433 (2002)) at a distance of less than 73 nm (Ref. 112). (Arnold, M. et al. Chemphyschem 5, 383—388 (2004)) A mechanism for sensing the spacing was indicated by studies at the single-molecule level, which showed that a single trimer of fibronectin type III domains 7-10 (with spacing of 50-60 nm) was preferentially bound to leading edges of active lamellipodia when 55-nm long talin dimers were present (Brock, A. et al. Langmuir 19, 1611— 1617 (2003); Jiang, G., Giannone, G., Critchley, D.R., Fukumoto, E. & Sheetz, M. P. Nature 424, 334-337 (2003)). Once spatially organized by crosslinking, either through talin or (X- actinin (Calderwood, D.A. Biochem. Soc. Trans. 32, 434-437 (2004); Critchley, D.R. Biochem. Soc. Trans 32, 831-836 (2004); Ginsberg, M. H. , Partridge, A. & Shattil, S. J. Curr. Opin. Cell Biol. 17, 509—516 (2005)), these oligomeric sites recruit and activate other components to form focal contacts (Zaidel-Bar, R., Cohen, M., Addadi, L. & Geiger, B. Biochem. Soc. Trans. 32, 416-420 (2004); Arnold, M. et al. Chemphyschem 5, 383-388 (2004); Coussen, F., Choquet, D., Sheetz, M. P. & Erickson, H.P. J. Cell ScL 115, 2581-2590 (2002); Delanoe-Ayari, H., Al Kurdi, R. , Vallade, M. , Gulino-Debrac, D. & Riveline, D. Proc. Natl Acad. ScL USA 101, 2229-2234 (2004); Cluzel, C. et al. J. Cell Biol. 171, 383-392 (2005)). The size of those adhesion-site complexes is limited by the size and shape of the external surface contacts (Wang, N. , Ostuni, E., Whitesides, G. M. & Ingber, D. E. Cytoskeleton 52, 97-106 (2002); Lehnert, D. et al. J. Cell Sci. 117, 41-52 (2004); Gallant, N. D. , Michael, K. E. & Garcia, A. J. MoI. Biol. Cell 16, 4329- 4340 (2005)) and focal adhesion kinase (FAK) might regulate their turnover (IHc, D. et al. Nature 311, 539-544 (1995); Kirchner, J. , Kam, Z. , Tzur, G. , Bershadsky, A. D. & Geiger, B. J. Cell ScL 116, 975-986 (2003); Chen, B. H. , Tzen, J. T. , Bresnick, A. R. & Chen, H. C. J. Biol. Chem. 277, 33857-33863 (2002); Dixon, R. D. et al. Structure 12, 2161-2171 (2004); Yamamoto, D. et al. Cell Signal 15, 575-583 (2003)).
[0077] In addition, studies on single adhesive islands revealed that focal adhesions localize asymmetrically along the periphery of the small islands that experienced the highest tensional stress (Chen, C. S. , Alonso, J. L. , Ostuni, E. , Whitesides, G. M. & Ingber, D. E. Biochem. Biophys. Res. Commun. 307, 355-361 (2003)). [0078] Force-activated mechanoresponses
[00791 Considering the large number of mechanosensory motifs, there are potentially several pathways and engineering paradigms by which mechanical signals could be transduced into biochemical signals. In many examples, the mechanotransduction process is position- and time- dependent. The robust behaviour of the mechanical aspects of the development of most organisms belies a signalling system that is highly engineered and remarkably reproducible, despite the fact that many signalling pathways are activated. The final integrated response is often tuned to give an important functional behaviour. The cellular response to rigid versus soft surfaces are considered, but other mechanoresponsive pathways that are involved in the integrated responses to overall size and shape of the cellular environment have been at least partially identified (Numaguchi, Y. et al. Angiogenesis 6, 55-64 (2003).
[0080] The response of cells to substrate rigidity in cell culture is cell-specific and seems to be correlated with the rigidity of the native environment of the cell, which can vary significantly between different tissues (Engler, A. J. et al. J. Cell Biol. 166, 877-887 (2004); Georges, P. C. & Janmey, P. A. J. Appl. Physiol. 98, 1547-1553 (2005)). It is still unclear why some cells normally require a rigid surface for growth, whereas others thrive on soft surfaces. At the physical level, transduction of substrate rigidity into a biochemical signal involves either measuring the compliance of the external linkages or measuring how much they are spatially displaced for a given force.
[0081] Changing the rate-of-force increase. The rigidity of the substrate might tune binding strength of the cell by changing the rate-of-force increase acting on the receptor-ligand bond. It has been postulated that there is a rate-dependent increase in the time required for receptor— ligand unbinding and therefore a higher matrix rigidity would increase the bond lifetime (Evans, E. Anmi. Rev. Biophys. Biomol. Struct. 30, 105-128 (2001)). In addition to the stiffness of the linkage, some bonds in adhesion complexes might strengthen if strained (catch bonds). Therefore, physical mechanisms could be sensitive to the rate of increase in the force exerted on external ligands, as determined by its tether compliance.
[0082] Measurement of displacement for a given force. Alternatively, the binding of an external ligand or a mechanical deformation could initiate the assembly of two different complexes in the cell, one that depends on the force because it is linked to both the cytoskeleton and the external ligand and a second that is only linked to a separate extracellular site or a curved membrane edge (FIG. 4). The first complex can translocate with the rearward-moving actin filaments, whereas the second complex remains stationary. When the cytoskeleton pulls on the ligand, the rigidity of the external ligand linkage will determine how far the cytoskeleton-linked complexes will be displaced in a given time period before reaching isometric conditions and whether the force will be sufficient to activate any of the force sensors through the exposure of otherwise cryptic peptide sequences. For soft substrates, the separation between the fixed and moving components is greater, and might be sufficient to prevent the sensor from interacting with the stationary component (FIG. 4).
[0083] Other relative changes in spacing might occur that are due to partial protein unfolding. Based on observations with laser tweezers, it became clear that changes in force (of ~10 pN) over a timescale of 1 second or over a length of 50-100 nm (for example, a molecular level) can give rise to rigidity responses (Giannone, G. & Sheetz, M.P., Trends Cell Biol, (in the press); Georges, P.C. & Janmey, P.A., J. Appl. Physiol. 98, 1547-1553 (2005)). Several observations indicate that the timescale of 1 second is important. For example, the normal rearward transport rate of actin (60 nm second"1; Ref. 134) will move sites by 50—100 nm in approximately 1 second. Body motions such as walking, heart rates and other activities that produce tissue contractions are also on the timescale of approximately 1 second (Giannone, G. et al. Cell 116, 431-443 (2004)). Furthermore, analyses of the reaction of endothelial cells to magnetic beads show dramatic differences with oscillations in force on a frequency of about 1 Hz (Ref. 135) (Mack, P. J. , Kaazempur-Mofrad, M. R. , Karcher, H. , Lee, R. T. & Kamm, R. D. Am. J. Physiol. Cell Physiol. 287, C954-C962 (2004)) Oscillations in force on a 1 -second timescale may be able to produce a rigidity response and such oscillations in vivo could result from the normal contractile activity of tissues and tissue rigidity.
[0084] Different cells often have different responses to matrix rigidity (Discher, D. E. , Janmey, P. & Wang, Y. L. Science 310, 1139-1143 (2005)). For example, neurons have a preference for soft surfaces, whereas fibroblasts grow better on rigid surfaces Georges, P. C. & Janmey, P. A. J. Appl. Physiol. 98, 1547-1553 (2005)). The tyrosine kinase, FAK, has been shown to be involved in the pathway that senses the rigidity of polyacrylamide surfaces that are coated with collagen (Wang, H.B., Dembo, M., Hanks, S.K. & Wang, Y. Proc. Natl Acad. ScL USA 98, 11295-1 1300 (2001)), whereas the Src kinases are involved in sensing rigidity on surfaces that are coated with fibronectin (Jiang, G., Huang, A.H., Cai, Y., Tanase, M. & Sheetz, M. P. Biophys. J. 9 Dec 2005 {biophysjΛ 05.072462 v I)) Furthermore, an important substrate for the Src-family kinases, pl30Cas, shows a marked increase in phosphorylation on cell or cytoskeleton stretch (Tamada, M., Sheetz, M.P. & Sawada, Y. Dev. Cell 7, 709-718 (2004). Several studies have indicated that tyrosine kinases and phosphatases have a crucial role in the sensing of rigidity. Extracellular signal-regulated kinases (ERKs) and Rho constitute part of an integrated mechanoregulatory circuit that links matrix stiffness, through integrin clustering, to cytoskeletal tension and, ultimately, regulation of tissue phenotype (Silver, F. H. & Siperko, L. M. Crit. Rev. Biomed. Eng. 31, 255-331 (2003); Ingber, D. E. Proc. Natl Acad. Sd. USA 102, 11571-11572 (2005); McBeath, R., Pirone, D.M., Nelson, C. M., Bhadriraju, K. & Chen, CS. Dev. Cell 6, 483-495 (2004); Civelekoglu-Scholey, G. et al. J. Theor. Biol. 232, 569-585 (2005); Shiu, Y. T. et al. Biophys. J. 86, 2558-2565 (2004)).
[0085] During cell spreading or migration, cells are continually encountering new ligands. As cells stabilize in a tissue, they become less dynamic, however, there is a continuous turnover of contact and cytoskeletal proteins on the timescale of minutes or less (Zaidel-Bar, R. , Cohen, M., Addadi, L. & Geiger, B. Biochem. Soc. Trans. 32, 416-420 (2004); Von Wichert, G., Haimovich, B., Feng, G.S. & Sheetz, M. P. EMBOJ. 22, 5023-5035 (2003); Wehrle-Haller, B. & Imhof, B. Trends Cell Biol. 12, 382-389 (2002); Machacek, M. & Danuser, G. Biophys. J. 90, 1439-1452 (2006); Cluzel, C. et al. J. Cell Biol. 171, 383-392 (2005); Webb, D. J. et al. Nature Cell Biol. 6, 154—161 (2004)). As cellular contacts are maintained for days and possibly longer, new contacts must form as the older ones are lost. Those new contacts can be tested for rigidity and, therefore, the cells can continually sample the rigidity of their environment. Indeed, during spreading and migration, cells generate periodic contractions of their lamellipodia on rigid, but not soft, substrates (Giannone, G. et al. Cell 1 16, 431-443 (2004)).
|0086] It has been shown that some cells lose their shape sensitivity to substrate rigidity once they come into contact with other cells (Yeung, T. et al. Cell Motil. Cytoskeleton 60, 24-34 (2005)). Cell-cell binding can overwrite cell-substrate-induced signalling, hi tissues, cellular- level mechanosensing, transduction and response processes can maintain the proper physical homeostasis, which is markedly altered in cancers (Paszek, M. J. et al. Cancer Cell 8, 241—254 ' - (2005)).
[0087] Global regulation of cell function
[0088] At the cellular level, mechanical signals can have profound effects that lead to cell death, growth or differentiation. Because the effects are extremely important, cells not only constantly monitor the mechanical environment but they also actively remodel their environment. Initially, cells will sense the mechanical features of their environment, which will cause rapid motility and signalling responses. As the cell pulls on the environment, it will modify the extracellular matrix and will create new signals, such as those originating from fibronectin unfolding. Intracellular signals will alter the expression pattern of the cell and, over time, the cellular forces and cellularly generated matrices will change the cell shape. At any stage, extracellular signals, such as hormones or external mechanical stimuli, can cause acute changes that will set off a further round of cell and matrix modifications. It is therefore not surprising that attempts to engineer tissue scaffolds have often been confounded by adverse cell behaviours, especially at later time points (Giannone, G. et al. Cell 1 16, 431—443 (2004); Ratner, B. D. & Bryant, S. J. Annu. Rev. Biomed. Eng. 6, 41-75 (2004); Langer, R. & Tirrell, D. A. Nature 428, 487-492 (2004)) It is therefore useful to discuss the overall process, from mechanosensing to protein expression, in terms of associated time-dependencies (FIG. 5)-. [0089] Initial interaction and mechanosensing events occur on the subsecond to second timescale. Early cell responses take seconds to minutes and involve cytoskeletal redistribution, reinforcement of linkages and changes in cell motility. These changes entail activation of actin- filament extension, the recruitment of myosin molecules to generate cell contractions, consolidation of adhesive contacts by the recruitment of other receptors and cytoplasmic molecules to support the myosin contraction forces, and sometimes a global cellular response, such as a burst in Ca2+ signalling or general contraction.
[0090] As the cell generates forces on the proteins that link the ECM to the cytoskeleton, the responses of the mechanosensitive sites to the forces and compliance or changes in local curvature will cause a secondary cellular response. Examples of such reiterative processes include cell polarization and the size-dependent phagocytosis of large particles that involves a specific mode of actin-dependent membrane extension that conforms to the shape of the particle being phagocytosed (Kwiatkowska, K. & Sobota, A Bioessays 21, 422-431 (1999). In the case of cell polarization, uniaxial stretching, fluid shear or rigidity gradients are often sufficient to cause polarization (Katsumi, A. et al. J. Cell Biol. 158, 153-164 (2002); Helmke, B. P. & Davies, P. F. Ann. Biomed. Eng. 30, 284-296 (2002); Lo, C. M. , Wang, H. B. , Dembo, M. & Wang, Y. L. Biophys. J. 79, 144-152 (2000)) and the extracellular matrix guides the orientation of the cell-division axis (Thery, M. et al. Nature Cell Biol. 7, 947-953 (2005)). The assembly of the actin cytoskeleton can be mechanically triggered. Uniaxial stretch suppresses lamellipodia in a directional fashion (Katsumi, A. et al. J. Cell Biol. 158, 153-164 (2002)), whereby Rac activation depends on the direction of the tension (Katsumi, A. et al. J. Cell Biol. 158, 153—164 (2002); Machacek, M. & Danuser, G. Biophys. J. 90, 1439-1452 (2006). During these early mechanosensing and response events, there is often insufficient time for the cell to synthesize new proteins.
[0091] Although the physical properties of the ECM determine the initial rigidity response, cell-generated forces on matrices align and stiffen them (Storm, C. , Pastore, J. J. , MacKintosh, F. C. , Lubensky, T. C. & Janmey, P. A. Nature 435, 191-194 (2005)) and partially unfold ECM proteins in a continuous feedback loop that is regulated by cell contractility (Barker, T. H. et al. SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity. J. Biol. Chem. 280, 36483-36493 (2005); Gao, M. et al. Structure and functional significance of mechanically unfolded fibronectin type 1111 intermediates. Proc. Natl Acad. Sci. USA 100, 14784-14789 (2003); Zhong, C. et al. J. Cell Biol. 141, 539-551 (1998)). The extent to which the matrix proteins are unfolded should regulate the exposure of their cryptic peptide sequences or alter the configuration of exposed recognition sites, therefore giving the cell the ability to self-regulate, through contractions and signalling processes (reviewed in Refs 8,148). (Vogel, V. Annu. Rev. Biυphys. Biomol. Struct. 35, June 2006 (doi:
10.1 146/αnnMrev.Z>/op/iy.s.35.102013);Vogel, V. & Baneyx, G. Annu. Rev. Biomed. Eng. 5, 441- 463 (2003). Mechanical stress can also upregulate the production of ECM proteins indirectly, by stimulating the release of a paracrine growth factor, or directly, by triggering an intracellular signalling pathway that activates gene expression (Chiquet, M. , Renedo, A. S. , Huber, F. & Fluck, M. Matrix Biol. 22, 73-80 (2003)). External forces or changes in cell contractility will further change the configuration of exposed sites. A quasi-, although highly dynamic, steady state is often reached that involves cycles of protein expression, modification and finally turnover with further regulation by external chemical and physical stimuli. [0092] The local response to rigidity and form may have an effect on cell shape and gene expression. As noted above, force on peripheral contacts can produce signalling molecules that could translocate to the nucleus. Alternatively, the plasma membrane is physically linked with the nuclear membrane (Ingber, D. E. Tensegrity II. How structural networks influence cellular information processing networks. J. Cell Sci. 1 16, 1397-1408 (2003); and it might be possible that physical factors and nuclear deformation could regulate gene expression a mechanism which is yet identified. There has been relatively little discussion of the fact that the forces act on peripheral adhesion complexes, and therefore the associated spatial displacements or the effect on protein unfolding are higher there than at the nuclear membrane. Because forces are dissipated from the sites of local impact at the periphery of the nucleus, it is likely that the mechanism of mechanosensing is amplified more efficiently at the periphery and therefore has the greatest effect on the peripheral contacts.
[0093] Transcription factors that are recruited to the adhesion sites could have an important role in translating the physical stimulus that is sensed at the periphery into biochemical signals that alter gene expression. Transcription factors might be modified in a force-dependent manner and transported to the nucleus; for example, paxillin is modified at focal-contact sites and is then transported to the nucleus (Woods, A. J. et al. J. Biol. Chem. 277, 6428-6437 (2002)). This mechanism provides an obvious way to transform force on specific intracellular-adhesion sites to a change in protein expression.
[0094] Recent microarray studies confirm that changes in cell shape correlate with several changes in gene expression (Dalby, M.J., Riehle, M.O., Sutherland, D.S., Agheli, H. & Curtis, A. S. Eur. Cell Mater. 9, 1—8 (2005)). Important shape-dependent changes in gene expression occurred for proteins of the cytoskeleton, proliferation, transcription, translation, ECM production and inter- and intracellular signalling complexes. As the cells rounded up, genes encoding a few small G proteins were among those that were downregulated. Tyrosine kinases that interact with G proteins of the Ras family to stimulate cell proliferation and differentiation are also downregulated, as is α-actinin (Dalby, M.J., Riehle, M.O., Sutherland, D. S., Agheli, H. & Curtis, A. S. Eur. Cell Mater. 9, 1-8 (2005)). Many of the tyrosine kinases and phosphatases that have been linked to changes in cell and tissue shape are also linked to the early events of force and rigidity sensing (reviewed in Ref. 25) (Giannone, G. & Sheetz, M. P. Trends Cell Biol. (in the press)). These observations indicate that the overall cell shape that results from the local responses to force and geometry over time, and the integration of those responses, have an important role in the regulation of gene expression. For a synthetic surface, however, it is not just the geometry and rigidity but also its surface chemistry that ultimately controls the composition and conformation of surface-adsorbed proteins and, therefore, the integrin-mediated cell- signalling processes (Keselowsky, B. G. , Collard, D. M. & Garcia, A. J. Proc. Natl Acad. ScL USA 102, 5953-5957 (2005)).
[0095] There is a continual feedback between cell sensing of force, rigidity or form and the cell contractility that, together with biochemical signals, coregulates cell and tissue shape and, ultimately, the shape of the organism. New nanotechnologies will enable us to test the molecular mechanisms of mechanosensing and transduction. Important questions include a further definition of the local responses to mechanical and geometrical forces and the investigation of signal propagation within the cell, including within the nucleus, to control gene expression. Tissue scaffolds that are engineered at the micro- and nanoscale will enable a better understanding of the force- and geometry-sensing mechanisms, which then must be linked with the cellular-response pathways for both short- and long-term morphological changes. [0096] Cell behavior may be determined by cell dynamics and the state of the cellular environment. The latter parameters can allow a better understanding of the feedback mechanism between cell contractility and mechanosensing, as well as the role of pulsatory activity in cell function and replication. Such studies can also be motivated by the need for better tissue scaffolds, and implantable materials (Vogel, V. & Baneyx, G. Annu Rev Biomed Eng 5, 441-63 (2003)). Elucidating the dynamic nature of the interactions of the cell with its environment can clarify the requirements for better biomedical devices and drive the subsequent phases of research in engineered materials and technologies.
[0097] To study the mechanotransduction pathways under conditions approaching in vivo situations, the effect that forces of controlled magnitudes and frequencies have on cells can be investigated in engineered environments of different rigidities. Cell responses to mechanical perturbations both locally (sub-micron scale) and globally (whole-cell assays) on such engineered environments can be quantified. In one embodiment, the mechanical perturbations can be generated using one of the following non-limiting examples of devices: pipet-assisted manipulation device, laser tweezer, optical trap, magnetic field generator, or a substrate stretching device, wherein a stationary clamp is attached to a first end of the substrate and a mobile clamp is attached to a second end of the substrate. For example, these devices can physically manipulate an engineered environment, such as a cell growth substrate or scaffold of the invention.
[0098] Some non-limiting examples of cell responses: cell growth; cell differentiation; apoptosis; cell movement/cytokinetics; and cell morphology changes. In one embodiment, cell growth can be assessed via measuring a gross change in cell size, an increase in cell proliferation, an increase in focal adhesion assembly, or a combination thereof. In another embodiment, cell differentiation can be determined via examining the expression of target genes utilizing standard molecular biology methods practiced in the art. In a further embodiment, apoptosis can be investigated via examining cell viability or the expression and/or downregulation of target genes. In yet another embodiment of the invention, cell movement can be assessed via motility assays utilized in the art. In other embodiments, cell morphology changes can be examined via light, fluorescent, or electron microscopy methods utilized by one skilled in the art.
[0099] "A cell growth substrate of the invention can be permeable to liquids, gases, and cellular by-products. It can comprise a growth medium in addition to a cross-linked polymer, wherein increased strain on the polymer does not substantially increase rigidity of the polymer, and wherein the polymer has an elastic modulus (E) of about 0.1 kPa to about 10.0 kPa (E is described in Engler at al., (2004) Biophys J S6: 617-28, which is hereby incorporated by reference). Polymers are long chain organic molecules that are assembled from smaller molecules called monomers. Polymers comprise many repeating monomer units in long chains, and can be classified as synthetic or natural polymers (for example, those of biological in nature that can comprise proteins, carbohydrates, and nucleic acids). In one embodiment, the polymer is cross-linked and can comprise natural polymers and their derivatives, synthetic polymers and their derivatives, or a combination thereof. These natural polymers can be anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers.
[00100] Non-limiting examples of anionic polymers can include hyaluronic acid, alginic acid, carageenan, chondroitin sulfate, dextran sulfate, and pectin. Some examples of cationic polymers, include but are not limited to, chitosan or polylysine. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).
[00101] Examples of amphipathic polymers can include, but are not limited to collagen, gelatin, fibrin, and carboxymethyl chitin. Non-limiting examples of neutral polymers can include dextran, agarose, or pullulan. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Λ/m NY Acad Sd 944: 62- 73).
[00102] Synthetic polymers or derivatives thereof (for example, polyesters) can also be used to generate the cell growth substrate of the invention..Non-limiting examples of polyesters can include polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyacrylamide, and polydimethylsiloxane. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).
[00103] Using expertise in condensed matter physics with a focus in magnetism and magnetic materials, as well as interdisciplinary training in materials science, microfabrication, and cell biology, systems can be engineered for measuring the limits of cellular responses to mechanical perturbations. For example, a magnetic tweezers apparatus generates forces as large as 20 nN with frequencies from 0 to 3 kHz, values that match and exceed those found in tissues. This system allows application of local forces at the position of interest: lamellipodium, lamella, and perinuclear region, via beads (for example, magnetic beads) attached to specific receptors on the cellular dorsal surface.
[00104] In one embodiment, the bead can be a magnetic bead. In another embodiment, the diameter of the magnetic bead can be greater than or equal to about 0.1, 0.2, 0.3, 0.5, 0.6, 0.75, 0.8, 0.9, 1.0, 2.0, 2.5, 3, 4, or 5 μm in diameter. In a further embodiment, the diameter of the bead is about 2.7 μm in diameter.- In some embodiments, the cell growth substrate described above also comprises a magnetic material, such as a magnetic bead, wire and the like. Non- limiting examples of magnetic material include nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt. [00105] The trajectory of the beads in the example above is the result of the interplay between the magnetic force and the force exerted by the cell on the bead. For example, fϊbronectin-coated beads were placed on laminin coated glass substrates and mouse embryonic fibroblasts (MEFs) were subsequently allowed to spread. When the protruding edge of a cell made contact with a bead, initial adhesive contacts formed and the beads were displaced by the rearward actin flow in a radial trajectory towards the nucleus (FIG. 6A). Once the beads were observed to engage in rearward motion, a constant level of force was applied via magnetic tweezers (FIG. 6B). [00106] A range of cellular responses to the localized force was observed (such as cell growth; cell differentiation; apoptosis; cell movement/cytokinetics; and cell morphology changes). At relatively low force, F<500 pN, aside from an immediate visco-elastic response (see also Matthews, B. D., et al., J Cell Sci 119, 508-18 (2006)), the velocity of the bead returned to a constant value equal on the average to the pre-stress value. Very high forces detached the beads from the cells, but they were able to withstand ~10 nN for ~5 sec before breakage. In the intermediate 0.7-10 nN force regime, the onset of an adaptive phase when force is applied was observe, followed by restoration of constant bead velocity (FIG. 6C). For example, this is illustrated in FIG. 6C where 1.0 nN was applied.
[00107] The adaptive phase is defined by the presence of fluctuations in the bead's velocity and direction of movement, and can last between 20 and 140 seconds. Since the magnetic force is constant, the variations in bead velocity indicate variations in the cell force. This can be interpreted as a cyclic testing of the site of mechanical stimulation. As the cell adapts to the local stress, it generates pulsatory traction forces at the site of the mechanical signal, i.e. the bead. This is reminiscent of the periodic lamellipodial contractions in spreading and migrating cells (Dobereiner, H. G., et al.,. J Appl Physiol 98, 1542-6 (2005); Dubin-Thaler, B. J., Giannone, G., Dobereiner, H. G. & Sheetz, M. P . Biophys J 86, 1794-806 (2004); Giannone, G. et al. (2004). Cell 116, 431-43; Giannone, G. & Sheetz, M. P. Trends Cell Biol (2006)). [00108] It has been shown that -cells sense and respond to the rigidity of their environment by periodically pulling back on the advancing cellular edge. The magnetic bead assay can mimic the manner in which the ECM applies forces on cells in tissues. In addition to the evidence from the bead trajectory under external force, the dynamics of adhesion proteins is needed for understanding the mechanism of force transduction. When MEF cells expressing α— Actinin- GFP (FIG. 7A) were stimulated with large forces, a flow of this actin-associated protein was observed towards the bead. Maximum accumulation (seen as the red region in FIG. 7B occurred during the adaptive phase. While the magnetic force on the bead was maintained constant for periods exceeding the duration of the adaptive phase, the intensity of fluorescence decreased (FIG. 7B) as the beads recovered their constant rearward movement. Oscillatory local stimuli have recently been utilized to determine if the adaptive phase is stimulated by short pulses, thereby enabling the cell to sustain higher forces in the long term. This would allow the study of the cellular response (such as cytoskeletal reorganization) to pulsatory mechanical signals. The rigidity of the substrate can also be decreased to the point where normal cells will stop growing. By applying local modulated forces on the dorsal side via magnetic beads or nanowires, the activation of mechanosignaling can be controlled.
[00109] Whole cell assays can allow the cellular adaptive response to be studied when the environment (adhesive substrate) has different rigidities or is pulsating. In the first generation of actuated surfaces, two assays can be utilized: active and stretchable substrates. Active substrates (such as cell growth substrates) can be made with embedded magnetic materials (such as magnetic) nanowires that can be actuated in non-contact mode by means of an external magnetic field (FIG. 8). In one embodiment, the nanowires comprise a magnetic material. Some non- limiting examples of magnetic material include nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt. In a particular embodiment of the invention, the magnetic nanowires were nickel nanowires. In other embodiments, the diameter of the magnetic wire can be greater than or equal to about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm in diameter. In a particular embodiment, the diameter of the wire is about 300 nm in diameter. In some embodiments, the length of the magnetic wire (for example, the nanowire) can be greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μm in length, hi another embodiment, the length of the magnetic wire is about 30 μm in length. In a further embodiment, nanowire is about 300 nm in diameter, 30 μm long, and is comprised of nickel.
[00110] Synthetic biodegradable, matrixes, such as the cell grwoth substrate of the invention described in Example 1 or the microfence scaffold described in Example 2, can be manufactured using synthetic polymers (such as those described above), in addition to naturally occurring, absorbable materials, such as polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide- lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester), catgut suture, collagen, silk, chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid, elastin, and the like. The flexibility in their design allows the synthesis of a wide range of polymers with varying mechanical, biologic, and degradation properties. For instance, their mechanical and degradation properties can be manipulated by changing the polymer molecular weight during synthesis, and can thus be tailored to fit a particular application. Methods for fabricating 3D scaffolds using polymers and biomaterials are well established in the art. For review, please see Tsang and Bhatia, (2006) Adv. Biochem. Eng./Biotechnol. 103:189-205; Wang et al., (2006) Biomaterials 27: 6064-82; Khademhosseini et al., (2006) Proc. Natl. Acad. Sci. 103(8): 2480-7; and Leong et al., (2003) Biomaterials 27:. 6064-82, which are all incorporated by reference in their entirety.
[00111] An active substrate can be fabricated via suspending the wires in unpolymerized cell growth substrates (for example those made of polyacrylimide gels) and aligning them by a uniform magnetic field. Ferromagnetic nanowires are highly responsive to magnetic fields (Tanase, M. et al., (2001) Nano Letters 1, 155-158) and can be directed to self-assemble in arrays perpendicular to the gels' surface. Polymerization is then induced, and the wires remain in this configuration even after the aligning field is removed. In another embodiment, the active substrate can be fabricated via suspending magnetic beads in unpolymerized cell growth substrates (for example those made of polyacrylimide gels) and aligning them by a uniform magnetic field.
[00112] For example, soft gels can be employed that do not support normal cell growth (FIG. 8B). An external oscillating magnetic field will tilt the embedded magnetic nanowires, and transmit local oscillatory displacements at the surface of the gels and at the same time to the basal side of the cells (FIG. 8C). In preliminary tests, the displacements achieved at the surface of the gels were on the order of hundreds of nanometers. According to the model (FIG. 4), the cells may be able to sense the local displacements as sites of increased rigidity (FIG. 8D). This assay may allow the spatial correlation of mechano-response, and its controlled initiation. By varying the frequency and amplitude of the magnetic field, the stress at the surface of the gels can be reliably modulated, and the density of nanowires in the array determines the distance between the wires and ultimately the number of oscillating sites per cell. In varying these parameters, the mechano-induced cellular functions can be controlled. [00113] To address the cell in its entirety, substrates can be developed (such as those described above) that support many cells while being stretched. Cells can be plated on soft gels that will be subject to oscillatory stresses. A system that provides a stress gradient within the same substrate can therefore generate parallel data for cells in different mechanical environments. The invention provides an apparatus for applying a mechanical perturbation to a cell (for example, a cell in vitro), wherein the apparatus comprises the cell growth substrate described above in addition to an external force generator associated with the substrate. In various embodiments, the apparatus can further comprise a means for quantitating a cellular response to the mechanical perturbation.
[00114] Some non-limiting examples of cell responses can include cell growth; cell differentiation; apoptosis; cell movement/cytokinetics; and cell morphology changes. In one embodiment, cell growth can be assessed via measuring a gross change in cell size, an increase in cell proliferation, an increase in focal adhesion assembly, or a combination thereof. For example, gross changes in cell size can be determined with microscopy methods (light, fluorescence, electron, and the like) by obtaining cell size measurements. Cell proliferation can be examined via FACS analysis, cell density readings (i.e., ODβoo absorption readings), or other methods commonly used in the art. In another embodiment, cell differentiation can be determined via examining the expression of target genes (for example, via examining mRNA or protein levels) utilizing standard molecular biology methods practiced in the art. In a further embodiment, apoptosis can be investigated via examining cell viability or the expression and/or downregulation of target genes (for example, via examining mRNA or protein levels). In yet another embodiment of the invention, cell movement can be assessed via motility assays utilized in" the art. In other embodiments, cell morphology changes can be examined via light, fluorescent, or electron microscopy methods utilized by one skilled in the art. [00115] hi one embodiment of the invention, the external force generator is a means for physically manipulating the cell growth substrate. Some non-limiting examples of external force generating devices include a pipet-assisted manipulation device, laser tweezers, a magnetic twisting cytometry device, an optical trap, a magnetic field generator, and a substrate-stretching device (for example, a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate) (Sniadecki et al., (2006) Annals Biomed Eng 34(1): 59-74). . . .
[00116]- For example, large rectangular soft gels are fully released from their polymerization templates and anchored between a mobile and a fixed clamp (FIG. 9A). The latter one is positioned diagonally across the gel and with respect to the mobile clamp, such that the length of substrate suspended between the two varies incrementally from one side of the gel to the other. Strains were achieved that varied from 10 to 100% across the same gel. Elastic materials such as these gels undergo changes in strain (e=ΔL/ ) linearly proportional to the change in the applied
/*-o load (or stress σ = Force/ ). The rigidity of the material is given by Young's modulus o/ .
[00117] However, all such materials have an elastic limit beyond which the response to stress is non-linear and the rigidity of the material increases. As such, it is important to verify whether the rigidity of the gels remained unchanged when the gels were stretched. For example, large soft gels (22x50x1 mm, 8% acrylimide 0.03% bis) were placed under a load constant in time, such that the strain varied from 20 to 100% across the 22 mm width of the gels. No significant difference was observed in the spreading area of RPTPα +/+ cells plated for 12 hrs on soft gels under 100% (FIG. 9B), 20% (FIG. 9C), and zero strain (FIG. 9D). This indicates that the cells did not detect increased rigidity in the elongated substrates. For comparison, FIG. 9E shows cells plated on un-stretched rigid gels (8% acrylimide 0.2% bis).
[00118] A purpose of the whole-cell assay consisting of stretchable substrates is to investigate the long-term effects of pulsatory signal in soft environments on cell proliferation and migration. As such, the invention provides a method for stimulating growth of a cell in vitro. Here, cells can be plated on or in the cell growth substrate described above, the substrate containing the cell is perturbed (for example, mechanically, electrically, or magnetically), and the growth of the cell of the cell is detected, wherein the perturbation of the substrate stimulates the growth of the cell. The invention also provides a method for stimulating cell differentiation in vitro where cells can be plated on or in the cell growth substrate described above, the substrate containing the cell is perturbed (for example, mechanically, electrically, or magnetically), and the differentiation of the cell of the cell is detected, wherein the perturbation of the substrate stimulates the differentiation of the cell.
[00119] As described above, an elastic modulus (E) is the mathematical description of an object or substance's tendency to be deformed along an axis when an opposing force is applied along that axis. (E) describes tensile elasticity and thus reflects the measure of the stiffness of a given material. It is defined as the ratio of tensile stress to tensile strain and can be experimentally determined from the slope of a stress-strain curve created during tensile tests conducted on a sample of the material (described in Engler et al., (2004) Biophys J 86: 617-28, which is hereby incorporated by reference). In one embodiment of the invention, the cell growth substrate can have an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. For example, if a substrate has an elastic modulus of about 3 kPa, the cell growth substrate can promote a fibroblast cellular response (such as growth, differentiation, apoptosis, cytokinetics, morphological changes, and the like). If the substrate has an elastic modulus of about 2 kPa, the substrate can promote a myoblast cellular response, such as those previously described. If the substrate has an elastic modulus of about 1 kPa, the cell growth substrate can promote a neuronal cellular response, such as those described above. [00120] In one embodiment, the growth of the cell can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or a combination thereof. Examples of methods used to detect cell growth have been described above. In another embodiment, differentiation of the cell can be detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or a combination thereof. For example, the -expression of target genes can be ascertained by examining mRNA or protein-levels utilizing standard molecular biology and biochemical methods practiced in the art. Cell morphology changes in addition to changes in size can be examined via light, fluorescence, or electron microscopy methods utilized by one skilled in the art.
[00121] Mechanical perturbations can stimulate a cellular response. In some embodiments of the invention, perturbing the cell growth substrate entails subjecting the substrate harboring cells to an external force generator previously described. In one embodiment, the cell can be excised from a tissue (for example muscular tissue, such as skeletal, cardiac, or smooth; neuronal; connective; epithelial; or haemopoietic). The tissue can come from a cultured cell line or an animal (for example a mammal, such as a dog, cat, human, bird, and the like). [00122] The invention also provides for a method of promoting the growth of a cell in vivo. The method can comprise the ex vivo creation of a matrix from the cell growth substrate described above followed by insertion of the matrix into the body of a subject and subsequently applying a magnetic field to the subject, wherein the application of the magnetic field can promote the growth of the cell. In addition, the invention provides for a method of treating a wound, where the method can comprise the ex vivo creation of a matrix from the cell growth substrate described.above followed by insertion of the matrix into the body of a subject and subsequently applying a magnetic field to the subject, wherein exposure to the magnetic field can result in the proliferation of cells. In some embodiments, the subject can be an animal (for example a mammal, such as a dog, cat, human, horse, cow, sheep, rabbit, bird, and the like). [00123] In one embodiment of the invention, the cell growth substrate can have an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In other embodiments, the substrate can have an elastic modulus of about 3 kPa, wherein the cell substrate can promote a fibroblast cellular response. In further embodiments, the substrate can have an elastic modulus of about 2 kPa, wherein the substrate can promote a myoblast cellular response. In particular embodiments, the substrate can have an elastic modulus of about 1 kPa, wherein the cell substrate can promote a neuronal cellular response. [00124] Mechanical perturbations can stimulate a cellular response. In some embodiments of the invention, perturbing the cell substrate entails subjecting the substrate harboring cells to an external force generator previously described. The growth of the cell can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or a combination thereof. Examples of methods used to detect cell growth have been described above. Cell differentiation can be detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or a combination thereof. For example, the expression of target genes can be ascertained by examining mRNA or protein levels utilizing standard molecular biology and biochemical methods practiced in the art. Cell morphology changes in addition to changes in size can be examined via light, fluorescence, or electron microscopy methods utilized by one skilled in the art.
EXAMPLES
[00125] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate the exemplary modes of making and practicing the present invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods may be utilized to obtain similar results.
EXAMPLE 1 - RPTPα is required for the fibroncctin-specific rigidity response in hippocampal neurons
[00126] Loss of receptor-like protein tyrosine phosphatase alpha (RPTPα) in knockout animals results in severe hippocampal abnormalities, disturbance of learning and memory, and decreased anxiety. Mechanistic studies in fibroblasts link this enzyme to reinforcement of matrix contacts, force transduction, and matrix rigidity response, but its function in neurons is unknown.
[00127] In this example, RPTPα was shown to mediate fibronectin-specific rigidity responses in hippocampal neurons in an SFK-dependent process that leads to the recruitment of fibronectin and pl30Cas phosphorylation at the leading edge of the growth cone. The results show that the rigidity of a cell growth substrate can be manipulated to affect a cellular response, such as cell growth and differentiation (for example, as shown with neurons). Here, RPTPα is reported to be required for reinforcement of fibronectin-cytoskeleton bonds and the rigidity response in hippocampal neurons. Neurite differentiation and extension is inhibited on rigid fibronectin matrices in wild type hippocampal neurons, whereas neurons lacking RPTPα differentiate and extend neurites rapidly on both rigid and soft surfaces. The RPTPα dependence of the rigidity response in neurons is fϊbronectin-specifϊc/and requires αvβ6 integrin clustering at the leading edge of the growth cones. As in fibroblasts, RPTPα is necessary for the recruitment of the Src- family kinase, Fyn, to the leading edges as well as the phosphorylation of pl30Cas.
[00128] Materials and methods
[00129] Primary hippocampal neuron culture- Hippocampi were dissected from the brains ofRPTPα+/+ and RPTPα-/- neonate mice (Pl) and incubated for 30 minutes in 2.5% trypsin solution. Upon trypsinization neurons were dissociated through a series of the diameter- decreasing pipettes and resuspended in Neurobasal-A medium supplemented with 2% B-27 supplement, 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (all materials from Invitrogen). Neurons were plated on coverglass or gels that were pre-incubated with 20μg/mL polylysine and subsequently coated with 50μg/mL laminin (BD Biosciences) or 50μg/mL fibronectin (Roche).
[00130] Laser trap bead assays- Silica beads (0.64μm in diameter) were activated with avidin as described previously (Jiang, G., et al., (2003). Nature 424, 334-337) and coated with lOμg/ml biotinylated full-length FN (Roche) or lOμg/ml biotinylated full-length VN (BD Biosciences). As a control, beads were coated with biotinylated BSA (Sigma). Optical gradient laser trap set at 10OmW (40pN/μm) (Axiovert TV 100; Carl Zeiss Microimaging, Inc) was equipped with a 10Ox objective, and calibrated as described previously (Choquet, D., et al., (1997). Cell 88, 39-48). The beads were held at the smooth lamelipodium-like edges of the growth cones for approximately 3 -5 s, and then, the laser trap was turned off to check for binding. If the bead was bound, the trap was turned back on and the rearward movement of the bead was recorded using a cooled CCD camera. The fraction of the beads bound and moving rearward was calculated as mean + standard error for at least three independent experiments and statistical significance of the results confirmed by t-test (p<.01). The total number of beads included in analysis was at least 35 beads for each condition. The rearward movements of the beads were further analyzed using Nanotracker plug-in, with a tracking accuracy of 3-5nm for 0.64 μm beads. The MSD values were calculated using an algorithm modified from (Qian, H., et al., (1991). Biophys J 60, 910-921).
100131] Neurite extension on polyacrylamide substrates-The full-length FN-coated or LN- coated polyacrylamide substrates were prepared as described previously (Pelham, R. J., Jr., and Wang, Y. (1997). Proc Natl Acad Sci USA 94, 13661-13665). The flexibility of the substrate was manipulated by maintaining the total acrylamide concentration at 5% while varying the bis- acrylamide component between 0.25% (rigid), 0.1% (medium) and .025% (soft) (E=7.5kPa, E=4kPa and E=SOOPa, respectively) (Engler, A., et al., (2004). Biophys J86, 617-628). The uniformity of coating on the substrate surface was examined by coating the gels with proteins conjugated to Cy5 fluorophore (Amersham Biosciences) according to manufacturer's instructions and visualized by confocal microscopy. Experiments were performed 36h after the neurons were plated on the polyacrylamide gels. Neurite extension was quantified for at least 50 neurites for each condition and statistical significance of the results confirmed by t-test (p<.01). Data is presented as mean+standard error of at least two independent experiments. In experiments with SFK inhibitor, lOμM SU6656 (Calbiochem) was added after neurons were adhered to the substrate, and further incubated for total time of 36h. [00132] Antibodies- In this example, the following antibodies were used: a mouse monoclonal anti-RPTPα antibody (BD Transduction Laboratories), a mouse monoclonal anti- (Xvββ antibody (USBiological), a mouse monoclonal anti-Tau antibody (Biosource), an affinity purified polyclonal rabbit anti-phoshoY165Cas antibody (Cell Signaling Technology), a mouse monoclonal anti-Fyn antibody (Chemicon), goat anti-mouse Ig conjugated with Alexa 477 (Molecular Probes), a goat anti-rabbit Ig conjugated with Alexa477 (Molecular Probes), and goat anti-mouse Ig conjugated with Alexa 568 (Molecular Probes). To block binding of FN-coated beads, anti-ctsβi (Chemicon), anti-αv (BD Pharmingen), and anti-αvββ (USBiological) antibodies were added to the medium at 10mg/ml final concentration 30min prior to the bead assay experiments. GPen (.5mM) was used for blocking Ovβ3 integrins as described previously (Von Wichert, G., et al., (2003). J Cell Biol 161, 143-153).
[00133] Immunocytochemistry- Hippocampal neurons were plated onto FN/LN-coated coverglass or FN/LN-coated polyacrylamide gels. After 36h incubation for the described time, cells were fixed in 3.7% formaldehyde and permeabilized with 0.1% Triton. Fixed cells were incubated with primary antibodies (described above) for Ih followed by washing and incubation with appropriate fluorescent secondary antibodies (also described above). Fluorescent signals from all samples were visualized by confocal microscopy.
[00134] Microscopy and analysis- Images of immunofluorescently stained samples were acquired using a Fluoview confocal microscope (Olympus, Melville, NY) equipped with 4Ox, 6Ox, and 10Ox objectives. Analysis of acquired images was performed with the image analysis program, ImageJ (by W. Rasband (NIH, Bethesda, MD http://rsb.info.nih.gov/ImageJ).
[00135] Results
[00136] Reinforcement of integrin-cytoskeleton bonds is impaired in RPTP(X-/- neurons interacting with fibronectin, but not vitronectin [00137] Extracellular matrix proteins bind to specific integrin receptors on the cell surface, triggering a variety of signaling events that result in the formation of integrin-cytoskeleton bonds (Giancotti, F. G., and Ruoslahti, E. (1999). Integrin signaling. Science 285, 1028-1032). Next, these bonds can be "reinforced" by force-dependent recruitment of various focal contact proteins. Reinforcement of the integrin-cytoskeleton bonds is critical for the response to the matrix rigidity (Choquet, D., et al., (1997). Cell 88, 39-48; Giannone, G. et al. (2004). Cell 116, 431-43; Kostic and Sheetz, (2006) MoI Biol Cell. 17(6):2684-95). Since the RPTPα-/- fibroblasts were defective in the reinforcement of the FN- and VN-coated beads, the reinforcement of the integrin-cytoskeleton bonds may also have been impaired in RPTPα-/- neurons. To test this possibility, the laser tweezers assay was used. The beads coated with ECM proteins, were held by the optical trap at the leading edge of the growth cones to mimic interactions between advancing growth cones and the matrix (FIGS 1 OA-B). [00138] Since the defects in RPTP α-/- fibroblasts were most pronounced on FN, deficiency in neuron reinforcement of the FN-coated beads was also examined. The beads were placed at the edge of lamelipodium-like extensions of the growth cones with a laser tweezers. After a short period of time (3-5s), the beads were released from the laser trap to check for binding. When beads bound to the surface of the growth cone, the laser trap was turned back on and the rearward movement of the bead was recorded and further analyzed.
[00139] Most of the binding events purportedly occur through integrins, since the nonspecific binding of BSA-coated beads materialized at low levels in both control and knockout neurons (21.4+2.5% and 17.1+1.1%, respectively). Given that RPTPα is downstream of integrin activation by ECM, the bead binding frequency perhaps may not be affected by the absence of RPTPα. Indeed, no difference in binding frequencies of FN-coated beads was observed between RPTPa+/+ and RPTPα-/- growth cones (65.5+6.6% and 64.4+3.4%, respectively) (FIG. 10C). Fibronectin bead reinforcement was decreased by two-fold but bead binding was unaltered on RPTPα-/- neurons relative to controls (FIG. 10C). In contrast, vitronectin bead reinforcement and binding were unaltered on RPTPα-/- neurons relative to controls (FIG. 10D). [00140] Further, the rearward movement of bound beads was analyzed and the frequency of the reinforcement events was determined. Two major types of events were identified: some beads were moving rearward and once they reached the edge of the trap, they were pulled back into the center of the trap (designated as breaking events, FIG. 10A); others continued moving past the edge of the trap and toward the proximal portion of the growth cone, and occasionally continued up the axon (designated as reinforcement, FIG. 10B). The fraction of breaking events was reciprocally proportional to the rigidity of the trap and hence to the rigidity response (Jiang, G., et al., (2003). Nature 424, 334-337). As predicted, the number of breaking events was significantly higher in RPTPα-/- neurons than in controls (Fig. 10C). Therefore, the reinforcement of FN-clustered integrin-cytoskeleton bonds was impaired in RPTPα-/- growth cones, indicating that the rigidity response was impaired as well. [00141] The effect of RPTPα deletion on reinforcement was further determined by quantifying the diffusivity of the beads through calculation of the mean square displacement (MSD). MSD is used as a measure of the stiffness of the bead-cytoskeleton contact and is inversely proportional to the bead reinforcement (Choquet, D., et al., (1997). Cell 88, 39-48; Qian, H., et al., (1991). Biophys J 60, 910-921). Individual trajectories of the beads were generated (FIG. 1OE, 10F), and MSD was determined as described previously (Qian, H., et al., (1991). Biophys J 60, 910-921). The average MSD of the beads moving rearwards was determined during the initial period of time after the beads moved outside of the trap. The average MSD of the beads was two to threefold higher during early rearward movement on RPTPα-/- growth cones compared to RPTPa+/+ growth cones (FIG. 10G). MSD was on average higher for FN-coated beads bound to RPTPα-/- growth cones compared to RPTPa+/+ growth cones (FIG. 10G); however, no significant difference was observed for VN-coated beads (FIG. 10H).Thus, the greater bead diffusion along perpendicular axis in RPTPα-/- growth cones supports the hypothesis that RPTPα is required for reinforcement of integrin-cytoskeleton bonds. [00142] Since vitronectin (VN) binds strongly to some of the FN-binding integrins, the role of RPTPα in the signaling pathway stimulated by VN was also examined via experiments described above except that VN-coated beads were used (FIG.10D, 10H). No differences in reinforcement or binding frequencies were observed between knockout and wild type neurons. Hence, RPTPα may be involved in a signaling pathway upregulated by the activation of (a) FN-specific integrin(s).
[00143] FN-stimulated RPTPa signaling is activated through avβ6 integrin [00144] The particular integrin involved in the RPTPα-mediated reinforcement in neurons was investigated next. The expression of a variety of FN-specific integrin subunits was reported in the hippocampus (Pinkstaff, J. K., et al., (1999). J Neurosci 19, 1541-1556), including αvβe integrins (Chan, C. S., et al., (2003). J Neurosci 23, 7107-7116), whose expression was previously believed to be limited to epithelial cells. Since αvβ3 integrin was previously implicated in RPTPα— signaling, and was expressed at very low levels in the brain, different integrin subunits were hypothesized as being responsible for RPTPα activation in the rigidity response of the hippocampal neurons. Several candidates were tested by measuring the effect of blocking antibodies on reinforcement in the bead assay. Controls for each antibody were performed with BSA-coated beads. [00145] As an additional control, GPen, a short peptide that specifically inhibits Ovβj" integrins, was used. Addition of GPen had no effect on FN-coated bead binding and reinforcement. Further, a blocking antibody for ctsβi did not influence the behavior of the FN- coated beads. Interestingly, both anti-Ovββ and anti-0Cv integrin blocking antibodies reduced the binding frequencies threefold compared to antibody-free controls (FIG. 1 IA). Therefore, the binding levels in the presence of these antibodies were only slightly higher than the nonspecific binding level determined for BSA-coated beads (24.2+1.9% for anti-αv and 31.8+4.8% for anti- ctvββ, compared to 21.4+2.5% for BSA-coated beads). The reinforcement frequency (determined as ratio between the number of reinforced beads and the number of bound beads) was also significantly reduced (33.7+5.3% for anti-oCy and 35.7+10.0% for anti-ctvβό), and corresponded to the reinforcement levels in RPTPα-/- neurons (35.1+3.2%)(F1G. 1 IB). Thus, αvββ integrins may be involved in RPTPα-mediated reinforcement of FN-cytoskeleton bonds and FN rigidity response in the growth cones of hippocampal neurons.
IOO146| To further explore the role of αvββ integrin in this process, the effect of ECM substrates on αvβ6 integrin localization was examined. Neurons were plated on laminin (LN) and FN-coated glass for 48h, and then visualized αvββ integrins by immunofluorescence. On FN, otvββ integrins were concentrated at the edge of the growth cone (FIG. 1 IE). In contrast, on LN, ctvββ was concentrated at the cell body with lower levels along the axon, and very low levels in the growth cones (FIG. 1 IF). Interestingly, RPTPα was abundantly present in the growth cones of neurons plated both on FN and LN. (FIG. 1 1C, 1 ID). Thus, an increased fraction of OCvββ integrins accumulated at the edge of the growth cone upon interaction with FN may cause RPTPα activation, leading to the upregulation of the downstream rigidity response pathway. [00147] RPTPcc^ neurons are deficient in FN-specific rigidity response [00148] Fibronectin and laminin show differential distribution in the mammalian brain during development (Chun, J. J., and Shatz, C. J. (1988). J Cell Biol 106, 857-872; Hagg, T., et al., (1989). Neuron 3, 721-732). The upregulation of FN expression in the brain has been also reported in pathological conditions such as epileptic seizures (Hoffman, K. B., et al., (1998). Brain Res 812, 209-215) and Sturge- Webber syndrome (Comi, A. M., et al., (2003). Pediatr Res 53, 762-769), and FN is known to ameliorate the effects of ischemic stroke (Sakai, T., et al., (2001). Nat Med 7, 324-330). Since RPTPα is required in the reinforcement of the FN- cytoskeleton bonds in both fibroblasts and neurons, as well as in the rigidity response in fibroblasts, the effect of FN substrate rigidity on neurite extension in RPTPα -/- hippocampal neurons was examined.
[00149] It was important to determine first if rigidity and deletion of RPTPα altered the stages of differentiation (Dotti, C. G., et al., (1988). JNeurosci 8, 1454-1468): stage 1 is characterized by the absence of neurites; at stage 2, neurites of approximately equal lengths are extended (FIG. 12A); and at stage 3, the significantly longer axons are differentiated (FIG. 12B). Neurons were isolated from the brains of neonate mice (Pl) and plated on FN -coated polyacrylamide gels of varying rigidities. After 48h of incubation in serum-free medium, the lengths of extended neurites were measured and the differentiation stages of the neurons were determined (FIG. 12C). While RPTPa+/+ neurons differentiated faster on soft than stiffer FN-coated substrates (38.7%+4.8% neurons at stage 3 on rigid, 39.5%+3.7% on intermediate, and 62.7%+2.3% on soft), RPTPα-/- showed no preference for soft substrates (62.0%+6.5% neurons at stage 3 on rigid, 64.1%+6.6% on intermediate, and 66.3%+5.6% on soft) and differentiated at a rate similar to wild type neurons plated on soft FN-coated gels. Furthermore, the average lengths of the neurites (both axons and dendrites) were also reflective of the absence of a FN rigidity response in the absence of RPTPα. The soft substrate stimulated neurite extension in RPTPa+/+ neurons,
while the neurite outgrowth in RPTPa-/- neurons appeared independent of substrate rigidity (FIG. 12D). In addition, the neurites of RPTPα-/- neurons were on average longer and wavier
than those of RPTPa+/+ neurons (FIGS. 12D, 12E, 12F).
[OOISO] Whether the absence of a rigidity response in RPTPα-/- neurons was limited to FN- coated substrates was investigated next. On LN-coated substrates of different rigidities, RPTPα -/- neurons showed a significant rigidity response (FIG. 13). In both control and RPTPα -/- neurons, the increasing rigidities of LN-coated substrates inhibited both neurite elongation and axon differentiation. There was no difference evident in the LN rigidity response with the loss of RPTPα. Therefore, RPTPα may mediate the FN-specific rigidity response, while rigidity of LN matrices affects (an) alternate rigidity response pathway(s). [00151] SFK activation andpl30Cas phosphorylation are regulated by RPTPa in the FN rigidity response at the leading edge of the growth cone
[00152] Since SFKs, and in particular Fyn, have been previously implicated as RPTPα substrates in a variety of processes including the FN rigidity response in fibroblasts, SFKs may be involved in this process in neurons as well. Therefore, the effect of a broad SFK inhibitor (10μM SU6656) on the neurite extension and rigidity response to FN-coated substrates was examined. Similar to RPTPα-/- neurons, neurons cultured in the presence of the SFK inhibitor, showed no preference for the soft FN matrices, and there was no difference in neurite extension between substrates of different rigidities in the presence of inhibitor (FIG. 14A). However, the average neurite length was reduced in the presence of the inhibitor compared to both control and RPTPα-/- neurons, probably due to inhibition of various regulatory pathways involving SFKs. Although SFK inhibition had a negative effect on axon differentiation, the rigidity-dependence of differentiation was also abolished similar to RPTPα-/- neurons (FIG. 14B). Thus, it appeared that in addition to being involved in pathways regulating neurite extension (Robles, E., et al., (2005). JNeurosd 25, 7669-7681), SFK(s) are indispensible in the RPTPα-mediated FN-rigidity response.
[00153] Next, the effect of FN rigidity on localization of Fyn and its direct substrate p 130Cas was determined. Although pl30Cas was known as an indispensable component in the regulation of actin cytoskeleton organization, focal contact formation, and migration of fibroblasts (Cary et al., (1998). J Cell Biol. 140(l):211-21; Cho and Klemke (2000). J Cell Biol. 149(l):223-36; Honda et al., (1999.) Biochem Biophys Res Commun. 262(l):25-30), its role in neuronal motility has been poorly understood. However, this docking protein was highly expressed in the brain and it appeared to be required for the elongation of the cerebellar granule cell axons (Huang, J., et al., (2006). MoI Biol Cell J 7, 3187-3196). In addition, recent studies have identified pi 30Cas as a critical force-transducing molecule (Sawada et al., (2006). Cell. 127(5): 1015-26). [00154] Therefore, whether the substrate rigidity affects the distributions of Fyn and pl30Cas, which are potential downstream targets of RPTPα, was further examined. In RPTPa+/+ neurons, Fyn accumulation at the leading edge of the growth cones was greater on rigid FN substrates than on soft (FIG. 14C). In contrast, only low Fyn accumulations were observed in RPTPα-/- neurons regardless of substrate rigidity (FIG. 14D). This result indicated that Fyn recruitment to the leading edge was stimulated by substrate rigidity-dependent RPTPα activation.
[00155] Similarly, there was a decrease in the level of phosphorylated pl30Cas with a decrease in substrate rigidity (FIG. 14E), which was congruent with the stretch-dependence of pl30Cas activation previously shown in fibroblasts (Tamada, M., et al., (2004). Dev Cell 7, 709- 718). In RPTPα-/- neurons, only low levels of phosphorylated pl30Cas were detected at the edges of the growth cones -regardless of substrate rigidity (FIG. 14F). Thus, the formation of a force-transducing integrin-RPTPα complex at the leading edge followed by Fyn-mediated, force- dependant pl30Cas phosphorylation may be required for the FN rigidity response in neurons.
[00156] Discussion
[00157] In this example, dissociated hippocampal neuronal cultures displayed different growth properties on polyacrylamide gels of different rigidities. Given that RPTPα-/- mice display a severe hippocampal phenotype (Petrone, A., et al., (2003). Embo J 22, 4121-4131), and that RPTPα was implicated in force transduction and the rigidity response in fibroblasts (Jiang, G., et al., (2006). Biophys J 90, 1804-1809; Von Wichert, G., et al., (2003). J Cell Biol 161, 143- 153), RPTPα ablation may affect the rigidity response in neurons as well. Consistent with this hypothesis, RPTPα-/- neurons lacked the ability to distinguish between FN-coated substrates of varying rigidities, unlike RPTPa+/+ neurons which differentiated faster and grew longer neurites on softer than on stiffer substrates, hi contrast, the LN rigidity response was not affected by the absence of RPTPα, indicating FN specificity of the integrin(s) that activate RPTPα. [00158] Further, the correlation between the rigidity response and the reinforcement of integrin-cytoskeleton bonds was confirmed (Choquet, D., et al., (1997). Cell 88, 39-48). Motile growth cones were logical candidates to test for the impairment of reinforcement. Laser tweezers-held, ECM-coated beads were used to mimic interactions between substrate and motile growth cones, and determined that RPTPα-/- growth cones were impaired in FN-specific reinforcement. Further experiments with blocking antibodies indicated that FN binding to αvββ integrins was required for RPTPα activation. Interestingly, different integrins are required in processes such as radial migration (Anton, E. S., et al., (1999) Neuron 22, 277-289) and long- term potentiation (Kramar. E. A., et al., (2003). J Biol Chem 278, 10722-10730; Kramar, E. A., et al., (2006). Proc Natl Acad Sd USA 103, 5579-5584), which are severely impaired in RPTPα-/- mice. A specific role for αvββ integrins or the ββ subunit has not been elucidated previously in hippocampus development. While the FN-null mutation is lethal at an early embryonic stage (E8.5), null mutations for individual FN-specifϊc integrin subunits exhibit less severe phenotypes (Yang, J. T., et al., (1999). Dev Biol 215, 264-277). This has been explained by redundant roles that various integrins play in the process of cell adhesion. Interestingly, αvββ appears to be indispensable in the FN rigidity response. This finding suggests a possibility that integrin subunits might be non-redundant in fine-tuning of the cell adhesion and motility, as well as other functions such as LTP.
[00159] Finally, the RPTPα-mediated rigidity response in hippocampal neurons was shown to be SFK-dependent and that Fyn, as well as its direct substrate pl30Cas localize to the leading edge of the growth cones in a rigidity-dependant manner. The role of Fyn in the neuronal rigidity response might seem contradictory, since the broad inhibitor of SFKs had not only inhibited the rigidity response, but also overall neurite extension. This can be explained by a specific and non-redundant role that Fyn plays in the rigidity response. Therefore, the molecular mechanism of the FN rigidity response in neurons may be mediated through a pathway similar to the one proposed in fibroblasts (Jiang, G., et al., (2006). Biophys J 90, 1804-1809; Kostic and Sheetz, (2006) MoI Biol Cell. 17(6):2684-95). The model is illustrated in FIG. 15. It shows that rigidity could be sensed by the relative displacement of RPTPα-immobilized Fyn and a liganded integrin complex with pl30Cas that depends upon the rigidity of the surrounding matrix. In RPTPa+/+ neurons, the rigidity of the matrix triggers force-dependent activation of the RPTPα, followed by activation of Fyn, that consequently phosphorylates stretch-sensitive pl30Cas. This results in the further recruitment of the focal contact proteins causing the reinforcement of the interaction between the growth cone and the substrate. This reinforcement has a negative effect on the neurite extension. On the soft matrices, the force exerted by the actin-myosin network in response to the substrate rigidity, does not reach critical threshold necessary for the reinforcement of the FN-cytoskeleton bonds and subsequent focal contact formation. Therefore, the neurite extension is stimulated on soft matrices.
[00160] This model proposes that Fyn plays a critical role through its immobilization by palmitate groups to lipid domains near the leading edge. This is speculative but the phenotype of the Fyn knockout mice is similar to that of the RPTPα-/- in several respects (Grant et al., (1992) Science 258(5090): 1903-10). Similarly, pl30Cas is an important component in many motility pathways including rigidity response (Kostic and Sheetz, (2006) MoI Biol Cell. 17(6):2684-95; Tamada, M., et al., (2004). Dev Cell 7, 709-718; Vuori and Ruoslahti (1995) J Biol Chem. 270(38):22259-62); however, pl30Cas-/- mice die in utero before the brain has functionally developed making it difficult to determine the effect of pl30Cas ablation on brain development and function (Honda et al., (1998) Nat Genet. 19(4):309-l 1). Nevertheless, studies in dissociated cerebellar neurons showed that pl30Cas is required for neurite extension (Huang, J., et al., (2006). MoI Biol Cell 17, 3187-3196). Further, phosphorylation of p 130Cas has been recently shown to be directly related to force transduction (Sawada et al., (2006). Cell. 127(5):1015-26). Once phosphorylated, pl30Cas could signal to a variety of different pathways that normally promote growth and not differentiation. Thus the model is plausible but much more is needed to prove the exact roles of the components. [00161] It is perhaps surprising that the same molecular components would be implicated in the rigidity responses of such different cells as fibroblasts and neurons, particularly since in the two cases the cells have different rigidity responses. In fibroblasts, the response to the rigid matrix is needed for growth and motility, whereas, in neurons, the response to rigid matrix inhibits differentiation and mobility of the growth cones. In both cases, the response to rigid matrix appears to promote proliferation, and not differentiation. The response to soft matrices is apoptosis in the case of the fibroblasts and increased differentiation in the case of neurons. This can be explained by differences in the differentiation level and the motile structures present in these cell types. Neuronal growth cones are smaller than lamellae of the fibroblasts and they are known to pull the neurites forward. Reinforcement of the integrin-cytoskeleton bonds and focal contact formation stabilizes the lamellipodia and supports cell spreading in fibroblasts (Choquet, D., et al., (1997). Cell 88, 39-48; Giannone, G. et al. (2004). Cell 116, 431-43). Growth cones distinguish FN from LN by forming contacts on FN similar to focal contacts characterized in fibroblasts (Gomez, T. M., et al., (1996). J Neurobiol 29, 18-34). The stiff substrates may support formation of these focal contacts, which in turn stabilize interactions between growth cones and the substrate. This might result in reduced velocity of the growth cone progression and eventually in shorter neurites (FIG. 15). Similar rigidity responses could lead to different cellular responses in different cell backgrounds.
[00162] Thus, the FN-rigidity response in neurons may require RPTPα activation through CCvβό integrins, and subsequent recruitment of Fyn and its substrate pi 30Cas to the leading edge. This pathway appears to be critical for matrix rigidity-dependent regulation of neurite extension and axon differentiation, which can explain at least some of the abnormal aspects of hippocampal structure and function in RPTPot-/- mice. Since FN plays an important regulatory role in both normal development and variety of pathological processes in the brain, one could use these findings as a basis for further understanding of the basis for those diseases.
EXAMPLE 2 - Microfences as Active Surfaces for Cell Growth and Maintenance [00163] Active Fences Coated with Electrospun Fibers
[00164] We.have fabricated fences from PDMS using molds that were microfabricated by - standard microfabrication techniques (Tsang and Bhatia, (2006) Adv. Biochem. Eng./Biotechnol. . 103:189-205; Khademhosseini et al., (2006) Proc. Natl. Acad. ScL 103(8): 2480-7; Folch and Toner, (2000) Annu Rev BiomedEng. 2:227-56; Voldman et al., (1999) Annu Rev Biomed Eng. 1:401-25). Typically, fences are 1--2 micrometers in thickness, 5-10 micrometers in height, and up to lmm in length with spacing of 20 to 100 micrometers (see FIG. 16). For active fences, it is planned to have Piezo actuators (Pϊezo Systems, Cambridge, MA) or magnetic wires in the fences that will move them maximally 1-2 micrometers. Fibers will be electrospun of silk or other polymers and stretched over the fences in one-dimensional arrays (FIG. 16). Fiber diameters of 100 to 1000 nm will be used and fibers will be coated with growth factors, adhesion proteins, extracellular matrix molecules or fragments, or any combination thereof. In a typical experiment, cells will be applied to the fibers and followed over time to determine growth and differentiation patterns along with forces and morphology. [00165]

Claims

WHAT IS CLAIMED:
1. A cell growth substrate comprising:
a) a cross-linked polymer, wherein increased strain on the polymer does not substantially increase rigidity of the polymer, and wherein the polymer has an elastic modulus (E) of about 0.1 kPa to about 10.0 kPa; and
b) a growth medium,
wherein the substrate is permeable to liquids, gases, and cellular by-products.
2. The substrate of claim 1, wherein the polymer is not collagen, not hyaluronic acid, not polydimethylsiloxane (PDMS), and not a carbohydrate based gel.
3. The substrate of claim 1, wherein the polymer comprises an anionic polymer, a cationic polymer, an amphipathic polymer, a neutral polymer, a synthetic polymer, or any combination thereof.
4. The substrate of claim 3, wherein the cationic polymer comprises chitosan or polylysine.
5. The substrate of claim 3, wherein the amphipathic polymer comprises gelatin, fibrin, or carboxymethyl chitin.
6. The substrate of claim 3, wherein the neutral polymer comprises dextran, agarose, or pullulan.
7. The substrate of claim 3, wherein the synthetic polymer comprises a polyester or derivative thereof.
8. The substrate of claim 7, wherein the polyester comprises polyethylene glycol,
- polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, or polyacrylamide.
9. The substrate of claim 1, wherein the polymer is functionalized with chemical groups.
10. The substrate of claim 1, further comprising a magnetic material, a Piezo actuator, or a combination thereof.
11. The substrate of claim 10, wherein the magnetic material comprises nickel or iron.
12. The substrate of claim 10, wherein the magnetic material is in the form of a bead.
13. The substrate of claim 12, wherein the bead is about 0.1 μm to about 5 μm in diameter, about 0.2 μm to about 4 μm in diameter, about 0.3 μm to about 3 μm in diameter, about 0.4 μm to about 2 μm in diameter, or about 0.5 μm to about 1 μm in diameter.
14. The substrate of claim 13, wherein the bead is about 2.7 μm in diameter.
15. The substrate of claim 10, wherein the magnetic material is in the form of a nanowire.
16. The substrate of claim 15, wherein the nanowire is about 2-50 μm in length, about 3-40 μm in length, about 4-30 μm in length, about 5-20 μm in length, or about 6-10 μm in length.
17. The substrate of claim 15, wherein the nanowire is about 30 μm in length.
18. The substrate of claim 15, wherein the nanowire is about 100-500 nm in diameter, about 150-450 nm in diameter, about 200-400 nm in diameter, or about 250-350 nm in diameter.
19. The substrate of claim 15, wherein the nanowire is about 300 nm in diameter.
20. The substrate of claim 1 , wherein the elastic modulus is about 0.3 kPa to about 8 kPa, about 0.5 kPa to about 7 kPa, about 0.6 kPa to about 6 kPa, about 0.7 kPa to about 6 kPa, about 0.8 kPa to about 5 kPa, about 0.9 kPa to about 4 kPa, or about 1.0 kPa to about 3 kPa.
21. An apparatus for applying a perturbation to a cell in vitro, the apparatus comprising:
a) the substrate of claim 1 ; and
b) an external force generator associated with the substrate.
22. The apparatus of claim 21, wherein the external force generator comprises an electrical force, a magnetic force, a mechanical force, or a combination thereof.
23. The apparatus of claim 21, wherein the external force generator comprises a pipet- assisted manipulation device, a laser tweezer, an optical trap, or a magnetic field generator.
24. The apparatus of claim 21 , wherein the external force generator stretches the substrate.
25. The apparatus of claim 24, wherein the substrate is stretched intermittently.
26. The apparatus of claim 21, wherein the external force generator comprises a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate.
27. The apparatus of claim 21 , further comprising a means for quantifying a cellular response to the perturbation.
28. The apparatus of claim 27, wherein the cellular response quantified is cell growth, cell differentiation, apoptosis, cell movement, cell proliferation, cell morphology changes, or a combination thereof.
29. A scaffold for supporting cells, the scaffold comprising:
a) portions of the substrate of claim 1 ; and
b) fibers stretched over the substrate portions.
30. The scaffold of claim 29, wherein the portions are strips in the form of a fence.
31. The scaffold of claim 30, wherein the fence is about 0.5 μm to about 3 μm thick, about 0.6 μm to about 2.5 μm thick, about 0.7 μm to about 2 μm thick, about 0.75 μm to about 1.5 μm thick, or about 0.8 μm to about 1 μm thick.
32. The scaffold of claim 30, wherein the fence is about 2 μm thick.
33. The scaffold of claim 30, wherein the fence is about 3 μm to about 12 μm in height, about 4 μm to about 11 μm in height, about 5 μm to about 10 μm in height, about 5.5 μm to about 9 μm in height, or about 6 μm to about 8 μm in height.
34. The scaffold of claim 30, wherein the fence is spaced about 10 μm to about 200 μm apart, about 15 μm to about 150 μm apart, about 20 μm to about 100 μm apart, or about 25 μm to about 50 μm apart.
35. The scaffold of claim 30, wherein the fence is spaced about 25 μm apart.
36. The scaffold of claim 30, wherein the fence is less than about 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 μm in length.
37. The scaffold of claim 29, wherein the fiber is absorbable.
38. The scaffold of claim 29, wherein the fiber comprises polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide-lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester), catgut suture, collagen, silk, chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid, elastin, or any combination thereof.
39. The scaffold of claim 29, wherein the fiber is about 50 nm to about 1500 nm in diameter, about 100 nm to about 1250 nm in diameter, about 200 nm to about 1000 nm in diameter, about 250 nm to about 900 nm in diameter, about 300 nm to about 800 nm in diameter, about 350 nm to about 750 nm in diameter, about 400 nm to about 700 nm in diameter, or about 500 nm to about 600 nm in diameter.
40. The scaffold of claim 29, wherein the fiber is coated with a growth factor, an extracellular matrix molecule, or a combination thereof.
41. The scaffold of claim 40, wherein the extracellular matrix molecule comprises hyaluronic acid, collagen, chondroitin, or a combination thereof.
42. A method for stimulating a cellular response in vitro, the method comprising:
a) plating a cell on or in the substrate of claim 1 or the scaffold of claim 29;
b) perturbing the substrate or the scaffold containing the cell; and
c) detecting the cellular response,
the perturbation of the substrate or the scaffold stimulating the cellular response.
43. The method of claim 42, wherein the perturbation comprises a mechanical perturbation, an electrical perturbation, a magnetic perturbation, or any combination thereof.
44. The method of claim 43, wherein perturbing comprises subjecting the substrate or the scaffold to the external force generator of claim 21.
45. The method of claim 44, wherein the external force generator stretches the substrate or the scaffold.
46. The apparatus of claim 44, wherein the substrate or scaffold is perturbed intermittently.
47. The apparatus of claim 44, wherein the substrate or scaffold is perturbed up to 10 times per hour, up to 5 times per hour, up to 2 times per hour, or 1 time per hour.
48. The apparatus of claim 44, wherein the substrate or scaffold is perturbed up to 10 times per day, up to 5 times per day, up to 2 times per day, or 1 time per day.
49. The apparatus of claim 44y- wherein the substrate or scaffold is perturbed up to 10 times per week, up to 5 times per week, up to 2 times per week, or 1 time per week.-
50. The method of claim 42, wherein the cellular response is cell growth, cell differentiation, apoptosis, cytokinetics, morphological changes, or a combination thereof.
51. The method of claim 42, wherein the cellular response is detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, mRNA expression levels, a change in cell morphology or a combination thereof.
52. The method of claim 42, wherein the substrate or the scaffold has an elastic modulus of about 3 kPa.
53. The method of claim 52, wherein the substrate or the scaffold promotes a fibroblast cellular response.
54. The method of claim 42, wherein the substrate or the scaffold has an elastic modulus of about 2 kPa.
55. The method of claim 54, wherein the substrate or the scaffold promotes a myoblast cellular response.
56. The method of claim 42, wherein the substrate or the scaffold has an elastic modulus of about 1 kPa.
57. The method of claim 56, wherein the substrate or the scaffold promotes a neuronal cellular response.
58. The method of claim 42, wherein the cell is obtained from a tissue.
59. The method of claim 42, wherein the cell is a primary cell, is a cell from a cell culture that has been passaged, or is a cell from a cell line.
60. A method for promoting growth of a cell in vivo, the method comprising:
a) inserting the scaffold of claim 29 into a subject; and
b) applying a magnetic field to the subject,
wherein the application of the magnetic field promotes growth of the cell.
61. The method of claim 60, wherein the scaffold has an elastic modulus of about 0.3 kPa to about 8 kPa, about 0.5 kPa to about 7 kPa, about 0.6 kPa to about 6 kPa, about 0.7 kPa to about 6 kPa, about 0.8 kPa to about 5 kPa, about 0.9 kPa to about 4 kPa, or about 1.0 kPa to about 3 kPa.
62. The method of claim 60, wherein the scaffold has an elastic modulus of about 3 kPa.
63. The method of claim 62, wherein the scaffold promotes a fibroblast cellular response.
64. The method of claim 60, wherein the scaffold has an elastic modulus of about 2 kPa.
65. The method of claim 64, wherein the scaffold promotes a myoblast cellular response.
66. The method of claim 60, wherein the scaffold has an elastic modulus of about 1 kPa.
67. The method of claim 66, wherein the scaffold promotes a neuronal cellular response.
68. The method of claim 60, wherein cell growth comprises wound healing in a subject.
69. The method of claim 68, wherein the subject is a mammal.
0. The method of claim 68, wherein the subject is a human, a dog, a cat, a mouse, a rat, a horse, a pig, a cow, or a bird.
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US9157070B2 (en) 2010-04-12 2015-10-13 Spiber Technologies Ab Methods and combination comprising eukaryotic cells and recombinant spider silk protein
US10156561B2 (en) 2012-10-05 2018-12-18 The Regents Of The University Of California Mechanical stress response analysis of cells and tissues
WO2019025070A1 (en) 2017-07-31 2019-02-07 Università Degli Studi Di Genova A three-dimensional hydrogel scaffold for cell culturing and a method for the production thereof
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US9157070B2 (en) 2010-04-12 2015-10-13 Spiber Technologies Ab Methods and combination comprising eukaryotic cells and recombinant spider silk protein
CN102650077A (en) * 2011-02-24 2012-08-29 中国科学院合肥物质科学研究院 Chitosan-finishing iron nanowire, method for producing same and application thereof
US10156561B2 (en) 2012-10-05 2018-12-18 The Regents Of The University Of California Mechanical stress response analysis of cells and tissues
WO2019025070A1 (en) 2017-07-31 2019-02-07 Università Degli Studi Di Genova A three-dimensional hydrogel scaffold for cell culturing and a method for the production thereof
WO2022250614A3 (en) * 2021-05-28 2023-01-12 National University Of Singapore Cell-culture platform and a magnetic material for such a platform

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