WO2008127295A2 - Auto-assemblage guidé de matrices à structure nanométrique ordonnée utile pour le génie tissulaire - Google Patents

Auto-assemblage guidé de matrices à structure nanométrique ordonnée utile pour le génie tissulaire Download PDF

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WO2008127295A2
WO2008127295A2 PCT/US2007/022397 US2007022397W WO2008127295A2 WO 2008127295 A2 WO2008127295 A2 WO 2008127295A2 US 2007022397 W US2007022397 W US 2007022397W WO 2008127295 A2 WO2008127295 A2 WO 2008127295A2
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particles
fibrin
enzyme
protein
beads
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WO2008127295A9 (fr
WO2008127295A3 (fr
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Donald E. Ingber
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Boston Childrens Hospital
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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
    • C12N5/0075—General culture methods using substrates using microcarriers
    • 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
    • C12N2533/50—Proteins
    • C12N2533/56—Fibrin; Thrombin
    • 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
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C12N2535/10—Patterned coating

Definitions

  • the invention relates to methods and compositions for tissue engineering, wound healing and regenerative medicine.
  • ECM extracellular matrix
  • Ordered ECM structures are important for guiding cellular behavior in vivo, such as orienting cell motility during neurogenesis[5], epitheliogenesis[6] and angiogenesis[7]. These findings have been attributed to a mechanism termed "contact guidance"[8], in which anisotropic material properties can elicit a directional cellular response[9].
  • the nanometer scale structure of materials also determines their mechanical behavior[10]. The ability to develop biocompatible materials or artificial ECMs with defined nanostructure can therefore have significant value for tissue engineering and regenerative medicine.
  • biomolecules including peptides, lipids, and enzymes have been used to self-assemble gel networks for tissue engineering and drug delivery applications[21-26].
  • Described herein are methods for producing structures useful, for example, for tissue engineering.
  • the methods described herein involve an enzyme activity immobilized on a surface, which is placed in contact with a solution comprising polypeptide monomers.
  • the activity of the enzyme modifies the polypeptide monomers to promote polymerization of the monomers to form polymer structures associated with the surface.
  • the methods described herein provide a guided self-assembly of polypeptide structures.
  • the self assembly is guided to form regular lattices or other specific structures by specifying the arrangement of the surfaces to which the enzyme, optionally with one or more co-factors, is attached.
  • a structure comprising enzyme-associated particles and a protein polymer assembled adjacent thereto, wherein protein monomer precursors of the protein polymer are substrate for the enzyme, and wherein the activity of the enzyme upon the monomer precursors induces the polymerization of the protein biopolymer.
  • the particles are superparamagnetic.
  • the protein polymer is a fibrin protein polymer.
  • the particles are arranged in a substantially regular pattern.
  • the pattern is a substantially regular lattice.
  • the lattice is a triangular lattice.
  • the substantially regular pattern is achieved by applying a magnetic field to the particles.
  • the particles comprise thrombin.
  • the structure further comprises a viable cell.
  • the cell can be prokaryotic or eukaryotic. In one embodiment, the cell is a mammalian cell.
  • the fibrin protein is deposited in fibrils that are predominantly aligned along the main axis between adjacent particles.
  • a structure comprising superparamagnetic particles and a fibrillar biopolymer deposited thereupon, wherein said particles are arranged in a substantially regular lattice arrangement.
  • the paramagnetic particles comprise thrombin.
  • the structure further comprises a viable cell.
  • the cell is prokaryotic or eukaryotic.
  • the cell is a mammalian cell.
  • the fibrin protein is deposited in fibrils that are predominantly aligned along the main axis between adjacent particles.
  • the substantially regular lattice arrangement comprises a triangular, square, or chained lattice arrangement.
  • a method of preparing a protein polymer structure comprising: a) providing a plurality of particles comprising an enzyme; b) contacting, under conditions sufficient for catalytic activity of the enzyme, the particles and a precursor polypeptide monomer that is a substrate for the enzyme, wherein the enzyme catalyzes a modification of the precursor polypeptide monomer to a polypeptide monomer that polymerizes with itself or with another polypeptide, wherein the contacting results in such polymerizing, thereby forming a protein polymer structure comprising the particles.
  • the protein polymer structure comprises an ordered periodic structure.
  • the particles comprise superparamagnetic particles.
  • the method comprises exposing the particles to a magnetic field.
  • the enzyme comprises thrombin.
  • the precursor polypeptide monomer comprises fibrinogen.
  • the protein polymer structure comprises a fibrin lattice structure.
  • the method further comprises contacting the structure with a viable cell.
  • a method of producing an ordered periodic structure comprising paramagnetic beads and fibrin protein deposited thereupon, the method comprising: subjecting a suspension comprising thrombin-coated paramagnetic particles and a solution comprising fibrinogen to a magnetic field that draws the thrombin-coated paramagnetic particles to the air-liquid interface of the suspension, and maintaining the field for a time sufficient to permit the assembly of a fibrin lattice between the particles, wherein the paramagnetic particles and fibrin lattice form an ordered periodic structure comprising paramagnetic beads and fibrin protein deposited thereupon.
  • the method further comprises contacting the ordered periodic structure with a viable cell.
  • the term "predominantly,” when used in reference to the alignment of fibrils means that a given fibril is at least 2.5 times more likely to be aligned on the main axis between particles, as opposed to being perpendicular to the main axis.
  • protein monomer refers to a polypeptide that forms a repeating unit in a biopolymer assembled by polymerizing such monomer.
  • protein monomer precursor refers to a monomelic (i.e., not polymeric) form of a polypeptide that is the substrate for an enzyme that modifies the polypeptide in a manner that promotes the self- assembly of the modified monomers (now protein monomers) into a polymeric structure.
  • self-assembly refers to the polymerization of protein monomers that occurs after the enzyme modification of protein monomer precursors to a form that can polymerize (i.e., to protein monomers). While self-assembly refers most particularly to the an assembly process that does not require participants other than enzyme- modified (or "primed") protein monomers, the term “self-assembly” does not exclude the possible presence or requirement for one or more co-factors that aids the assembly process, e.g., by increasing the kinetics or by providing an enzyme co-factor necessary for the action of the enzyme, such as ATP or GTP, among others.
  • protein polymer refers to a polymer comprising monomers of a polypeptide.
  • a "polymer” includes at least 5 molecules of a protein monomer, preferably at least 10 monomer molecules, at least 20 monomer molecules, at least 30 monomer molecules, at least 50 monomer molecules, at least 75 monomer molecules, at least 100 monomer molecules, at least 200 monomer molecules, or at least 300 monomer molecules or more.
  • a polypeptide itself is a polymer of amino acids, but it is not a protein polymer.
  • a polypeptide becomes part of a protein polymer when it is assembled with at least 5 like molecules or more (a homopolymer) or becomes assembled in a repeating (i.e., three or more repeats, e.g., 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 repeats or more) structural assembly with different polypeptide monomers (a heteropolymer or co-polymer).
  • the term "induces the polymerization” means that an enzyme modifies a protein monomer or its precursor in a manner that promotes the self-assembly of a polymer of such modified protein monomers.
  • substantially regular refers to a pattern or arrangement in which particles are spaced at regular distances and angles from each other in 2D or 3D space.
  • regular distances is meant distances that vary by 10% or less, preferably 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or by even smaller variations, e.g., 0.1%, 0.01%, or less.
  • regular angles is meant angles that vary by 10% or less, preferably 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or by even smaller variations, e.g., 0.1%, 0.01%, or less.
  • the term "ordered periodic structure” refers to a structure that is comprised of a repeating pattern (i.e., at least 5 repeats, preferably at least 25, at least 50, at least 100 or more repeats) of a sub-structure.
  • the sub-structure can be a triangular subunit, and the ordered periodic structure is comprised of such triangular subunits packed together in space, for example, such that at the center of such a pattern, each side of a triangular subunit is shared by a said of another triangular s ⁇ bunit.
  • Subunits of e.g., rectangles, hexagons, or other regular polygons can be the basis of a repeating subunit in an ordered periodic structure.
  • the repeating subunit can include a 3D arrangement, e.g., a pyramid or a geodesic dome, among others.
  • microbead refers to a particle, preferably comprised of substantially biologically inert material in the size range of approximately 10 nm to approximately 25 ⁇ m. Preferred sizes are 100 nm to 5 ⁇ m. Microbeads can be made of any number of non-protein polymers, e.g. acrylonitrile polymer, or of super-paramagnetic materials ("superparamagnetism” refers to materials which become magnetic in the presence of an external magnet, but revert to a non magnetic state when the external magnet is removed).
  • the term "predominantly aligned” means that it is at least 2.5 times more likely that a protein fibril will be aligned on the main axis, as opposed to being perpendicular to the main axis between two particles.
  • Figure 1 shows a graph showing thrombin enzymatic activity 4.5 ⁇ m diameter magnetic microbeads covalently linked to thrombin (Thrombin Beads) compared to supernatant washes (Supernatants) and uncoated samples (Control Beads).
  • Figure 2 shows that fibrin clot size depends on bead concentration
  • photographs are shown of 35 mm plastic dishes containing fibrin gels formed by 7.8 X 10 4 (b), 1.9 X 10 5 (c), 6.7 X 10 5 (d), and 4.0 X 10 6 (e) thrombin-coated beads per ml of fibrinogen solution. Clot size increased as the bead concentration was raised.
  • Figure 3 shows that fibrin fibrils nucleate at the surface of the thrombin-coated beads.
  • Figure 4 shows a schematic diagram of the magnetic bead array guidance system.
  • Figure 5 shows phase contrast micrographs of bead patterns (a,c,e) and histograms of the inter-bead spacing distribution (b,d,f) within samples.
  • Highly ordered microbead arrays were created with magnetic guidance when applied to beads present either in PBS (a,b) or and fibrinogen solution (c,d) with the external magnet system, while random bead distributions were formed in fibrinogen solution (e,f) in the absence of an applied magnetic field.
  • Figure 6 shows low (a) and high (b) magnification confocal microscopic images of fibrin scaffolds created with a magnetically-oriented magnetic microbead array, and the a diagram showing the grid overlay system used for computerized morphometry (c).
  • the high magnification view was composed by overlaying 7 confocal Z-slices, each 1 ⁇ m apart (bead diameter, 4.5 ⁇ m).
  • Figure 7 shows fluorescence micrographs of HMVE cells cultured on the fibrin scaffolds (green) with defined nanoscale structure created using the magnetic fabrication technique and stained for F-actin (violet) and nuclei (blue), a) Cells adhered and spread well on these scaffolds, and remained viable after more than 2 days of culture, b) A single cell spread over many bead diameters which exhibits a radial array of actin stress fibers at a peripheral adhesion site near a bead (arrow) that is surrounded by a similar radial array of fibrin nanofibrils in the clot below, c) A region where multiple cells are spread over the fibrin clot; arrow indicates a small membrane extension containing two actin stress fibers that extend in parallel with underlying oriented fibrinogen nanofibrils that extend radially from the surface of a nearby magnetic bead.
  • the methods described herein take advantage of the action of an enzyme on a protein monomer to induce polymerization of the monomer.
  • the enzyme When the enzyme is associated with a surface, polymerization of the monomers occurs at, and extending away from, the surface to form protein structures associated with the surface.
  • the surface is, for example, a nano- or micro-bead, the polymers can form between the beads to generate structures that are influenced by the arrangement of the beads.
  • the enzyme- associated bead guides the assembly of protein structures that can have any 2- or 3- dimensional structure, defined by the arrangement of the beads.
  • the self assembly is guided to form regular lattices or other specific structures by specifying the arrangement of the surfaces to which the enzyme, optionally with one or more co-factors, is attached.
  • lattices of polymers formed from polypeptide monomer subunits that self-assemble into polymeric structures at a desired site, and preferably, with a particular spatial geometry are described.
  • the methods described herein can be applied to a range of polypeptide polymerization systems, each generally characterized in that the process of polypeptide monomer subunit polymerization is catalyzed by an enzyme reaction that modifies the monomer subunit in a manner that promotes ordered aggregation of the modified polypeptide monomers into polymeric forms.
  • biopolymer lattices produced by these methods, including, for example, fibrin lattice structures.
  • the structures described herein can be generated in vitro or in vivo. While the polymerization of fibrin microfilament structures is described in the Examples herein, other polymer systems can be used to generate these and other structures in a similar manner by immobilizing an enzyme and, if necessary or desired, any co-factor(s) either necessary for the polymerization or that influence, e.g., the efficiency of such polymerization. Upon contact with the enzyme, immobilized, for example, on a microbead, polypeptide monomers are modified such that they polymerize.
  • cytoskeletal polymers such as actin, tubulin, and vimentin, among others. See, e.g., Kawamura et al., 1970, J. Biochem. 67: 437-457 and U.S. Patent No. 7,192,702, which describe in vitro actin polymerization, and Kuriyama, 1975, J. Biochem. 77: 23-31 and Sandoval et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75: 3178-3182, which describe tubulin polymerization in vitro.
  • the enzyme modification can be, as non-limiting examples, cleavage of a precursor monomer (by a protease), or attachment or removal of a moiety, e.g., attachment of a phosphate (by a kinase enzyme) or removal of a phosphate (by a phosphatase enzyme).
  • the enzyme can be native to the species in which it will ultimately be used, e.g., a human enzyme for use in human systems, or it can be heterologous. Enzymes applicable to the methods described herein can be wild-type enzymes from any species, e.g., bacterial or fungal enzymes, up through enzymes from higher eukaryotes, e.g., mammals.
  • the enzyme can be a recombinantly altered enzyme.
  • Alterations might include, for example, alterations that provide altered (e.g., broadened, narrowed, or shifted) substrate specificity and/or alterations that provide altered reaction kinetics.
  • a wide range of enzyme alterations affecting these and other properties of the enzyme e.g., stability, thermal activity profiles, etc.
  • the alteration of enzymes to provide desired effects is well within the abilities of one of skill in the art.
  • Such structures include protein polymer structures assembled by the guided self-assembly approach provided herein.
  • the protein polymer structures can include, for example, structures comprising microbeads that were coated with enzyme, e.g., magnetic microbeads, that have become integrated into the protein polymer structures.
  • the structures can further include, for example, cells that are or have become associated with the polymeric structures.
  • the structures are substantially regular structures, and even more particularly the structures can be where enzyme-associated beads have been arranged with a particular 2D or 3D geometry prior to and/or during protein polymerization.
  • Fibrin plays a critical role in tissue development, hemostasis, angiogenesis, and wound repair.
  • Fibrin clots (3D matrices composed of fibrin) are formed when thrombin — a downstream enzyme in the coagulation cascade — cleaves the plasma glycoprotein fibrinogen to release fibrin protein.
  • Continuous thrombin activity increases the concentration of soluble fibrin monomers and induces them to self-assemble into linear polymers called protofibrils.
  • Protofibrils subsequently aggregate laterally and become covalently bonded to form fibrin fibers and lattices that make up the backbone of the fibrin clot[27].
  • Fibrin clots form physiologically in response to blood vessel damage where they immediately prevent bleeding, and later promote angiogenesis and tissue repair[28]. They also form at sites of bone fracture where they facilitate regeneration in the fracture callus, and nerve cells can use fibrin clots as an ECM bridge to repair severed nerves.
  • fibrin plays a critical role in many natural healing processes, it has been investigated extensively as a biological scaffold for bone[29, 30], cartilage[31], neural[32], adipose[33], and blood vessel[34, 35] regeneration. These studies revealed that the structure and morphology of fibrin networks (i.e., fiber size, branching, fiber spacing) influence their physical properties (e.g., viscoelasticity[36]) and biological functionality[37], including their ability to support nerve growth[38], leukocyte migration[39], and capillary morphogenesis[40-42] in vitro.
  • physical properties e.g., viscoelasticity[36]
  • biological functionality[37 including their ability to support nerve growth[38], leukocyte migration[39], and capillary morphogenesis[40-42] in vitro.
  • Described herein is a method to spatially constrain the fibrin self-assembly process on the nanometer to micrometer scale by using magnetic fields to orient thrombin-coated magnetic microbeads. While enzymes are often coupled to magnetic beads for biochemical applications[43], their use to catalyze the formation of ECMs with oriented microstructure is new.
  • an external magnetic field was used to organize superparamagnetic microbeads coated with thrombin into a two-dimensional (2D) periodically-ordered geodesic array at the air-liquid interface of a fibrinogen solution. The external magnetic field controls the lattice shape and spacing.
  • Tosyl-activated superparamagnetic beads (4 X 10 7 beads in 100 ⁇ l; 4.5 ⁇ m diameter; Dynal Biotech, Oslo, Norway) are washed with phosphate buffered saline (PBS) without calcium or magnesium (Invitrogen Corporation, Carlsbad, CA), reconstituted in carbonate buffer (pH 9.4), and combined with 300 ⁇ l of bovine plasma thrombin (50 U/ml; Sigma) containing 0.1% bovine serum albumin (BSA; Intergen, Purchase, NY).
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the beads After incubation for 24 hrs at 4°C, the beads are washed with 0.1% BSA in PBS and resuspended in 100 ⁇ l of the BSA solution. Control beads are prepared by leaving thrombin out of the 0.1% BSA solution.
  • enzyme e.g., thrombin or other protein-monomer- modifying enzyme can be associated covalently or non-covalently with the surface or bead.
  • Methods for the covalent and non-covalent association of enzyme proteins with solid supports, including microbeads, are well known to those of skill in the art.
  • H-D-Phenylalanyl-L-pipecolyl-L- arginine-p-nitroaniline dihydrochlorine H-D-Phe-Pip-Arg-pNA «2 HCl; Diapharma Group, Inc., West Chester, OH
  • H-D-Phe-Pip-Arg-pNA «2 HCl; Diapharma Group, Inc., West Chester, OH
  • Fibrinogen from bovine plasma (Sigma) is reconstituted at 10 mg/ml in 0.9% (w/vol) NaCl, sterilized by filtration through a Nalgene (0.22 ⁇ m) filter, and dispensed into aliquots for storage at -20 0 C.
  • fibrinogen conjugated to Alexa Fluor-488 (10 mg/ml; Molecular Probes, Eugene, OR) is mixed 1 :1000 with the unconjugated fibrinogen solution prior to use.
  • Photomicrographs of fibrin fibril nucleation from the surface of the beads are obtained with a small number (3.8 x 10 4 ) of beads using differential interference contrast (Nomarski) imaging every 30 sec for 40 min with an Eclipse 2000-E inverted microscope (Nikon) equipped with a Photometries CoolSnap HQ digital camera (Roper Scientific, Inc., Duluth, GA) and IPLab image acquisition and processing software (Vaytek). Additional images are obtained on a DM IRE2 confocal microscope with a TCS SP2 acousto- optical beam splitter (Leica, Northvale, NJ).
  • the magnetic array system is composed of a Neodymium- Iron-Boron (NdFeB) Nickel coated ring magnet (0.750" OD x 0.410" ID x 0.375" thick, 0.5 T peak field at the magnet surface, Master Magnetics, Castle Rock, CO) placed on top of an iron washer (0.750" OD x 0.125" ID x 0.125” thick). Resultant fibrin clots are fixed and images are recorded on the Eclipse 2000-E microscope. The fibrinogen and CaCl solutions are in a 150 Mm NaCl, 10 mM HEPES (Sigma) buffer (pH 7.4) for this part of the study only.
  • NdFeB Neodymium- Iron-Boron
  • Ideal magnetic field-induced bead arrays used as controls are formed with uncoated beads in PBS solution (i.e., rather than fibrinogen solution) maintained at 23 °C for 1 hr.
  • a thresholding IPLab script is used to identify the coordinates of the bead centers in photomicrographs from three different regions of three separate samples, and a program written in MatLab computer software (The Math Works, Inc., Natick, MA) determines the distance between each bead and its three nearest neighbors.
  • fibrin fibril orientation To analyze the nanostructure of the fibrin gels, 143 ⁇ l fibrinogen solution (10 mg/ml) is combined with 5 ⁇ l 0.1% Triton-X-100, and 2 ⁇ l thrombin coated beads (1.9 X 10 beads/ml). The solution is placed on a Lab-Tek glass slide, and incubated at 37°C for 7-14 hours, approximately 2 cm below the bottom of the magnet.
  • the resultant fibrin array clots are then incubated in EBM-2 medium (Cambrex Bio Science Walkerville, Inc., Walkerville, MD) for 5-20 hr at 37 0 C with 5% CO 2 , fixed, and mounted under CoverWell imaging chamber gaskets (Molecular Probes) with Fluoromount- G (Southern Biotechnology Associates, Inc., Birmingham, AL).
  • Z-stacks of confocal images identifying triangular bead arrangements are flattened into a composite projection image to capture the fibrin clot 3D architectural features.
  • a grid of squares (Fig. 6c) is overlaid on these images either with its long axis parallel to a line drawn between the beads comprising the triangle or with the long axis parallel to a line drawn between one bead and the mid-point of the opposite side linking the two remaining beads of the triangle.
  • Fibrin fibril intersections with the vertical (V) and horizontal (H) grid lines, and values for the total lengths of these lines (TV and TH, respectively), are used to calculate an orientation ratio (V/TV: H/TH). Orientation ratios are obtained from four different locations in two separate bead arrays.
  • HMVE Human microvascular endothelial cells from neonatal dermis (passage ⁇ 8, Cascade Biologies, Inc., Portland, OR) are cultured in EBM-2 supplemented with microvascular endothelial cell growth medium (EGM-2 MV). Cultures are maintained at 37°C in a humidified atmosphere with 5% CO 2 .
  • clots are seeded onto fibrin clot bead arrays (6,000-25,000 cells/ml) created using the magnetic fabrication technique; after 2 days, clots are washed with EGM-2 MV, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton-X, 0.1% BSA in PBS prior to staining of actin with Alexa Fluor-594 phalloidin and nuclear staining with 4',6-diamidino-2-phenylindole (DAPI, 1 ⁇ g/ml in PBS, Molecular Probes).
  • DAPI 4',6-diamidino-2-phenylindole
  • the methods described herein can be used to generate protein polymer structures, and particularly ordered protein polymer structures.
  • Such structures can be used, for example, for tissue repair, e.g., by forming a clot-like structure at the site of blood vessel damage. Because the arrangement of the structure's polymers can be guided into an ordered form, e.g., a lattice, the number of possible shapes and arrangements of the polymeric structures is essentially limited only by the number of different arrangements one can impose on the enzyme-associated surfaces or beads.
  • the ability to direct the generation of a polymeric structure at a desired location at will can be exploited therapeutically.
  • the formation of a structure in vivo at a desired location can be guided or directed by placement of a magnetic probe by catheter.
  • the magnetic probe collects enzyme- associated microbeads at the site where the probe is placed by catheter, and the enzyme directs the modification of monomer polypeptides at that location.
  • the high local concentration of modified monomer results in the assembly of polymers in the location of the beads held in place by the magnetic probe.
  • the structure Upon release of the magnetic field, the structure remains in place for therapeutic function.
  • Microneedle magnetic catheter probes and methods for their use are described, e.g., in WO 2006/039675, titled “Apparatus and Method for Nanomanipulation of Biomolecules and Living Cells,” which is incorporated herein in its entirety by reference.
  • a protein monomer can be engineered as a fusion with a therapeutic moiety.
  • a high local concentration of the therapeutic moiety can be achieved by stimulating the polymerization of the fusion protein at the site of an enzyme- coated microbead, and the microbeads can be drawn to or concentrated at a desired site, e.g., by placement of a magnetic probe by catheter as discussed above.
  • the bead/protein polymer structure remains, and the therapeutic moiety can have its effect on surrounding target tissue(s).
  • the catheter can actually remove the assembled structure if so desired after a period of time.
  • Cells sense and respond to nanoscale cues on the surface of planar adhesive scaffolds, such as variations in substrate topography[50], surface roughness[51] and adhesion ligand organization[52]. It is likely that cells within living tissues also sense and respond to 3D nanoscale features of physiological ECMs. However, developing biomaterials with defined spatial presentation of these signals on a submicron length scale in order to control cell function has proven to be technically challenging.
  • a novel system in which enzyme-coated magnetic microbeads that were magnetically arranged into ordered periodic structures were used to catalyze the formation of a natural fibrin biomaterial with defined anisotropy composed of nanometer sized fibrillar components.
  • Active thrombin chemically cross- linked to the surface of magnetic microbeads, induced fibrin clot formation as a function of bead concentration.
  • time-lapse and confocal microscopy confirmed that fibrin fibrils nucleated primarily from the region around the surface of the beads and extended out in a radial direction within these gels.
  • the fibrin nanofibrils that subsequently polymerized from the beads preferentially oriented along the main bead-bead axes in a triangulated geodesic pattern.
  • These scaffolds were biocompatible and cultured cells could sense and responds to nanoscale structures created within the clots, as detected by co-alignment of actin stress fibers and underlying fibrin fibrils.
  • Example 1 Bead-immobilized thrombin is active
  • Magnetic microbeads (4.5 ⁇ m diameter) that were coated with thrombin (7.4 x 10 "9 Units/bead) via tosyl cross-linking were found to retain high levels of thrombin activity when analyzed using an amidolytic assay to measure the level of cleavage of the synthetic tripeptide substrate, S-2238 (Fig. 1).
  • control beads that lacked thrombin had virtually no activity, and analysis of bead washes confirmed that thrombin-coated beads that were washed more than four times failed to release any significant soluble thrombin activity (Fig- 1).
  • the thrombin enzyme was able to maintain its catalytic activity after being chemically coupled to the surface of the magnetic microbeads using tosyl cross-linking chemistry.
  • the immobilized enzyme cleaved significant quantities of thrombin-specific chromogenic substrate and formed macroscale fibrin clots when incubated in fibrinogen solution. Even though only 2% of the thrombin provided during the coupling reaction remained bound to the beads following extensive washes, a relatively low concentration of beads ( ⁇ 10 6 AnI) was sufficient to create robust clots. Fibrin clot formation was clearly due to thrombin immobilized on the beads, and not soluble enzyme, because an equal volume of bead supernatant was not able to form a clot.
  • Nomarski microscopy was carried out to analyze the spatial pattern of clot formation.
  • Time-lapse recording revealed that the first fibrils appeared near the surface of each bead within 10 min after fibrinogen addition, and that these fibrils continued to grow and extend until they eventually coalesced with fibrils extending from the surface of neighboring beads to form a dense fibrillar lattice (Fig. 3a-d).
  • a representative confocal image of a clot formed in this manner clearly shows individual fibrin fibrils extending radially from the surface of each bead (Fig. 3e).
  • beads, with bead spacing of R also produces a bead-bead repulsion force, F m (/?) « — .
  • the beads should self-assemble at equilibrium into 2D regularly spaced triangular lattices (diagrammed in Fig. 4) as a result of a force balance between inter- bead repulsion due to their parallel magnetization and the concurrent force that pulls all the beads towards the center of the ring magnet[47-49].
  • the nanometer scale fibrin fibrils that polymerized from the beads preferentially oriented along the bead-bead axes and hence, formed a similar triangulated pattern (Fig. 6a,b).
  • an orientation ratio was determined with a grid placed either along the main bead-bead axis or at a 30° angle to that axis (i.e., along a line stretching from a bead to the mid point of the opposite side of the equiangular triangle formed by three adjacent beads; Fig. 6c).
  • fibrin fibrils were almost 3 times (2.64 ⁇ 0.31) more likely to be aligned along the main axis between adjacent beads than along perpendicular grid lines.
  • fibrin fibrils were predominately oriented along the bead-bead axis throughout the lattice, even though fibrin monomers released by thrombin cleavage were initially in solution (i.e., only the catalytic thrombin enzyme was immobilized on the beads).
  • fibrin lattices with defined structure on the nanometer scale can be constructed magnetically.
  • the peak field and gradient at the sample can be simply adjusted by changing the distance between the magnet and the sample: higher magnetic fields yield closer-packed lattices.
  • the lattice shape can be altered by varying the angle between the magnetic field and the 2D plane of the air-liquid interface[46, 47, 49]. Low angles yield chains, and high angles yield triangular lattices. In the present study, an ideal triangulated lattice was formed when the external magnet was oriented centrally and perpendicular to the interface.
  • HMVE cells were cultured on the fibrin matrices formed by the magnetically-induced arrays of thrombin-coated beads to investigate biocompatibility with the engineered substrate and to examine cell-biomaterial interactions.
  • Cells adhered and spread well on the substrates after 4 hrs and remained healthy for at least 2 days in culture (Fig. 7a), thus confirming the biocompatibility of the scaffolds.
  • Fig. 7b Internal actin stress fibers also aligned radially in these regions mimicking the underlying pattern of fibrin fibrils (Fig. 7b).
  • a novel fibrin-based biomaterial with defined anisotropy composed of oriented nanoscale fibrils.
  • These biocompatible materials were fabricated through magnetically-guided assembly of a 2D array of thrombin-coated magnetic microbeads that formed oriented fibrin fibrils through enzyme-initiated molecular self assembly in the local microenvironment of each bead.
  • These enzymatically-active beads may be used to form ordered scaffolds for a variety of applications including, analysis of cell and tissue development, tissue engineering and regenerative medicine, hemorrhage control, and vascular occlusion therapy for aneurysms or cancer, as well as microelectronics.
  • This magnetically-guided, biologically-inspired microfabrication system is unique in that large scaffolds may be formed with relatively little starting material, and thus it can be useful for in vivo tissue engineering applications.

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Abstract

L'invention concerne des procédés de production de structures utiles, par exemple, pour le génie tissulaire. De manière générale, les procédés décrits dans le présent document font appel à une activité enzymatique immobilisée sur une surface, qui est mise en contact avec une solution comprenant des monomères de polypeptides. L'activité de l'enzyme modifie les monomères de polypeptides pour favoriser la polymérisation des monomères afin de former des structures polymères associées à la surface. Les procédés décrits permettent ainsi un auto-assemblage guidé de structures de polypeptides. Selon certains modes de réalisation préférés, l'auto-assemblage est guidé pour former des structures régulières ou d'autres structures spécifiques en spécifiant la disposition des surfaces sur lesquelles l'enzyme, éventuellement avec un ou plusieurs co-facteurs, est fixée.
PCT/US2007/022397 2006-10-20 2007-10-22 Auto-assemblage guidé de matrices à structure nanométrique ordonnée utile pour le génie tissulaire Ceased WO2008127295A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015130745A1 (fr) * 2014-02-26 2015-09-03 Brigham And Women's Hospital, Inc. Système et procédé d'auto-assemblage magnétique
US10928404B2 (en) 2014-02-26 2021-02-23 The Brigham And Women's Hospital, Inc. System and method for cell levitation and monitoring

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US7615373B2 (en) * 1999-02-25 2009-11-10 Virginia Commonwealth University Intellectual Property Foundation Electroprocessed collagen and tissue engineering
US6811776B2 (en) * 2000-12-27 2004-11-02 The Regents Of The University Of Michigan Process for ex vivo formation of mammalian bone and uses thereof
US7338657B2 (en) * 2001-03-15 2008-03-04 Biosphere Medical, Inc. Injectable microspheres for tissue construction
US6993406B1 (en) * 2003-04-24 2006-01-31 Sandia Corporation Method for making a bio-compatible scaffold

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015130745A1 (fr) * 2014-02-26 2015-09-03 Brigham And Women's Hospital, Inc. Système et procédé d'auto-assemblage magnétique
US10208282B2 (en) 2014-02-26 2019-02-19 The Brigham And Women's Hospital, Inc. System and method for magnetic self-assembly
US10928404B2 (en) 2014-02-26 2021-02-23 The Brigham And Women's Hospital, Inc. System and method for cell levitation and monitoring
US12066445B2 (en) 2014-02-26 2024-08-20 The Brigham And Women's Hospital, Inc. System and method for cell levitation and monitoring

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