Classifications

• • 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
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
  • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
  • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/20—Pills, tablets, discs, rods
  • A61K9/2004—Excipients; Inactive ingredients
  • A61K9/2022—Organic macromolecular compounds
  • A61K9/2031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyethylene oxide, poloxamers
  • A61K9/204—Polyesters, e.g. poly(lactide-co-glycolide)
• • 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
  • A61L27/58—Materials at least partially resorbable by the body
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
  • A61L2300/412—Tissue-regenerating or healing or proliferative agents
  • A61L2300/414—Growth factors

Definitions

• the present invention relates to the use of three-dimensional microrod scaffolds for the temporal release of growth factors useful in tissue regeneration, engineering, and treatment of disorders.
• a major challenge in engineering tissues is mimicking the complex cellular organization and function of the native tissues of the human body.
• Tissue structure and function are very highly interrelated so that cellular and macromolecular organization of the tissue often brings about mechanical and biological functionality.
• it is the circumferential arrangement of smooth muscle fiber layers that allows for change in the caliber in the lumen of blood vessels (Fawcett, 1986); the wickerwork pattern of collagen fibers in the skin give it mechanical strength (Alberts, 1994); the polygonal phenotype and complex arrangement of hepatocytes are essential for proper liver function (Boron, 2003); and the spiraling parallel arrangements of myocytes in the ventricle eject blood (Streeter, 1979; Sommer, 1995). Without proper cellular organization, an artificial tissue does not function adequately.
• a more promising approach involves myocytes grown on collagen networks that are mechanically paced to form 3D electrically connected strips that have been grafted in vivo (Zimmerman, 2002). This is appealing because it recreates trabeculae ensheathed with fibroblasts and endothelium ⁇ 100 ⁇ m in diameter. However, it is not easy to see how this approach could be scaled up for surgery.
• the recent exterior "chain-mail jacket” approach (Zimmerman, 2006) is fraught with practical difficulties for use in human patients.
• This model has an outer layer of connective tissue that might well cause fibrosis and prevent the myocytes of the graft from connecting directly with the healthy heart of the host. Without a good electrical connection the electrocardiogram would show incomplete synchronous activity. The cell source also remains an unresolved challenge.
• the present invention provides a scaffold comprising non-degradable or biodegradable microrods and optionally a carrier.
• the carrier is a matrix.
• the matrix is selected from the group consisting of: collagen, gelatin, gluten, elastin, albumin, chitin, hyaluronic acid, cellulose, dextran, pectin, heparin, agarose, fibrin, alginate, carboxymethylcellulose, matrigel, hydrogel and organogel.
• the microrods modulate the local microenvironment of a cell, alter a cell's cytoskeletal architecture, alter a cell's proliferation, including but not limited to promoting or suppressing cell proliferation, or regulating cellular organization, structure, phenotype or function.
• the microrods are biodegradable.
• the microrods are porous, have a textured surface or are porous and have a textured surface.
• the carrier is aqueous. In still other aspects, the carrier is saline or a buffer.
• the microrods are synthesized from one or more polymers.
• polymer is selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), poly(e-caprolactone) (PCL), poly(ethylene glycol) diacrylate (PEGDA), and SU-8.
• the microrods are synthesized from one or more copolymers.
• the copolymer is selected from the group consisting of poly(lactide-co-glycolide) (PLGA) and poly(DL-lactide-co-e-caprolactone) (DLPLCL).
• the microrods are on average, each about 1 micron to 1,000 microns in length.
• the microrods have a range of stiffness from about 1 kPa to about IMPa. - A -
• the microrods have a shape of a regular or irregular polyhedron.
• the microrods have a three-dimensional shape selected from the group consisting of: rod, cube, cone, cylinder, sphere, spiral, deltoid, asteroid, rhombus, parallelogram, trapezoid, cuboid, pyramid, prism, tetrahedron, pentahedron, hexahedron, septahedron, octahedron, nonahedron and decahedron, and irregular cross- sections.
• the microrods are associated with a biomolecule.
• the biomolecule is a protein in the insulin-like growth factor (IGF) family of proteins, an isoform of the IGF family of proteins, an E-domain peptide of the IGF family of proteins, or an Ea, Eb, or Ec domain peptide of the IGF family of proteins.
• IGF insulin-like growth factor
• the biomolecule is mechano-growth factor (MGF).
• MGF mechano-growth factor
• the MGF is stabilized or native.
• the biomolecule is an E- domain peptide of MGF or a biologically active fragment of MGF.
• the biomolecule is associated with the microrods by covalent interaction. In another embodiment, the biomolecule is elutable from the microrod.
• the microrods are associated with a targeting molecule that interacts with target cells expressing a binding partner for said targeting molecule.
• the targeting molecule is selected from the group consisting of: an organic compound, a drug, a peptide hormone, a cell adhesion molecule, a cell adhesion molecule ligand, an antibody immunospecific for an epitope expressed on a surface of a target cell type, a growth factor, a growth factor receptor expressed on the surface of a target cell type, a growth factor that modifies stem cell behavior, an anti-cancer agent, an anti-apoptosis agent, an agent that depresses fibrosis, an agent that builds blood vessels, an agent that suppresses cell proliferation, a cytokine, a chemokine and an anti-inflammatory agent.
• the microrods are associated with cells in vivo.
• the microrods are associated with progenitor cells in vivo.
• the cells are cardiac cells, fibroblasts, cardiac myocytes, bone marrow mesenchymal cells, endothelial cells, smooth muscle cells, vascular cells, cardiac stem/progenitor cells, neuronal cells, neuroendocrine cells, skeletal muscle cells, bone cells, tendon cells, connective tissue cells, stem cells, or fibroblasts.
• the microrod scaffold is an injectable composition or is surgically implantable.
• Methods are also contemplated by the present invention, hi one embodiment, a method for repairing damaged tissue comprising the step of administering the microrod scaffold to damaged tissue in an amount and over a time effective to repair damaged tissue is provided.
• the damaged tissue is muscle tissue or arises from a muscular disorder.
• the damaged tissue is cardiac tissue or arises from trauma.
• the damaged tissue is neuronal tissue or arises from a neurological disorder.
• the damaged tissue is neuroendocrine tissue or arises from a hormonal disorder such as diabetes.
• the damaged tissue is selected from the group consisting of skeletal, bone, tendon and connective tissue or arises from growth abnormalities, osteoporosis, fractures, and ischemic damage due to peripheral vascular disease.
• a method for promoting cell survival of differentiated cells comprising the step of administering the microrod scaffold to damaged tissue in an amount and over a time effective to promote cell survival is provided.
• a method for promoting differentiation of a stem/progenitor cell comprising the step of administering the microrod scaffold to damaged tissue in an amount and over a time effective to promote differentiation is provided.
• a method for promoting cell proliferation of a stem/progenitor cell comprising the step of administering the microrod scaffold to damaged tissue in an amount and over a time effective to promote cell proliferation is provided.
• Fig 1. shows a bright-field image of polymer microrods on wafer showing some released while others are still attached.
• Fig 2. shows microprojection effects on immature stem cell DNA replication as well as range and rate of beating.
• Fig 3. shows the effect of microrod stiffness on 3T3 cell proliferation in a 3D gel.
• Fig 4. shows the effect of microrods on mouse bone marrow mesenchymal cell (mBMSC) proliferation.
• Fig 5. shows microrods (lOO ⁇ m long) retard the proliferation of fibroblasts and increase myocyte size compared to growth in Matrigel alone.
• Fig 6. shows a time course of degradation of a PGA micropeg as example for expected microrod degradation.
• Fig 7. shows myocyte-positive mouse embryonic stem cell derived cardiomyocytes (ESCM) cells.
• Fig 8. shows encapsulation efficiency of drug ( ⁇ g) of PLGA particles (0.4 dL/g) synthesized using acetone or acetonitrile as the solvent.
• Fig 9. shows peptide release from biodegradable microstructured films.
• Fig 10. shows gene expression analysis in neonatal rat ventricular myocytes (NRVM) grown for 7 days on MGF E-domain eluting sheets.
• Fig 11. shows quantification of MGF and IGF-I isoform expression in the mouse heart following myocardial infarct using real time RT-PCR.
• Fig 12. shows expansion of stem cells in mouse heart with MGF treatment after myocardial infarction.
• Fig 13. shows pressure-volume loops from instrumented mice 2 weeks post myocardial infarction with and without systemic delivery of MGF E domain peptide.
• Fig 14. shows apoptosis analysis of mouse cardiac myocytes post-infarct.
• Fig 15. shows fluorescent images of SU-8 microrods injected into mouse tibialis anterior (left) and myocardium (right) for 2-days. - -
• microrod is a generic description that in only one aspect is a "rod-like” product. The term does not, however, imply that all microrods have the same shape, much less that they are all rod-like; despite the name, microrods of the scaffold can have any three dimensional shape.
• microrods of the scaffold are cubes, cones, cylinders, spheres, rods, cuboids, pyramids, prisms, tetrahedrons, pentahedrons, hexahedrons, septahedrons, octahedrons, nonahedrons or decahedrons.
• microrods having one or more different shapes are contemplated in additional aspects of the scaffold. Accordingly, in various aspects, microrods have a three-dimensional shape of any regular polyhedron, any irregular polyhedron, and combinations thereof.
• the shape of a microrod is, in some ways dictated by the intended use. That is to say, certain microrod shapes may be more desirable for specific tissues, specific locations in specific tissues or specific modes of administration or implantation.
• an injectable scaffold may require microrods of a shape that is amenable to the flow in an injection stream.
• microrods having an increased surface area are beneficial. Surface area of any microrod is increased by synthesizing the microrod having a textured surface. Alternatively, microrods can be synthesized to be porous as another means to increase surface area. In still another alternative, the microrods are synthesized in such a way that they are both textured and porous.
• microrods are either non-degradable, partially biodegradable, wholly biodegradable, or combinations and/or mixtures thereof.
• the microrods are synthesized from one or more polymers, one or more copolymers, one or more block polymers (including di-block polymers, tri-block polymers, and/or higher multi-block polymers), as well as combinations thereof.
• Useful polymers include but are not limited to poly(methyl methacrylate), polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), poly-caprolactone, and elatin/caprolactone, collagen-GAG, collagen, fibrin, poly( anhydrides), poly(hydroxy acids), poly(ortho esters), poly(propylfumerates), polyamides, polyamino acids, polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes and polysaccharides, polypyrrole, polyanilines, polythiophene, polystyrene, polyesters, non-biodegradable polyurethanes, polyureas, poly(ethylene vinyl acetate), polypropylene, polyethylene, polycarbonates, poly(ethylene oxide), polydioxanone, "pseudo-polyamino acid” polymer based on tyrosine, tyrosine-derived polycarbonate poly(DTE
• the microrods are formed from one or more phospholipids.
• 2- methacryloyloxyethyl phosphorylcholine (MPC) one or more cationic polymers (poly(a-[4- aminobutyl]-L-glycolic acid), or one or more silicone-urethane copolymers.
• microrods in the scaffold are formed from co-polymers of any of the above, mixtures of the above, and/or adducts of the above. The worker of ordinary skill will readily appreciate that any other known polymer is suitable for making microrods of the instant scaffolds.
• microrods of the invention are synthesized, physical properties of the microrods can be designed and controlled.
• microrods are on average, each about 0.01, about 0.05, about 0.1, about 0. 5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 1 15, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, _ -
• microrods of the scaffold have a cross-sectional area of about A microns times B microns, wherein A and B are independently selected from about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 305, about 310, about 315,
• the microrods are designed to have a specific stiffness which provides extracellular attachment and force transmission. Forces generated by cells are important in tissue morphogenesis and have been shown to affect a number of cellular processes including: assembly and organization of the extracellular matrix, gene transcription, cell motility, growth, differentiation, apoptosis, and signal transduction (reviewed in Chicurel, 1998; Shyy and Chien, 2002; Samarel, 2005). Matrix rigidity also affects cellular phenotype (Pelham and Wang, 1997): on more compliant substrates, cells were less spread, focal adhesions were irregular, and motility rates were higher than on stiffer substrata.
• Substrate rigidity also can be manipulated to direct cell movement (Lo, 2000) and to optimize differentiation and myofibril assembly in skeletal myotubes (Engler, 2004) and stem cell phenotype and differentiation (Engler, 2006).
• Physical forces encountered by cardiac cells in vivo include active force production, gain and loss of adhesion, membrane stretch, and compression due to changes in ventricular cavity pressure.
• the molecular systems through which cells convert mechanical cues from the extracellular matrix, ECM, into intracellular signals have been the subject of active investigation (Wang and Ingber, 1994; Ingber, 2003, 2006; LeIe, 2006).
• Cardiomyocytes adhere to their ECM through membrane-associated structures known as costameres, vinculin-rich complexes that simultaneously engage ECM filaments through transmembrane integrin receptors and - -
• FAs focal adhesions
• these channels serve as a foundation for complex feedback relationships between mechanical inputs and cellular contractility; for example, externally applied mechanical loads feed back through activation of Rho GTPase to increase FA density, which in turn supports the development of additional contractile myofibrils (Sharp, 1997; Torsoni, 2005).
• additional contractile myofibrils Sharp, 1997; Torsoni, 2005.
• microrods of the scaffolds have a stiffness of about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 305, about 310, about 315, about 320, about 325
• the scaffold comprising the microrods also includes a carrier.
• the nature of the carrier if present, is in certain aspects dictated by the intended use of the scaffold, and in other aspects, the means by which the scaffold in placed in a target tissue.
• the carrier is simply water, and in one aspect pharmaceutical grade water.
• the carrier is a buffer, and in certain aspect, the buffer is pharmaceutically acceptable. Buffers in the scaffold include, but are not limited to, saline, glycine, histidine, glutamate, succinate, phosphate, acetate, aspartate, or combinations of any two or more buffers.
• any pharmaceutically acceptable buffer is contemplated for use in the scaffold as a carrier.
• the carrier if present, is a matrix.
• a matrix is useful as a carrier in those instances when the scaffold is required to maintain some degree form, regardless of the pliability or permanence of the form.
• the matrix is viscous, yet still flowable, and in other aspects, the matrix is solid, semi-solid, gelatinous or of any density in between.
• the matrix is collagen, gelatin, gluten, elastin, albumin, chitin, hyaluronic acid, cellulose, dextran, pectin, heparin, agarose, fibrin, alginate, carboxymethylcellulose, matrigel, hydrogel organogel or mixtures and/or combinations thereof.
• any pharmaceutical grade matrix is amendable for use in a scaffold of the invention.
• Scaffolds of the invention are useful simply as microrods, or as microrods and a carrier in view of the fact that the microrods are capable of influencing the local environment of target cells, and in certain instances, alter cellular cytoskeletal architecture, alter cellular proliferation, suppress cellular proliferation, promote cellular proliferation or regulate cellular organization, structure, phenotype and/or function.
• the scaffold is utilized to further modulate a biological process in or around a target cell or tissue type - -
• the scaffold further comprises one or more biomolecules which has the ability to modulate one or more desired biological effects.
• a biomolecule is any naturally-occurring or synthetic chemical compound that is produced by or can play a role in the processes of living cells. Accordingly, biomolecule includes but is not limited to naturally-occurring and synthetic nucleic acids, including DNAs, RNAs, antisense RNAs, siRNAs, ribozymes, oligonucloetoides; lipids, including but not limited to phospholipids, glycolipids, steriods, sterols, cholesterol, prostaglandins, leukotrienes; carbohydrates, including but not limited to sugars, oligosaccharides, polysaccharides, monosaccharides, di saccharides; polypeptides, including but not limited to amino acids, peptides, enzymes, hormones, cytokines, chemokines, growth factors, vitamins, neurotransmitters; therapeutics, including but not limited to analgesics, antibiotics, anticancer agents, anti-inflammatory agents, anti-apoptotic agents; drugs, including but not limited to an
• the biomolecule is a protein in the IGF family of proteins.
• the biomolecule is IGF or a biologically active fragment thereof, including, but not limited to mechano-growth factor (MGF) or a biologically active fragment thereof.
• MGF mechano-growth factor
• a IGF E-domain peptide is the Ea, Eb or Ec domain or a biologically active fragment thereof.
• MGF E-domain peptide is the biologically active fragment thereof.
• the E-domain of MGF is produced by the heart within days after stress but not bound by circulating binding proteins and thus is available to potentiate the actions of local IGF-I.
• MGF is up-regulated in skeletal muscle under conditions of increased growth (Yang, 1996, 2002; McCoy, 1999), and it is a splice variant of IGF-I only produced by the tissue at times of stress. While IGF-I functions as an endocrine factor secreted by the liver, it also functions as a paracrine/autocrine growth factor expressed in non-hepatic tissues mediating regenerative processes thus having the potential for undesirable effects for therapeutic application (Russell, 1985; Vetter, 1986). Multiple IGF-I isoforms are expressed in different tissues that arise by alternate splicing from a common translated preprohormone.
• the preprohormone is cleaved by cellular endoproteases leaving the prohormone (mature 70 amino acid peptide + E-domains), which is further cleaved to yield identical mature peptides from all isoforms but different E-domains (Fo yt, 1991).
• One isoform, the IGF-IEa has a similar structure to the major endocrine form produced by the liver and has been given - 1 -
• IGF-IEb muscle-liver type IGF-I and muscle IGF-l .
• IGF-IEb muscle-liver type IGF-I and muscle IGF-l .
• MGF muscle-liver type IGF-I
• MGF-IEb muscle-liver type IGF-I
• MGF-IEb muscle-liver type IGF-I
• MGF-IEb muscle-liver type IGF-I
• MGF-IEb muscle-liver type IGF-I and muscle IGF-l .
• MGF IGF-IEb
• E-domain peptides are chemically modified to increase the molecule's stability, preserve its activity, or resistance to degradation.
• native IGF, MGF and/or E-domain peptides refers to non-stabilized or not otherwise modified MGF.
• the biomolecule is associated with the microrods by covalent interaction, and in other aspects the biomolecule is associated with the microrods by non- covalent association.
• a covalent interaction one or more biomolecules are directly attached to the microrod through any suitable means.
• one or more biomolecule is attached to the microrod through a space or linker that has no biological activity itself, or through a second biomolecule which possesses the same or a different biological activity compared to the first biomolecule.
• the biomolecule is elutable from the microrod.
• Elutable in various aspects means that the biomolecule can be separated from the microrods through, for example, simply diffusion, cleavage of a covalent bond, dissociation or some other type of interaction.
• the biomolecule in various aspects, is released in a controlled manner and in other aspect, the release is bolus in nature.
• the microrods are associated with a targeting molecule that interacts with a target cell or tissue expressing a binding partner for said targeting molecule.
• the targeting molecule is without limitation and for purposes of exemplification only, a cell adhesion molecule, a cell adhesion molecule ligand, an antibody immunospecific for an epitope expressed on the surface of a target cell type, or any member of a binding pair wherein one member of the binding pair is expressed on the target cell or tissue of interest.
• the microrods are associated with cells in vitro and in vivo.
• the microrods are mixed with cells and/or modified in such a way that the microrods attract cells from the environment, whether in vivo or in vitro.
• the cells associated with the microrods are of the same type as the target tissue or cell type for which the microrods are intended for use.
• the cells are stem cells, muscle cells, cardiac cells, smooth muscle cells, - -
• bladder cells brain cells, kidney cells, muscle (skeletal, smooth and cardiac) cells, neuronal cells, liver cells, pancreatic cells, and skin cells, as well as cells associated with tendons, cartilage and bone.
• the cells may be, but are not limited to: stem cells, embryonic, adult or progenitor cells; muscle cells, cardiac, smooth or skeletal myocytes, myoblasts, myoepithelial, myofibroblasts, myoendothelial or pericytes; bladder epithelial cells; brain cells, neuron, glial, epithelial or oligodendrocytes; kidney cells, parietal, podocytes, proximal tubule brush border, Loop of Henle thin segment, thick ascending limb, distal tubule, cortical collecting duct, medullary collecting duct or interstitial; neuronal cells, neurons, Schwann, basket, Betz, Purkinje, pyramidal, Renshaw, granule, anterior horn, motor neurons or alpha motor neurons; liver cells, hepatocytes, cholangiocytes or ovalocytes; pancreatic cells, Islet, alpha, beta, delta or polypeptide producing; skin cells, epidermatitis, fibroblast
• compositions comprising the scaffold of the invention are designed based on the intended use of the scaffold, for example, with respect to the target tissue or cell type and/or the route or means of administration.
• the route or means of administration is an overriding factor is preparation of the scaffold or a composition comprising a scaffold.
• Administration of the scaffold compositions may be accomplished in a number of ways including, but not limited to, surgical implantation, injection, parenteral delivery, including intramuscular, subcutaneous, intramedullary as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, intestinal administration, topical, inhalation, transdermal, transmucosal, buccal, pulmonary, sublingual, oral, rectal or vaginal. Osmotic mini-pumps and timed-released pellets or other depot forms of administration may also be used.
• Methods are also contemplated by the present invention.
• a method for repairing damaged tissue comprising the step of administering an aforementioned composition to damaged tissue in an amount and over a time effective to stimulate tissue repair.
• Methods of the invention are useful for the treatment of disorders such as, but not limited to, muscular disorders such as, but not limited to all forms of muscular dystrophy, atrophy, cachexia, sarcopenia and weakness of urethral sphincter muscle.
• the damaged tissue is cardiac tissue, wherein the method are useful for the - 1 -
• the damaged tissue is neuronal tissue, and methods are useful for the treatment of, among other disorders, neurological disorders (loss of neurons due to damage or maintenance of CNS, stroke), and neurodegenerative disorders (ALS, Parkinson's Alzheimer's).
• the damaged tissue is neuroendocrine tissue, and methods of the invention are useful for the treatment of, among other disorders, hormonal disorders (diabetes).
• the damaged tissue is skeletal, bone, tendon and connective tissue. And methods of the invention are useful for the treatment of, among other disorders, growth abnormalities, osteoporosis, and fractures.
• Ischemic damage due to peripheral vascular disease affects many of the tissues in body mentioned above, as well as others. All tissues damaged by ischemic disease would benefit from treatments with the invention.
• the invention provides methods for use of the microrod scaffold for veterinary uses.
• animal husbandry the administration of drugs to animals involves a great deal of handling which is laborious for the handler and stressful for the animals. Often animals must be brought in from long distances making prolonged or frequent treatment difficult if not prohibitive. In some cases the stress levels caused by handling stock can impair the performance of the treatment, this is particularly true in deer. Many drags are given in slow release capsules that can cause problems when still remaining in the animal at slaughter. (United States Patent 6,669,682)
• the present invention contemplates uses of the microrod scaffold for biomolecule delivery in veterinary applications. - -
• the invention provides methods for use of the microrod scaffold for tissue engineering.
• Tissue engineering often involves delivering a gel material such as hydrogel into a defective area of the body or to a body area where repair or reconstruction is desired.
• the gel material can be used as a support matrix for the growth of surrounding tissue cells.
• microrods are also dispersed in the gel material and injected or surgically implanted into the body along with the gel material to promote tissue regeneration, promote or suppress cell proliferation, alter cytoskeletal architecture, modulate the local environment of a cell or otherwise regulate cellular organization, structure, phenotype or function.
• the condition of skin tissue is affected by factors such as humidity, ultraviolet rays, cosmetic compositions, aging, diseases, stress and eating habits. As the result, various skin troubles can arise.
• the skin also becomes less resilient with age as illustrated by the formation of wrinkles. Aging is generally associated with the thinning and general degradation of skin. As the skin naturally ages, there is a reduction in the number of cells and blood vessels that supply the skin. There is also a flattening of the dermal-epidermal junction that results in weaker mechanical resistance of this junction. As a consequence, older persons are more susceptive to blister formation in cases of mechanical trauma or disease processes. (United States Patent 7,196,162).
• the skin also contains an elaborate network of elastin fibers that is responsible for maintaining its elastic properties. With excessive exposure to sunlight the elastic fiber system becomes hyperplastic, disorganized and ultimately disrupted. This process is known as actinic elastosis and it is the principal cause of wrinkling, discoloration and laxity of the skin in the exposed areas of the body. As new fibroblasts, endothelial cells and keratinocytes form, the skin can repair itself. However, the skin becomes less able to do so as it ages. Therefore, agents that can accelerate the growth and repair of prematurely aged skin are needed. (United States Patent 7,196,162)
• the invention contemplates methods for treating symptoms of aging where the microrod scaffold is administered in an amount and over a time effective to reverse or treat the symptoms of aging. - -
• compositions of the invention are also used for anabolic therapeutics.
• the invention is used to treat anabolic disorders including but not limited to, andropause, adisopogenital syndrome, functional metrorragia, fibroma and endometriosis as well as asthenia, osteroporosis, senescence and metabolic perturbations after prolonged treatment with corticotheraphy.
• Methods are also contemplated for anabolic therapeutics.
• the invention is used for treating anabolic disorders by administering an anabolically effective amount of the microrod scaffold over a time effective to reverse the effects of the anabolic disorder (United States Patent 4,431,640).
• Methods are also provided for promoting survival of differentiated cells comprising the step of administering a scaffold to damaged tissue in an amount and over a time effective to stimulate tissue repair.
• Methods are contemplated for promoting differentiation of a stem/progenitor cell comprising the step of administering an aforementioned composition to damaged tissue in an amount and over a time effective to stimulate tissue repair, is provided.
• a method for promoting cell proliferation of a stem/progenitor cell comprising the step of administering an aforementioned composition to damaged tissue in an amount and over a time effective to stimulate tissue repair.
• PLGA Poly(lactide-co-glycolide)
• PLA poly(lactic acid)
• PGA poly(glycolic acid)
• PLGA for drug delivery usually consists of the amorphous racemer D 5 L-PLA, which allows for more homogeneous drug dispersions (Jain, 2001).
• the PLA:PGA monomer ratio dictates the physical and chemical characteristics of the copolymer.
• the PLA methyl group (-CH3) renders the copolymer more hydrophobic and less crystalline than PGA, and thus it degrades slower.
• the ester bonds of the polymer backbone are hydrolyzed to form soluble oligomers and monomers that then enter the citric acid cycle (Shive, 1997).
• MGF E-domain peptide molecules in the PLGA MRS core are protected from enzymatic and chemical degradation until they are released from the matrix. This protection within the MRS permits the delivery of insoluble drugs and, most importantly, increases the chances that an otherwise unstable peptide can be delivered to its site of action.
• Microstructures with the volume (cross-sectional area 15xl5 ⁇ m2, length 100 ⁇ m) but varying in stiffness (tensile moduli 50 to 500 MPa) are used and fabricated as described below. All experiments are repeated with 5 primary cultures for statistical analysis.
• PLGA/PCL blends are initially chosen because this material can easily be microfabricated, is biocompatible, has a tunable modulus, and can degrade and release drug over a suitable time period ( ⁇ 2 weeks). Specifically, the effects of MRS made from polymer blends of varying stiffness are compared. The stiffness of the polymer MRS can be modulated in the proposed range by varying the PLGA co-polymer ratio and incorporation of a less stiff PCL component. This systematic tuning in mechanical properties was recently demonstrated by Sung (2005).
• PDMS masters are created from silicon wafers supporting SU-8 microrods.
• Microtranfer molding is then used to form the structures in PLGA and PLGA-PCL blends. Specifically, PLGA is dissolved in solvent and deposited on the PDMS mold and placed under vacuum to displace trapped air. A silicon wafer is placed over the mold and the mold is inverted, placed under pressure, and cured. Once cured, the mold is peeled off.
• One disadvantage of using microtransfer molding is the residual thin film of polymer interconnecting each individual microrod. This film can be removed by etching or by applying excess pressure to the mold to separate MRS from the film. In this manner, several batches of MRS may be generated from a single master. Using these preparation techniques, 50/50 PLGA microstructures with moduli ranging from 500 MPa to 1300 MPa were obtained. Furthermore, peptides may be added to the prepolymer solution and incorporated into the MRS during the transfer process.
• Tensile properties of the polymer blends used for the MRS can be measured using a method adapted from Huang (2006). Effects of polymer composition on mechanical properties are assessed through uniaxial cyclic loading using an electromechanical testing system. Uniform individual films (200 ⁇ m thick) are stretched to 10% strain, allowed to recover for 2 min, and loaded again to 0.3 N to failure. Linear (Young's) moduli, maximum stresses, yield strains and recovery percentages (working strains) are determined from the stress-strain curves. All experiments are repeated three or more times with triplicate samples.
• NRVF Neonatal rat ventricular fibroblasts
• MRS MRS concentrations of cells and of the MRS.
• NRVF Neonatal rat ventricular fibroblasts
• Three seeding concentrations of the NRVF 250, 500 and 1,000 Xl 03/ ⁇ m3 of Matrigel
• various MRS concentrations from 0, 5,000, 10,000 and 20,000 rods/microliter of gel are used.
• NRVM are terminally differentiated cells that no longer have the capacity to divide. Instead, NRVM are known to respond to increased workload, or increased reactive forces, by becoming larger (hypertrophy) and expressing a more mature contractile phenotype. Hypertrophy and maturation of the muscle phenotype increases proportionately when NRVM are exposed to embedded MRS of varying stiffness. Maturation of a muscle phenotype is not simply an increased size, but involves myofibrillar organization, contractile isoform expression as well as myriad calcium handling and energetic modifications.
• MRS with stiffness varying from 50MPa to 500MPa or cells grown in the Matrigel alone is used to analyze the effect of microrod stiffness on tissue architecture of NRVM and NRVF.
• Myofibrillar organization is assessed morphologically using confocal microscopy to identify well-organized sarcomeres by myosin heavy chain (MF-20) and ⁇ -actinin antibodies followed by counterstaining with FITC-phalloidin for anti-sarcomeric and F-actin.
• the Z- stack mode permits analysis of 3D structural features to determine the extent of longitudinally running myofibrils vs. the non-striated stress cables (Motlagh, 2003).
• NRVM maturation is characterized by the molecular diversity of myofibrillar isoforms, e.g. the troponin I (TnI) isoform transformation from the slow skeletal (ssTnl) to the cardiac (cTnl) isoform and from the ⁇ - to ⁇ -myosin heavy chain (MHC) (Schiaffmo, 1996).
• TnI troponin I
• ssTnl slow skeletal
• cTnl cardiac
• MHC ⁇ - to ⁇ -myosin heavy chain
• Microrods were created in a variety of polymeric materials, including biodegradable materials, using soft lithographic techniques previously developed (Deutsch- Snyder and Desai, 2001; Tao and Desai, 2005). Using this approach, rod-like structures were molded from a master template in a biodegradable polymer such as poly-lactic-acid, poly- glycolic acid or co-polymers whose features slowly degrade over time when incubated in warm, physiologic solution. MRS were designed to be -100 ⁇ m long with a 15 ⁇ m x 15 ⁇ m cross-section (Fig 1).
• PDMS poly-dimethyl-silicone
• Fibroblasts also interact with SU-8 microrods suspended in gel (Fig 3) and PEGDA microrods of two different concentrations (Fig 3). In a lmm-thick layer of Matrigel, fibroblasts migrated and spread in a manner typical in culture. However, inclusion of microstructures dramatically changed both the appearance and proliferation of the fibroblasts. Polarized fibroblasts interacted with microrods by attaching, spanning to another microrod, or growing alongside with the long axis of the cell following the long axis of the rod. A few cells had no interaction with the microrods.
• mBMSC Mouse bone marrow mesenchymal cells
• Fig 4A Phase contrast images show that mBMSC migrated to and aggregated around microrods of higher stiffness than the surrounding gel, i.e. mechanotaxis with different morphology.
• the WST-I assay determined the relative absorbance over Matrigel alone (intensity of experimental wells - intensity of Matrigel only) and thus, relative cell number after 1 and 5 or 7 days of culture (Fig 4 B, C). Both 50 and lOO ⁇ m microrod scaffold blunt proliferation is detected, but this effect was greater for longer microrods.
• fibroblasts formerly rat ventricular fibroblasts (NRVF) interacting with polymer microrods suspended in gel were carried out (Fig 5).
• NRVF neonatal rat ventricular fibroblasts
• Fig 5A fibroblasts migrated and spread in a manner typical in culture
• Fig 5B inclusion of microstructures dramatically changed both the appearance and proliferation of the fibroblasts.
• Polarized fibroblasts interacted with microrods by attaching, spanning to another microrod, or growing alongside with the long- axis of the cell following the long-axis of the rod (Fig 5B). A few cells had no interaction with microrods.
• NRVM neonatal rat ventricular myocytes
• Microengineered systems can be made to degrade over an intended time period. Given the appropriate biomaterial one can observe long-term maintenance of cellular organization with and without structural cues. The ability to microfabricate structures out of biodegradable materials (a PLGA co-polymer blend) (Figs 1 & 2; Snyder and Desai, 2001) and have achieved short-term cell organization has been demonstrated.
• biodegradable materials a PLGA co-polymer blend
• MRS are manufactured from biodegradable materials instead of traditional micro fabrication polymers such as PDMS or SU-8 to assess survival of tissue architecture after the topography disappears.
• Factors such as molecular weight, exposed surface area and crystallinity affect degradation rates (Lanza, 2000).
• processing conditions have can alter degradation characteristics of a material. For instance, heat molding can accelerate the degradation rate (von Oepen, 1992).
• Substantial degradation of a microstructure takes about a week in PGA and 3 weeks to a month on PLA (Snyder 2001). Therefore, a PLGA copolymer blend is used.
• T he stability of MRS are tested in three ways: 1) by looking at wet and dry weight over time; 2) by determining the feature size using scanning electron microscopy over time. To determine biodegradation, initial length, width and thickness of the MRS (approximately 100 microns long and 15x15 ⁇ m cross section) are measured. Next, initial weight of the substrata are measured; and 3) MRS are suspended in 3D gels with PBS (pH ⁇ 7).
• the dishes are placed in a 37 0 C incubator and the PBS changed regularly to simulate tissue culture conditions. Scaffolds are observed for weight, thickness, width and length loss at different time intervals every 24 hours (Kim, 2003; Wilson, 2002). This is repeated every 3 days over the 2-week period.
• Biodegradable, biocompatible, deformable grooved substrata (lO ⁇ m trench and mesa, 5 ⁇ m deep groove, 15-20 ⁇ m thick layer) were prepared from PLA, PGA, or PLGA.
• the surface was sterilized in 70% ethanol, coated with laminin and seeded with NRVM at 500,000/cm 2 . This membrane floats so it is weighted down.
• the NRVM were fixed and processed for immuno-localization. Degradation of microtexture was seen when incubated in DMEM culture media at 37 0 C over a period of 7 days. The initial height was 8.5 ⁇ m as seen in the X-profile. After 4 days, the height decreased to 6.5 ⁇ m. Finally at 7 days, features were ⁇ 4.6 ⁇ m in height.
• Such slowly degrading microstrueture was useful in our tissue engineering or drug delivery platforms (Fig 6).
• the well-established Rl ES cell line is used and can be grown indefinitely to derive a heterogeneous cell population, including cardiomyocytes (ESCM), see reviews (Boheler, 2002; Wobus, 2002; Boheler, 2003; Caspi and Gepstein, 2004; Wobus and Boheler, 2005).
• the wild type Rl cells are used to form an embryoid body by the hanging drop method and suspended for 2 days. Cells are dispersed and plated and after 2-3 days, clusters of beating cells are dissected, dispersed by 15-20 minutes in collagenase, spun and replated on gelatin coated dishes. These are allowed to attach, and additional media is added. Cells are cultivated and observed over the following week, and used over 2 - 4 weeks.
• a heterogeneous cell population of cells is derived that includes cardiomyocytes.
• ESCMs are in the 1 O ⁇ m size range and, based on our previous findings, microstructured features in the cell size range are recognized and effective in regulation.
• ESCM are grown in 3D gel with or without MRS. Thus, cell responses can be separated due to the MRS stiffness from those due to the 3D gel.
• Cells are grown on non-textured, flat PLGA surfaces as an additional 2D control. Gene expression is attainable with RT-PCR with specific primers used on cells isolated by laser trap.
• MRSs of different stiffnesses are used to quantify blunting of proliferation of non-cardiomyocytes from the heterogeneous population of ES cells, using desmin and ⁇ -actinin as muscle specific markers to identify the ESCM.
• BrDU is used to compare the extent of cell proliferation of cells near the 3D microscaffolds with those distant or grown on flat surfaces.
• the WST-I colorimetric assay is used to assess the overall proliferation of cells on MRS in 3D gel vs. gel alone. All experiments are repeated at least five times for statistical analysis.
• Beating rates provide indices of contractile maturity and are used to guide study of development and organization of the sarcomeric structures.
• Cells are fixed, permeabilized and stained and traditional immunochemistry used to compare cells near the 3D MRS gels with those in 3D gel alone.
• Myofibrils and sarcomeres are evaluated by myosin, a- actinin - 2 -
• the progression of early contractile isoforms is well known to provide useful information about the maturation of the myocyte early in cardiogenesis (Schiaffino, 1996; Westfall, 1996).
• the maturation level of ESCMs is assessed using quantitative RT- PCR to determine the levels of cTnl and ssTnl isoform expression in ESCMs at 2 and 7 days after exposure to MRS on varying stiffness.
• the sensitive immunochemical approach assesses distribution of proteins (such as phospholamban, calsequestrin) in ES cells at various locations near or distant from MRS.
• proteins such as phospholamban, calsequestrin
• the phospholamban transcript is present several days after the initiation of spontaneous contractions, while the SR calcium binding protein calsequestrin (Casq2) is expressed at a much later time point (-day 14 after EB plating).
• the physiologic transcript profile in vivo is mirrored for the proteins, such as phospholamban, and Casq2 (Fu, 2005).
• Data suggest that SR maturation occurs only with continued in vitro developments (Boheler, 2002; Yang, 2002; Sauer, 2001).
• Topography may provide a spatial and physical niche that mimics a remodeling process for many proteins that occurs with the heart's maturation (Spach, 2000; Angst, 1997), e.g. gap junctions and connexin 43 expression increases sharply during embryonic stages (Fishman, 1991). Focal adhesions are detected (n-cadherin, vinculin, paxillin), and connexin 43.
• Cardiac stem cells were derived from the Rl ES cell line and grown on microtextured surfaces and had distinct morphologies depending on attachment to the micro- projections (Fig 7).
• CM proliferation occurs during early stages of differentiation and spontaneous contractions are maintained for weeks.
• Cells on flat surface had a flattened morphology and tended to beat arrhythmically and much more slowly (ranging from 7-30 bpm).
• ESCM attachment of ES cells to the microprojections allowed ESCM to be derived and affects contractile properties.
• This Example permits verification of optimal formulation of MRS, time-release properties, ratios of MRS/myocyte, dosage of MGF.
• Biodegradable, PLGA-based microspheres have already demonstrated their potential for GF delivery in tissue equivalents (background).
• Protein release profiles from PLGA microspheres result from the interplay of drug diffusion through the polymeric time-evolving matrix, internal morphology of the system, and polymer erosion (Crotts, 1998; Ungaroa, 2006). In aqueous environments, water rapidly hydrates the particle and drug diffusion occurs through the innate PLGA micropores (angstrom- or nanometer-dimension) and the macroporous structure of the particle.
• MGF loaded PLGA MRS The methods used to create MGF loaded PLGA MRS are similar to those described for unloaded MRS above.
• MGF-PLGA solution is deposited on the PDMS mold and placed under vacuum to displace trapped air.
• MGF-PLGA solution is obtained by dissolving MGF and PLGA in acetonitrile to give the desired concentration.
• Excess PLGA is removed from the surface and a silicon wafer is placed over the mold. The mold is inverted, placed under pressure (50-150 psi) and cured at 65°C. Once cured, the PDMS mold is peeled off resulting in freestanding MRS. It is important to note that the solvent used (acetonitrile) and processing conditions are compatible with preserving MGF bioactivity as shown by the Goldspink lab as shown in preliminary data.
• the incorporation (encapsulation) efficiency measures the amount of peptide loaded into PLGA MRS in relation to the initial amount of peptide in the solvent during MRS fabrication.
• MGF-loaded PLGA MRS are solubilized in appropriate solvents, which liberate the incorporated MGF molecules.
• the released MGF is analyzed using a customized Enyzme Linked Immunosorbent assay (ELISA). This parameter is necessary in order to calculate the equivalency of MRS and MGF doses.
• ELISA Enyzme Linked Immunosorbent assay
• the MGF-loaded PLGA MRS is suspended in 1 mL of sterile PBS supplemented with 5 wt % penicillin/ streptomycin and place in a 37°C incubator for the duration of the study. At intervals of 1, 2, 4, 6, and 24 hours and daily thereafter, 1000 ⁇ L of eluant is collected and replaced by 1000 ⁇ L of new sterile PBS. The removed eluant is stored in two separate volumes of 250 and 750 ⁇ L for ELISA and bioactivity assays, respectively, at -8O 0 C until analyzed.
• MGF and IGF from MRS may permit myocytes to thrive under normal and even under hypoxic conditions. This is tested by use of the prototype MRS with the desired physiologic release profile described above to have an initial bolus within 12 hours followed by 2 week sustained release. Both the native and a stable form of the MGF E- domain, 24 amino acids, are used first and act as paracrine/autrocrine growth factors for muscle. NRVM are grown in 3D gel and introduce variable quantities of different formulations for the native or stable MGF-loaded MRS, with the necessary controls. Empty MRS are used to control for cytotoxity by the PLGA vehicle.
• Peptide in free solution or delivered via the MRS permits evaluation of the effect of the time-release properties of the MRS MGF level.
• the same amino acids in MGF are used in scrambled order as a negative control.
• Dishes without cells are used to confirm characteristic release properties after storage.
• NRVM are harvested at day 1, 7 or 14 after GF addition for assays to study short and long term effects on gene expression and apoptosis by biological evaluation methods described below. Five different cell isolations are used for statistical evaluations.
• the NRVM are isolated and plated in 3D Matrigel as above.
• a 100ng/ml bolus of stabilized or native MGF E-domain peptide is pipetted directly to the media, or we add the required number of MRS loaded to attain a similar total load.
• Sufficient MGF can be readily loaded into each MRS so that the quantity delivered can regulate the dosage for physiologic responses.
• Approximately two million NRVM is used per dish in culture. Calculations and data suggest that a ratio of 1 MRS per 1000 myocytes is sufficient, so we begin in that range.
• the native MGF peptide degrades rapidly in vitro with the majority being lost by 30 minutes and all by two hours in plasma at 37 0 C. Thus, the native MGF delivered in vivo would degrade before it reached its destination in underperfused regions of the heart.
• FITC labeled MGF is used in the MRS to characterize the time course of MGF release. Fluorometric measurements of FITC levels in the supernatant media are taken every hour for 6 hours and less frequently for the next two weeks. As a control, MRS alone is used to confirm the profile of MGF release seen in the acellular experiments described above. Thus, time profiles are collected for native or stable MGF- MRS with and without NRVM growing. - -
• Differences in elution of GF makes is measured using phase microscopy for tissue architecture of living cells in the 3D composite and also use confocal microscopy with Z- stack to determine the distribution of proteins with specific antibodies. A number of different acrylamide/bisacrylamide ratios are used to optimally separate the extracted proteins. Proteins are transferred to nitrocellulose and probed with specific antibodies using standard western blotting techniques (Goldspink 2004). Quantitative real time RT-PCR is also used to assess changes in muscle gene expression normalized to the housekeeping genes (such as GAPDH or L7) with SYBR Green detection in the LightCycler thermocycler (Roche Diagnostics).
• RNA is extracted from cells using Trizol and 100 ng of total RNA is used in each RT-PCR reaction. Quantification of the RT-PCR reaction is based upon a series of in vitro transcribed mRNA standards prepared for each gene and run along side to develop a standard curve as previously published (Goldspink, 2004). Primers for both the a- and ⁇ - myosin heavy chains are used as indices of contractile protein expression, and the ⁇ -subunit of L-type calcium channel for rhythmicity of beating.
• PI Propidium iodide
• PI can be used to assess dead cells. PI intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. PI is dissolved in buffer at 1 ⁇ g/ml. To assess viability of cell sample, 2 ⁇ L PI stock solution are added to each well and mixed well. Samples are kept in solution at 4°C protected from light until analysis on the flow cytometer.
• IGF-I has a potent effect on many stem cells, particularly those in skeletal and cardiac muscle and therefore, is tested on stem cells. It is likely that MGF is also active alone, or that it works together with IGF-I . Therefore, as described above, both MGF E- domain and IGF-I elution on mouse embryonic stem cells are tested. For normal heart development to proceed, complex interactions must take place among cardiac-restricted transcription factors to regulate early processes of commitment and differentiation, and to promote maturation (Garcia-Martinez and Schoenwolf, 1993; Olson and Schneider, 2003; Kelly and Buckingham, 2002). Early cardiac lineage markers, Nkx2.5, GATA-4, IsI-I are assayed after MGF treatment. The molecular markers for maturation of the contractile phenotype are TnI contractile isoforms.
• ESCM with unloaded MRS or treated with MGF alone, IGF- 1 alone, or MGF and IGF- 1 together are compared to determine the effects on their proliferative properties, apoptosis, differentiation and maturation ability. These experiments are used to determine the time course of cardiac lineage commitment and maturation of cells. Gene expression is attainable with RT-PCR with specific primers. Immunochemistry gives individual ES cell information. At least 5 cultures are assessed for statistical analysis.
• PCR analysis is used for early cardiac lineage markers, Nkx2.5, GATA-4, IsI-I for cells at various time points after exposure to different stiffness MRS. Comparisons are made at 2 and 7 days after GF treatment. Individual cells are also identified to determine the proportion that are muscle-positive (ESCM) in the whole population. The heterogeneous cells are fixed, permeabilized, stained and used to compare cells with or without GF MRS over the experimental time course. DNA synthesis in cells is quantified by BrDU incorporation and cells are co-stained with muscle-specific markers (desmin and ⁇ -actinin) to identify the ESCM sub-population. Random fields are counted for total BrDU stained cells and those that are also muscle positive.
• ECM muscle-positive
• Contractile maturation of ESCM is assessed as using quantitative RT-PCR to determine the levels of cTnl and ssTnl isoform expression in ESCMs at 2 and 7 days after treatment with MGF.
• Proliferation of mES progeny is determined with or without GF by the WST-I colorimetric assay.
• Apoptosis is assessed by Western blotting for activity of Cas ⁇ ase-C by 18kD cleavage fragment, annexin V and PI staining.
• Stem cell proliferation and cytotoxicity of ESCM in the presence/absence of MGF MRS is determined by cell number assessed by propidium iodide.
• NRVM showed that a IHz cycle 20% strain was the required stimulus for MGF expression, while the 10% strain at IHz was insufficient. Thus, the expression of MGF from stressed myocytes in vitro occurred after the first 2 days of extreme work. Rest and strain groups were the same for housekeeping gene, L7, and IGF-IEa.
• Treatment of hearts with the MGF E-domain peptide following infarction induces proliferation of stem cells with early cardiac lineage.
• Controlled delivery and release of the MGF E-domain peptide via the MRS may induce proliferation and mobilization of the cardiac stem cell pool and in conjunction with the 3D structure of the MRS, promotes engraftment and regeneration of new cardiac muscle cells.
• This combined approach of using the 3D structure and the elution of a physiologically relevant growth factor from the MRS serves to promote stem cell therapy to rescue the infarcted heart.
• MGF eluting MRS may provide a means by which resident cardiac stem cells may be supported for therapeutic use in cardiac muscle regeneration.
• the MGF E-domain eluting MRS is administered following a myocardial infarct and determine the effects at 2 days and 2 weeks post- infarct.
• the effects of localized MGF E-domain delivered via MRS on the cardiac stem cell population size, distribution and interaction with the 3D MRS at 48 hours post-treatment is assessed, as is the expression of cardiac specific lineage proteins and cell-to cell contact after degradation of the MRS at 2 weeks post-treatment.
• mice are anesthetized with methoxyflurane inhaled in a closed chamber and intubated with an 18-gauge angiocatheter. Surgical anesthesia is maintained using 0.5% isoflurane delivered through a vaporizer with a mixture of 95%oxygen/5%carbon dioxide connected in series to a rodent ventilator with the stroke volume set at 0.2 to 0.4 ml and a respiration rate of 125 breaths/min. A left thoracotomy is performed to expose the heart and the pericardium ruptured. The heart is exteriorized and the left coronary artery ligated to produce myocardial infarction. To administer the MRS therapy, the operator is blinded to which treatment is being delivered.
• mice Animals are allowed to recover in a heated cage before being returned to the animal facility.
• MGF-E-domain peptide are delivered via subcutaneous infusion using Alzet mini-osmotic pumps (100 / ⁇ l/pump) implanted immediately after the infarct for 2 weeks. Pumps are loaded with peptide (lmg/kg/day) dissolved in 5% mouse serum in saline and are surgically implanted and closed with a wound clip.
• mice are euthanized with isoflurane (2%); the hearts within each group are processed and systematically analyzed as follows. ⁇ 00130] 1 , lmmimohistochemical analysis on sections examines the expression of stem cell markers and expression of cardiac lineage specific proteins (5 hearts/group).
• Resident stem cells are isolated from the cardiac myocyte depleted cell suspensions prepared by collagenase digestion of the heart (5 hearts/group).
• Routine histology is useful to determine the inflammatory responses at day 2 after injection of the MRS in vivo. Tissue architecture in the injection sites is also assessed for fibrosis and normal myocyte features at all time points. Sections are blocked with serum before incubation with primary antibodies to label stem cell markers (Sca-1, c-kit, CD45, CD34, CD31 , (Pharmingen) cell cycling (Ki67 and BrdU), lineage specific proteins (Nkx2.5, GAT A4, IsI- 1), cTnl, ssTnl, connexin 43, smooth muscle actin, and Von Willebrand factor. Confocal microscopy is used to visualize fluorescent conjugated secondary antibodies.
• Controlled delivery and release via the MRS may provide an efficacious means for supplying the MGF E-domain peptide to potentiate the actions of the paracrine/autocrine IGF-I and prevent apoptosis. Promoting cell survival and preventing cell death in cardiac myocytes and cardiac stem cells means more are viable and thus the need to normalize wall stress is minimized, thereby preventing hypertrophy. These changes prevent the remodeling of the heart and lead to long-term improvement in ventricular function.
• the experimental approach is to administer the MGF E-domain eluting MRS following a myocardial infarct and determine the effects at 2, 4 and 10 weeks post- infarct.
• These time points are critical in the mouse model since they represent reproducible and measurable events that without treatment, herald the onset of the hypertrophic response (2 wks), compensatory hypertrophy (4 wks) and the decompensation to failure (10 wks).
• At each time point post-intervention we perform a complete analysis of the cardiac hemodynamic, geometric, biochemical and molecular data. These measures are determined relative to the administration and the actions of the MRS with or without the MGF E-domain peptide, which we predict serve as negative controls.
• mice within each cohort are systematically analyzed to ensure correspondence between, hemodynamic, geometric and biochemical data collection.
• the methodological approaches detailed below are first for the assessment of cardiac function in vivo and then for the molecular and biochemical studies.
• Echocardiography is used to examine both the structural and functional changes within each group.
• ventricular tissue is used for quantification of the infarct size, histological analysis near MRS injection site, analysis of apoptosis using TUNEL staining and immunohistochemical staining.
• RNA is extracted for analysis of gene expression using real time RT-PCR and protein expression evaluated using Western blot analysis.
• Echocardiography is used to examine the geometric changes of the heart.
• Transthoracic two-dimensional targeted M-mode and pulsed-wave Doppler echocardiography is performed with a 15-MHz linear array transducer attached to a Sequoia C256 system. Images of the left ventricle, LV, are taken from the parasternal short axis view at the level of the papillary muscles and LV internal dimensions are measured at the end of diastole and systole according to the American Society of Echocardiography leading-edge method on the M-mode tracings (Sahn, 1978).
• the LV fractional shortening (FS, %) is calculated from digital images as (LV end-diastolic dimension — LV end-systolic dimension) / LV end- diastolic dimension x 10-2.
• Stroke volume (SV) is calculated as product of main pulmonary artery (MPA) mean velocity time integral (VTI) and corresponding MPA mean cross sectional area. Multiplying SV by heart rate yields cardiac output (CO). All calculations are made from at least three consecutive cardiac cycles (Goldspink, 2004).
• mice After euthanasia of the mice with an overdose of isofturane (2%), the following structural, biochemical and molecular assays are done. The hearts are removed for determination of the heart weight to body weight ratio, followed by subsequent molecular and biochemical analysis. Mice are divided into two subsets (6 in each group). In one group, hearts are fixed and used for histological analysis of the effects of the MRS in the tissue, quantification of infarct size, analysis of apoptosis with TUNEL staining. In the remaining group, myocytes are freshly isolated and processed for analysis of apoptosis using FACS plus gene and protein expression analysis. Infarct quantification
• the abdominal aorta is cannulated, the heart(s) arrested in diastole with KCl, and then perfused with 10% (vol/vol) formalin at a pressure equal to the in vivo measured end- diastolic pressure.
• the left ventricular intracavity axis is measured, and three transverse slices from the base, mid-region, and apex are fixed, dehydrated and embedded in paraffin for sectioning. Sections are incubated with triphenyltetrazolium chloride (TTC) (20 mins, 37 0 C), which permits clearer discrimination of the various regions.
• TTC triphenyltetrazolium chloride
• the tissue in the center of the infarct zone is white, whereas the healthy tissue distal to the infarct zone has the typical deep red appearance.
• the mid-section is used to measure left ventricular wall thickness and chamber diameter.
• the infarct size is determined by planimetry of the scarred portion of the ventricle and expressed as a percent of scar/viable myocardium yielding the extent of fibrosis. Histology and immunochemistry is performed as described above.
• Apoptosis is observed using TUNEL staining in situ (apoptosis detection kit from CardiacTACS, Trevigen, Inc).
• pimonidazole hydrochloride are used as a marker of hypoxia (Hypoxyprobe TM-I Kit, Chemicon International). Animals will be injected with pimonidazole hydrochloride (60 mg/kg body weight), 90 minutes before sacrifice. Sections from these hearts are incubated with hypoxyprobel- Mabl for 40 minutes at room temperature (working dilutions 1 :50) and with goat anti-mouse IgG antibody FITC for 60 minutes (Sigma). Based on the hypoxia marker, pimonidazole hydrochloride, areas are designated as ischemic and periischemic for the MI group as previously published (Zampino, 2006). Western blotting and quantitative RT-PCR
• a number of different acrylamide/bisacrylamide ratios are used to optimally separate various proteins. Proteins are transferred to nitrocellulose and probed with specific antibodies using standard western blotting techniques. Membranes are incubated in Blocking Buffer consisting of 5% nonfat dry milk in TBS (2OmM Tris-HCl, pH 7.6, 137mM NaCl) for Ih. Affinity-purified antibodies against phospho-Akt (Ser473/308), phospho-Bad (Serl36), activated Caspase-3 C (18kD cleavage fragment), total Akt, FOXC ⁇ , Bad (Cell Signaling), incubated overnight at +4 0 C.
• Blocking Buffer consisting of 5% nonfat dry milk in TBS (2OmM Tris-HCl, pH 7.6, 137mM NaCl) for Ih.
• RNA is extracted using Trizol and a 100 ng of total RNA is used in each RT-PCR reaction (Goldspink, 2004).
• the stabilized peptide was delivered systemically (2 weeks) via mini-osmotic pumps implanted prior (12 hrs) to coronary artery ligation in mice. Whether the E-domain peptide is sufficient to either expand the resident population of stem cells or act as a mobilization/ homing factor attracting stem cells to the heart was determined. A side population of stem cells in the heart was identified defined by efflux of Hoechst and sensitivity to verapamil treatment (Asakura, 2002). Based upon reports in the literature, these cells usually constitute 0.03-0.06% of the total digest (Oh 2003).
• mice Four groups of mice are used: Sham, MI, Sham+MGF (E-domain peptide, lmg/Kg/day), and MI+MGF (Fig 13A).
• MI Sham+MGF
• MI+MGF MI+MGF
• Boheler KR ES cell differentiation to the Cardiac Lineage. In Methods in Enzymology. Editors: P.M. Wassarman and G.M. Keller, Volume 365, Chapter 16, pp.228- 241, 2003.
• IGF-I myocardial insulin-like growth factor 1
• Cardioprotective c-kit+ cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines. J Clin Invest. 2006 July 3; 116(7):
• Foyt HT LeRoith D, Roberts CT. Differential association of insulin-like growth factor I mRNA variants with polysomes in vivo. J Biol Chem. 1991 Apr 15;266(l l):7300-5.
• Gray DS Tien J, Chen CS, Repositioning of cells by mechanotaxis on surfaces with micropatterned Young's modulus. J Biomed Mater Res A. 2003 Sep l;66(3):605-14.
• Jain RA The manufacturing techniques of various drag loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials. 21(2000) 2475-90.
• Senyo SE Koshman YE, Russell B, Stimulus interval, rate and direction differentially regulate phosphorylation for mechanotransduction in neonatal cardiac myocytes.
• Microsphereintegrated collagen scaffolds for tissue engineering Effect of microsphere formulation and scaffold properties on protein release kinetics. Journal of Controlled Release. 113, 2006, Pages 128-136.
• Wobus AM and Boheler KR Embryonic stem cells - Prospects for developmental biology and cell therapy. Physiol Rev. 2005; 85(2):635-678.

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